iVeuroscience Vol. 17. No. 4, pp. 1243-1255, 1986 Printed in Great Britain

0306-4522/86 $3.00 + 0.00 Pergamon Press Ltd

0 1986 IBRO

AUTORADIOGRAPHIC STUDY OF THE DISTRIBUTION OF [3H]y -AMINOBUTYRATE-ACCUMULATING

NEURAL ELEMENTS IN GUINEA-PIG INTESTINE: EVIDENCE FOR A TRANSMITTER FUNCTION OF

y-AMINOBUTYRATE

A. KRANTIS, D. I. B. KERR and B. J. DENNIS Department of Physiology, University of Adelaide, Adelaide, South Australia 5000, Australia

Abstract-High affinity uptake, and the distribution of ‘H-radiolabelled y-aminobutyrate (GABA), cis-3-aminocyclohexanecarboxylic acid, p-alanine, proline, and leucine have been examined auto- radiographically in laminar preparations of the myenteric plexus from the guinea-pig intestine. Following labelling with [3H]proline and [3H]leucine, which are incorporated into neurons, silver grains were concentrated over recognisable perikarya in the ganglia and meshworks of the plexus, whilst [)H]GABA labelled a smaller proportion of neurons and their processes. Specificity of labelling in the sites of [‘HIGABA-uptake was established using combinations of labelled and unlabelled GABA, /I-alanine, and cis-3-aminocyclohexanecarboxylic acid, substrates for gial or neuronal high affinity GABA uptake systems. Only myenteric neurons and their processes were labelled significantly by [3H]GABA and its analogue cis-3-[3H]aminocyclohexanecarboxylic acid. Using autoradiographs of laminar preparations and paraffin sections, [‘HIGABA labelling was found over nerve fibre bundles that could be traced from their ganglionic origins through the interconnecting meshworks of the myenteric plexus into the innervation of the deep muscular plexus of the circular muscle layer where GABA is evidently concerned with prejunctional modulation of transmitter release.

The extensive but selective distribution of [)H]GABA high affinity uptake sites in neural elements of the guinea-pig myenteric plexus is consistent with GABA being an enteric neurotransmitter.

High affinity uptake of y-aminobutyrate (GABA) has been characterised in the central nervous system,“,” where it occurs in a variety of different neuron types and associated glia,56,58 and is evidently concerned in the limiting or termination of GABA-mediated ac- tions.’ These high affinity transport systems in neu- rons and glia differ in their substrate specificities,2*~5s allowing differentiation of their respective uptake sites, where the uptake of GABA, or [3H]GABA, into neurons can be prevented by L-2,Cdiaminobutyric acid (L-DABA),~O cis-3-aminocyclohexanecarboxylic acid (ACHC),’ and nipecotic acid48 which also par- tially inhibits glial uptake, whilst p-alanine is taken up exclusively by glia and is an inhibitor of the glial uptake of GABA and [3H]GABA.27*39s57 Such specificity of labelling with [3H]GABA, and its pre- vention by specific inhibitors of the high affinity transport system, has formed the basis for the auto- radiographic demonstration of GABAergic neurons in the central nervous system, all the available evi- dence indicating that high affinity neuronal GABA uptake sites are an exclusive property of neurons that employ GABA as a neurotransmitter.29,M Using auto- radiography, sites for the high affinity uptake of

Abbreviations: ACHC, cis-3-aminocyclohexanecarboxylic acid; L-DABA, L-2,4-diaminobutvric acid: GABA. y-aminobutyrate.

[3H]GABA have also been demonstrated in cultured myenteric neurons,30,31,34,35 and in neuronal elements in laminar preparations of the guinea-pig intestine,4 it again being concluded that these neurons are GABAergic.29.30.” Further direct evidence for high affinity uptake sites for GABA in the myenteric plexus has been provided by uptake and release studies using [3H]GABA in isolated intestinal prepa- rations from the guinea-pig, where preincubation with the neuronal uptake blockers (DABA, ACHC, or nipecotic acid) prior to loading with [3H]GABA, leads to a significant reduction in the subsequent stimulus evoked release of tritium.30*40x4’,42,61 Further- more, GABA-induced ileal contractile responses are potentiated by these uptake blockers,“2 indicative of neuronal GABA uptake sites in close proximity to the neurons responding to GABA. That these myenteric neuronal uptake sites are indeed associated with GABAergic neurons is strongly indicated by the demonstration of GABA-induced ileal responses that result from the release of endogenous GABA, a release that can be manipulated by alteration of GABA metabolism in the intestine.4’.42 A GABAergic innervation in the plexus is also implied by the alteration of motility in the intestine following the application of GABA-antagonists.46,52sM Thus it is to be anticipated that GABA-uptake sites in the enteric nervous system will be associated with GABAergic

1243

I244 A. KRANTIS et ul.

neurons in the myenteric plexus, as has been found in the central nervous system.

Both [“H]GABA and [‘H]ACHC are accumulated specifically into central GABAergic neurons,34 whilst [-‘HI@-alanine is a specific substrate for high affinity transport into glia:27.2’ we have therefore employed these agents together with specific inhibitors of high affinity neuronal or glial GABA uptake to further characterise the high afKnity uptake of GABA into neuronal and gliaI17 (Schwann-cellS) elements of the myenteric plexus as revealed by autoradiography

applied to laminar and paraffin sectioned prepara- tions of the intestine. Such laminar preparations,

consisting of the stretched myenteric plexus attached to the longitudinal muscle layer, allow a ready exam-

ination of the disposition and ramifications of neural elements within the plexus meshworks.7~‘~~‘5~21~3~~M~s9

EXPERIMENTAL PROCEDlJRES

Preparations of the myenteric plexus were obtained from freshly killed guinea-pigs of either sex, weighing 25&400 g. Segments of small and large intestine 8-.lOcm long were quickly removed and placed in Krebs solution (37&C), gassed with a mixture of 95% Oz and 5% CO,. The segments were cleared of contents, after which they were kept in gassed Krebs solution containing amino-oxyacetic acid 10. 5 M, to inhibit GABA-transaminase. Amino- oxyacetic acid was present in the solution throughout the subsequent processing which was as follows:

1. Laminar-stretch preparations. Longitudinal muscle with attached myenteric plexus was dissected free from segments of the large or small intestine.’ After being cut into short lengths the individual tissues were pre-incubated for 10 min in 350 ~1 Krebs (37“C) containing amino-oxyacetic acid, after which labelling of neuronal high affinity uptake sites was carried out in the presence of appropriate uptake inhibitors at IO-‘M. The tissues were incubated for a further 5min with shaking after which tritiated GABA (66Ci/m mole; 2,3-[‘HIGABA) was added to give a final concentration of 5 x IO-’ M. Tissues were incubated with shaking for 20 min and then washed for I min in fresh Krebs solution, placed in the stretched state serosa-down on subbed slides. fixed with 4% gIutaraldehyde-phosphate buffer (pH 7.6), air dried, and ~nalIy dehydrated and de- fatted preparatory to being treated for autoradiography.

Other laminar-stretch preparations were similarly treated again with uptake inhibitors present at 10. ‘M where appropriate, using a final concentration of IO “M of: [3H]p-alanine, [‘HIACHC, L-[‘Hlproline or L-[‘Hlleucine.

2. Short segments of whole intestine, 223 cm long were treated for [‘H]GABA autoradiography as described above: however, before fixation a glass rod was positioned through the lumen to maintain the integrity of the tubular nature of the intestinal segment. Following fixation, the preparations were removed from the glass rod, washed. dehydrated and processed for paraffin sectioning. Sections (20pm) cut at various orientations were brought down to HzO, mounted on subbed slides and processed for autoradiography.

For light microscopic autoradiography, the slides with either stretch preparations or paraffin sections were re- hydrated, dipped in Ilford K, nuclear emulsion. and stored at 4°C for 1421 days in light-tight boxes. They were then developed for 3Amin in Kodak D,, developer at 20°C. washed and fixed for 8 min in 30% sodium thiosulphate and prepared for light microscopic examination. Some prepara- tions were also counterstained with neutral red or cresyl violet before coverslipping, to reveal the underlying cyto- architecture. Suitable controls showed that positive or nega-

tive chemography, or physical artifact, did not contribute to the observed patterns of silver grains in the autorndiographs observed under dark field or transmitted light microscopy.

As part of a mo~hologi~l study, some iaminar prepara- tions were similarly dissected and then treated by a modification of the Gabella’” reduced nicotinamide-adeninc dinucleotide-diaphorase method for neurons, which aided identification of particular cell types accumulating radio- labelled amino acids.

RESULTS

The localization of [‘Hlproline and [3H]leucine in lam- inar preparations of the ileum

Various cell types, including neurons and glia, of the central nervous system accumulate proline and leucine which, if radiolabelled, can be demonstrated using autoradiography. *.I4 In this study, laminar preparations of the ileum displayed extensive uptake of these tritiated a-amino acids into the myenteric plexus, after only 20min incubation. Numerous dense accumulations of silver grains were present over each ganglion of the myenteric plexus (Figs 1,2) where the overall appearance resembled that of a myenteric plexus stained for neurons by the reduced nicotinamide-adenine dinucleotide- diaphorase method.‘6.‘9 These dense accumulations of silver grains were evidently over neurons, as many as 20 being labelled in each ganglion, including those with an oval-shaped soma, as well as Dogiel type I and type II cells.13~2’

Dense accumulations of silver grains were also found scattered along the course of the primary and secondary meshworks but these were more often punctate rather than giving the appearance of hbres and “tracts” as seen with 13H]GABA labelling (Figs 5,6). Some of the labelled structures occurring within the fibre bundles of the primary and secondary

meshworks were evidently cell bodies since soma, dentrites and emergent axon could be discerned (Fig. 3a). These particular cells bore a strong resemblance to Dogiel type II multipolar neurons found in nearly all ganglia,24.55 an example of which is shown here stained by the reduced nicotinamide-adenine dinucleotide-diaphorase method (Fig. 3b). These “ex- traganghonic” neurons were found to be a mean 30 pm long and 15 pm wide, similar to those reported by Cook and Burnstock. Along with these densely labelled structures there were numerous others, smaller and more faintly labelled, found over the ganglia and along the course of the primary and secondary meshworks, in autoradiographs from f3H]proline and f3H]leucine, but not [3H]P-alanine- treated tissues (Fig. 15).

In the tertiary plexus, dense accumulations of silver grains occurred at intersections of the meshwork. These were more common with [3H]proline or [‘HJieucine than with [-‘H]GABA (Fig. 4), and are proposed to be neurons since DABA prevented their labelling with 13H]GABA. In the present experiments, the distribution of silver grains associated with up-

[3H]GABA-autoradiography in the gut 1245

take of these tritiated a-amino acids did not resemble autoradiographs of myenteric plexus treated with [3H]GABA in that the distinctive labelling of “tracts” and fibres, seen with the latter particularly in the tertiary plexus, was absent when the [3H]o:-amino acids were used, although some axons could be seen outlined with silver grains for short distances in the primary plexus and ganglia (Figs 1, 2, 3a).

Localization of [‘HIGABA in myenteric laminar prep- arations

Autoradiographs from ileal myenteric laminar preparations were similar whether the tissues were incubated with [‘H]GABA (5 x 10e9 M) alone or in the presence of p-alanine ( 10m3 M). The characteristic meshworks of the myenteric plexus’3.M,55 were re- vealed by the silver grains which formed in most part, only a diffuse cover over the ganglia and primary plexus (Pp) giving a lightly shaded appearance whilst the secondary (Ps) and tertiary (Pt) meshworks were in places outlined by more dense accumulations of silver grains resembling distinct tracts (Fig. 6). Trails of silver grains were also evident over fibre bundles of the primary meshwork, often as single fibres coursing through a ganglion. No dense accumu- lations of silver grains were observed in regions where the longitudinal muscle was devoid of myenteric plexus, neither was there labelling over adherent circular muscle fibres or of blood vessels.

