Effects of botulinum neurotoxin type A on the expression of gephyrin in cat abducens motoneurons

Effects of Botulinum Neurotoxin TypeA on the Expression of Gephyrinin Cat Abducens Motoneurons

BERNARDO MORENO-LOPEZ,1,2 ROSA R. DE LA CRUZ,2 ANGEL M. PASTOR,2

JOSE M. DELGADO-GARCIA,2 AND FRANCISCO J. ALVAREZ1*1Department of Anatomy, Wright State University, Dayton, Ohio, 45435

2Laboratorio de Neurociencia, Facultad de Biologıa, Universidad de Sevilla,41012-Sevilla, Spain

ABSTRACTIn this study, we investigated the effects of long-term synaptic blockade on postsynaptic

receptor clustering at central inhibitory glycinergic synapses. High doses of botulinumneurotoxin type A injected in the lateral rectus muscle completely abolishes inhibitorypostsynaptic potentials onto abducens motoneurons within 2 days postinjection, and transmis-sion remains blocked for at least 2 months. Using this model, we analyzed the expression ofgephyrin, a glycine receptor clustering protein, on the membrane of motoneuron somata afterbotulinum neurotoxin type A injection in their target muscle. Immunofluorescence or electronmicroscopy immunohistochemistry revealed gephyrin-immunoreactive clusters (most , 0.5µm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructur-ally, presynaptic terminals containing flattened synaptic vesicles (F terminals) were foundassociated with multiple gephyrin-immunoreactive postsynaptic densities (average 1.24gephyrin clusters/F1 profile). No significant changes in gephyrin-immunoreactive clusterswere observed at 5 days postinjection, but we found significant reductions (25–40%) in thedensity of gephyrin clusters 19 and 35 days postinjection. Hence, the physiological alterationsreported in this model precede structural changes on postsynaptic receptor cluster density.The decrease in gephyrin-immunoreactive clusters was paralleled by reductions in synapticcovering (F1 terminals per 100 µm of membrane). Presumed inactive F1 terminals thatremained attached to the motoneuron surface displayed normal gephyrin-immunoreactiveclusters; however, the pre- and postsynaptic membranes in between synaptic active zonesfrequently appeared separated by enlarged extracellular spaces. We concluded that postsynap-tic receptor cluster dissolution seemed more directly related to terminal retraction than toinactivity alone. J. Comp. Neurol. 400:1–17, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: synaptic plasticity; postsynaptic density; ultrastructure; synaptology

Activity-dependent structural reorganizations of postsyn-aptic densities and the neurotransmitter receptor clusterscontained within them have been implicated in modula-tion of synaptic efficacy (Siekevitz, 1985; Calverly andJones, 1990; Geinisman et al., 1993; Lisman and Harris,1993; Jones and Harris, 1995). At central synapses, mostresearch effort has concentrated on the effects of synapticactivity or denervation on postsynaptic structure andreceptor clustering. Little is known, however, about thesignificance of synaptic disuse (without denervation) onthe structural maintenance of postsynaptic receptor clus-ters in the brain. To address this issue, we used here arecently developed experimental model to obtain long-term blockade of central synapses on abducens motoneu-rons using botulinum neurotoxin type A (BoNT).

BoNT is a well-known presynaptic blocker of acetylcho-line release at the neuromuscular junction (Cull-Candy etal., 1976; Dolly, 1992). When injected into a muscle, BoNTis internalized by motoneuron terminals where it func-tions as an endopeptidase, cleaving protein components ofthe synaptic machinery responsible for vesicle docking and

Grant sponsor: NIH; Grant number: NS 33555; Grant sponsor: DGCIT(Spain); Grant number: PB93–1175; Grant sponsor: CICYT (Spain); Grantnumber: SAF96–0160; Grant sponsor: NATO Collaborative Research;Grant number: CRG-960221.

*Correspondence to: Francisco J. Alvarez, Department of Anatomy,Wright State University, Dayton, OH, 45435. E-mail: [email protected]

Received 22 August 1997; Revised 12 May 1998; Accepted 16 May 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 400:1–17 (1998)

r 1998 WILEY-LISS, INC.

exocytosis (Blasi et al., 1993; Dolly et al., 1994). As a result,BoNT induces a characteristic flaccid paralysis of theaffected muscle. In addition to the peripheral effects, wehave recently shown that BoNT injected at doses ten timeshigher than needed for the paralysis of the lateral rectusmuscle can alter the firing and synaptic properties ofabducens motoneurons (Moreno-Lopez et al., 1994, 1997;Pastor et al., 1997). Inhibitory synaptic activity on abdu-cens motoneurons was affected rapidly, being virtuallyextinguished in 2–3 days and for up to 2 months postinjec-tion. This synaptic blockade occurs without obvious signsof degeneration in the presynaptic terminals, although apartial retraction of synaptic terminals has been observed(Pastor et al., 1997). Excitatory synapses were also af-fected but with a slower time course and greater variabil-ity from motoneuron to motoneuron. The mechanism ofaction of these central effects has not been fully character-ized, although these could be a direct central action of thetoxin following its retrograde transport to the motoneuroncell bodies and transynaptic relocalization to inhibitoryterminals in direct synaptic contact with the motoneurons(Wiegand et al., 1976; Wellhoner, 1989; Moreno-Lopez etal., 1997; Pastor et al., 1997). Such a mechanism wouldresemble the transynaptic action of retrogradely trans-ported tetanus toxin, another clostridial toxin that sharesmany similarities with BoNT in its mechanism of actionand tertiary structure (Mellamby, 1984; Montecucco andSchiavo, 1993). This approach allowed us to investigatedirectly the effects that long-term synaptic inactivitymight have on the postsynaptic receptor ensembles atinhibitory synapses in the adult brain, in vivo. Specifically,we have analyzed the expression of immunoreactivity forgephyrin, a postsynaptic glycine receptor clustering pro-tein, at inhibitory synapses onto abducens motoneurons.

