Brain Research, 561 (1991) 128-138 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391170396

128

BRES 17039

Quantitative analysis and postsynaptic targets of GABA-immunoreactive boutons within the rat trigeminal motor nucleus Sikha Saha 1, Kwabena Appenteng 1 and Trevor F.C. Batten 2 Departments of 1Physiology and 2CardiovascularStudies, University of Leds, Leeds (U.K.) (Accepted 21 May 1991)

Key words: y-Aminobutyric acid; Electron microscopy; Trigeminal; Motoneuron; Synapse

We have used the post-embedding immunogold labelling method using antibodies to y-aminobutyric acid (GABA) to obtain quantitative data on the distribution, frequency, postsynaptic targets and ultrastructural characteristics of GABA-immunoreactive (GABA-IR) boutons in the trigeminal motor nucleus of rats. We have also combined this method with horseradish peroxidase tracing to obtain specific evidence for termination of some GABA-IR boutons onto identified jaw-elevator motoneurones. Twenty-eight percent of all synapses in the motor nucleus involved GABA-IR boutons. Seventy-three percent of the GABA-IR boutons formed axo-dendritic synapses, 13% axo-somatic synapses and 14% axo-axonic synapses. Ninety-three percent of GABA-IR boutons formed symmetrical synapses. Overall, 58% of all boutons contained only flattened vesicles, while 26% contained round vesicles and 16% a mixture of vesicle types. Measurements of bouton cross sectional area, apposition length, and active zone length were obtained from serial reconstructions of 15 GABA-IR boutons and 30 unlabelled boutons. In each case mean values for GABA-IR boutons were significantly smaller than those for nonlabelled boutons. INTRODUCTION Thus far two sources of monosynaptic excitatory input onto jaw-elevator m o t o n e u r o n e s have been examined in detail, and in each case the amplitude of the unitary excitatory postsynaptic potentials (EPSPs) appears to be considerably less than that observed at the m o r e c o m m o n l y studied synapse of hindlimb muscle spindle afferents onto hindlimb m o t o n e u r o n e s . F o r example, elevator muscle spindle afferents elicit unitary EPSPs o f m e a n amplitude 18/~V in elevator m o t o n e u rones (cat2), while interneurones situated in the region immediately caudal to the trigeminal m o t o r nucleus elicit EPSPs of less than 30/~V (rat4). In contrast, lumbosacral spindle afferents elicit unitary EPSPs of m e a n amplitude 100/~V in h o m o n y m o u s m o t o n e u r o n e s (for review see ref. 20). The i m m e d i a t e question p r o m p t e d is why should the excitatory effects on elevator m o t o n e u r o n e s be so much w e a k e r than those on hindlimb m o t o n e u tones? A m o n g the possibilities that could account for this difference is that excitatory synapses onto trigeminal m o t o n e u r o n e s are subject to a tonic inhibition. The inhibition could be exerted on the presynaptic terminals onto m o t o n e u r o n e s , on the m o t o n e u r o n e s themselves, or perhaps even at both sites.

A m o n g the strong possible candidates for the role of inhibitory transmitter is y-aminobutyric acid ( G A B A ) . T h e r e is a wealth of evidence suggesting that G A B A may be a m e d i a t o r of inhibitory synaptic actions in a n u m b e r of different areas of the central nervous system (CNS), and that it exerts its actions via at least two different types of receptors 8'1°'12"16. Light microscopical (LM) studies have revealed that the trigeminal m o t o r nucleus is innervated by fibres which are immunoreactive for glutamate decarboxylase (GAD29), the enzyme involved in G A B A synthesis, and so this result implies that G A B A - i m m u n o r e a c t i v e ( G A B A - I R ) fibres m a y terminate within the trigeminal m o t o r nucleus. H o w e v e r , the postsynaptic targets of the G A B A - I R fibres were not identified, nor is there any quantitative data on the frequency of occurrence of either G A D or G A B A - I R synaptic b o u t o n s within the trigeminal m o t o r nucleus. It has therefore not been possible to assess the importance of G A B A inputs in the control of masticatory m o t o n e u r o n e behaviour. The p r o b l e m s are confounded by the additional fact that there have b e e n no electrophysiological studies dealing with the specific question of whether G A B A can act as a neurotransmitter within the trigeminal m o t o r nucleus. Quantitative electron microscopical ( E M ) immunohis-

Correspondence: K. Appenteng, Dept. of Physiology, Univ. of Leeds, Leeds LS2 9NQ, U.S. Fax: (44) (0532) 334381.