Very dense accumulations of silver grains were found aggregated in patches over about half the ganglia and scattered along the course of the primary, secondary and tertiary meshworks, particularly at junctions of the network (Figs 5, 6). In some in- stances, these very dense accumulations were recog- nisably localised over not only the soma but also dendrites and the emergent axon of individual neu- rons within ganglia (Figs 7-l 3). The types of neurons labelled with [3H]GABA included those with an elongate soma (maximum dimensions 50pm long and 15 pm wide) more characteristic of Dogiel type II cells,‘3,‘6 particularly those with a basal dendritic cluster and offset nucleus (Fig. 13), and others with one or more long emergent process (Figs 7-9). These cell types in the ganglia were all clearly dis- tinguishable from those with the “cogwheel’ dendritic pattern of Dogiel type I cells,‘3.55 some of which were labelled by [3H]leucine or [3H]proline. We were un- able to distinguish specific extraganglionic cell types labelled by [‘HIGABA within the meshworks, al- though intense labelling of structures (mean length 25 pm, width 15 pm) was evident at some inter- sections of the tertiary meshwork (Fig. 5). These no longer labelled with [3H]GABA following treatment with L-DABA or nipecotic acid, neither were they labelled by [3H]/j’-alanine.

Little difference was apparent between tissues treated with [3H]GABA in the presence or absence of b-alanine. The even, light, diffuse cover of silver

grains over ganglia and meshwork of the primary plexus persisted in the presence of fi-alanine.

Effects of L-2,4diaminobutyric acid, nipecotic acid and cis-3-aminocyclohexanecarboxylic acid on [3H]GABA uptake

In tissues treated with [3H]GABA in the presence of L-DABA (10e3 M), nipecotic acid (10m3 M), or ACHC (10 - ’ M), there was almost a complete lack of dense accumulations of silver grains over the plexus (Fig. 14). At the concentrations used, nipecotic acid appeared to be more effective than L-DABA as an inhibitor of GABA uptake into myenteric neurons. Such silver grains as were present formed a diffuse cover, more obvious over the ganglia and primary meshwork. This could be distinguished from the cresyl violet counterstaining, particularly by the use of dark field illumination, and is said to be character- istic of glial uptake of [3H]GABA resulting in a faint labelling of glial ceIlss8

Uptake of cis-3-[3H]aminocyclohexanecarboxylic acid into laminar preparations of colon and ileum

ACHC is specifically taken up by neurons through the GABA-uptake carrier,3*50 hence, [3H]ACHC would be expected to be incorporated into putative GABAergic myenteric neurons. Incubation with [3H]ACHC (5 x 10m9 M) yielded autoradiographs showing discrete accumulations of silver grains over the ganglia (Fig. 16). These closely resembled the labelled structures found with [‘Hlproline, [3H]leucine or [3H]GABA (Fig. 17) but again only very faint labelling of processes was visible, presumably due to the incubation period being too short. Nevertheless, some 5-10 such structures could be counted over each ganglion, which represents a significant proportion, lO-15%, of the neurons present in any one gan- glion.20

[3H]p-Alanine uptake into the myenteric plexus

The nature of the distribution of the uptake sites for [3H]p-alanine (IO-* M) which is substrate specific for transport into glia5” lacked any of the features of [3H]GABA uptake. Such labelling as did occur was not obviously accumulated over any cell bodies, glial or neuronal. Where silver grains were present, they were localized to a light diffuse cover over the ganglia and, occasionally, over the secondary but not the tertiary meshworks (Fig. 15). This was in sharp contrast to the accumulations of silver grains seen over neurons of the primary meshwork and distinct tracts within all three meshworks in [3H]GABA treated tissue, indicating that [3H]/?-alanine was not taken up by neuronal elements.

Incubating the tissue with [3H]p-alanine in the presence of L-DABA (10m3 M), which we observed to markedly reduce the uptake of [3H]GABA, did not alter the distribution of [3H]fi-alanine in the plexus.

1246 A. KRANTlS et al.

Figs l-17. Light microscopic autoradiographs showing uptake of ‘H into guinea-pig ileal myenteric plexus. Scale bars = 50 pm.

Fig. 1. [‘H]Leucine (lo-* M). Labelled cell bodies are present throughout the plexus, particularly in ganglia (G) and the primary (Pp) and secondary (Ps) meshworks.

Fig. 2. [3H]Proline (10-s M). Labelled cells are found throughout the ganglia (G) and the course of the primary (Pp) and secondary (Ps) meshworks. Labelled structures (B) can also be identified at intersections

of the tertiary plexus (Pt).

Fig. 3(a). [‘H]Leucine (10-s M). Dense accumulations of silver grains can bc seen over a ganglion (G), as well as the soma and emergent axons of extraganglionic neurons (N) within fasciculi of the primary plexus (Pp). (b) Light micrograph of guinea-pig ileal myenteric plexus stained by the modified reduced nicotinamide-adenine dinucleotide phosphate-dehydrogenase method. A neuron (N) with long dendrites

and single axon characteristic of Dogiel type II neurons can be seen within a ganglion (G).

Fig. 4. [3H]Leucine (lo-* M). The secondary (Ps) and tertiary (Pt) meshworks can be seen with a diffuse cover of silver grains. Dense accumulations of silver grains (B) are present over intersections of the tertiary

plexus (Pt).

Fig. 5. Accumulations of silver grains associated with [‘HIGABA uptake in the guinea-pig ileal myenteric plexus. Heavily labelled tracts and bodies (B) are present in the secondary (Ps) and tertiary (Pt) meshworks. [rH]GABA (5 x 10e9 M) and p-alanine (IO-) M) were present in the incubation medium. Pp,

primary plexus.

