Biology of the Ceil. Volume 63, 1988

COLOCALISATION DU GABA ET DE LA DOPAMINE DANS LES SYSTEMES NEURONAUX TUBERO-HYPOPHYSAIRES ET TUBER0-1NFUNDIBULAIRES. D~monstration par double

m~rauaqe par l'or collo~dal dans plusieurs esp@ces. S. SCHIMCHOWITSCH, P. VUILLEZ. M.3. KLEIN et M.E. STOECKEL. Laboratoire de Physiolosie, URA CNRS 30g. 21 rue Ren~

Descartes 6W000 Strasbourg FRANCE. D0abondantes prolections GABAeruiques et dopaminergiques sont d@crites dana Is

zone externe de l'@minence m@diane (EM) et dana le lobe neuro-interm~diaire (LNI) de i'hvpophyse. Le lobe interm@diaire (LI) des L~porid@s. d@pourvu des deux types d'innervation, constitue la seule exception connue chez fez Mammif~res. Lee obser- vations inuRunocvtochimiques sur coupes semi-fines s@ri@es montrant une distribution similaire des deux types de pro~ections dane I'EM et dens le LNI chez la Grenouille et chez plusieurs esD~ces de Mammif~res (Rat, Souris. Cobave, Chat, Lapin) posent le probl~me de la colocalisation du GABA et de la dopamine dana ces axones.

Une m@thode de double maruuage "recto verso" par lea anticorps anti-GABA et anti- tvrosine hvdroxylase (TH, enzyme de s.vnth@se de la dopamine), mis en @vidence par des anticorps secondaires coupl@s • des particules d'or colloidal de 25 et de 5 nm, a ~t@ appliqu~e sur des coupes fines de tissus fixes par un m@lange paraformald~hyde- qlutarald~hvde (1:1) et inclus dans le Lowicrvl.

Comme nous l'avions d@jA montr@ chez le rat*. la colocalisation des zmmunor~activit~s GABA et TN s'observe svst~matiquement dane lee axones innervant le LI. saul chez le. Lapin. Dans la zone externe de I~EM chez routes lee esp~ces ~tudi~es. l'immunor~activit@ GABA eat d~tect@e dane tous lee axones marauds par l'anticorps anti-TH slots ~u'un certain nombre d'axones r@agissant avec l'anticorDs anti-GABA ne sont pas immunor~actifs pour la TH. La Dr@sence de GABA dane lee fibres dopaminergiques du svst@me hypothalamo-hypophysaire paraTt @tre un ph@nom@ne constant dont la siunification fonctionnelle reste & Dr~ciser. " VUILLEZ P., CARBA30 PEREZ S. and STOECKEL M.E. Neurosci. Lett. (1987) 79. 53-58.

THE RETROGRADE TRANSPORT OF MONOCLONAL ANTIBODIES RAISED AGAINST VASOPRESSIN INJECTED INTO THE RAT NEUROHYPOPHYSIS. AN ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL STUDY. Arlette BURLET, BriEitte FERNETTE Jean-Pierre NICOLAS and Claude BURLET. LaboratoiredeBioloEieCellulaire, IR3ERMU308, 38rueLiomois, F-54000N/~

Our previous studies have demonstrated that monoclonal antibodies (MAbs) raised against vasopressin (VP) injected into the paraventricular nucleus of the rat, penetrated into the VP neurons and were carried to the fibres of the median eminence. These results could be explained by the expres- sion of the VP precursor at the level of the plasma membrane of the neuron. To confort this hypothesis, MAbs-VP were injected into the neurohypophysis of normal and dehydrated Lon E Evans (LE) rats, and in normal and DDAVP-treated Brattleboro (di/di) rats. All the animals were killed 18h after the MAbs in- Jection. Fixed thick sections of brain were stained directly for the immunoenzymatic detection of in- Jected MAbs. After araldite embedding, the ultra-thin sections were simultaneously treated for the in- tensification of the DAB reaction and for the detection of endogenous neurohypophyslal peptldes (VP or oxytocln, OT) by the gold labelled IEG.

The VP-MAbs are transported retrogradely and are stained in the median eminence and magno- cellular neurons of LE rats. These labelled neurons are found in the ante- and retro-chlasmatic subdi- visions of the supraoptic nuclei, in the paraventrlcular nuclei, in individual magnocellular neurons and in most of the accessory groups of the anterior hypothalamus, the suprachiasmatic nuclei being devoid of staining. At the electron microscope level, the injected VP-MAbs are stained in dense vesi- cles of various size. These vesicles are very numerous in the neuron cytoplasm but also in the Intra- nucleus dendrites and proximal axons. Most of marked neurons contain specific gold labelling of VP neu- rons. In MAbs labelled processes, the endogenous VP is identified both in non-labelled vesicles and in labelled vesicles, In the median eminence of the LE rat, the injected MAbs are located in neurotu- bules and in dense vesicles. The co-existence of endogenous VP can be detected in the marked fibres. In di/di rat, VP-MAbs load the basement membrane of the nerve fibres and there are neither dense vesi- cle nor ueur,~tubt, le labellings.

These data show that the retrograde transport of VP-MAbs occurs mainly in the VP neurons.

REGIONAL DISTRIBUTION OF ASPARTATE-IMMUNOREACTIVE STRUCTURES IN THE"INTRAEPIDERMIC NERVOUS SYSTEM OF SAGITTA SETOSA. Michel DUVERT (1), Ghlslaine CAMPISTRON (2) and Brigltte ONTENIENTE (3). (1) Laboratoire de Cytologie and (3) de Physiologie des Interactions Cellulaires, UA CNRS 339, Av. des Facultds, 33405 Talence cedex, (2) Laboratoire de Neuroimmunologie, IBCN CNRS, 1 rue Camille Saint-SaUna, 33077 Bordeaux cedex, France,

In Sagitta setosa, the locomotor muscle is made of four quadrants of alternating A and B fibers which are under cholinergic control. The nervous system is an intraepidermic network without any topographical correlation with the architecture of the underlying muscle.

Various neuromediators were assayed by pharmacological (*) and cytochemical techniques in order to determine their putative implication in the modulation of the contraction, We report here the first results obtained with a anti-aspartate antiserum.

Sagitta setosa were fixed in 5% glutaraldehyde in sodium cacodylate buffer 0,1M, pH 7.4 and incubated with anti-aspartate serum (1:3000) at 4°C for 12 h. The reaction was revealed by an indirect anti-peroxidase technique using peroxidase-labeled Fab fragments. Controls were performed with an anti-aspartate serum previously adsorbed with an excess of immunogen.

An intense labeling is observed in discrete areas of the peripheral nervous system : i) in the ventral ganglion ; ii) within the nervous plexus localized between the head and the ganglion ; iii) in the posterior lateral nerve trunks and the associated nerve plexuses. These plexuses extend on both sides of the nerve trunks over about a Quarter of each muscular quadrant. The immunoreactive fibers stop abruptly, clearly delineating the aspartate- positive part of the nervous intraepidermic network.

Our observations point out two pecularities of the neuromuscular system of Sagitta setosa : i) there is no topographical correlation between nerve endings and the architecture of the locomotor muscle ; ii) neuromediators have a discrete regional distribution within the nervous plexus.

(°) Duvert M. and Savineau J.-P., (1986) Tissue and Cell, 18:937-952

22a