Most inhibitory synapses on cat abducens motoneuronsare glycinergic (Spencer et al., 1989; Lahjouji et al., 1996).The postsynaptic densities of glycinergic synapses areenriched with gephyrin, a receptor clustering protein thatanchors postsynaptic glycine receptors by cross-linkingtheir b subunit to the underlying peripheral cytoskeleton(Kirsch et al., 1991; Meyer et al., 1995). Clustering ofgephyrin at postsynaptic sites is a necessary step for theformation of a glycine receptor cluster in the postsynapticdensity. Gephyrin clusters appear during synaptogenesisjust prior to the formation of glycine receptor clusters, andif gephyrin clustering is prevented using antisense meth-ods, blocking synaptic activity, or disrupting Ca21 entrythrough L-type channels, glycine receptors will not clusterpostsynaptically (Kirsch et al., 1991; Kirsch and Betz,1998). Hence, gephyrin-immunoreactive clusters are asuitable indicator of receptor clustering in the postsynap-tic density, and at least during development their forma-tion is dependent on synaptic activity. Gephyrin has alsobeen suggested to interact with g-aminobutyric acidA(GABAA) receptors in certain synapses of the brain andretina (Triller et al., 1987; Kirsch and Betz, 1993; Sassoe-Pognetto et al., 1994, 1995; Bohlhalter et al., 1994; Cabotet al., 1995). In other somatic motoneurons (spinal), gephy-rin-immunoreactivity has been shown to map specificallythe localization and structural organization of postsynap-tic glycine receptor clusters (Triller et al., 1985, 1990; Toddet al., 1995, 1996; Alvarez et al., 1997); however, the exactrelationship between postsynaptic gephyrin immunoreac-tivity and nonglycinergic inhibitory synapses has not beenstudied for abducens motoneurons, and this issue was not

addressed here. In our approach, therefore, gephyrinimmunolocalization should be interpreted as a marker forthe structural integrity of postsynaptic receptor clusters atglycinergic, and perhaps also other inhibitory synapsesafter BoNT-induced synaptic block. Abducens motoneu-rons were identified either by their immunoreactivity tocalcitonin gene-related peptide (CGRP) or by retrogradetransport of horseradish peroxidase (HRP) injected in thelateral rectus muscle. The results presented here havebeen previously reported in abstract form (Moreno-Lopezet al., 1996).

MATERIALS AND METHODS

Eight cats were used in this study. Experiments on liveanimals were carried out in Spain according to presentlegislation for the use of laboratory animals (BOE 67/8509–8512, 1988). All experimental procedures have been de-scribed in detail in earlier publications (Alvarez et al.,1997; Moreno-Lopez et al., 1997; Pastor et al., 1997).Briefly, BoNT (provided by Dr. J.O. Dolly, Imperial College,London, UK) was injected (3 ng/kg; 5 µl total volume) inthe left lateral rectus muscle of cats anesthetized withketamine (35 mg/kg, i.m.). Successful injections wereconfirmed by the total paralysis of the muscle and subse-quent loss of eye abduction. Abducens motoneurons wereretrogradely labeled with HRP in animals used for elec-tron microscopy purposes. Twenty-four hours before kill-ing the animals for electron microscopy, they were reanes-thetized with ketamine (35 mg/kg, i.m.), and both lateralrectus muscles were injected with a 20% HRP solution(Grade I, Boehringer Mannheim, Barcelona, Spain) pre-pared in 2% dimethylsulfoxide. All animals were deeplyanesthetized with sodium pentobarbital (50 mg/kg, i.p.)before being perfused transcardially with fixative. Forlight microscopy, three animals were perfused with 4%paraformaldehyde in 0.1 M sodium phosphate buffer (PB,pH 7.4), 5, 19, and 35 days after injection of BoNT. Forelectron microscopy, five animals were perfused with 4%paraformaldehyde and 0.2% glutaraldehyde in PB at 5(n 5 1), 19 (n 5 1), and 35 (n 5 3) days postinjection. Thebrainstem was removed and postfixed in the same fixativefor 1–2 hours at 4°C, and then tissue blocks were stored in15% sucrose at 4°C. Transverse sections (40–50 µm)through the abducens region were obtained by using avibratome for electron microscopy or a sliding freezingmicrotome for light microscopy.

Light microscopy

Sections were collected in 0.01 M phosphate buffer, pH7.1–7.3, with 0.09% saline and containing 0.1% TritonX-100 (PBS/TX). The sections were blocked with normalgoat serum (1:10 in PBS/TX) and then placed in a mixtureof primary antisera containing a mouse monoclonal anti-gephyrin antibody (Boehringer Mannheim; 1:100 dilution)and a rabbit polyclonal CGRP antibody (Peninsula Labs.,Belmont, CA; 1:2,000 dilution). Some sections were incu-bated in a different antibody mixture containing a ratmonoclonal anti-choline acetyltransferase (ChAT) anti-body (Boehringer Mannheim; 1:5 dilution) and a rabbitpolyclonal anti-CGRP antibody (Peninsula Labs.; 1:2,000dilution). Sections were incubated in primary antisera for2 days at 4°C. Thereafter, the sections were washed inPBS/TX and incubated for 2 hours at room temperature ina mixture of a 1:50 dilution of goat anti-mouse IgG (or

2 B. MORENO-LOPEZ ET AL.

anti-rat IgG) conjugated with fluorescein isothyocianate(FITC, Jackson Labs., West Grove, PA) and a 1:50 dilutionof goat anti-rabbit IgG conjugated with tetramethylrhoda-mine isothyocianate (TRITC, Jackson Labs.) in PBS/TX.Finally, the sections were washed in PBS and mountedwith Vectashield (Vector Labs., Burlingame, CA).

Sections were analyzed by using a BH2 Olympus micro-scope fitted with appropriate filters (Chroma Technology,Brattleboro, VT) for independent or simultaneous visual-ization of FITC and TRITC fluorescence. Images werecaptured by using a CCD integrating camera (Dage 72)coupled through a framegrabber (digitization 8 bits/pixel;Imascan Mono-D, Imagraph, Chelmsford, MA) to Image-Pro analysis software (Media Cybernetics, Silver Spring,MD) running in a Pentium PC. Gephyrin-immunofluores-cent clusters were identified and counted by eye on thecomputer screen. The number of gephyrin immunofluores-cent clusters per 100 µm of linear membrane was obtainedfrom images of motoneurons captured in single focalplanes, approximately at the level of the midregion of theircell bodies. Several focal planes (four to eight) werecaptured from 16 control and 16 experimental motoneu-rons at each postinjection time. Linear densities fromdifferent focal planes were averaged to obtain a singleestimate for each motoneuron. The number of gephyrin-immunofluorescent clusters per 100 µm2 of surface areawas obtained by superimposing images from several ‘‘top-surface’’ focal planes (three to five) of 12 control and 12experimental motoneurons at each postinjection time.Image superimposition of these focal planes resulted in a2D projection of a partial region of the surface membraneshowing the distribution of gephyrin clusters. The area ofthe reconstructed surface was estimated from the 2Dimage. This method underestimates the true membranesurface area; however, we can assume a similar underesti-mation in control and experimental (BoNT-treated) moto-neurons. The depth (D) of a single focal plane imagedthrough our 1003 oil-immersion objective was calculatedto be around 0.3–0.4 µm (D 5 l4n[sin a/2]2 ; l 5 illumina-tion wavelength; n 5 refractive index of the mediumbetween the specimen and the objective; a 5 the half-angleof the cone of light entering the objective). Effects of BoNTtreatment were expressed as the percentage of changecompared with control motoneurons (100 3 [control clus-ter density - experimental cluster density]/control clusterdensity).