Fig. 6. Autoradiograph of a myenteric plexus from the guinea-pig ileum treated with 5 x 1O--9 M GABA in the presence of lo-) M /I-alanine, showing the distribution of [‘HIGABA uptake sites within the ganglia (G), and meshworks (primary Pp, secondary Ps, tertiary Pt). Dense accumulations of silver grains occur

over neurons (N) within the ganglia. Counter stained with neutral red.

Figs 7-13. Dense accumulations of silver grains can be seen over both the soma and emergent processes of neurons (N) within seven different ganglia (G) of the guinea-pig ileal myenteric plexus treated for [)H]GABA (5 x lO-9 M) autoradiography. p-Alanine (10m3 M) was present in the incubation medium. Pp,

primary plexus.

Fig. 14. Autoradiograph of a guinea-pig ileal myenteric plexus treated with [3H]GABA (5 x lO--9 M) in the presence of L-DABA (10e3M) which prevents neuronal uptake of GABA. The ganglia (G) and primary meshwork (Pp) are readily identifiable, counter stained with cresyl violet, but with only a very

diffuse cover of silver grains in the absence of [3H]GABA uptake.

Fig. 15. Light microscopic autoradiograph of guinea-pig ileal myenteric plexus after incubation in [3H]/3-alanine (5 x 10e9 M). Silver grains are almost totally absent; only the ganglia (G) and beginnings of the associated primary plexus (Pp) are delineated, due in most part to the counter-staining with cresyl

violet.

Fig. 16. Reversed-positive autoradiograph of a guinea-pig ileal myenteric plexus treated with [‘HIACHC (5 x 10m9M). The ganglion (G) contains numerous clearly labelled neurons that have taken up this

neuronal-specific compound.

Fig. 17. Autoradiograph of guinea-pig ileal myenteric plexus after incubation in [‘HIGABA (5 x IO-’ M) in the presence of /?-alanine (lO-3 M). Many dense accumulations of silver grains can be seen located over neurons in the ganglia (G); the number of labelled neurons is comparable with that in the immediately

previous figure.

Figs 18-24. Light microscopic autoradiographs of guinea-pig intestine treated with [3H]GABA (5 x 10m9M) in the presence of p-alanine (IO-‘M). Sections (20pm) were prepared in paraffin and

counter stained with cresyl violet or neutral red. Scale bars = 50 pm.

Fig. 18. Transverse paraffin sections prepared from ileum. (a) The layers of the intestine wall, longitudinal muscle (lm), myenteric plexus containing ganglia (G), circular muscle (cm), mucosa-lumen (muc) are identified by the cellular stain. Silver grains associated with 1H uptake are present over the myenteric plexus and circular muscle only. Ganglionic neurons can be identified, some of which show [3H]GABA

labelling. (b) Detail of the mucosal layer, no labelling with [3H]GABA is to be seen.

Fig. 19. Transverse paraffin section prepared from the guinea-pig distal colon. Cresyl violet stained cells characteristic of the different muscle and nerve layers can be seen. Silver grains are present only over the myenteric plexus and circular muscle. Labelled nerve bundles appear deep to the ganglion within the circular muscle (cm). In the ganglion (G) labelled neurons (N) can be identified, some with occasional

emergent processes. Im, longitudinal muscle.

Fig. 20. Transverse paraffin sections prepared from the guinea-pig ileum. A neuron (N) intensely labelled with [3H]GABA is visible in the ganglion (G). Emergent processes can be distinguished. No counter stain.

Figs 21-23. Transverse paraffin sections prepared from the guinea-pig ileum. Accumulations of silver grains occur over the ganglia (G) and associated nerve bundles (nb). Unlabelled cells can be seen lying near intensely labelled neurons (N). Dense clusters of silver grains also occur over the circular muscle (cm).

The serosa and longitudinal muscle (lm) are free of [‘H]GABA labelling.

Fig. 24. Transverse paraffin sections of the guinea-pig distal colon. Sections were cut obliquely across the width of the colon wall. Silver grains occur over distinct tracts running parallel to the circular muscle (cm) fibres, seen here stretching obliquely across the section. No labelling is found in the longitudinal layer (Im),

but silver grains appear over neurons within the ganglia (g). Neutral red counterstaining.

Figs l-5.

Figs 14-18.

Figs 19-24.

[3H]GABA-autoradiography in the gut 1251

Localisation of [‘H]GABA in parafin sections of colon and ileum

Autoradiographs of transverse paraffin sections from the guinea-pig ileum and distal colon, labelled with [3H]GABA (5 x 10m9 M), displayed an extensive distribution of label within the ganglia, and, in addition, a widespread distribution of [‘H]GABA uptake sites in the meshworks of the plexus, and particularly in the circular muscle layer. By contrast, accumulations of silver grains associated with J3H]GABA uptake were almost completely absent from the serosa, longitudinal muscle, submucosa, muscularis mucosae or villi as seen in counterstained preparations (Figs 18a, b).

A diffuse cover of silver grains occurred over the ganglia, reminiscent of labelled neuropil, together with dense accumulations of silver grains clearly over intraganglionic cells including their dendrites (Figs 19, 20). Almost all ganglia as seen in paraffin sections contained labelled cells, and in many instances more than one labelled cell was present. In addition, it was possible to discern regions devoid of silver grains resembling unlabelled cells outlined by densely la- belled neuropil (Figs 21, 22).

A conspicuous feature was the accumulation of silver grains over structures, presumably fibre bun- dles, extending from the ganglia and the inter- connecting primary meshwork into the circular mus- cle layer (Figs 21-23). These trails of silver grains were found only over the outer half of the circular muscle layer, there being no label present in the innermost part of this layer or the submucosal plexus, which is characteristic of myenteric nervous inner- vation of the circular muscle via the deep muscular plexus. There were also many punctate aggregations of silver grains scattered irregularly within the same region (Figs 19,21,22). These started abruptly for the most part some SK100 pm from the myenteric plexus layer, apparently representing transversely cut la- belled fibre bundles of the deep muscular plexus. This notion was reinforced by the observation of many trails of silver grains running parallel to the circular muscle fibres in sections cut obliquely across the width of the colon wall (Fig. 24).

