Journal für Hirnforschung: Band 19, Heft 1 1978 [Reprint 2021 ed.] 9783112522042, 9783112522035

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ISSN 0021-8359

Journal fur Hirnforschung mm m

Begründet von Cécile und Oskar Vogt Herausgeber: J. Anthony, Paris • A Hopf, Düsseldorf W.Kirsche, Berlin • J.Szentägothai, Budapest Schriftleitung: W.Kirsche, Berlin Wiss. Sekretär: J.Wenzel, Berlin

Akademie-Verlag • Berlin EVP25.-M -32105

Begründet von Cécile und Oskar Vogt Unter Mitwirkung des Cécile und Oskar Vogt Instituts für Hirnforschung in Düsseldorf und der Arbeitsgemeinschaft für vergleichende Neuroanatomie der Fédération mondiale de Neurologie (World Fédération of Neurology) Herausgeber: H . ADAM, Salzburg—Wien

O. S.

A D R I A N O W , Moskau J . A N T H O N Y , Paris J . A R I É N S K A P P E R S , Amsterdam E. C R O S B Y , Ann Arbor A . D E W U L F , Corbeck-Lo J . E S C O L A R , Zaragoza R. H A S S L E R , Frankfurt a. M.

E.

HERZOG,

Santiago

A. HOPF, Düsseldorf

J . J A N S E N , Oslo W. K I R S C H E , Berlin J . K O N O R S K I , Warschau S T . K Ö R N Y E Y , Pees M . M A R I N - P A D I L L A , Hanover-New Hampshire J . M A R S A L A , Kosice H. A. M A T Z K E , Lawrence D. M I S K O L C Z Y , Tirgu Mures G . P I L L E R I , Waldau-Bern T. O G A W A , Tokyo

Le JOURNAL F Ü R HIRNFORSCHUNG publiera des études sur la morphologie normale (anatomie, histologie, cytologie, microscopie électronique, histochimie), sur le développement du sytsème nerveux ainsi que des études anatomiques expérimentales. On acceptera aussi des travaux du caractère de la coopération entre des domaines différents à condition qu'ils contiennent des résultats morphologiques obtenus par les méthodes de la neuromorphologie et de la neurophysiologie ou de la neuropharmacologie et de la neurochimie. Les travaux doivent contenir des acquisitions nouvelles sur l'action réciproque entre la structure et la fonction. Des études neuropathologiques seront seulement acceptées quand elles contribuent à la conaissance des structures normales, des changements structurels ou de leur signification fonctionelle. Des études sur la localisation cérebrale de phénomènes expérimentaux ou cliniques d'exicitation ou de déficit (doctrine des localisations) seront également publiées par le JOURNAL F Ü R HIRNFORSCHUNG. Üne partie spéciale sera réservée à la neurobiologie comparée.

Bezugsmöglichkeiten des „Journal für Hirnforschung".

B . REXED, Upsala

Bestellungen sind zu richten

M. VOGT, Cambridge

— in der DDR an eine Buchhandlung oder an den AkademieVerlag, D D R - 108 Berlin, Leipziger Straße 3 — 4

H. S T E P H A N , Frankfurt a. M. J . S Z E N T Á G O T H A I , Budapest W. J . C. V E R H A A R T , Leiden F. W A L B E R G , Oslo K. G. W I N G S T R A N D , Kopenhagen E . W I N K E L M A N N , Leipzig W . W Ü N S C H E R , Berlin A . D . Z U R A B A S H V I L I , Tbilissi

Im JOURNAL F Ü R HIRNFORSCHUNG werden Arbeiten aus dem Gesamtgebiet der normalen Morphologie (Anatomie, Histologie, Cytologie, Elektronenmikroskopie, Histochemie) und der Entwicklungsgeschichte des Nervensystems unter Einschluß experimentell-anatomischer Arbeiten veröffentlicht. Es werden auch Arbeiten multidisziplinären Charakters aufgenommen, sofern sie morphologische Ergebnisse beinhalten, die mit neuromorphologischen und neurophysiologischen oder neuropharmakologischen bzw. neurochemischen Methoden gewonnen wurden und einen Erkenntnisgewinn hinsichtlich der Wechselwirkung zwischen Struktur und Funktion beinhalten. Neuropathologische Arbeiten werden nur angenommen, wenn sie Beiträge zur normalen Struktur, den Strukturwandlungen oder deren funktionellen Bedeutungen enthalten. Zum Publikationsgebiet des JOURNAL F Ü R HIRNFORSCHUNG gehören auch Arbeiten, die sich mit der Zuordnung experimenteller Reiz- und Ausfallerscheinungen bzw. klinischen Symptomen zu bestimmten Strukturen • des Gehirns („Lokalisationslehre") befassen. Als spezielles Publikationsgebiet ist die vergleichende Neurobiologie vorgesehen.

The JOURNAL F Ü R HIRNFORSCHUNG will publish studies on normal morphology (anatomy, histology, cytology, electron microscopy, histochemistry), on the development of the nervous system, as well experimental anatomical studies. Papers of multidisciplinary character will also be included so far as they contain morphological results which were obtained using neuromorphological and neurophysiological or neuropharmacological and neurochemical methods and provide further information on the interaction between structure and function. Neuropathological studies will only be published of they contribute to the knowledge of normal structures structurals changes or their functional significance. Papers dealing with the cerebral localization of experimental excitation and deficit phenomena or clinical symptoms (localization theory) will also be published by the J O U R N A L F Ü R HIRNFORSCHUNG. A special part of the publication is reserved for comparative neurobiology.

— im sozialistischen Ausland an eine Buchhandlung für fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb — in der B R D und Westberlin an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, Erich Bieber, 7 Stuttgart 1, Wilhelmstraße 4 - 6 — in Österreich an den Globus-Buchvertrieb, 1201 Wien, Höchstädtplatz 3 — im übrigen Ausland an den internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Republik, D D R - 701 Leipzig, Postfach 160, oder an den Akademie-Verlag, D D R - 108 Berlin, Leipziger Straße 3 bis 4 — in den übrigen westeuropäischen Ländern an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, Erich Bieber GmbH, CH - 8008 Zürich/Schweiz, Dufourstraße 51

Journal für Hirnforschung Herausgeber: Im Auftrag des Akademie-Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, D D R - 1 0 8 Berlin, Leipziger Straße 3 — 4; Fernruf: 2236 221 und 2236 229; Telex-Nr.: 114420. Bank: Staatsbank der DDR, Berlin, Kto.-Nr.: 6836-26-20712. Schriftleitung: Prof. Dr. sc. med. W. Kirsche, Berlin. Veröffentlicht unter der Lizenznummer 1326 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckhaus „Maxim Gorki", D D R - 74 Altenburg. Erscheinungsweise: Das Journal für Hirnforschung erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 180,— M zuzüglich Versandspesen (Preis für die D D R 150,— M); Preis je Heft 30,— M (Preis für die D D R 2 5 , - M). Bestellnummer dieses Bandes 1018/19 © 1979 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 60315

Inhaltsverzeichnis Band 19 — 1978 Dieser Band enthält 337 Abbildungen und 48 Tabellen

E . : s. unter

BOGONEZ,

L. M

GARCÍA-SEGURA,

423

BRAUER, K . , W . SCHOBER a n d E . W I N K E L M A N N :

Phy-

logenetical changes and functional specializations in t h e dorsal lateral geniculate nucleus (dLGN) of mammals. W i t h 16 figures 177 BRAUER,

K.: s. unter

LENKOV,

D. N

415

BRAUER, K . , L. LEIBNITZ, L . W E R N E R u n d E .

WINKEL-

MANN, Axonähnliche Gliafortsätze in der Pars ventralis des Corpus geniculatum laterale (CGLv) Mit 5 Abbildungen 533 s. unter

BRÜCKNER, G.:

LENKOV, D . N

415

und C. P F I S T E R , Fluoreszenzhistochemische und neurohistologische Untersuchungen zur adrenergen Innervation des Cortex pyriformis der R a t t e . Mit 5 Abbildungen 101

DANNER, H.,

Untersuchungen zur Embryonalentwicklung des Gehirns von Scyliorhinus canicula (L.) I. Bildung der Hirngestalt, Migrationsmodi und -phasen, Bau des Zwischenhirns. Mit 7 Abbildungen und 1 Tabelle 313

FARNER, H . - P . :

II. Das Tectum opticum und dessen Stratifikation. Mit 7 Abbildungen 333 I I I . Das optische System und angrenzende Nuclei im mesencephalen Tegmentum. Mit 5 Abbildungen 405 FERRES-TORRES, FRÖHLICH, J . :

R . : s. unter

s. unter

FROTSCHER, M.,

MEYER,

G

371

RUMMELFÄNGER, H

K . SCHARMACHER

und

291 M.

SCHARMA-

CHER, Zur umweltabhängigen Differenzierung von Pyramidenneuronen im Hippocampus (CA 1) der R a t t e . Die Differenzierung von apikalen Seitendendriten und Basaldendriten. Mit 7 Abbildungen und 4 Tabellen 445 GARCÍA-SEGURA, L . M.,

C. RODRIGUEZ-GONZALEZ

and

J . M. G O N Z A L E S - R O S , The effect of early experience on t h e exploratory behaviour, learning ability and on t h e synaptosomes of t h e mice brain. W i t h 4 figures and 1 table GARCÍA-SEGURA, BOGONEZ,

L. M.,

R . MARTINEZ-RODRIGUEZ,

R . MARTINEZ-MURILLO

and

A.

1. The range of dendrites of t h e central region neurons. W i t h 2 figures . . . 2. The range of dendrites of t h e paramedial region neurons. W i t h 3 figures 3. The range of dendrites of t h e lateral region neurons. W i t h 3 figures 4. The range of dendrites of the lateral horn neurons. W i t h 2 figures

145

HAJDU, F.:

s. unter

MADARASZ, M

159

HAJDU, F.:

s. unter

MADARASZ, M

193

TÖMBÖL, T

203

HAJDU,

F . : s. unter

C., und E. S C H U L Z : Quantitative Untersuchungen an Sternzellen im Bereich der cingulären Rinde der R a t t e . Mit 9 Abbildungen und 1 Tabelle 519

HERRMANN,

HEUMANN,

D.: s. unter

75

295

T . : s. unter

GROTTEL,

K

379

T.: s. unter

GROTTEL,

K

433

HOFMAN,

T.: s. unter

GROTTEL,

K

439

M.: Morphogenesis of cerebral m a t r i x ectopies in h u m a n fetus and newborn. W i t h 12 figures and 4 tables 485

HRABOWSKA,

IBANEZ,

A. C., S. unter Ruiz, B. F s. unter

ITAKURA, T . :

TOHYAMA,

J . : s. unter

479

M

NARKIEWICZ,

165 0

133

and J . N A I T O : Variations of the dog cerebral sulci, compared in particular with those of t h e cat. W i t h 7 figures 457 and

Afferent fiber connections from t h e lower brain stem to the r a t cerebellum by the horseradish peroxidase method combined with MAO staining, with special reference to noradrenergic neurons. W i t h 8 figures and 3 tables N . SHIMIZU:

TÖMBÖL, T

145 159

KINNEY,

GERLE,

J., s. unter

MADARÀSZ, M

193

TÖMBÖL, T

203

and T . H O F M A N : Dendritic range-of the neurons of t h e intermediate gray a t t h e levels of t h e first and second lumbar segment of t h e spinal cord in the cat.

GROTTEL, K

HOFMAN,

MADARÀSZ, M

GROTTEL, K . ,

301

HOFMAN,

J . : s. unter

.

G

KIMOTO, Y . , K . SATOH, T . SAKUMOTO, M . TOHYAMA

E.

TOLE-

GARCIA-SEGURA/L. M.

s. unter

HOFMAN, T . :

J . : s. unter

s. unter

LEUBA,

D., G. L E U B A and Th. R A B I N O W U C Z , Postn a t a l development of t h e mouse cerebral neocortex. IV. Evolution of t h e total cortical volume, of the population of neurons and glial cells. W i t h 3 figures and 2 tables 385

HEUMANN,

GERLE,

M.:

539

TÖMBÖL, T

GERLE,

J.

439

s. unter

KIMOTO, Y . :

GONZALES-ROS,

433

KAWAMURA, K . ,

: Evidences for the existence of glycoproteins and mucopolysaccharides metabolic cycles in the cerebellum of birds and mammals. W i t h 11 figures and 2 tables 423

s. unter

KOZIK, M. B

379

HAJDU, F.:

JURANIEC,

DANO

GERLE, J . :

s. unter

GROTTEL, K . :

295

75

s. unter

TOHYAMA, M

F . C.: An experimental Study of the Central Gustatory P a t h w a y s in the Monkey, Macaca mul a t t a and Cercopithecus aethiops. W i t h 19 figures

85 165

21

KIP, G.: Qualitative und quantitative Untersuchungen des Corpus geniculatum laterale an einer ontogenetischen Reihe von männlichen Tupaia belangen. Mit 22 Abbildungen und 4 Tabellen . . . 345 Jänos Szentägothai zur Vollendung des 65. Lebensjahres. Mit 1 Foto 189

KIRSCHE, W . :

Inhaltsverzeichnis B a n d 19 and K . G R O T T E L : Alterations of dendritic spines following intoxication b y mercury phenylacetate. W i t h 3 figures and 1 table 539

KOZIK,M. B.,

s. unter

LEIBNITZ, L . :

LENKOV, D . N.,

BRAUER, K

E . WINKELMANN,

533 K . BRAUER

and

G., D . H E U M A N N and Th. R A B I N O W I C Z : Postn a t a l development of t h e mouse cerebral neocortex. I I I . Some dynamical aspects. W i t h 3 figures and 2 tables 301

LEUBA,

G.: s. unter

HEUMANN, D

LODHA,

M.: s. unter

SOOD,

MADARÁSZ, M . ,

s. unter

P. 0

TÖMBÖL, T

unter

MARTINEZ-RODRIGUEZ,

GARCIA-SEGURA, L . M .

s; unter

s. unter Ruiz,

5

SOMOGYI,

Gy.: s. unter

TÖMBÖL, T

203

423

K

457

B. F

479 The

Distribution of Axon Terminals with Flattened Vesicles in t h e Nuclei of the Amygdaloid Body of t h e Cat. W i t h 11 figures and 1 table 133 C.: s. unter

DANNER,

H

101

J . C.: The Tectum opticum of Pantodactylus schreiberii Wiegmann (Teiidae, Lacertilia, Reptilia). W i t h 12 figures 109

QUIROGA,

RABINOWICZ,

T h . : s. unter

LEUBA,

RABINOWICZ,

T h . : s. unter

HEUMANN,

RODRIGUEZ-GONZALES, C.:

G

s. unter

L. M RUIZ,

301 D

385

GARCÍA-SEGURA,

75

B. F., I . S. N A J E R A and A. C. I B A N E Z : An ultrastructural study of the subependymal plate in the hamster. W i t h 7 figures 479

SOOD,

P. P. and M. L O D H A : A comparative histochemical mapping of ATPase and 5-Nucleotidase in the medulla oblongata, spinal cord and cerebellum of mouse. W i t h 28 figures and 2 tables

H., und J . F R Ö H L I C H : Autoradiographischer Nachweis von tritiiertem Cholinchlorid im Hirngewebe. Eine Methode zum lichtoptischen Nachweis im Paraffinschnitt. Mit 1 Abbildung . 291 T . : s. unter

SAKUMOTO, T . ,

s. unter

KIMOTO,

5

U.: Development of the sensory systems in the larval and metamorphosing European grass frog (Rana temporaria L.). W i t h 17 figures and 3 tables 543

SPAETI,

SCHARMACHER, K .

SCHULZ,

und

E . : S. unter

s. unter

M.:

s. unter

SCHOBER, W . :

FROTSCHER, M .

.

.

445

BRAUER, K

HERRMANN,

177

C

519

T h . : Experimental alterations in number and length of different membrane complexes on axosomatic contacts in the t r o u t (Salmo irideus, Gibbons 1855). W i t h 10 figures and 7 tables . . . .

TAKAHASHI, Y . : TAMBOISE,

Y

TOHYAMA,

85 M

165

s. unter

T . : s. unter

TOHYAMA,

SAVY,

M

45 165

C

469

TÖMBÖL, T . , M . MADARÁSZ, F . H A J D U , G y . SOMOGYI,

J.

G E R L E , Quantitative histological studies on the lateral geniculate nucleus in the cat. I. Measurements on Golgi material. W i t h 12 figures . . . . 1 4 5 TÖMBÖL, T . :

s. unter

MADARÁSZ, M

159

TÖMBÖL, T . :

s. unter

MADARÁSZ, M

193

TÖMBÖL,

T.,

M.

MADARÁSZ,

G y . SOMOGYI,

F.

HAJDU,

: Quantitative histological studies on the lateral geniculate nucleus in t h e cat. IV. Numerical aspects of t h e transfer from retinal fibers to cortical relay. W i t h 4 figures 203 J. GERLE

TOHYAMA, M . :

s. unter

KIMOTO, Y

85

TOHYAMA, M . , K . SATOH, T . SAKUMOTO, Y . KIMOTO,

Y.

and T . I T A K U R A : Organization and projections of the neurons in t h e dorsal tegmental area of t h e rat. W i t h 8 figures . 165 TAKAHASHI, K . YAMAMOTO

TOLEDANO, WELENXO,

A.: s. unter

GARCÍA-SEGURA, L .

M

J . : I n memoriam Marian Chomiak

WERNER, L.:

RUMMELFÄNGER,

SAKUMOTO,

145 193

GARCÍA-SEGURA,

NARKIEWICZ, O., J. JURANIEC a n d T . WRZOLKOWA:

PFISTER,

85

TÖMBÖL, T

159

und R . F E R R E S - T O R R E S : Quantitative altersabhängige Variationen der Dendritenspines im Hippocampus (CA 1, CA 3 und Fascia Dentata) der Albinomaus. Mit 5 Abbildungen 371

NAJERA, I. S.:

KIMOTO, Y

Gy.: s. unter

SOMOGYI,

MADARÁSZ, M

MEYER, G.,

KAWAMURA,

s. unter

SHIMIZU, N . :

MADARÁSZ, M

423

J . : S. unter

Divergent responses to thyroid hormone t r e a t m e n t of t h e different secondary germinal layers in t h e postnatal r a t brain. W i t h 11 figures 395

SERESS, L . :

Gy.: s. unter

203

L. M

NAITO,

C. et E. T A M B O I S E : Effets cytologiques de divers fixateurs sur les neurones du ganglion de Gasser du foetus de rat. Avec 15 figures et 2 tableaux . 469

SCHUSTER,

s. unter R.:

165

Gy.: s. unter

MAI, J . K.: The Accessory Optic System and t h e RetinoHypothalamic System. A Review. W i t h 2 figures and 1 table 213 MARTINEZ-MURILLO, R . :

85

M

SOMOGYI,

and

TÖMBÖL, T

KIMOTO, Y TOHYAMA,

SOMOGYI,

T . T Ö M B Ö L : Quantitative histological studies on t h e lateral geniculate nucleus in t h e cat. II. Cell numbers and densities in t h e several layers. W i t h 3 figures and 3 tables 159 I I I . Distribution of different types of neurons in t h e several layers of LGN. W i t h 6 figures . . . . 1 9 3 M A D A R Á S Z , M . , S.

s. unter

K . : S. unter

385

145

MADARÁSZ, M . , J . G E R L E , F . H A J D U , G y . SOMOGYI

SAVY,

G.

Vesicle size in basic classes of synapses in t h e r a t ' s lateral geniculate nucleus. W i t h 415 3 figures BRÜCKNER,

LEUBA,

SATOH, K . : SATOH,

s. unter

BRAUER, K

423 . . .

.289 533

WINKELMANN,

E . : s. unter

BRAUER,

K

177

WINKELMANN,

E . : s. unter

LENKOV,

D. N

415

WINKELMANN,

E . : S. unter

BRAUER,

K

533

WRZOLKOWA,

T.: S. unter

NARKIEWICZ, 0

133

Inhaltsverzeichnis B a n d 19 WÜNSCHER, W . : I n memoriam Richard Arwed Pfeifer. Mit 1 Foto YAMAMOTO, K . : s. u n t e r TOHYAMA, M

SCHIERHORN, H . :

1 165

Qualitative und quantitative Untersuchungen des Corpus mamillare an ontogenetischen Reihen von männlichen Tupaia belangen und SPFKatzen. Mit 17 Abbildungen und 4 Tabellen . . 497

ZEIMER, H . :

W . KIRSCHE:

Christian

Gottfried

Ehrenberg zum 100. Todestag

384

SCHMIDT, J . : Nonstriatal Dopaminergic Neurons . . SCHÖNHEIT, B . : S. N . OLENEV:

(Das Gehirn in der

.188

Ent-

wicklung. Zelluläre, molekulare und genetische Aspekte der Neuroembryologie). (russ.) . . . . 484 SCHULZ, E . : Architectonics of the Cerebral Cortex. . . 421 A. F . : S U C H E N W I R T H , R . : Taschenbuch der klinischen Neurologie. 404

SCHULZE, H .

Buchbesprechungen BRAUER, K . :

DUVERNOY,

H. M.:

Human

SCHULZE, H .

Brainstem

Vessels

532

Paraneurons. New Concepts on Neuroendocrine Relatives. Proceedings of the Symposium on t h e Paraneurons, Morioka, Japan, April 1st, 1977 468

DÖCKE, F . :

HECHT, K.: Brain and H e a r t Infarct MATTHIES, H . - J . : GARRATINI, S . ; PUJOL, J . F . ;

542 SAMO-

NIN, R. (Hrsg.) Interactions between putative neurotransmitters in t h e brain 384 OEHME, P . : Substance P. Nobel Symposium 37. . . . 1 8 8 Principles of Elektrolocation and J a m m i n g Aviodance in Electric Fish 404

RÜDIGER, W . : HEILIGENBERG, W . :

A. F . : T r e a t m e n t of Neuromuscular. . . 444

SCHULZE, H . A . F . : REDING, R i c h a r d u n d G ü n t h e r LANG

(Hrsg.): Schädel-Hirn-Trauma und Kombinationsverletzungen 576 STOBBE, H . :

KÖLMEL,

Fluid Cells

H. W.:

Atlas of Cerebrospinal 576

UNGER: Advance in Neurosurgery, Vol. 4. L u m b a r Disc. Adult Hydrozephalus 394 Afferent and Intrinsic Organization of Laminated Structures in t h e Brain. 7th International Neurobiology Meeting 438

WENZEL, J . :

WINKELMANN, E . : Physiology and Pathobiology of Axons. Edited by Stephen P. WAXMAN 496

ISSN 0021-8359

Journal für Hirnforschung ° \mm n®

DÄmüit m a l e s J®y maßfìw-INfe yroM®ß©gS(i Begründet von Cécile und Oskar Vogt

Redaktion: J. Anthony, Paris • A. Hopf, Düsseldorf W Kirsche, Berlin J.Szentägothai, Budapest Wis&Sekretär: J.Wenzel, Berlin

Akademie-Verlag • Berlin EVP25.-M-32105

Begründet von Cécile und Oskar Vogt Unter Mitwirkung des Cécile und Oskar Vogt Instituts für Hirnforschung in Düsseldorf und der Arbeitsgemeinschaft für vergleichende Neuroanatomie der Fédération mondiale de Neurologie (World Fédération of Neurology) Herausgeber: H . ADAM, S a l z b u r g — W i e n O . S . ADRIANOW, M o s k a u J . ANTHONY, P a r i s J . ARIÉNS KAPPERS,

Amsterdam

E . CROSBY, A n n A r b o r A . DEWULF, C o r b e c k - L o J . ESCOLAR, Z a r a g o z a R . HASSLER, F r a n k f u r t a . M . E . HERZOG, S a n t i a g o A . HOPF, D ü s s e l d o r f J . JANSEN, Oslo W . KIRSCHE, B e r l i n J . KONORSKI, W a r s c h a u ST. KÖRNYEY, P é c s

Le J O U R N A L F Ü R H I R N F O R S C H U N G publiera des études sur la morphologie normale (anatomie, histologie, cytologie, microscopie électronique, histochimie), sur le développement du système nerveux ainsi que des études anatomiques expérimentales. On acceptera aussi des travaux du caractère de la coopération entre des domaines différents à condition qu'ils contiennent des résultats morphologiques obtenus par les méthodes de la neuromorphologie et de la neurophysiologie ou de la neuropharmacologie et de la neurochimie. Les travaux doivent contenir des acquisitions nouvelles sur l'action réciproque entre la structure et la fonction. Des études neuropathologiques seront seulement acceptées quand elles contribuent à la conaissance des structures normales, des changements structurels ou de leur signification fonctionelle. Des études sur la localisation cérebrale de phénomènes expérimentaux ou cliniques d'excitation ou de déficit (doctrine des localisations) seront également publiées par le J O U R N A L F Ü R HIRNF O R S C H U N G . Une partie spéciale sera réservée à la neuro-, biologie comparée.

M. MARIN-PADILLA, Hanover-New Hampshire J . MARSALA, K o s i c e H . A . MATZKE, L a w r e n c e

D. MISKOLCZY, Tirgu Mures G. PILLERI, Waldau —Bern

T . OGAWA, T o k y o B . REXED, Upsala H . STEPHAN, F r a n k f u r t a. M . J . SZENTÁGOTHAI, B u d a p e s t W . J . C. VERHAART, L e i d e n M . VOGT, C a m b r i d g e . F . WALBERG, Oslo K . G . WINGSTRAND, K o p e n h a g e n E . WINKELMANN, L e i p z i g W . WÜNSCHEK, B e r l i n A . D . ZURABASHVILI, T b i l i s s i

I m J O U R N A L F Ü R H I R N F O R S C H U N G werden Arbeiten aus dem Gesamtgebiet der normalen Morphologie (Anatomie, Histologie, Cytologie, Elektronenmikroskopie; Histochemie) und der Entwicklungsgeschichte des Nervensystems unter Einschluß experimentell-anatomischer Arbeiten veröffentlicht. E s werden auch Arbeiten multidisziplinären Charakters aufgenommen, sofern sie morphologische Ergebnisse beinhalten, die mit neuromorphologischen und neurophysiologischen oder neuropharmakologischen bzw. neurochemischen Methoden gewonnen wurden und einen E r kenntnisgewinn hinsichtlich der Wechselwirkung zwischen Struktur und Funktion beinhalten. N^uropathologische Arbeiten werden nur angenommen, wenn sie Beiträge zur normalen Struktur, den Strukturwandlungen oder deren funktionellen Bedeutungen enthalten. Zum Publikationsgebiet des J O U R N A L F Ü R H I R N F O R S C H U N G gehören auch Arbeiten, die sich mit der Zuordnung experimenteller Reiz- und Ausfallerscheinungen bzw. klinischen Symptomen zu bestimmten Strukturen des Gehirns („Lokalisationslehre") befassen. Als spezielles Publikationsgebiet ist die vergleichende Neurobiologie vorgesehen. T h e J O U R N A L F Ü R H I R N F O R S C H U N G will publish studies on normal morphology (anatomy, histology, cytology, electron microscopy, histochemistry), on the development of the nervous system, as well experimental anatomical studies. Papers of multidisciplinary character will also be included so far as they contain morphological results which were obtained using neuromorphological and neurophysiological or neuropharmacological and neurochemical methods and provide further information on the interaction between structure and function. Neuropathological studies will only be published of they contribute to the knowledge of normal structures structurals changes or their functional significance. Papers dealing with the cerebral localization of experimental excitation and deficit phenomena or clinical symptoms (localization theory) will also be published by the J O U R N A L F Ü R H I R N F O R S C H U N G . A special part of the publication is reserved for comparative neurobiology.

Bezugsmöglichkeiten des „Journal für Hiniforschung". Bestellungen sind zu richten — in der D D R an eine Buchhandlung oder an den AkademieVerlag, D D R - 108 Berlin, Leipziger Straße 3 - 4 — im sozialistischen Ausland an eine Buchhandlung für fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb / — in der B R D und Westberlin an eine Buchhandlung oder an die Auslieferungsstelle K U N S T UND W I S S E N , Erich Bieber, 7 Stuttgart 1, Wilhelmstraße 4 - G — in Österreich an den Globus-Buchvertrieb, 1201 Wien, Höchstädtplatz 3 — im übrigen Ausland an den Internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Republik, D D R - 701 Leipzig, Postfach 160, oder an den Akademie-Verlag, D D R - 108 Berlin, Leipziger Straße 3 bis 4

Journal für Hirnforschung Herausgeber: Im Auftrag des Akademie-Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, D D R - 1 0 8 Berlin, Leipziger Straße 3 - 4 ; Fernruf: 2 20 04 41; Telex-Nr.: 114420; Postscheckkonto: Berlin 350 21. B a n k : Staatsbank der D D R , Berlin, K t o . - N r . : 6836-26-20712. Chefredaktion: Prof. Dr. J . Anthony, Paris; Prof. Dr. A. Hopf, Düsseldorf; Prof. Dr. W. Kirsche, Berlin; Prof. Dr. J . Szentdgothai, Budapest. Wissenschaftlicher Sekretär: Dr. Jürgen Wenzel, Berlin. Veröffentlicht unter der Lizenznummer 1326 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckhaus „Maxim Gorki", D D R - 74 Altenburg. Erscheinungsweise: Das Journal für Hirnforschung erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 180,— M zuzüglich Versandspesen (Preis für die D D R 150,— M); Preis j e Heft 3 0 , - M (Preis für die D D R 2 5 , - M). Bestellnummer dieses Heftes 1018/19/1. © 1977 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 60315

ISSN 0021-8359

Journal

. Hirnforsch. 19 (1978) 1 - 3

Internationales Journal für Neurobiologie

f Q y

Hirnforschung

Heft 1 • 1978 - B a n d 19

michnbahn

Tractus opticus Capsula

interna

?