Electron microscopy

Sections for ultrastructural analysis were first pro-cessed for histochemical detection of retrogradely trans-ported HRP by using 3,38-diaminobenzidine tetrahydrochlo-ride (DAB, Sigma, St. Louis, MO). Sections used mainly forquantitative purposes were cryoprotected in 25% sucroseand 10% glycerol and freeze-thawed in liquid nitrogen tomaximize detection sensitivity and penetration of thereagents. Sections used for qualitative analysis of ultra-structural details were not freeze-thawed and showedconsiderably less immunostaining penetration but bettermorphological preservation. All the sections were blockedin normal goat serum and incubated overnight (4°C) withanti-gephyrin antibodies (1:100 dilution in PBS). Immuno-reactive sites were then revealed with ABC-peroxidasekits (Vector Labs.). Peroxidase histochemistry was carriedout in 0.02% DAB and 0.01% H2O2 for 10 minutes. Thenthe sections were osmicated, dehydrated, counterstained

with uranyl-acetate, and flat-embedded as previously de-scribed (Alvarez et al., 1997). Ultrathin sections wereanalyzed by using a Philips 201 electron microscope at60 Kv.

For quantitative purposes, we concentrated our analysisat the top and bottom surfaces of the immunostainedsections to minimize the incidence of false negatives due tolack of antibody penetration. Measurements of the densityof gephyrin-immunoreactive clusters were obtained bycounting all gephyrin-immunoreactive clusters found insingle cross sections of 26–27 abducens motoneuron cellbodies at each postinjection time and dividing these num-bers by their cell perimeters. Gephyrin-immunoreactivepostsynaptic densities in control and treated motoneuronswere photographed at 15,0003 magnification and thenegatives scanned and digitized by using an HP4c scanner(Hewlett-Packard, Palo Alto, CA). The incidence of ultra-structurally different classes of terminals on abducensmotoneurons was analyzed in one cat prepared so as tooptimize ultrastructure (Cat #2). Measurements of gephy-rin-immunoreactive postsynaptic length and cell perim-eters were obtained by using Image-Pro (Media Cybernet-ics) analysis software.

Statistical analysis

Data were grouped for motoneurons ipsilateral to theBoNT injection (experimental side) and contralateral moto-neurons (control side) at each different survival period (5,19, and 35 days). Data from the six groups were comparedby using ANOVA tests for each of the following measure-ments: 1) linear density of gephyrin-immunoreactive(gephyrin-IR) clusters estimated with fluorescence micros-copy, 2) surface density of gephyrin-IR clusters estimatedwith fluorescence microscopy, 3) linear density of gephy-rin-IR clusters estimated with electron microscopy, and 4)linear density of boutons containing flat or pleomorphicsynaptic vesicles and gephyrin-immunoreactive postsynap-tic densities (F1) estimated with electron microscopy(Table 3, Fig. 7A). Numerical data sets in the resultssection are described as mean 6 SD; in the histograms,error bars represent the standard error of the mean(SEM). All data sets met the ANOVA test assumptions(significance was set at P , 0.01 to find deviations fromnormality in data distributions and equal variance crite-ria). Significance level for the ANOVA tests was set at P ,0.001. When significant differences were found, a pairwisemultiple comparison procedure (Tukey test) was per-formed. Significance between group pairs was set at P ,0.01 for fluorescence data and P , 0.05 for electronmicroscopy data. Pairwise comparisons of data from experi-mental and control sides of Cat #2 were performed byusing t-tests with a significance level of P , 0.05. Allstatistical tests were conducted by using SigmaStat ver. 2.0.

RESULTS

Light microscopy

Abducens motoneurons were identified in control andexperimental sides by their immunoreactivity to CGRP(Fig. 1A,B,D). Double immunocytochemical localization ofChAT and CGRP revealed that more than 80% of ChAT-IRmotoneurons displayed CGRP immunoreactivity, and allCGRP-IR neurons also contained ChAT immunoreactivity(Fig. 2A,B). CGRP immunolabeling provided a brighterand more slowly fading signal than ChAT immunolabel-

GEPHYRIN-IR IN BoNT-TREATED MOTONEURONS 3

ing. Thus, we found CGRP to be a convenient marker forunambiguous identification of motoneurons in abducensnuclei. Gephyrin-IR covering was analyzed in abducensmotoneurons identified by their CGRP immunoreactivityusing dual-color immunofluorescence (Figs.1D, 2C,D).

CGRP-IR abducens motoneurons were always sur-rounded by small gephyrin-IR clusters (,0.5 µm in diam-eter, Fig. 1C). Control motoneurons displayed 51.45 6 3.45(mean 6 SD, n 5 48) gephyrin-IR clusters per 100 µm oflinear membrane. Gephyrin-IR clusters were frequentlyorganized in rosettes, best visualized in en face views ofthe surface membrane (Fig. 1D and inset). Patches of

surface membrane in 36 control motoneurons were par-tially reconstructed by superimposing several focal planes(see Materials and Methods). We found 51.42 6 7.98gephyrin-IR clusters per 100 µm2 in these reconstructedpatches of surface membrane.

After injection of 3 ng/kg BoNT in the lateral rectusmuscle, the number of gephyrin-IR clusters on the motoneu-ron somatic membrane was reduced at long (19 and 35days), but not short (5 days) survival times (Figs. 3, 4). Atthe longest survival time used in this study (35 days),gephyrin-IR clusters on abducens motoneurons from theexperimental side frequently displayed weaker immunofluo-