DISCUSSION

Autoradiography in larninar preparations

The present study confirms and extends our earlier observations” that many structures labelled with [3H]GABA can be detected in the myenteric plexus by autoradiography, using dissected “laminar” prepara- tions of the intestinal wall dipped in sensitive nuclear emulsion. Such laminar preparations of the myenteric plexus present essentially a single layer of myenteric neurons that are not entirely enclosed by glial or satellite cells,*’ whilst the plexus itself is comprised of interconnected tubular structures, somewhat raised above the underlying longitudinal muscle,** and con-

tained within a thin basal laminar through which ganglionic neurons and their processes pro- trude.5~6J7~55 Thus the innervation of the longitudinal muscle is confined to the superficially placed my- enteric plexus, ramifying over the innermost surface of the longitudinal layer without penetrating it, in contrast to the circular layer where myenteric pro- cesses penetrate and cross the entire thickness. It is this superficial layer that is exposed upon dissecting away the underlying circular layer to provide the laminar preparations used here, and in consequence the sensitive nuclear emulsion is brought closer to the sources of /I-emission from labelled myenteric neu- rons than at first might seem possible. However, the full extent of labelling may not be revealed by this method, due to the poor penetrating power of the /I-emissions and the inability to bring the emulsion uniformly close to labelled structures. Nevertheless, in favourable preparations such as Fig. 6, the overall appearance of the autoradiographs is rather similar to that seen with methylene-blue staining (un- published observations) or immunohistochemical studies’ of similar laminar preparations. In many instances, considerable detail of dendrites and other processes could be seen as trails of silver grains, many more labelled structures were seen when using [3H]leucine or [3H]proline instead of [3H]GABA or [3H]ACHC, although fewer fibres labelled with the a-amino acids than with GABA or ACHC. With [3H]a-amino acids as the label, the number of dense accumulations of silver grains found in each ganglion approached 20 which is considerably less than the often stated cell population of 60-100.20 Thus the highest number of cells labelled with [3H]GABA or [3H]ACHC, (8/ganglion) may still represent an under- estimate of the number of GABAergic cells present in the ganglia, but we are confident that the many dense accumulations of silver grains in our autoradiographs represent labelling of a significant proportion of superficial neurons and their processes in the ganglia and meshworks of the dissected plexus, a conclusion reinforced by the similar labelling of neurons found in autoradiographs prepared from labelled paraffin sections of the intestinal wall where there can be no question of limitation in the penetrating power of the jI-emissions. Most of the radiolabelled cell bodies were in ganglia but many dense accumulations were also found over the interconnecting fibre bundles (fasciculi) of the primary and secondary meshworks comprising the nerve plexus. Cell bodies have been identified histochemically’6.20 in these “fasciculi”; however, in contrast to the elongate extraganglionic neurons that have been reported in the primary fasciculi,60 intensely labelled cells evident here within the primary fasciculi of [‘H]cc-amino acid treated ileum were morphologically similar to those hitherto described only in ganglia (cf. Fig. Sa with Fig. 29 of Schofield”). Dense accumulations of silver grains were also found at intersections of the tertiary mesh- work, and although more of these structures labelled

1252 A. KRANTIS et al.

with [‘Hlproline or [3H]leucine than with [3H]GABA, a significant number did label with the latter. The disposition and triangular shapes of these labelled cells strongly resemble the interstitial cells of Caja14 (Figs 572, 573) that are now thought to be interposed between nerve terminals and the smooth muscle of the intestine.10,6’,66 We never observed labelling of these structures with [3H]GABA in preparations treated with neuronal uptake blockers for GABA, strongly indicative that these structures are neurons. It is concluded that all three divisions of the my- enteric plexus contain cells that are very likely GABAergic neurons since they no longer labelled with [3H]GABA after treatment with inhibitors of neuronal high affinity GABA uptake.

Distribution of [3H]umino acids

In all the autoradiographs of tissue incubated with

any of the tritiated amino acids there was a diffuse cover of silver grains over the plexus, particularly over the ganglia and the primary and secondary

meshworks but not the smooth muscle itself. Numer- ous non-neuronal elements are associated with the myenteric p1exus,24,25,55 the most frequent being glial cells” or Schwann-cells,’ however there was little noticeable difference in the appearance of this diffuse silver grain cover between preparations labelled with [3H]GABA alone, or in the presence of /I-alanine, a specific glial cell uptake blocker for GABA, which strongly suggests that this labelling was not associ- ated with glia. Indeed, many of the cell bodies and processes of neurons in the ganglia are bare of ‘glia’, and, although satellite nuclei outnumber neurons several fold,20 there was no dense [3H]/3-a1anine label- ling with silver grains outlining glial cell soma as is

seen with [3H]/I-alanine autoradiographs in other nervous tissue.27.39.5s The diffuse cover of silver grains

over ganglia seen in this study may well be in part due to labelling of nerve terminals on neurons receiving GABAergic input within the ganglia. In this regard, the ganglia, as seen in paraffin sections of both the ileum and colon wall, displayed considerable labelling of regions surrounding unlabelled cells, characteristic of labelled neuropil.

