Gaba-like immunoreactive terminals on lumbar motoneurons of the adult cat. A quantitative ultrastructural study

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NeuroscienceResearch 24 (1996) 123-130

Gaba-like immunoreactive terminals on lumbar motoneurons of the adult cat. A quantitative ultrastructural study J. Destombes*, G. Horcholle-Bossavit, M. Simon, D. Thiesson VRA

CNRS

1448, VniversirC

Rent? Descartes,

45 rue des Saints-P&es.

75 270 Paris Cedex 06. France

Received 15June1995;accepted 29 September 1995

Abstract The aim of this ultrastructural study was to analysequantitatively the distribution of y-aminobutyric acid (GABA)-like immunoreactivityin axon terminalsapposedto somaticand proximal dendritic membranesof cat motoneuronsin lumbar column 2. Preembedding immunocytochemistrywasusedto count the GABAergic terminalscontactingprofilesof eighteenC-Xand six ymotoneurons.Of the 1293terminalscountedon the somaticand proximal dendritic compartmentsof cr-motoneurons,197were GABAergic. In contrast,a total numberof only 62 terminalswerecountedon y-motoneurons,of which 8 wereGABAergic. These populationsof GABAergic terminalswerelessnumerousthan the populationof glycinergicterminalsobservedin a previousstudy. The morphometriccharacteristics of GABAergic synapses wereanalyzedusingpostembedding immunocytochemistry.Most of the GABAergic terminalscontainedpleomorphicvesicles(F-type boutons,flattenedor pleomorphicvesicles).All terminalspresynaptic (P boutons)to large terminalscontaining sphericlevesicles(M-type boutons,characteristicof a-motoneurons),were GABAimmunopositive.Theseresultssuggestthat there are different distributionsof the GABAergic control of excitability on y- and (Ymotoneurons.GABA appearsto be strongly involved in post-synapticinhibition of cu-motoneurons, whereasy-motoneuronsreceivevery few GABAergic inhibitory inputs. Morphological correlatesof GABAergic presynapticinhibition wereseenon (Y-but not on y-motoneurons. Keywords:

GABAergic terminals;Ultrastructure; Immunocytochemistry;Alpha-motoneurons;Gamma-motoneurons;

1. Introduction

In cat peroneal motor nuclei, the somatic and proximal dendritic compartments of a-motoneurons consistently receive more F-type (flattened or pleomorphic vesicles) than S-type (spherical vesicles) synaptic boutons (Destombes et al., 1992a). The large complement of F-type. boutons, which are considered as inhibitory synapses (Uchizono, 1966) could include GABAergic terminals (Magoul et al., 1987; Holstege

and Calkoen, 1990; Ramirez-L&n and Ulfhake, 1993; Shupliakov et al., 1993) since GABA is involved in several inhibitory mechanisms of the mammalian spinal cord. Electrophysiological and pharmacological data * Corresponding author,Tel.: +331 42862139;fax: +33 1 49 27 90 62.

suggested that GABA acts on cat ar-motoneurons, both directly (Kellerth and Szumski, 1966; Curtis et al., 1971; Cullheim and Kellerth, 198 1; Schneider and Fyffe, 1990) and through presynaptic inhibition which controls the transmission of Ia afferent information to cy-motoneurons (Eccles et al., 1961, 1963; Curtis and Lodge, 1982; Curtis et al., 1982). In a recent study of cat (Y-and y-motoneurons located in lumbar column 2, known to contain flexor and peroneal motoneurons (Romanes, 195 1; Horcholle-Bossavit et al., 1988), preembedding immunochemistry was used to show that one third of the F-type terminal complement of these motoneurons are glycinergic (Destombes et al., 1992b). The aim of the present work was to provide a quantitative assessment of GABAergic terminals and to compare their distribution to that of glycinergic terminals in the same motoneuron populations. In addi-

01158~0102/96/s15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 016%0102(95)00980-8

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tion, the postembedding technique was used to examine the morphometric characteristics of GABAergic synapses. The results show that, on somatic and proximal dendritic membranes of cr-motoneurons, about 20% of Ftype boutons and all P terminals contain GABA. The mean number of GABAergic terminals per motoneuronal somatic profile was 7.6 for 01- and 1.3 for ymotoneurons. The role of GABA in the control of inhibition appears to be weaker for y- than for (Ymotoneurons. 2. Methods