129 t o c h e m i c a l studies, such as h a v e b e e n p e r f o r m e d in t h e visual c o r t e x 7, spinal c,o r d 21'37, basilar p o n t i n e n u c l e u s 27, h y p o t h a l a m u s 14, a n d n u c l e u s tractus solitarius 24, c a n b e used

to o b t a i n

data

on

the distribution,

frequency,

p o s t s y n a p t i c targets a n d u l t r a s t r u c t u r a l characteristics o f specific sets o f G A B A - I R

b o u t o n s in d i s c r e t e a r e a s o f

t h e C N S . This g e n e r a l a p p r o a c h has b e e n g r e a t l y facilit a t e d by t h e i n t r o d u c t i o n o f t h e p o s t - e m b e d d i n g i m m u n o g o l d l a b e l l i n g m e t h o d using a n t i b o d i e s to G A B A 9'34. W e h a v e u s e d this m e t h o d to o b t a i n d a t a o n t h e distribution of the GABA-IR

i n p u t to t h e t r i g e m i n a i m o t o r

nucleus o f the rat. W e h a v e also c o m b i n e d this m e t h o d with horseradish peroxidase

(HRP)

tracing to o b t a i n

specific e v i d e n c e f o r t e r m i n a t i o n o f s o m e G A B A - I R boutons onto jaw-elevator motoneurones. A preliminary abstract o f this w o r k has b e e n p u b l i s h e d p r e v i o u s l y 32.

MATERIALS AND METHODS

Fixation Rats in the weight range 200-300 g were anaesthetised with a mixture of haiothane in air and perfused through the ascending aorta with 100 ml of a Tyrode's solution (pH = 7.4), then with 400 ml of fixative, which was either 5% glutaraldehyde or 2.5% glutaraldehyde-0.5% paraformaldehyde. Both fixatives were freshly made in 0.1 M phosphate buffer (pH = 7.4) and chilled to 4 °C before use. The brains were quickly removed after fixation and sliced transversely into 1-2 mm thick blocks, which were then postfixed in the same fixatives for either 2 h (5% glutaraldehyde) or 18 h (2.5% glutaraldehyde-0.5% paraformaldehyde).

Tissue preparation for EM Ten rats were used for this part of the study, and the tissue blocks from each were rinsed thoroughly in 0.1 M phosphate buffer and then sectioned on a vibratome at a thickness of 200/~m. They were post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 20 rain, and dehydrated through graded alcohols and flat-embedded on glass slides under Thermanox plastic coversllps in Spurr's resin. After curing the resin at 60 °(2 for 24 h, the coverslips were peeled off and the resin wafer removed from the slide. The areas containing trigeminal motoneurones were examined under LM, cut out and remounted onto fresh resin blocks. Some semi-thin (1-2 /~m) resin sections were cut for orientation purposes and were collected onto gelatinised glass slides. Ultrathin sections of approximately 100 um thickness were collected onto 200 mesh uncoated nickel grids.

EM immunohistochemistry Sections were initially etched with 10% hydrogen peroxide for 15 rain to remove the resin and osmium from the surfaces of the sections, rinsed thoroughly with filtered deionised water and then rinsed with 0.1 M tris-saline buffer (TBS: pH = 7.6, filtered using a 0.4/~m filter). Sections were pre-incubated for 30 min with 10% pre-immune goat serum in 0.1 M TBS supplemented with 1% bovine serum albumen, 0.2% sodium azide, 0.1% Triton-X-100 and 0.2% EM grade gelatine. The grids were then placed for 24 h at 4 °C in drops of rabbit anti-GABA serum diluted 1:500-1:1000 in the supplemented TBS. Production and characterisation of the antiserum has been described previously by Seguela et ai. 33 and Buijs et al.9. The grids were then rinsed twice in the supplemented TBS and placed in drops of immunogold reagent. This consisted of a 1:20 dilution of colloidal gold particles (10 nm diameter) coated with goat-anti-rabbit IgG (Biocell) in supplemented TBS (pH = 8.2). Following this incubation the grids were thoroughly washed