Fig. 1. Gephyrin-IR cluster distribution on abducens motoneuronsidentified by their calcitonin gene-related peptide (CGRP)-immunore-activity. A: Low magnification view of the abducens region. VIIgindicates the genu of the facial nerve. TRITC immunolabeling (red)indicates CGRP-IR abducens motoneurons. B: Magnification of theboxed neuron in A. C: One midfocal plane of the same neuron showingFITC immunolabeling (green) for gephyrin. B and C correspond to thesame focal plane. D: Computer-generated superimposition of variousfocal planes through the same abducens motoneuron. The superimpo-sition of gephyrin-IR clusters at various focal planes on the top half ofthis cell produces an ‘‘en face’’ 2D view of gephyrin-immunofluorescentclusters. The cell also shows intracytoplasmatic CGRP immunolabel-ing (in red). Inset: High magnification view of rosette organizations(area boxed in D). These images were obtained by using routine

epifluorescence visualization of dual color-immunostained 50-µm sec-tions with a 1003 oil objective (n.a. 5 1.32 and collar to reduceout-of-focus fluorescence). Black and white (B/W) immunofluorescenceimages were captured through a Dage 72 low-light integrating videocamera and then pseudocolored with Image-Pro software (MediaCybernetics) to reflect accurately the immunofluorescence observed inthe tissue (inset is shown in the B/W image). Integrating time wasmanually adjusted for optimal sensitivity and contrast. In D, theimaging software was used to superimpose a midfocal plane image ofCGRP-immunofluorescence with a series of optical sections of gephyrin-immunofluorescence (obtained manually). The latter were used toreconstruct the gephyrin covering of the top surface of this motoneu-ron. Pixel size 5 0.16 µm. Scale bars 5 200 µm in A, 20 µm in B–D,2 µm in inset.


rescence compared with the control side (Fig. 3E,F). ANOVAanalysis indicated the presence of statistically significantdifferences (P , 0.001) on the linear and surface density ofgephyrin-IR clusters between different groups of motoneu-rons (Table 1). Pairwise multiple comparisons (Tukey test;significance level P , 0.01) were performed to establishwhich groups were significantly different. Gephyrin-IRclusters were significantly depleted by 38.7% in linearcovering or 28.8% in surface area covering after 19 days

postinjection (1003 [control density—experimental density]/control density). Thirty-five days postinjection, we mea-sured a 29.9% depletion in linear covering and a 24.4%reduction of surface area covering by gephyrin-IR clustersin experimental motoneurons. Differences between controlmotoneurons at 5, 19, and 35 days, control and experimen-tal neurons at 5 days, and experimental motoneurons at 19and 35 days did not reach statistical significance (Table 1,Fig. 4).

Fig. 2. Colocalization of choline acetyltransferase (ChAT) andcalcitonin gene-related peptide (CGRP) in cat abducens motoneurons.A,B: Photomicrographs of the same field visualized for ChAT FITC-immunofluorescence (A) and for CGRP TRITC-immunofluorescence(B). VIIg indicates the position of the genu of the facial nerve. VIindicates fascicles of the intramedullary portion of the abducensnerve. Most abducens motoneuron somata lie in between these twonerve bundles and contain both ChAT and CGRP (e.g., arrows).

C,D: Expression of gephyrin immunoreactivity by CGRP-immunofluo-rescent abducens motoneurons. In C, a number of CGRP-immunofluo-rescent motoneurons are visualized by using TRITC fluorescence. D isthe same field photographed under an FITC filter block to revealgephyrin immunofluorescence. Selected cells surrounded by gephyrin-immunofluorescent clusters (asterisks in D) correspond to CGRP-immunofluorescent abducens motoneurons shown in C. Scale bars 5100 µm in B (also applies to A), 50 µm in D (also applies to C).


Fig. 3. Effects of BoNT on the expression of gephyrin-IR clusters inabducens motoneurons. A,C,E: Control motoneurons photographed 5,19, and 35 days after application of BoNT in the contralateral lateralrectus muscle. B,D,F: Motoneurons photographed 5, 19, and 35 daysafter application of BoNT in the ipsilateral lateral rectus muscle. Nodifferences were found at short survival times (5 days); 19-day

postinjection gephyrin-IR clusters were similar in size and brightnessin control (con) and experimental (exp) sides; however, their numberappeared to be diminished after BoNT treatment. After 35 days, thedensity and intensity of gephyrin-immunofluorescent clusters weredecreased. Scale bar 5 10 µm (applies to all).

Electron microscopy

An ultrastructural study was carried out to analyzegephyrin-IR clusters and their associated synaptic termi-nals in control and experimental abducens motoneurons.Abducens motoneurons were identified by the retrogradetransport of HRP from the lateral rectus muscle (Fig. 5).Around 85% of terminals containing flattened or highlypleomorphic synaptic vesicles displayed gephyrin immuno-reactivity in their postsynaptic densities; these terminalswill be referred to as F1 terminals (Fig. 6). The remainingterminals (,15%) containing this synaptic vesicle typewere immunonegative and will be referred to as F2. Onlyvery rarely were membrane patches of DAB precipitatefound unrelated to synaptic active zones and frequently itwas difficult to resolve unambiguously whether they repre-

sented DAB precipitate translocated from nearby HRPgranules or gephyrin immunoreactivity. However, theirfrequency was similar in experimental and control sides.Finally, a third class of terminal was identified by theircomplement of predominantly spherical synaptic vesicles(S terminals). S terminals were never associated withgephyrin-IR postsynaptic densities. S terminals repre-sented around 30–40% of all synaptic terminals found incontact with control abducens motoneuron somata.

Control abducens motoneurons displayed an average of11.30 6 5.38 gephyrin-IR postsynaptic clusters per 100 µmof linear membrane (n 5 79 somatic cross sections). Thisestimate is in good agreement with our immunofluorescentcounts considering that the depth of focus of our immuno-fluorescent images is between 0.3 and 0.4 µm, whereas thethickness of an ultrathin section is 0.075–0.085 µm. Hence,counts at a single focal depth using light microscopy,sample approximately 4–5 times more membrane perlinear unit than ultrathin sections visualized under elec-tron microscopy. At the electron microscopic level we found9.06 6 4.01 F1 terminals per 100 µm of linear membrane(n 5 79 somatic profiles). Whenever an F terminal dis-played no synaptic specialization in the cross sectionsampled for quantitative analysis, it was clearly identifiedas F1 or F- in serial sections and included in the quantifi-cation. On the sample of F1 terminals displaying synapticactive zones we found 1.24 6 0.19 postsynaptic gephy-rin-IR clusters per F1 bouton profile (range 1–4). Thesefigures do not represent the total number of active zonesper F1 bouton, since individual boutons were not recon-structed through serial sections.

Experimental abducens motoneurons also displayed ge-phyrin-IR postsynaptic densities (Figs. 7, 8) and the sametypes of terminals found in control motoneurons but indifferent relative proportions (Table 2). We analyzed oneexperiment quantitatively (Cat #2) 35 days postinjection.

