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Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon
Journal of the Autonomic Nervous System 68 Ž1998. 33–42
Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon Anthony Krantis ) , Kim Nichols, William Staines DigestiÕe Diseases Research Group, Department of Cellular and Molecular Medicine, UniÕersity of Ottawa, 451 Smyth Road, Ottawa, Ont., Canada K1H 8M5 Received 11 April 1997; revised 15 September 1997; accepted 25 September 1997
Abstract GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide ŽNO. is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase ŽGABA-T. immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: Ži. neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, Žii. neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and Žiii. neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis. q 1998 Elsevier Science B.V. Keywords: GABAergic; Nitric oxide ŽNO.; NO synthase; Somatostatin; Enkephalin; GABA-transaminase ŽGABA-T. human colon; Immunohistochemistry; NADPH-diaphorase
1. Introduction The emerging neurochemical evidence for an extensive enteric neural and endocrine distribution of GABA, together with its well described actions in the mammalian gut, supports the proposal that the enteric GABAergic system is vast with respect to its distribution, types of communication and potential functions. GABAergic neurons and fibres have been identified in the myenteric and submucosal nerve layers of the guinea-pig and rat gut by 3 H-GABA high affinity autoradiography, as well as by ) Corresponding author. Present address: Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ont., Canada K1H 8M5. Tel.: q1 613 5625800 Ext. 8399; fax: q1 613 5625434; e-mail: [email protected].
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 7 . 0 0 1 1 3 - 6
immunohistochemical andror histochemical analysis of GABA and its synthetic enzymes w10,35,45,47,54x. Endocrine-like cells in the rat stomach and intestine have also been found to contain GABA and high affinity GABA uptake sites w10,29,31x and it is proposed that GABA in the gut may function as a neurotransmitter, hormone and paracrine agent. We have employed an immunofluorescence technique w29x used in the CNS for localizing the GABA-catabolizing enzyme 4-amino-butyrate:2-oxoglutarate transaminase ŽGABA-T., to identify GABAergic neurons w41,50x in the human colon w42x. We found GABAergic neurons in both the myenteric and submucosal ganglionated nerve networks and GABAergic nerve fibre projections within the smooth muscle layers. This distribution of GABAergic neural elements in human tissue is similar to that seen in the rodent gut w28,33,34,36,42,53x.
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GABA exerts actions on motor neurons via two distinct populations of neural receptors, the excitatory GABA A and the prejunctional inhibitory GABA receptors respectively w4,5,11,12,48,49x. GABA A receptors may also modulate acid secretion w22,27x and epithelial H 2 O and electrolyte transport w25,39x. Centrally, the range of signalling across GABAergic synapses is often enhanced by co-storage and release of neuropeptides such as somatostatin and neuropeptide Y w1,2x. Similar insight with respect to enteric neuro-humoral signalling is limited. In the rat colon a circuit consisting of GABA- somatostatin- and enkephalin-containing noncholinergic interneurons is proposed to play a key role in the regulation of descending relaxation, i.e. activation of somatostatinergic neurons by muscle stretch, leads to inhibition of enkephalin neurons and, in turn, disinhibition of GABAergic interneurons and motor neurons containing both vasoactive intestinal peptide ŽVIP. and nitric oxide ŽNO. w16,20,40x. This latter detail is unlikely to be important in the human colon since VIP and NO synthase do not colocalize with single neurons w40x. Pharmacological data suggest that GABA, somatostatin and enkephalin interneurons function within neural circuits controlling NO inhibitory motor neurons, but the anatomical correlate to these findings is limited. Supportive data reveal that in the rat colon, almost half of the myenteric nerve cell perikarya, including 35% of the NO synthesizing neurons contain GABA A receptors w38x. A variety of enteric interneurons control the circuits underlying peristalsis, consisting of an ascending phase regulating motor neurons that release acetylcholine to cause contraction w6,17x and a descending relaxation phase that involves motor neurons releasing VIP andror NO, or adenosine triphosphate w6,20x. We recently reported w42x that GABAergic neurons within the myenteric and submucosal nerve layers of the human colon contain NO synthase ŽNOS. establishing that nitrergic interneurons exist in the human colon. Enteric neurons often colocalize a number of putative transmittersrmodulators and this provides an opportunity for physiologically and morphologically distinct subpopulation of neurons to be further categorized by the neuroactive substances they contain w7x. Using a triple-labelling strategy which involved double-labelling immunohistochemistry combined with the histochemical demonstration of NADPH diaphorase-related
NOS activity in the human colon we demonstrate that GABAergic neurons comprise non-overlapping populations of somatostatin immunoreactive, enkephalin immunoreactive and NOS containing neurons.