Distribution qf’ [3H]GABA and [3H]ACHC

For the main part, among the cells only two morphological variants were found to label with [‘HIGABA or [jH]ACHC. One of these was unipolar with axon clearly labelled, as in Figs 7, 10, 16 (cf. Caja14 Fig. 569), the other, more common, had a cell body with a brush-like basal dendritic arborisation as in Fig. 13 (cf. Cajak4 Fig. 569). The functional significance of these [3H]GABA labelled cell types is not clear, since there is considerable difficulty in correlating the structure and function of cells on the basis of morphological considerations alone, as can be seen in the claims of motor or association func- tions for Dogiel type I cells, and sensory or choliner-

gic motor for type II cells.‘.‘b.55 The very dense labelling of the tertiary plexus with [3H]GABA oc- curred over typical GABA uptake sites in that it was prevented by ACHC, L-DABA or nipecotic acid, all of which are effective inhibitors of neuronal uptake of GABA.36,58 Since GABA, receptors have been de- scribed on the cholinergic innervation of the smooth muscle,2~23.38~s2~53 the heavy labelling of tertiary mesh- work would be explained in part if high affinity GABA uptake sites were associated with a GABAergic innervation concerned with GABA, receptor-mediated depression of cholinergic trans- mission in the motor innervation of the gut.

In paraffin sections of the intestine wall, silver trails from labelled tertiary bundles of the myenteric plexus could be traced into the circular muscle layer. Recip- rocal connections, made between the submucous plexus and the ganglia of the myenteric plexus, penetrate across the width of the circular muscle layer and also give rise to a deep muscular plexus inner- vating the circular muscle. ‘8~33 Stout branches leaving the myenteric plexus were labelled by [3H]GABA, but ran only over the outer half to two-thirds of the circular muscle layer. Here again, uptake sites associ- ated with GABAergic innervation related to a GABA, receptor-mediated modulation of the choli- nergic innervation of the circular muscle could well account for the heavy [‘HIGABA labelling of the tertiary meshwork, more particularly where found coursing through the circular muscle layer in parallel with the muscle fibres characteristic of nervous ramifications (deep muscular plexus) within the circu- lar muscle. Enkephalins also inhibit electrically evoked contractions of the smooth muscle in the guinea-pig ileum 64.6s by a prejunctional action on motor nerves releasing acetylcho1ine,‘~4’.5’~” and the distribution of enkephalin immunoreactive fibres within the circular muscle as seen in sections of the guinea-pig ileumj9 bears a striking resemblance to the autoradiographs of [‘HIGABA uptake sites in this study. Thus the two modulating innervations of the motor nerves may well run in parallel, or GABA and enkephalin may even exist as co-transmitters.

GABA may be an enteric neurotransmitter

There are so many similarities between the neural uptake of [3H]GABA, and its prevention by these specific uptake blockers. in the central nervous sys- tem and in the enteric nervous system, as seen both in uptake and release studies and here with auto- radiography, that it is most likely that GABA is a specific substrate for the transport carrier in cnteric neurons just as in central neurons. Evidently, the properties of enteric GABAergic neurons are very similar to those in the central nervous system in relation to uptake mechanisms, metabolism and pharmacology. Whilst uptake sites for GABA are thought, in general, to be confined to GABAergic neurons,2y.X4 this may be in error. and the presence of such sites is at best only supportive evidence of a

[3H]GABA-autoradiography in the gut 1253

transmitter role for GABA in either central or enteric

neurons. [Although, more recently, Ottersen (per-

sonal communication) has observed GABA-like im- munoreactivity in neurons of the myenteric plexus, confirming that it does contain GABAergic neurons.] As has been established in the mammalian central nervous system, more than one morphological type of enteric nerve cell possessed a GABA high affinity uptake mechanism, which, together with the estab- lished actions for endogenous and applied GABA at separate specific receptor populations within the my- enteric plexuq4* raises the possibility that GABA subserves several functions in the mammalian enteric nervous system. In particular, GABAergic neurons could be involved in nerve pathways mediating reflex peristalsis, for GABA has pharmacological actions involving the intrinsic inhibitory and excitatory in- nervation underlying this activity.45.47.52 Such a possi- bility is supported by the recent demonstration that GABA antagonism markedly alters spontaneous in- testinal motility54 as well as peristaltic actions in the

isolated colon of the guinea-pig as measured by faecal pellet propulsion. W* More recently, Jessen et al.,30

using electron-microscopic autoradiography, have shown that [3H]GABA uptake sites are restricted to nerve bundles within the plexus on the surface of the longitudinal muscle layer, there being no uptake sites associated with the innervation of the intestinal smooth muscle itself. The present study confirms this, and extends these observations to the circular muscle, from which we conclude that the cholinergic inner- vation of both circular and longitudinal intestinal muscle is subject to modulation by a prejunctional GABAergic innervation acting through GABA, re- ceptors capable of depressing cholinergic trans- mission.2,52

Taken together with the pharmacological, bio- chemical and autoradiographic evidence3’,49,6’,62 for the localization of GABA and its metabolic enzymes in mammalian myenteric neurons, the present evi- dence reinforces the likelihood that GABA is a neurotransmitter in the myenteric plexus.

1.

2.

3.

4. 5.

6.

7.

8.

9.

10.

11. 12.

13.

14.

15.

16. 17. 18. 19. 20. 21. 22. 23.

24. 25.

26. 27.