in deionised, filtered water. Sections were contrast stained with uranyl acetate and lead citrate prior to EM examination. The control performed for the immunohistochemical method was to leave out the primary antibody incubation step but to carry out all subsequent steps. The only staining then seen was the occasional clumping of a few gold particles over some myelin sheaths.

HRP tracing Four of the 10 rats were used for a combined HRP and GABAimmunohistochemical study. The rats were anaesthetised with pentobarbitone (36 mg/kg i.p.) and the skin over the masseter muscle incised. Forty to 80/d of a 40/~g//d solution of HRP (Sigma Type VI) was injected into the masseter muscle, the skin closed and the animals allowed to recover from the anaesthetic. After survival times of 24-48 h the animals were anaesthetised with a mixture of haiothane in air and perfnsed through the ascending aorta with a Tyrode's solution and then with 400 ml of the 5% glutaraldehyde fixative described above. The brains were removed and postfixed in the same fixative for 2 h and then sectioned at 50 #m on a vibratome. Sections were processed to reveal HRP activity using the tetramethyl-benzidine method described by Mesulam ~. The only modification of the method employed was the use of a pH 4.8 acetate buffer for the postreaction rinse, as this resulted in better ultrastructural preservation ~. The TMB reaction product was stabilized by reacting it with DAB and cobalt acetate as described by Rye et al. 31. Sections containing the trigeminal motor nucleus were then processed for EM analysis as described above.

Tissue preparation for LM Tissue to be examined under LM was sectioned on a vibratome at a thickness of 50 /~m, collected in 0.1 M phosphate buffer, washed in TBS and then incubated for 30 min with 10% pre-immune goat serum in the supplemented TBS. This was followed by a 24 h incubation at 4 °C in the anti-GABA serum at the dilutions described above. The antibody-antigen reaction was then visualised using a biotinylated anti-rabbit second antibody (1:50 dilution; Amersham) and streptavidin-HRP (1:200 dilution; Amersham). By way of control some sections were not incubated in the primary antibody but otherwise treated as above. No staining was subsequently seen in these sections. Semi-thin resin sections were stained by the same procedure.

EM Data analysis (a) Criteria for positive staining. A structure was considered to be positively labelled if the density of gold particles over it was at least 5 times that over surrounding non-immunoreactive structures. (b) Identification of synapses. The criteria used were the presence of an electron dense thickening associated with presynaptic vesicles as described by Gray 19. (c) Classification of postsynaptic targets of boutons. Somata were identified by the presence of dense stacks of rough endoplasmic reticulum and a nucleus. Axons were differentiated from dendrites by the absence of endoplasmic reticulum and ribosomes, and boutons identified by the presence of densely packed vesicles coupled with the absence of endoplasmic reticulum.

(d) Classification of GABA-IR boutons on basis of vesicle types. We performed a systematic determination of the vesicle types present in a random sample of GABA-IR boutons. Vesicles were classified as being either flattened or round (ovoid), and the relative proportion of each type in selected boutons was determined. Boutons could then be classified into 3 separate types; (1) those containing mainly flattened vesicles, (2) those containing mainly round, or (3) those containing a mixture of flattened and round vesicles.

