GABA-like immunoreactivity of identified interneurons in...

Cell Tissue Res (1988) 254:331-340

and Tissue R e s e a r c h �9 Springer-Verlag 1988

GABA-Iike immunoreactivity of identified interneurons in the flight system of the locust, Locusta migratoria R. Meldrum Robertson and Leon Wisniowski Department of Biology, McGil[ University, Montreal, Quebec, Canada

Summary. The transmitter content of identified inhibitory interneurons in the flight system of the locust, Locusta migratoria, has been characterized using antibodies raised against protein-conjugated gamma aminobutyric acid. Identified flight neurons were filled with the fluorescent dye, Lucifer Yellow. Serial sections of dye-filled neurons were incubated with an antibody to gamma aminobutyric acid which was subsequently tagged with a fluorescent marker. Excitatory motoneurons to wing muscles and 13 flight interneurons (3 excitatory, 7 inhibitory, and 3 with unknown synaptic effect) were examined. Neither the motoneurons nor any of the 3 excitatory interneurons contained immunoreactive material. Six of the 7 inhibitory interneurons did contain immunoreactive material. All the neurons which contained immunoreactive material and whose synaptic effect is known were inhibitory. We conclude that most of the inhibitory flight interneurons which have been described use gamma aminobutyric acid as their transmitter. Interestingly, at least 1 set of interneurons known to be inhibitory does not use gamma aminobutyric acid. We predict that the 2 interneurons which do contain immunoreactive material and whose synaptic effect is not yet known will be found to have inhibitory roles in the operation of the flight circuitry.

Key words: GABA Interneurons Flight Immunohisto- chemistry - Locusta migratoria (Insecta)

It is clear that a partial understanding of the neural mechanisms controlling behavior can be obtained by describing circuits of synaptic interactions between neurons in the central nervous system (e.g. Roberts and Roberts 1983; Eaton 1984; Selverston 1985). Not all behaviors are equally amen- able to analysis in terms of neuronal circuitry. One behavior which is particularly suitable for such an approach is locust flight (Robertson 1986; Reichert and Rowell 1986). Many flight interneurons have now been identified (Robertson and Pearson 1983; Reye and Pearson 1987), and a simple circuit of flight interneurons which can account for several features of the motor pattern for flight has been described (Robertson and Pearson 1985). This circuit operates with both excitatory and inhibitory synaptic interactions, but inhibitory ones predominate. Some of the inhibitory con-

Send offprint requests to. R. Meldrum Robertson, Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

nections can be blocked with picrotoxin and are reduced in strength by the postsynaptic injection of chloride ions (Robertson and Pearson 1985). These observations and the knowledge that gamma aminobutyric acid (GABA) under- lies much inhibitory transmission in the insect nervous system (Hildebrand 1982; Callec 1985; Watson and Burrows 1987; Robertson 1987) led to the proposal that the inhibitory flight interneurons may be GABAergic (Robertson and Pearson 1985).

GABA conjugated to proteins with glutaraldehyde can be used to generate antibodies which can locate GABAergic neurons in nervous tissue (Storm-Mathisen et al. 1983). This technique has been used successfully several times in insect nervous systems (e.g. Ammermiiller and Weiler 1985; Bicker et al. 1985; Hoskins et al. 1986; Watson and Bur- rows 1987). Indeed maps of the distribution of GABA-like immunoreactivity in the thoracic and abdominal nervous system of the locust, Schistocerca gregaria, have recently been published (Watson 1986; Watson and Pflfiger 1987). The work described in this paper was designed to test the hypothesis that inhibitory flight interneurons are GA- BAergic. Immunocytochemical techniques were used to determine whether identified flight interneurons show GABA- like immunoreactivity. In addition, the ability to predict the synaptic function of specific interneurons in the flight circuit, when physiological information on their function is not yet available, is useful for directing subsequent experi- ments. Examination of interneuronal structure has some predictive power (Pearson and Robertson 1987). Knowl- edge of the transmitter content of identified interneurons would indicate the possible role of the interneurons more directly.