REFERENCES

Ambache N. and Freeman M. A. (1968) Atropine-resistant longitudinal muscle spasms due to excitation of non-cholinergic neurons in Auerbach’s plexus. J. Physiol., Land. 199, 705-727. Bowery N. G., Doble A., Hill D. R., Hudson A. L., Shaw J. S., Turnbull M. J. and Warrington R. (1981) Bicuculhne-insensitive GABA receptors on peripheral autonomic nerve terminals. Eur. J. Pharmac. 71, 53-70. Bowery N. G., Jones G. P. and Neal M. J. (1976) Selective inhibition of neuronal GABA uptake by cis-1,3- aminocyclohexane carboxylic acid. Nature, Lond. 264, 28 l-284. Ramon y Cajal S. (1911) Histologic du SystPme Nerveux de I’Homme et des VertPbrPs, Vol. II. Paris, Maloine. Cook R. D. and Burnstock G. (1976) The ultrastructure of Auerbach’s plexus in the guinea-pig intestine. 1. Neuronal elements. J. Neurocyt. 5, 171-194. Cook R. D. and Burnstock G. (1976) The ultrastructure of Auerbach’s plexus in the guinea-pig intestine. II. Non-neuronal elements. J. Neurocyr. 5, 196206. Costa M., Buffa R., Furness J. B. and Solcia E. (1980) Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations. Histochemistry 65, 157-165. Cowan W. M., Gottlieb D. I., Hendrickson A. E., Price J. L. and Woolsey T. A. (1972) The autoradiographic demonstration of axonal connections in the central nervous system. Brain Res. 37, 21-51. Curtis D. R., Game C. J. A. and Lodge D. (1976) The in vi00 inactivation of GABA and other inhibitory amino acids in the cat central nervous system. Ex>. Brain Res. 25, 413428. Daniel E. E.. Helmv-Elkholv A. and Janer L. P. (1983) Neither a ourine nor VIP is the mediator of inhibitorv nerves of opossum oesophageal smooth muscle. J. Physiol. k, 243-266. De Feudis F. V. (1975) Amino acids as central neurotransmitters. Ann. Rev. Pharmac. 15, 1055130. Diab I. M., Dinerstein R. J., Watanabe M. and Roth L. J. (1976) [‘HIMorphine localization in myenteric neurons. Science 193, 689-69 1. Dogiel A. S. (1899) tiber den Bau der Ganglien in den Geflechten des Darmes und der gallenblase des Menschen und der Saugethiere. Arch. Anat. Physiot. 5, 130-159. Droz B. and Leblond C. P. (1963) Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography. J. camp. Neurol. 121, 3255346. Furness J. B. (1970) The origin and distribution of adrenergic nerve fibres in the guinea-pig colon. Histochemie 21, 2955306. Gabella G. (1969) Detection of nerve cells by a histochemical technique. Experientiu 25, 218-219. Gabella G. (1971) Glial cells in the myenteric plexus. Z. Nafurforsch. 26b, 244-245. Gabella G. (1972) Innervation of the intestinal muscular coat. J. Neurocyr. 1, 341-361. Gabella G. (1976) Structure of the Autonomic Nervous System. Chapman and Hall, London. Gabella G. (1979) Innervation of the gastrointestinal tract. Inc. Rev. Cytol. 59, 1299193. Gabella G. and Costa M. (1969) Adrenergic innervation of the intestinal smooth musculature. Experientia 25,395-396. Gershon M. D. (1981) The enteric nervous system. Ann. Rev. Neurosci. 4, 227-272. Giotti A., Luzzi S., Spagnesi S. and Zilleti L. (1983) GABA, and GABA, receptor-mediated effects in guinea-pig ileum. Br. J. Pharmac. 78, 469478. Gunn M. (1959) Cell types in the myenteric plexus of the cat. J. camp. Neural. 111, 83-100. Gunn M. (1968) Histological and histochemical observations on the mventeric and submucous olexuses of mammals. J. Anat. 102, 223-239. - Hill C. J. (1927) A contribution to our knowledge of the enteric plexus. Phil. Trans. R. Sot. Lond. B215, 3555387. Hosli E. and Hosli L. (1980) Cellular localization of the uptake of [3H]fl-alanine in cultures of the rat central nervous system. Neuroscience 5, 1455152.

1254 A. KRANTIS et al.

28. Iversen L. L. and Johnston G. A. R. (1971) GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors. J. Neurochem. 18, 1939-1950.

29. Iversen L. L. and Kelly J. S. (1975) Uptake and metabolism of y-aminobutyric acid by neurons and glial cells. Biochem.

