Partial Glycinergic Denervation Induces Transient Changes in the Distribution of a Glycine Receptor-Associated Protein in a Central Neuron

The Journal of Neuroscience, January 1992, 72(l): 116-131

Partial Glycinergic Denervation Induces Transient Changes in the Distribution of a Glycine Receptor-associated Protein in a Central Neuron

Tania Seitanidou, Marie-Anne Nicola, Antoine Triller, and Henri Korn Laboratoire Neurobiologie Cellulaire, INSERM U261, Dkpartement des Biotechnologies, lnstitut Pasteur, 75724 Paris Cedex 15, France

The effect of partial glycinergic denervation on the cellular had no effect on either the distribution of the surface redistribution of the 93 kDa peripheral polypeptide associated ceptor clusters, or the 93 kDa peripheral protein linked to with the glycine receptor was studied at the level of the these receptors. teleost Mauthner cell, an identified neuron of the goldfish Taken together, our results suggest that the ultrastructural brain (Carassius auratus). Previous studies using monoclo- distribution of the glycine receptor complex is regulated by nal antibodies raised against purified glycine receptors and “trophic” factors rather than by transmitter-evoked synaptic immunoperoxidase staining have shown that these proteins activity. are localized in clusters on the entire surface of this neuron. Specifically, the 93 kDa polypeptide was situated only on At most chemical synapses,cytoplasmic proteins are associated the cytoplasmic side of the postsynaptic membrane facing with receptor molecules. The best-documentedcase is that of active zones. the 43 kDa protein that is linked to the nicotinic receptor at the Unilateral electrolytic lesions of the vestibular complex neuromuscularjunction (Froehner et al., 1981; St.John et al., caused the degeneration of some glycinergic afferents to 1982; Sealock et al., 1984; Bridgman et al., 1987). It has been this neuron. When the first signs of this response appeared, recently confirmed that this protein induces the clustering of 3 d after the surgery, there was also a change in the ultra- ACh receptorswhen coexpressedwith it in oocytes (Froehner structural distribution of the 93 kDa polypeptide in the deaf- et al., 1990) or in transfected fibroblast cell lines (Phillips et ferented cell. The synaptic protein apposed to degenerating al., 1991; seealso Cartaud et al., 1981; Rousseletet al., 1982; axons did not spread onto adjacent extrasynaptic mem- Burden et al., 1983;Peng and Froehner, 1985;Bloch and Froeh- branes, and it disappeared a few hours after the disruption ner, 1987), implying that its function doesnot require the pres- of its presynaptic element. At the same time, a cytoplasmic enceof nerve inputs per se.However, despiteextensive studies immunoreactivity appeared as randomly distributed clusters of the effects of denervation on peripheral nicotinic receptor in the deafferented Mauthner cell; these aggregates, not (Jacob and Berg, 1987, 1988; Sargent and Pang, 1988; for re- seen in control preparations, were never found inside mem- views, seealso Fambrough, 1979; Schuetze and Role, 1987), brane-bound organelles. In some preparations these clus- nothing is known about the possibleinfluence of nerve injury ters were localized along arrays at a relatively constant dis- on the cellular distribution of the 43 kDa-associated protein. tance from the plasma membrane. The intracellular Such is also the casefor anchoring proteins in the CNS. immunoreaction product was found in the soma and the ini- Until recently, morphological data following selective dener- tial part of the dendrites, gradually decreasing in number vations have beendifficult to obtain in the vertebrate brain, for and intensity toward the extremities of these processes. At two reasons.One is that central pathways are intermingled, and later postoperative stages, 10-l 5 d after surgery, the 93 few of them can be severedin isolation. The secondis the lack kDa immunoreactivity remained only at postsynaptic mem- of specific antibodies, which are required for detailed immu- branes facing intact terminals. Similar alterations following nocytochemical studies and ultrastructural analysis of central denervation were observed in reticular neurons, at the level synapses.An exception is the Mauthner (M)-cell inhibitory net- at which degenerating presynaptic terminals were also de- work, part of which belongs to the vestibulo-vestibular com- tected. missuralpathway (Zottoli and Faber, 1980; Triller and Korn, In contrast, continuous 3-d blockade of synaptic transmis- 198l), that has beencharacterized electrophysiologically (Faber sion by strychnine, an antagonist of the glycine receptor, and Kom, 1973; Kom and Faber, 1976). Its terminals are glycinergic (Faber and Kom, 1980, 1988), and their effect on the M-cell is blocked by strychnine (Faber and Kom, 1988). Received Apr. 24, 1991; revised June 25, 1991; accepted Aug. 14, 1991. The glycine receptor (GlyR) is a ligand-gatedion channelthat We thank H. Betz and his colleagues (Zentrum fur Molekulare Biologie, Hei- mediatesCl-dependent inhibitory currents in the CNS (Kom et delberg) for providing the monoclonal antibodies, and P. Caramelle for technical assistance and photography. T.S. is the recipient of CEE Fellowship Contract al., 1990). It is antagonized by strychnine (reviewed in Kom SC1000304. This work was supported by a grant from DRET (No. 90.069). and Faber, 1990). This property has been used to purify the Correspondence should be addressed to Antoine Triller, Laboratoire Neuro- receptor complex from the mammalianspinal cord and to dem- biologie Cellulaire, INSERM U261, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France. onstrate that the protein contains three polypeptides of 48, 58, Copyright 0 1992 Society for Neuroscience 0270-6474/92/120116-16%05.00/O and 93 kDa. The first two are transmembrane polypeptides The Journal of Neuroscience, January 1992, L?(l) 117