In memoriam Richard Arwed Pfeifer In diesem Jahr gedenken wir des 100. Geburtstages von Richard Arwed P F E I F E R , dessen Name mit der Entwicklung der Hirnforschung und der Nervenheilkunde eng verbunden ist. Der Lebensweg und der Bildungsgang P F E I F E R S sind für seine Zeit ungewöhnlich gewesen. Als Sohn einer kinderreichen Bergmannsfamilie in Brand bei Freiberg in Sachsen am 21.11. 1877 geboren, besuchte er nach der Volksschule von 1892 bis 1898 das Lehrerseminar in Nossen. Danach trat P F E I F E R als Hilfslehrer in den Schuldienst ein. E r wurde ohne. Abitur auf Grund vorzüglicher Zeugnisse zum Universitätsstudium der Pädagogik im Interesse der Erlangung einer höheren Berufsausbildung zugelassen. Von 1901 bis 1907 studierte P F E I F E R Naturwissenschaften und Mathematik an der Universität Leipzig. Himforschuug, Bd. 19, Heft 1

Im Jahre 1905 promovierte er bei dem Psychologen zum Doktor der Philosophie. Nach Ablegung der Staatsprüfung für das höhere Lehramt wirkte P F E I F E R als Oberlehrer in Rochlitz, Bautzen und Dresden. Sein Studium leitete einen bedeutsamen Schritt für sein weiteres Leben ein. Durch seine Doktorarbeit „Über Tiefenlokalisation von Doppelbildern" kam er mit dem Physiologen HERING und dem Psychiater FLECHSIG in Berührung. Diese Gelehrten erkannten P F E I F E R S hohe Begabung für Naturwissenschaften und Medizin. Besonders FLECHSIG regte den jungen Oberlehrer an, Medizin zu studieren. P F E I F E R selbst fühlte sich zum Arzt berufen. E r verließ 1910 den Schuldienst und bestand als 34j ähriger die Reifeprüfung am Realgymnasium Dresden-Blasewitz. AnWUNDT

1

2

WÜNSCHER,

W.

schließend erfolgte in Leipzig und München sein Medizinstudium. In dieser Zeit vertiefte P F E I F E R , der 1915 sein ärztliches Staatsexamen ablegte, seine Beziehungen zu den Arbeiten FLECHSIGS, dem ersten Direktor der 1882 gegründeten Universitätsnervenklinik Leipzig. FLECHSIG vertrat den besonders von G R I E SINGER postulierten Standpunkt „Geisteskrankheiten sind Krankheiten des Gehirns." Entsprechend dieser Auffassung erhielt die Nervenklinik den Vorläufer des späteren Hirnforschungsinstitutes, das hirnanatomische Laboratorium. Hier führte P F E I F E R seine mye ogenetisch-anatomischen Untersuchungen für seine Dissertation „Über den feineren Bau des Zentralnervensystems eines Anencephalus" durch, mit der er 1915 zum Doktor der Medizin promovierte. Nach Beendigung des ersten Weltkrieges und des militärärztlichen Dienstes in einem Lazarett für Hirnverletzte trat P F E I F E R 1919 in die Universitätsnervenklinik Leipzig ein. Er übernahm 1920 nach dem Ausscheiden von FLECHSIG die Leitung des hirnanatomischen Laboratoriums. Seine Ernennung zum außerplanmäßigen außerordentlichen Professor für Psychiatrie und Neurologie erfolgte im Jahr 1924. Unter dem Einfluß der Lehre FLECHSIGS wandte sich zunächst weiteren myelogenetisch-anatomischen Forschungen zu. Es entstand die myeloarchitektonische Bearbeitung des zentralen Abschnittes der Hörleitung — mit dieser Arbeit habilitierte er sich 1920 — der Seh- (1925) und der Tastleitung (1934). In Erweiterung seines Arbeitsgebietes publizierte P F E I F E R eine Pathologie der Seh- (1930) und eine solche der Hörstrahlung (1936) einschließlich der kortikalen Seh- und Hörsphäre. Untrennbar ist sein Name mit der Angioarchitektonik des Gehirns verbunden. Mit einem von ihm erarbeiteten Verfahren gelang es P F E I F E R , eine Darstellung der Gehirngefäße in einer Vollständigkeit zu erreichen, die heute noch unübertroffen ist. Seine Angioarchitektonik hat den Ruf P F E I F E R S als Hirnforscher in der Welt besonders bekannt gema;ht. P F E I F E R führte den Nachweis, daß die Gefäße des Gehirns ein enges zusammenhängendes Netz bilden. Damit war die bis dahin allgemein anerkannte Lehre von den Cohnheimschen Endarterien und die daraus gezogenen Schlüsse für die Entstehung der Hirnerweichung unhaltbar geworden. Die ersten Ergebnisse seiner angioarchitektonischen Untersuchungen veröffentlichte P F E I F E R 1927, in einem Jahr, das für ihn von hoher Bedeutung war. Er erhielt die erste planmäßige außerordentliche Professur für Hirnforschung in Deutschland und seine Wirkungsstätte hieß seit dem 8. 6. 1927 „Hirnforschungsinstitut der Universität Leipzig". P F E I F E R vertrat stets die Ansicht, daß die HirnPFEIFER

forschung sowohl der „zweckfreien" Grundlagenforschung als auch der Bearbeitung von der Klinik gestellter Probleme zu dienen habe. In dieser Meinung wurde er durch seine jahrzehntelange Tätigkeit als Nervenarzt bestärkt. Unter den Kliniknachfolgern FLECHSIGS, B U M K E und SCHRÖTER, leitete P F E I F E R außerdem die Kinderabteilung der Nervenklinik. Hier wurde seine Auffassung von den psychischen Störungen und Erkrankungen des Menschen und deren Entstehen wesentlich mitbestimmt. P F E I F E R warnte seine Schüler vor unzulässiger Vereinfachung im Sinne eines psychophysischen Parallelismus. Er wies auf das Multifaktorielle im Ursachen- und Bedingungsgefüge der psychopathologischen Zustände hin. Für die psychiatrische Forschung forderte er die Berücksichtigung der naturwissenschaftlichen, psychologischen und pädagogischen Erkenntnisse. Seine Vorbildung als vorzüglicher Lehrer und sein warmherziges Wesen, die sich in seinem geschickten Umgang mit psychisch Kranken kundgaben, machten ihn frühzeitig auf die Bedeutung psychosozialer Einflüsse für das Auftreten psychischer Störungen und Erkrankungen aufmerksam. Seine Tätigkeit in der Kinderabteilung veranlaßte P F E I F E R , eine Lehre der medizinischen Pädagogik zu schaffen, deren Grundsätze noch heute ihre Gültigkeit besitzen. Ein weiterer Höhepunkt des arbeitsreichen Lebens P F E I F E R S lag in der Zeit nach dem zweiten Weltkrieg. Am 4. 12. 1943 zerstörte der britische Luftangriff die Nervenklinik und das Hirnforschungsinstitut der Universität. In neugewonnenen Räumen wurde am 1. 7. 1947 das Hirnforschungsinstitut praktisch wieder gegründet, für dessen Aufbau P F E I F E R kein persönliches Opfer scheute. Durch die Errichtung einer neuropathologischen Prosektur wurden auch im Interesse der Klinik die Aufgaben des Institutes erweitert. Die Kräfte von P F E I F E R wurden in besonderem Maße durch die Leitung der Universitätsnervenklinik gefordert, die ihm als 69jähriger im Feburar 1946 kommissarisch übertragen wurde und die er bis zum 31. 5. 1952 innehatte. Mit der Rekonstruktion dieser Klinik erwarb sich P F E I F E R einen weiteren bleibenden Verdienst. Trotz aller Belastungen widmete er sich seinen wissenschaftlichen Untersuchungen, wie die in diesen Jahren erschienenen Veröffentlichungen belegen. P F E I F E R war ein gütiger Lehrer, der seinen Schülern aufgeschlossen begegnete. Sein hohes ärztliches Ethos, sein pädagogisches Geschick und sein Forscherdrang sind für seine Umgebung stetige Eindrücke. Die ihm gebührende Anerkennung fand in den ihn zuteil gewordenen Ehrungen auch äußerlich ihren Niederschlag. Am 17.3. 1932 erfolgte die Wahl P F E I F E R S zum Mitglied der Deutschen Akademie der Naturforscher (Leopoldina) und am 8. 11. 1949 zum

In memoriam R .

Mitglied der Sächsischen Akademie der Wissenschaften. Die Verleihung des Nationalpreises der Deutschen Demokratischen Republik im Jahre 1954 stellte die Krönung seiner Wertschätzung dar. Nach langer, schwerer, mit der ihm eigenen Geduld getragenen Krankheit verstarb P F E I F E R am 15. 3.

A. PFEIFER

3

1957. In seinem kurz bemessenen Ruhestand konnte er noch die von ihm angeregte Abtrennung des Hirnforschungsinstitutes von der Nervenklinik und seine Verselbständigung erleben. W . WÜNSCHER

l*

J . Hirnforsch. 19 (1978) 5 - 2 0

D e p a r t m e n t of Zoology, U n i v e r s i t y of U d a i p u r , U d a i p u r - 313001, I n d i a

A comparative histochemical mapping of ATPase and 5-Nucleotidase in the medulla oblongata, spinal cord and cerebellum of mouse. B y P . P . SOOD a n d M a n j u l a L O D H A

W i t h 28 figures a n d 2 tables (Received 19 t h J a n u a r y 1977)

Summary: I n t h e present c o n t r i b u t i o n a c o m p a r a t i v e s t u d y of histochemical m a p p i n g of t h e d i s t r i b u t i o n of adenosine t r i p h o s p h a t a s e a n d 5-nucleotidase in t h e hind b r a i n of mouse has been made. There a r e m a n y similarities a n d dissimilarities between t h e distribution of these t w o enzymes in various nuclei, t r a c t s a n d fiber b u n d les. T h e n o t e w o r t h y differences are as follows: — 1. T h e AP, N D N V , N I , N N H , N O AD, NOAM, N O I , N P C , T S a n d SG are v e r y intensely positive for A T P a s e whereas, in 5-NUC s t u d y none of these nuclei d e m o n s t r a t e s i n t e n s i t y of such degree. 2. Nucleus a m b i g u u s is intensely positive for A T P a s e a n d is completely negative for 5-NUC. 3. T h e nucleus n. facialis is intensely positive for A T P a s e a n d is m o d e r a t e l y positive for 5-NUC. 4. NC, GN, P N , P C I a n d T C are completely negative for 5-NUC. I n A T P a s e p r e p a r a t i o n s only G N is n e g a t i v e a n d t h e rest of t h e areas d e m o n s t r a t e i n t e n s i t y of various degrees. Along w i t h these differences, similarities in t h e i n t e n s i t y of b o t h enzymes in various nuclei, t r a c t s a n d fibrous bundles also exist. An a t t e m p t has been m a d e t o correlate all t h e aforesaid differences a n d similarities in t h e distribution of these t w o enzymes w i t h t h e f u n c t i o n a l n a t u r e of t h e various areas of hind b r a i n of mouse.

Acknowledgement

Material and Methods

T h e a u t h o r s express their sincere g r a t i t u d e t o Dr. H . B. TEWARI, Senior Professor, D e p a r t m e n t of Zoology, Univ e r s i t y of U d a i p u r for general supervision a n d p r o v i d i n g t h e l a b o r a t o r y facilities.

Locally collected y o u n g mice were used in this s t u d y . A f t e r decapitation, t h e h i n d b r a i n w a s dissected o u t a n d fixed for 18 h o u r s in 1 0 % n e u t r a l formalin chilled a t 4°C. 40 ¡i serial sections were c u t on a freezing microtome, washed in distilled w a t e r a n d t h e n i n c u b a t e d for l 1 / 2 h o u r s a t 37 °C for 5-nucleotidase a n d A T P a s e ( W A C H S T E I N a n d M E I S E L , 1957 techniques). P r o p e r controls were also m a d e simultaneously. T h e nuclei, t r a c t s a n d fibrous b u n d l e s were identified on t h e basis of S I D M A N e t al. (1971).

Introduction The present work is a continuation of the efforts made by this laboratory to describe and compare the distribution of various oxidative and hydrolytic enzymes in the hind brain of mouse (SOOD and BOHRA, 1977; and S O O D and MULCHANDANI, 1977). Earlier, quite a number of studies have been made on the distribution of ATPase and 5-nucleotidase in the hind brain of rat and squirrel monkey (TEWARI and B O U R N E , 1963a, b; N A N D Y and BOURNE, 1964; SHANTHA e t a l . , 1 9 6 7 ; MANOCHA e t a l . , 1 9 6 7 ; MANO-

CHA, 1970). Comparative histoenzymological mapping of ATPase and 5-nucleotidase is lacking in literature, hence the present authors were prompted to take up study of histochemical mapping of these enzymes in serial sections of medulla oblongata, spinal cord and cerebellum. Furthermore, this study also provides information on the exact location of these enzymes in the various nuclei, tracts and fibrous bundles of the hind brain of mouse.

Results A. Spinal

Cord:

Much variation in the activities of 5-NUC and ATPase has been observed in spinal cord of the mouse. As for example, the nucleus dorsalis (ND; Fig. 14); Nucleus ventromedialis (NV; Fig. 14); Nucleus n. accessorii (NNA; Fig. 14) and nucleus reticularis (NRS; Fig. 14) are intensely positive for 5-NUC, whereas the same nuclei are moderately positive for ATPase (ND, NV, NNA, NRS; Fig. 1). A reverse condition is seen in substantia gelatinosa which is very intensely positive for ATPase (SG; Fig. 1) and moderately positive for 5-NUC (SG; Fig. 14). The nucleus cornucommissuralis dorsalis (DCN; Figs. 1,14); nucleus proprius cornus dorsalis (DN; Figs. 1, 14) and nucleus cornucommissuralis ventralis (VCN; Figs. 1, 14) are moderately positive for both enzymes. Nucleus posteromarginalis

6

Sood, P. P., and M. Lodha

RS

NC NN A VCN

Fig. 1. Transverse section of spinal cord (ATPase reaction).

NN H

NINH

NCOV TSV

NOA

©

M

NRP

Fig. 2. Transverse section of medulla oblongata at the level of area postrema (ATPase reaction).

NCOD

NRO © NS N A

NCOV

TC

NOAM

(PN; Fig. 1) and nucleus cervicalis lateralis (NC; Fig. 1) are mildly positive for ATPase and completely negative for 5-NUC (PN, NC, Fig. 14). It is interesting to note that unlike other nuclei, nucleus gracilis is almost negative for both the enzymes (GN; Figs. 1, 14). B.

Cerebellum:

In ATPase preparations, amongst the 3 layers of cerebellum, the most intense reaction is seen in the molecular layer (ML; Fig. 11) and the Purkinje layer (PL; Fig. 11). On the other hand, the granular layer shows moderate activity (GL; Fig. 11). The white matter demonstrates mildly positive fibers (NFL;

Fig. 3. Transverse section of medulla oblongata passing at the level of nucleus ambiguus (ATPase reaction).

Fig. 11). Like ATPase preparations the molecular layer is also intensely positive for 5-NUC (ML; Fig. 21) and the granular layer exhibits moderate activity (GL; Fig. 21), whereas the Purkinje layer demonstrates mild activity (PL; Fig. 21). The white matter (NFL) shows negligible activity. The central lobes of cerebellum show stronger activity of 5-NUC than the peripheral lobes (see Figs. 22, 23). C. Medulla

Oblongata:

Nucleus n. hpyoglossi:

In the ATPase preparations the boundaries of this nucleus are very sharp due to its intense activity (NNH; Figs. 2 — 5). The same nucleus is mode-

Histochemical mapping of the CNS

7

NCOP

NOAD

NCOV

Fig. 4. Transverse section of medulla oblongata at the region of maximum development of olivary complex (ATPase reaction).

NVM

Fig. 5. Transverse section of medulla oblongata at the level of nucleus paragigantocellularis lateralis (ATPase reaction).

NCOV NPH

Fig. 6. Transverse section of medulla oblongata at the level of nucleus prepositus hypoglossi (ATPase reaction).

rately positive for 5-NUC (NNH; Figs. 16, 17). In histological studies, two cell groups — a rostromedial group and a lateral group, have been identified. In both the histoenzymological preparations, however, the reactions are uniformly distributed, hence it has not been possible to differentiate these two cell groups. Nucleus interfascicularis n. hypoglossi:

The neurons of this nucleus are arranged along the intramedullary hypoglossal nerve. The nucleus de-

monstrates intense activity both for 5-NUC (NINH; Fig. 16) and ATPase (NINH; Figs. 2, 3, 12, 13). Further, the boundaries of the nucleus are not distinct in the present study, as all the surrounding nuclei also demonstrate activity of the same nature. Nucleus

intercalatus:

This nucleus is located between the nucleus n. hypoglossi and the nucleus dorsalis n. vagi. In ATPase preparations all these demonstrate intense activity of same nature, therefore, a distinct interca-

8

Sood, P. P., and M. Lodha

Fig. 7. Transverse section of medulla oblongata at the level of substantia grisea centralis medullae oblongatae (ATPase reaction). Fig. 8. Transverse section of medulla oblongata at the level of nucleus cuneatus lateralis (ATPase reaction). Fig. 9. Transverse section of medulla oblongata at the level of facialis nerve (ATPase reaction). Fig. 10. Transverse section of medulla oblongata passing through the nucleus n. facialis (ATPase reaction). Fig. 11. Section of cerebellum. Note intense activity in molecular layer (ML) and Purkinje layer (PL). The granular layer (GL) is moderately positive and nerve fiber layer (NFL) demonstrates mild activity (ATPase reaction).

latus nucleus does not appear in our study (NI; Fig. 2). The nucleus is moderately positive for 5-NUC (NI; Fig. 16). Like ATPase, in this enzyme too, nucleus intercalatus does not appear as a distinct nucleus since the other two surrounding nuclei are also moderately positive (i. e. NDNV and NNH; Fig. 16). Nucleus dorsalis n. vagi:

This nucleus is situated above the nucleus intercalatus and consists of a characteristic concentration of the nerve cells. Such concentration of cells is clearly seen in 5-NUC preparations (NDNV; Fig. 28). These cells show intense activity for 5-NUC. In ATPase preparations (NDNV; Fig. 2) the nucleus is characterised by very strong activity in the nerve cells as

Histochemical mapping of the CNS

well as in the neuropil, therefore, even at high magnification the cellular arrangement is not distinct (NDNV; Fig. 13). Area postrema:

The area postrema, situated on both sides of the 4th ventricle and dorsal to the nucleus tractus solitarius, is very intensely positive for ATPase (AP; Fig. 2). Further, its boundaries with nucleus dorsalis n. vagi are so closely mixed up due to intense reaction in both the nuclei that it is not possible to identify the area postrema as a distinct nucleus (see Fig. 2). The area postrema demonstrates a moderate activity for 5-NUC (AP; Fig. 16). Unlike ATPase, in 5-NUC study the boundaries of area postrema are clearly marked out from nucleus dorsalis n. vagi as the latter shows more intense activity than the former (see Fig. 16). Nucleus

It consists of medium to large multipolar cells with long dendrites placed in the reticular formation. The nucleus ambiguus is intensely positive for ATPase (NA; Figs. 3, 7,13) and completely negative for 5-NUC (NA; Figs. 18, 23). hypoglossi:

The nucleus prepositus is a triangular-shaped nucleus, situated dorsal to the rostral end of nucleus n. hypoglossi in the posterior sections of medulla oblongata. This nucleus is clearly seen in the histoenzymological preparations and is intensely positive both for ATPase (NPH; Fig. 6) and 5-NUC (NPH; Figs. 18, 20). Nucleus

trigemini:

This nucleus has irregular and loose outline and extends throughout the length of medulla oblongata. In the posterior sections of medulla oblongata, the nucleus is very easily identifiable in all histoenzymological preparations. The nucleus is strongly positive for ATPase (NSV; Figs. 2 - 5 , 12) and 5-NUC (NSV; Figs. 15 — 18, 28). Both these enzymes are mainly concentrated in neuropil. Nucleus tractus solitarius:

This nucleus, lies lateral to the dorsal nucleus of vagus, is intensely positive for ATPase (NTS; Figs. 4, 6, 7) and moderately positive for 5-NUC (NTS; Figs. 19, 20, 23). The boundaries of the nucleus are clearly visible in 5-NUC reactions as the surrounding nuclei show more intense activity (see Figs. 19, 20, 23). Nucleus tractus spinalis n. trigemini

ambiguus:

Nucleus prepositus

Nucleus tractus spinalis

vestibularis:

In the present study only the nuclear vestibularis medialis, lateralis and spinalis are recognised. All these three nuclei are intensely positive for ATPase (NVL, NVM, NVS; Figs. 4, 5, 7 - 1 0 ) as well as for 5-NUC (NVL, NVM, NVS; Figs. 18, 2 3 - 2 5 , 27). Nucleus n. facialis:

This nucleus shows oval to ovoid shape outline with welldefined boundaries. In the histoenzymological preparations the nucleus n. facialis is clearly visible (Figs. 8, 22, 23). I t is strongly positive for ATPase (NNF; Fig. 8) and moderately positive for 5-NUC (NNF; Figs. 22, 23). Further, in histological studies, it is demonstrated that its neuropil is permeated by longitudinal fiber bundles particularly at its lateral part. Such fibers are not seen in 5-NUC study. However, in some sections there appears to be some ATPase positive fibers crossing through the nucleus n. facialis (see Fig. 8).

9

oralis:

This nucleus lies on the ventro-lateral side in some anterior sections of medulla oblongata and like nucleus tractus spinalis trigemini, demonstrates intense activity both for ATPase (NO; Figs. 8,10) and 5-NUC (NO; Figs. 23, 24, 25). Cochlearis complex:

Two cochlear nuclei are identified in present study namely Nucleus cochlearis dorsalis (NCD) and ventralis (NCV). Nucleus cochlearis dorsalis is an elongated and oval-shaped nucleus whereas the ventral nucleus is ovoid and extends to the caudal end of pons. In ATPase preparations both the cochlear nuclei are intensely positive (NCD, NCV; Figs. 7, 9, 10). In contrast to this, the 5-NUC shows a mild activity (NCD, NCV; Figs. 2 2 - 2 5 ) . Nucleus olivaris complex:

The complex consists of nucleus olivaris accessorius dorsalis (NOAD) and medialis (NOAM), nucleus olivaris inferior (NOI) and superior (NOS). These nuclei are composed of relatively small to medium sized cells with numerous short branching dendrites. In this study nucleus olivaris accessorius medialis (NOAM; Figs. 2 - 4 , 1 2 ) , dorsalis (NOAD; Figs. 4 - 7 ) , inferior (NOI; Fig. 4) and superior (NOS; Fig. 9) are very intensely positive for ATPase whereas, these nuclei are mildly positive for 5-NUC (NOAM, NOAD, NOI; Figs. 16—18, 28) except nucleus olivaris superior which shows intense reaction (NOS; Figs. 25, 26). The boundaries of these nuclei, in both the cases, are clearly marked out. Nucleus cuneatii medialis and lateralis:

In ATPase preparations the nucleus cuneatus lateralis is moderately positive (NCL; Figs. 6, 8, 9). The neuro-

10

Sood, P. P., and M. Lodha

NCOD NCOV NINH Fig. 12. High magnification of a section of medulla oblongata passing through nucleus n. hypoglossi. Note intense activity of ATPase in neuropil of various nuclei (ATPase reaction).

NOAM NCM

NINH

Fig. 13. High magnification of figure 3. Note intense activity of ATPase in cytoplasm and nucleoli of the neurons of various nuclei (NINH, NNA, NS, NCM). Further note positive activity in the neuropil of all the nuclei.

pil and the neurons show positive reaction. 5-NUC preparations also demonstrate similar activity (NCL; Figs. 19, 20). The nucleus cuneatus medialis lies dorsal to the nucleus parvocellularis compactus and has irregular obundaries. This nucleus is intensely positive for

ATPase (NCM ; Figs. 2, 12, 13) and moderately positive for 5-NUC (NCM; Fig. 16). Nuclei raphe:

In the present study two parts of nuclei raphe — namely nuclei raphe obscurus (NRO) and nuclei raphe

Histochemical mapping of the CNS

Fig. 14. Transverse section of spinal cord demonstrating 5-NUC activity. Fig. 15. Transverse section of medulla oblongata through the posterior region (5-NUC reaction). Fig. 16. Transverse section of medulla oblongata at the level of area postrema (5-NUC reaction). Fig. 17. Transverse section of medulla oblongata at the region of maximum development of olivary complex (5-XUC reaction).

magnus (NRM) which lie along the midline of the medulla oblongata, have been identified. The former extends throughout the length of medulla while the latter appears in the anterior sections only. In histo-

11

enzymological preparations both these nuclei are intensely positive for ATPase (NRO, NRM; Figs. 2 - 1 0 ) and mildly positive for 5-NUC (NRO, NRM; Figs. 1 5 - 1 8 , 22-25). Nucleus

sub-trigeminalis:

The nucleus lies ventral to the nucleus tractus spinalis n. trigemini (NSV) in the medial sections of medulla oblongata. Its boundaries are clearly seen in ATPase (NS; Figs. 2 - 4 , 1 3 ) , as well as in 5-NUC (NS;Figs. 18, 28) reactions since the nucleus demonstrates more intense activity for both the enzymes than the surrounding areas.

12

Sood, P. P., and M. Lodha

NVM NPH

Fig. 18. Transverse section of medulla oblongata at the level of nucleus ambiguus (5-NUC reaction).

NOAD

Fig. 19. Transverse section of medulla oblongata at the level of nucleus paragigantocellularis lateralis (5-NUC reaction).

Fig. 20. Transverse section of medulla oblongata passing through nucleus prepositus hypoglossi (5-NUC reaction). Nucleus centralis medulla

oblongatae:

Two sub-divisions of this nucleus, generally known as nucleus centralis medullae oblongatae pars dorsalis (NCOD) and pars ventralis (NCOV), have been recognised in the central region of reticular formation (Koikegami, 1957). Like other reticular nuclei the NCOD and NCOV also demonstrate intense activity for both the enzymes. NCOV; Figs. 2 - 5 , 1 2 , 1 5 - 1 7 , 28). Nucleus reticularis

medullae

oblongatae:

Histologically, the reticular formation throughout the medulla oblongata can be differentiated into four main nuclei — namely formatio reticularis, nucleus

reticularis paramedianus, nucleus reticularis gigantocellularis and nucleus reticularis lateralis. a) Nucleus reticularis gigantocellularis is located rostral to the nucleus reticularis paramedianus and has characteristic large cells with elongated dendrites, as well as medium-sized and small cells. This nucleus is intensely positive both for ATPase (NG; Figs. 6, 8) and 5-NUC (NG; Figs. 20, 22, 26). b) Nucleus paragigantocellularis dorsalis: According to OLSZEWSKI and B A X T E R ( 1 9 5 4 ) the dorsal area above the nucleus reticularis gigantocellularis which contains hardly any gigant cells, is known as nucleus

Histochemical mapping of the CNS

13

Fig. 21. Transverse section of cerebellum demonstrating intense activity of 5-NUC in molecular layer (ML), moderate activity in granular layer (GL) and mild activity in nerve fiber layer (NFL) and Purkinje layer (PL). Fig. 22. Transverse section of medulla oblongata passing through nucleus raphe magnus (5-NUC activity).

NRM

Fig. 23. Transverse section of medulla oblongata passing through the anterior region of medulla oblongata (5-NUC reaction).

paragigantocellularis dorsalis. Like other reticular nuclei it demonstrates intense activity both for 5-NUC (NPD; Figs. 19, 20, 22, 26) and ATPase (NPD; Figs. 6, 8). c) Nucleus paragigantocellularis lateralis is located ventrolaterally and is oval to ovoid-shaped. It is distinguished from the rest of the reticular nuclei by strong ATPase reactions (NPL; Figs. 5, 6, 8). In 5-NUC preparations the nucleus is also intensely positive (NPL; Figs. 19, 20). d) Nucleus reticularis paramedianus is located on either side of nucleus raphe. Its cells are parallely

arranged with the nucleus raphe. The nucleus is moderately positive for ATPase (NRP; Figs. 2, 13) as well as for 5-NUC (NRP; Figs. 15, 16, 28). Substantia

grisea centralis medullae oblongatae :

The nucleus lies at the mid-dorsal area of medulla oblongata. In the present study there is no clear-cut demarcation of the boundaries of this nucleus as the nucleus and the surrounding areas are intensely positive for ATPase (SGO; Fig. 7). In 5-NUC preparations the substantia grisea centralis medullae oblongatae is moderately positive (SGO; Fig. 23) and similar is the condition of its surrounding nuclei.

14

Sood, P. P., and M. Lodha

NVM

Fig. 24. T. S. through nucleus n. facialis (5-NUC reaction) .

•vlRM

NVM NVL

NO NOS Fig. 25. T. S. through most anterior region of medulla oblongata (5-NUC reaction).

NRM Nucleus

parvocellularis:

The nucleus consists of densely packed cells (NPC; Figs. 2, 12, 16). In the anterior sections the cells of the nucleus are loosely arranged and its position is somewhat lateral (NP; Figs. 6, 8, 20, 22, 23). As far as the intensity of the ATPase is concerned, the nucleus parvocellularis compactus is very intensely positive (NPC; Figs. 2,12,13) as compared to nucleus parvocellularis (NP; Figs. 6, 8). In 5-NUC preparations the intensity is the reverse; here the nucleus parvocellularis demonstrates stronger activity than the nucleus compactus. In other words the nucleus parvocellularis is intensely positive (NP; Figs. 20,

22, 23) and the nucleus parvocellularis compactus is mildly positive for 5-NUC (NPC; Fig. 16). Facial nerve —

VII:

This nerve is intensely positive for ATPase (NF; Fig. 9) and moderately positive for 5-NUC (NF; Figs. 22, 24, 26, 27). Tracts:

Though in histological studies a large number of tracts have been identified in the hind brain of mouse (see Sidman et al., 1971) but in our histoenzymological preparations only few tracts are seen. These are

Histochemical mapping of the CNS

15

Fig. 26. High magnification of a section of medulla oblongata at the level of nervus facialis. Note intense activity of 5-NUC in the cytoplasm and nucleoli of the neurons of various nuclei (5-NUC reaction).

Fig. 27. High magnification of a section of medulla oblongata at the level of nervus facialis (5-NUC reaction).

— tractus spinocerebellaris dorsalis (TSD) and ventralis (TSV), tractus spinalis n. trigemini (TST), tractus corticospinalis (TC) and tractus solitarius (TS). Of these five tracts, in ATPase preparations, the most intense reaction is seen in tractus solitarius (TS; Fig. 4), whereas the same tract is moderately positive for 5-NUC (TS; Fig. 17). Tractus spinalis n. trigemini is moderately positive for ATPase (TST ; Figs. 2 - 7 , 10) and mildly positive for 5-NUC (TST; Figs. 22, 24, 25, 28). The tractus spinocerebellaris dorsalis and ventralis are moderately positive for ATPase (TSD, T S V ; Figs. 2, 13) and intensely posi-

tive for 5-NUC (TSD, TSV; Fig. 28). Finally, the tractus corticospinalis demonstrates moderate activity for ATPase (TC; Figs. 3, 6, 12) while the same tract is completely devoid of 5-NUC activity (TC; Figs. 16, 17, 19, 20, 23, 26). Discussion Evaluating the different patterns of enzymatic localization in the hind brain of mouse, it appears that in general the activity of ATPase is more intense in most of the nuclei and tracts than 5-NUC activity,

16

Sood, P. P., and M. Lodha

NDNV

NCOD

NCOV

Fig. 28. High magnification of a section of medulla oblongata at the level of nucleus olivaris accessories medialis (5-NUC reaction) . T a b l e 1. Showing the various nuclei, figure numbers and the intensity of the enzymes. Nuclei

Abbreviation

ATPase activity Intensity Fig. No.

5-Nucleotidase activity Intensity Fig. No.

1.

2.

3.

4.

5.

Area postrema Nucleus ambiguus Nucleus cornucommissuralis dorsalis Nucleus cochlearis dorsalis Nucleus cervicalis lateralis Nucleus cuneatus lateralis Nucleus cuneatus medialis Nucleus centralis medullae oblongatae pars dorsalis Nucleus centralis medullae oblongatae pars ventralis Nucleus cochlearis ventralis Nucleus cornucommissuralis ventralis Nucleus dorsalis Nucleus dorsalis n. vagi Facial nerve V I I Nerve fiber layer Nucleus gracilis Nucleus reticularis gigantocellularis Nucleus intracalatus Nucleus interfascicularis n. hypoglossi Nucleus n. accessorii Nucleus n. facialis Nucleus n. hypoglossi Nucleus tractus spinalis n. trigemini oralis

AP NA DCN

+ + + + + -|

2

+ -

NCD NC NCL NCM NCOD

+ + —|H— + + + +

7,9,10 1 6,8,9 2,12,13 2,3 — 5,12

—+ + + + +

22 — 25 14 19,20 16 15 — 17,28

NCOV

+ +

2,4,5,12

+ +

15-17,28

NCV YCN

+ + -|—

9, 10 1

- + + -

24 14

ND NDNV NF NFL GN NG

-|— + + + + + —

+ + + + + -

+ +

I 2,13 9 II 1 6,8

+ +

14 28 22,24,26,27 21 14 20,22,26

NI NINH

+ + + + +

2 2,3,12,13

+ + +

16 16

NNA NNF NNH NO

H— + + + + + + +

1 8 2-5 8,10

+ + + +

14 22, 23 16,17 23-25

3, 7, 13 1

6.

+ -

+ +

16

18, 23 14

Histochemical mapping of the CNS

17

Continuation T a b l e 1 : Nuclei 1. Nucleus olivaris accessorius medialis Nucleus olivaris inferior Nucleus olivaris superior Nucleus parvocellularis Nucleus parvocellularis compactus Nucleus paragigantocellularis dorsalis Nucleus proprius cornus dorsalis Nucleus prepositus hypoglossi Nucleus posteromarginalis Nucleus paragigantocellularis lateralis Nucleus raphe magnus Nucleus raphe obscurus Nucleus olivaris accessorius dorsalis Nucleus reticularis paramedianus Nucleus reticularis Nucleus sub-trigeminalis Nucleus tractus spinalis trigemini Nucleus tractus solitarii Nucleus ventromedialis Nucleus vestibularis lateralis Nucleus vestibularis medialis Nucleus vestibularis spinalis Granular layer Molecular layer Pedunculus cerebellaris inferior Purkinje layer Substantia gelatinosa Substantia grisea centralis medullae oblongatae Tractus corticospinalis Tractus solitarius Tractus spinocerebellaris dorsalis Tractus spinalis n. trigemini Tractus spinocerebellaris ventralis

Abbreviation 2.

ATPase activity Intensity Fig. No. 4. 3.