TABLE 1. Number of Gephyrin-IR Clusters in Control and BoNT-TreatedMotoneurons Estimated Using Immunofluorescence1

Days

5 19 35

Density of gephyrin-IRclusters per 100 µmof linear membrane

Control 53.87 6 3.30 (16) 50.91 6 2.79 (16) 49.59 6 2.89 (16)Experimental 55.81 6 2.76 (16) 31.22 6 5.71 (16)* 34.74 6 5.41 (16)*

Density of gephyrin-IRclusters per 100 µm2

of surface membraneControl 48.86 6 7.59 (12) 53.04 6 8.54 (12) 52.36 6 7.80 (12)Experimental 56.38 6 11.63 (12) 37.79 6 7.40 (12)* 39.57 6 7.68 (12)*

1Data represent the mean 6 SD. The number of sampled motoneurons is shown inparentheses for each group. For linear measurement, each motoneuron was sampledthrough several optical cross sections. For surface density measurements the top-surface membrane of sampled motoneurons was partially reconstructed through severalfocal planes to obtain a density surface estimate. BoNT, botulinum neurotoxin; IR,immunoreactivity.*Statistically significant differences from control (P , 0.001, ANOVA test; P , 0.01Tukey test).

Fig. 4. Quantification of gephyrin (geph) FITC-immunofluorescentclusters at 5, 19, and 35 days postinjection with BoNT. A: Time courseof changes in the mean number of clusters per 100 µm of somaticperimeter in control (solid bars) and after treatment (open bars). Errorbars indicate the standard error of the mean (SEM). Controls at eachstage were taken from the uninjected side. Asterisks indicate signifi-cant differences with respect to their control groups (P , 0.001,

ANOVA; P , 0.01, Tukey test pairwise comparisons). Sixteen motoneu-rons were sampled in each group. Each motoneuron was analyzedthrough four to eight focal planes. B: Same as A but the number ofclusters refers to 100 µm2 area of membrane after partially reconstruct-ing patches of surface membrane through several optical sections.Twelve motoneurons were sampled in each group. Asterisks indicatestatistically significant differences as in A.


This experiment was prepared so as to optimize ultrastruc-tural preservation (see Materials and Methods), and synap-tic boutons could be unambiguously classified as F or Saccording to their vesicle content. Care was taken toanalyze only section depths with abundant immunoreactiv-ity. In this animal the proportion of axosomatic F1 termi-nals was lower and the proportion of S terminals washigher in experimental motoneurons (n 5 9 somatic pro-files) compared with control (n 5 9 somatic profiles; Table2). Overall, experimental abducens motoneurons werecontacted by fewer synaptic boutons than control motoneu-rons (26.4% depletion, P , 0.05, t-test, Table 2) butdifferent terminal types were lost in different proportions.Synaptic linear frequency (number of terminals/100 µm oflinear neuron perimeter) was reduced by 38.1% for F1terminals, 20.2% for F2 terminals, and 10.9% for Sterminals (Table 2). Differences in synaptic linear fre-quency reached statistical significance for F1 terminalsbut not for F2 or S terminals (t-test, P , 0.05). Theseobservations can be best explained by a preferential detach-ment of F1 terminals from the cell somata of abducensmotoneurons after BoNT injection. Further evidence (Figs.7, 8; Pastor et al., 1997) also suggests that the adherence ofsynaptic boutons to the postsynaptic membrane afterBoNT treatment is less impaired in S terminals.

In a different series of animals prepared such thatimmunolabeling detection was optimized, we analyzedchanges in gephyrin-IR cluster number in treated vs.control abducens motoneurons after 5, 19, or 35 dayspostinjection (Fig. 9 and Table 3). ANOVA test indicatedthe existence of statistical significant differences betweengroups (P , 0.001). Statistically significant reductions

(Tukey test, P , 0.05) in the number of gephyrin-IRclusters on experimental motoneurons compared withtheir controls were found at 19 days (31.2% decrease) and35 days (40.5% decrease; Fig. 9 and Table 3). The differ-ences between controls, and between control and experi-mental motoneurons, at 5 days did not reach statisticalsignificance (P . 0.05). The number of terminals presynap-tic to immunolabeled postsynaptic densities (F1) wasanalyzed with similar procedures and found to be reducedin parallel. At 19 days we measured a 32.8% reduction inF1 terminals, and after 35 days they were reduced by39.0%. We did not observe any change in the ratio ofimmunolabeled postsynaptic densities per terminal at anypostinjection time (1.24 6 0.19 in controls; 1.22 6 0.23 at 5days after BoNT injection; 1.26 6 0.16 at 19 days; 1.23 60.19 at 35 days). The results suggest that the depletion ofgephyrin-IR postsynaptic clusters occurs in parallel with areduction of F1 boutons and is not caused by a reduction ofgephyrin-IR postsynaptic densities per terminal or thedisappearance of gephyrin immunoreactivity from thepostsynaptic density.

The density of both gephyrin-IR clusters and F1 termi-nal covering did not decrease secondary to an increase inmotoneuron surface induced by the BoNT treatment.Although the perimeter of the somatic profiles sampledincreased slightly in experimental motoneurons (Table 3),this increase was relatively small and just barely signifi-cant at 5 days, being not significant at 19 and 35 days, thetime points at which we found large changes in synapticdensity covering. Our perimeter measurements at theultrastructural level parallel previous estimates of so-matic cross-sectional areas sampled from semithin

Fig. 5. Low power electron micrograph of an abducens motoneuron identified by the presence ofretrogradely transported HRP ‘‘granules’’ (arrowheads). A presynaptic terminal associated with postsyn-aptic gephyrin-IR clusters is indicated (arrows). This area is enlarged in Figure 6A. The area marked withan asterisk is enlarged in a serial section in Figure 6B. Scale bar 5 10 µm.


Fig. 6. Ultrastructural characteristics of gephyrin-IR clusters andtheir associated F1 presynaptic terminals in control abducens moto-neurons. A: A synaptic terminal (T) containing flattened synapticvesicles establishes synaptic contact with the motoneuron soma (S)through two independent small active zones that postsynapticallydisplay DAB precipitate (arrows) indicative of gephyrin immunoreac-tivity. C: A higher magnification of the top synapse in A is shown in anadjacent serial section; arrowheads indicate the presynaptic cluster ofsynaptic vesicles. B: High magnification of a serial section through the

area labeled with an asterisk in Figure 5 shows a terminal (T1)establishing an small immunoreactive synapse (arrow) with the soma(S) of the abducens motoneuron. A second terminal nearby (T2),establishes a larger immunoreactive synapse (large arrow) with adendrite (D). D: A higher magnification of the top synapse in B isshown in a nonadjacent serial section; arrowheads indicate thepresynaptic cluster of synaptic vesicles. Asterisks indicate regionswith diffusion-artifacts of the DAB precipitate. Scale bars 5 0.5 µm inA,C (applies to B,D, respectively).