(reviewed in Betz and Becker. 1988).,, whereas the 93 kDa subunit tronhvsioloaical recordinns in order to determine if in those conditions is a nonglycosylated peripheral membrane protein (Schmitt et the antagonist had reduced or suppressed the M-cell responses to syn- aptically released glycine. Three of them were investigated 5-6 hr after al., 1987). Among the monoclonal antibodies (mAbs) raised a unique dose, the remaining 4 (two of which were recorded on both against the purified rat GlyR, the 4a mAb specifically binds to M-cells) were subjected to a chronic treatment for 3 d before physio- the 48 kDa or o-subunit, while the 5a and 7a ones bind to the logical analysis. 93 kDa protein (Pfeiffer et al., 1984). Electrophysiological recordings. Fish treated with strychnine as de- The 48 kDa subunit, which bears the antagonistic binding scribed above were anesthetized with MS222 and immobilized with site (Pfeiffer et al., 1984), and the 93 kDa subunit have been Flaxedil(1 &m body weight). The preparation and basic physiological techniques were similar to those employed before (Kom and Faber, detected at restricted sites facing presynaptic active zones in the 1976). The M-cell was identified on the basis of its stereotyped response adult brains of the rat (Triller et al., 1985; Altschuler et al., to antidromic stimulations of its axon in the spinal cord (Furshpan and 1986; Van den Pol and Gores, 1988) and goldfish (Triller et al., Furukawa, 1962) and extra- or intracellular activities were monitored 1986; Seitanidou et al., 1988, 199 l)..Furthermore, Western blot with low-resistance (2-5 MQ) microelectrodes. The latter were filled with K-acetate or with 3 M KC1 in order to maximize and therefore analysis has shown that the antibodies recognize only antigens detect possible inhibitory postsynaptic potentials (IPSPs) since in this of similar molecular weight in the rat and goldfish brain and neuron resting membranes at Cl- equilibrium potentials are close to that they are colocalized regionally with strychnine-binding sites each other (Furukawa and Furshnan. 1963). At the end of some re- (Becker-et al.. 199 1). - cording sessions, the resting membrane conductance was measured on ’ Taking advantage of these probes, we have investigated the current traces evoked by d& and hyperpolarizing command pulses and obtained as previously (Faber and Kom, 1986) with single-electrode role of innervation on the ultrastructural distribution of the voltage-clamp recordings (Axon Instruments) with chopping rates in M-cell postsynaptic 93 kDa polypeptide. Our results indicate the range of 16-33 Hz. that even a mild denervation results in striking alterations of Immunocytochemical techniques. Animals were anesthetized as above this protein’s cytoplasmic localization that are different from and injected intramuscularly with 0.5 ml Flaxedil. For electron mi- those reported in the neuromuscular junction. In contrast, a croscopy, they were perfused through the heart with a mixture of 4% chronic and massive block of synaptic activity has no detectable paraformaldehyde (PFA) and 0.1% glutaraldehyde in 120 mM phosphate buffer (PB), pH 7.4, for 20 min, followed by 4% PFA alone. The brains influence on this protein and the associated glycine receptors. were removed, postlixed overnight in 4% PFA in PB, and sectioned Parts of these results, which do not support the concept that with a Vibratome. The slices (80 pm) were reacted with 0.1 mM lysine synaptic transmission controls the stability of junctional recep- for 30 min and were incubated overnight at room temperature in the tors, have been presented briefly in a previous communication 7a mAb diluted 1: 100 in phosphate-buffered saline (PBS). This antibody recognizes different antigenic sites on the 93 kDa polypeptide (Pfeiffer (Seitanidou et al., 1990). et al., 1984; Triller et al., 1985). The binding sites were detected as described elsewhere (Seitanidou et al.. 1988) usinn the ABC (Vectastain kit) method (Hsu et al., 198 1). The ‘avidin-biotm-HRP complex was Materials and Methods revealed after addition of 0.03% diaminobenzidine tetrachloride (DAB) Our experiments were based on the notion that glycinergic commissural in 0.05 M Tris. uH 7.4. and H,O,. The formation of the HRP dark interneurons innervate both Mauthner cells in the goldfish brainstem reaction product- was controlled- under light microscopic observation. (Triller and Kom, 198 1). The tissue was then postfixed in buffered 2% OsO,, dehydrated in graded Electrolytic lesions. Adult goldfish (Carassius auratus) 13-l 5 cm in ethanol, and flat-embedded in Araldite. Sections with a silver interfer- body length were used. They were anesthetized with 0.30% 3-aminoben- ence color were counterstained with lead citrate and viewed with a zoic acid ethyl ester (Sigma). When gill movement ceased, they were Philips EM 10 electron microscope. transferred to a surgical chamber where 0.20% of the same anesthetic For light microscopy, the fish were only perfused with 4% PFA in was recirculated through the mouth and over the gills during surgery. 120 mM PB, pH 7.4, for 15 min. After dissection, the brains were Once a fish was immobilized, the cerebellum and the anterior part of immersed in 40% sucrose in PBS overnight. Thick 60 pm sections were the vagal lobe were exposed. The cerebellum was pushed forward and collected in 0.25% ammonium chloride in PBS, to block free aldehyde the dura mater over the fourth ventricle was removed. During this groups. After washing in PBS, the slices were put through a series of procedure, both otic capsules remained intact but the semicircular canals ethanol solutions at 25%, 40%, 50%, 40%, and 25% in order to increase of the lesioned site were often disrupted. An insulated tungsten electrode immunoreactivity. The tissue was allowed to react overnight with the (tip diameter, < 1 rm) was then introduced in the medulla, at the most 4a mAb that snecificallv recoanizes the 48 kDa DolvDeDtide of the GlvR anterior part of the right cerebellar crest. A positive current of 0.005- (Pfeiffer et al.; 1984). The amibody was diluted in-PBS (1:250) in the 0.05 mA was applied for 15-30 set, with the expectation that lesions presence of 0.12% gelatin and 0.25% Triton X-100. After extensive produced by similar anodal intensities are reproducible in size and shape rinsing with PBS, the sections were successively incubated with a bio- (Moore, 1981). The electrode was removed and the cerebellum was tinylated horse anti-mouse IgG and biotin-streptavidine-fluorescein returned to its original position. A piece of parafilm slightly larger than complex (Amersham), diluted in PBS (1:200), for 2 hr. The slices were the size of the hole was placed on the skull, sealed around its edge with mounted with Mowiol (Hoechst, Frankfurt) and examined with a con- acrylic glue, and covered with dental cement. The fish was then respired focal scanning laser microscope (CSLM; Phoibos 1000, Molecular Dy- continuously with tap water for 15 min and returned to the aquarium. namics, Sunnyvale, CA, USA). We obtained high-resolution digitized After variable survival times, the location of the lesion and its extent images, with a pixel size of 0.15 pm, by using a 100x objective (NA were verified in transverse thick sections (80 pm) embedded in araldite 1.3). The visualization of large surfaces of cell membrane was obtained (see below). by look-through projections (Carlsson and Aslund, 1987) of consecutive As summarized in Table 1, brains from 31 lesioned fish, 10 intact digital sections. ones, and 5 sham-operated control animals were processed for electron Morphometrical data. The ratio of degenerating somatic fibers to the microscopy. The ultrastructural distribution of the 93 kDa protein was total number of presynaptic afferents was measured on random ultrathin analyzed after different postoperative intervals of 2-15 d, as listed in sections from three experimental animals, 3 d after lesion, using the Table 1. M-cell’s axon cap as a landmark for this neuron’s soma. Endings still Chronic block ofglycine receptors by strychnine. As in previous studies in contact with the M-cell membrane, or within a maximum distance (Faber and Kom, 1982, 1988), glycinergic receptors were functionally of 3 pm of it, were counted in order to guarantee that the profiles were blocked by injecting intramuscularly 5 &gm body weight of strychnine indeed presynaptic to this cell. sulfate diluted in 0.9% saline solution. The animals received a dose of The proportion of intact small-vesicle boutons (SVBs) apposed to 93 strychnine every 6 hr for l-3 d. A total of 24 adult goldfish were treated kDa-immunoreactive postsynaptic densities versus the total number of in this manner: 7 of them were used to visualize the localization of the contacts was calculated from surface sections ofthree deafferented M-cells, 93 kDa polypeptide with electron microscopy, and 8 were used to an- after a survival time of 3-5 d, and on four animals injected with strych- alyze the a-subunit distribution. The remaining 9 were used for elec- nine for 3 d. 118 Seitanidou et al. l Induced Changes in Glycine Receptor Distribution