Materials and methods

Animals

Locusts, Locusta migratoria, were obtained from a crowded colony established at McGill University. Both males and females were used with no difference in the results obtained.

Intracellular staining and identification of interneurons

We used a deafferented preparation of the locust which was capable of expressing the centrally generated flight rhythm (Robertson and Pearson 1982). After a dorsal dissection, the meso- and metathoracic ganglia were supported

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Fig. 1 A-D. Distribution in mesothoracic and metathoracic ganglia of neuronal somata immunoreactive to GABA antibody. A Transverse section through medial posterior group in metathoracic ganglion, Bright rhodamine fluorescence of secondary antibody is clear. Solid arrow indicates cell body of unidentified interneuron which did not show specific labelling. This neuron heavily filled with Lucifer Yellow (also see Fig. 2). B Transverse section through lateral anterior group in mesothoracic ganglion. Open arrows indicate limit of neuropil. Note staining of fine processes in neuropil. C Transverse section through medial ventral group in mesothoracic ganglion. D Distribution map of immunoreactive somata in meso- and metathoracic ganglia. LeJ? of figure indicates clusters of somata on dorsal surface, whereas right of figure indicates somata on ventral surface. In this and all subsequent micrographs, dorsal is uppermost. In this and all subsequent drawings anterior is uppermost. LAG, lateral anterior group; L D G lateral dorsal group; M D G medial dorsal group; meso mesothoracic ganglion; meta metathoracic ganglion; M P G medial posterior group; M V G medial ventral group. Scale bars: 100pro in A, B, C; 200pm in D

A

D M n

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Fig. 3A-C. Excitatory motoneuron to first basalar muscle of mesothoracic segment does not show GABA-Iike immunoreactivity. A Drawing of Ist basatar motoneuron in mesothoracic ganglion. Cell body lies on ventral surface. B Transverse section of Lucifer Yellow-filled cell body of motoneuron. C Same section viewed to visualize rhodamine. Note no immunoreactive fluorescence of this motoneuron. Open arrow indicates position of soma. In B and C margin of ganglion (bottom offigures) is ventral surface, and lateral is to left, Scale bars: 200 ~tm in A; 100 ~m in B and C

Fig. 2A, B. Contaminat ion of fluorescent markers. A Intense Lucifer Yellow fluorescence of unidentified interneuron in MPG of metathoracic ganglion. Soma and some neuronal branches in neuropil are evident. Neuron could not be identified because 3 neurons were filled together in this ganglion and it proved impossible to resolve branching patterns. B Same section viewed with rhodamine optics to locate antibody fluorescence. Open arrow indicates cell body filled with Lucifer Yellow and visible in A. Note some rhodamine fluorescence discernible in A (filters configured to visualize Lucifer Yellow), but Lucifer Yellow fluorescence is only faintly discernible in B (filters configured to visualize rhodamine). See also Fig. 1 A which is higher power view of same section. Cell body indicated by solid arrow in Fig. 1 A. Margin of ganglion (bottom of figures) is ventral surface and filled neuron is just to left of midline, Scale bar. 100 ~tm in A and B

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302

Fig. 4A-C. Interneuron 302 shows GABA-like irnmunoreactivity. A Drawing of interneuron 302 in mesothoracic ganglion. Cell body lies on ventral surface. B Transverse section of Lucifer Yellow-filled cell body of 302 in mesothoracic MPG. C Same section viewed to visualize rhodamine fluorescence. Note that soma of 302 (open arrow) contains irnmunoreactive material. For comparison, asterisk indicates soma which does not show specific labelling. In B and C, margin of ganglion (bottom of figures) is ventral surface, and lateral is to left. Scale bars: 200 lam in A; 100 grn in B and C