Four adult cats were deeply anesthetized with sodium pentobarbital(50 mg/kg), and fixed by transcardiac perfusion with a solution of l-2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The L7 segment of the spinal cord was removed, postfixed in the same fixative for 2 h and kept overnight in a 30% PB saccharose solution at 4°C. Transverse sections (50 pm) were cut with a vibratome (Lancer) and processed for immunocytochemistry. The preembedding method was applied in two cases and the postembedding method in the two others. 2.1. Preembedding immunocytochemical

labeling

We have used a commercially available antibody obtained from rabbits after immunization against the conjugates: GABA-Glutaraldehyde-Carriers (Biosoft). The specificity of this antibody was determined by radioimmunological tests. The cross-reactivity ratios for the following compounds: fl-alanine, glycine, taurine, aspartate and glutamate were, respectively, 1: 175, 1:795, 1:17 700, 1:50 000 and 1:50 0000 (Seguela et al., 1984). Free-floating sections were incubated in 5% normal goat serum (NGS) in PB saline containing 0.1% TritonXl00 (PBST, pH 7.6) for 1 h and placed overnight at 4°C in a polyclonal GABA antiserum diluted I:3000 or 1:5000 in PBST containing 1% NGS. After this primary serum incubation, the sections were rinsed in PBST and transferred to the secondary antibody solution, biotinylated anti-rabbit IgG (diluted 1:60 in PBST) for 1 h. The sections were then washed and incubated for 1 h with the Avidin-Biotin-Peroxidase complex (Vectastain ABC reagent, Vector). The enzymatic reaction was carried out using 0.01% hydrogen peroxide and 0.025% 3-3’-diaminobenzidine, in 0.05 M Tris-buffer (pH 7.6). The reaction time was checked under the light microscope and was found to range between 5 and 10 min. Control sections incubated using the same protocol, but omitting the primary antiserum, were completely devoid of specific immunolabeling. Sections of cerebellar cortex, where structures containing GABA immunoreactivity have been extensively described (Somogyi et al.,

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1985), were processed with the same method and examined by light microscopy for positive staining: GABA immunoreactivity was observed in the soma of Golgi and basket cells and in the varicosities surrounding Purkinje cell bodies. Sections were washed in 0.1 M PB (pH 7.4) and postfixed in 1% osmium tetroxide for 45 min, stained in an aqueous solution of uranyl acetate, dehydrated with graded ethanol and flat embedded in Epon between plastic coverslips. Blocks were trimmed out from the dorsolateral part of the ventral horn and glued to Epon cylinders. As immunolabeling was restricted to the superficial portion of tissue, ultrathin sections were taken close to the surface of the block and collected on collodion coated copper slot grids. The labeling of terminal profiles was checked on at least two consecutive ultrathin sections. 2.2. Postembedding immunocytochemical

staining

Ultrathin sections collected on butvar coated nickel slot grids were treated according to De Zeeuw et al. (1988). After preincubation in 5% NGS, sections were incubated overnight at 4°C with a polyclonal GABA antiserum (Biosoft) diluted 1:1000 in Tris-buffered saline: 0.05 M (pH 7.6) containing 0.1% Triton X-100 (TBST). After rinsing with TBST (pH 8.2) the sections were incubated for 1 h with goat-anti-rabbit antibody coupled to 15 nm gold particles (Biocell), diluted 1:40 in TBST. The ultrathin sections were subsequently stained with uranyl acetate and lead citrate. A terminal profile was considered immunopositive for GABA if the density of gold particles within the boundaries of the terminal was at least 4-5 times higher than that found in background and surrounding structures. The labeling of terminal profiles was checked on at least two consecutive ultrathin sections. Ultrathin sections were examined using an electron microscope (Siemens Elmiskop 102 or Jeol CX 100). Microphotographs (magnification: x 20 000) of synaptic profiles were taken of the ultrathin sections of postembedded material. Each immunopositive synaptic profile was examined for vesicle content and synaptic densities. Lengths of apposition and lengths of active zones were measured and systematically compared to those of terminals observed in standard material. 3. Results