Table 1. Presynaptic degeneration pattern and distribution of the 93 kDa polypeptide in control and in deafferented M-cell

Num- Degenerating ber of profiles 93kd-IR fish Plasma membrane (46 Survival Lesion local- Intra- total) time ization in VN s NC cellular N D F 2 2d + - - - + - - 3 3d + + + + + + + 4 3d + - + + + - - 6 4d + - + + + - - 5 Sd + - + + + - - 3 6d + - + + + - - 2 Id + - - + + - - 3 10d + - - - + - - 3 15d + - - - + - - 2 3d sham-operated - - - + - - 3 4d sham-operated - - - + - - 10 not operated - - - + - - A total of 46 animals were used: 3 1 with unilaterally lesioned vestibular nuclei, 5 sham-operated, and 10 unoperated fish were analyzed. In these selected animals, the lesion was precisely localized in the vestibular nuclei at the level of the M-cell. A representative example of such lesion is illustrated in Figure 1A. Two categories of degenerating profiles were observed: endings synapsing (S) with the M-cell, and fibers not in contact (NC) with it. These axons were in the M-cell’s synaptic bed, at a distance less than 10 Mm from the plasma membrane. The 93kd-IR was intracellular or associated with plasmalemma. In the latter category, the 93kd-IR was apposed to normal (N) or degenerating (D) endings, or facing free (F) extracellular space.

Electron microscopy sections obtained in the same conditions were the rostrocaudaland 450 pm in the horizontal and vertical axes. used to estimate the length of the postsynaptic membrane labeling. The The central cavity, where the tissue was completely destroyed, extent of the HRP reaction product was directly measured from micro- wasno more than 200 hrn in diameter (in this and other prep- graphs (42,000 x). The surface area of the GlyR clusters labeled with the 4a mAb was quantified on confocal micrographs. All these mea- arations as well). Damagedtissue, encircled by a narrow rim of surements were carried out using a digitizing pad connected to a mi- gliosis, was present 360 pm posterior to the M-cell’s lateral crocomputer. dendrite, disappearingat the level of the tip of its ventral dendrite. In all material selectedfor the present study, the lesion was located in the vestibular complex, either just above the Results secondarygustatory and descendingtrigeminal tracts (Fig. MB) Behavioral changesin operatedanimals or, more medially, between the ventricle and the outer limit of Immediately after recovery from anesthesia,all fish with uni- the brain (not shown). laterally damagedvestibular nuclei were unable to swim along As schematizedin Figure 1B, the damagedarea was situated a straight line. They swam with disordered movements in an at the entrance of the eighth nerve above the descendingtri- arc bent toward the lesion. When still, they usually remained geminal tract and close to the lateral dendrite of the M-cell. on the affected side,near the bottom of the tank. Two days after Thus, it is possiblethat in some experiments, the current had surgery, they exhibited normal posture and equilibrium at rest, reachedthe extremity of this process,which penetratesinto the and they beganto swim normally at 4 d. Finally, their swimming vestibular nuclei in this region. In order to ensure that any and resting behavior had completely recovered by the tenth morphological changesfollowing surgery were due to causes postoperative day. In contrast, the startle reflex provoked by other than (1) the degenerationof the M-cell itself or (2) the tapping on the aquarium was always elicited, even immediately disappearenceof the eighth nerve endings, one of the major after the operation. excitatory inputs of the ipsilateral M-cell, we have restricted Sham-operatedcontrol fish had normal swimming responses our study to the M-cell contralateral to the lesion. However, in even though they remained immobile at the bottom of the three preparations with intact ipsilateral M-cells, identical re- aquarium during the first postoperative days. sults were obtained from eachcommand neuron, on either side of the brain. Unilateral lesion of vestibular complex and Three days after the operation, there was an anterogradede- degenerationpattern generation of approximately 10% of the presynaptic afferents at In every operated animal selectedfor this study (Table l), the the level of the M-cell soma. As depicted in Figure lC, this lesion was restricted to the vestibular complex and affected all phenomenon concerned crossedsecond-order neurons whose four vestibular nuclei. Its maximum dimensionsnever exceeded axons project ipsi- and contralaterally, sincecommissural neu- 800 pm in the longitudinal and 600 pm in the dorsoventral and rons project bilaterally onto the soma and within the axon cap lateromedial axes. of both M-cells, to the reticular formation and to the contra- A representative photomicrograph of a transverse thick sec- lateral vestibular complex. tion at the level of the lesion obtained 3 d after surgery is shown The morphology of the investigated M-cell was compared in Figure 1A. Its shapewas ovoid, extending up to 640 pm in with that of the unoperatedside within thoseof control animals, The Journal of Neuroscience, January 1992, 12(l) 119

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Figure 1. Extent of electrolytic lesions. A, Photomicrograph of a frontal thick (80 pm) section along the medial line of the brain, at the level of the vestibular nuclei, showing the lesion (broken line) produced by a cathodal current. Its boundaries are designated by a marked scar (arrows). Scale bar, 500 pm. II, Transverse section through the same lesion (stippled area), indicating its relation to adjacent structures at the level of the Mauthner cell. C, Drawing of commissural glycinergic vestibular neurons synapsing on both M-cells. Vestibular complexes ipsi- [N. Vest (ipsi)] and contralateral [N. Vest (contra)] to the lesion are surrounded by dotted circles. Note that efferent processes (solid lines) terminating on both M-cells [M cell (ipsi, contra)] and in the reticular nuclei (N. Ret) can be affected by a lesion of the vestibular nucleus. Axons issued by vestibular neurons contralateral to the lesion (broken lines) are also shown. AC, axon cap; CB, cerebellum; DT, descending trigeminal tract; EG, eminentia granularis; MC, Mauthner cell; MLF, medial longitudinal fasciculus; RN, reticular nuclei; TGS, secondary gustatory tract; VW, fourth ventricle. 120 Seitanidou et al. l Induced Changes in Glycine Receptor Distribution