on a stainless steel platform and superfused with saline (in mM: 147NaC1, 10KC1, 4CAC12, 3NaOH, 10 HEPES buffer). Neurons were penetrated in their neuropil segments with glass microelectrodes filled at the tip with the fluorescent dye, Lucifer Yellow CH (Aldrich, Milwaukee; 4% in distilled water) and back-filled with 0.5 M LiCI. The elec- trode resistance was between 100 and 150 MegOhms. Intra- cellular recordings were taken from the neurons during the expression of the flight rhythm (initiated by stimulation of the wind-sensitive head-hairs) and then the neurons were filled with Lucifer Yellow by the passage of 5 10 nA of hyperpolarizing current for about 10 min. The ganglia were excised and fixed for 1 h in a mixture of 4% paraformalde- hyde and 0.25% glutaraldehyde. They were then dehydrate- d in an ethanol series and cleared in methyl salicylate. They were viewed in whole mount using a Wild M5 binocular dissection microscope fitted with an epifluorescence illumi- nator (Wild-Leitz). The neuronal structures were drawn in plan view using a drawing tube. Neurons were identified on the basis of their physiological and morphological prop- erties and were numbered according to Robertson and Pear- son (1983).

Irnmunocy tochemistry

We used antibodies raised in rabbits against GABA conjugated with glutaraldehyde to either bovine serum albumin (GABA-BSA) or keyhole-limpet hemocyanin (GABA- KLH). These antibodies and the protein-GABA conjugate were provided by T. Kingan (University of Arizona, Tuc- son) and the methods for their preparation are described

elsewhere (Hoskins et al. 1986). We also used anti-GABA- BSA prepared in a similar way (Wenthold et al. 1986) and provided by R.J. Wenthold (NIH, Bethesda). The patterns of immunoreactive staining obtained with each of these antibodies were similar, but we found that the anti-GABA- KLH gave better results. All the results described here were obtained using anti-GABA-KLH. Unless otherwise noted, all other reagents were purchased from Sigma Chemical Co. (St. Louis, Missouri).

After each neuron was drawn, the ganglia were taken from methyl salicylate back to absolute ethanol, infiltrated with xylene, and embedded in paraffin wax. Serial sections of the ganglia were cut at 10 p.m and mounted on slides using an albumin/glycerol section fixative (Fisher Scientific, Montreal). The sections were dewaxed with xylene and re- hydrated to phosphate-buffered saline containing 0.5% Tri- ton X-100 (PBS). Non-specific staining was reduced by in- cubating the sections for 30 rain in 1% ethanolamine (Fish- er) in PBS followed by washing with PBS and incubation with 10% normal goat serum in PBS for 30 rain. Sections were incubated overnight (4 ~ C, on a rotary shaker) with GABA antisera diluted 1:250, 1:500, 1 : 1000, or 1:2000 in PBS and containing 0.02% sodium azide. Best results were obtained using anti-GABA-KLH at 1:250 dilution. To locate the position of the bound GABA antibody, the sections were washed several times with PBS and then incubated for 1 h with TRITC-conjugated goat-anti-rabbit secondary antibody (1:50 in PBS containing 0.02% sodium azide). The sections were then washed thoroughly with PBS and mounted in fluoromount.

Sections were viewed and photographed using a Leitz

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Fig. 5A-C. Interneuron 513 shows GABA-Iike immunoreactivity. A Drawing of interneuron 513 in metathoracic ganglion. Cell body lies on ventral surface. B Transverse section of Lucifer Yellow-filled cell body of 513 in metathoracic MPG. Note that intense rhodamine fluorescence seen in C causes some contamination of image. C Same section viewed to visualize rhodamine fluorescence. Note that soma of 513 (open arrow) contains immunoreactive material. For comparison, asterisk indicates soma which does not show specific labelling. In B and C, margin of ganglion (bottom of figures) is ventral surface and lateral is to right. Note relatively high background fluorescence caused by glutaraldehyde fixation. Scale bars: 200 l.tm in A; 100 ~tm in B and C