(e) Determination of proportion of GABA-IR boutons in the trigeminal motor nucleus. A total of 12 grids containing immuno-stained sections from 6 different animals were randomly selected prior to initiai examination of the material under EM. Each grid was then placed into the EM and the beam randomly focused onto two squares in the grid containing sections from the trigeminal motor nucleus. All bou-

130 tons forming synapses within these squares were then surveyed and the numbers of GABA-IR and non-immunoreactiveboutons counted. (39 Quantitative reconstructions of single boutons. Serial reconstructions of boutons were performed in order to obtain reliable measurements of bouton cross-sectional area, apposition length, and length of active zone. Boutons were photographed at a magnification of 10,000-20,000x and reconstructions made from the enlarged negatives at final overall magnifications of 78,Mf)-l10,600x. Reconstructions were only performed for those boutons where we obtained a minimum of at least 4 consecutive serial sections which passed through a point of maximum bouton diameter. Commonly at least 5 sections were used for most reconstructions and on occasions 7 sections. Measurements of bouton parameters were performed using a digitising tablet in association with a Video#an image analysis system (Kontron). Each measurement was repeated 6 times and the average then taken to give the final value. RESULTS O u r first step was to ascertain that we could obtain discrete staining at the LM level in areas well k n o w n to contain G A B A immunoreactive structures. For this we focused our attention particularly o n the cochlear nucleus and superior olive because of their proximity to the trigeminal motor nucleus, but other areas examined ineluded the cerebellum, vestibular nuclei, and visual cor-

tex. Fig. 1 shows an example of labelling of n e u r o n a l structures in the superior olive (Fig. la). U n d e r the same conditions no labelled cells were seen in the trigeminal motor nucleus but there was a profusion of labelled fibres within the nucleus. The degree of fibre labelling was such that semi-thin resin sections (1-2 /zm) were required for clear visualisation of individual fibres and their varicosities (Fig. lb). No labelled cells were seen in the trigeminal motor nucleus in these semi-thin sections. In 50/~m LM sections labelled cells were seen in the nearby trigeminal main sensory nucleus and in the ventral part of the rostral pole of the trigeminal nucleus oralis. The dorsal part of this pole of the trigeminal nucleus oralis has been shown to contain interneurones, many of which make excitatory monosynaptic connections onto jaw-elevator m o t o n e u r o n e s 1"3. We did not search for labelled neurones in trigeminal areas more than 1 m m caudal to the trigeminal motor nucleus. Using the post-embedding E M immunogold staining method, G A B A - I R boutons were seen to make either axo-axonic (Fig. 2), axo-dendritic (Fig. 3a,c) or axo-somatic (Fig. 3b,d) contacts with various postsynaptic targets in the motor nucleus. Typically axo-axonic synapses

Fig. 1. LM views of GABA-IR structures in (a) the superior olive, and (b) the trigeminal motor nucleus. Section in (a) is a 50/~m vibratome section, and in (b) 1-2/,m semi-thin resin section. Note in (b) the profusion of GABA-IR fibres (arrows) around the unlabelled soma (S) of a neurone in the motor nucleus. Note labelling of somata in (a) but not in (b). Bars = 50/tm.

131

Fig. 2. Axo-axonic contacts formed by GABA-IR boutons in the trigeminal motor nucleus, a: a GABA-IR bouton (GT) synapses (open arrowhead marks point of contact) onto an unlabeUed axon profile (AX) which in turn synapses onto a dendrite (D; filled arrowhead marks point of contact), possibly by an electrical synapse. Note that another GABA-IR bouton also synapses onto the same dendrite (small arrow marks point of contact) but by means of a classical chemical synapse, b: high power view of contact formed by GABA-IR bouton onto the unlabelled axon. The asterisk in GT marks a dense cored vesicle ad the curved arrow heads in AX mark examples of 'coated vesicles'. Note that 'coated vesicles' are also present in GT. c: contact formed by a GABA-IR bouton (GT) onto another unlabelled axon (AX) which in turn synapses onto the soma (S) of a neurone. Open arrowheads mark points of synaptic contact. A second GABA-IR bouton can be seen synapsing with the soma of the same neurone. Additional GABA-IR axons not forming synapses onto structures in the micrograph are labelled (G). d: high power view of contact formed by the GABA-IR bouton in (c) onto the unlabelled axon. Note the presence of 'coated' vesicles in AX (curved arrowheads). Bars = 0.5 gin.