3.1. Classification of terminals Terminals apposed to motoneurons were identified on the criteria we previously applied to our standard electron microscopy material (Destombes et al., 1992a), namely, size of terminal profile, number and shape of synaptic vesicles, densities associated with synaptic

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membranes and post-synaptic structures. S boutons were filled with numerous spherical vesicles, Their active zones had relatively wide synaptic clefts and were asymmetrical due to thick postsynaptic membrane densities. F boutons contained pleomorphic and flattened vesicles, and occasional dense core vesicles. The thin pre- and post-synaptic densities appeared as symmetrical layers on both sides of synaptic clefts. C boutons were the largest (4-9 pm), and contained spherical vesicles. The characteristic feature of these boutons was a subsynaptic cistern apposed directly to the postsynaptic membrane. M boutons appeared as large terminals (3-6 pm) contacting dendritic membranes, filled with spherical vesicles, forming prominent asymmetrical synapses, and characterized by multiple active zones with subsynaptic Taxi bodies in regular rows under the postsynaptic membrane. These dense bodies consist of material resembling that of postsynaptic densities. P boutons were small terminals apposed on M-type boutons, containing irregularly shaped vesicles. As it was not possible to systematically examine serial sections, synaptic specialization was not demonstrated for every terminal profile. Two categories of profiles were taken into account: (1) boutons exhibiting the three classical features of synapses (Fig. 1): clustering of vesicles near the presynaptic membrane, densities apposed to pre- and/or postsynaptic zones and synaptic cleft; (2) terminal profiles in close apposition with the motoneuron membrane over a minimal length of 0.5 pm and filled with synaptic vesicles. Using these criteria, the proportions of the different types of terminals were in the same range as in standard material (Destombes et al., 1992a) and in material treated for GABA immunocytochemistry.

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3.2. Identification

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of motoneurons

Motoneuron profiles were identified according to classical ultrastructural features (Conradi, 1969; Johnson, 1986). Distinction between (Y-and y-motoneurons was made not only by size but also using the three criteria previously established by Destombes et al., (1992a), namely, (1) ol-motoneurons have many more synaptic terminals than y-motoneurons; (2) a-motoneurons are contacted by four types of boutons, F-, S-, C- and M-type, whereas y-motoneurons only have F and S boutons; (3) the nucleolus of ar-motoneurons has a characteristic ‘lacy’ aspect contrasting with the compact nucleolus found in y-motoneurons. The distinction between y-motoneurons and interneurons of a similar size was unambiguous because the latter contained little cytoplasm, a rich Golgi apparatus, a poorly organized endoplasmic reticulum and had invaginated nuclei (Johnson and Sears, 1988). Labeled and unlabeled terminal profiles were counted on motoneuron cell bodies and dendrites in continuity with the soma and in addition on dendrites of CXmotoneurons which were identified by the presence of characteristic C-boutons. 3.3. Quantification of GABA-like minals in preembedded material

immunoreactive

ter-

GABA-immunoreactive terminals observed on the somatic and proximal dendritic membranes of lumbar motoneurons were intermingled with non-immunoreactive boutons (see Destombes et al., 1992a for definitions of somatic and proximal dendritic compartments). Labeled profiles were occasionally seen on dendritic spine-like protrusions (Fig. 1) or on more distal dendrites of motoneurons. In total, 877 terminals of the F-, S-, and C-types were counted on the somatic compartments of 18 CYmotoneurons (Table 1). In addition, 416 terminals were counted on longitudinally sectioned dendritic profiles seen in continuity with 11 somatic profiles and on 4 transverse sections of dendrites (range of diameters, Table 1 Frequency of the different types of GABA-labeled terminals on the somatic membranes of 18 lumbar o-motoneurons

Fig. 1. GABAergic terminals on lumbar a-motoneurons. Two labeled F boutons (1, 2) establishing symmetrical synaptic contacts (arrows) with a dendrite (D). Terminal 1 is enveloping a dendritic ‘spine-like protrusion’. Bar: 1 pm.