Figure 7


sections and in a larger number of abducens motoneurons(Pastor et al., 1997). In this study a small increase in cellarea was reported after a similar BoNT treatment. Thisincrease was barely significant at some time points withinthe first 2 weeks of treatment and then reverted to controlsize at 30 days (Pastor et al., 1997).

No statistically significant differences were found in thesize of gephyrin-IR clusters measured in electron micro-graphs of experimental and control abducens motoneurons(compare Figs. 6 and 7). As previously reported for spinalneurons (Alvarez et al., 1997), somatic gephyrin-IR postsyn-aptic clusters were considerably smaller than those lo-cated in medium-caliber dendritic segments (distal) foundin the adjacent neuropil even if associated with the sameF1 bouton (Fig. 10A). Cross sections of gephyrin-IR clus-ters on the soma of control motoneurons measured 0.38 60.14 µm (n 5 84; range 0.14–0.76 µm), and 0.32 6 0.10 µm(n 5 73; range 0.17–0.74 µm) on experimental motoneu-rons 35 days postinjection (t-test, P . 0.05). Decreases inimmunoreactivity intensity were difficult to assess at theultrastructural level, mainly because small differences inthe relative distance to the surface of sampled synapsesinside the immunostained 50-µm vibratome section mayhave large effects in the intensity of immunostainingdisplayed. Other factors related to the nature of immunocy-tochemical reactions also contribute to generate nonlineari-ties in the relation between the intensity of DAB precipi-tate and the amount of antigen present in the tissue.

BoNT treatment also induced a partial detachment ofthose synaptic boutons that remained in contact with cellsomata. Loss of adherence between synaptic boutons andthe postsynaptic membrane was seen as a widening of theintercellular space interposed between pre- and postsynap-tic membranes. Widened intercellular spaces were muchmore frequently observed in the experimental side than inthe corresponding control side (Figs. 7, 10B,C). A detailedquantitation of this ultrastructural alteration has alreadybeen published by Pastor et al., (1997). Here we confirmedthat they are more abundant in F terminals comparedwith S terminals (i.e., Fig. 10C) and that the immunoreac-tive characteristics of postsynaptic densities opposed toF1 terminals displaying widened separations of the pre-

and postsynaptic membranes are not different from con-trol F1 terminals. The motoneuron cytoplasm frequentlycontained submembrane cisterns underneath widened in-tercellular spaces (Fig. 7B). Glial processes were onlyrarely found between pre- and postsynaptic membranes(see Fig. 8). Interestingly, many partially detached termi-nals were in contact with the postsynaptic membraneexclusively through their synaptic specializations, andthese remained immunoreactive to gephyrin in their post-synaptic sides (Fig. 8).

Some large-caliber dendrites could be identified as proxi-mal dendrites because they contained retrogradely trans-ported HRP granules (Fig. 10B,C). Proximal dendriteswere contacted by synaptic terminals of similar character-istics to the cell soma. Although no detailed quantitativestudy was performed on proximal dendrites they seemedmore densely covered by synaptic terminals than motoneu-ron somata. Ultrastructural alterations of proximal den-dritic synapses induced by injection of BoNT resembledthose described in the soma.

DISCUSSION

The major finding of the present study is a reduction inthe number of gephyrin-IR clusters and F1 terminals onabducens motoneurons after long periods (19 and 35 days)of synaptic inactivity following injection of a large dose(3 ng/kg) of BoNT in their target muscle. This reductionmight in part explain the long-term blockade (2–3 months)of inhibitory synaptic activity found on abducens motoneu-rons after BoNT injections (Moreno-Lopez et al., 1994,1997; Pastor et al., 1997). However, the time course andextent of the observed physiological alterations is quitedifferent from the observed effects on gephyrin-IR clustersand associated presynaptic terminals. Previous physiologi-cal results indicate that inhibitory synaptic transmissionis rapidly affected after injection of a similar large dose ofBoNT (Moreno-Lopez et al., 1994, 1997; Pastor et al.,1997). In alert cats, BoNT produces a significant reductionof motoneuron inhibition during off-directed saccades asearly as 2–5 days postinjection (Moreno-Lopez et al., 1994,1997). From the second week postinjection onward, pausesin motoneuron firing during off-directed saccades areabolished, suggesting the impairment of synaptic transmis-sion from inhibitory reticular burst neurons onto themotoneuron (Escudero and Delgado-Garcıa, 1988). Simi-larly, diminished modulation of motoneuron firing duringhead rotation indicates almost total suppression of vestibu-lar inhibitory synaptic input by 1 week postinjection(Moreno-Lopez et al., 1997). Both vestibular and reticularinhibition on abducens motoneurons have been proposedto use glycinergic mechanisms (Spencer et al., 1989).Moreover, intracellular recordings in anesthetized ani-mals showed complete abolition of inhibitory postsynapticpotentials from the ipsilateral vestibular system on abdu-cens motoneurons just 2 days after the injection of thesame dose of BoNT in the lateral rectus muscle (Pastor etal., 1997). In contrast, the present morphological dataindicate that decreases in gephyrin-IR cluster density onthe surface of BoNT-treated motoneurons are significantonly several days after the inducement of chronic block ofglycinergic synapses by BoNT. In addition, reductions ingephyrin-IR cluster covering were never as complete asthe observed decrease in synaptic function (even at the

Fig. 7. Ultrastructural characteristics of F1 terminals synapsingon abducens motoneurons ipsilateral to the BoNT-treated muscle (35days postinjection). The most apparent oddity of F1 terminals in theexperimental side was the appearance of widened intercellular spaces.Presynaptic vesicle accumulations and ultrastructural characteristicsof the presynaptic terminals were not qualitatively different fromcontrol F1 terminals (i.e., Fig. 6). Gephyrin immunoreactivity appearsslightly reduced compared with the control, but a number of technicalfactors can also contribute to this result (i.e., different sampling depthin 50-µm section or plane of section). A: F1 terminal (T) profile withtwo synaptic active zones opposed by gephyrin-IR postsynaptic densi-ties (arrows) on the plasma membrane of an abducens motoneuron(MN). Pre- and postsynaptic membranes are separated by widenedgaps of intercellular space. Intercellular gaps are more apparent thanon control motoneurons. B: Submembrane cisternae (arrowheads) arefrequently associated with regions of the postsynaptic membranewhere the presynaptic terminals have detached from the motoneuron(MN) surface. Arrows indicate postsynaptic densities with gephyrinimmunoreactivity. C: F1 terminal with three independent synapticactive zones displaying gephyrin-IR postsynaptic densities (arrows).The motoneuron (MN) membrane invaginates in the region of detach-ment between pre- and postsynaptic membranes (asterisk). Scalebar 5 0.5 µm (applies to all).


longest time period examined, 35 days postinjection).Therefore, we conclude that BoNT-induced alterations inglycinergic synaptic function are more pronounced andprecede structural changes affecting the number of postsyn-aptic receptor clusters.