NOAM

+++

2 - 4 , 12

+++

++ +++

4 9 6, 8 2, 12, 13

++ ++

NPD

++

6, 8

++

19, 20, 22, 26

DN

+ -

1

+ -

14

NPH PN NPL

++

++

+ +

6 1 5, 6, 8

18, 20 14 19, 20

NRM NRO NOAD

++ ++ +++

8-10 2 - 7 , 9, 10 4, 7

NRP

+ -

2, 13

+ -

15, 16, 28

NRS NS NSV

+ ++ ++

1 3, 4, 13 2 - 5 , 12

++ ++ ++

14 18, 28 1 5 - 1 8 , 28

NTS NV NVL

+ + + ++

4, 6, 7 1 7, 9, 10

+ ++ ++

19, 20, 23 14 2 3 - 2 5 , 27

NVM NVS GL ML PCI

+ + + + +

4, 5, 7 - 1 0 4, 5 11 11 6, 7

+ + + +

18, 2 3 - 2 5 , 27 18 21 21 17, 20, 22

PL SG SGO

+ + +++

++

11 1 7

TC TS TSD

+ +++ + -

3, 6, 12 4 2, 13

TST TSV

+ + -

2, 3, 5 - 7 , 10, 13 2

NOI NOS NP NPC

++ +

-

+

+ + + +

5-Nucleotidase activity Intensity Fig. No. 5. 6. -

+

-

+

-

+

++ -

+

-

+

-

+

-

+ + + +

+ + -

+ ++ -

+

++

1 6 - 1 8 , 28 17, 18 25, 26 20, 22, 23 16

22, 24, 25 1 5 - 1 8 , 23, 25 16-18

21 14 23 16, 17, 19, 20, 23, 26 17 28 22, 24, 25, 28 28

Note: For abbreviations see table II.

as for example few nuclei (AP, NDNV, NI, NNH, NOAD, NOAM, NOI, NPC, TS), are very intensely positive and in such nuclei it has not been possible to distinguish the various elements of the area. It is interesting to note that such a condition is not seen in 5-NUC preparations. In all we have studied fifty-six nuclei, tracts and fibrous bundles (seeTable 1), out of which eleven areas are very intensely positive Hirnforschung, Bd. 19, Heft 1

for ATPase and none for 5-NUC; twenty-seven are intensely positive for ATPase and twenty three for 5-NUC; fourteen nuclei are moderately positive while three are mildly positive for ATPase. In 5-NUC preparations sixteen nuclei are moderately positive and eleven are mildly positive. One nucleus of spinal cord, i.e., nucleus gracilis, is negative for ATPase while the number of negative nuclei is six in 5-NUC 2

18

Sood, P.,P. and M. Lodha

study. Thus an overall picture makes it clear that intensity of ATPase in various nuclei, tracts and fibrous bundles is stronger than 5-NUC (see Table 1). The ATPase activity appears in glial cells, blood vessels, nerve cells, neuropil and nerve fibers. In other words most of the structural elements of the medulla oblongata are positive for ATPase. In nerve cells the activity of ATPase is restricted mainly to neuronal membranes and nucleoli. There appears to be little activity in the cytoplasm while the nuclei are generally free from enzymatic activity. Further, the neuropil is more strongly positive as compared to neurons. Earlier BARRON and TUNCBAY (1962) and NANDY and BOURNE (1964) have made similar observations. In ATPase preparations few nerve fibers and tracts demonstrate moderate to intense activity. Most of the nerve fibers, fibrous bundles and tracts could not be identified in the present series of sections as either they are so intense that they have been mixed up with intensely positive nuclei or are completely devoid of enzymatic activity. Though detailed studies about the presence of ATPase and 5-NUC in tracts and fibre bundles of hind brain of mammals are not available in literature, yet few attempts have been made to demonstrate these enzymes in fibrous bundles and tracts of the central nervous system of other vertebrates. Earlier, TEWARI and SOOD (1974) and SOOD and TEWARI (1972 a; 1976) have demonstrated intense activity of ATPase and 5-NUC in various olfactory tracts of frog and toad. SOOD and TEWARI (1972b) have also seen intense activity of 5-NUC in lateral and medial olfactory tracts in the

olfactory bulb of mouse. However, TEWARI and RAJBANSHI (1972) found negligible activity of 5-NUC and ATPase in the tracts of hind brain of Saccobranchus fossilis, though they found intense activity of 5-NUC and mild activity for ATPase in the 10th cranial nerve. In literature there are different opinions about the presence of 5-NUC and ATPase in the white and grey matter. In biochemical studies, great species variability in the regional distribution of 5-NUC in brain tissue is suggested by the ratio of the activity in white matter to that of grey matter. REIS (1937) found this ratio is 1.0 to 4.2 for man, 1.0 to 0.9 for rabbit and 1.0 to 0.4 for calves. In histochemical studies of 5-NUC and ATPase NAIDOO (1962) found enzyme activity exclusively in myelinated fibers in mice. TEWARI and BORNE (1963 a) showed intense activity of 5-NUC in the cerebellar white matter of rat. BARRON and TUNCBAY (1964) found 5-NUC in the glia of white matter of cats, with much higher activity in the neuropil of grey matter. Intense activity of 5-NUC in the white matter of mouse brain is also seen by (SCOTT 1967). Similarly, intense activtiy of ATPase in the white matter of rat has been reported by ToRACK (1965) and MANOCHA (1970). Further, BONTING et al. (1961) demonstrated a large amount of Na—K ATPase in the grey matter of nervous tissue. Since the reticular formation is formed by intermingling of grey and white matter and all above data including present study support our view that both the enzymes are present in the grey and white matter of the brain, intense activity of 5-NUC and ATPase in reticular nuclei is expected. Further, the reticular

Table II Showing the differences of the distribution of ATPase and 5-NUC in the various layers of cerebella of different mammals. Name of Authors

Molecular layer

Purkinje layer

Granular layer

Nerve fiber layer

ATPase

5-NUC

ATPase

5-NUC

ATPase

5-NUC

ATPase

5-NUC

Becker et al. (1960) Rat. Tewari and Bourne (1963 a, b) Rat. Shantha et al. (1967) Squirrel monkey Scott (1967) Mouse

+ + + -

NA NA

+ + + +

NA NA

NA + -

NA NA

NA

NA NA + +

+ -

H— + to + + to + Show +ive NA & — ive bands

+-

- +

+ -

+ -

Scott (1967) Cat.

NA

Manocha (1970) Squirrel monkey Present study Mouse Abbreviations :

NA

+ -

Very Intense NA

NA

+ -

++

+ +

+ +

to + + Low to NA moderate activity

Low to NA moderate NA - + to + - + + -

to + + —ive except nerve fibers Negative

NA

Moderately strong

NA

Negative

NA

+ -

NA - +

+ -

-

+ + + = Very intensely positive ; H- + = Intensely positive

H— —.+ NA

= Moderately positive; = Mildly positive; = Not attempted.

= Negative;

+

Histochemical mapping of the CNS nuclei a r e b o t h sensory a n d m o t o r in n a t u r e (CAJAL, 1 9 1 1 ) , therefore, t h e m o t o r a n d sensory functions of 5 N U C a n d A T P a s e in t h e r e t i c u l a r f o r m a t i o n seems t o b e negligible,

instead the

e n z y m e s in t h e

mouse

b r a i n m a y b e c o n c e r n e d w i t h general m e t a b o l i s m . I n cerebellum, intense a c t i v i t y of A T P a s e a n d 5 N U C is seen in m o l e c u l a r l a y e r , m o d e r a t e

activity

in g r a n u l a r l a y e r a n d mild t o negligible a c t i v i t y in w h i t e m a t t e r . H o w e v e r , t h e a c t i v i t y of 5 - N U C a n d A T P a s e in P u r k i n j e l a y e r is quite different.

The

P u r k i n j e cells d e m o n s t r a t e intense a c t i v i t y for A T P a s e a n d mild a c t i v i t y for 5 - N U C . W h i l e c o m p a r i n g t h e a c t i v i t y of 5 - N U C a n d A T P a s e of m o u s e cerebell u m w i t h o t h e r m a m m a l s like squirrel m o n k e y (SHANTHA e t al. 1 9 6 7 ; MANOCHA, 1 9 7 0 ) ; r a t (TEWARI a n d BOURNE, 1 9 6 3 a, b) a n d c a t (SCOTT, 1 9 6 7 ) , large n u m b e r of differences in t h e i n t e n s i t y of b o t h t h e e n z y m e s in v a r i o u s l a y e r s of t h e cerebellum a r e d e t e c t e d (see T a b l e I I ) , which suggest t h a t t h e r e is a species v a r i a t i o n of t h e distribution of these e n z y m e s in t h e differ e n t l a y e r s of cerebellum of m a m m a l s . T h e f u n c t i o n a l significance of 5 - N U C in t h e cerebellum or in a n y o t h e r n e r v o u s tissue is n o t well known. T h e role of 5 - N U C in inhibition a n d e x c i t a t i o n o r in afferent o r efferent functions h a s been e x p e c t e d by

SCOTT ( 1 9 6 7 ) ,

though

he

doubts

the

relation

of this e n z y m e in either of t h e s e pairs of functions. T h e o t h e r role of 5 - N U C , like nucleic a c i d c a t a b o l i s m a n d b r e a k d o w n of N A D P (DIXON a n d W E B B , 1 9 6 4 ; HARDONK, (REIS,

1968),

1951),

in

the

in t r a n s p o r t

regulation

of

mechanism

glycolysis (HARDONK,

1 9 6 8 ) , in cell g r o w t h (EKER, 1 9 6 5 , 1 9 6 6 ; FRITZSON, 1 9 6 7 ; HARDONK a n d KOUDSIAAL, 1 9 6 8 ) , in different tissues h a s been r e p o r t e d . B u t how far 5 - N U C in t h e cerebellum is c o n c e r n e d w i t h t h e s e functions or w i t h e x c i t a t i o n a n d inhibition, is n o t y e t k n o w n .

References K . D. and T. O. T U N C B A Y : Phosphatase in cuneate nuclei after brachial plexectomy. Arch. Neurol. (Chic). 7, 2 0 3 - 2 1 0 (1962). B A R R O N , K . D. and T . O. T U N C B A Y : Phosphatase histochemistry of feline cervical spinal cord after brachial plexectomy. J . Neuropath, and Exper. Neurol. 23, 368 (1964).

BARRON,

BECKER, N. H.,

S . GOLDFISCHER,

W . Y . SHIN

and

A.

B.

The localization of enzyme activities in the rat brain. J . Biophys. Biochem. Cytol. 8, 649—663 (1960). B O N T I N G , S. L . , K . A . S I M O N and M . H. H A W K I N S : Studies on sodium-potassium activated adenosine triphosphatase. I. Quantitative distribution in several tissues of the cat. Arch. Biochem. 95, 4 1 6 - 4 2 3 (1961). C A J A L , S. R . Y . : Histologie du systeme nerveux de l'homme NOVIKOFF:

et des vertebres. Norbert Maloine, Paris, 2 vols (1911). and E . C . W E B B : Enzymes, 2nd edition. Longmans, Green and Co. Ltd., London (1964). EKER, P . : Activities of thymidine kinase and thymidine DIXON, M..

19

deoxyribonucleotide phosphatase during growth of cells in tissue culture. J . biol. Chem. 240, 2607 — 2611 (1965). EKER, P . : Studies on thymidine kinase of human liver cells in culture. J . biol. Chem. 241, 659 — 662 (1966). F R I T Z S O N , P . : Dephosphorylation of pyrimidine nucleotides in the soluble fraction of homogenates from normal and regenerating rat liver. Eur. J . Biochem. 1, 12 — 20 (1967). H A R D O N K , M. J . : I. Distribution of 5'-nucleotidase in tissue of rat and mouse. Histochemie. 12, 1 — 17 (1968). H A R D O N K , M. J . and J . K O U D S T A A L : I I . The significance of 5'nucleotidase in the metabolism of nucleotides studied by histochemical and biochemical methods. Histochemie. 12, 1 8 - 2 8 (1968). K O I K E G A M I , H . : On the correlation between cellular and fibrous patterns of the human brain stem reticular formation with some cytoarchitectonic remarks on the other mammals. Acta Medica et Biologica5, 21 — 72 (1957). M A N O C H A , S . L . , T . R . S H A N T H A and G . H. B O U R N E : Histochemical studies on the spinal cord of squirrel monkey (Saimiri seiureus). Exp. Brain Res. 3, 25 — 39 (1967). M A N O C H A , S . L . : Histochemical distribution of alkaline and acid phosphatase and adenosine triphosphatase in the brain of squirrel monkey (Saimiri seiureus). Histochemie. 21, 2 2 1 - 2 3 5 (1970). N A N D Y , K . and G . H. B O U R N E : Adenosine triphosphatase and 5-nucleotidase in spinal cord — Histochemical study of localization in rat spinal cord. Archives of Neurology. 11, 5 4 7 - 5 5 3 (1964). N A I D O O , D . : The activity of 5-nucleotidase determined histochemically in the developing rat brain. J . Histochem. Cytochem. 10, 4 2 1 - 4 3 4 (1962). OLSZEWSKI, J . and D . B A X T E R : Cytoarchitecture of the human brain stem. J . B . Lippincott Company, Philadelphia (1954). R E I S , J . L . : Über die spezifische Phosphatase der Nervengewebe. Enzymologia. 2, 110 — 116 (1937). R E I S , J . L . : The specificity of phosphomonoesterases in human tissue. Biochem. J . 48, 548 — 551 (1951). S C O T T , T. G.: The distribution of 5'-nucleotidase in the brain of the mouse. J . Comp. Neurol. 129, 97 — 108 (1967). S H A N T A , T. R., K . I I J I M A and G. H. B O U R N E : Histochemical studies on the cerebellum of squirrel monkey. Acta Histochem. 27, 1 2 9 - 1 6 2 (1967). S I D M A N , R . L., J . B . A N G E V I N E and E . T. P I E R C E : Atlas of the mouse brain and spinal cord. A Commenwealth Fund Book. Harvard Univ. Press, Cambridge, Massachusetts. pp. 1 - 2 6 1 (1971). S O O D , P . P . and M . H. B O H R A : Histochemical mapping on the distribution of simple esterase and acetylcholinesterase in the medulla oblongata, cerebellum and spinal cord of mouse. J . Hirnforschung 18, 75—87 (1977). S O O D , P . P . and M . M U L C H A N D A N I : A comparative histoenzymological mapping on the distribution of acid and alkaline phosphatases and succinic dehydrogenase in the spinal cord, and medulla oblongata of mouse. Acta Histochem. 60, 1 8 0 - 2 0 3 (1977). S O O D , P. P. and H. B . T E W A R I : Histochemical mapping of the distribution of acid phophatase, 5-nucleotidase and nonspecific esterase in the forebrain of the toad. Brain Res. 38, 4 0 7 - 4 2 0 (1972a). S O O D , P. P., and H. B . T E W A R I : Study on the distribution of some hydrolytic enzymes and their functional significance in the olfactory bulb of mouse. Acta Histochem. 43, 2 1 8 - 2 3 4 (1972b). S O O D , P. P., and H. B . T E W A R I : Histochemical mapping of the distribution of acid phosphatase and 5-nucleotidase in 2*

20

Sood, P. P., and M. Lodha

the forebrain of frog (Rana tigrina). J . Hirnforschung. 17, 289-303 (1976).

TEWARI, H. B., and G. H. BOURNE: Histochemical studies

on the distribution of alkaline and acid phosphatases and 5-nucleotidase in the cerebellum of rat. J . Anat. (London) 97, 6 5 - 7 2 (1963a).

TEWARI, H. B., and G. H. BOURNE : Histochemical studies

on the localization of adenosine triphosphatase in the cerebellum of the rat. J . Histochem. Cytochem 11, 246—256 (1963 b).

TEWARI, H . B . ,

and

V . K . RAJBANSHI:

Histological

TORACK, R. M. : The relationship between adenosine triphosphatase activity and triethyltin toxicity in the production cerebral edema of the rat. The American J . of Pathol. 46, 245-261 (1965). WACHSTEIN, M., and E. M E I S E L : Histochemistry of hepatic phosphatases at the physiologic pH with special reference to the demonstration of bile canaliculi. Amer. J . Clin. Path. 27, 1 3 - 2 3 (1957).

and

histochemical studies in the hind brain of a freshwater teleost, Saccobranchus fossilis. Ann. Histochem. 17, 1 — 25 (1972). TEWARI, H. B., and P. P. SOOD: Histochemical mapping of the distribution of adenosine triphosphatase, succinic dehydrogenase and non-specific esterase in the fore-brain of frog (Rana tigrina). J . Hirnforschung. 15,129 — 142 (1974).

Authors address: D r . P . P . SOOD,

Department of Zoology, University of Udaipur, Udaipur-313001, India.

J . Hirnforsch. 19 (1978) 21 - 4 3

Department of Anatomy, University of Alabama in Birmingham

An Experimental Study of the Central Gustatory Pathways in the Monkey, Macaca mulatta and Cercopithecus aethiops B y Francis Cleveland

KINNEY,

Ph.

D.1

With 19 figures (Received 20 t h January 1977)

Summary: 1. The cortex at the base of the central fissure, that is the fronto-parietal operculum, represents primary sensory receptive cortex for gustatory modalities. 2. This primary receptive cortex for taste is linked to the anterior Island of Reil by short inter- and intracortical association fibers. Thus the anterior island is a gustatory association area involved in the subjective recognition of gustatory modalities. 3. The nucleus ventralis posteromedialis pars parvocellularis of the dorsal thalamus is the thalamic receptive nucleus for gustatory impulses in the macaque. 4. Fibers which originate at rostral glossopharyngeal levels from the dorsal visceral gray terminate in the contralateral nucleus ventralis posteromedialis pars parvocellularis.

Acknowledgements I am especially grateful and appreciative for the constant guidance and direction so freely given by my teacher, Dr. Elizabeth C . C R O S B Y . I am indebted to Dr. Earl G . H A M E L , J r . , chairman of my graduate committee, for his continual help and support. For his contributing to the Department of Anatomy of the University of Alabama in Birmingham all the rhesus monkeys used in this study, I owe a special thanks to Dr. Leon H. S C H M I D T , of the Southern Research Institute.

Introduction Relatively little research has been completed to determine and document the central (neural) gustatory pathways in primates and man. This is surprising in view of the obvious importance of taste. The loss of taste or one of the gustatory sensations (such as the ability to recognize salty, sweet, sour or bitter substances) may, if no peripheral lesion can be located, be indicative of a lesion within the central nervous system. If the pathways involved in transmitting gustatory impulses to higher centers, including the cerebral receptive and association cortices for the cortical recognition of taste, are known, then the loss of taste or better still the loss of one of the 1

This investigation was completed as partial fulfillment for the Doctor of Philosophy Degree in Anatomy at the University of Alabama in Birmingham.

modalities of taste will give the clinician a clue as to the possible level of the lesion. The cell bodies of gustatory fibers are found in the geniculate ganglion of the facial nerve, the inferior (opetrosal) ganglion of the glossopharyngeal nerve, and the inferior (or nodose) ganglion of the vagus nerve. The central processes of these fibers enter the brain stem (at pons and medulla levels) and synapse, at or near their level of entrance, in the nucleus (dorsal visceral gray) of the tractus solitarius (Schwartz, Roulhac, Lam and O'Leary, 1951, man). The secondary projections to higher centers from the nuclei of the tractus solitarius which together receive both special and general visceral afferent fibers are not well documented. I t has been reported that some secondary fibers concerned with taste relay by way of the contralateral medial lemiscus, or adjacent fibers, in guinea pig (ALLEN, 1923) to the " . . . nucleus ventralis et lateralis thalami" and thence by thalamic sensory radiations to the cerebral cortex (von B E C H T E R E W , 1 9 0 8 — 1 9 1 1 ) . P E N F I E L D and RASMUSSEN ( 1 9 5 0 ) traced gustatory impulses to the fronto-parietal operculum at the base of the central fissure for the cortical recognition of taste. GORSCHKOW ( 1 9 0 1 ) , a student of VON BECHTEREW, carried gustatory impulses to the potential island region in the dog. Terminal degeneration within the contralateral nucleus ventralis posteromedialis pars parvocellularis resulting from an electrolytic stereotaxic lesion of the nucleus of the tractus

22

Kinney, F . C.

solitarius at the level of the glossopharyngeal nerve in monkey NR 1 (of this study) confirms the work of others that this is the thalamic receptive nucleus in regard to gustatory sensibilities. The question also arises as to the identification of specific levels within the central nervous system at which gustatory impulses come into consciousness. BRADLEY (1963) performed a number of experiments

on monkey (Macaca mulatto) which involved the removal of cortical areas and the placing of lesions in thalamic and superior collicular levels. Preoperatively and postoperatively monkeys were given banana sprinkled with quinine. Following removal of the cortex at the base of the central fissure and its opercular extension one monkey continued to respond to the quinine but did so more slowly than observed preoperatively. With lesions involving the superior colliculus of the midbrain there was a great reduction in response but again the animal rejected the quinine. This would indicate, perhaps, that awareness of bitter gustatory sensation comes into consciousness at some level other than those ablated by Bradley. There are few reported clinical cases which support the available anatomical evidence for the central gustatory pathways in man, two of which are reported below. ADLER (1934) reported a case in which a patient had a glioblastoma of the third ventricle which initially involved the most rostral medial poition of the nucleus ventralis posteromedialis of the dorsal thalamus. Contralateral to the side of the lesion the patient had a loss of all gustatory sensation except a slight r e c o g n i t i o n of b i t t e r . SHENKIN a n d LEWEY

(1943)

reported a case in which the patient had epileptic seizures preceded by an aura in which he experienced a sour-bitter taste. At operation a vascular anomaly was discovered in which the vessels located in the lateral fissure were greatly dilated, the point of greatest dilatation being over the most inferior portion of the postcentral gyrus. The patient had a loss of sweet perception over the entire side of the tongue contralateral to the lesion. While the above two clinical cases concur with the available information on the central connections of secondary and tertiary gustatory pathways, it is apparent from the literature that further documentation for these pathways is necessary. A more complete review of the pertinent literature is included in the Discussion. Materials and Methods Normal

Material

A large portion of t h e brain stem of one m o n k e y was used t o s t u d y t h e normal configuration of t h e fiber connections a n d nuclear groups extending f r o m c a u d a l medullary levels t o rostral inferior collicular levels. T h e brain stem was embedded

in p a r a f f i n a n d cut transversely o n a r o t a r y microtome a t t w e n t y microns. Alternating sections were stained b y t h e Weil-Weigert technique for myelinated fibers a n d w i t h thionin for t h e nuclear groups.

Experimental

Material

One green m o n k e y (Cercopithecus aethiops) a n d four rhesus m o n k e y s (Macaca mulatto) were used in t h e experimental studies. Sour a n d b i t t e r were t h e t a s t e modalities tested. E a c h m o n k e y was tested several times b o t h preoperatively a n d postoperatively a f t e r h a v i n g fasted for a m i n i m u m of twelve hours. Initially, when testing for t h e ability t o recognize sour substances, a piece of b a n a n a which h a d been soaked for a m i n i m u m of twelve hours in reconstituted lemon juice ( " R e a L e m o n " ) was offered t o each animal. Subseq u e n t l y a piece of lemon-treated b a n a n a or a slice of lemon was given t o each animal when testing his responses t o sour substances. A piece of b a n a n a w i t h quinine sulfate powder sprinkled within its center was offered t o each m o n k e y t o t e s t his ability t o recognize b i t t e r substances. T h e response t o t h e sour a n d b i t t e r substances of each m o n k e y were consistent w i t h those reported on particular days in t h e Results section. Records were k e p t of t h e responses of t h e m o n k e y s t o these t w o substances and, whenever possible, responses were documented photographically with an eight or sixteen millimeter movie camera. All surgical procedures were performed under sterile technique. An electrolytic stereotaxic lesion, based on t h e f i g u r e s o f A T L A S a n d INGRAM ( 1 9 3 7 ) , w a s p l a c e d i n t h e n u c -

leus of t h e t r a c t u s solitarius a t t h e level of t h e glossopharyngeal nerve in one m o n k e y so t h a t degeneration studies could confirm t h e rostral projection of those secondary g u s t a t o r y fibers f r o m t h e nucleus of t h e t r a c t u s solitarius t o t h e nucleus ventralis posteromedialis p a r s parvocellularis of t h e dorsal t h a l a m u s . E i t h e r left or right craniotomies or sequentially b o t h were p e r f o r m e d in t h e remaining monkeys a n d cortical lesions using either surgical suction or cauterization were placed a t t h e base of t h e central fissure in t h e fronto-parietal operculum. Following surgery, 300,000 units of sterile procaine penicillin G suspension were administered intramuscularly for a period of five d a y s t o minimize f u r t h e r t h e possibility of infection. Postoperatively t h e monkeys were not tested for their responses t o t h e sour a n d b i t t e r substances u n t i l t h e period of antibiotic t h e r a p y was completed. I m m e d i a t e l y u p o n recovery f r o m surgery, a n d t h e r e a f t e r until t h e t i m e of sacrifice, a n y behavioral changes or n e u rological deficits p r e s e n t were recorded. E a c h m o n k e y was n o t sacrificed for a m i n i m u m of fourteen d a y s t o assure a sufficient a m o u n t of degeneration of t h e p a t h w a y s affedted. Prior t o perfusion-fixation each a n i m a l was anesthetized w i t h k e t a m i n e hydrochloride a n d his anterior chest wall incised t o expose t h e pericardial cavity. T h e left ventricle was cannulated a n d 0.9% saline followed b y 10% formalin b u f f e r e d w i t h calcium c a r b o n a t e or cacodylate was g r a v i t y fed t h r o u g h t h e cardiovascular system. I m m e d i a t e l y following perfusion-fixation each brain was removed a n d placed in 10% formalin buffered w i t h calcium c a r b o n a t e or cacodylate. T h e gross locations of cortical lesions were determined on p o s t m o r t e m . T h e location a n d e x t e n t of t h e electrolytic stereotaxic lesion in t h e m o n k e y (monkey N R 1) in which such a lesion was placed a n d t h e connections f r o m this lesion a n d f r o m t h e lesion of t h e fronto-parietal operculum of m o n k e y N R 2 were determined histologically b y

Central Gustatory Pathways in the Monkey the F I N K and H E I M E R silver impregnation technique for degenerating axons ( 1 9 6 7 ) , the D E O L M O S and I N G R A M silver impregnation technique ( 1 9 7 1 ) and the use of thionin stain.

Results The protocols and histological examination of the lesions and resulting degeneration where available are presented. Monkey NR 1 Preoperative Testing: 20th, 21st and 22nd of May 1975. The monkey was slower to reject the quinine banana than was observed in other monkeys prior to their operative procedures. She did not hesitate to eat a slice of lemon. Operative Protocol: Female. Weight — 4.5 kg. 23 May 1975. The skin and underlying connective tissue were reflected from the superior medial aspect of the left side of the head. A burr hole was drilled through the bone to the dura at the stereotaxic coordinates: A 0.7 and L5. The electrode was inserted through the cortex of the left cerebral hemisphere at the stereotaxic coordinates: A 0.7, L 5 and H —12. A stimulus of 0.1 volt produced ispilateral movement of the lower face at the corner of the mouth confirming that the tip of the electrode was in the motor nucleus of the facial nerve. In attempting to locate the nucleus of the tractus solitarius, it was considered essential as an important anatomical landmark to locate stereotaxically the motor nucleus of the facial nerve. Once stereotaxic placement of the electrode in this nucleus was verified by appropriate response to electrical stimulation the electrode was moved laterally (0.5—1.0 millimeters) and superiorly (1—2 millimeters) to the approximate anatomical location of the nucleus of the tractus solitarius (dorsal visceral gray) that receives those primary special visceral afferent fibers for taste which enter the brain stem in the facial nerve. Using a unipolar electrode, an electrolytic stereotaxic lesion was placed at this point. This area is continuous longitudinally with those parts of the nucleus of the tractus solitarius which receive primary gustatory fibers entering the brain stem by way of the glossopharyngeal and vagus nerves. These coordinates were then transposed caudally and longitudinally within the pons and the medulla to coincide with the known anatomical relationships of the nucleus of the tractus solitarius at these levels. A lesion was then placed in the nucleus of the tractus solitarius at the level of entrance of the glossopharyngeal nerve to the medulla. Four electrolytic stereotaxic lesions with a current of 3.5 ma for a period of 30 seconds were placed in monkey NR 1 at the following coordinates: Lesion 1, A 0.7, L 4.5 and H —10; Lesion 2,

23

A 0.7, L 5.0, and H - 1 0 ; Lesion 3, P 2.6, L 3.5 and H - 1 3 and Lesion 4, P 2.6, L 4.5 and H - 1 2 and H -13. Postoperative Notes: Immediately upon recovery from anesthesia the monkey exhibited a rotary nystagmus which lasted for approximately twentyfour hours. She held her head at an angle toward the side of the lesion until the time of sacrifice. The monkey continually held on to the side of the cage from the time of recovery from the anesthesia until her demise. Postoperative Testing: The monkey responded exactly as she had done preoperatively. She responded slowly to the quinine-treated banana, finally rejecting it. The monkey did not hesitate to eat the slice of lemon. Sacrificed: 6 June 1975. Description of stereotaxic lesions and resulting degeneration: The brain stem of this monkey, from caudal medullary levels through rostral thalamic levels, was frozen with solid carbon dioxide and sliced on a sliding microtome at 25 microns. Every fifth section was stained with the D E OLMOS and INGRAM ( 1 9 7 1 ) silver impregnation method for degenerating axons. Adjacent sections through the levels of the lesions and through the thalamus were stained with thionin. Light microscopy revealed that lesions 1 and 2 (Figure 1) were placed at the level of the entering rootlets of the facial nerve in the caudal on-third of the pons. The lesions involved the superior vestibular nucleus at this level and extended from the floor of the fourth ventricle superiorly to the middle of the lateral reticular formation inferiorly. The lesion did not directly involve the nucleus of the tractus solitarius at this level; however, the lesion was so extensive that many secondary gustatory fibers which may have crossed to the opposite side were most probably interrupted in their contralateral passage and indeed the secondary gustatory tract which had been traced from medullary levels was observed to become larger and more discrete at this level (Figure 2). Lesions 3 and 4 were placed at rostral glossopharyngeal levels in the medulle. Lesion 3 (Figure 1) involved the deep white matter of the cerebellum and degenerating axons were traced into the left superior cerebellar peduncle. Lesion 4 (Figure 3) involved the medial, lateral and inferior vestibular nuclei as well as the nucleus of the tractus solitarius (dorsal visceral gray) at the level of the most rostral portion of the glossopharyngeal nerve. At the level of the lesion in the medulla a small, degenerating finely medullated bundle of fibers is present just dorsal to the medial lemniscus. At caudal pontine levels this same bundle becomes larger and more discrete (Figure 1). It is difficult to follow the

24

Kinney, F. C.

secondary gustatory tract in its rostral projection to the contralateral side of the brain since it crosses at an oblique angle to reach its destination just dorsal to the medial lemniscus, for only a few fine fibers can be traced directly across the midline. However, as the medial lemniscus assumes its characteristic horizontal plane in the rostral pons, the secondary gustatory fibers are seen to maintain their medial and dorsal position in relationship to the medial border of the medial lemniscus. At rostral inferior collicular levels degenerating axons of the secondary gustatory tract are still dorsal to the medial border of the medial lemniscus in close relationship to the ventral portion of the central tegmental bundle within which there is a small degenerating descending component which represents a path resulting from the lesion of the left brachium conjunctivum. At the junction between the inferior and superior colliculi (Figure 4) secondary gustatory fibers interdigitate with the most lateral portion of the decussating degenerating ascending axons of the brachium conjunctivum (primarily dentatorubrothalamic tract). These secondary gustatory fibers cannot be clearly distinguished (Figure 5) from this last mentioned path until their respective terminations within the dorsal thalamus. Many fibers of the ascending component of the brachium conjunctivum terminate within the rostrally located small-celled portion of the red nucleus, whereas other components within this bundle project directly to the nucleus ventralis lateralis of the dorsal thalamus. Within the medially and rostrally located parvocellular portion of the nucleus ventralis posteromedialis (arcuate nucleus of many authors) of the dorsal thalamus terminal degeneration of the secondary ascending gustatory fibers was observed. The level of termination within the dorsal thalamus of ascending degenerating axons of the secondary gustatory tract is shown in Figure 6. Terminal degeneration within the most rostral portion of the nucleus ventralis posteromedialis pars parvocellularis is seen in Figure 7; and terminal degeneration of the ascending component of the brachium conjunctivum in the nucleus ventralis lateralis can be observed in Figure 8. Monkey NR 2 Operation NR One

Preoperative Testing: 27 April 1975 and 28 April 1975. Monkey NR 2 ate the lemon-soaked banana without hesitation. The animal bit into but did not continue to eat a piece of lemon. The monkey immediately rejected the quinine-treated banana upon tasting the quinine.