Number of terminals observed

% of the total

Number of unlabeled terminals

Number of labeled terminals

% of labeled terminals

F-type = 631 S-type = 193 C-type = 53

71.9 22.0 6.0

496 191 53

135 2 -

21.4 I.0 -

740

137

15.6

Total = 877

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Table 2 Frequency of the different types of GABA-labeled terminals on 15 proximal dendritic profiles of lumbar a-motoneurons Number of terminals observed

% of the total

Number of unlabeled terminals

Number of labeled terminals

% of labeled terminals

F-type = 302 s-type = 99 C-type = 15

12.5

243 98

59

19.5

23.1

3.6

Total = 416

1

1.0

15 60

356

16.8

5-12 pm) which could be identified as a-motoneuron dendrites by the presence of C-type terminals (Table 2). In these two samples, the proportions of F-type terminals were, respectively, 7 1.9 and 72.5% whereas those of S-type were 22.0 and 23.7%. Similar percentages were found in standard material (Destombes et al., 1992a) indicating that the imrnunohistochemical procedures did not significantly alter the ultrastructural features used to characterize the terminal types. The numbers of labeled and unlabeled terminals counted on the cell bodies of the 18 a-motoneurons are given in Table 3: the percentages of GABA-positive terminals were in a range of 9.5-25.4% (mean 15.6%). The corresponding figures were 12.8-34.1% (mean 16.8%) for the terminals counted on proximal dendritic compartments. Table 3 Numbers of labeled and unlabeled terminals on the cell bodies of 18 a-motoneurons Moto- Unlabeled terminals Labeled terTotal neuron ~ minals S-type F-type C-type ~ S-type F-type 1 2 3 4 5 6 7 8 9

10 11 13 14 15 16 17 18

9 4 12 14 5 4 16 18 8

42 16 24 29 15 23 30 22 42 22

4 2 3 2 3 2 5

15

31

I

12.0 8

9 22 23 20 35

23 2 2

11

10 4 13 19 12

40 37

1 5 2

I 3 5 8

I I -

9 6 9 5 7 6 6 9 8 8 2

-

16 10 6 4 6 7 6

64 28 47

48 40 36 46 48 73

40 63 7

42 41 29 57 71 63

% of labeled terminals 14.0 21.4 19.1 10.4 20.0 16.6 15.2 18.7 10.9 20.0 25.4 41 23.8 14.6 13.8 10.5 9.8 9.5

Fig. 2. Presynaptic GABAergic terminals on an M terminal. Two GABAergic boutons (arrows) apposed to an unlabeled M bouton contacting a proximal dendrite (D). The M bouton shows several active zones underlined by Taxi bodies (triangles). Bar: I pm.

Most of the GABA-immunoreactive terminals were identified as F-type boutons because they had symmetrical synaptic specializations showing densely packed pleomorphic and clear vesicles. Of the 631 F-type terminals counted on somatic compartments, 135 (21.4”/) were GABA-immunoreactive and the proportion was similar for the proximal dendritic compartment with 59 of 302 (19.5O/) F-type terminals displaying GABA immunoreactivity (Tables 1 and 2). In contrast, of the 292 S-type boutons counted on both somatic and dendritic compartments, only 3 were labeled and immunoreaction product was absent from all of 68 C-type boutons (Tables 1 and 2). M-type boutons, mainly observed on proximal dendrites, were usually contacted by one or two P terminals. None of the 20 M boutons observed showed any labeling but all of the 14 axo-axonic P-boutons apposed to them were irmnunopositive (Fig. 2). In the present material, the sample of y-motoneurons was smaller than that of a-motoneurons since they correspond to only 30-40% of the lumbar motor pools (Horcholle-Bossavit et al., 1988). In addition, ymotoneurons receive less synaptic contact than (Ymotoneurons. They had very few GABA-immunoreactive terminals and, in a total of 62 boutons counted on the somatic and dendritic membranes of 6 ymotoneurons, only 8 F-type terminals showed characteristic labeling (12.9%). The mean number of GABA positive terminals per somatic profile on y-motoneurons was 1.3, as opposed to 7.6 per profile on omotoneurons. 3.4. Morphological features of GABA like immunoreactive synapsesexamined in postembeddedmaterial