It is noteworthy that at the neuromuscular junctionBoNT does not completely block neurotransmitter releasebut seems to decrease the sensitivity for Ca21 of the releasemechanism. Therefore BoNT quite effectively blocks stimu-lus-evoked release of acetylcholine and significantly re-duces, but does not abolish completely, spontaneous re-lease (Cull-Candy et al., 1976; Molgo et al., 1990). BoNT-poisoned motor end-plates do not degenerate, at least inthe short term, but enlarge and sprout, forming newsynaptic contacts with the muscle fiber (Duchen, 1971). In

parallel, acetylcholine receptors clustered postsynapti-cally become more mobile and there is also de novosynthesis of acetylcholine receptors. As a consequence thepostsynaptic receptor cluster area enlarges and extrasyn-aptic acethylcholine receptors emerge leading to the phe-nomenon of ‘‘supersensitivity’’ in the muscle fiber (Thesleff,1960; Pestronk et al., 1976; Chee Yee and Pestronk, 1987).In the present report we could not directly assess terminalsprouting and the new formation of inhibitory synapses onabducens motoneurons; however, the size of the postsynap-tic receptor cluster did not seem to enlarge over the timeperiods examined. It is also unknown whether spontane-ous neurotransmitter release persists at BoNT-‘‘silenced’’central inhibitory synapses. It is possible that the persis-tence of some ‘‘quantal’’ release from presynaptic termi-

Fig. 8. An axon terminal (T) making a synapse onto a BoNT-treated motoneuron soma (S) that appears almost completely de-tached from the postsynaptic membrane except at the synaptic activezone (long arrow). The postsynaptic region shows gephyrin-immunore-

activity. The open intercellular space (asterisks) between the pre- andpostsynaptic membranes is partially occupied by a glial (g) extension(short arrow). Arrowheads indicate a granule with retrogradely trans-ported HRP. Scale bar 5 0.5 µm.


nals could aid in the maintenance of the postsynapticstructure and receptor cluster in the absence of impulse-evoked release. Nevertheless, total pharmacological blockof Mauthner cell glycinergic synapses (with strychnine),maintained for up to 3 days, did not appreciably changethe structure or density of postsynaptic glycine receptors(Seitanidou et al., 1992). Unfortunately, it was technicallynot possible to maintain strychnine poisoning for longerperiods of time in the study of Seitanidou et al. (1992). Inthis same study a rapid retraction of gephyrin-IR patchesfrom the plasma membrane was observed (3 days) follow-ing anterograde degeneration of presynaptic glycinergicterminals. Disappearance of gephyrin-IR patches corre-lated with the glial engulfment and complete detachmentfrom the Mauthner cell membrane of most degeneratedterminals. Interestingly, synaptic boutons in contact withthe postsynaptic membrane, even at advanced stages ofdegeneration, displayed postsynaptic gephyrin-IR clus-ters, although these were of slightly less diameter andimmunoreactivity. Overall these results suggest that thepostsynaptic receptor cluster, at least at inhibitory syn-apses on motoneurons and the Mauthner cell, constitutes aquite robust structure that shows little change whenchallenged by inactivity or manifest presynaptic degenera-tion, as long as the presynaptic terminal remains attachedto the postsynaptic element.

New interesting results by Kirsch and Betz (1998) wererecently reported while this manuscript was under revi-sion. These authors found that blocking glycinergic neuro-transmission in cultures of spinal neurons, by using eithertetrodotoxin or strychnine, prevents the clustering ofgephyrin and glycine receptors at postsynaptic sites whenthe treatment is performed early during synaptogenesis.In contrast, after maturation of the postsynaptic cluster,similar treatments for a period of 1 week in culture had noeffect, hence giving further support to the view thatinactivity has little influence on the stability of the postsyn-aptic receptor cluster at matured glycinergic synapses.

We found no alterations in the number of clusters perterminal or size of the clusters, despite the decrease intotal number of gephyrin-IR clusters on the membranesurface of treated motoneurons at 19 and 35 days postinjec-tion. There was a good correlation between the loss of F1

terminals and gephyrin-IR clusters, suggesting that detach-ment of the presynaptic terminal occurs concurrently withthe dissolution of the postsynaptic gephyrin-IR patch. Theadhesion properties of the presynaptic terminal seemed tobe lost or reduced with inactivity, and this was visualizedas widened intercellular spaces between the pre- andpostsynaptic membranes in regions outside synaptic spe-cializations. Whether these widened spaces are artifacts offixation or do exist in vivo is not known. However, theywere larger and more frequently found in experimentalmotoneurons than in control motoneurons. Their incidenceincreased more rapidly in F1 terminals than in otherterminal types and this might be related to the observedpreferential loss of F1 terminals from the motoneuronsurface compared with F2 or S terminals. In addition,submembrane cisternae seemed to associate with postsyn-aptic membranes opposite to detached presynaptic mem-branes. We therefore conclude that widened spaces mightbe a reflection of alterations in the adhesive properties ofthe pre- and postsynaptic membranes. In a previousultrastructural study (Pastor et al., 1997) a detailedquantitative analysis of these ultrastructural alterationswas provided. The percentage of detached membrane insynaptic appositions after BoNT treatment was 32% for Fterminals and 20% for S terminals. In contrast, F and Sterminals sampled in the control side only exhibited 4% oftheir membrane detached from the motoneuron surface(Pastor et al., 1997). These figures represent averages ofthe whole synaptic terminal population and variabilityfrom terminal to terminal was also found. The quantita-tive data described by Pastor et al. (1977) can be easilyextrapolated to the F1 terminals studied here because thegreat majority of postsynaptic densities in relation with Fterminals were found to be immunoreactive. It is possiblethat an activity-dependent loss of the adhesive propertiesof the extrajunctional membrane is directly responsible foran increase in synaptic stripping that might explain inturn parallel reductions in both the number of membranegephyrin-IR clusters and F1 terminals. Using inverte-brate models, it has been suggested that neurotransmitterrelease or other activity-dependent mechanisms can modu-late the levels of adhesion molecules at particular locationsof the neuronal membrane and that this influences pat-terns of synapse formation and elimination (Bailey andKandel, 1993; Zhu et al., 1995).