Operative Protocol: Male. Weight — 3.0 kg. 29 April 1975. The skin and temporalis muscle were reflected on the left side of the head. A left craniotomy was performed and the bone and dura overlying the base of the central fissure and the superior and middle temporal gyri were removed. A large lesion which involved the base of the precentral gyrus, the base of the postcentral gyrus, the adjacent superior temporal gyrus and a small portion of the middle temporal gyrus (Figure 9) was placed with surgical suction. Massive bleeding of the middle cerebral artery resulted. The bleeding was controlled with absorbable gelatin sponge (Gelfoam, Upjohn) and as soon as the surgical area was dry the wound was closed. Postoperative Notes and Testing: The monkey showed a marked paralysis of the entire right side of the body. However, by 29 July 1975, this monkey had apparently fully recovered from his paralysis. He was tested for his responses to quinine and bitter substances on this date. The monkey rejected the quinine-treated banana more slowly than prior to surgery and he no longer found the slice of lemon objectionable, for he ate the entire slice without hesitation. Operation NR Two

Operative Protocol: Male. Weight — 2.9 kg. 19 August 1975. A right craniotomy was performed and the bone overlying the base of the central fissure and the adjacent superior temporal gyrus was removed. The dura was incised with a cutting needle and reflected. Using a micropipette for surgical suction, the cortex at the base of the central fissure, its opercular surface and the adjacent superior temporal gyrus were removed. Little bleeding occurred and the wound was closed. Postoperative Notes and Testing: The monkey fully recovered from this operation with no apparent neurological deficits. Following this second operation little difference could be detected in the taste responses of this monkey. The response to the quinine-treated banana was the same as had been observed following the first operation in that the monkey still rejected the quinine more slowly than had been observed prior to any surgical procedure and he still ate the slice of lemon. The location and extent of the lesions in both hemispheres are seen in Figures 9 and 10. Sacrificed: 1 September 1975. Description of resulting degeneration from the lesion in the right cerebral hemisphere: The right cerebral hemisphere of monkey NR 2 was sliced in a coronal plane on a sliding microtome at 30 microns after having been frozen with solid carbon dioxide. Every fifth section was stained by the D E OLMOS and I N GRAM (1971) silver method for impregnating degenerat-

Central Gustatory Pathways in the Monkey

ing axons. Adjacent sections were stained with thionin. The level of the lesion at the base of the central fissure can be observed in Figure 11. The base and the inferior portion of the opercular surface were ablated as well as the adjacent portion of the superior temporal gyrus. From the lesion in the frontal and parietal opercula fine degenerating axons can be traced which course through the extreme capsule to end in the dorsal anterior island (Figure 12) thus linking the bases and opercular surfaces of the pre- and postcentral gyri to the island by short association bundles. Degenerating axons from neurons in the frontal and parietal lesions can also be traced to the nucleus ventralis posteromedialis of the dorsal thalamus (Figures 13 and 14). These axons course dorsal to the claustrum, and, accompanying the thalamocortical sensory radiations (Figure 15), pass through the posterior limb of the internal capsule to gain the thalamus. Terminal degeneration is abundant in the nucleus ventralis posteromedialis with only a few fine fibers terminating in the nucleus ventralis posteromedialis pars parvocellularis. Cytolysis of the cells of the nucleus ventralis posteromedialis as well as massive gliosis is clearly evident thus indicating retrograde degeneration from the cortical lesions. Even though degenerating axons could be traced to the parvocellular portion of the nucleus ventralis posteromedialis little if any retrograde degeneration is observable in this portion of the nucleus. Monkey NR 3 Preoperative Testing: 3 January 1975. Monkey NR 3 was tested for taste. He hesitantly rejected the lemon-soaked banana after he tasted it. He dropped the piece of lemon as soon as he tasted it. He expectorated the quinine-treated banana as soon as he had tasted the quinine. Operative Protocol: Male. Weight — 7.0 kg. 4 January 1975. An incision of approximately six centimeters slanting posterior superiorly to anterior interiorly was made above the left ear. It was necessary to excise a large portion of the temporalis muscle in order to have an adequate visual field. As much bone as possible was removed overlying the left temporal lobe and the inferior aspects of the rostral parietal lobe and the caudal frontal lobe. The dura was incised with a cutting needle and reflected with a scapel to expose the lateral fissure and the superior, middle and part of the inferior temporal gyri of the left temporal lobe. In an attempt to remove the island and the gustatory association areas a large portion of the temporal lobe was removed with suction. Little bleeding oc-

25

curred. The wound was packed with Gelfoam and was closed as soon as the surgical area was dry. Postoperative Notes and Testing: On 5 January, 1975 the monkey was eating, taking water and sitting erect. By 31 January 1975, the monkey's incision was completely healed and he appeared to have no neurological deficits. On 2 February 1975 monkey NR 3 was tested for his responses to sour and bitter substances. The animal did not, as had been previously observed, find the slice of lemon objectionable for he ate the entire slice. He rejected the quininetreated banana but did so slightly more slowly and with less vehemence than had been observed prior to any surgical procedure. The location and extent of the lesion was confirmed on postmortem and can be observed in Figure 16. Sacrificed: 12 February 1975. Monkey NR 4 Cercopithecus aethiops Operation NR One

Preoperative Testing: 12 August 1974. Monkey NR 4 was offered a piece of quinine-treated banana which he expectorated as soon as he tasted the quinine. Following his rejection of the quinine banana the animal did not hesitate to eat a piece of untreated banana. He was then offered a piece of lemon-treated banana which he smelled, dropped, then tasted and finally rejected. Operative Protocol: Male. Weight — 5.6 kg. 13 August 1974. The skin and temporalis muscle were reflected on the left. A left craniotomy was performed in which the bone and dura overlying the base of the central fissure were removed. The cortex at the base of the precentral gyrus and at the base of the postcentral gyrus and the opercular surfaces of these two cortical areas were removed by cauterization in the left cerebral hemisphere. Postoperative Notes: Monkey NR 4 fully recovered from this operation with no permanent neurological deficit. For the first two weeks following surgery a slight paresis of the right upper extremity was evident. Operation NR Two

Preoperative Testing: 26 November 1974. Monkey NR 4 was given a piece of lemon-soaked banana which he sniffed, tasted, then dropped and finally ate. The animal was then offered a piece of quinine-treated banana which he expectorated immediately upon tasting the quinine. Operative Protocol: 27 November 1974. The skin and temporalis muscle were reflected on the right. A right craniotomy was performed in which the bone and dura

26

Kinney, F. C.

overlying the base of the central fissure were removed. By cauterization a large lesion was placed in the cortex at the base of the precentral gyrus and at the base of the postcentral gyrus and also the opercular surfaces of these two gyri. There occurred a large amount of hemorrhage due to involvement of branches of the middle cerebral artery. The bleeding was controlled with Gelfoam, cotton and suction. The wound was packed with Gelfoam and as soon as the surgical area was dry the wound was closed. Postoperative Notes and Testing: 10 December 1974. Monkey NR 4 was tested for taste. He did not hesitate, as had been previously observed, to eat the lemon-treated banana. The monkey rejected the quinine-treated banana, but did so more slowly than had been previously observed. His behavior indicated that there had been a loss of some taste discrimination following the removal of the cortices at the base of the central fissure and of the opercular surface of this area bilaterally. The locations of the cortical lesions were confirmed on postmortem and are shown in Figure 17 and Figure 18.

complications arising from this disease. His brain was removed and placed in 10% formalin buffered with calcium carbonate. Figure 19 shows the location and extent of the cortical lesion in the left hemisphere. Discussion and review of the literature Many previous investigators 1934;

(ALLEN, 1 9 2 3 ;

W A L K E R , 1 9 3 8 ; BLUM, W A L K E R a n d

ADLER, RUCH,

1 9 4 3 ; PATTON, R U C H a n d W A L K E R , 1 9 4 4 a n d ABLES

and BENJAMIN, 1 9 6 0 ) have concluded that the nucleus ventralis posteromedialis, and/or its most medial portion (pars parvocellularis), of the dorsal thalamus is responsible for the thalamic reception of gustatory stimuli. Thalamocortical connections between the medial portion of the nucleus ventralis posteromedialis (i. e. parvocellular portion of this nucleus as described by OLSZEWSKI, 1 9 5 2 ) and the fronto-parietal operculum have frequently been demonstrated in the macaque (WALKER, 1 9 3 4 ; L E GROS CLARK, 1 9 3 7 a n d LOCKE, 1 9 6 7 ) .

CHOW and PRIBRAM ( 1 9 5 6 ) observed, based on retrograde degeneration studies following the placing of lesions at various cortical locations in the macaque, Monkey NR 5 that the nucleus ventralis posteromedialis projected Preoperative Testing: 27 December 1974. Rhesus to the operculum and to the anterior island. Based monkey NR 5 was offered a piece of quinine-treated on experimental evidence in the macaque, ROBERTS banana which he expectorated immediately upon and AKERT ( 1 9 6 3 ) reported that it was the anterior tasting the quinine. He bit into but did not continue island which received primary afferent connections to eat a slice of lemon. Unlike Monkey NR 4 (Cerco- from the nucleus ventralis posteromedialis pars pithecus aethiops) and the older rhesus monkey NR 3, parvocellularis and not the fronto-parietal operboth of whom had initially rejected, rather hesi- culum. B E N J A M I N , EMMERS and BLOMQUIST ( 1 9 6 8 ) tantly, the banana which had been soaked for twelve and BENJAMIN and BURTON ( 1 9 6 8 ) reached the hours in reconstituted lemon juice, the young rhesus conclusion that there are only sustaining projections did not hesitate to eat the lemon-treated banana. of gustatory modalities to somatic sensory area I Operative Protocol: Male. Weight — 2.9 kg. 30 De- in the squirrel monkey and that both the anterior cember 1974. The skin and temporalis muscle on the island and the cortical area in somatic sensory left were reflected. A left craniotomy was performed in area I which receives gustatory modalities must which the bone and dura overlying the base of the be removed surgically in order to result in retrograde left central fissure were removed. Cauterization was degeneration in the nucleus ventralis posteromedialis utilized to remove the cortex at the base of the central pars parvocellularis of the dorsal thalamus. fissure and the opercular surface of this area. Little Using evoked potential techniques (BLOMQUIST, bleeding occurred. As soon as the surgical area was dry BENJAMIN and EMMERS, 1 9 6 2 ) the dorsal thalamus the wound was closed. in the squirrel monkey was explored bilaterally Postoperative Notes: Monkey NR 5 recovered from with recording electrodes for responses to electrical this operation with no apparent neurological deficits. stimulation of the chorda tympani nerve, the lingualOn 2 February 1975 monkey NR 5 was tested for his tonsilar branch of the glossopharyngeal nerve and responses to sour and bitter substances. He expecto- the lingual nerve. The responses of the two nerves rated the quinine-treated banana as soon as he relaying gustatory modalities were reported to be tasted the quinine. He bit into but did not continue to largely ipsilateral with meager contralateral projeceat a piece of lemon. He did not hesitate to eat a tions, whereas the lingual nerve was reported to have a mainly contralateral projection. piece of lemon-treated banana. On 5 February 1975 monkey NR 5 was stricken Considering the available literature, there can be with shigellosis and on 14 February 1975 died from little question that the nucleus ventralis posterome-

Sacrificed: 11 December 1974.

Central Gustatory Pathways in the Monkey

dialis pars parvocellularis receives secondary gustatory projections from that part of the nucleus of the tractus solitarius (dorsal visceral gray) which is found at the level of the facial and glossopharyngeal nerves in the pons and medulla. In the present study such projections were confirmed by the rostral projection of secondary gustatory fibers to the contralateral nucleus ventralis posteromedialis pars parvocellularis in monkey NR 1. The neurophysiological evidence for the ispilaterality, contralaterality or bilaterality of the secondary projections of the dorsal visceral gray is conflicting. The evidence from monkey NR 1 is in accord with the observations that the lingual-tonsilar nerve has a primarily contralateral cortical (and therefore thalamic) projection(BENJAMIN a n d P F A F F M A N N , 1 9 5 5 ; YAMAMOTO a n d K A W A M U R A ,

1 9 7 5 ) . Certainly the clinical evidence (which will be discussed later in this report) overwhelmingly favors thalamic and cortical projections of gustatory modalities which are primarily contralateral in termination. During the latter part of the nineteenth century and throughout the first part of this century, it was believed by most observers that the cortical localization for gustatory impulses was in the temporal lobe in close association with afferent olfactory impulses. F E R R I E R ( 1 8 8 6 ) concluded in experimental procedures on two monkeys that bilateral lesions of the lower temporo-sphenoidal lobe resulted in abolition of smell and taste over the entire tongue. F E R R I E R thus felt ... the gustatory centres are situated at the lower extremity of the temporosphenoidal lobes in close relation with those of smell. Ferrier failed to mention, however, that the adjacent parietal lobe had also been damaged in his experiments, as is shown in Figure 101 of his book. The significance of this fact was also noted by B O R N S T E I N ( 1 9 4 0 a). K E N N E D Y ( 1 9 1 1 ) considered gustatory auras and olfactory hallucinations to be important tools in diagnosing uncinate fits and temporal lobe lesions, linking gustatory impulses with olfactory impulses in the temporal lobe. C U S H I N G ( 1 9 2 2 ) discussed ten of fifty-nine clinical cases in which there were temporal lobe lesions. Of these only two patients had gustatory auras; one patient thought he had smelled and tasted peaches on one occasion and insisted that he smelled and tasted roasted peanuts on another, and another patient complained of gustatory sensations which were peppermint in nature. V I L L I G E R ( 1 9 2 5 ) stated " . . . the gustatory center has not been definitely located but probably adjoins that for smell," and G R I N K E R ( 1 9 3 7 ) considered the hippocampal formation to have both olfactory and gustatory functions. B O R N S T E I N ( 1 9 4 0 a) in an extensive paper, reviewed

27

the clinical and experimental evidence for the temporal lobe theory for the cortical localization of taste and concluded that there was insufficient evidence to support this theory. Three clinical cases, each one of which was the result of a bullet wound to the left parietal bone, were discussed by B O R N S T E I N (1940b). The first case resulted in a lesion of the middle third of the pre- and postcentral gyri. The patient had an ageusia for sweet, sour, salty and bitter on the contralateral side of the tongue except for hypogeusia for salty substances on the contralateral tip. Taste was intact or only slightly impaired on the ipsilateral side of the tongue. The second case resulted in a lesion which was located in the face and tongue areas of the pre- and postcentral gyri, i. e., the upper border of the posterior frontal operculum and the parietal operculum. The patient exhibited an ageusia for sweet, sour and weak bitter solutions on the contralateral border of the tongue with slight hypogeusia for salty. On the contralateral tip there was a hypogeusia for sweet, salty and sour and on the contralateral base only sour and sweet were impaired. The third patient developed a parietal opercular syndrome not from the bullet wound itself but rather from the transplantation of a bone graft over the pre- and postcentral gyri which resulted in a subdural hematoma over this area. There was a hypogeusia for sweet, salty and bitter and a slight hypogeusia for sour on the contralateral side of the tongue. The border of the tongue on the contralateral side was chiefly involved. Gustatory disturbances were slight on the ipsilateral side of the tongue. S H E N K I N and L E W E Y ' S clinical case (1943) in which there was an involvement of the most inferior portion of the postcentral gyrus also indicated this area as a possible site for the cortical localization of gustatory sensations. GORSCHKOW ( 1 9 0 1 ) postulated that gustatory impulses were localized in the potential island region in the dog. A D L E R ( 1 9 3 5 ) gave support to the possibility that the island may be the cortical area responsible for the cortical recognition of gustatory impulses. She discussed a case in which a tumor grew into the island from the superior temporal lobe. The patient had a hypogeusia on the tongue contralateral to the cortical lesion. P E N F I E L D and E R I C K S O N ( 1 9 4 1 ) cited a clinical case in which the patient had seizures preceded by a gustatory aura of an indescribably nature. "This patient had a well circumscribed slowly growing astrocytoma of the island of Reil." P E N F I E L D and E R I C K S O N concluded that taste was probably represented cortically at the base of the pre- and postcentral gyri in close association with the cortical areas for the jaws and the tongue. P E N F I E L D and J A S P E R ( 1 9 5 4 ) , based on their observations of clinical cases, stated " . . . gustatory sensation has a representation

28

Kinney, F. C.

in the cortex which seems to be closely related to salivation, to the alimentary system, and to second sensory representation. It is located beneath the fissure of Sylvius and within the circular sulcus." Gustatory impulses have been localized in the opercular insular junction and in the floor of the anterior island by P E N F I E L D and RASMUSSEN (1950). PATTON and RUCH (1946) concluded in a series of cortical ablation experiments on monkeys and chimpanzees that taste deficits only occurred when the buried cortex of the fronto-parietal operculum was invaded. After recognizing that Gerhardt had described an area of granular cortex of sensory type in the chimpanze which was located deep in the frontal operculum bordering on the circular fissure, RUCH and PATTON (1946) attempted destruction of this area. Successful ablation was achieved in only one monkey resulting in a severe taste impairment in regard to bitter solutions. These investigators expanded their previous conclusions by stating that taste is localized in the parainsular operculum and not in the free cortex of the operculum. BAGSHAW and PRIBRAM ( 1 9 5 3 ) observed a lowering in quinine acceptance thresholds in monkeys in which the anterior insula or both the anterior island and the anterior supratemporal plane had been surgically ablated. Lesions of the anterior island plus operculum resulted in moderate decreases in quinine thresholds. Lesions of the insula, the operculum and the anterior supratemporal plane together resulted in marked and prolonged ageusia. B R A D L E Y (1963), in his experiments on macaques, considered the cortex at the base of the central fissure and the opercular surface of this area to be important for normal taste discrimination. He, furthermore, suggests from his experiments that taste may have a representation at mid-brain levels. Bilateral cortical representation in the squirrel monkey for all three nerves innervating the tongue, that is, the chorda tympani branch of the facial nerve, the lingual-tonsilar branch of the glossopharyngeal nerve, and the lingual branch of the trigeminal nerve, was demonstrated by BENJAMIN et al. (1968) by the utilization of evoked potential techniques. The cortical area found to be responsive to electrical stimulation was in somatic sensory area I at the base of the pre- and postcentral gyri. The major projection, however, of the chorda tympani and lingual-tonsilar nerves was found to be ipsilateral. In a related study which also utilized evoked potential techniques, a responsive locus for taste was located in the most anterior opercular-insular cortex (BENJAMIN and BURTON, 1968). Only stimulation of the ipsilateral chorda tympani nerve and the ipsilateral lingual-tonsilar nerve was effective.

Chemical stimulation of the tongue and/or electrical stimulation of the ipsilateral chorda tympani nerve produced evoked potentials in neurons of the nucleus ventralis posteromedialis pars parvocellularis in the rat and marmoset (GANCHROW and ERICKSON, 1 9 7 2 ) . Antidromic stimulation of these same cells by electrical stimulation of the cortical projection area of the chorda tympani nerve indicated direct thalamocortical projections of taste neurons and direct projections from the cortex to the cells of the thalamus which receive gustatory impulses. Based on cytoarchitectural studies in the squirrel monkey, SANIDES ( 1 9 6 8 ) concluded that there was a surface cortical gustatory area in somatic sensory area I and also a deep pure gustatory area located in the frontal operculum and bordering on the insula. In cat, PATTON and AMASSIAN ( 1 9 5 2 ) reported a bilateral cortically responsive area to electrical stimulation of the chorda tympani nerve which was superior to the rhinal fissure and rostral to the anterior ectosylvian fissure. The insula as well as other cortical areas were unresponsive to the stimulation. RUDERMAN et al. ( 1 9 7 2 ) obtained responses of neurons in the nucleus ventralis posteromedialis pars parvocellularis of the cat while stimulating the anterior portion of the tongue with citric acid solutions. Light brush strokes of various facial regions resulted in evoked potentials which were recorded from electrodes in the nucleus ventralis posteromedialis. Electrolytic lesions of the nucleus ventralis posteromedialis pars parvocellularis resulted in cortical degeneration in either the lateral or both blanks of the presylvian sulcus. Electrolytic lesions that were placed in the nucleus ventralis posteromedialis resulted in cortical degeneration in the coronal gyrus. Thus separate cortical projection areas for taste and facial tactile impulses were delineated. The cortical area responsive to mechanical stimulation of the ipsilateral chorda tympani nerve in the cat was located by BURTON and EARLS ( 1 9 6 9 ) in an area which extended from the coronal gyrus anteriorly to the orbital sulcus posteriorly and lay dorsal to the rhinal fissure. This active surface cortex was located within somatic sensory area I for the tongue. ZOTTERMAN ( 1 9 5 8 , cat) reported evoked potentials from cortical cells which responded only to taste stimuli. The responses were not to specific taste modalities. COHEN et al. ( 1 9 5 7 ) explored the cortical projection area of the tongue (orbital surface of the cerebral hemisphere) in cat, with recording electrodes. This area was responsive to electrical stimulation of the chorda tympani and lingual nerves and also to thermal, mechanical and gustatory stimulation of the tongue. Some cortical cells were responsive to

Central Gustatory Pathways in the Monkey

electrical stimulation of both chorda tympani and lingual nerves. Cortical cells which responded to mechanical stimulation did not respond to gustatory or thermal stimulation. Evoked potentials were recorded from five cortical cells which responded only to gustatory modalities, but again the cells were not specific in their response to particular gustatory sensations. LANDGREN ( 1 9 5 7 ) reported 2 7 single cortical cells (out of 101 cortical cells from which evoked potentials were recorded) which responded to more than one type of sensory stimulation. Cells were found which responded to combinations of touch, stretch, cooling, warming and various gustatory stimuli. Various sensory modalities including taste, therefore,were seen to converge on single cortical cells. Evoked potential studies were utilized by BENJAMIN and PFAFFMANN ( 1 9 5 5 ) to locate the cortical receptive zones for the glossopharyngeal and chorda tympani nerves in the rat. The chorda tympani nerve was found to be represented bilaterally in the cerebral cortex whereas the glossopharyngeal nerve only had a contralateral cortical representation. Bilateral ablations of this cortical area resulted in permanent taste deficits. The authors stated that the boundaries of the lesions included the anterior insular cortex as delimited by Krieg in the rat. Bilateral removal of a small area of cortex in the rat just dorsal to the rhinal fissure in somatic sensory area I produced partial taste deficits (BENJAMIN and A K E R T , 1 9 5 9 ) . Inclusion of an adjacent fringe area in bilateral ablations caused severe gustatory impairments. This cortical area corresponded to the cortex which was responsive to stimulation of the glossopharyngeal and chorda tympani nerves as determined by evoked potential techniques. Ablation of all neocortex with the exception of this focal gustatory area had no effect upon the taste discrimination of the animals. Electrical stimulation of the chorda tympani, glossopharyngeal and lingual nerves in rabbits (YAMAMOTO and KAWAMURA, 1 9 7 5 ) demonstrated a primarily ipislateral responsive area in the insular cortex for the chorda tympani nerve, a primarily contralateral response in the same cortical area for the glossopharyngeal nerve and predominantly contralateral projection in somatic sensory area I for the lingual nerve. All projections, however, were bilateral. The cortical projection area for the chorda tympani nerve was found rostral to the projection for the glossopharyngeal nerve and partially overlapped it and the lingual nerve projection area partially overlapped the dorsal aspect of the chorda tympani projection area. The possible existence of a subcortical and even subthalamic localization of taste responses in carni-

29

vores was demonstrated by MACHT ( 1 9 5 1 ) . Mesencephalic and bulbospinal cat preparations rejected quinine, saline and acetic treated substances at the same concentrations as normal rejection thresholds. In the present study lesions of the cortex at the base of the central fissure and its opercular surface resulted in taste deficits. A unilateral destructive lesion of this area resulted in a definite slowing of the taste responses of one monkey and a loss of some discrimination in regard to sour substances in two out of three monkeys. Destructive lesions placed bilaterally in the cerebral cortex at the base fo the central fissure and extending into the opercular surfaces of the precentral and postcentral gyri resulted in a slowing of the response to quinine in one monkey. More importantly the loss of a more delicate type of discrimination, in regard to sour substances, was observed with monkey NR 4 (Cercopithecus aethiops) following bilateral ablation of this area. It is evident from work of others and from the present study that the cortex at the base of the central fissure extending into the fronto-parietal operculum is of particular importance in the reception of gustatory modalities and, being directly continuous with primary sensory cortex, represents primary receptive sensory cortex for taste. The functional significance of this area as it relates to the anterior island has, however, remained somewhat obscure. Experimental observations have repeatedly shown that the anterior island in primates and man is involved in the recognition of gustatory modalities. The coronal sections of monkey NR 2 indicate that the cortex at the base of the central fissure and continuing into the fronto-parietal operculum is linked to the dorsal anterior island by short association bundles which course through the extreme capsule. These short association bundles, then, relate gustatory modalities from their primary receptive cortex to the association cortical areas involved in the recognition of gustatory impulses, that is the anterior dorsal part of the Island of Reil. E L L I O T SMITH ( 1 9 0 7 ) traced, in man, fibers from the lateral olfactory tract into the anterior island. CROSBY, HUMPHREY and L A U E R ( 1 9 6 2 ) stated that "The olfactory stalk has been traced into the frontal end of the island on the pyriform side. This relation is seen in the His embryological models." Thus, the anterior island, which receives both olfactory and gustatory impulses, is a correlative cortical area for special visceral afferent sensations and allows for subjective discrimination in regard to these sensations. Special visceral sensations are in turn correlated with other known general visceral functions of the island. P E N F I E L D and RASMUSSEN ( 1 9 5 0 ) elicited a number

30

Kinney, F . C.

of visceral sensations upon electrical stimulation of the insula in awake patients. Upon stimulation of various locations within the insula, patients reported a curious disagreeable taste, the sensation of nausea, vomiting, a feeling of illness, an aura of attack with swallowing and mouth opening and the desire to defecate. KAHN et al. (1.969) discussed several clinical cases involving the insula. One patient complained of a bitter taste in his mouth which preceded convulsions. The patient had a tumor of the anterior island. Another patient had a four-year history of convulsions during which he complained of nausea and often vomited. At operation an arteriovenous malformation was discovered which had a focal point in the parietal operculum and the posterior island. A third patient had auras of abdominal pain which frequently preceded convulsions. A right temporo-parietal cyst was found at operation. The location of the lesion

suggested that stimulation in the posterior island region resulted in abdominal pain. KAHN et al. (1969) suggested that there is some evidence that primary gustatory cortex is connected to the uncus region by fascicles which pass through the island. Such connections could account for rep o r t e d cases (KENNEDY, 1 9 1 1 a n d CUSHING, 1922) in

which gustatory auras preceded convulsions which were the result of lesions located primarily in the temporal lobe. Uncinate fits which are preceded by auras of an olfactory nature are the result of lesions located primarily in the region of the amygdala; or along the lateral olfactory tract and perhaps the anterior perforated space. However, uncinate fits which are preceded by gustatory auras might result from lesions located largely in the anterior island but which involve those fascicles which connect the island with the uncus region.

r

'

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mk^

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- V *

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Fig. 1. Photomicrograph of a transverse section through the caudal one third of the pons of Monkey N R 1. Stereotaxic electrolytic lesions 1 and 2 at the level of the entering rootlets of the facial nerve on the left side are clearly evident. The arrow in the upper left quadrant of the photomicrograph indicates the position of stereotaxic electrolytic lesion number 3 which involved the deep white matter of the cerebellum. The area ecnlosed in black (shown at a higher magnification in Figure 2) indicates the position of the secondary ascending gustatory tract just dorsal to the medial lemniscus. De Olmos and Ingram preparation, x 6.3

List of Abbreviations Used in Figs. 1, 2 and 3 GN V I I InfO ML MLF Pyr

Genu of Seventh (Facial) Cranial Nerve Inferior Olive Medial Lemniscus Medial Longitudinal Fasciculus Pyramid

SGTr NTS NIX IV V

Secondary Gustatory Tract Nucleus of the tractus solitarius Ninth (Glossopharyngeal) Nerve Fourth Ventricle

C f i i t m l G u s t a t o r y P a t h w a y s ili t h e M o n k e y

I'ig.

Photomicrograph

degenerating

»1: t h e a r e a o u t l i n e d i n l i l a c k i n F i g u r e 1.1 ( M o n k e y

1). T h e a r r o w s p o i n t t o sonic; of

axons ot the secondary ascending gustatory tract. 'Hie secondary ascending gustatory

tract

31

the

passes in a

rostra! longitudinal direction through t l i e l i r a i n stem and the d e g e n e r a t i n g ;i\ons are t h e r e f o r e cut transversely. I )i: Oitnos a n d I n g r a m p r e p a r a t i o n .

:!:!

¡1. P h o t o m i c r o g r a p h of a t r a n s v e r s e s e c t i o n t h r o u g h t h e

rostral

medulla of

Monkey

X l t 1. 'I'he. size,

a n': e x t e n t of t h e fourth s t e r e o t a x i c e l e c t r o l y t i c lesion piaceli in M o n k e y N K I a r e c l e a r l y e v i d e n t . I V Olmos ami Ingram preparation, x

fi.il

relationships

32

Kinney, F. C.

Fig. 4. Photomicrograph of a transverse section through the junction between the inferior and superior colliculi of Monkey NR 1 at the level of the decussation of the brachium conjunctivum. The area outlined in black indicates the position of the secondary gustatory tract at this level. De Olmos and Ingram preparation, x 6.3 Fig. 5. Photomicrograph at a higher magnification of the area enclosed in black in Figure 4 (Monkey NR 1). The secondary gustatory tract interdigitates with degenerating fibers of the decussating brachium conjunctivum and cannot be distinguished from them until their respective levels of termination within the dorsal thalamus. The arrows indicate some of the fibers of the secondary gustatory tract as they swing superiorly to interdigitate with the fibers of the brachium conjunctivum. De Olmos and Ingram preparation, x 100 List of Abbreviations Used in Figs. 4 and 5 CA Cerebral Aqueduct DBC Decussation of the Brachium Conjunctivum

MLF Medial Longitudinal Fasciculus SGTr Secondary Gustatory Tract

Central Gustatory Pathways in the Monkey

Himforschung, Bd. 19, Heft 1

3

33

34

Kinney, F. C.

Fig. 6. Photomicrograph of a transverse section through the dorsal thalamus at the most rostral level of the nucleus ventralis posteromedialis pars parvocellularis of Monkey NR 1. Terminal degeneration within the nucleus ventralis posteromedialis pars parvocellularis (outlined in black) of the secondary gustatory tract is shown at a higher magnification in Figure 7. Terminal degeneration of the brachium conjunctivum (dentatorubrothalamic tract) in the nucleus ventralis lateralis is outlined in black and shown at a higher magnification in Figure 22. De Olmos and Ingram preparation, x 6.3 Fig. 7. Photomicrograph of the nucleus ventralis posteromedialis pars parvocellularis (outlined in Figure 6, Monkey NR 1) demonstrating terminal degeneration of the secondary gustatory tract. De Olmos and Ingram preparation, x 32 Fig. 8. Photomicrograph of the nucleus ventralis lateralis (outlined in Figure 6, Monkey NR 1) of the dorsal thalamus illustrating terminal degeneration of the ascending component of the brachium conjunctivum. De Olmos and Ingram preparation, x 32 List of Abbreviations Used in Figs. 6, 7 and 8 CN VL

Centromedian Nucleus Nucleus Ventralis Lateralis

VPMpc III V

Nucleus Ventralis Posteromedialis pars parvocellularis Third Ventricle

Central Gustatory Pathways in the Monkey

.•-j'r-?

3®

35

36

Kinney, F. C.

Fig. 9. Left cerebral hemisphere of Monkey NR 2. The large coitieal lesiön which iuvelvuQ the-bases of the pre- and postcentral gyn, at the base of the central fissure (CF), and tHe adjacent superior and satddie temporal gyri is clearly ap$>.3sfe»t. Fig. 10. Right cerebral hemisphere of Monkey NR % illustrating the cortical 3$sioh which involved the fronto-parietal opea> culum at the base of the central fissure (CF), its opercular surface and the adjacent portion "of the superior temporal gyrus.