Figure 2. Distribution of degenerating profiles on the contralateral M-cell following short postoperative intervals. A, Somatic membrane of M-cell from unoperated animals with its typical investment by afferent terminals. Note the labeled postsynaptic differentiations (arrows) of two synaptic contacts. B, Diagram of the distribution of degenerating profiles (shaded area) at the peripheral part of the axon cap, on the soma (S) and on small ventral dendrites (SW). C, Electron micrographs of the M-cell axon cap, 3 d after a unilateral lesion of the vestibular complex. The peripheral part (PP) of the axon cap is occupied by necrotic debris of fibers (single arrowheads), some of which are surrounded by a lamina of glia (double The Journal of Neuroscience, January 1992, 12(l) 121 on semithin sections. The shape, size, position, and orientation cisternae. For a very brief period of time, labeled membrane of its dendrites and axon remained unaltered after denervation. patches were left deprived of any presynaptic element, apposed The cell nucleus was always located centrally and somewhat to glial processes or to necrotic debris. Randomly distributed dorsomedially, close to the axon hillock. The nuclear outline glial processes, next to intact terminals, which themselves were was smooth and slightly infolded with no sign of chromatolysis apposed to a 93kd-IR site, were directly in contact with the (referenced in Zottoli et al., 1984). At the EM level, the peri- M-cell somatic membrane over long distances, a feature never karyal and dendritic cytoplasm appeared normal, containing observed in nonoperated animals. their usual supply of organelles. Intracellular 93kd- IR Ultrastructural distribution of degenerated axons and fate of A striking modification of the 93 kDa protein’s expression took synaptic GlyR-associated 93 kDa protein on the M-cell place in the cytoplasm of the denervated M-cell (Fig. 3A; see Previously, in nonoperated fish, the 93 kDa immunoreactivity also Table 1) under the form of an immunoreactivity on the (93kd-IR) was only detected on synaptic membranes, in ap- third day that persisted up to the seventh postoperative day. position to presynaptic release sites (Seitanidou et al., 1988). This staining was concentrated at discrete loci, where it had a The immunoreactive product was contiguous to SVBs at the circular or oval shape. The cytoplasmic electron-dense precip- soma (Fig. 2A) and dendrites, and to almost all unmyelinated itate was never observed inside membrane-limited structures club endings (UCEs) in the peripheral part of the axon cap. such as mitochondria, Golgi and Golgi vesicles, rough and In control animals (n = 4), 38.5 f 3.1% of the somatic ter- smooth reticulum, or lysosomes. Rather, as revealed by high minals presynaptic to the M-cell were apposed to 93 kDa-stained magnification (Fig. 30, the oxidized DAB at the periphery of postsynaptic densities. This number decreased 3 d after lesion the cluster appeared to be related to the cytoskeletal domain (n = 3), when only 3 1.1 f 3.1% of intact endings were in front and had the form of small dots. Cistemae of agranular endo- of immunolabeled membrane. The afferent inputs on the two plasmic reticulum or small vesicles found at the border of the main dendrites remained unaffected. immunoreaction product were stained along their entire perim- As shown in Table 1, degenerating profiles were either in eter or just a part of it. In this case, the reaction product was direct contact with their target, or slightly detached from the associated only with the cytoplasmic surface of their membrane. plasmalemma of the postsynaptic cell. After the third postop- Most often clusters of the intracellular 93 kDa protein were erative day, the profiles were rarely still attached to the M-cell. randomly distributed in the cytoplasm of the M-cell, in front However, scarce degenerating fibers could be observed in the of any type of presynaptic ending, at varying distances from the synaptic bed until 7 d after surgery. The distribution of degen- plasma membrane, including a juxtaposition (Fig. 30. How- erated terminals was analyzed at 3 dafter lesion. As schematized ever, in three preparations obtained 3 d or 4 d postoperatively, in Figure 2B, the deafferentation of the M-cell was restricted to they were aligned at l-5 pm from the somatic membrane (Fig. the soma, the small ventral dendrites, and the peripheral part 4). This ordered distribution was almost parallel to the plas- of the axon cap, whose morphology appeared otherwise normal. malemma for at least one-third of its perimeter. At this time, many dark axonal profiles surrounded by astro- In the principal dendrites of the M-cell, where degenerating cytic processes were found in the peripheral part of the axon fibers were not encountered, the cytoplasmic immunoreactivity cap (Fig. 2C), but never in its central core. The lesioned endings was nevertheless present (data not shown), although less pro- were occasionally in contact with cap dendrites present in this nounced than that at the somatic level. In the lateral and in the region. As shown in Figure 20, they were also present at the ventral dendrite of deafferented M-cells, the occurrence of pre- somatic level of the M-cell, either in direct contact with it, or cipitate diminished progressively with distance from the soma in its synaptic bed at some distance (2-5 pm) from the plas- and was absent at their extremities. In these processes, the cy- malemma. Dark terminals, still attached to the M-cell (Fig. 2D), toplasmic staining was dispersed within the cytoskeletal ele- were considered as an early sign of degeneration, which preceded ments and not associated with the membranes of the endo- complete enwrapment by glial processes. plasmic reticulum. To determine if the affected afferents were normally apposed The effect of presynaptic deprivation was also studied 1O-l 5 to the 93 kDa protein, tissues were immunolabeled with the 7a d after surgery. During this late postoperative interval, degen- mA6. Again, two distinct types of degenerating profiles were erating terminals were never present close to the M-cell. The observed in the synaptic bed: (1) electron-dense boutons, at- presynaptic bed was comparable to that of the control animals. tached to the M-cell and engulfed by glial cell processes (Fig. Nevertheless, terminal-free regions were apparent, with afferent 2D), their corresponding postsynaptic membrane being often fibers spaced farther apart in the extracellular space. The only immunolabeled (Fig. 20 inset); the staining in front of the de- antigenic determinant recognized by the 7a mAb during this generating or intact presynaptic endings never outflanked the period was that of membranes postsynaptic to afferent processes extrasynaptic membranes; and (2) dark fibers (Fig. 20) in the filled with pleiomorphic vesicles. synaptic bed, disconnected from the M-cell and completely en- As already described, the cytoplasmic 93kd-IR was almost sheathed by a lamina of reactive astrocytic cells with numerous exclusively localized on nonmembranous structures. However

c arrowheads). In contrast, the central core is unaffected. D, Degenerating axonal profiles (arrowheads) in the synapticbed of andapposed to (arrows) the M-cell soma.Same survival time as in C. Note stainedglycine receptors (crossed arrow) in front of a terminal bouton undergoingdark degeneration,still in contact with the M-cell. Inset, Highermagnification. A, axon; CC, centralcore; DL, lateraldendrite; DV, ventral dendrite; n, nucleus.Scale bars: A, C, and D, 2 pm; inset, 500 nm. 122 Seitanidou et al. l Induced Changes in Glycine Receptor Distribution