consisted of a small G A B A - I R b o u t o n presynaptic to a much larger unlabelled axon. T h e postsynaptic structure in axo-axonic synapses in turn synapsed either onto a dendrite o r a cell body, usually by a classical chemical synapse (Fig. 2c), but occasionally by a presumptive electrical synapse (Fig. 2a). Irrespective of the type of contact formed, all G A B A - I R b o u t o n s were invariably presynaptic to non G A B A - I R structures (Figs. 2 and 3). A x o - a x o n i c synapses m a y p r o v i d e a means o f presynaptic m o d u l a t i o n of transmission o n t o neurones, whereas the axo-dendritic and axo-somatic m a y be the basis for postsynaptic effects. W e d e t e r m i n e d the total p r o p o r t i o n of synapses in the m o t o r nucleus involving G A B A - I R b o u t o n s using the p r o c e d u r e described in section ( ' e ' ) o f Materials and Methods. W e surveyed 375 synapses and found that 106 (28%) involved G A B A - I R boutons, while 269 (72%) in-

volved non G A B A - I R boutons. The relative frequency of axo-axonic, axo-dendritic and axo-somatic synapses involving G A B A - I R b o u t o n s was assessed in a s e p a r a t e survey. The sample for this consisted o f 314 G A B A - I R boutons, of which 229 (73%) were found to form axodendritic synapses, 42 (13%) axo-somatic synapses and 43 (14%) axo-axonic synapses. Ninety-three p e r c e n t of G A B A - I R b o u t o n s f o r m e d symmetrical synapses, b u t we could not distinguish b e t w e e n symmetrical and asymmetrical contacts in the remaining 7% of cases (e.g. Fig. 3b,c). Specific evidence as to the identity o f some o f the structures contacted b y G A B A - I R b o u t o n s was o b t a i n e d by combining G A B A - i m m u n o h i s t o c h e m i s t r y with the r e t r o g r a d e labelling o f elevator m o t o n e u r o n e s with HRP. Fig. 4 shows examples of synapses f o r m e d by G A B A - I R b o u t o n s onto the s o m a t a (Fig. 4) and dendrites (Fig. 5)

132

Fig. 3. Axe-dendritic (a,c) and axe-somatic (b,d) contacts formed by GABA-IR boutons (GT) within the trigeminal motor nucleus. Sites of synaptic contact marked by open arrowheads. The boutons in (a)(b) and (d) form symmetrical synapses but the synapse in (c) cannot be categorised with certainty as either symmetrical or asymmetrical. Note the presence of ‘coated’ vesicles in (b) and (d) (curved arrow heads), and dense cored vesicles in (a) (filled arrowheads). Boutons in (a) and (c) classified as containing round vesicles, bouton in (b) contains mixed vesicles, and bouton in (d) flattened vesicles. D, dendrite; S, soma. Bars = 0.5 pm.

of HRP labelled elevator motoneurones. A practical difficulty encountered here was that processing of the tissue for HRP labelling resulted in a decrease in the intensity of the subsequent labelling. We were therefore unable to quantify the relative proportion of synapses formed by GABA-IR boutons on labelled motoneurone dendrites and somata. Our assumption is that the majority of synapses formed onto neurones within the motor nucleus must be onto motoneurones, as few interneurones have been reported within the motor nucleus. On this basis the high number of GABA-IR synapses formed onto dendrites is perhaps not surprising given that the dendritic surface area accounts for 99% of the total surface area of individual motoneurones28. A qualitative impression given in Fig. 2 is that there may be pronounced differences in size of GABA-IR boutons and that some may be considerably smaller than non GABA-IR boutons. However, questions as to differences in bouton size are best resolved by making measurements from serially reconstructed boutons, as this provides the only sure means of avoiding spurious dif-

ferences due to boutons being sectioned at different points. Fig. 6 illustrates the change in bouton size in a series of 5 consecutive serial sections through a GABA-IR terminal. Similar reconstructions have been obtained from 14 other GABA-IR boutons and also from 30 unlabelled boutons. All the reconstructed boutons synapsed onto either dendrites or somata, and so our sample may suffer from the bias of excluding boutons forming axo-axonic synapses. Fig. 7A shows the distribution of values of cross-sectional areas obtained for both GABA-IR (filled bars) and unlabelled boutons (open bars). The mean cross-sectional area for unlabelled boutons was 1.99 pm2 (range = 0.62-4.92; S.D. = 1.05) and 1.26 pm2 for GABA-IR boutons (range = 0.69-1.89; S.D. = 0.39), the difference in the means being statistically significant (P < 2%). Apposition length is a commonly measured parameter which is taken as a reflection of the extent of the synaptic contact formed by a bouton on a postsynaptic structure (Fig. 7B). Here again there was a difference in values obtained for the two sets of boutons with the mean for unlabelled bou-