Central Gustatory Pathways in the Monkey

37

'it

ExtrCap

CI Is E x t Cap LF

Fig. 11. Photomicrograph of a coronal section through the level of the right cortical lesion of Monkv XK 2. As can be observed, the deep white m a t t e r of this cortical area as well as the fronto-parietal operculum was involved in the lesion. Fine degenerating axons can be traced through the extreme capsule into the dorsal anterior part of the Island of Keil. ])e Olmos and Ingram preparation, x Fig. 12. Photomicrograph a t a higher magnification of t h e area outlined in black in Figure 11 (Monkey X k 2). Preterminal and terminal degeneration can be observed throughout t h e field. De Olmos and Ingram preparation, x 230 List of Abbreviations I'sed in Figs. 11 and 12 CI Claustruni Kxt Cap External Capsule Kxtr Cap Kxtreme Capsule

FPC)

l'ronto-Parietal operculum

Is

Island of Keil

I-F

Lateral Fissure

STG

Superior Temporal Gyrus

38

Kinney, F. C.

Fig. 13. Photomicrograph of a coronal section through the dorsal thalamus of Monkey NR 2. Terminal degeneration from the lesion in the fronto-parietal operculum can be observed within the nucleus ventralis posteromedialis and a few fine fibers can be traced to the parvocellular portion of this nucleus. De Olmos and Ingram preparation. X 6.3 Fig. 14. Photomicrograph at a higher magnification of the area outlined in black in Figure 27 (Monkey NR 2). Massive terminal degeneration can be seen in the nucleus ventralis posteromedialis. The arrows indicate the terminal degeneration of a few fibers within the parvocellular portion of the nucleus ventralis posteromedialis. De Olmos and Ingram preparation. X 21.6 Fig. 15. Photomicrograph of degenerating corticothalamic fibers which accompany the sensory radiations (thalamocortical fibers) as they pass through the posterior limb of the internal capsule to gain entrance to or exit from the dorsal thalamus (Monkey NR 2). De Olmos and Ingram preparation. X 32 List of Abbreviations Used in Figs. 13, 14 and 15 Cn CT ICapPl

Centromedian nucleus Corticothalamic fibers Internal Capsule Posterior Limb

VPM VPMpc

Nucleus Ventralis Posteromedialis Nucleus Ventralis Posteromedialis pars parvocellularis

Central Gustatory Pathways in the Monkey

V '¿"J,',•• •

39

40

Kinney, F. C.

Fig. 16. The left cerebral hemisphere of Monkey NR 3. The arrow indtimlss tte lesion of the postcentral gyrus -aM its opercular surface. Fig. 17. The left cerebral hemisphere of Cercopithecus aethiops. The cortical lesion at the base of the pre- and postcentral gyri is clearly evident. Arrow indicates the central fissure (CF).

Central Gustatory Pathways in the Monkey

41

Tig 18. The right cerebral hemisphere of Cerùopitheùus caetkiops. The cortical lesion at thé base of the pre- and postcentral gyri. which also include the adjacent superior temporal gyrus, is clearly evident. Pig. 19. The left cerebral hemisphere of Monkey NR 5. The cortical lesion at the base of the central fissure and the adjacent superior temporal gyrus is clearly shown.

42

Kinney, F. C.

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tions in gustation. Brain Res., 36, 2 8 9 - 3 0 5 (1972). GORSCHKOW, J. P.: Über Geschmacks- und Geruchszentren in der Hirnrinde Inaug.-Diss., St. Petersburg (Russian) Abstract in Neurol. Zentralblatt, 20, 1 0 9 2 - 1 0 9 3 (1901). GRINKER, R. R.: Neurology. Charles C Thomas, Springfield (1937). KAHN, E . A.,

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TAREN: Correlative Neurosurgery. (2nd Ed.) Charles C Thomas, Springfield (1969). KENNEDY, F.: The symptomatology of temporosphenoidal tumors. Arch. Int. Med., 8, 3 1 7 - 3 5 0 (1911). LANDGREN, S.: Convergence of tactile, thermal and gustatory impulses on single cortical cells. Acta Physiol. Scand., 110, 2 1 0 - 2 2 1 (1957). L E G R O S C L A R K , W . E . : The connections of the arcuate nucleus of the thalamus. Proc. Roy. Soc. London, Ser B, 123, 1 6 6 - 1 7 9 (1937). LOCKE, S.: Thalamic connections to insular and opercular cortex of monkey. J. Comp. Neurol., 129, 219 — 240 (1967). M A C H T , M . B.: Subcortical localization of certain " t a s t e " responses in the cat. Fed. Proc., 10, 88 (1951). OLSZEWSKI, J . : The Thalamus of the Macaca mulatta. Karger, Basel (1952). PATTON, H . D . , a n d V . E . AMASSIAN: C o r t i c a l p r o j e c t i o n of

chorda tympani nerve in cat. J. Neurophysiol., 15, 245 — 250 (1952). PATTON, H . D . , a n d T . C. RUCH: T h e r e l a t i o n of t h e f o o t of

the pre and postcentral gyrus to taste in the monkey and chimpanzee. Fed. Proc., 5, 79 (1946). PATTON, H . D . ,

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A. E. WALKER:

Experi-

mental hypogeusia from Horsley-Clarke lesions of the thalamus in Macaca mulatta. J. Neurophysiol., 7,171 — 184 (1944). P E N F I E L D , W . , and T. C. E R I C K S O N : Epilepsy and cerebral localization. Charles C Thomas, Springfield (1941). P E N F I E L D , W . , and H. J A S P E R : Epilepsy and the Functional Anatomy of the H u m a n Brain. Little, Brown and Co., Boston (1954). P E N F I E L D , W . , and T. R A S M U S S E N : The Cerebral Cortex of Man. Macmillan, New York (1950). ROBERTS, T., and K. AKERT: Insular and opercular cortex and its thalamic projection in Macaca mulatta. Schweiz. Arch. Neurol. Neurochir. Psychiat., 92, 1 - 4 3 (1963). RUCH, T . C., a n d H . D . PATTON: T h e r e l a t i o n of t h e d e e p

opercular cortex to taste. Fed. Proc., 5, 89 — 90 (1946). RUDERMAN, M . I.,

A.R.MORRISON,

and

P.J.HAND:

A

solution to the problem of cerebral cortical localization of taste in the cat. Exp. Neurol., 37, 522 — 537 (1972). SANIDES, F . : The architecture of the cortical taste nerve areas in squirrel monkey (Saimiri sciureus) and their relationship to insular, sensorimotor and prefrontal regions. Brain Res., 8, 8 7 - 1 2 4 (1968).

Central Gustatory Pathways in the Monkey SCHWARTZ, H . G . ,

G . E . ROULHAC, R . L . LAM,

and

J.

L.

Organization of the fasciculus solitarius in man. J . Comp. Neurol., 94, 2 2 1 - 2 3 9 (1951). S H E N K I N , H . A., and F . H . L E W E Y : Aura of taste preceding convulsions associated with a lesion of the parietal operculum: Report of a case. Arch. Neurol. Psychiat., 50, 3 7 5 - 3 7 8 (1943). V I L L I G E R , E . : Brain and Spinal Cord. (3rd Ed., W . H . F . Addison, Editor) J . B . Lippincott Co., Philadelphia. 4925. VON B E C H T E R E W , W . : Die Functionen der Nervencentra. Fischer, J e n a ( 1 9 0 8 - 1 9 1 1 ) . W A L K E R , A. E . : The thalamic projection to the central gyri in Macacus rhesus. J . Comp. Neurol., 60: 161 — 184 (1934). W A L K E R , A. E . : The Primate Thalamus. The University of Chicago Press, Chicago (1938). O'LEARY:

43

and Y . K A W A M U R A : Cortical responses to electrical and gustatory stimuli in the rabbit. Brain Res., 94, 4 4 7 - 4 6 3 (1975). Z O T T E R M A N , Y . : Studies in the nervous mechanism of taste. Exp. Cell Res., Supp 5, 5 2 0 - 5 2 6 (1958).

YAMAMOTO, T . ,

Author's

address:

Francis Cleveland K I N N E Y , Ph. D . Department of Anatomy University of Alabama in Birmingham B o x 317, University Station Birmingham, Alabama 35294

J.

Hiriiforsch.

19 (1978) 45 - 73

Anatomisches I n s t i t u t des Bereiches Medizin (Charité) der Humboldt-Universität Berlin (Director: Prof. Dr. sc. med. W . K I R S C H E )

Experimental alterations in number and length of different membrane complexes on axosomatic contacts in the trout (Salmo irideus, Gibbons 1855) By Thomas

SCHUSTER

W i t h 10 figures and 7 tables (Received 12 t h April 1977)

Summary: 1. Subjects of this research are t h e mixed synapses between secondary vestibular axon terminals and perikarya of t h e oculomotor nucleus of t h e t r o u t (Salmo irideus, G I B B O N S 1855). These mixed synapses of characteristic shape and extraordinary size show three different types of special membrane complexes: active zones, gap junctions and desmosome-like structures. 2. Quantitative estimations regarding t h e plasticity of these mixed synapses have been performed on ultrastructural level. Test condition was a different labyrinthine receptor stimulation. For this purpose the fish had t o swim for a period of 5 or 14 days a) in violantly streaming and edding water or b) in steadily running water (a: "stressed fish", b : "immobile fish"). After counting t h e number and measuring t h e length of t h e different membrane complexes the " p o r t i o n " of t h e membrane complexes (product of number and length) has been estimated. 3. The obtained results are in t h e control group as follows: portion of active zones a t t h e contact zone — 15.8%, portion of gap junctions — 6.4%, portion of desmosome-like structures — 6.6%, total portion of all membrane complexes a t t h e contact zone — 28.8%. The mean length of t h e different membrane complexes are: active zones — 0.33 ¡xm, gap junctions — 0.23 |im, desmosome-like structures — 0.22 (Jim. 4. The changes of the different membrane complexes in relation to test condition and time are t h e following: The portion of active zones of 14-d-immobile fish covers 8.8% of t h e contact zone. This value increases t o 26% in the 14-d-stressed fish. By contrast, t h e portion of desmosome-like structures and gap junctions falls from 13.2% or 17%, respectively (immobile fish) to 6.2% or 3.6%, respectively (stressed fish). Comparing the total portions of all membrane complexes it is seen t h a t t h e 14-d-groups (stressed and immobile fish) show a higher proportion of membrane specialization t h a n the control group (39% in immobile fish, 3 5 . 8 % in stressed fish, in contrast t o 28.8% in the control group). 5. Regarding t h e positioning of t h e three different membrane specializations, as a rule, a close vicinity of gap junctions and desmosome-like structures is striking. This fact correlates with t h e quantitative changes of t h e portions (see above) and is in accordance with the literature. 6. W i t h regard to these results the experimental conditions, especially t h e influence of stress factors are discussed. Difficulties in differentiating t h e three types of membrane specializations are mentioned. 7. Changes in active zone portions are discussed in relation to a neuromodulating function of ACh, t h e likely transmitter a t these chemical synapses. The active zone increase in stressed fish m a y be interpreted as the effect of an functional "overloading". 8. Comparing t h e metabolic dependence of both chemical and electrotonic transmission gap junctions are supposed to be more independent transmission structures, their changes may represent a compensatory mechanism in metabolic transport processes. 9. Juxtaposition of desmosome-like structures and gap junctions as well as t h e simultaneous changes of b o t h structures support the idea of a functional relationship between them.

Zusammenfassung: 1. Untersuchungsobjekt dieser Arbeit waren die „gemischten" Synapsen, die zwischen sekundären vestibulären Axon-Terminalen und Perikarya des Okulomotorius-Kerns der Forelle (Salmo irideus, G I B B O N S 1855) ausgebildet werden. Diese außerordentlich großen und charakteristisch gestalteten K o n t a k t e besitzen drei unterschiedliche Typen von Membranspezialisierungen : Aktivzonen, "gap junctions" und desmosomen-artige Strukturen. 2. Die quantitativ-elektronenmikroskopischen Untersuchungen galten der Plastizität dieser gemischten Synapsen, die mittels unterschiedlich starker Reizung der Labyrinth-Rezeptoren getestet werden sollte. Zu diesem Zweck h a t t e n die Fische 5 bzw. 14 Tage a) in stark strömenden und wirbelndem Wasser („Belastungstiere") oder b) in ruhig und gleichmäßig fließendem Wasser zu schwimmen („Ruhetiere"). Nach Zählung und Messung der speziellen Membrankontakte wurde aus dem P r o d u k t beider der „Anteil" des entsprechenden Membrankomplexes an der Gesamtlänge der Kontaktzone bestimmt.

46

Schuster, Th. 3. In der Kontrollgruppe wurden folgende Werte ermittelt: Anteil der Aktivzonen an der Kontaktzone — 15,8%, gap junctions — 6,4%, desmosomen-artige Strukturen — 6,6%, Gesamtanteil aller speziellen Membrankomplexe — 28,8%. Durchschnittliche Länge der verschiedenen Membrankomplexe: Aktivzonen — 0,33 ¡¿m, gap junctions — 0,23 (im, desmosomen-artige Strukturen — 0,22 (im. 4. Die Änderung der Anteile der Membrankomplexe in Abhängigkeit von den Versuchsbedingungen: Der Anteil der Aktivzonen beträgt bei 14-d-Ruhetieren 8,8%. E r steigt bei 14-d-Belastungstieren auf 26% an. Im Gegensatz dazu sinkt der Anteil der desmosomen-artigen Strukturen und gap junctions von 13,2 bzw. 17% bei Ruhetieren auf 6,2 bzw. 3,6% bei 14-d-Belastungstieren. Der Gesamtanteil aller speziellen Membrankomplexe liegt sowohl bei 14-d-Belastungstieren (35,8%) als auch bei 14-d-Ruhetieren (39%) über dem Wert der Kontrolltiere (28,8%). 5. Im Hinblick auf die Lage-Beziehungen der drei unterschiedlichen Membrankomplexe zueinander war die häufige, enge räumliche Nachbarschaft von gap junctions und desmosomen-artigen Strukturen auffällig. Dieser Befund stimmt mit den quantitativen Veränderungen des Anteils beider Strukturen, die simultan erfolgen, überein und steht mit den Angaben aus der Literatur in Einklang. 6. Die Ergebnisse werden zunächst in Hinblick auf mögliche Einflüsse von Streß-Faktoren diskutiert. Weiterhin wird auf die Schwierigkeiten bei der Differenzierung der drei speziellen Membrankomplexe eingegangen. 7. Die Veränderungen des Anteils der Aktivzonen werden in Zusammenhang mit einer neuromodulatorischen Funktion des wahrscheinlichen Transmitters ACH betrachtet. Der Anstieg des Aktivzonen-Anteils bei Belastungstieren wird als das Resultat einer Reizüberlastung ("overloading") angesehen und umgekehrt. 8. Beim Vergleich der Stoffwechsel-Abhängigkeit der chemischen und der elektrotonischen Transmission scheinen gap-junction-Strukturen weniger abhängig zu sein; die entgegengesetzt zu den Aktivzonen erfolgende Änderung des Anteils der gap junctions könnte als ein Kompensationsmechanismus beim interneuronalen Stofftransport angesehen werden. 9. Enge Nachbarschaft von gap junctions und desmosomen-artigen Strukturen sowie simultane Änderung des Anteils beider Strukturen unter Belastung sprechen für einen funktionellen Zusammenhang zwischen beiden.

Introduction Two levels exist at which synapses influenced by experience may be examined morphologically: 1st — new formation or involution of synapses (changes in number), 2nd — modulation of specialized structures of individual synapses. If both changes in the number of synapses and changes in the structure of existing synapses are regarded as changes in efficiency of transmission we are able to examine what has been term e d " s y n a p t i c p l a s t i c i t y " (BLOOM1970; CRAGGl972a;

STREIT et al. 1972). In this research only changes in the number of synapses have been controlled. Whereas quantitative data on chemical synapses (active zones) are available, until now the plasticity of gap junctions or "mixed synapses" (contacts showing both active zones and gap junctions) has not yet been investigated electron microscopically. This fact contrasts with the occurence of these special synaptic contacts. Gap junctions — the morphological correlate of electrotonic transmission — are not only common in lower vertebrate nervous tissue, they have been found in mammalian C. N. S. (SOTELO 1 9 7 5 ) , in p r i m a t e n e o c o r t e x (SLOPER 1 9 7 2 ) a n d i n m a n

(MOLLGARD 1975). Mixed synapses are also present throughout the vertebrate phylum (Table V, see below). These findings are not in agreement with the assumption that gap junctions may be important only in lower vertebrates. Because of their extraordinary size and characteristic shape the mixed synapses in trout oculomotor nucleus ("giant synapses" — KIRSCHE 1967) are especially suitable for studying quantitative changes

in synaptic activity electron microscopically. Three types of special membrane complexes occur at these axosomatic contacts between secondary vestibular axon terminals and oculomotor perikarya: active zones, gap junctions and desmosome-like structures. To examine the plasticity of these complex mixed synapses the question should be answered in what way a functional "overloading" or "underloading" of the labyrinthine receptors changes number or size of the three different membrane complexes. Some of these results have been partly reported (SCHUSTER 1 9 7 6 , 1977).

Materials and methods Material

and experimental

conditions:

Rainbow trouts (Salmo irideus, G I B B O N S 1855) ranging from 11 to 14 inches in length were used. No special selection was made in relation to sex. In order to achieve relatively natural stimulation conditions for the stimulation of the labyrinthine receptors the fish had to swim for two longer periods (5 days or 14 days) in water moved to a different degree. A high and long-lasting stimulation of the labyrinthine receptor cells should be accomplished by swimming in violantly streaming and edding water. To put this into effect the trouts were held in a wire net cage (22 x 24 inches wide and a water depth of about 22 inches) which was placed in a sluice. The success of feeding was douptful under the conditions of the violantly streaming water in the small cage. In the following these fish will be named "stressed" animals. A low stimulation (with few and weak stimuli per time unit) should be achieved by swimming in steadily running water. For this purpose the trouts were held in a channel about 2 m long and 50 cm wide. The water depth here was

Experimental alteration in mixed synapses about 10 cm. These fish will be named "immobile" ones in the following. All other conditions like water composition, 0 2 -content etc. were the same for all groups examined. The mean temperature was 7°C. One point of uncertainty was the feeding of the trouts. In spite of regular feeding it may be, that the stressed group (partly) did not get enough feed because of the restricted food intake. Therefore a difference in nutritional condition of stressed fish as against the immobile ones may exist. The food was the same for all groups and the same as before the beginning of the experiment. Five groups of fish were examined: control group (normally held fish in the pools), 5 days and 14 days stressed animals, 5 days and 14 days immobile animals. All tests were carried out within a period of three weeks in February/March. It should be mentioned, that in the group "14 days stressed fish", which covered 12 animals at the beginning of the experiment, 4 animals died within this period. This fact will be discussed below.

The total number of fish available for the electron microscopic analysis was 33 (Table I). In contrast to the stressed fish all trouts swimming in the steadily running water survived, but their behaviour was changed: the fish were swimming slowly and they could be seized without difficulty.

T a b l e I. Number of animals in the experiment.

below the ventricular surface. For further investigation blocks were used only from the dorsomedial region of the oculomotor nucleus i. e. the region of the so-called claw-shaped giant synapses (KIRSCHE 1 9 6 7 , SCHUSTER 1 9 7 1 ) .

Fixation, staining and microscopy: Blocks were fixed in cold glutaraldehyde (2.5%) solution for 12 hrs. The buffer used was 0.1 M cacodylate adjusted to a pH of 7.4. After an extensive washing procedure in cold buffer the material was postfixed with osmium tetroxide in cacodylate buffer for one hour and again washed repeatedly. The fixed blocks of tissue were then dehydrated in seven 5 to 20 min steps in graded acetone. Embedding was in micropal. The fixation effect varied greatly. Some pieces were well fixed, whereas others showed heavy destructions of the tissue. Thick sections (1 ¡xm) for light microscopy were stained with hot 1% methylene blue in 1% borax. Thin sections showing silver interference colors were cut with glas knives by a L K B ultramicrotome (Ultrotome III). Sections were stained with lead citrate and uranyl acetate (REYNOLDS 1963). Tissue was examined with a TESLA BS 613 microscope.

number of trouts

group

control 5 days 14 days 5 days 14 days

47

immob. immob. stressed stressed

at the begin- number of fish which ning of experiment died within test period

at the end of experiment

6 5 6 9 12

6 5 6 8 8

1 4

Preparation: The preparation was performed without anesthetizing and perfusion to largely exclude short-term effects on the morphological substrate. The fish were sacrificed by decapitation. The time for removing the brain and excising blocks of tissue was within one minute. The procedure of preparation was as follows: The upper part of the skull was removed to expose the brain. Then the tectum was removed and an aqueous glutaraldehyde solution (2.5%) was applied rapidly for a first fixation by injecting cautiously into the midbrain ventricle. After amputating the nerves and blood vessels in the base of the brain it was put into an ice-cooled Petri dish filled with fixing solution. A razor blade was used to obtain two slices each 1 mm thick of the tegmental oculomotor region. 4 cubes each 1 mm3 were excised from the region just

Criticism of preparation and, fixation method: The advantages of the preparation method used here are the absence of influences caused by narcotics or by stress (operation) which may occur during the perfusion of animals. The risks are perhaps the occurence of hypoxia during preparation and the distorsion of the brain mass by cutting the tissue. However, glutaraldehyde used as primary fixative has a relatively fast penetration rate (PETERS 1970), and the nervous structures which have to be preserved lie just below the ventricle surface. The results of this preparation method were quite different. According to the definition of a "good fixation" by P A L A Y et al. (1962) only "good fixated" material has bsen used. Shrinkage of tissue: For the quantitative estimations (see below) the influence of shrinkage produced by fixation, dehydratation and embedding has not been taken into account. But because of the equal treatment of tissue in all groups tested the shrinkage is the same and its possible influence is negligible from this reason. Counting and measuring Electron micrographs with a magnification of 100.000 x were used. After measuring the length of the contact zone number and lenght of active zones

48

Schuster, Th.

vestibular axon terminal. As a rule, the knobs are in contact with the large oculomotor somata or with the dendritic main trunk (for details see SCHUSTER 1971, 1973). Only this type of ending was investigated. The contacts between these large terminals and the oculomotor perikarya form the following three specialized membrane complexes: active zones, gap junctions and desmosome-like structures. These three types are evident in both perfused and non-perfused material (normally kept fish) (Figs. 1 and 2). Likewise they can be found in stressed and immobile fish (Figs. 3 and 4). The three membrane complexes have been differentiated by means of the following morphological ciriteria: 1) Active zones: Pre- and postsynaptic thickening of varying degree, gap widening and local accumulation of clear synaptic vesicles are typical features. But the individual active zones show many variations: Statistics The membranes may be curved (Figs. 2 b, 3g, 5 a) Five animals per group were analyzed. Only blocks of or straight (Figs, l b , c, 3d), or they may be folded the dorsomedial region of the oculomotor nucleus were (Figs. 5 c, f). The asymmetrical membrane thickenused (by identifying the claw-shaped giant synapses). ing may be well visible (Figs. 5 a, d) or less well The axosomatic contacts of about 3—4 cells per block (Figs, lb—d). Clearly symmetrical synaptic contacts were investigated, at least contacts of 2 0 — 2 5 cells (GRAY-type II, GRAY 1959) have not been observed per group. No special random-sampled method was at these axosomatic contacts; they have been rarely used. seen in the neighbourhood (Fig. 3 b). The concenAfter a first estimation of mean values and errors tration of round clear vesicles varies between a local a runaway-test (HULTZSCH 1 9 6 1 ) was applied to eli- accumulation in close contact with the presynaptic minate single values indicating a striking deviation membrane including omega-figures (Figs, lc, 3f, 5 a, from the mean. This single value xg was eliminated b, d) and a loose distribution of synaptic vesicles if the following condition had been fullfilled: among the the presynaptic axoplasm (Fig. 2d). Large granular vesicles of different degrees of filling \x, - x\ > ¿|S.D.| occur in a small amount (Figs, l b , e, 3c, d, 5a, b, e) where x is the mean value, k a factor depending on n or they are absent (Figs, lc, 2e, 5d). and significance, and S.D. standard deviation. Regularly spaced dense projections according to Mean values and errors (standard deviation S.D. and GRAY and GUILLERY (1966) most clearly visible after standard error of mean S.E.M.) were then calculated PTA-treatment, were also noted. Sometimes the dense without using transformation methods. The test for projections were seen extremly clear (Fig. 3d). They significance was made by means of variance ana- were also discernible in tangentially sectioned synaplysis. ses (Fig. 1 g). But the presentation of these dense ° projections varied considerably. In some special membrane complexes which seem to be desmosomeResults like structures, "dense projection"-like particles have also been observed (Figs, le, f, 2e). Qualitative characteristics 2) Gap junctions: The neuronal gap junctions Membrane complexes observed here are characterized by the apposition In the trout oculomotor nucleus lightmicroscopically of the two membranes to within 20—40 A (in agreelarge endings of characteristic size and shape are ment with R E V E L and KARNOVSKY 1967); SOTELO visible (HORSTMANN 1 9 5 3 / 5 4 - 1 9 5 6 ; KIRSCHE 1 9 6 7 ) . (1975) measured 15—20 A). The overall thickness of The so-called claw-shaped type of these "giant synap- the apposed membranes falls in the range of 150 to ses" (KIRSCHE 1 9 6 7 ) occurs only in the dorsomedial 170 A. Because of the fixation method used here region of the nucleus (KIRSCHE 1 9 6 7 ; SCHUSTER 1 9 7 1 ) . instead of a gap a median dense line is visible in The claw-shaped end-apparatus consists of many large perpendicular sections (BRIGHTMAN and R E E S E 1969). end-knobs, arising by branching of the secondary Therefore two main features are useful in identifying gap junctions and desmosome-like junctions were estimated. Because of tangential or oblique sections of the contact zones a clear differentiation between the different membrane complexes was not always possible. After counting the different membrane specializations their number has been correlated with a standardized contact zone of 5 [xm by transformation in order to compare all data from contact zones of length differing to a great extend. To get a clear picture of the true relations the term "portion of membrane complexes" has been introduced. This "portion" has been estimated as the product of length and number (mean values). The portion therefore means the t o t a l length of a certain membrane complex along the contact zone either in [Am per 5 fxm contact zone or in %.

Fig. 1. Perfused fish. Axosomatic contact zones between secondary vestibular axon terminals and oculomotor somata. a) The contact zone shows from right to left an active zone (A), gap junctions (arrows) and desmosome-like structures (D). 46 000 x . b) and c) Desmosome-like structures between two (peripheral) active zones, b) 78 000 x , c) 95 000 x . d) Two gap junctions (arrows) in close vicinity to a desmosome-like structure, to the right hand an active zone. 120 000 X. e) A desmosome-like structure (in the centre of the contact zone) showing "dense projections" which seem to be spaced regularly. 70 000 X. f) and g) Similar dense projections at a partly tangentially sectioned membrane contact. See Fig. 2e and f. 65 000 x . Himforschung, Bd. 19, Heft 1

4

50

Schuster, Th.

Fig. 2. Normally kept fish, decapitated, non-perfused. a) b) c) d) e)

Desmosome-like structures, in the centre of the contact zone arranged in juxtaposition to a gap junction. 8 2 0 0 0 X . An active zone and one large gap junction (with clear "synaptic" vesicles), partly rotating into a tangential plane. 57 000 x Detail from b). 160 000 x . Desmosome-like structures and "omega"-figures (arrow). 7 7 0 0 0 X . and f) Desmosome-like structures showing "dense projections" in a regular pattern (arrows), e) 9 0 0 0 0 x . f ) 7 6 0 0 0 X .

Experimental alteration in mixed synapses

tS*.ssy ^: & Jb;

InE *! £

4*

51

52

Schuster, Th.

Fig. 3. Stressed fish. a) b) c) d) e) f)

From left to right: desmosome-like structure, gap junction and active zone. 106000X. GRAY-type-II-synapse (to the upper right). 9 0 0 0 0 x . The three different types of membrane specializations. 45 000 x . Clearly visible dense projections at an active zone. 54000 x . The section of the contact zone often shows one membrane specialization following the other. 42 000 X. Gap junctions in close vicinity with desmosome-like structures. The fine structure of the gap junctions is partly visible. 136000X. g) An active-zone and an extensive perpendicularly sectioned gap junction contacted by clear synaptic vesicles. 135 000 x .

Experimental alteration in mixed synapses

53

54

Schuster, Th.

Fig. 4. Immobilized fish. a) All special membrane complexes. From left to right: both small gap junctions and desmosome-like structures alternatively arranged. A large gap junction and a desmosome-like structure in the middle of the contact zone: see inset (b). a) 3 8 0 0 0 X . b) 80000X. c) and d) gap junctions are often observed, c) 41000 X d) 45 000 X. e) The figure illustrates the difficulties in differentiating clearly between active zones and desmosome-like structures. 62 000 x . f) An arrangement of desmosome-like structures without the presence of other types of membrane specializations. 65 000 x .

Experimental alteration in mixed synapses

55

56

Schuster, Th.

Fig. 5. Evaginations and indentations. a) A somatic evagination. The contacting membranes are forming active zones. 5-d-immobile fish. 53 000 X. b) A somatic evagination with active zone and gap junction. 14-d-immobile fish. 79000 x . c) Evagination, totally surrounded by the axon terminal. The membranes are forming several active zones and gap junctions. 14-d-immobile fish. 32 000 x . d) An axonic invagination into the soma. The contact zone shows a number of active zones which are partly obliquely sectioned. 14-d-stressed fish. 40 000 x . e) A "finger-like" protrusion into the axon terminal. 14-d-stressed fish. 41000 X. f) Invagination of an axon terminal into the soma of an oculomotor neuron showing active zones with a high accumulation of clear synaptic vesicles. 14-d-stressed fish. 40 000 x .