or thoplan fluorescence microscope equipped with filters to visualize Lucifer Yellow and rhodamine fluorescence. By switching filters it was possible to determine in the same section whether the sectioned cell body of a neuron filled with Lucifer Yellow also showed GABA- l ike immunoreactivity as determined by rhodamine fluorescence of the secondary ant ibody. The quali ty of the results, in terms of the contras t between specific staining and background staining, was variable in different preparat ions. A high level of background staining was caused both by background fluorescence (i.e., fluorescence in the absence o f any rhodamine marker) and by non-specific labelling, and was probably a result of glutaraldehyde fixation and the high concen- t ra t ion of pr imary an t ibody necessary for the immunof luor- escence technique. Specific labelling was recognized relative to a variable background and not as an absolute level of fluorescence. In the major i ty of cases it was possible to distinguish the same pat tern of specific labelling. The few preparat ions with which this was impossible were discarded. The overall pa t tern of GABA- l ike immunoreact ivi ty in the meso- and metathoracic ganglia was determined graphical ly by reconstructing the serial sections.

To test the specificity o f the procedure, some sections of par t icular prepara t ions were processed omit t ing the primary ant ibody and some were processed using pr imary an- t ibody which had been previously incubated overnight at 4 ~ C with protein-conjugated G A B A (10 lag per ml o f diluted antibody). These steps eliminated specific labelling.

Specific clusters of immunoreact ive neuronal somata were named according to Watson (1986) as follows: LAG lateral anterior group; LDG lateral dorsal group; L VG lateral ventral group; MDG medial dorsal group; MPG medial poster ior group; and M VG medial ventral group.

Results

General distribution of immunoreactive staining

The G A B A - K L H ant ibody labelled numerous neuronal somata throughout the meso- and metathoracic ganglia. La- bel was confined to the cytoplasm of each neuron, and when the nucleus occupied the complete thickness of the section, it was obviously unlabelled. Even the weak background fluorescence appeared stronger in the cytoplasm

336

A j

Fig. 6A-C. Interneuron 520 shows GABA-like immunoreactivity. A Drawing of interneuron 520 in metathoracic ganglion. Cell body lies on ventral surface. B Transverse section of Lucifer Yellow-filled cell body of 520 in metathoracic MPG. C Same section viewed to visualize rhodamine fluorescence. Note that soma of 520 (open arrow) contains immunoreactive material. In B and C, margin of ganglion (bottom of figures) is the ventral surface, and lateral is to right. Scale bars: 200 lam in A; 100 lain in B and C

than the nucleus of unlabelled somata. The neurons were grouped for the most part in well-defined clusters located in specific regions of the layers of cell bodies on the dorsal and ventral surfaces of the ganglia (Fig. 1 a~c). The immunoreactive neuronal somata in the ventral, medial, and posterior region (MPG) of the ganglia tended not to be as tightly clustered, and neurons without label were found in- terspersed with labelled neurons in this region. In this region we also found more neurons which showed an interme- diate level of staining than in the other regions. The map of the distribution of the groups (Fig. 1 d) corresponds very closely to the map described for immunoreactive somata in the thoracic ganglia of a different species of locust, Schistocerca gregaria, (Watson 1986) such that the same groups could be identified and named accordingly. Immu- noreactive labelling was also found associated with the fine neuronal processes throughout the neuropil (Fig. I b).

Irnmunoreactive staining of identified neurons

We found that intense rhodamine fluorescence of the secondary antibody occasionally could contaminate faintly the image obtained when the microscope filters were configured to visualize Lucifer Yellow fluorescence (Fig. 2). However, it was always possible to identify the neuron which had been filled. Intense Lucifer Yellow fluorescence resulted in contamination so faint under rhodamine optics that the possibility of obtaining false positive results could be ruled

out (Figs. 1 a, 2). For each identified neuron the same clear positive or clear negative result was seen in at least 3 different preparations.