Clear differences in densities of gold particles ap-

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Fig. 3. Immunogold labeling of GABAergic boutons. A: GABAergic bouton (I) containing pleomorphic vesicles and establishing symmetrical synaptic contact (black arrow) with a dendrite (D). Gold particles are numerous on vesicle clusters and mitochondria. A nearby unlabeled bouton (2) contains round vesicles. B: GABAergic bouton (I) containing pleomorphic vesicles and establishing two symmetrical synaptic contacts (black arrows) with a dendrite (D) and a dendritic ‘spine-like protrusion’ (sp) which is contacted by an unlabeled terminal (3) containing pleomorphic vesicles.The dendrite is also contacted by an unlabeled bouton (2) showing an asymmetrical active zone and Taxi bodies. A myelinated fiber (Ax) is labeled by numerous gold particles. Bar: 1 pm.

Fig. 4. Presynaptic GABAergic terminal on an M terminal. GABAergic bouton labeled with gold particles (star) contacting an unlabeled M bouton showing several actives zones (triangles). (D): proximal dendrite. Note the location of gold particles on the mitochondrion. Bar: 1 pm.

Fig. 5. GABAergic P boutons (stars) apposed to an M terminal located on an axon hillock (AH). Bar: 1 pm.

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peared between labeled and unlabeled terminals (Fig 3). Gold particles were numerous on vesicle clusters and on mitochondria. Grain density varied in a range of 25-85 particles/pm2 on labeled profiles as opposed to 2-l 1 particles/pm2 for background and unlabeled structures. A large majority of labeled F boutons contained numerous irregularly shaped or flattened vesicles and consistently formed symmetrical synaptic junctions (Fig. 3A and B). Labeled P-boutons apposed to M terminals were small, contained pleomorphic vesicles and their active zones were short (Fig. 4). Occasionally, labeled P-boutons were seen on the axon hillock of an a-mototoneuron (Fig. 5). The lengths of terminal appositions were measured in samples taken from two types of material (F boutons in non-stained material and labeled F boutons in postembedded material). The mean apposition lengths of GABA-positive terminals were: 1.99 f 0.54 pm (range l-4.5 pm, n = 130) in postembedded material, compared to 2.38 f 0.92 pm (range l-5.5 pm, n = 282) for F-boutons in standard material. The mean lengths of active zones measured in 107 labeled boutons from postembedded material and in 109 F-type boutons from standard material were, respectively, 0.34 f 0.13 and 0.37 f 0.12 pm. Large GABA-positive boutons often had several active zones of a similar size. The percentages of boutons exhibiting two or three actives zones were 19% and 15%, respectively, in the two samples. All examined P boutons apposed to M terminals were immunopositive (Figs. 4, 5).

4. Discussion Pre- and postembedding methods were used in the present study for two complementary approaches. With the preembedding method, the labeled terminals can be identified and counted on motoneuronal profiles, allowing a comparison to be made with a previous quantification of glycinergic terminals obtained using the same method (Destombes et al., 1992b). It is known that, in prembedded material, the penetration of antibodies is restricted to the superficial portion of sections. But the fact that all the P boutons observed in our prembedded material were GABA-immunoreactive indicates that restriction of antibody penetration does not bias the quantitative assessment of GABA-positive terminals and allows a direct comparison to be made with the previous study of glycinergic terminals. In prembedded material, however, synaptic complexes were not evident on every terminal profile of a particular plane of section. Postembedding immunocytochemistry on ultrathin sections was therefore used in order to gain morphometric details about GABA-positive synapses. In our material, most GABA-like immunoreactive terminals on lumbar motoneurons contained irregularly