Figure 3. Redistribution of the 93kd-IR, detected by the 7a mAb after partial glycinergic denervation of the M-cell soma. A, Appearance of intracellular immunoreactivity at early postoperative intervals. The reaction product (triangles) is in the cytoplasm of the M-cell, 3 d after surgery. Note the membane labeling (single arrows) in apposition to some presynaptic fibers and the lack of immunoreactivity of the Golgi apparatus (double arrow). B, Higher magnification of the intracellular staining, illustrating that the internal immunoreactivity has a globular shape. There is a dark reaction product at the border of the staining associated with cytoskeletal elements, ribosomes (triangles), and the smooth endoplasmic reticulum (arrowhead). C, Staining in proximity of the M-cell plasmalemma; the extracellular space is occupied by glial cells, which are directly apposed to the plasma membrane. Scale bars: A and B, 500 nm; C, 250 nm. in a few instances, it was found near the Golgi apparatus or The 93 kDa polypeptide was affected, as describedabove. At associated with the cytoplasmic side of peripheral vesicles of early stagesafter surgery, the immunoreactivity waspresent not the Golgi complex. only at synaptic membranesin front of intact endings,but also in the form of clusters,randomly distributed within the somatic Efect of denervation on the reticular neurons cytoplasm (Fig. 5) and the initial part of the dendrites. Layered Cells lying close to the medial and distal parts of the M-cell’s astrocytic processesoccupied the extracellular spaceand were ventral dendrite were examined since they are also targets of occasionally in contact with the noninnervated plasmalemma, the commissuralvestibular interneurons(Triller and Korn, 1981). interspersedbetween afferent terminals. Some neuronswere di- The Journal of Neuroscience, January 1992, 72(l) 123

Figure 4. Evidencethat the cytoplasmic darkreaction product (arrowheads) is locatedat a constantdistance from the deafferentedsomatic M-cell membrane,3 d afterlesion. Scale bar, 4 pm.

rectly in contact with electron-dense presynaptic endings, un- dergoing a degenerative process and facing immunolabeled post- Block of the GlyR complex by strychnine synaptic membranes. The evolution in this transient cytoplasmic Swimming behavior of animals injected with strychnine was staining was identical to that of the M-cell. severely affected. At approximately 3 min after the first injec- tion, the fish was stationary in a straight posture. The startle Extent of 93 kDa labeling on postsynaptic membranes responsedid not habituate and 10 responsesin seriescould be In order to establish if the length of the synaptic labeling was elicited at brief intervals by taps on the aquarium. After the modified after lesions, the extent of the corresponding immu- secondinjection, the animals were lying on one side, on the noprecipitate was compared from data collected in six control bottom of the aquarium. At this stage,the stimulus triggered a and six operated animals. As indicated by the histograms (Fig. strong contraction and a tremor of the muscles,but the animals 6A,,B,,C,), obtained from random EM sections of lesioned an- were unable to swim. imals, the mean length of labeled specialized membranes was 0.452 pm (SD = 0.149 pm; n = 75) on the cap dendrites, 0.76 1 Block of synaptic responses by strychnine pm (SD = 0.3 13 pm; n = 74) on the ventral and lateral dendrites, Intracellular recordingsdemonstrated that chronic injections of and 0.653 pm (SD = 0.265 pm; n = 104) on the soma. In strychnine significantly impeded the M-cell responsesto acti- unoperated animals (Fig. 6A,,B,,C,), the extent of the staining vation of the recurrent collateral network that otherwise re- was about the same. More specifically, it averaged 0.447 Frn mained essentiallyunaltered. The experimental design(Fig. 7A) (SD = 0.137 pm; n = 62), 0.728 Mm (SD = 0.229 pm; n = 130), and the basic principles usedto reach this conclusion were as and 0.656 pm (SD = 0.290 pm; n = 182) at the corresponding follows. regions of the M-cell. First, after ortho- or antidromic firing of the M-cell, the pre- In contrast, the length of patches linked to degenerating so- synaptic volleys in the terminals of collateral interneurons ap- matic terminals was reduced to 0.504 pm (SD = 0.157 Mm; n pear, in the axon cap, as a positive potential (Fig. 7B,), the so- = 25) (Fig. 60), that is, 77% that ofthe control. These data were called extrinsic hyperpolarizing potential or EHP (Fumkawa obtained from stained postsynaptic densities in apposition to and Furshpan, 1963). In normal conditions, this volley evokes electron-dense endings still attached to the M-cell, one of which the glycine-mediated postsynaptic inhibition of the M-cell (ref- is shown in Figure 6E. erenced in Faber and Kom, 1982) and both disappearat stim- 124 Seitanidou et al. l Induced Changes in Glycine Receptor Distribution

Figure 5. Partially denervated reticular neuron, at early (3 d) postoperative intervals: antigenicity to the 7a mAb at normal synaptic contacts (arrows) and in the cytoplasm (triangles). Scale bar, 1 pm. ulation rates of >4/sec (Fig. 7B,) due to a marked frequency and its underlying conductancechange were blocked by the drug, sensitivity of the synapsesintercalated along the collateral path- as shown by (1) the absenceof Cl--dependent voltage change way (Faber and Korn, 1978). Second, the peak postsynaptic after firing of the M-cell, in recordingsobtained with KC1 elec- inhibitory conductance change, G,,, (or G,,,, in the case of the trodes (not shown), and (2) estimatesof the parameter r, which inhibitory conductance underlying the collateral IPSP), can be was almost identical in a given cell, at slow (Fig. 7BJ and at determined by measuringits shunting effect on the height of a high (Fig. 7BJ frequenciesof antidromic stimulations, that is, testing antidromic spike. Briefly, treating the M-cell membrane when the reduction of the test height spike could be attributed as having a resting conductance G,, in parallel with the inhib- to refractoriness alone. Also, when the strength of the spinal itory channelsand in serieswith the driving force for chloride, stimuluswas increased, the test spikecould summatewith added led to the expressions EPSPs(Fig. 7&), a situation never encountered in the caseof compositeexcitatory and inhibitory responses(Korn and Faber, G(IPSP) = (r/l - r)G,, (1) 1975). with Pooled (n = 9) mean values of r in absenceor in presenceof collateral input were almost identical, 0.13 rt 0.07 (*SD, range, r=(V- V)/v, (2) 0.03-0.23) and 0.15 + 0.06 (*SD; range, 0.05-0.24), respec- where r is the fractional reduction of the spike, and V and V’ tively; the latter value indicates considerably lessreduction in are the amplitudes of the control and test spikes,respectively the test spikeamplitude, at the peak of the anticipated collateral (for derivations and justifications, see Faber and Korn, 1982). IPSP, than the N-70% in normal conditions, as shown, for In practical terms, Vand V” can be assessedfrom the onset to comparison,in Figure 7C,,C, (seealso Fukami et al., 1965;Korn the peak of the corresponding action potentials (Fig. 7B,,B,). and Faber, 1976). At all M-cells subjectedto strychnine injections for 6 hr (n = 3) or 3 d (n = 6), the waveform and amplitude of the EHP were Morphological observations normal (Fig. 7B,), indicating that the presynaptic volley reached The distribution of the a-subunit of the GlyR was examined the endings of the collateral neurons and that, as expected, it with CSLM, 2 d (n = 2 fish) or 3 d (n = 6) after the beginning vanished during high-frequency stimulation rates (Fig. 7BJ. In of strychnine treatment. The immunoreactivity pattern of the contrast, and although the antidromic spike evoked by threshold surfacereceptors (Fig. 8A,) did not differ from that of control spinal stimulation was normal (Fig. 7B,), the collateral IPSP (n = 10) animals (Fig. 8B,; seealso Triller et al., 1990). Fluo-