30

31

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

Pharmac. 24, 933-938. Jessen K. R., Hills J. M., Dennison M. E. and Musky R. (1983) y-Aminobutyrate as an autonomic neurotransmitter: release and uptake of [3H] y-aminobutyric in guinea pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography. Neuroscience 10, 1427-1442. Jessen K. R., Mirsky R., Dennison M. E. and Burnstock G. (1979) GABA may be a neurotransmitter in the vertebrate peripheraf nerves. Nature 281, 71-74. Jessen K. R., Saffrey M. J., Van Noorden S., Bloom S. R., Polak J. M. and Burnstock G. (1980) Immunohist~hemi~l studies of the enteric nervous system in tissue culture and in situ: localization of vasoactive intestinal peptide (VIP), substance-P and enkephalin immunoreactive nerves in the guinea-pig gut. Neuroscience 5, 1717-1735. Jessen K. R., Polak J. M., Van Noorden S., Bloom S. R. and Burnstock G. (1980) Peptide-containing neurons connect the two ganglionated plexuses of the enteric nervous system. Nature, Lond. 283, 391--393. Jessen K. R., Saffrey M. J., Baluk P.. Hanani M. and Burnstock G. (1983) The enteric nervous system in tissue culture III. Studies on neuronal survival and the retention of biochemical and mo~hological di~erentiation. Bra&r Res. 262, 4962. Jessen K. R., Saffrey M. J. and Burnstock G. (1983) The enteric nervous system in tissue culture. 1. Ceil types and their interactions in explants of the myenteric and submucous plexuses from guinea-pig, rabbit and rat. Brain Res. 262, 17-35. Johnston G. A. R. (1976) Physiological pharmacology of GABA and its antagonists in the vertebrate nervous system. In GABA in Nervous System Function (eds Roberts E., Chase T. N. and Tower D. B.), pp. 395411. Raven Press, New York. Johnston G. A. R. (1978) Neuropharmacology of amino acid inhibitory neurotransmitters. Ann. Rev. Pharmac. Tox. 18, 269-289. Kaplita P. V., Waters D. H. and Triggle D. J. (1982) y-Aminobutyric acid action in guinea-pig ileal myenteric plexus. Eur. J. Pharmac. 79, 43-5 I. Kelly J. S. and Dick F. (1976) Differential labelling of gliai cells and GABA-inhibitory interneurons and nerve terminals following the mi~oinjection of 13H]~-alanine, [3H]DABA and [3H]GABA into single folia of the cerebellum. Cold Spring Harbour Symposia on Quantitative Biology 30, 933106. Kerr D. 1. B. and Krantis A. (1983) Uptake and stimulus-evoked release of [-‘HI-p-aminobutyric acid by myenteric nerves of guinea-pig intestine. Br. J. Pharmac. 78, 271-276. Kerr D. I. B. and Ong J. (1984) Evidence that ethylenediamine acts in the isolated ileum of the guinea-pig by releasing endogenous GABA. Br. J. Pharmae. 83, 1699177. Kerr D. I. B. and Ong J. (1984) GABA and GABA-receptors in the enteric nervous system. Neuropharmacofogy 23, 835-836. Kleinrok A. and Kilbinger H. (1983) y-Aminobutyric acid and cholinergic transmission in the guinea-pig ileum. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 322, 216220. Krantis A. and Kerr D. 1. B. (1981) Autoradiographic localization of [‘Hjgamma-aminobutyric acid in the myenteric plexus of the guinea-pig small intestine. Neurosci. Lett. 23, 263-268. Krantis A. and Kerr D. I. B. (1981) GABA induced excitatory responses in the guinea-pig small intestine are antagonized by bicuculline, picrotoxinin and chloride ion blockers. Naunyn-Schmiedeberg’s Arch. exp. Purh. Pharmak. 317, 257-261. Krantis A. and Kerr D. I. B. (1981) The effect of GABA antagonism on propulsive activity of the guinea-pig large intestine. Eur. J. Pharmac. 76, 111-I 14. Krantis A., Costa M., Furness J. B. and Orbach J. (1980) y-Aminobutyric acid stimulates intrinsic inhibitory and excitatory nerves in the guinea-pig intestine. Eur. J. Pharmac. 67, 461-468. Krogsgaard-Larsen P. and Johnston G. A. R. (1975) Inhibition of GABA uptake in rat brain slices by nipecotic acid. various isoxazoles and related compounds. J. Neurochem. 25, 7977802. Miki Y., Taniyama K., Tanaka C. and Tobe T. (1983) GABA, glutamic acid decarboxylase, and GABA transaminase levels in the myenteric plexus in the intestine of humans and other mammals. J. Neurochem. 40, 861-865. Neal M. J., Cunningham J. R. and Marshall J. (1979) The uptake and radioautographical localization in the frog retina of r3H] (f) amin~yclohexane carboxylic acid, a selective inhibitor of neuronal GABA transport. Bruin Rex 176, 285-296. North R. A,, Katayama Y. and William J. T. (1979) On the mechanism and site of action of enkephalin on single myenteric neurons. Brain Res. 165, 67-77. Ong J. and Kerr D. I. B. (1983) GABA,- and GABA,-receptor-mediated modification of intestinal motility. Eur. J. Pharmac. 86, 9-l 7. Ong J. and Kerr D. I. B. (1983) Interactions between GABA and S-hydroxytryptamine in the guinea-pig ileum. Eur. J. Pharmac. 94, 3055312. Ong J. and Kerr D. I. B. (1984) Evidence for a physiological role of GABA in the control of guinea-pig intestinal motility. Neurosci. Lett. SO, 339-343. Schofield G. C. (1968) Anatomy of muscular and neural tissues in the alimentary canal. In Handbook of Physiology, Section 6, Vol. IV, pp. 15791627. American Physiological Society. Schon F. and Iversen L. L. (1972) Selective a~umuIation of [)H]GABA by stellate cells in rat cerebellar cortex in oivo. Brain Res. 42, 503-507. Schon F. and Kelly J. S. (1974) The characterization of [IH]GABA uptake into the satellite glial cells of the sensory ganglia. Brain Res. 66, 289-300. Schon F. and Kelly J. S. (1975) Selective uptake of [3H]/-alanine by glia: association with the glial uptake system for GABA. Brain Res. 86, 243-257. Schultzberg M., Hijkfelt T., Nilsson G., Terenius L., Rehfeld J. F., Brown M., Elde R., Goldstein M. and Said S. (1980) Distribution of peptide- and Cat~hoIami~e-containing neurons in the gastrointestinal tract of rat and guinea-pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine fi-hydroxylase. Neuroscience 5, 689.--744.

[‘HIGABA-autoradiography in the gut 1255

60. Takeo 0. and Sugai N. (1974) Morphology of extraganglionic fluorescent neurons in the myenteric plexus of the small intestine of the rat. J. camp. Neural. 158, 109-120.

61. Taniyama K., Kusunoki M., Saito N. and Tanaka C. (1982) Release of y-aminobutyric acid from cat colon. Science 217, 1038-1040.

62. Taniyama K., Kusunoki M., Saito N. and Tanaka C. (1983) GABA evoked ACh release from isolated guinea-pig ileum. Life Sci. 32, 2349-2353.

63. Thuneberg L., Rumessen J. J. and Mikkelsen H. B. (1982) The interstitial cells of Cajal: intestinal pacemaker cells? In Motility of the Digestiue Tract (ed. M. Wienbeck), pp. 115-122. Raven Press, New York.

64. Van Nueten J., Van Ree J. M. and Van Houtte P. M. (1977) Inhibition by Met-enkephahn of peristaltic activity in the guinea-pig ileum, and its reversal by naloxone. Eur. J. Pharmac. 41, 341-342.

65. Waterfield A. A., Smockum R. W. J., Hughes S. J., Kosterlitz H. W. and Henderson G. (1977) In vitro pharmacology of the opioid peptides, enkephalins and endorphins. Eur. J. Pbarmac. 43, 107-116.

66. Yamamoto M. (1977) Electron microscopic studies on the innervation of the smooth muscle and the interstitial cell of Cajal in the small intestine of the mouse and rat. Arch. Histol. jap. 40, 171-201.

(Accepted 20 October 1985)