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shaped or flattened vesicles, termed F-type terminals. They resembled the F boutons described on cat sacral motoneurons (Ramirez-Leon and Ulfhake, 1993). Pleomorphic or flattened vesicles were also observed in GABAergic synapses of rat lumbar (McLaughlin et al., 1975; Holstege and Calkoen, 1990) and trigeminal motoneurons (Saha et al., 1991). The presence of numerous axo-somatic and axodendritic GABAergic terminals on a-motoneurons indicates that GABA plays a significant role in postsynaptic inhibition. A possible origin for these GABAimmunoreactive terminals could be the GABAergic intemeurons located throughout the dorsal horn laminae (Barber et al., 1982). In addition, the presence of GABA immunoreactive cell bodies and dendrites observed in the vicinity of cat lumbar motoneurons was also reported for the human spinal cord @Valdvogel et al., 1990). Some of the GABAergic terminals could be responsible for the inhibitory mechanisms that are suppressed by bicuculline, a specific GABA antagonist. Thus, three GABAergic inhibitory responses were reported in cat lumbar motoneurons, namely (1) a component of stretch-activated inhibition (Kellerth and Szumski, 1966); (2) recurrent inhibition from a specific sub-population of Renshaw cells (Cullheim and Kellerth, 1981; Schneider and Fyffe, 1990); (3) inhibition of a-motoneurons by a population of interneurons (class II interneurons of Rudomin, 1990) which could also be involved in presynaptic inhibition (see infra). A supraspinal origin for some of the GABAergic terminals found on lumbar motoneurons is suggested by the presence of GABAergic neurons in the caudal medullary raphe (Millhorn et al., 1987), in the ventromedial reticular formation of the lower brainstem (Holstege, 1991) and in the lateral vestibular nucleus (Blessing et al., 1987; Walberg et al., 1990) which are known to send projections to the ventral spinal cord. GABA-immunoreactive terminals corresponded to an average 16.2% of the total terminal complement on the somatic and proximal dendritic compartments of cat lumbar ar-motoneurons (Tables 1 and 2). A similar study on a group of motoneurons with different function and location indicated that 25% of the vesicle-containing axonal profiles were GABAergic (Ramirez-Leon and Ulfhake, 1993) with a preferential location on medium and large dendrites. A comparison with the proportions of glycinergic terminals counted in a study using a similar methodology (Destombes et al., 1992b) indicates that glycinergic terminals accounted for 33% of the Ftype terminal population as opposed to an average of 16.2% GABAergic F boutons (range 9.5-34.1%). These proportions are in agreement with a recent light microscopic study which provided a quantitative estimate of the somatic covering of cat lumbar a-motoneurons by immunoreactive puncta (Shupliakov et al., 1993). These authors showed that glycinergic terminal-like structures

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are the most numerous, covering 26-42% of the somatic membrane, while GABAergic terminals only cover lo-24% of the cell body surface. Pharmacological data (Kmjevic et al., 1977) suggest that glycine could exert fast, potent and sustained inhibition, whereas GABA could have slow, discrete long-duration effects subtly modulating the conductance of the motoneuronal membrane. In the rat spinal cord it has been shown that GABA-immunoreactive terminals are apposed to postsynaptic glycine receptors present on the motoneuronal membrane (Triller et al., 1987). This suggests that GABA could mediate postsynaptic inhibition by acting on chloride ionophores associated to both GABA,,, and glycine receptors. Interactions between the two inhibitory neurotransmitters could be expected since coexistence of GABA and glycine in neurons of the rat dorsal horn has recently been reported (Todd and Sullivan, 1990) and GABAA and glycine receptors are widespread on rat motoneuronal surfaces (Seitanidou et al., 1988). In addition, the coexistence of glycine and GABA in nerve terminals on cat spinal motoneurons has recently been reported @mung et al., 1994; Tall and Holstege, 1994). Axo-axonic synapses modulating primary sensory afferent excitability are a common feature in the spinal cord of vertebrates from the lamprey (Christenson et al., 1991, 1993) to mammals. Axo-axonic contacts with GABA immunoreactivity in the presynaptic elements were described in the rat motor neuropil (Holstege and Calkoen, 1990). In the cat spinal cord, the morphological counterpart of presynaptic inhibition was suggested to be the P boutons which establish synaptic contacts with large S- or M-type terminals. The latter are considered to be the endings from Ia afferents which monosynaptically excite ar-motoneurons (Conradi, 1969; Pierce and Mendell, 1993). Our observations show for the first time that P boutons apposed to M terminals are GABA-immunoreactive and support the hypothesis that GABA mediates presynaptic inhibition of Ia afferent connections. The absence of P and M boutons on y-motoneurons is in agreement with the lack of Ia excitation on these neurons. However, GABA is also present in presynaptic synapses located on terminals of Ia afferents contacting interneurons which mediate nonreciprocal inhibition (Maxwell et al., 1990, Jankowska, 1992). The paucity of GABAergic terminals on y-motoneurons, similar to that of glycinergic terminals (Destombes et al., 1992b), could account for the fact that y-motoneurons display a high excitability under many experimental conditions (Kemm and Westbury, 1978) and discharge for long periods at high rates up to 100 discharges/s during locomotion in the decerebrated cat (Murphy et al., 1984). Low synaptic covering on the somatic and proximal dendritic membranes on ymotoneurons is well known (Lagerback, 1985; Destom-