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Fig. 4. Low (a) and high (b) power views of the symmetrical synaptic contact (open arrowhead) formed by a GABA-IR bouton (GT) onto the soma of an HRP labelled elevator motoneurone (MN). HRP reaction product marked by asterisks. Bouton classified as containing round vesicles.

tons being 1.63 # m (range = 0.75-2.73; S.D. = 0.48) and 1.21 # m for G A B A - I R boutons (range = 0.41-2.33; S.D. 0.45; Fig. 7B). However, apposition length overestimates the functional extent of synapses as no distinction is made between electron dense and non-electron dense regions of synapses. A better parameter to use is the length of the active zone, as this measures just the length of the electron dense portions of the synapse (Fig. 7C). Values of active zone length for unlabelled boutons ranged from 0.58-2.26 ~ m (mean = 0.96; S.D. = 0.35)

and from 0.23-1.3 # m (mean = 0.77; S.D. = 0.36; Fig. 7C) for G A B A - I R boutons, the difference being significant at the 5% level. Thus G A B A - I R boutons are both smaller and make less extensive synapses than unlabelled boutons. Finally, we examined the vesicle types present in individual sections through G A B A - I R boutons and then used this to classify the boutons into o n e of the 3 categories described in Materials and Methods (see section 'd'). The justification for this was that in the serial re-

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Fig. 5. Low (a) and high (b) power views of the symmetrical synaptic contact (open arrowhead) formed by a GABA-IR bouton (GT) onto the dendrite (D) of an HRP labelled elevator motoneurone. HRP reaction product marked by asterisks. Bouton classified as containing mixed vesicles.

constructions we found that the type of synaptic vesicles within individual boutons remained constant from section to section (Fig. 6), implying that the synaptic vesicle type within boutons could be reliably determined from single sections 11. Fig. 8 shows the relative incidence of the 3 categories of boutons in synapses formed by G A B A - I R endings. Overall 58% (183/314) of boutons contained flattened vesicles, and so would correspond to classical inhibitory terminals ss, while 26% (82/314) contained round vesicles and 16% (49/314) a mixture of ves-

icle types. Boutons containing flattened vesicles accounted for 79% of endings forming axo-axonic synapses and 67% of endings making axo-somatic contacts. However, only 57% of axo-dendritic contacts were formed by boutons containing flattened vesicles. Dense cored vesicles were found in 52 of the 314 G A B A - I R boutons surveyed and were present in boutons containing either flattened, round or mixed vesicles (Fig. 2b, 3a and 6). The incidence of dense cored vesicles in the endings forming different contacts was 19% for axo-axonic, 14% for axo-

135 somatic and 17% for axo-dendritic. Unlike synaptic vesicles, dense cored vesicles appear to be segregated within the bouton, and so their true incidence can only be reliably estimated from serially reconstructed boutons (Fig. 6; also ref. 14). In our sample of reconstructed axo-dendritic and axo-somatic boutons the incidence of dense cored vesicles was 33%. In addition, many G A B A - I R

and unlabelled boutons also contained what we have termed 'coated' vesicles (e.g. Figs. 2 and 3). These do not appear to be the same as classic coated vesicles known to be involved in pinocytosis or membrane capture, as the vesicles in our material had both a dense matrix and thicker wall. We are not aware of previous reports of similar vesicles in boutons but our assumption is that our 'coated' vesicles may be a type of synaptic vesicle. DISCUSSION

Fig. 6. Serial sections through a GABA-IR bouton which formed an axo-dendritic contact (arrows mark limits of active zone of synapse) within the trigeminal motor nucleus. Numbers denote order of sections. The bouton reaches its maximum diameter in 3. The bouton can be classified in all sections as one containing round vesicles. Note also the presence of dense cored vesicles in some sections (arrowheads in 1,2,3 and 4).