Figure 6. Morphometric data relative to the extent of the 93kd-IR on M-cell postsynaptic membrane, 3-5 d after surgery. A-C, Histograms of the length of the patches of 93 kDa polypeptide that were apposed to the indicated regions of the M-cell in operated (left) and in control (right) animals with corresponding means (m), SDS, and sample sizes (the first parameter was not significantly modified, as indicated by Student’s t tests). 0, Frequency distribution of the 93kd-IR size at synaptic contacts of degenerating endings in the somatic region. E, Electron micrograph showing a dark terminal bouton still in contact with the postsynaptic cell. Distance between bars, 0.45 pm. LENGTH OF 93 KD-IR APPOSED TO INTACT TERMINALS

DENERVATED A 2 CONTROL

axon-cap 16 m=0.452 k 0.149 m = O-447_+ 0.137 12: 12 n =75 n ~62 a- a

0 20 i L D 0 0.4 0.8 1.2 1.6 2P” D- 0.8 1.2 1.6 2 pm

dendrites m ~0,761 k 0.313 m= 0.728+ 0.229 n t-t=74 n=130 6 l-L

0 0.4 0.8 1.2 1.6 2 v-n ii8 c 2 25 40 20 soma 30 m=0.653 kO.265 m =0.656 ho.290 15 20 n ~182 10 5 10

0 0 ~ 0 0.4 0.8 1.2 1.6 2 ,prn 0 0.4 0.8 1.2 1.6 2 pm

LENGTH OF 93 KD-IR APPOSED TO DEGENERATING TERMINALS u 8- m z soma .-2 6- 2 m ~0.504 4 0.157 8 4- rT n =25

5 2- m.1 d o- .- = ,"'I' "I 0 0.4 0.8 1.2 1.6 2 w-n 126 Seitanidou et al. l Induced Changes in Glycine Receptor Distribution

5/set

msec ’

l/4 set 1 --- -- l/4 set S/set

r = 0.14 r= 0.13

: I;\ CTRL i/4 set 1 6 C i/4 set C 5/set n n n 2 A tl

Figure 7. Electrophysiologicalanalysis of strychnineeffects on glycinergictransmission. A, Diagramof the experimentalarrangement and the synapticrelations investigated, representing one classof intemeurons,the collateralneurons, which mediatefeedback inhibition of the M-cell (shadedarea). Responses were monitored inside (in) or outside(out) the neuron,that is, in the axon cap (broken line), whichwas located on the basisof its characteristicresponses to antidromicactivation of the M-axon (Stint. Ax.). B, Resultsobtained after 3 d of chronicinjection of strychnine.B, and BZ,Extracellular recordings of the M-cell spike(truncated, except in inset in BJ followedby a typical presynapticvolley (B,, arrow), whichdisappeared (BJ at increasedstimulation rates. B,-B,, Intracellularresponses to single(BJ andpaired (B,-B6) antidromicstimulations at the indicatedfrequencies. Note that the fractionalreduction of the secondspike height, r (see text) wasnearly the same,whether the inhibitory networkwas activated (B,) or not (B,), indicatingin both casesa lackof inhibitory responseof the M-cell. With strongerstimulus intensities, the test spikedid summatewith evokedEPSPs (B,, crossed arrow). C, Sameprotocol as above, in an untreated(control) preparation, with indicated modificationsof r dueto the collateralIPSP (C,) or to refractorinessalone (C,). Eachtrace in B and C is the averageof four sweeps;recordings wereobtained with K-acetate-filledelectrodes. rescentclusters separated by unlabeled membranepatches were tions (seeFig. 2A). Furthermore, the average diameter of the present in the entire M-cell plasmalemma.The high-resolution stained somatic contacts (mean +- SD = 0.639 f 0.232 pm; n CSLM pictures allowed us to demonstratethat the size of each = 105) remained unchanged. In contrast to the denervation individually labeled microdomain remainedunchanged. As il- effect of the distribution on the 93 kDa peripheral protein, in- lustrated in Figure 8A, and B,, the mean surfacearea of somatic tracellular antigenicity wasabsent in strychnine-treated M-cells. clusterswas 0.53 pm2 (SD = 0.41 pm*; n = 5 16) in strychnine- Finally, the distribution of the GlyR complex on reticular neu- injected animals and 0.51 pm* (SD = 0.43 pmZ; n = 970) in rons was comparableto that in the M-cell. controls. Thesemeans were not significantly different (p < 0.05, Student’s t test). Discussion At the ultrastructural level, the localization of the 93 kDa The major findings of this study, summarizedin Table 1, were protein at synaptic contacts persistedthe first (n = l), the second the following: (1) the short persistenceof the postsynaptic 93 (n = 2), and the third (n = 4) day of strychnine application (Fig. kDa protein at degeneratingterminals and its transient appear- 8C). The overall shapeand the number (39.3 + 1.71%; n = 4) ante as clustersin the cytoplasm of the deafferentedneurons, of labeled synapseswere identical to those of control prepara- and (2) the absenceof significant modifications of the GlyR and The Journal of Neuroscience, January 1992, 72(l) 127

A 2 B 2 100 I, GlyR4a mAb GlyR4a mAb strychnine control rn 80 I E m=0.53*0.41 pm* I m=0.51 kO.43 pm* g 60 0 n-516 n=970 6 40 M 6 = 20