Experimental alteration in mixed synapses

M> 1 '

57

58

Schuster, Th.

gap junctions: First, an association of electron dense or semidense material lies in the cytoplasm on either side of the two apposed membranes (cytoplasmic fuzz). Second, the presence of a 100 A hexagonal lattice is an important feature of the junction. In perpendicular sections therefore bands occur at an interval of about 85—100 A (probably the center-tocenter distances of the interplasmic channels) (Figs. 2 c, 3f, g). The identification of the hexagonal arrays in tangential sections ("honeycomb" pattern — ROBERTSON 1970) was difficult with this preparation method. Only if the gap junction first sectioned perpendicularly is continuously rotating into a tangential plane one may be sure to call the whole complex a gap junction. Commonly on the axoplasmic side of the gap junction vesicles are present, but in a small number (Figs. 2 a, b, f, 3f, g, 4 b). Not only clear synaptic vesicles but also large dense core vesicles occured above these junctions (Figs, l a , 3e, 4 a, c). Pedicle-like connections between the clear synaptic vesicles and the "presynaptic" membrane were observed (Figs. 3f, g, 4a). 3) Desmosome-like structures: The gap widening occurs in the range of the active zone gap with little amount of electrondense material within the gap. Behind the membrane on each side electron-semidense material of a different degree is visible in the cytoplasm (Figs, lb—d, 2a, d—f, 4a—b, f). Clear spherical ("synaptic") vesicles do not occur in a high accumulation, but they may be present in a small number (Figs. 2a, d, 4f). Omegafigures ( A K E R T 1973) sometimes are visible at these desmosome-like structures (Fig. 2d). These figures seem to be formed by spiny vesicles (ANDRES 1964; G R A Y and W I L L I S 1970; SCHUSTER 1974). Two points should be stressed here: A) Because of the often similar picture of active zones and desmosome-like structures, at least at these contacts — gap widening, slow and unclear membrane thickening — only one characteristic difference exists: a high accumulation of vesicles at the chemical synapses in contrast to the absence or low accumulation of vesicles at desmosome-like structures. For this reason K R I E B E L et al (1969) speak of desmosome-like complexes both "with closely associated presynaptic vesicles" and "desmosome-like complexes that lacked any accumulation of synaptic vesicles". B) It should be remembered, that clear spherical vesicles occur at all membrane complexes mentioned above, but in a different number. Positioning

of the membrane

complexes

The three membrane complexes occur in a different manner along the sectioned contact zone between

axon terminals and perikaryon. In the electron micrograph they may lie separated and independent from one another. On the other hand these structures seem to be organized in ensembles. So for instance desmosome-like structures sometimes occur in a high number alongside the contact zone, only interrupted by short intervals at which the membranes show their normal picture, whereas other specializations are absent (Fig. 4f). But as a rule, one may say that there exists a close vicinity between gap junctions and desmosome-like structures regardless of a single or multiple presence of the latter one (Figs. Id, 2a, f, 3e, f). In such cases the gap-j unction-like regions lie between the gap-widened desmosome-like complexes (Fig. 4 b). In the majority these gap junction regions are very small. The fact of the close local relations of the two membrane specializations is supported by the quantitative estimations (see below). Evaginations

and

indentations

As noted by previous investigators the perikaryal surface of the fish oculomotor neuron sometimes show pockets or indentations into which terminals protrude (WAXMAN and PAPPAS 1971) (Figs. 5d, f). Axons deeply invaginating into the soma are also reported by B E N N E T T et al (1967 d) in the relay nucleus of a gymnotid fish. On the other hand evaginations of the soma surface similar to those in the chick ciliary ganglion (SZENTAGOTHAI 1964; TAKAHASHI and HAMA 1 9 6 5 ; TAKAHASHI 1 9 6 7 ; CANTINO a n d MUGNAINI

1975) are visible, surrounded by terminals (Figs. 5 a, b, c, e). But these formations are scarcely seen in control animals. They were found in stressed fish (Figs. 5 a, d, e, f) as well as in immobile ones (Figs. 5 b, c). In contrast to the "soma-spikes" in chicks (TAKAHASHI 1967) shape and size of the evaginations are irregular. Besides they are too scarce to speak of genuine somatic thorns or spines described for instance in the goldfish tangential nucleus (HINOJOSA 1973) or in the cerebellar central nuclei of the cat (ANGAUD and SOTELO 1973). Unlike short dendritic expansions mitochondria do not occur in the cytoplasm of the evaginations. Both soma indentations and evaginations form synaptic contacts with the adjacent terminals. Active zones and gap junctions are visible in the electron micrographs. In the indentations active zones seem to be in the majority. The distances between the single synaptic contacts are very small (Figs. 5d, f). The somatic evaginations reveal active zones and gap junctions (Figs. 5 a, b, c). In contrast to WAXMAN and PAPPAS (1971) desmosome-like structures were not observed; this is in agreement with the chick ciliary neuron evaginations (CANTINO and MUGNAINI 1975).

Experimental alteration in mixed synapses

59

N U M B E R per 5/u m contact zone

ACTIVE ZONES significant differences:

432-

14"d"im.»Control

H H111 14 d 5d immobile fish

Control

5-d"str.»14d-str. Control" I4"d-str.

5d 14 d stressed fish

DESMOSOME-LIKE STRUCTURES significant differences: 14"d~im." Control 5Td"im. "Control

14 d 5d immobile fish

Control

5d 14 d stressed fish

GAP JUNCTIONS significant differences: 14-d-im.» Control 14 d im. * 5~d~im. 5~d"im." Control Control' 14-d-str. 14 d 5d immobile fish

Control

p i g

ß

N u m b e r

of

the

three

different membrane

complexes per 5 jj.m contact zone in the five test groups. E r r o r s are expressed as S.E.M. Values see Table II.

5d 14 d stressed fish

Indentations and evaginations without synaptic specializations have never been seen.

membrane complexes (product of lenght and number, see material and methods) at the contact zones yields the following results (Table I I I ) : The "total portion" Quantitative estimations of the three membrane complexes at the contact zone The results of the quantitative analysis are summa- (control group) is about 30%. This means: 30% of the rized in tables I I — I V and figures 6 — 1 0 . The mean length of each contact zone are covered by membrane values of the l e n g h t of the three membrane com- specializations. Active zones cover 16%; gap juncplexes (control group) are (Table I I ) : active zones tions and desmosome-like structures each covers — 0.33 [xm (n = 116), desmosome-like structures — about 6.5% of the lenght of the contact zone. There0.22 |xm (n = 113), gap junctions — 0.23 FXM (n = 91). fore the relation between the three types in the The variability is high. The longest diameters of active control group is: active zones : desmosome-like zones (measured in stressed fish) were 1.10 (xm and structures : gap junctions = 2.5 : 1 : 1. These values 1.20 [¿m. The longest measured gap junction (control deviate from those cited by SCHUSTER ( 1 9 7 3 ) (active group) was 1.50 [xm. The mean values of the n u m - zones : gap junctions = 4 : 1 ) . This latter relation b e r s of the three different membrane complexes per had been obtained from 7 analyzed contact zones 5 ¡xm contact zone are in the control group: active (of anaesthetized and perfused fish) as compared zones — 2.42, desmosome-like structures — 1.52, with about 60 ones now. Moreover at that time no gap junctions — 1.60 (of about 60 analyzed contact differentiation had been made between active zones and desmosome-like structures. Adding the two zones). The estimation of the "portion" of the specialized values of active zones and desmosome-like structures

60

Schuster, Th.

T a b l e II. Number and length of the different membrane specializations in the different test groups. Results and significances (p). Values are expressed as means ± S. E. M. group

feature active zones

desmosome-like structures

gap junctions

number

length

number

length

number

length

P 1 [%]

5

1

1

5

1

NS

14-d-im. fish

1.85±0.16

0.24 ± 0.01

3.31 ± 0.20

0.20 ± 0.01

4.05 ± 0.28

0.21 ± 0.01

P [%]

NS

NS

NS

NS

1

1

5-d-im. fish

1.99 ± 0.22

0.24 ± 0.01

2.64 ± 0.26

0.18 ± 0.01

2.86 ± 0.26

0.15 ± 0.01

P [%]

NS

1

1

1

1

1

control

2.42 ± 0.16

0.32 ± 0.01

1.52 ± 0.18

0.22 ± 0.01

1.60 ± 0.17

0.23 ± 0.01

P [%]

NS

NS

NS

NS

NS

NS

5-d-str. fish

2.81 ± 0.19

0.32 ± 0.01

1.23 ± 0.20

0.21 ± 0.01

1.66 ± 0.16

0.23 ± 0.02

P [%]

1

NS

NS

NS

1

5

14-d-str. fish

3.54 ± 0.13

0.37 ± 0.01

1.91 ± 0.14

0.22 ± 0.01

1.02 ± 0.13

0.18 ± 0.01

NS

NS

1

1

1

P 2 [%]

5

number = number per 5 |im contact zone. p 1 = p between 14-d-immobile fish and control. p 2 = p between 14-d-stressed fish and control. NS = non significant.

L E N G T H injum ACTIVE ZONES

• Bill

0,4 0,3

02 0,1

14 d 5d immobilefish

Control

significant differences: 1 4 - d - i m . "Control 5 " d " i m . «Control Control "14-d~str.

5d 14 d stressed fish

DESMOSOME'LIKE STRUCTURES signicant differences:

0,4 0,3

14"d"im."Control 5 - d " i m . "Control

Q21 0.1 5d 14 d immobilefish

Control

5d 14 d stressed fish

GAP JUNCTIONS significant differences: 14-d"im. " 5 - d - i m . 5-d"im.«Control 5-d-str.«14-d-str. ControL"14-d"str. 14 d 5d immobile fish

Control

5d 14d stressed fish

published here we come to a ratio of 3.5:1. This ratio is not much different from that of 4:1. The changes of the different membrane complexes in relation to test conditions and time are as follows (Tables I I I and IV) : Clear changes are visible in the numbers of membrane complexes (Fig. 6). Whereas not all groups show significant differences from one another, a distinct trend seems to be outlined for all membrane complexes: The number of active zones decreases in immobile fish and increases in stressed fish. By contrast, the numbers of desmosome-like complexes and gap junctions increase in immobile fish. In stressed fish the numbers of both structures do not change markedly. The values of lengths alter as follows (Fig. 7) : The length of active zones is decreased in immobile fish but increased in stressed fish. The lenght of both desmosome-like structures and gap junctions changes only to a small extend. In contrast to the changes in their numbers the picture of these alterations does not show any clear trend. A better representation of the "plasticity" of the different membrane specializations is evident by introducing again the "portion" of membrane specializations into the quantitative analysis (Figs. 8a—c; Table III). Accordingly the portion of active zones Fig. 7. Length of the three different membrane complexes in |j.m. Errors are expressed as S.E.M. Values see Table II.

Experimental alteration in mixed synapses

I total portion of all special membrane

61

complexes in the length of the contact zone [%]

Ik d immobile

5 d immobile

H

control

5d

stressed

14 d

stressed

8a

Fig. 8. Portions (number x length) of the different membrane complexes in % (at the left corner of each column). Values see Table I I I . For explanation see text.

I portion of special membrane

%d immobile

5 d immobile

complexes

in the length of the contact zone[%]

[s

30

B

177

control

5d

stressed

U d stressed active 8b

zones

ap junctions+

desmosome-l.struct.

M portion of the different membrane complexes in the length of the contact zone/%/

5 d immobile

^

b

II

IH

control 5 (¡stressed 74 d stressed

9 la¥

[ ^ l H I ^ ^ H H ^ H E l H active zones

I I desmosome-1, struct.

• gap

junctions

8c

Portion of the different membrane complexes at the contact zone [ % ]

Ud

immobile fish 'Active zones.

5d

control

5d

stressed fish

s Desmosome"like structuresj

Kd

Gap junctions

Fig. 9. The percentual values of the different membrane complexes in the five test groups. The curves clearly show the alterations. See text. Values see Table III.

Experimental alteration in mixed synapses

63

Changes of the portion of the different membrane complexes in % Values of control = 0 %

Active zoneSj

^ ^ ^ Desmosome-like structures;

Gap junctions

g. 10. Percentual changes of the portions of the different membrane complexes (values of the control group — 0). Values see Table IV. For further explanation of figs. 9 and 10 see text.

T a b l e I I I . Portion of the three special membrane complexes (number x length) of the contact zone length in |xm per 5 ¡j.m contact zone and in % . type of membrane complex

portion 14-d-immobile fish

active zones desmosome-like str. gap junctions desmosome-like str. and gap j unctions active zones, gap junctions and desmosome-like str.

5-d-immobile fish

|un

%

¡¿m

/o

0.44 0.66 0.85 1.51

8.8 13.2 17.0 30.2

0.47 0.47 0.42 0.89

9.4 9.4 8.4 17.8

1.95

39.0

1.36

27.2

of 14-d-immobile fish covers 8.8% of the contact zone. This value increases to 26% in the group of stressed fish (Fig. 8a). By contrast, the portions of' desmosome-like structures and gap junctions fall from 13.2% or 17%, respectively (immobile fish) to 6.2% or 3.6%, respectively (stressed fish) (Figs. 8c, 9, 10).

control

5-d-stressed fish

14-d-stressed fish

/o

fun

%

¡im

%

0.79 0.33 0.32 0.65

15.8 6.6 6.4 13.0

0.89 0.25 0.33 0.58

17.8 5.0 6.6 11.6

1.30 0.31 0.18 0.49

26.0 6.2 3.6 9.8

1.44

28.8

1.47

29.4

1.79

35.8

(jtm

At this point we should briefly return to the local relations (positioning) of desmosome-like structures and gap junctions mentioned above. The quantitative changes of both specialized membrane structures seem to correlate with what has been said previously because their changes occur fairly simultaneously (Fig. 8 c). Summarizing both the percentual values

64

Schuster, Th.

T a b l e IV. Changes in the portion of the three membrane complexes in % (control values = 0). type of

changes in %

membrane complex

fish

14

_d_im

5

_d_i m .

control

figh

5-d-str. figh

14-d-str. fish

active zones

-44.3

-40.6

0

+13.6

+64.5

desmosomelike str.

+100.0

+42.5

0

—28.8

-6.1

gap junctions

+165.5

+31.2

0

+3.1

-43.7

of desmosome-lilce structures and gap junctions it is obvious that an active-zone-increase from 8.8 to 26% (from 14-d-immobile fish to 14-d-stressed fish) is contrasted with a decrease of desmosome-like structures and gap junctions from 30.2 to 9.8% (Fig. 8b). Comparing now the total portions of all membrane complexes (Fig. 8 a ; Table III) it is seen that the 14-dgroups (stressed and immobile fish) show a higher proportion of membrane specializations than the control group (39% in 14-d-immobile fish, 35.8% in 14-d-stressed fish, in contrast to 28.8% in the control group). This means that there is a difference of about 10% between the 14-d-groups and the control. Figure 10 and table IV show these changes on the basis that the values of the control group are = 0. From these curves we can see changes in an enormous range especially with regard to the values of desmosomelike structures and gap junctions. Discussion Experimental

conditions

According to GRONOW ( 1 9 7 4 ) , the experimental conditions to which both stressed and immobilized fish were exposed, have to be regarded as stress situations. This means a general mobilization of the fish organism by psychic excitation, increased muscle activity, hunger, infection and so on. Like in mammals an increased brain activity may be expected (HAMBERGER et al. 1 9 6 9 ) .

The possible known stress factors in fish are (GRONOW 1 9 7 4 ; JACOB 1 9 7 4 ) :

a) stressed fish: — hunger, produced by a restricted food supply — the keeping in the narrow net cage (the small size of the reservoir) — wounds on mouth, fins and skin caused by injuring at the sides and corners of the wire net cage — intensive muscle activity

— an increased susceptibility to infections resulting from the stress conditions, wounds etc. b) immobilized fish: — decreased 0 2 -content of the water in the channels because of its slow and steady flowing — the keeping in the relatively small channel — an increased susceptibility to infections produced by the stress conditions. Total exhaustion (by long-lasting muscle activity) is likely one reason for the death of some 14-d-stressed fish (Doi 1932). Another reason may be the increased susceptibility to bacterial attack or virus infection caused by the stress conditions. I t is interesting to note in this connection that the lysozyme level of the blood in so-called "hunger-stress-trouts" reaches its maximum after a period of 14 days (ZARSKE 1975). The lysozyme level of the blood is an expression of the bacterial loading. I t may well be that there exist other but unknown stress factors. We should bear in mind that a number of "stressors" (stress factors — S E L E Y E 1936, 1950) alter the hormonal situation of the fish and the tone of the sympathetic nervous system which influences the metabolic conditions, the oxygen uptake, the protein biosynthesis and so on (BRETT 1964; RANDALL and STEVENS 1967; GRONOW 1974). Hormonal effects on the growth of synapses for instance are discussed by CRAGG (1972a). Therefore the following fact should be taken into consideration: The plasticity of the different membrane specializations at these vestibulo-ocular "giant synapses" may be not only the result of varied labyrinthine influences. Because of the different stress factors of the experimental conditions the results are perhaps influenced in an unknown way. Discussion of the qualitative

results

Regardless of the difficulties in differentiating the membrane specializations three membrane complexes have been distinguished at these contacts: active zones, gap junctions and desmosome-like structures. Concerning the gap junctions it would perhaps be better to speak of gap-j unction-like complexes because it is not always certain that "true" gap junctions have been counted. So for instance very short sections of gap junctions (within the range of about 0.01—0.03 ¡im) between adjacent desmosomelike structures are not always to be clearly identified (BENNETT 1973 b). From a functional point of view it seems possible that there exist gap junctions not only of different shape and size (spots, belts, etc.) but also of differing structural principles (STAEHELIN 1974). BENNETT (1973 a,b) introduced the term "class of gap junctions". If the main function of the

Experimental alteration in mixed synapses

gap junctions is the electrotonic transmission, this function would be safeguarded by the presence of the small gap widening to prevent lateral current leakage ( R O B E R T S O N 1 9 7 0 ) . The wide-spread labile membrane appositions ( B E N N E T T et al. 1 9 6 7 d; BRIGHTMAN and R E E S E 1 9 6 9 ) as a result of fixation artifacts do not show any characteristic fine structural elements. Occulding junctions (true "tight junctions") have so far rarely been observed in nervous tissue. They have been found in addition to desmosome-like junctions in the Nc. oliv. inf. ( K I N G and A N D R E Z I K 1 9 7 5 ) , at the receptor cells of the acoustolateralis system ( B E N N E T T 1 9 7 1 ) and at the olfactory epithelium (BRIGHTMAN and R E E S E 1 9 6 9 ) . Neither they have been seen in mixed synapses nor in the special case of the oculomotor region. Another point of uncertainty is the desmosomelike structure and their distinction from active zones lacking high vesicle accumulation, respectively. These difficulties have been mentioned previously (PALAY

1956;

a n d GUILLERY GRAY a n d

ROBERTSON et al. 1 9 6 3 ;

COLONNIER

1 9 6 4 ; TAKAHASHI a n d HAMA

GUILLERY 1966;

1965;

BENNETT et al. 1967 c ;

et al. 1 9 6 8 ) . This problem is of special interest in studying embryonic, immature and cultured nervous tissue ( B U N G E et al. 1 9 6 7 ; A G H A J A N I A N KRIEBEL

and

BLOOM

1967;

BLOOM a n d

AGHAJANIAN

HAMORY 1 9 6 9 ; L A R R A M E N D I 1 9 6 9 ; MUGNAINI

GUILLERY

1967,

1969;

GUILLERY

COLONNIER 1 9 7 0 ) . According to the authors the filamentous contacts unlike the desmosome-like complexes show fine filaments and tubular profiles either on both sides or together with granular dense bodies on the postsynaptic component. They can often be seen adjacent to active zone contacts. For this reason it has been suggested that these contacts are either stages of formation or destruction of active zones, or both active zones and filamentous contacts are distinct structural specializations with different aspects of synaptic function. Since 1 9 7 0 G U I L L E R Y and COLONNIER as well as B R A U E R and coworkers ( 1 9 7 4 ) have only made a distinction between active zones and symmetrical filamentous contacts. In other regions of the C.N.S. no further subdivisions have been made among the "non-synaptic" junctional complexes. In the case of the trout oculomotor nucleus a further differentiation among the desmosome-like complexes was not possible either. The combined occurrence of the three complexes active zone, gap junction and desmosome-like strucHirnforschung, Bd. 19, Heft 1

(HINOJOSA a n d ROBERTSON 1 9 6 7 ; HINRICHSEN YASHIKI

and

and

1970; S O T E L O and I L I N A S 1972) — nearly always show desmosome-like complexes (Table V). The combination of active zones and desmosome-like structures with the exclusion of gap junctions also exists ( A N G A U T and S O T E L O 1973: cat cerebellar central nuclei — active zones and attachment plates; CORDULA N I T S C H and B A K 1974: rabbit hippocampal mossy fiber giant boutons — active zones and "desmosomes or attachment plates"; R O S E M A R I E D U N N 1975: desmosome-like plaques flanking the synapses of the outer hair cells in cat cochlea). By contrast, gap junctions combined with desmosome-like structures without active zones on the same contact zone occur much more frequently in the vertebrate C.N.S.

1969,

et al. 1 9 7 3 ) . In the lateral geniculate nucleus of cats and monkeys G U I L L E R Y and COLONN I E R furthermore differentiated between desmosomelike and filamentous contacts (COLONNIER and G U I L 1964;

et al. 1 9 7 0 ; SOTELO a n d PALAY 1 9 7 0 ; SOTELO

TAXI

LARRAMENDI

1972; WENZEL

LERY

ture along the same contact zone has been repeatedly described in the vertebrate C.N.S. For the desmosome-like structure several terms have been used ("desmosomoid differentiations" — R O B E R T S O N 1965; "attachment plaques" — WAXMAN and P A P P A S 1968; SOTELO and P A L A Y 1970; "puncta adhaerentia" — N A K A J I M A 1974; "desmosome-like plaques" — R O S E M A R I E D U N N 1975). Contact zones with the combination of the two complexes active zone and gap junction — the so-called mixed synapses ( P E T E R S

1968;

1 9 7 0 ; KAWANA e t al. 1 9 7 1 ; D E L CERRO a n d S N I D E R

65

1968;

1969b;

KRIEBEL

HINOJOSA

et

1973;

al.

1968,

SOTELO

and 1969;

et al.

1 9 7 4 ; CANTINO a n d MUGNAINI 1 9 7 5 ; P A P P A S e t

al.

1975; SOTELO 1975; SOTELO et al. 1976). Whether the latter type of contact is concerned or the apparently much more common mixed synapse including desmosome-like structure — the close vicinity of desmosome-like complexes and gap junctions is striking ( B E N N E T T et al. 1 9 6 7 b and d; H I N O JOSA a n d

ROBERTSON

1967;

K R I E B E L et al.

1968,

1 9 6 9 ; S O T E L O a n d T A X I 1 9 7 0 ; W A X M A N a n d PAPPAS 1 9 6 9 , 1 9 7 1 ; PAPPAS a n d WAXMAN 1 9 7 2 ;

NAKAJIMA

1 9 7 4 ; SOTELO e t a l . 1 9 7 4 ; CANTINO a n d

MUGNAINI

1975;

SOTELO

1975).

This agrees with the results

presented here. In some electron micrographs such a juxtaposition also occurs between active zones and desmosome-like structures ( N A K A J I M A 1974) as mentioned in the case of the filamentous contacts (see above). In Fig. 14 of N A K A J I M A ' S paper (1974) for instance along the contact zone the following complexes occur without intervals of "normally" non-specialized membranes: active zone — punctum adhaerens — gap junction — punctum adhaerens — active zone. But all these combinations are quite in the minority compared with the combination of gap junctions and desmosome-like structures. The fact of the close vicinity of the two membrane specializations gap junction and desmosome-like structure which has been observed so 5

66

Schuster, T h .

T a b l e V. Contact areas with three different types of specialized membrane complexes (active zones, gap junctions, desmosome-like structures) in the vertebrate C.N.S. — data from the literature. animal

region

mode of contact

reference

ammocoetes of the lamprey (Lampetra planeri)

vestibular nuclei

axosomatic

STEFANELLI a n d

electric fish (Gymnotus carabo)

magnocellular mesencephalis nucl.

axosomatic

SOTELO e t al.

spiny boxfish (Chilomycterus schoepfi)

oculomotor nucleus

axodendritic

PAPPAS a n d WAXMAN

Salvelinus pluvius (teleost)

oculomotor nucleus

axosomatic

YASHIKI

goldfish (Carassius aur.)

tangential nucleus

axosomatic

HINOJOSA

Spheroides mac., Chilomyct. oculomotor nucleus sch., Myoxocephalus (teleosts)

axosomatic, axodendritic

K R I E B E L et al.

goldfish (Carassius aur.)

Mauthner cell

axosomatic, axodentritic axoaxonic

NAKAJIMA

frog

spinal cord intermediate gray matter

axodendritic

SOTELO a n d T A X I

CARAVITA

1970 1975

1972

1969a 1973 1969

1974

1970

chick

ciliary ganglion, ciliary neurons

axosomatic

BRIGHTMAN a n d R E E S E

chick

ciliary ganglion, ciliary neurons

axosomatic

CANTINO a n d MUGNAINI

rat

lateral vestibular nucleus

axosomatic

SOTELO a n d PALAY

rat

descending vestibular nucleus

axosomatic

SOTELO

1969 1975

1970

1975

T a b l e V I a. Lengths of active zones — some data from the literature. animal

region

length in jjtm

reference

Formica rufa, Camponodus ligniperdus (invertebrates)

synaptic glomeruli in the neuropil of the corpora pedunc.

0.1 — 0.25

STEIGER

trout

oculomotor nucleus, axosomatic giant synapses

0.35

SCHUSTER

cat

cerebellar central nuclei, mediumsized boutons on large neurons

0.15 — 0.3

ANGAUT a n d SOTELO

1967

1973

1973

T a b l e V I b. Lengths of gap junctions in nervous tissue — some data from the literature. length in ¡xm

animal

region

Carassius auratus

laterale dendrite of the Mauthner cell synaptic discs of the club endings

0.2-0.5

Gymnotus carabo

cerebellum, gap junctions between mossy fiber terminals and granular cell dendrites

0.08-0.2

Carassius auratus

Nc. vest, tang., 1st) caliciform endings 2nd) trilobated endings 3rd) club endings

chick

Nc. vest, tang., gap junctions of the spoon synaptic endings ventral cochl. nc. vestibular nuclei inf. oliv. nc., dendrodendritic gap junctions

rat rat cat

reference ROBERTSON 1963,

et al.

ROBERTSON

1963

SOTELO a n d LLINAS

HINOJOSA

1972

1973

0.14-0.33 0.2-0.36 0.08-0.22 0.04-0.47

HINOJOSA a n d

ROBERTSON

1967

0.1-0.2

SOTELO e t al.

0.05-0.4

SOTELO

0.05 — 0.2

SOTELO e t al.

1976

1975 1974

Experimental alteration in mixed synapses

often support the idea of a functional relation between them. This is in agreement with the quantitative results. Discussion of the quantitative results Lenght and number of special membrane

complexes

The mean values of the length of the different membrane complexes measured are within the range of figures cited in the literature (Table V i a , b). A problem in this connection is whether the measured length always represents a diameter of the sectioned complex. Especially large figures suggest that the frequent disc-like shapes of the active zones or gap junctions are replaced by other forms, for instance by horseshoe-like configurations in the case of active zones ( A N D R E S 1 9 7 5 ) or belts in the case of gap junctions (BRIGHTMAN and R E E S E 1 9 6 9 ; F R I E N D and G I L U L A 1 9 7 2 ; ZAMPIGHI a n d ROBERTSON 1 9 7 3 ) .

The longest synaptic structure measured was a 1.5 /«n gap junction section. According to the information from the literature gap junctions up to 10 [Am have been observed (Table VII). The longest measured active zone was 1.2 (z.m. Active zones "more than one micron" have also been described (SOTELO 1 9 6 9 ) .

either of gap junctions / desmosome-like structures (stressed fish) or of active zones (immobile fish). In each case the process of new-formation is more pronounced than the involution process. Therefore the total portion of membrane complexes increases in both groups, 14-d-stressed and 14-d-immobile fish, compared with the control. Two points should be emphasized here: 1) The "possible receptive area" of this axosomatic contact seems to be so large as to permit a high variability in the number of special membrane complexes. 2) Two opposite processes of formation and involution of special membrane complexes occur under the experimental conditions without an equilibrium existing between both processes. The changes in the different membrane complexes Active

of special membrane

complexes

The total portions of all special membrane complexes range from 29% (control group) to 39% (14-d-immobile fish) or 36% (14-d-stressed fish), respectively. This is an increase in the total portion of about 1/4 in each of the two 14-d-test groups, both in complete contrast as regards their test conditions. If transformed from the contact z o n e into the contact a r e a these values would be much higher. Therefore a new-formation of special membrane complexes has to be stated under the test conditions described above. Differentiating now among the three membrane complexes it becomes clear that this new-formation of contacts concerns only active zones (stressed fish) or gap junctions / desmosome-like structures (immobile fish). This process of new-formation of special complexes is in contrast with an involution process

zones

Changes in number, length or size of active zones have often been observed (CRAGG 1969; MBLLGARD et al. 1971; BLOOM 1970 lit.; A K E R T 1973). Their number increases during maturation ( V O E L L E R et al. 1963; O C H I 1 9 6 7 ; A G H A J A N I A N a n d BLOOM 1 9 6 7 ; L A R R A MENDI

Total portion

1969;

CRAGG

1972 b ;

HINDS

1975;

MARIA

and Ross 1975; W E S T R U M 1975; P O K O R N Y and H A M O R I 1976), following learning experiments ( W E N Z E L 1974 lit.) and reinnervation ( M A T T H E W et al. 1976b), on stimulation (MANINA 1970, 1971, 1976; W H I T T A C K E R et al. 1975) etc. The change in the portion of chemical synapses is often in a good correlation with environmental conditions. MANINA (1970) holds the view that a sharp increase in the number of active zones following experimental neurosis in dog's C.N.S. ("hypersynapsy") is the result of a "functional overloading". The changes in the portion of active zones of the oculomotor mixed synapses are accompanied by simultaneous changes in the number of mitochondria in the secondary vestibular axon terminals, i.e. an increased portion of active zones in a stressed fish group is followed by an increased mitochondrial CASERTA

T a b l e V I I . Maximal length of gap junctions in nervous tissuedata from the literature. animal

region

mormyrid fish

spinal and medullary electroup to more than motor nuclei medullary electromotor nuclei > 3 UM1 axodendritic gap junctions > 1 (jtm oculomotor nucleus 1.2 (¿m 1.9 (xm1 spinal electromotor neurons cervical spinal cord, ventral horn 1 ¡jtm

gymnotid fish Electrophorus trout gymnotid fish frog 1

67

length

reference 1 0 (IM

BENNETT

et al. 1967b

BENNETT e t al. 1 9 6 7 a MESZLER e t a l . 1 9 7 2 SCHUSTER 1 9 7 3 PAPPAS e t al.

1975

SOTELO a n d T A X I

measured in the presented electron micrographs 5*

1970

68

Schuster, Th.

density and vice versa (OSSYRA et al. 1977). Likewise the density of clear synaptic vesicles of the presynaptic active zone areas of these contacts is changed in dependence on the test conditions (FENSKE et al. 1978). These facts reflect the close functional relations between presynaptic mitochondrial pool (as energy producting machine and Ca++ accumulation system), transmitter storage and releasing system and chemical transmission (LEHNINGER 1970; SIMKISS 1974; Joo and PARDUCZ 1976). The likely transmitter of this transmission system is acetylcholine ( P F I S T E R et al. 1 9 7 7 ) . The close relation between ACh level and cholinergic nerve cell condition is well known (MC INTOSH 1 9 4 1 ; EVANS and SAUNDES 1 9 6 7 ; WHITTACKER et al. 1 9 7 5 Lit.). On the other hand ACh is suggested to act in central cholinergic synapses more as a neuromodulator (controlling the membrane level) than as a transmitter for rapid transmission of direct signals ( K R N J E V I C 1 9 7 1 ; B E R N A R D I et al. 1 9 7 6 ) . Such a behaviour has been described for other transmitter substances too (BLOOM 1 9 7 0 ) . The assumption of a modulating function of ACh and active zones at these contacts is supported by the fact that until now only in the case of the chick ciliary mixed synapse a biphasic action potential has been recorded which shows a short latency electrically mediated spike followed by a longer lasting chemically mediated signal (MARTIN a n d P I L A R 1 9 6 3 a a n d B ; H E S S e t a l . 1 9 6 9 ; LANDMESSER a n d PILAR 1 9 7 2 ) .

Supposing a function in neuromodulation an increase of active zones at these mixed synapses may be explained as a result of an "overloading": The cell react in a compensatory mechanism by creating stable conditions for a higher membrane threshold in order to diminish the number of signals. In such large nerve cells with high voltage thresholds electrotonic transmission by gap junctions is the most efficient way to exicte these cells (ZUCKER 1972). Gap

junctions

Electrotonic coupling of homonymous fish oculomotor somata by way of presynaptic fibers has been demonstrated ( K R I E B E L et al. 1 9 6 8 ; WAXMAN et al. 1 9 6 8 ; KIDOKORO 1 9 6 9 ) . The regulation of rapid eye movements is probably the aim of the quick and synchronous activity mediated by electrotonic coupling ( K R I E B E L et al. 1 9 6 9 ; KORN and B E N N E T T 1 9 7 5 ) . In Astroscopus the motoneurons of the oculomotor nucleus are coupled directly with each other by gap junctions ( B E N N E T T and PAPPAS 1 9 6 7 ) . The synchronization in the electric organ discharge therefore seems to be improved. Investigations in the electromotor system of electric gymnotid fish show a closer coupling in cases in which more gap junctions are

present ( B E N N E T T et al. 1 9 6 7 d). According to PAPPAS and WAXMAN ( 1 9 7 2 ) it seems possible to speak of a correlation between the degree of electrotonic coupling and the frequency and size of gap junctions. The increase of the number of gap junctions in the immobile fish group therefore would suggest a higher degree of electrotonic coupling. Looking at the changes of gap junctions in the stressed fish compared with those of the control group, no significant changes are seen or only small ones. This would suggest: No changes occur in the degree of electrotonic transmission. Comparing the efficiency of both chemical and electrotonic transmission B E N N E T T (1972 a, b) holds the view that the latter may be more important under "stress" conditions (like hypoxia) than under ordinary circumstances because a number of metabolicdependent transmission steps is absent at the electrical synapse. Under hypoxia, for instance, the neuronal protein synthesis is blocked up (ALBRECHT and SMIALEK 1974). Probably the chemical transmission would be more concerned than the electrotonic transmission. This is due not only to the energy consumption but also to the transmission steps directly or indirectly dependent on the protein synthesis (for instance the transmitter storage and releasing system). Is this mixed synapse perhaps able to a certain degree to regulate the activity of both transmission types in dependence on the metabolic situation (as a compensatory mechanism)? As a hypoxia is likely to exist in immobile fish the increase of gap junctions then would give a possible explanation. SOTELO et al. (1976) showed that deafferentiation in mammalian C.N.S. does not alter the gap junction structure. The authors also observed "new localizations of gap junctions" in the deafferentiated region. This behaviour of gap junctions would differ from that of active zones after deafferentiation (MATTHEW et al. 1976 a, b). These findings seem to support the idea of a better resistivity of the gap junction structures compared with active zones. Another point of view is the role of gap junctions in metabolic transport processes. Ions, small molecules and larger molecules up to an ion M.W. of about 640 are able to cross these junctions ( B E N N E T T 1973a). The function of ion and molecule transfer via the hydrophilic gap junction channels has been discussed by several authors (SOTELO and PALAY 1970; ASADA

and

BENNETT

1971;

PAPPAS

et

al.