None of the excitatory motoneurons to wing muscles which were filled showed immunoreactive staining (Fig. 3). In contrast, the common inhibitor motoneurons, which can be identified by their characteristic size, position in the ganglia, and direction of the primary neurite (Watson et al. 1985; Watson 1986), did show immunoreactive staining. In 1 preparation, this was confirmed by filling and process- ing a common inhibitor motoneuron in the mesothoracic ganglion with Lucifer Yellow (not shown).

We examined the GABA-like immunoreactivity of 13 flight interneurons of which 7 were inhibitory, 3 were excitatory, and 3 had an unknown synaptic effect. The synaptic effects of the interneurons were determined in previous ex- periments involving simultaneous intracellular recordings of either 2 interneurons or an interneuron and a motoneuron. A connection between 2 neurons was judged probably to be monosynaptic if their processes overlapped, and if the synaptic delay (i,e., after subtracting conduction delay) between presynaptic spike and postsynaptic potential was constant with a value of lms or less (Robertson and Pear- son 1985; Pearson and Robertson 1987). Two of the iner- neurons examined (401, 501) exist as sets of serial homo- logues in 4 fused neuromeres, and one (201) is known to be represented multiply in the same hemiganglion (i.e. at least 2 with the same morphology and physiology) (Robert-

A

3 o , - L

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Fig. 7A-C. Interneuron 301 shows GABA-like immunoreactivity. A Drawing ofinterneuron 301 in mesothoracic ganglion. Cell body lies on ventral surface. B Transverse section of Lucifer Yellow-filled cell body of 301 in mesothoracic MPG. C Same section viewed to visualize rhodamine fluorescence. Note that soma of 301 (open arrow) contains immunoreactive material. For comparison, asterisk indicates soma which does not show specific labelling. In B and C, margin of ganglion (bottom of figures) is ventral surface, and lateral is to left. Note relatively high background fluorescence caused by glutaraldehyde fixation. Scale bars: 200 ~tm in A; ! 00 gm in B and C

son and Pearson 1983). There is so far no known difference between the members of a particular set. The question of whether individual members of a set might be distinguish- able in terms of their GABA-like immunoreactivity was not directly addressed in this study. However, when different members o f a particular set were examined, they each showed the same reaction with the GABA-antibody. Fig- ures 4~7 show examples of identified flight interneurons which showed positive reactions with the G A B A antibody, whereas Figures 8 and 9 show examples of flight interneurons which showed negative reactions with the G A B A antibody. Table I lists the results for all of the interneurons and relates this to the synaptic effect of the interneurons. None of the interneurons which were known to be excitatory (201, 506, 701) were labelled with the GABA antibody. All of the interneurons which showed GABA-like immunoreactivity and whose synaptic effects were known (301,302, 501, 511, 513, 520)were inhibitory. One interneuron (401) was known to be inhibitory, but was not labelled with the G A B A antibody. Of the interneurons with unknown synaptic effect one (207) showed a negative reaction to the G A B A antibody, and the other 2 (507, 538) showed a positive reaction.

Discussion

In this paper we have described the pattern of GABA-like immunoreactive labelling of neuronal somata in the meso-

and metathoracic ganglia o f the locust, Locusta migrator&. Also, using the technique of double-marking, both with the intracellular injection of Lucifer Yellow and with the fluorescent marker o f the antibody, we have described the GABA-like immunoreactivity of 13 identified flight interneurons. For the following reasons we are confident that the antibodies raised against G A B A - K L H and GABA-BSA are labelling the somata and processes of inhibitory interneurons which use G A B A as a transmitter: (1) localization of GABA-like immunoreactivity in nervous tissue using antibodies against protein-conjugated G A B A has been established since 1983 as a useful technique (Storm-Mathisen et al. 1983), and the antibodies we used have been used to reveal GABAergic neurons in a variety of other systems (e.g. Bicker et al. 1985; Hoskins et al. 1986; Wenthold et al. 1986; Johnson and Stretton 1987); (2) the pattern of labelling described here in the meso- and metathoracic ganglia was identical using 2 different antibodies (anti-GABA- K L H and anti-GABA-BSA) from different sources, and this pattern is very similar to the pattern of GABA-like immunoreactivity in the thoracic ganglia o f a closely related species o f locust revealed with a different antibody to GABA-BSA (Watson 1986); (3) omission of the primary antibody from the immunocytochemical procedure, or pre- absorption of the primary antibody with protein-conjugated G A B A prevented any specific labelling of neurons; and (4) excitatory neuromuscular neurotransmission in insects is probably glutamatergic (Piek 1985) and the excitato-