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bes et al., 1992a) but information about the more distal regions are still lacking. In this regard, functional differences between (r- and y-motoneurons were explained by a comparative analysis of their dendritic morphologies and electrotonic architectures (Moschovakis et al., 1991; Burke et al., 1994). In addition to the inhibitory supraspinal tracts which exert a parallel control of y- and ar-motoneurons (see supra), some of the GABAergic inhibitory effects on ymotoneurons could originate from specific central sites, for instance the reticular zone of the substantia nigra (Wand et al., 1981) and contribute to dissociate y- from o-activity. Acknowledgments The authors wish to thank Dr. L. Jami for valuable discussion and for critical reading of the manuscript and Dr. G. Butler-Browne for scrutinizing the English. Thanks are also due to Mr. G. Masquelier for excellent photographic work. This research was supported by the Fondation pour la Recherche Medicale. References Barber, R.P., Vaughn, J.E. and Roberts, E. (1982) The cytoarchitectonic of GABAergic neurons in the rat spinal cord. Brain Res., 238: 305-328. Blessing, W.W., Hedger, SC. and Oertel, W.H. (1987) Vestibulospinal pathway in rabbit includes GABA-synthesizing neurons. Neurosci. Lett., 80: 158-162. Burke, R.E., Fyffe, R.E.W. and Moschovakis, A.K. (1994) Electrotonic architecture of cat gamma motoneurons. J. Neurophysiol., 72: 2302-23 16. Christenson, J., Bongianni, F., Grillner, S. and Hiikfelt, T. (1991) Putative GABAergic input to axons of spinal interneurons and primary sensory neurons in the lamprey spinal cord as shown by intracellular lucifer yellow and GABA immunohistochemistry. Brain Res., 538: 313-318. Christenson, J., Shupliakov, O., Cullheim, S. and Grillner, S. (1993) Possible morphological substrates for GABA-mediated presynaptic inhibition in the lamprey spinal cord. J. Comp. Neurol., 328: 463-472. Conradi, S. (1969) Ultrastructure and distribution of neuronal and glial elements on the motoneurone surface in the lumbosacral spinal cord of the adult cat. Acta Physiol. Stand. Suppl., 332: S-48. Cullheim, S. and Kellerth, J.-O. (1981) Two kinds of recurrent inhibition of cat spinal a-motoneurons as differentiated pharmacologically. J. Physiol., 312: 209-224. Curtis, D.R. and Lodge, D. (1982) The depolarization of feline ventral horn group Ia spinal afferent terminations by GABA. Exp. Brain Res., 46: 215-233. Curtis, D.R., Duggan, A.W., Felix, D. and Johnston, G.A.R. (1971) Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat. Brain Res., 32: 69-96. Curtis, D.R., Lodge, D., Bomstein, J.C., Peet, M.J. and Leah, J.U. (1982) The dual effects of GABA and related amino acids on the electrical threshold of ventral horn group Ia spinal afferent terminations in the cat. Exp. Brain Res., 48: 387-400. De Zeeuw, CL, Holstege, J.C., Calkoen, F., Ruigrok, T.J.H. and Voogdt, J. (1988) A new combination of WGA-HRP anterograde tracing and GABA immunocytochemistry applied to afferents of the

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