Our results seem to indicate that the postsynapticreceptor cluster of central inhibitory synapses disorganizejointly with, or rapidly following, synaptic terminal detach-ment. A few ultrastructural studies have neverthelessdescribed postsynaptic densities that remain on the plasmamembrane for variable periods of time after degenerationand detachment of the presynaptic terminal (Sotelo, 1968;Gentschev and Sotelo, 1973). These examples were ob-tained in the peripheral cholinergic synapse betweenpreganglionic and postganglionic sympathetic cells and incentral glutamatergic synapses between auditory affer-ents and ventral cochlear neurons. It is, however, un-known whether postsynaptic densities independent ofpresynaptic terminals do contain neurotransmitter recep-tor clusters. It is quite possible that different types ofsynapses disorganize postsynaptic receptor aggregationswith different time courses, perhaps underlying differentmechanisms of formation and elimination. For example,acetylcholine postsynaptic receptor clusters are never

TABLE 2. Synaptology of Control and Experimental AbducensMotoneurons (Cat #2; 35 Days Postinjection)1

F1 F2 S No.2

Proportions of differenttypes of terminals

Control (%) 51.6 14.3 34.1 114Experimental (%) 42.1 15.8 42.1 126

F1 F2 S All No.3

Number of terminalsper 100 µm oflinear membrane

Control(mean 6 SD) 8.13 6 3.55 2.18 6 1.24 5.32 6 2.36 15.63 6 5.46 9

Experimental(mean 6 SD) 5.03 6 1.69* 1.74 6 0.78 4.74 6 2.87 11.50 6 2.58* 6

1F1, a presynaptic terminal containing flattened or pleomorphic synaptic vesicles andimmunoreactive postsynaptic densities; F2, a presynaptic terminal containing pleomor-phic or flattened synaptic vesicles and nonimmunoreactive postsynaptic densities; S, apresynaptic terminal containing mainly spherical synaptic vesicles.2Total number of terminals.3Number of motoneurons analyzed.*Statistically different from control (P , 0.05, t-test).


completely eliminated in denervated adult skeletal muscle(Frank et al., 1975; Porter and Barnard, 1975), survive forvariable periods of time on denervated ciliary ganglionneurons (Jacob and Berg, 1988), and are quickly disorga-nized on denervated frog cardiac ganglion neurons (WilsonHorch and Sargent, 1996).

In conclusion, gephyrin-IR clusters on the motoneuronmembrane were reduced only after long periods of BoNT-

induced synaptic inactivity and this reduction occurred inparallel with a decrease in the number of presynapticterminals. Our results seem to indicate that the disorgani-zation of postsynaptic receptor clusters is perhaps morestrongly related to presynaptic terminal detachment thanto synaptic inactivity per se.

ACKNOWLEDGMENTS

We acknowledge Dr. J.O. Dolly (Imperial College, Lon-don) for kindly providing BoNT, Dr. Robert E.W. Fyffe forcritical reading of earlier versions of the manuscript, andDianne E. Dewey for proofreading the final manuscript.

TABLE 3. Number of Gephyrin-IR Clusters and F1 Terminals in Controland BoNT-Treated Motoneurons Estimated Using Electron Microscopy1

Days

5 19 35

Density of gephy-rin-IR clustersper 100 µm oflinear mem-brane


Number of F1 ter-minals per 100µm of linearmembrane


Average perimeter(µm) of profilessampled2

Control 127.27 6 22.50 (26) 132.30 6 21.61 (27) 139.46 6 31.06 (26)Experimental 151.24 6 25.80 (26)* 150.26 6 20.93 (27) 141.11 6 25.20 (26)

1Data represent mean 6 SD. In parentheses is the number of individual somatic crosssections (profiles) analyzed for each group (usually at midsomatic level).2Each profile (cross section) was obtained from a different motoneuron at approximatelymidnuclear level, but not all of them contained a nucleolus.*Statistically significant differences from control, (P , 0.001 ANOVA; P , 0.05 Tukeytest for the density of gephyrin cluster and number of F1 terminals; P , 0.002 ANOVA;P , 0.05 Tukey test for differences in average profile perimeter).

Fig. 10. A: F1 terminal contacting both the soma of abducensmotoneurons (S) and a small-caliber dendrite (D) in the surroundingneuropil. Synaptic contacts on the somatic membrane have smallactive zones and gephyrin-IR clusters (lower arrow). Synaptic contactson small caliber dendritic regions (distal) frequently display largeactive zones and gephyrin-IR clusters (top arrows). B: High magnifica-tion electron micrograph of an F terminal (F) establishing contact withan abducens motoneuron dendrite (D) through two immunoreactive,synaptic active zones (arrows). Retrogradely transported HRP gran-ules are located outside the field of view. An enlarged intercellularspace (asterisk) is apparent between dendritic and axonal membranes;however, the terminal remains attached at the points of synapticcontact and maintains its postsynaptic complement of gephyrin immu-noreactivity. C: Low magnification electron micrographs of a proximaldendrite (D, arrowheads point to retrogradely transported HRPgranules), surrounded by immunoreactive (double arrows) and nonim-munoreactive (arrows) synaptic terminals. Enlarged intercellularspaces (asterisks) are more commonly found associated with F (1)terminals than with S terminals. Scale bars 5 1 µm in A, 0.5 µm in B,1 µm in C.

Fig. 9. Quantification of gephyrin (geph)-IR clusters and F1terminals with electron microscopy. A: Mean number of F1 boutonscounted per 100 µm of motoneuron perimeter 5, 19, and 35 days afterinjection of BoNT. Control motoneurons contralateral to the injectedeye are represented by solid bars. Experimental motoneurons (ipsilat-eral side) are indicated with open bars. Error bars indicate thestandard error of the mean (SEM). Controls at each stage were takenfrom the uninjected side. Significant reductions in the number of F1terminals surrounding motoneurons somata were obtained at 19 and

35 days (P , 0.001, ANOVA test; P , 0.01, Tukey test pairwisecomparisons). Data were obtained from single cross sections of 26abducens motoneurons sampled in each group. When an F bouton didnot display a synaptic specialization in the sampled cross section, itwas clearly identified as F1 or F- in serial sections. B: Mean number ofgephyrin-IR clusters per 100 µm of linear membrane of the samemotoneurons. Data from control and experimental motoneurons ap-pear represented as in A. Statistical significance as in A.


Figure 10

This work wa supported by NIH grant NS 33555 to F.J.A.,DGCIT (Spain) PB93–1175 to J.M.D.-G., CICYT (Spain)SAF96-0160 to A.M.P., and NATO Collaborative Researchgrant CRG-960221 to F.J.A. and R.R.C.

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Effects of botulinum neurotoxin type A on the expression of gephyrin in cat abducens motoneurons