338

Fig, 8A-C. Interneuron 506 does not show GABA-like immunoreactivity. A Drawing of interneuron 506 in metathoracic ganglion. Cell body lies on dorsal surface. B Transverse section of Lucifer Yellow-filled cell body of 506. C Same section viewed to visualize rhodamine fluorescence. Note that soma of 513 (open arrow) does not show specific labelling. Immunoreactive soma in metathoracic LDG can be seen (solid arrow). In B and C, margin of ganglion (top ~?ffigures) is dorsal surface, and lateral is to right. Scale bars: 200 lain in A; 100 lain in B and C

ry motoneurons to wing muscles were not labelled with the antibody, whereas common inhibitor motoneurons are GABAergic (Watson 1986) and they were labelled with the antibody.

For the most part the results reported here are unre- markable. They were generally predictable and serve to confirm what previous results had indicated. Nevertheless a few points are worth further discussion. It is perhaps not surprising that the general pattern of GABA-like immunoreactivity of neuronal somata in the thoracic ganglia of Locusta migratoria was very similar to what has been described for Schistocerca gregaria (Watson 1986). Indeed, this observation lends some support for the idea, often put into practice, that results obtained with one of the species are readily transferrable to the other. In some cases close comparison can justify this practice (e.g., Reye and Pearson 1987). However it would be wrong to assume that the labelling patterns are identical. The descriptions are at a gross level and the requisite detailed information which might allow such a conclusion is not yet available.

Establishing that a particular physiological connection between neurons is monosynaptic is notoriously difficult. Good evidence might include an electron microscopical demonstration of the anatomical characteristics of a direct connection such as has been done for connections from a sensory afferent to wing muscle motoneurons (Peters et al. 1985). In the absence of this evidence, and other evidence

(Berry and Pentreath 1976) which is difficult to obtain in insects, it is common to consider a connection as being monosynaptic if: (1) the processes of the 2 neurons branch in the same region of the neuropil at least at 1 place; and (2) the synaptic delay (observed latency minus conduction delay between recording sites) between presynaptic spike and postsynaptic potential is constant and 1 ms or less (Robertson and Pearson 1985; Pearson and Robertson 1987). So far, of 7 interneurons which have been judged previously to be inhibitory using the 2 criteria listed above, 6 show GABA-like immunoreactivity. G A B A is known only as an inhibitory neurotransmitter in insect nervous systems (Callec 1985), and thus the immunocytochemistry confirms that these 6 interneurons are indeed inhibitory. Therefore, this strong correlation supports the use of the above criteria to establish probable monosynapticity.

It is particularly interesting that 1 interneuron (401) does not show GABA-like immunoreactivity, but is thought to be inhibitory, using the same criteria as for the other interneurons (Pearson and Robertson 1987). One possible explanation is that in this particular case the observed inhibitory connections may not be monosynaptic and 401 may not be an inhibitory interneuron. However the physiological evidence suggesting that 401 is inhibitory is a good as it is for the other interneurons. The most likely explanation is that 401 is indeed inhibitory, but the inhibitory neurotransmitter used by interneuron 401 is not GABA. In