1971;

1973 a). An increase in the portion of gap junctions under extraordinary circumstances (as during relative immobilization) may mean a higher degree of exchange of substances to compensate losses in metabolic activities caused by hypoxia or other influences. But it should be remembered too BENNETT

Experimental alteration in mixed synapses

that the junctional permeability i.e. the transport capacity of gap junctions also depends on oxidative phosphorylation, on the intracellular Ca++ content etc.

(POLITOFF e t

al.

1969;

OLVEIRA-CASTRO

and

LOEWENSTEIN 1 9 7 1 ; R I E S K E 1 9 7 5 ) .

The mechanism of the formation of new gap junctions may take place based on the hypothesis that particles of both membranes are forming hemichannels which are linking up in a following stage forming intercytoplasmic channels. Two analogous models can be referred to: 1st — the formation of nystatin channels in a single bimolecular lipid membrane (BENNETT 1973 a), 2nd — the secretion of mucous vesicles in Tetrahymena (SATIR et al. 1972). Perhaps we have to regard the juxtapositional desmosome-like structures as such pre-gap-junctional structures, viz stages of accumulation of particles along both membranes, later forming hemichannels for bridging the gap. The time for new-formation of functioning gap junctions may range within minutes, because electrotonic coupling between reaggregated cells develops within a period of about 10 min (BENNETT e t al. 1 9 7 2 a, b ) . ITO a n d

LOEWENSTEIN

have shown that the formation of fully viable junctional passageways occurs within seconds (LOEWENSTEIN 1967; ITO and LOEWENSTEIN 1969). Assuming that under the present test conditions the nervous tissue of the immobile fish has been "damaged" to some degree (e.g. by hypoxia) it may be supposed that the cells try to compensate it by forming metabolic more "independent" transmission structures. In quite another context HULL and FULLER (1975) suppose from informations on the changed morphology of human central neurons that the functioning of maldeveloped neurons may be caused by a "fewer normal (and possibly more abnormal) synapses with each other". From this it can be concluded: This degree of damage must be reversible. The stage of a irreversible damage, for instance an injury of a cell, by contrast, would be followed by a decoupling (BENNETT 1972 a, b, 1973a, lit.). Following this hypothesis the increase in the portion of gap junctions is considered an adaptive cellular response to altered metabolic conditions. Desmosome-like structures

Two facts support the idea of a functional relationship between gap junctions and desmosome-like structures: 1) the close local relations of both structures (that is in accordance with the literature — see above), 2) the simultaneous changes of the portion of both structures in dependence on test conditions. For this reason it may be supposed that the desmosome-like structures are precursors of true gap junctions, i.e. places of accumulation of pre-gap-junctional material

69

in a second step forming gap junction structures (for the possible formation mechanisms see above). Previously existing gap junctions may act as inductors because of better metabolic conditions in the vicinity of the gap junctions. On the other hand, if desmosomelike structures are regarded as stages of distruction of gap junctions, it would be difficult to explain the simultaneous changes in the portions of both gap junctions and desmosome-like structures. The elucidation of the function of these structures is complicated by the following two facts: 1) Quantitative electron microscopic estimations of similar structures have been made only at contacts lacking gap junctions. 2) A lot of junctional structures has been demonstrated espically in ontogenetic research without clear morphological features for differentiating the structures.

Acknowledgement I am very grateful to Mrs. Elke R O E S E L E R and Mrs. Ursula for their great technical assistance in preparing the material. This research was supported by the Ministerium fur Wissenschaft und Technik der DDR. ZUHLKE

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T . S. BODENHEIMER.

and

D.

S. STAGE:

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STREIT, P.,

K . AKERT,

C. SANDRI,

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and

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Experimental alteration in mixed synapses and K . H A M A : Some observations on the fine structure of the synaptic area in the ciliary ganglion of the chick. Z. Zellforsch. 67, 1 7 4 - 1 8 4 (1965). V O E L L E R , K., G. D. P A P P A S , and D. P . P U R P U R A : Electron microscopic study of development of cat superficial neocortex. Exp. Neurol. 7, 107 — 130 (1963). W A X M A N , S . G . , and G . D. P A P P A S : Discussion on: Y . K I D O K O R O : Cerebellar and vestibular control of fish oculomotor neurones. I n : Neurobiology of cerebellar evolution and development. Editor: R . Llinas. Chicago: Amer. Med. Ass. 1969. Pp. 2 5 7 - 2 7 6 . W A X M A N , S. G., and G. D. P A P P A S : An electron microscopic study of synaptic morphology in the oculomotor nuclei of three inframammalian species. J . Comp. Neurol. 143, 4 1 - 7 2 (1971). W E N Z E L , J . : Morpho-funktionelle Untersuchungen zur Neuronenstruktur und synaptischen Organisation des Hippocampus. Diss. Dr. sc. med. Humbold-Universität 1974. WENZEL, J . , Mareike W E N Z E L , Gisela G R O S S E und W . K I R S C H E : Elektronenmikroskopische Untersuchungen zur Entwicklung der Synapsen in Explantatkulturen des Hippocampus bei Rattenembryonen. Z. mikrosk.-anat. Forsch. 87, 1 9 5 - 2 1 7 (1973). W E S T R U M , L . E . : Synaptic patterns in olfactory cortex of newborn and young rats. Anat. Ree. 181, 508 (1975). TAKAHASHI, K . ,

W H I T T A C K E R , V . P . , H . ZIMMERMANN, a n d M . J .

73

DOWDALL:

The biochemistry of cholinergic synapses as exemplified by the electric organ of Torpedo. J . Neural Transmission. Suppl. 12, 3 9 - 6 0 (1975). Y A S H I K I , K . : Electron microscopic observations of the nucleus oculomotorius in the teleostean brain. Yonago Acta . medica 13, 8 5 - 1 0 2 (1969a). Y A S H I K I , K . K . : Finestructure of the trigeminal nucleus in the teleostean brain. Yonago Actamedical3, 228 — 248 (1969b). Z A M P I G H I , G . , and J . D. R O B E R T S O N : Fine structure of the synaptic discs separated from the goldfish medulla oblongata. J . Cell Biol. 56, 9 2 - 1 0 5 (1973). Z A R S K E , A. : Untersuchungen am Serumlysozymgehalt von Salmo gairdneri (Richardson, 1836) unter extremen Bedingungen. Diplomarbeit 1975. Sektion Biowiss. der KMU Leipzig. ZUCKER,

R . S . : J . Neurophysiol. (1974).

35,

STAEHELIN

Author address: Dr. rer. nat. T h .

SCHUSTER

D D R - 1 0 4 Berlin Anatomisches Institut der Charité der Humboldt-Universität Philippstraße 12

599 (1972). Cited by

J. Hirnforsch. 19 (1978) 75 - 83

Instituto cajal; C.S.I.C.; Velazquez, 144; Madrid-6; Spain

The effect of early experience on the exploratory behaviour, learning ability and on the synaptosomes of the mice brain By L . M . GARCÍA-SEGURA, C . RODRIGUEZ-GONZALEZ a n d J . M . GONZALEZ-ROS

With 4 figures and 1 table (Received May 7, 1977)

Summary: This study deals with the effects that are produced by an enriched early experience on the mitochondrial fraction on the mice brain. The total content of lipids and proteins in mitochondrial fraction, the morphology of the mitochondries and the density of synaptosomes were investigated. A significant difference in the total quantity of lipids of the mitochondrial fraction and the density of synaptosomes was observed between mice subjected to an enriched environment and others subjected to an impoverished environment. These mice, which were subjected to a psychological study before proceeding to the extraction of the mitochondrial fraction, displayed distinct characteristics. The enriched group displayed a greater exploratory behaviour and a greater capacity to learn than the mice raised in the impoverished environment. Résumé: Dans ce travail on étudie les effets qu'une expérience précoce enrichie produit sur la fraction mitochondriale de l'encéphale du souris. On fait des recherches sur le contenu total de lipids et de protéines de la fraction mitochondriale, sur la morphologie des mitochondries et sur la densité de synaptosomes. On a remarqué une différence significative dans la quantité totale de lipides de la fraction mitochondriale et dans la densité de synaptosomes entre des souris soumis à une ambiance enrichie et d'autres soumis à une ambiance appauvrie. Ces souris, étudiés psychologiquement avant de procéder à l'extraction de la fraction mitochondriale, ont présenté des caractéristiques différentes. Le groupe enrichi a présenté un plus grand comportement explorateur et une plus grande capacité d'apprentissage que les souris élevés dans une ambiance appauvrie. S t i m u l a t i o n before r e a c h i n g a d u l t h o o d is v e r y i m -

1 9 7 5 a , 1 9 7 6 a , b ; BARLOW, 1 9 7 5 ; KOLATA, 1 9 7 5 ; BRA-

p o r t a n t for t h e n o r m a l d e v e l o p m e n t of t h e individual.

ZIER, 1975;MEISAMI, 1 9 7 5 ; BLAKEMORE, 1 9 7 5 ; K U F F -

T h e effect of e a r l y e x p e r i e n c e on t h e b e h a v i o u r a n d

LER a n d NICHOLLS, 1 9 7 6 ) .

c a p a c i t y t o l e a r n in a d u l t h o o d h a s b e e n s t u d i e d in

t h e s e a n d o t h e r experiences t h a t t h e n e r v o u s s y s t e m

v a r i o u s a n i m a l species including m a n (FREDERICSON,

e x h i b i t s a p l a s t i c i t y a t t h e morphological, physiolo-

1 9 5 1 ; MARX, 1 9 5 2 ; KING a n d GURNEY, 1 9 5 4 ; SEITZ,

gical a n d molecular level in response t o t h e environ-

1954,

m e n t in which t h e a n i m a l finds itself, which in t u r n

1959;

WEININGER,

1956;

BERSTEIN,

1957;

LEVINE, 1 9 5 7 a, b ; MONTGOMERY a n d ZIMBARDO, 1 9 7 5 ;

supposes a c o n s t a n t

ZIMBARDO a n d MONTGOMERY, 1 9 5 7 ;

conditions.

DAWSON a n d

HOFFMAN, 1 9 5 8 ; ADER, 1 9 5 9 ; HOFFMAN, 1 9 5 9 ; KING

One is led t o believe b y

adaptation

to

environmental

I n this w o r k a r e studied t h e effects p r o d u c e d b y

a n d ELEFTHERIOU, 1 9 5 9 ; EHRLICH, 1 9 5 9 , 1 9 6 1 ; D E -

living in a n

NENBERG a n d B E L L , 1 9 6 0 ; WOODS a n d al., 1 9 6 0 ; RO-

b e h a v i o u r a n d on t h e density of s y n a p t o s o m e s in t h e

SEN, 1 9 6 1 ; D E N E N B E R G a n d a l . , 1 9 6 2 ; D E N E N B E R G a n d

m i t o c h o n d r i a l f r a c t i o n in mice. I t w a s o b s e r v e d t h a t

SMITH, 1 9 6 3 ; SOSKIN, 1 9 6 3 ; D E NELSKY a n d DENEN-

t h e m i c e raised in a n e n v i r o n m e n t rich in stimuli

BERG,

environment

enriched in stimuli

on

1 9 6 7 a, b ; MCCALL a n d LESTER, 1 9 6 9 ; TEES,

displayed a g r e a t e r density of s y n a p t o s o m e s , t o g e t h e r

1 9 6 9 ; W E L L S e t al., 1 9 6 9 ) . T o g e t h e r w i t h t h e p s y c h o -

w i t h a n ability t o learn m o r e r a p i d l y a n d a m o r e li-

logical effects, h a v e been described t h e morphological,

vely exploratory behaviour.

histological, b i o c h e m i c a l a n d physiological differences in t h e b r a i n s of animals, which a f t e r b i r t h h a v e been s u b j e c t e d t o enriched o r i m p o v e r i s h e d e n v i r o n m e n t s (see reviews i n : BENNETT e t al., 1 9 6 4 ; LEVITSKY a n d BARNES, 1 9 7 2 ; ROSENZWEIGH a n d al., 1 9 7 2 ; DUYCKAERTS e t al., 1 9 7 2 ; FAIREN a n d VALVERDE, 1 9 7 3 ; HAMORI, 1 9 7 3 ; VAN HOF, 1 9 7 4 ; GAR&A-SEGURA, 1 9 7 4 ,

Material and Methods Psychological tests. — 120 mice aging reared in our laboratory, were taken Equivalent numbers of males and All of the mice were numbered and

one month, which were from the Balb/c batch. females were selected. by means of a table of

76

Garcia-Segura, L. M., C. Rodriguez-Gonzalez, and J. M. Gonzalez-Ros

random numbers, they were divided into two groups, both of which had equal numbers of males and females. One of the groups (30 males, 30 females) was divided between 5 boxes measuring 25 X 35 cm., 12 mice going into each box (6 males, 6 females). The other group was divide between 15 boxes measuring 25 X 35 cm., with 4 mice (2 males, 2 females) going into each box. The boxes which contained 12 mice also contained a collection of various objects (obstacles, small boxes, gangways, etc.), the relative positions of which were changed within the box 4 times a week. The mice remained in the boxes for a total of 60 days, which means that the tests began when the mice had reached an age of 3 months. All the mice were used for the tests. The exploratory behaviour of the mice was investigated by placing them in a plastic box measuring 25 x 50 cm., in which there were 3 transparent plastic tubes, 15 cm. in length and 5 cm. in diameter, which were located as indicated in figure 1. A fotometer was used to ensure that the intensity of light inside the tubes was the same as in the rest of the box. The number of times that each mouse put its head up to behind its ears inside each tube were then counted, using a time limit of 5 minutes for each mouse. Likewise the number of times that each mouse introduced its whole body were also counted. The exploration tests were done individually for each mouse and were carried out in the same test box. The measurements were taken in the course of one week during the same hours every day in order to avoid the influence of varying levels of wakefulness, and the mice were tested on a rota basis according to group and sex, so that every day the same number of mice from each group were being tested. Each time that a test on a mouse had been carried out, the box and tubes were washed with soap and water in order to avoid the distortion of the results caused by the possible presence of females which are in heat. The results were used to calculate the statistical probability of a mouse putting its head inside a tube and the probability of a mouse introducing its whole body. For this various tests were used. A complex maze (as that shown in figure 1), which was flooded with water, was used to test the ability of the mice to learn. The objective consisted in a platform above the level of the water. The time that each mouse took to reach the objective in a series of 10 consecutive tests was mesured, allowing a period of 2 minutes between each test. The maximum duration of each test was 1 minute. If the mouse did not reach the objective within this time limit a time of 60 seconds was recorded and the mouse was allowed to rest for a period of 2 minutes before being replaced it in the maze. Tissue preparation: isolation of a synaptosome-enriched fraction. — The brains of the mice were excised and rinsed in a cold 0,05 M Tris-sucrose 0,25 M buffer, pH 7.4. The pooled tissues were weighed and homogenized in a 30% buffer medium in a Potter-Elvehjem glass homogenizer with a teflon pestle, and filtered through two layers of cheesecloth. The filtrate thus obtained was centrifuged at 1476 Xg for 8 min. and the crude supernatant was used as the total homogenate. All operations were carried out at 0°—4°C. The total homogenate thus obtained was centrifuged at 8000 Xg for 10 min. The supernatant was separated and stored a short time under N2 at 0 °C until used. The sediment was resuspended in 5 ml buffer medium and centrifuged twice at 9000 Xg and 10000 Xg respectively. The sediment thus obtained, labelled as the 10000 Xg pellet, was used as the synaptosome-enriched fraction and resuspended in 5 ml of buffer medium in order to determine the quantity of proteins and lipids.

Proteins and lipids determinations. — The total quantity of proteins was determined using Lowry's method (1951). The total lipids were extracted using the Bligh and Dyer (1959) procedure. The lipid extracts were washed with 0.73% NaCl solution and the organic phase was separated, dried over anhidrous sodium sulphate and evaporated to a state of dehydration in a rotary evaporator under N2. The total quantity of lipids was determined gravimetrically. Electron microscopic study. — A part of the synaptosomeenriched fraction, obtained as indicated above, was resuspended in glutaraldehyde at 5% in Millonig buffer at 4°C and remained thus, being continually centrifuged at 10,000Xg in order to obtain a pellet which was rinsed in Millonig buffer with additional sucrose 0.25 M. Postfixation took place directly on the pellet using 0 4 0 s at 2% in Millonig buffer for l 1 / 2 hours. The material was embedded in Araldit (Fluka) after being dehydrated in acetone. Sections were stained with uranyl acetate and lead cytrate, supported on backing film, and observed with an JEOL 100-B transmission electron microscope. In order to make a statistical analysis a certain number of grids were selected at random and fields measuring 6 microns X 4.3 microns were photographed as described further on in this article.

Results Psychological tests. — (a) Exploratory behaviour: The group of mice subjected to the enriched environment, which shall be called group "E", displayed a greater degree of activity in the test box, while the mice subjected to the impoverished environment, group "I", showed a greater tendency to remain quietly in a corner of the box and deficate more frequently. This difference in behaviour between the two groups is also reflected in the findings of the test where the number of times the mice put their heads (or entire bodies) inside the tubes were counted, in which the mice from group " E " also displayed more iniciative. Each time a mouse put its head inside one of the tubes it scored one point and when it put its whole body inside, it scored two points. The number of points that group " E " obtained in this way was 829, while group " I " only obtained 475 points. A frequency table can be drawn up taking the number of mice that scored 0, 1, 2,..., n points. Having carried an %2 t e s t it was observed that the distribution obtained for group " E " differed from that obtained for group " I " by a level of signification p < 0.005. The advantage of using an %2 test is that it is not necessary to establish any hypothesis about how theoretic distributions behave, so that there are fewer imposed conditions that have to be met in order for the resulting statisticts to be considered to be reliable. (b) Ability to learn: The mice from group " E " displayed much more activity in the maze, with the result thet 80% reached the objective, while only 30% of the mice from group " I " succeded at the first

Environment, behaviour and synaptosomes in mice

77

1.0

'

1

f

e

0.5

• E

o I 0.1 10 c

I.

Fig. 1. Left: Box used to measure the exploratory behaviour. I t consists of three plastic transparent tubes. Mice were placed between the lower tubes. Right: Complex maze. The winning post is the square that is not ruled. Ruled objects are obstacles. Mice were placed between the lower obstacles.

1

2

3

4

5

6

supernatant E supernatant I mitochondrial E mitochondrial I

e

50

25

1

2

3

4

5

6

7

B

9

10

Fig. 2. Mice's percentage that were not able to resolve the maze in 10 consecutive trials. E : Mice E; I: Mice I.

attempt. In the successive attempts the performance of the mice from group " E " continued to be better that that of those from group " I " (Fig. 2). Thus at the fourth attempt all of the mice from group " E " were able to solve the problem presented by the maze, while 30% of the mice from group " I " still had not managed to dv so. The curves demonstrating the ability to learn according to the time taken by the mice to solve the problem of the maze also show significant differences between group " E " and group " I " (Fig. 3). Concentration of proteins and lipids in the synaptosome-enriched fraction: The concentration of the total quantity of proteins and the total quantity of

9

10

T a b l e I. proteins mg/ml

rm

*

Fig. 3. Mice E and I learning curve that was obtained by controling the time (T) they took to resolve the maze in 10 consecutive tests. Ordinate: 1—T.

100-

75-

7

6.64 7.28 11.8 10.0

lipids mg/ml

total volume ml

1.88

38 31 6

1.87 7.4 4.76

6

lipids in the mitochondrial fraction of the mice in group " E " and that of the mice in group " I " were measured. These measurements were also carried out in the supernatant. The results appear in table I. As can be observed in this table there is an important difference in the total concentration of lipids in the mitochondrial fraction, that of the mice from group " E " (7.40 mg/ml being greater than that of the mice from group " I " (4.76 mg/ml). Analysis of the synaptosome-enriched fraction using the electron microscope: The density of synaptosomes and the morphology of the mitochondries were studied. Two types of mitochondries were considered: the condensed type and the normal type. The relative proportion of these two types of mitochondries in the synaptosome-enriched fraction of the mice from group " E " and those from group " I " was then analysed. For this areas selected at random from the respective grids were photographed at the same magnification. The photographic plates were then developed. All those areas in which the preparation did not completely cover the picture were disgarded. Using the remaining plates the number of mitochondries of both types were counted. The researcher who counted the mitochondries was ignorant as to which group of mice the photographs belonged. Likewise

78

Garcia-Segura, L. M., C. Rodriguez-Gonzalez, and J . M. Gonzalez-Ros

66% 62% 52%

5 2%

48%

38%

5

< 5

E

34%

< 5

>5

E

I

>5

< 5

>5

Fig. 4. A : Field percentage with less and more than 5 condensed mitochondries. B: Field percentage with less and more than 5 normal mitochondries. C: Field percentage with less and more than 10 mitochondries. D : Field percentage with less and more than 10 synaptosomes. E = Mice E ; I = Mice I.

I

Fig. 4 b

Fig. 4 a

56%

54%

46°/o

44%

10

Fig. 4c 10

E

72%

28%

74%

26%

Fig. 4d < 1 0 > 10

< 10 >10

E

I

Environment, behaviour and synaptosomes in mice

the researcher who took the photographs did not know to which group of mice the grids belonged and manipulated the relays of the microscope which controlled the movements of the grid in such a way that the grid was moved at random. A distribution for the mice from group " E " reflecting the number of fields which contained 0, 1, 2, . . n condensed mitochondries was obtained, as was a similar distribution counting the normal mitochondries. The same was done for the mice from group " I " . Subsequently the two distributions were compared using an %2 test. Under these conditions it was found that the distributions of the condensed and normal mitochondries and the total of the two (the distribution of which was obtained by adding the two previous distributions) did not produce any appreciable difference between the mice from group " E " and those from group " I " . However, the distribution obtained for the two groups of mice, which considered the number of fields which contained 0 , 1 , 2 , . . . , « synaptosomes, were significantly different when they were compared using an yj- test, with a level of signification of p < 0.005 (Fig. 4). Figure 4 shows that in the grids belonging to the mice from group " E " there is a greater number of fields that posses a high number of synaptosomes ( > 10) than in those belonging to the mice from group from group " I " , in which the large majority of the fields in the girds thereof possess less than 10 synaptosomes. This indicates that the density of synaptosomes per field is greater in the grids from group " E " than in those from group " I " .

Discussion At present there is a lot of evidence indicating that the nervous system has an adaptative plasticity. There are certain structures, however, which appear to be less modificable than others in response to the environment. The system wihch has been subjected to greatest study with respect to its adaptative plasticity, is without doubt, the visual system. There have been described important changes in the morphology and size of the neurons of the lateral geniculate body and in the visual cortex, according to the visual stimuli that they are subjected to (VALVERDE, 1 9 6 7 , 1971;

GLOBUS a n d SCHEIBEL,

1967;

COLEMAN

R I E S E N , 1 9 6 8 ; RUIZ-MARCOS a n d VALVERDE,

and 1969;

FIFKOVA, 1 9 6 8 , 1 9 7 0 ; LUND a n d LUND, 1 9 7 2 ; B L A K E MORE

and

MITCHELL,

1973;

PARNAVELAS

et

al.,

1 9 7 3 ; FREEMAN a n d P E T T I G R E W , 1 9 7 3 ; W I E S E L a n d H U B E L , 1 9 6 3 , 1 9 7 4 ; VAN H O F , 1 9 7 4 ; BARLOW, 1 9 7 5 ; RYUGO e t a l . , 1 9 7 5 ; W I N F I E L D e t a l . , 1 9 7 6 ;

MEYER

et al., 1 9 7 6 ) . These morphological variations depen-

79

dant on stimulation can be co-related with other physiological and biochemical variations (WIESEL and HUBEL,

1963 a, b ;

1965;

HUBEL

HIRSCH a n d SPINELLI, and

BLAKEMORE,

and WIESEL,

1 9 7 0 , 1 9 7 1 ; VAN

1973;

PETTIGREW

et

1970;

SLUYTERS al.,

1973;

CHUNG e t a l . , 1 9 7 3 ; P E T T I G R E W a n d G A R E Y ,

1974;

B L A K E a n d HIRSCH, 1 9 7 5 ; CREUTZFELDT a n d H E G G E LUND,

1975;

FLAUDRIN

and

JEANNEROD,

1975;

ZABLOKA e t a l . , 1 9 7 5 ; S T R Y K E R a n d S H E R K ,

1975;

TRETTER et al., 1 9 7 5 ; IMBERT a n d BUISSERET, 1 9 7 5 ; LEVENTHAL

and HIRSCH, 1975; M U I R and Mitchell,

1 9 7 5 ; P E C K a n d BLAKEMORE, 1 9 7 5 ; GARCÍA-SEGURA,

1975b, c; CHAKRABARTI and DAGINAWALA, 1975, 1 9 7 6 ; DODWELL e t a l . , 1 9 7 6 ; FLANDRIN e t a l . , 1 9 7 6 ; F U K U I a n d VOGT, 1 9 7 6 ;

PETTIGREW a n d

KONISHI,

1 9 7 6 ; SILLITO, 1 9 7 6 ; SINHA a n d R O S E , 1 9 7 6 ; HOF-VAN

DUIN,

1976;

MAFFEI

and

VAN

FIORENTINI,

1976). At the same time these displays of plastic adaptability to the environment have not only been observed in the visual system, but also there exists data pertaining to other systems which supports the concept of an overall plasticity in the nervous system (BENNETT e t a l . , 1 9 6 4 ; DIAMOND e t a l . , 1 9 6 6 , HENDERSON,

1970;

LEVITAN e t a l . ,

1972;

1976;

ROSEN-

ZWEIGH e t a l . , 1 9 7 2 ; CHRONISTER e t a l . , 1 9 7 3 ; RAMIREZ e t a l . , 1 9 7 4 ;

CRAGG, 1 9 7 5 ; D E F E U D I S e t

al.,

1975a, b; 1976; GISIGER et al., 1975; MOORE and AITKIN,

1975;

SCHER e t a l . , SEGURA,

FELDMAN 1975;

a n d DOWD,

RYUGO e t a l . ,

1975;

1975;

FROT-

GARCÍA-

1974, 1975a, b, c, d, 1976a, b; DAVENPORT

et al., 1 9 7 6 ;

JOHNSON e t a l . , 1 9 7 6 ;

MEYER et

al.,

1 9 7 6 ; NEWCOMBE, 1 9 7 6 ; BONDAREFF a n d GEINISMAN,

1976). This plasticity has also been studied at synapses and synaptosome level. The importance of the synapses as an adaptable system responsible for important functions in the nervous system, like the learning, is begining to be analysed. These facts are directly connected with the prossesses of regeneration in the nervous system and the specificity of the neuronal connections. The data obtained in this particular study supports the hypothesis that the total number of synapses in the brain is dependent upon the environmental stimulation received. With respect to the psychological experiments, these demonstrate that environmental stimulation is capable of modifying the subsequent behaviour of the individual. Previous experiments have demonstrated that this environmental dependency also occurs in adulthood (INGLIS, 1975; GARCÍA-SEGURA, 1977). A methodological criticism towards the experimental plan that has been used for this study can be found in GARCÍASEGURA (1977). The results obtained therefrom may be interpreted as showing a modification in conduct caused by the environment in which the animals

80

Garcia-Segura, L. M., C. Rodriguez-Gonzalez, and J . M. Gonzalez-Ros

lived.

The

animals

which

were

subjected

to

an

e n v i r o n m e n t impoverished in stimuli were m u c h m o r e withdrawn and emotive, consequently they

tended

t o r e m a i n longer in one c o r n e r of t h e t e s t b o x . A t t h e s a m e t i m e t h e y showed t h e m s e l v e s t o h a v e

more

difficulties in solving t h e p r o b l e m of t h e m a z e t h a n t h e m i c e w h i c h h a d been s u b j e c t e d t o a m o r e sensorially enriched e n v i r o n m e n t . I n t h e s e l a t t e r m i c e t h e r e m a y h a v e o c c u r r e d t h e p h e n o m e n o n of a t r a n s fer of t h e i r ability t o l e a r n o b t a i n e d t h r o u g h

the

c h a r a c t e r i s t i c s of t h e i r previous e n v i r o n m e n t (GARCIASEGURA, 1 9 7 7 ) . T h e s e psychological results a p p e a r t o h a v e a m o r p h o l o g i c a l b a s i s : in t h e brains of t h e m i c e w h i c h were s u b j e c t e d t o a n impoverished

environ-

m e n t t h e r e were less synapses. W i t h r e s p e c t t o t h e b i o c h e m i c a l results it is e v i d e n t t h a t t h e r e is a clear difference in t h e lipidic c o m p o sition in t h e m i t o c h o n d r i a l f r a c t i o n s of t h e t w o g r o u p s of mice. H o w e v e r , n o difference w a s found b e t w e e n t h e t o t a l n u m b e r of mitochondries, n o r in t h e prop o r t i o n in t h e different t y p e s of mitochondries, b e t ween t h e t w o groups. T h e b i o c h e m i c a l

differences

w h i c h were found, therefore, a p p e a r t o b e due t o t h e s y n a p t o s o m e s . H o w e v e r , this f a c t requires subsequent confirmation. T o conclude, t h e results of this s t u d y provide one m o r e indication t h a t t h e s y n a p s e is a n element w i t h qualities of plasticity, d e p e n d a n t , t o a c e r t a i n e x t e n t , on e n v i r o n m e n t a l stimulus, a s a r e b e h a v i o u r a n d t h e ability t o learn.

T., and H . F. D A G I N A W A L A : Effect of unilateral visual deprivation and visual stimulation on the activities of alkaline phosphatase, acid phosphatase, Na+—K+ activated Mg++ catalysed adenosine triphosphatase and on the content of sodium and potassium ions of the optic lobe of adult pigeon. J . Neurochem., 24, 9 8 3 - 9 8 8 (1975).