The results of this study provide a quantitative assessment of both the G A B A innervation of the trigeminal motor nucleus and also morphometric data on synaptic boutons within the motor nucleus. Our main finding with regard to the extent of the GABAergic innervation is that approximately 28% of all synapses in the trigeminal motor nucleus involve G A B A - I R boutons. The high incidence of GABAergic synapses provides strong grounds for believing that G A B A may well be an important neurotransmitter within the motor nucleus. There is clearly now a need for electrophysiologieal studies aimed at demonstrating a G A B A action on cells in the motor nucleus and determining the types of G A B A receptors mediating these actions. Such studies have been performed on spinal motoneurones (e.g. ref. 16), but paradoxically there is no quantitative morphological data on the frequency of GABAergic innervation of spinal motoneurone pools. There is, however, quantitative data on the frequency of GABAergic synapses in the visual cortex and lamina II of the dorsal horn, both areas known to receive a rich GABAergic innervation. G A B A - I R boutons account for approximately 17% of all synapses in the visual cortex (cat7), while in the dorsal horn just under 40% of the peripheral processes in type I glomernli are G A B A - I R (rat37). More recently Decavel and Van den Po114 have reported that 49% of all boutons in the medial hypothalamus are GABA-IR. Both G A D - I R and G A B A - I R boutons have been reported to make axo-axonic, axodendritic and axo-somatic contacts within spinal motoneurone pools, and in this respect there would appear to be no difference in the postsynaptic targets within trigeminal and spinal motoneurone pools (rat2123). With respect to the hypothesis outlined in the introduction, we can say that our results allow for the possibility that activation of GABAergic neurones may provide a means of modulating, either directly or indirectly, transmission at synapses onto elevator motoneurones. Axo-axonic synapses appear to be a variable feature of the GABAergic input to different areas of the CNS. For example, such contacts have been observed in the

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Active zone (//,m) Fig. 7. Distribution of values of bouton cross-sectional area (A), apposition length (B) and active zone length (C) for GABA-IR (filled bars) and unlabelled (open bars) boutons forming synapses within the trigeminal motor nucleus. All values derived from measurements made on serially reconstructed boutons. Sample sizes; GABA-IR boutons, n = 15; unlabeiled n = 30.

trigeminal nucleus caudalis 5, the basilar pontine nucleus 27, hypoglossal nucleus (monkey36), and within the spinal cord in laminae II (8/26 G A B A - I R boutons: rata7), V125 and IX (9.2%, 82/779 G A B A - I R boutons21). In contrast, none have been found in the visual cortex (0/ 342 G A B A - I R boutons: cat 7) and only 1 (out of 420 G A B A - I R boutons examined) in the nucleus tractus solitarius 24. It would dearly be of interest to identify the source and transmitter specificity of the postsynaptic terminals in axo-axonic synapses in the trigeminal motor nucleus. In the lumbosacral cord, Maxwell et al. 25 have shown the G A B A - I R boutons make axo-axonic contacts with the terminals of mucle spindle afferents labelled by intracellular injection of HRP. Clearly a similar approach is required in the trigeminal motor nucleus. We did not observe any synapses between G A B A e r gic structures in our material nor have contacts between G A D - I R or G A B A - I R structures been reported in spinal m o t o n e u r o n e pools 21'23. In contrast, 11% of all G A B A e r g i c synapses in the visual cortex 7 and 9% of G A B A e r g i c synapses in the nucleus tractus solitarius 24 are onto either G A B A - I R dendrites or somata, and

some axo-axonic synapses in both the basilar pontine nucleus 27 and hypoglossal nucleus 36 are between G A B A e r g i c elements. Contacts between G A B A e r g i c structures have been suggested to represent a mechanism for achieving dis-inhibition 7, and the figures given above would suggest that this mechanism may be more prevalent in areas containing G A B A - I R neurones. Therefore the absence of such contacts within the trigeminal motor nucleus may simply be a consequence of the absence of G A B A - I R neurones within the motor nucleus. A c o m m o n feature of the G A B A - I R input to all areas of the CNS is that boutons predominantly contain flattened vesicles. O u r data represents a further example of these same features, and so far the only area deviating from this trend is the trigeminal nucleus caudalis. G A D - I R boutons in this area contain either round or a mixture of vesicle types but none contained only flattened vesicles 5. Approximately 15% of G A B A - I R boutons in the visual cortex contain either round or a mixture of vesicle types 7 and 42% of our sample of G A B A - I R boutons contained either round or mixed vesicles, so clearly a significant fraction of G A B A e r g i c bou-