339

Fig. 9A-C. Interneuron 401 does not show GABA-like immunoreactivity. A Drawing of interneuron 401 in metathoracic ganglion. Cell body lies on ventral surface. B Transverse section of Lucifer Yellow-filled cell body of 401. C Same section viewed to visualize rhodamine fluorescence. Note that soma of 401 (open arrow) does not show specific labelling. Immunoreactive somata in metathoracic MPG can be seen (e.g. solid arrow). In B and C, ventral is to bottom of figures and cell body of 401 is just to right of the midline. Scale bars: 200 lam in A; 100 ~tm in B and C

the lobster, none of the neurons in the s tomatogastr ic ganglion shows GABA- l ike immunoreact ivi ty (Cazalets et al. 1987) in spite o f the fact they are interconnected with inhibitory connections. Some of these inhibi tory interactions are thought to be mediated by glutamate and some by acetyl- choline (Eisen and Marde r 1982). In insects there is no evidence for an inhibi tory role of other substances, which is as convincing as the evidence for G A B A as an inhibi tory neurotransmit ter . However candidate substances which have at least been shown to have a hyperpolar izing effect on insect neurons are glutamate, glycine, taurine, and 5-hy- droxyt ryptamine (Usherwood et al. 1980; Pi tman 1985), and there is s tronger evidence for a central t ransmit ter role, albeit excitatory, for glutamate (Sombati and Hoyle 1984). The t ransmit ter content of interneuron 401 remains to be discovered.

Recently, it has been demonst ra ted that it is possible to predict the synaptic effect of some thoracic interneurons in the locust from certain features of their morphology (Pearson and Rober t son 1987). Interneurons which have

a ventro-medial ly located cell body, a la teral ly-bowed primary neurite, and an axon contralateral to the cell body were shown to be inhibitory. Not all inhibi tory interneurons share these structural features. It was suggested that the structural similarities have developmental origins, i.e. the similar interneurons originate from 1 or a few similar neu- roblasts. Interestingly, the only described inhibitory interneuron lacking the shared features of other inhibi tory interneurons is interneuron 401 (Pearson and Rober tson 1987). We have demonstra ted in this study that interneuron 401 is not GABAergic . Inhibi tory interneurons examined here which do possess the features (301,302, 501,511,513, 520) all showed GABA-l ike immunoreact ivi ty. F rom the limited evidence to date it seems that these structural features are predictive of GABAerg ic inhibi tory interneurons rather than just inhibi tory interneurons.

In conclusion, in the major i ty of cases we can confirm the hypothesis that inhibi tory flight interneurons are GABAergic . However, at least 1 inhibi tory flight interneuron does not show GABA-I ike immunoreact ivi ty. Based

340

Table 1. GABA-like immunoreactivity and synaptic effect of flight interneurons

Interneuron GABA-like Synaptic effect References immunoreactivity

201 no EXC a, d 207 no ? -- 301 yes INH a, b, d 302 yes INH a, d 401 no INH a, d 501 yes INH a, b, d 506 no EXC c, d 507 yes ? c 511 yes INH a, d 513 yes INH d 520 yes INH c, d 538 yes ~ - 701 no EXC a, d

Entries indicate whether a particular flight interneuron does or does not exhibit GABA-like immunoreactivity and relate this to the known synaptic effect of that interneuron. E X C - excitatory; I N H - inhibitory; ? not yet determined. References are to papers that describe either the structure or the synaptic output of the interneuron, a - Robertson and Pearson 1983; b Robertson and Pearson 1985; c - Reye and Pearson 1987; d Pearson and Robert- son 1987

o n o u r results , we p red ic t t h a t i n t e r n e u r o n s 507 a n d 538 will be f o u n d to be inh ib i to ry . N o p r e d i c t i o n s can be m a d e a b o u t i n t e r n e u r o n 207 w h i c h was n o t i m m u n o r e a c t i v e .

Acknowledgments . We are very grateful to T. Kingan and R.J. Wenthold for giving us the GABA antibody and for helpful sugges- tions on the immunocytochemical procedure. We thank R. Levine, G. Pollack, and S. Wang for their comments on a previous version of this paper. Supported by the Natural Sciences and Engineering Research Council of Canada and the Faculty of Graduate Studies and Research at McGill University.

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Accepted April 18, 1988

GABA-like immunoreactivity of identified interneurons in...

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