CHAKRABARTI,

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Instituto Cajal; C.S.I.C. Velazquez, 144 Madrid-6/Spain

J . Himforsch. 19 (1978) 8 5 - 1 0 0

Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka/Japan (Director: Prof. Dr. N. S H I M I Z U )

Afferent fiber connections from the lower brain stem to the rat cerebellum by the horseradish peroxidase method combined with MAO staining, with special reference to noradrenergic neurons By Yasuhiko

KIMOTO,

Keiji

SATOH,

Tetsuro

SAKUMOTO,

Masaya

TOHYAMA,

and Nobuo

SHIMIZU

With 8 figures and 3 tables (Received 30 th May 1977) Summary: The origin of cerebellar noradrenaline (NA) was investigated by means of the horseradish peroxidase (HRP) method, combined with the monoamine oxidase (MAO) staining method to identify the NA neurons. The present study gives clear cut evidence that the main source for cerebellar NA in the rat is the locus coeruleus (Lc), both the dorsal and ventral parts which innervate the entire cerebral cortex and hypothalamus respectively. The present study clearly demonstrates that pontine NA neurons such as the nucleus subcoeruleus (Sc) NA neurons also send their axons to the cerebellum. The fact that some neurons in the nucleus dorsalis nervi vagi and the nucleus commissuralis were labeled by H R P indicates strong possibility that A2 NA neurons also innervate the cerebellum, though direct proof was lacking in this study. Furthermore, some unknown afferent fibers to the cerebellum were found in this study. These are as follows : (1) non-noradrenergic neurons in the Sc area, (2) nucleus reticularis gigantocellularis and nucleus reticularis parvocellularis, (3) group 1 (of M E E S S E N and O L S Z E W S K I ) , (4) a cell group extending to the ventrolateral region of the reticular formation of the pons, (5) nucleus ambiguus, (6) nucleus nervi facialis. Résumé: L'innervation cérébelleuse des neurones contenant la noradrénaline (NA) ont fait l'object d'étude par la method de marquage rétrograde de la peroxidase (HRP), combiné à la réaction de la monoamine oxidase (MAO) pour identifier les neurones NA. Notre étude clairement revèle que la NA cérébelleuse chez le rat est fourni principalement par locus coeruleus (Lc). De plus, nos résultats ont distinctement certifié que les autres neurones NA, comme le neurones NA du noyau subcoeruleus (Sc), également innervent le cérébellum. Il faundrait noter que l'on a observé l'éxistence de la projection le cérébellum des neurones apparaissant dans le noyau dorsalis nervi vagi et la noyau commissuralis. Ce résultat suggère que des neurones NA A2 envoient également la projection NA vers le cérébellum, bien que la preuve directe soit manqué. Nous avons encore élucidé l'éxistence des projections non-aminergiques, vers le cérébellum des structures suivantes: (1) les neurones non-noradrenergique dans la région du Sc, (2) noyau réticularis gigantocellularis et noyau réticularis parvocellularis, (3) groupe 1 (d'après M E E S S E N and O L S Z E W S K I ) , (4) groupe cellulaire s'étendant dans la région ventro-laterale de la formation reticulée du pons, (5) noyau ambiguus, (6) noyau nervi facialis.

Abbreviations used in the text and figures A CC Ce Cl Cm Cp FLM FG Gp Lc LL LM nco Oi Os PCI PCS Ph Rgc R1

nucleus ambiguus canalis centralis cerebellum nucleus cuneatus lateralis nucleus cuneatus medialis nucleus corporis pontobulbaris fasciculus longitudinalis medialis fasciculus gracilis griseum pontis nucleus locus coeruleus lemniscus lateralis lemniscus medialis nucleus commissuralis nucleus olivaris inferior nucleus olivaris superior pedunculus cerebellaris inferior pedunculus cerebellaris superior nucleus prepositus hypoglossi nucleus reticularis gigantocellularis nucleus reticularis lateralis

Em Ro Rpc Rpm Rtp Sc Tc Ti Tm TM To Tp VI Vm Vsp IV VII X XII 1 X

nucleus raphe magnus nucleus raphe obscurus nucleus reticularis parvocellularis nucleus reticularis paramedianus nucleus reticularis tegmenti pontis nucleus subcoeruleus nucleus tractus spinalis caudalis nucleus tractus spinalis interpolaris nucleus motorius nervi trigemini tractus mesencephalicus nervi trigemini nucleus tractus spinalis oralis nucleus sensorius principalis nervi trigemini nucleus vestibularis lateralis nucleus vestibularis medialis nucleus vestibularis spinalis ventriculus quartus nucleus nervi facialis nucleus dorsalis nervi vagi nucleus nervi hypoglossi group 1 group x

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1 Fig. la, b, c, d, e, f, g and h. Diagrammatic representation showing localization of HRP containing neurons in the lower brain stem following injections of HRP into cerebellum, (a) Case 27. The injection site centered in lobus anterior, (b) Case 16. The injection site was declive, folium and tuber, (c) Case 32. The injection site centered in uvula and nodulus (d) Case 18. The injection site centered in lobulus ansiformis and lobulus paramedianus. (e) Case 14. The injection site was

Cerebellar NA innervation

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paraflocculus and flocculus. Blackened areas and surrounding shaded areas indicate the central and peripheral areas of the injection site. Small dots indicate H R P positive neurons of small size, and large dots indicate those of medium or large size. Triangular blackened dots indicate those of NA neurons, (f), (g) and (h) are low-power photomicrographs showing the spread and intensity of injected HRP. (f) Case 46. x 18 (g) Case 21. X 10 (h) Case 14. x 11.

88

Kimoto, Y., K. Satoh, T. Sakumoto, M. Tohyama, and N. Shimizu

Cerebellax N A innervation

Introduction

89

1974;

sections were rinsed thoroughly in buffer. And then they were incubated in the tryptamin-nitroblue tetrazolium solution for 60 min at 37°C. To compare the results of the double staining procedure, the neighboring 2 sections were stained for either H R P or MAO. The terminology used follows, unless otherwise stated, that

OLSON a n d F U X E , 1 9 7 1 ; SACHS e t a l . , 1 9 7 3 ; SEGAL

o f TABER ( 1 9 6 1 ) , VALVERDE ( 1 9 6 2 ) , a n d ZEMAN a n d I N N E S

et al., 1973), especially by its dorsal portion which also innervates monosynaptically the entire cerebral cortex (MAEDA et al., 1973; MAEDA and SHIMIZU,

(1963).

It has been believed that cerebellar noradrenaline (N A) might be supplied exclusively by locus coeruleus (Lc) (CHU a n d

BLOOM, 1 9 7 4 ; KOBAYASHI e t a l . ,

1 9 7 2 ; OLSON a n d F U X E , 1 9 7 1 ; SHIMIZU e t a l . , 1 9 7 4 ;

et al., 1974a; TOHYAMA et al., 1974b; 1971). However, our recent experimental study clearly revealed that cerebellar NA was supplied mainly by both the dorsal part of Lc and its caudal continuation, A 4 N A neurons (TOHYAMA, 1976). We further suggested another type of NA afferent fibers into the cerebellum, though several similar findings were reported (ANDEN and UNGERSTEDT, 1967; UNGERSTEDT, 1971). But precise origins of cerebellar NA still remain under debate. Recently a retrograde tracer technique with horseradish peroxidase (HRP) has been developed and this technique has been applied in determining the cells of origin at various central nervous system TOHYAMA

UNGERSTEDT,

(GRAYBIEL a n d HARTWIEG, 1 9 7 4 ; K U Y P E R S e t

al.,

and L AVAIL, 1 9 7 2 ) . Nevertheless by the H R P method alone, the H R P labeled neurons could not be identified as NA neurons. We recently succeeded in combining the H R P method and monoamine oxidase (MAO) staining method (GLENNER, 1957) by which NA neurons showed strong MAO activity (SATOH et al., 1 9 7 6 ) . Using this technique, the present study attempted to elucidate the detailed origins of cerebellar NA, and further aimed to detect important afferent fibers to the cerebellum from the lower brain stem. 1 9 7 4 ; LAVAIL

Materials and Methods More than 50 male rats (weighing 150 — 200 g) were used. Under the nembutal anesthesia, H R P (Sigma type VI, 30% solution in saline) was injected (0.2 (¿1) into various parts of the cerebellar cortex, using a micropipette system or a Hamilton 10 ¡J syringe. After the survival period of 24 h, the animals were perfused transcardially with a mixture containing 0.4% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2 — 7.3, 4°C). The brains were immediately removed and postfixed with the same mixture for 2 —4 h, followed b y washing overnight at 4°C in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. The brain stem and the cerebellum were frozen and serial frontal sections (30 ¡¿m thick) were cut, using a cryostat. The procedure for demonstrating the presence of H R P and/ or MAO was performed as in our previous study (SATOH et al., 1976). The activity of H R P and MAO was demonstrated on the same section (double staining) of an every-third section series. After immersion of sections in the 3,3'-diaminobenzidine containing hydrogen peroxide (66 (¿1/100 ml), the

Results The distribution and intensity of injected H R P in the cerebellum and labeled neurons in the lower brain stem in all experiments are summarized in Table 1, 2 and 3 respectively. [1] NA neurons The NA neurons labeled following injections of H R P in the following parts of the cerebellum are described first. (1) Lobus anterior; Following injections of HRP into lobus anterior H R P containing neurons were detectable in bilateral Lc, both in the dorsal and the ventral part (Table 2, Fig. l a , f). Although the HRP labeled neurons distributed evenly in the dorsal part and the ventral part of Lc, the number of labeled neurons was somewhat different in the rostral and the caudal part. Namely more labeled neurons were detectable in the caudal part than those in the rostral part. H R P containing neurons often occupied the subcoeruleus (Sc) area following such type of injection. However, interestingly enough, these H R P positive neurons were composed of non-noradrenergic neurons because these neurons never showed MAO activity (Fig. la). The HRP positive neurons were found in the nucleus reticularis lateralis (Rl) and its surrounding territories where Al NA neurons were located, but these labeled neurons did not show MAO activity, either. Accordingly all of them were not identified as NA neruons. In the region of the nucleus commissuralis (nco) which showed strong MAO activity, several HRP positive neurons were detectable (Fig. 3). In the areas where A 5 or A 7 NA neurons were situated, some labeled neurons were also found. But none of them showed MAO activity. (2) Declive, Folium vermis, Tuber vermis; This area belongs to the neocerebellum. A large number of NA neurons in Lc on both sides were labeled (Table 2, Fig. l b ) , a n d the number of these neurons was twice or three times as large as that found in the cases which received the HRP injections into lobus anterior. These neurons occurred throughout the entire extent of Lc and no difference in topography was found between the dorsal part and the ventral part. In the Sc area, few neurons were labeled by HRP.

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Fig. 4. A dark-field photomicrograph showing a lot of H R P positive neurons of 'group I' of M E E S S E N and O L S Z E W S K I following injection of H R P into the cerebellar nuclei, though there was a wide diffusion of H R P into the ipsilateral hemispherium. (Case 42). X 120.

graphy between the dorsal part and the ventral part (Table 2, Fig. I d , g, 2). The number and intensity of labeled neurons was greater than that of the vermis of the neocerebellum. In the Sc area, just ventral to Lc, there could be found some labeled neurons. In contrast to the cases described above, some of these H R P containing neurons in the Sc area simultaneously showed MAO activity (Fig. 2). In the A 1, A 2, A 5 and A 7 areas, no labeled neurons with MAO activity were seen. (5) Paraflocculus, Flocculus; Such type of H R P injection could label only a few NA neurons in bilateral Lc (Table 2, Fig. l e , h). Labeled neurons were found both in the dorsal part and in the ventral part of Lc. In the Sc area and also other regions where A 1, A 2, A 5 and A 7 NA neurons could be found, no H R P positive neurons occurred. (6) In three cases in which the spread of H R P was found in the whole part of the cerebellum, a lot of NA neurons were labeled throughout the entire extent of Lc. And also in the Sc area, many neurons containing H R P were found, and among them several neurons showed MAO activity at the same time. In the A 2 area, some neurons were also labeled. One or two labeled neurons in the A 5 and A 7 areas exhibited strong MAO activity at the same time. This fact might suggest that A 5 and/or A 7 NA neurons also partly take part in NA regulation in the cerebellum. However, as faint, diminished H R P activity was found also in the caudal part of the colliculus inferior and the dorsal part of the nucleus vestibularis lateralis (VI), whether these H R P positive neurons in the A 5 and A 7 areas innervate the cerebellum is open to argument. [II] Nonaminergic neurons Following injections of H R P to the cerebellar cortex,

various kinds of nonaminergic neurons in the lower brain stem were labeled (Table 1, 2, 3). (1) Reticular nuclei; Almost all cells in the nucleus reticularis tegmenti pontis (Rtp) were labeled following injections of H R P into the neocerebellum (Table 2, Fig. l b , d). Labeled neurons were smallsized in the medial part and medium-sized in the lateral part. The small-sized neurons were also seen in the lateral part. Small neurons medially and laterally merged with the cells of the nuclei pontis, so that the R t p can be considered to be the dorsal extention of t h e n u c l e i p o n t i s as c l a i m e d b y JANSEN a n d BRODAL

(1954). Compaired with the number of small-sized labeled cells in the medial part following H R P injections into declive, folium and tuber, the number of these cells following injections into lobuli simplex, ansiformis and paramedianus slightly decreased (Fig. l b , d). On the contrary, in the cases in which the injection site was restricted to lobus anterior, several small- or medium-sized neurons in the lateral part of the R t p were labeled (Fig. l a ) . And following injections into the archi- and paleocerebellum of lobus posterior, only a few neurons were labeled (Fig. 1 c, e). From the fact that the R t p neurons send fibers mainly onto the neocerebellum, it can be stressed that there exists a strong connection between the R t p and the neocerebellum. Another interesting fact was that cellular group 1 of MEESSEN a n d OLSZEWSKI w a s also l a b e l e d i n t h e

rat after the H R P injection into the neocerebellum (Table 2, Fig. I d ) . But this type of injection could label only a few neurons in this group. In the cases in which H R P was injected into the cerebellar nuclei, especially in the nucleus lateralis and the nucleus interpositus, a lot of medium-sized cells occurred in this area 'group 1', though in the hemispherium and in the nucleus medialis was seen a spread of H R P (Table 1, 2, Fig. 4). Caudal pole of labeled cells in

Cerebellar NA innervation

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'group 1' was located ventrolateral to FLM at the mid level of Lc. Rostrally these labeled neurons shifted ventrally but were discontinuous to the Rtp. In this study, it is clear that the neocerebellum receives afferent fibers from some neurons of 'group 1'. And also, as described above, there is a strong possibility that 'group 1' sends its fibers to the cerebellar nuclei. In the nucleus reticularis gigantocellularis (Rgc), neurons were labeled following the HRP injections into the cerebellum (Fig. 1). These labeled neurons in the Rgc could be subdivided into three groups. (1) A neuron group which is situated between the nucleus ambiguus (A) and efferent root of nervus hypoglossus. These neurons were labeled regardless of the difference of the injection sites. This group is composed of medium- and small-sized neurons. (2) A neuron group which occupied the area dorsal to the nucleus olivaris inferior (Oi). This neuron group was composed of medium-sized cells and labeled following injections into the lobus anterior. This neuron group was also labeled following injections into declive, folium and tuber, but the number of them decreased (Fig. 1 a, b). (3) A cellular band located just medial to the nucleus tractus spinalis n. trigemini (Fig. Id, 5). This cell band was composed of mediumsized cells and was labeled after injections into the hemispherium of lobus posterior (Fig. Id, e). This cell cluster lateroventrally fused with the subnucleus subtrigeminalis of the R1 and dorsally merged with the A. It is worthwhile to note that this cell band was labeled bilaterally following injections particularly into the hemispherium of the neocerebellum. Accordingly this cell group projects especially onto the hemispherium of the. neocerebellum, and as is reported below, it might have close relationship with the dorsal group of the A. Relatively many small-sized neurons, which belong to the nucleus reticularis parvocellularis (Rpc),

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Fig. 5. A dark-filed photomicrograph showing a cell band (arrows) located just medial to the nucleus tractus spinalis interpolaris (Ti). These neurons partly belong to the nucleus reticularis gigantocellularis (Rgc), and partly to the dorsal group of the nucleus ambiguus (A). They merged ventrolaterally with the subnucleus subtrigeminalis of R1. They were labeled by H R P particularly after injections into the hemispherium of the neocerebellum. (Case 18). X 75.

dorsal to the nucleus nervi facialis (VII), were labeled in the cases when HRP was injected into hemispherium of the neocerebellum (Table 2, Fig. 1 d). A lot of cells in the R1 were labeled (Table 2, Fig. 1 a—d). HRP labeled cells found following injections into lobus anterior slightly outnumbered those observed after injections into lobus posterior. They were composed of cells of two types, medium- and small-sized ones. It should be emphasized that smallsized cells, which might correspond to the subnucleus subtrigeminalis of R1 of TABER, were also labeled, and they were located just ventromedial to the tractus spinalis n. trigemini. (2) Branchial motor nuclei: nucleus ambiguus, nucleus nervi facialis, nucleus motorius nervi trigemini; Following HRP injections into the hemispherium of the neocerebellum, a group of fairly large cells, which is considered to correspond to the dorsal group of the A (Fig. Id, 5), was labeled bilaterally together with the cell band reported above. Another group of cells located ventrolateral to the cell band which corresponds to the medial group of the A, was never labeled following any type of injection. And also a few, fairly large motor cells in the VII were labeled bilaterally after HRP injections ipto the hemispherium of the neocerebellum (Fig. Id, 6). Recently E L L E R and CHAN-PALAY (1976) reported that the cells in the nucleus motorius n. trigemini were labeled following HRP injections into the nucleus lateralis, though we could not label the nucleus motorius n. trigemini in this study. Accordingly, it is clear that some of the branchiomotor neurons give rise to their axons to the cerebellum. But it is uncertain whether these axons to the cerebellum are axon collaterals of the cranial nerves. (3) Nucleus tractus spinalis nervi trigemini, nucleus sensorius principalis nervi trigemini; Following in

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Fig. 7. A dark-field photomicrograph of H R P positive neurons of the nucleus corporis pontobulbaris (Cp). They were located in the trigeminal motor nerve, rostrally shifted to the area just lateral to the lemniscus lateralis, caudally fused with 'group k' of M E E S S E N and O L S Z E W S K I . The nucleus sensorius principaris n. trigemini, which was situated dorsolateral to Cp, also contained H R P granules. (Case 21). X 75.

jections into lobus anterior, a few HRP containing neurons could be seen in the nucleus sensorius principalis (Tp) and nuclei tractus spinalis oralis, interpolarisandcaudalis(To,Ti,Tc) (Table 3, Fig. la). After injections into the archi- and paleocerebellum

Fig. 6. A dark-field photomicrograph of some H R P positive neurons in the nucleus n. facialis (VII). They were found after injections of H R P into the hemispherium of the neocerebellum. It seems that some of the branchiomotor neurons might give rise to fibers to the cerebellum, (cf Fig. 5, nucleus ambiguus.) (Case 18). x 100.

of lobus posterior vermis (pyramis, uvula, nodulus), small-sized labeled neurons were observed bilaterally in Tp, To, Ti and Tc. At the level of nervus hypoglossus small-sized labeled neurons were located in the ventral part of Tc and the part just adjacent to the tractus spinalis (Fig. 1 c). The HRP injections into declive, folium and tuber showed several small cells labeled. These neurons were situated in the ventrolateral part of the Ti, just medial to the tractus spinalis at the level of the rostral pole of the Oi. Rostrally these neurons in the To shifted to occupy the dorsomedial part (Fig. lb). A lot of small-sized cells in all of these sensory nuclei groups of the nervus trigeminus were labeled following injections into the hemispherium of the neocerebellum (lobulus simplex, lobulus ansif ormis, lobulus paramedianus), from the level of nervus hypoglossus to the level of Lc (Fig. 1 d). However, in addition to the small-sized cells, medium-sized neurons containing HRP were intermingled in the Ti and the Tc. It is worth mentioning that the number of labeled neurons ipsilateral to the injection site was larger than that of contralateral. After injections into paraflocculus and flocculus, several small neurons in the ipsilateral Tc and Ti were labeled, while the contralateral Tc and Ti were hardly labeled (Fig. 1 e). (4) Nucleus corporis pontobulbaris {Cp); Small-sized neurons, which extended to the ventrolateral region of the reticular formation of the pons and were considered to be the nucleus corporis pontobulbaris of TABER, were labeled bilaterallyin every case (Fig. la—e, 7, Table 2). Caudally, these neurons occupied the region ventral to the nucleus motorius n. trigemini (Tm) which corresponds to 'group k' of M E E S S E N and OLSZEWSKI, and were located in the radix of the trigeminal motor nerve. Rostrally, narrow strands of the labeled cells merged with the lateral region of the

Cerebellar NA innervation

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Fig. 8. A dark-field photomicrograph showing H R P positive neurons of 'group x' of B R O D A L and of its medial continuation. (Case 21). X 100.

griseum pontis (Gp) and extended forward along the lateral border of lemniscus lateralis (LL). (5) Vestibular nuclei; In almost all cases, the nucleus vestibularis medialis (Vm) and nucleus vestibularis spinalis (Vsp) were labeled bilaterally (Table 3, Fig. l a — e ) . B u t , following injections into paraflocculus and flocculus, only the ipsilateral Vsp was labeled (Fig. l e ) . Especially the H R P injections into pyramis, uvula and nodulus could label many more neurons in the V m and the Vsp, and the labeled neurons occurred throughout the extent of the Vm and the Vsp (Fig. l c ) . As for the nucleus vestibularis lateralis and the nucleus vestibularis superior, no H R P containing neurons could be found in this study. In almost all cases, a group of small-sized cells, which was located in the area between the caudal half of the Vsp and the rostral pole of the nucleus cuneatus lateralis (CI), was labeled. These neurons might topographically and cytoarchitecturally correspond to the cell 'group x ' of BRODAL in the cat (Table 3, Fig. l a — e , 8). Labeled cells were small-sized and situated ventral ot the rostral pole of the CI. At this level, however, it was almost impossible to separate the cell group from the CI. These cells extended to the level of the caudal half of the nucleus vestibularis lateralis (VI). Some of them occupied the area between the tractus spinalis 11. trigemini and the pedunculus cerebellaris inferior (PCI), and some of them on the margin of the PCI. Especially following injections into the neocerebellum or into paraflocculus and flocculus, several small-sized cells were labeled bilaterally just dorsal to Ti (Fig. I d , e, 8). while another type of injection failed to demonstrate them (Fig. l a — c ) . This cell group laterally merged with the 'group x ' . Furthermore, these H R P containing neurons in the 'group x ' rostrally shifted dorsalward and fused with small-sized H R P positive neurons in the Hirnforsclmng, Bd. 19, Heft *

area just dorsal to the PCI when the injection site was found in pyramis, uvula, nodulus, paraflocculus and flocculus. This neuron group might correspond to the cell 'group y' of BRODAL in the cat. Some smaller cells among the root fibers of nervus vestibulocochlearis, which represent the noyaux interstitiels du nerf veslibulaire of CAJAL (1909), were observed to contain H R P granules when H R P was injected into the neocerebellum. (6) Nucleus prepositus hypoglossi (Ph); Large cells belonging to this nucleus give fiber projection to the entire cerebellum (Table 3, Fig. l a — e ) . In addition to these large neurons, small cells of 'pars a' of MEESSEN and OLSZEWSKI were also labeled. Furthermore, medium-sized neurons ventral to the Ph were also labeled by H R P , which were located along the lateral margin of the fasciculus longitudinalis medialis (FLM). (7) Nucleus olivaris inferior (Oi); The distribution of H R P labeled neurons in the Oi differed according to the injection site of H R P (Fig. l a - e , Table 3). After the H R P injections into lobus anterior, several small cells were observed to contain the enzyme in the nucleus olivaris accessorius medialis. And also the H R P positive neurons in the nucleus olivaris principalis and the nucleus olivaris accessorius dorsalis occurred in one-third of the ventrolateral part of these nuclei (Fig. l a ) . Following injections of H R P into declive, folium and tuber, a cluster of H R P containing cells were demonstrated in the nucleus olivaris accessorius medialis, the ventral part of the nucleus olivaris principalis and the dorsomedial pole and ventrolateral pole of the nucleus olivaris accessorius dorsalis. It is worthwhile to note that the middle part of the nucleus olivaris accessorius dorsalis was scarcel labeled (Fig. l b ) . In the case, when the H R P solution was injected 7

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K moto; Y., K. Satoh, T. Sakumoto, M Tohyama, and N. Shimizu

into the hemispherium of the neocerebellum, cells in the contralateral Oi at the level of the caudal end of efferent roots of nervus hypoglossus were labeled. But only a few neurons in the caudal part of the nucleus olivaris accessorius medialis were labeled following such type of injection (Fig. Id). On the contrary, a lot of cells in the caudal part of the nucleus olivaris accessorius medialis were labeled when the injection site was limited to uvula. When the spread of H R P activity was found in paraflocculus and flocculus, several neurons in the nucleus olivaris accessorius medialis and the ventromedial part of the nucleus olivaris principalis were labeled contralaterally (Fig. le). To summarize, lobus anterior receives afferent fibers from the nucleus olivaris accessorius medialis, one-third of the ventrolateral part of the nuclei olivaris principalis and accessorius dorsalis; declive, folium and tuber from the nucleus olivaris accessorius medialis, the ventral part of the nucleus olivaris principalis and the nucleus olivaris accessorius dorsalis; uvula from the nucleus olivaris accessorius medialis; the hemispherium of the neocerebellum from almost all the nucleus olivaris inferior except the caudal part of the nucleus olivaris accessorius medialis. During the course of this investigation, some detailed reports were published (BRODAL et al., 1975;

Injections of H R P into the hemispherium of the neocerebellum succeeded in labeling the neurons in the Cm (Fig. Id). The above data partly confirmed the recent study concerning the projection of CI and Cm to the cereb e l l u m (RINVIK a n d WALBERG, 1 9 7 5 ) .

(10) Griseum pontis (Gp); Following the H R P injections into the vermis, small neurons in the medial and the lateral part of the Gp were labeled. And also some small neurons with H R P granules embracing the tractus corticospinalis were seen (Fig. la—e, Table 3). H R P containing cells located especially in the lateral and the medial part of the Gp were seen when H R P was injected into paraflocculus and flocculus (Fig. le). On the contrary, after the H R P injections into the hemispherium of the neocerebellum, labeled cells occurred especially in the intermediate zone of the Gp (Fig. 1 d). But rostrally at the caudal level of the decussatio pedunculorum cerebellarium superiorum almost all cells in the Gp were labeled. The number of labeled neurons was contralateally dominant (Fig. 1 d, e). In summary, cells in the intermediate zone of the Gp give riste to fibers onto the hemispherium of the neocerebellum, and the medial and the lateral part to the vermis and the hemispherium of the archi- and paleocerebellum.

BRODAL, 1 9 7 6 ; HODDEVIK e t al., 1 9 7 6 ) .

(S) Nucleus raphe; Some small-sized neurons loading H R P granules were scattered in the raphe region from the mid level of the Oi to the level of the VII in almost all cases (Table 2, Fig. la—e). Some of these cells belong to the nucleus raphe magnus (Rm) and the nucleus raphe obscurus (Ro). In rare cases, scattered H R P positive neurons were also found in small number in the nucleus raphe dorsalis and the nucleus centralis superior following H R P injections into the hemispherium of the neocerebellum. Although it is certain that some of the neurons in the raphe region contain serotonin, whether the labeled neurons in this study contain serotonin is not clear. (9) Nucleus cuneatus lateralis (CI), nucleus cuneatus medialis (Cm); In each case neurons in the CI were labeled bilaterally, but there existed difference in the number of the labeled neurons according to the injected lobules (Table 3, Fig. la—e). Following injections of H R P into the archi- and paleocerebellum besides paraflocculus and flocculus, labeled neurons occurred throughout this nucleus, while following injections into paraflocculus and flocculus the number of the labeled cells became far less. After injections of H R P into the neocerebellum, several neurons in the CI were labeled. And after injections into the hemispherium, ipsilateral labeled neurons outnumbered those in the contralateral side.

Discussion According to the previous investigations on cerebellar NA innervation, it has been believed that cerebellar NA is supplied ipsilaterally by Lc neurons, though several recent investigations stated the possibility of another type of NA afferent fibers from the lower brain stem in addition to Lc (ANDEN et al., 1967; UNGERSTEDT, 1 9 7 1 ; TOHYAMA, 1 9 7 6 ) . T h e

present

study furnishes the convincing evidence that Sc NA neurons also contribute to the regulation of cerebellar NA, though it is certain that Lc NA neurons and their caudal continuation, A 4 NA neurons, are the main source for cerebellar NA. Furthermore, the result of our study demonstrated that Lc NA neurons innervate bilaterally the entire cerebellar cortex, neo-, paleo- and archicorteces, while Sc NA neurons innervate bilateral neocortex. It is certain that sagittal cut of the cerebellum did not cause an accumulation of the fluorescent substances in the axons bilateral to the lesion. I t is possible that transection of terminal NA fiber does not cause the accumulation of NA in their terminal fiber system. Therefore the NA fiber system from Lc and Sc might be interlaced with one another within

Cerebellar NA innervation

the cerebellum on the level of the terminal fiber system. The present study furthermore furnishes an interesting fact that several cells in the nco, where strong MAO activity was found and according to the histofluorescence method A 2 NA neurons were distributed, were labeled by HRP after injections into lobus anterior and lobus posterior vermis (pyramis, uvula, nodulus). Although direct proof is lacking in this study, from the topographical analysis of these cells it is likely that A 2 NA neurons send their fibers to innervate the cerebellum, especially the archi- and paleocerebellum. It is not clear in this study whether the same neurons in Le, Se, A 4 and A 2 neurons which innervate the cerebral cortex and hypothalamus, simultaneously innervate the cerebellum. Our previous study elucidated that the same Lc and A 4 neurons that innervate monosynaptically the entire cerebral cortex give rise to their axons to innervate the cerebellum. Accordingly, it is possible that the same Sc and A 2 NA neurons, which innervate the hypothalamus and the spinal cord (SATOH et al., 1 9 7 7 ) , also give rise to their axons or axon colaterals to innervate the cerebellum. The precise role of NA in the cerebellum is still uncertain, although some suggestions that there is a close relationship between the NA nerve terminals and the Purkinje cells and the molecular layers of the cerebellum were made (HÒKFELT and F U X E , 1 9 6 9 ; BLOOM et al., 1 9 7 1 ; ADACHI et al., 1 9 7 5 ) . In order to determine whether NA neurons really take part in the regulation of the cerebellar function, attemps to clarify the morphological relationship of NA nerve terminals with another neuronal elements under electron microscope are needed. For electron microscopic vidualization of NA nerve terminals, pottassium permanganate fixation method has proved most suitable in the sympathetic NA nerve terminals and also in the central nervous system. By using this method, the fine structures of NA nerve terminals in the cerebellum will be reported in detail in the future. The evidence that NA neuron system appears at early stage during both ontogeny and phylogeny (ToHYAMA, MAEDA personal communication) suggests that NA system might play an important role during development of the cerebellum of the vertebrate brain. It should be noted that in the area where NA neuron groups were disseminated, such as Se, A 5, A 7 and also A 1, several neurons were labeled by HRP following various types of injections. However these labeled neurons were exclusively composed of nonnoradrenergic cells other than several neurons which contained HRP granules in the Sc area, because none

99

of the cells other than the aforementioned cells in the Sc area showed MAO activity. To summarize, the present study gives clear cut evidence that the main source for cerebellar NA is Lc, both dorsal and ventral part which innervate the entire cerebral cortex and hypothalamus respectively. The present study clearly demonstrate that pontine NA neurons such as Sc NA neurons also send their axons to the cerebellum. The fact that some neurons in the A 2 area were labeled by HRP indicates strong possibility that A 2 NA neurons also innervate the cerebellum, though direct evidence was lacking in this study. Besides the afferent system already well known, several unknown inputs to the cerebellum were first confirmed in this study. These neuron groups are as follows: (1) non-noradrenergic neurons in the Sc area, (2) nucleus reticularis gigantocellularis and nucleus reticularis parvocellularis, (3) group 1, (4) nucleus corporis pontobulbaris, (5) nucleus ambiguus, (6) nucleus nervi facialis. Since HERRICK suggested three hypotheses for cerebellar participation in movement (1924), several hypotheses have been postulated. These newly demonstrated afferent fibers to the cerebellum surely play an important role for functional localization of the cerebellum, though it is not clear at present. In addition to the above newly found inputs to the cerebellum, we confirmed the well known afferent systems. Some of these systems were also recently confirmed by E L L E R and CHAN-PALAY using the HRP method (1976). BRODAL defined Rtp, R1 and Rpm as precerebellar reticular nuclei (1957). The fact that these three nuclei were labeled by HRP following injections into the cerebellum supports his findings. Another major sources of inputs to the cerebellum postulated by many authors such as Gp, Oi and CI were also labeled. The afferents to the cerebellum that remain as other afferents including Tp, To, Ti, Tc, Ph, the raphe system and Lc were also labeled by HRP. However afferent system from the nucleus tractus mesencephalici n. trigemini and nucleus motorius n. trigemini to the cerebellum ( E L L E R and CHAN-PALAY, 1976) could not be confirmed in this study. References ADACHI, K . ,

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Demonstration of ascending projection from locus coeruleus by degeneration silver method. Exp. Brain Res. 20, 1 8 1 - 1 9 2 (1974). T A B E R , E.: The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat. J. comp. Neurol. 116, 27 — 69 (1961). T O H Y A M A , M.: Comparative anatomy of cerebellar catecholamine innervation from teleosts to mammals. J. Hirnforsch. 17, 4 3 - 6 0 (1976).

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