” i, 0:5 i 1.5 2 2.5 3 I.5 1 1.5 2 2.5 3 Surface area, pm*

Figure 8. Unaltered cellular distribution of the GlyR complex on the M-cell, after chronic application of strychnine. A and B, Effect of blockade of glycinergic transmission on the 48 kDa polypeptide. A, and B,, Confocal fluorescent images of the a-subunit on the soma (arrows) or on dendrites (crossed arrows), in treated (A,) and in control (B,) animals. A2 and B2, Fre- quency histograms of the surface areas of stained clusters obtained from the M-cell somatic region. Note that the means were not significantly different, as confirmed by a Student’s t test. C, Electron micrograph of somatic synaptic contacts (arrows) stained by the 7a mAb, with normal expression of the 93 kDa protein. Note the absence of immunoprecipitate in the cytoplasm. MC, M-cell soma. Scale bars: A, and B,, 6 cm; C, 725 nm. 128 Seitanidou et al. l Induced Changes in Glycine Receptor Distribution

of the 93 kDa protein distribution after chronic block of synaptic that reinervation of preexisting labeled membrane patches due transmission. Thus, our data suggest that factors, other than to collateral sprouting of proximal axons takes place during the glycine, associated with the afferent nerve endings control the first 2 weeks following our lesions, which is the longest time expression of these synaptic molecules. period covered by this study. Glycinergic nature of the degenerating endings Comparison with the nicotinic receptor-associated 43 kDa Different types of degenerative effects may coexist, such as those protein described in the axotomized M-cell, where terminal profiles Several lines of evidence suggest that membrane proteins are display reactive deafferentation, but with no signs of degener- involved in the anchoring and/or the maintenance of the re- ation (Wood and Faber, 1986). However, immunolabeling of ceptor clusters (Bloch and Froehner, 1987; Schmitt et al., 1987; the 93 kDa protein requires a fixative containing a lower con- Kordeli et al., 1989) via interactions with the cytoskeleton. No centration of glutaraldehyde (0.1%) than the 2.5% used by those conclusive data are yet available on the role of innervation on authors. In order to avoid dubious interpretations of our data, the distribution of the 43 kDa protein. Accumulation and clus- we considered as degenerated fibers only those containing a dark tering of the 43 kDa protein can occur in the absence of afferent electron-dense material. nerve (Peng and Froehner, 1985; Bloch and Froehner, 1987; The unilateral destruction of the vestibular nuclei caused the Kordeli et al., 1989), and a diffuse cytoplasmic form has been degeneration of no more than 10% of the terminal boutons on detected in developing electrocytes (Kordeli et al., 1989). Other the soma of the M-cell. All the morphological criteria used to studies have shown that denervation causes a threefold increase classify the afferents of the M-cell (Nakajima, 1974) could not in the amount of the mRNA for the 43 kDa protein in a leg be applied to these altered terminals; yet, their location on the muscle of the adult mouse (Frail et al., 1989). M-cell already suggested that they belonged to UCEs in the axon cap, and SVBs when in contact with the soma, outside this Possible function of the cytoplasmic 93 kDa polypeptide region. Excitatory second-order vestibular neurons that cross The transient appearance of the 93kd-IR in the cytoplasm of the midline have been reported in other species (Graf and Ezure, the denervated cells may result from (1) a decrease in the rate 1986) but excitatory endings, containing gap junctions and of its transport to the plasma membrane, (2) an increased rate identified as “club endings” (Nakajima, 1974) were never ob- of its synthesis, or (3) an alteration in any postranslational mod- served on the contralateral deafferented M-cell. Furthermore, ification that would affect the expression of the epitope. It may the degenerating fibers were found in regions where the 93 kDa also be the consequence of the 93 kDa protein recycling from polypeptide is expressed, and where activation of the crossed the deafferented synapses. A number of other receptors such as vestibular pathway produces a strong chloride-dependent in- those of NGF or epidermal growth factor are known to be re- hibition that is mediated by glycine (Kom and Faber, 1976; cycled with their ligands (for review, see Greene and Shooter, Triller and Kom, 198 1; Faber and Kom, 1982). However, elec- 1980; Goldstein et al., 1985). However, internalization seems trolytic lesions could have caused also retrogade damage to unlikely in our experiments, since the only labeled vesicles ob- contralateral neurons, some branches of which innervate the served occasionally were close to the Golgi apparatus, that is, M-cells. The influence of terminals other than glycinergic ones far from the somatic membrane. on the distribution of the 93 kDa polypeptide during the early A different mechanism for internalization of the components stage after lesion is unlikely, although heterologous regulation of the postsynaptic site has been reported in the ventral cochlear of neurotransmitter receptors has been reported in the CNS. In nucleus of the rat (Gentschev and Sotelo, 1973), where entire the rat brain, serotonergic deprivation results in an increase of free postsynaptic sites were eliminated by engulfment into the a-adrenergic receptors (Rappaport et al., 1985) and non-do- cytoplasm. In the M-cell, unstained internalized postsynaptic paminergic fibers modulate a D, receptor denervation super- densities were never observed. Furthermore, in the labeled cy- sensitivity (Herve et al., 1989). toplasmic cluster of the 93 kDa protein, membrane remnants were never visualized and intracellular clusters were detected Fate of postsytiaptic sites and of synaptic 93kd-IR in the principal dendrites where degenerating profiles were not The 93kd-IR facing dark degenerating terminals remained be- found. hind the apposed synaptic contacts. Its average length was only The pattern of the cytoplasmic staining of the 93 kDa protein slightly diminished compared with the control animals, 3 d after described in this work completely differs from that of the nic- disruption of the crossed inhibitory input. We never observed otinic receptor in the chicken ciliary ganglia (Jacob et al., 1986; changes in the density and/or small fragmentations of the syn- Jacob and Berg, 1987) and of the GABA receptor in the rat aptic 93 kDa clusters, although low quantities ofthis polypeptide cerebellum (Somogyi et al., 1989). In these studies, the intra- at extrasynaptic loci may have remained undetected with our cellular immunoreactivity was only associated with membrane- immunohistochemical technique. bound organelles like the endoplasmic reticulum or the Golgi A persistence of postsynaptic specializations has been re- apparatus, or the nuclear envelope. This difference was expected ported after primary deafferentation in the ventral cochlear nu- since transmembrane subunits, like those of glycine, ACh, and cleus of the rat (Gentschev and Sotelo, 1973), and after sec- GABA receptors, are thought to be synthesized and modified tioning the preganglionic fibers in frog sympathetic ganglia in the rough endoplasmic reticulum and sorted out in the Golgi (Sotelo, 1968). In our material, unoccupied stained postsynaptic apparatus (Rothman and Fine, 1980). Conversely, the 93 kDa densities that persisted only during the early phase of the de- polypeptide is a nonglycosylated peripheral membrane protein generative process seem to have shorter life spans. Reoccupation and should not follow the same biosynthetic or regulatory path- of vacant postsynaptic sites by a sliding process from adjacent way; it