137

Vesicle Types

Contact Types

F - FI.,~TTI~IED M " 14IXED R - ROUND

M

R

n=229

• i f53, AXON F

*XON

ao

M

n=42

67~

F

R

M

R

Fig. 8. Vesicle types in GABA-IR boutons. Boutons were classified on the basis of the shapes of vesicles that they contained. The incidence of boutons containing flattened (F), round (R) or a mixture (M) of flattened and round vesicles are shown separately for axo-axonic (top), axo-dendritic (middle) and axo-somatic (bottom) synapses.

tons in some areas do not contain just flattened vesicles. Differences in the vesicle types present in boutons may provide clues as to the origin of the G A B A e r g i c input. Local circuit neurones in the cortex are characterised by the presence of only flattened vesicles in boutons, and so G A B A - I R boutons containing either round or a mixture of vesicle types have been suggested to arise from outside the cortex 7. If we assume that similar rules apply outside the cortex, then one possibility is that the G A D - I R input to the trigeminal nucleus caudalis may arise from areas extrinsic to the nucleus or its immediate vicinity. The same argument would also lead to the conclusion that G A B A e r g i c boutons forming axo-axonic synapses within the trigeminal motor nucleus may be

REFERENCES 1 Appenteng, K. and Girdlestone, D., Transneuronal transport of wheat germ agglutinin-eonjugated horseradish peroxidase into trigeminal interneurones of the rat, J. Comp. Neurol., 258 (1987) 387-396. 2 Appenteng, K., O'Donovan, M.J., Somjen, G., Stephens, J.A. and Taylor, A., The projection of jaw elevator muscle spindle

drawn essentially from local circuit neurones, as almost all the boutons contain flattened vesicles. By implication, boutons giving rise to either axo-dendritic or/and axosomatic synapses may be drawn both from local circuit and extrinsic neurones. The G A B A e r g i c neurones in the trigeminal main sensory nucleus and the ventral portion of the trigeminal nucleus oralis could be considered candidates for the role of local circuit neurones. It may be possible to distinguish subpopulations of G A B A - I R boutons by determining which putative neurotransmitters are co-localised within particular boutons. G A B A e r g i c neurones in different areas of the CNS are also known to be immunoreactive for choline acetyltransferase z3, monoamines 3°'39 and various neuropeptides 15'35, all of which have been localised within fibres in the trigeminal m o t o r nucleus 17. We have no specific evidence for the co-localisation of other putative neurotransmitters in our sample of G A B A - I R boutons, but the fact that 33% of serially reconstructed boutons contained dense cored vesicles suggests that a significant number of G A B A - I R boutons may indeed contain other transmitters. The evidence is that dense cored vesicles are c o m m o n in boutons containing either catecholamines or neuropeptides 6 and, at least in the raphe nuclei, from where a serotoninergic input to the trigeminal motor nucleus is known to originate ~7, the serotonin appears to be present in the dense cored vesicles of G A B A e r g i c boutons 18,30. Previous morphometric data on boutons has generally been based on measurements on single sections through terminals with the result that while the maximum values reported serve as reliable estimates, the minimum values would be distorted by spurious variations introduced by differences in sectioning. The areas of individual vesicles contained in G A B A - I R boutons have been determined 7, but there appear to have been no systematic attempts to determine the apposition length, active zone length or cross-sectional area of G A B A - I R boutons. Therefore our finding that all these parameters are significantly smaller for G A B A - I R (cf unlabelled boutons) boutons remains to be confirmed.

Acknowledgements. We thank the MRC for support. This work formed part of the PhD thesis of S. Saha.

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