is most probably synthesized by free polyribosomes. The intact terminals (Gentschev and Sotelo, 1973) was never ob- staining of the Golgi by the 7a mAb might result from an early served at early or late postoperative intervals. Thus, it is unlikely association at this level of the cytoplasmic 93 kDa polypeptide The Journal of Neuroscience, January 1992, 72(l) 129 to the glycine receptor transmembrane subunits, before their The effectiveness ofthe chronic block of synaptic transmission translocation and their subsequent insertion into the plasma was ascertained using Equation 1 to estimate G,,.. With param- membrane. It is also possible that proximal vesicles and Golgi eters r obtained at slow stimulation rates, the mean collateral membranes, or even ribosomes, are stained, due to absorption conductance equaled 1.9 f 0.9 @Ls(n = 5) which is already 30% of translocated DAB precipitate during the enzymatic immu- of that in control conditions (Faber and Kom, 1982; Kom and noreaction. Faber, 1990). This estimate dropped to 0.4 + 0.35 $S (*SD; n Since intracellular clusters of 93kd-IR were not apparent in = 5; range, O-1.0 PS) when correcting r for refractoriness. Pre- the axon hillock or the M-cell axon, passive diffusion seems vious recordings of pairs of neurons and quanta1 analysis sup- unlikely to account for their transport in the dendrites. Rather, port the notion that the effect of strychnine was postsynaptic, their location at a fixed distance from the plasma membrane since in acute experiments, the drug reduced the quanta1 con- (Fig. 4) suggests the involvement of the cytoskeleton. An ex- ductance with little influence on the presynaptic releasing pa- ample of a similar mechanism of a nonvesicular transport of rameters n and p (Faber and Kom, 1988). Whatever its origin, molecules by the cytoskeleton was reported in cultured hippo- our data indicate that a larger fraction of glycinergic junctions campal neurons where newly synthetized mRNA is transported was involved by prolonged applications of strychnine than after to the dendrites (Davis et al., 1987). Such an active transport denervation. may contribute to selectively direct and segregate proteins at specialized regions, such as surface microdomains of central Absence of morphological effects of the “jiunctional” neurons. A subplasmalemmal synaptic molecule, like the 93 denervation kDa polypeptide, should be only localized at restricted areas of The specificity of strychnine’s action on the glycinergic post- the plasma membrane, associated with the appropriate receptor, synaptic sites has been recognized (Young and Snyder, 1973, and functionally match with its corresponding presynaptic el- 1974; Faber and Kom, 1988) and its effects on other receptor ement. Along this line, in multinucleated muscle fibers from systems occur at concentrations above its kd at the inhibitory mature animals, ACh receptors are preferentially transcribed GlyR by a factor of 100-50 x lo6 (Baron and Guth, 1987). and synthetized by a few nuclei at synaptic areas (Merlie and Thus, although there is a GlyR less sensitive to strychnine in Sanes, 1985; Fontaine et al., 1988). the spinal cord of cats (Ryall et al., 1972) in neonatal rats (Becker et al., 1988; Hoch et al., 1989), in primary cultures of Chronic block of synaptic responses fetal motoneurons (Hoch et al., 1989) and in medullary neurons It has long been shown that strychnine blocks the collateral IPSP (Lewis et al., 1989) it is unlikely that their presence accounts (Furukawa et al., 1963) a property extensively used to dem- for the failure of the antagonist to reproduce the morphological onstrate that, although GABA receptors are present on the M-cell effects of denervation, since the M-cell somatic IPSPs and in- (Diamond and Roper, 1973; Diamond et al., 1973; Faber and hibitory postsynaptic currents are completely abolished by low Korn, 1980) glycine is the major transmitter operant at the doses of strychnine (Faber and Kom, 1988). Furthermore, the somatic level (Mazliah and Werman, 1974; Faber and Korn, staining of the GlyR 48 kDa channel-forming polypeptide was 1982) including at synapses issued by the commissural inter- not hampered by strychnine. This was probably due to the fact neurons (Faber and Kom, 1988). Also, despite suggestions that that the epitope of this immunoglobulin is located within the in other systems, strychnine may affect responses mediated by first 100 amino acids of the 48 kDa chain and is different from GABA (Davidoff et al., 1969), the latter are not changed in this the antagonist binding site (Schroder et al., 199 1). neuron by doses of the drug that block the collateral IPSP (Di- As for the neuronal nicotinic receptor, denervation of the amond et al., 1973). cardiac ganglion neurons causes a decrease of the total number Recovery of synaptic potentials from a block by strychnine of the surface ACh receptors (Sargent and Pang, 1988) and that is exceptional during acute experiments and was only observed of the chick ciliary ganglion produces a decline in the number when low amounts of the antagonist were injected iontophoreti- of intracellular receptors, leaving unaffected the postsynaptic tally in the axon cap to verify that the background synaptic ones (Jacob and Berg, 1987, 1988). noise is inhibitory in the M-cell (Kom and Faber, 1990) yet These data, and our results, contrast with those obtained at prolonged exposure of neurons to strychnine has not been at- the neuromuscular junction or on the electrocytes, where de- tempted in vivo for behavioral and/or physiological studies of nervation (Bourgeois et al., 1973, 1978a; Fambrough, 1974; its long-term effects that in the present study remained confined Loring and Salpeter, 1980) as well as a postsynaptic blockade to the GlyRs. Indeed, spike amplitudes (30-50 mV) and resting of the receptors by d-tubocurarine (Berg and Hall, 1975) or potential (75-80 mV) were as in normal M-cell somata (Faber a-bungarotoxin (Chang et al., 1975), and presynaptic inhibition and Kom, 1978). EPSPs unmasked by the relief of inhibition by botulinum toxin (Chang et al., 1975; Bourgeois et al., 1978b) were also the same (Furukawa et al., 1963) as well as the re- all caused the appearence of extrajunctional ACh receptors over fractoriness of test spikes during paired stimulations at closed the entire myofiber surface. These treatments were reported to intervals. Only G,, the input conductance, was unusually ele- accelerate the turnover of the junctional ACh receptors (Loring vated since it averaged 13.8 + 1.65 PS (*SD; IZ = 5; range, 1l- and Salpeter, 1980; Avila et al., 1989; Fumagalli et al., 1990). 15 PS), which is higher than the 6.08 $!i and 6.2 &i in current- However, their number revealed after labeling with 1251-oI-bun- and voltage-clamp, respectively (Faber and Kom, 1982, 1988). garotoxin remained intact 18 d after denervation (Porter and This value returns to the lowest ranges of the early estimates of Barnard, 1975). Several factors that interfere with the synthesis M-cell input resistances (Furshpan and Furukawa, 1962; Fu- or clustering of ACh receptors have been recently identified, kami et al., 1965). The resting M-cell’s conductance is domi- including the calcitonin gene-related peptide (Laufer and Chan- nated by a voltage-dependent K+ conductance (Faber and Kom, geux, 1987) or agrin, that is released from the presynaptic nerve 1986), and a nonspecific effect of strychnine on the cell’s mem- ending (Godfrey et al., 1984; Nitkin et al., 1987) and whose brane is the most likely explanation for this finding. staining intensity decreases after denervation (Reist et al., 1987). 130 Seitanidou et al. * Induced Changes in Glycine Receptor Distribution

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