2. Materials and methods By consent Žunder approval of the Research Ethics Board of the Children’s Hospital of Eastern Ontario, CHEO. tissue specimens Žeach approximately 2 = 1 cm in dimension. were taken from the surgically resected normoganglionic segments of sigmoid colon obtained from four infants Žages 8 to 20 months. diagnosed with Hirschsprung’s disease, undergoing surgery for bowel obstruction at CHEO. Preoperative diagnosis included clinical, radiological and anorectal manometric assessment. Close cooperation and coordination between the pediatric pathologist at the time of diagnosis ensured the correct handling of tissue. The removed tissue examined in this study included only normal, ganglionated intestine. Tissue taken from full thickness resection margins were received at the time of surgery in a freshly prepared Kreb’s bicarbonate solution. These tissues were then processed for histochemistry: tissue segments were washed in ice cold 0.1 M sodium phosphate buffered saline ŽPBS, pH 7.4., pinned out on styrofoam and fixed for 2 h at 48C in 4% paraformaldehyde and 0.4% picric acid in 0.16 M sodium phosphate buffer ŽPB, pH 7.0.. The colon segments were kept at 48C in PB ŽpH 7.2. containing 10% sucrose and 0.1% sodium azide for a minimum of 24 h prior to sectioning. Serial frozen transverse or cross sections were taken at a 6 m m thickness and mounted onto glass slides. Three triple-labeling cytochemical strategies were employed in this study. Slide mounted frozen serial sections from each patient tissue sample Ž3 slidesrtissue sample. were reacted for immunofluorescent demonstration of GABA-T and Som, or GABA-T and Enk, or Som and Enk and then treated for NADPH diaphorase histochemistry. In some cases, sections were reacted singly for GABA-T immunofluorescence followed by NADPH diaphorase histochemistry. Three serial sections Ž1, 2 and 3. were treated in the respective order by incubating in Ži. rabbit antiGABA-T Ž1:400. and monoclonal mouse anti-Som Ž1:200;
Fig. 1. Micrographs of cross sections of the myenteric nerve layer and smooth muscle layers of the human colon. Ža. Intensely GABA-T immunoreactive nerve cells are present in a ganglion of the myenteric plexus ŽMp.. Within the circular muscle Žcm. intensely labelled fibre bundles Žlarge arrowheads. are distributed throughout the muscle. Žb. Micrograph of the same section showing intensely Enk immunoreactive cells in the ganglion. Enk immunoreactive fibres in the circular muscle layer were less intensely labelled. Bar s 100 m m. Fig. 2. Micrographs of myenteric plexus sections showing a single ganglion. Intensely GABA-T immunoreactive cells are shown with arrows Ža. Enk immunoreactivity Žb. and NO synthase related NADPH-diaphorase staining Žc. are presented alongside. GABA-T cells colocalizing Enk Ž`. and GABA-T cells stained with NO synthase related NADPH-diaphorase Ž^. are shown. Bar s 100 m m. Fig. 3. Micrographs of intensely GABA-T immunoreactive myenteric ganglion cells Ža. and nerve fibres Žarrows. in the longitudinal Žlm. and circular Žcm. muscle Žb.. In Žb., the varicose nature of these GABA-T positive nerve fibres is evident. Bar s 100 m m.
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A. Krantis et al.r Journal of the Autonomic NerÕous System 68 (1998) 33–42
Žii. rabbit anti-Enk Ž1:400. and monoclonal mouse anti-Som Ž1:200. and Žiii. rabbit anti-GABA-T Ž1:400. and mouse anti-met-Enk Ž1:200. in PBS Žphosphate buffered saline, pH 7.2. containing 0.3% Triton X-100 for 18 h at 48C. Following a 15 min wash in PBS, each of the three serial tissue sections were incubated in fluorescein isothiocyanate ŽFITC.-conjugated donkey anti-rabbit Ž1:20. ŽAmersham, Ont., Canada. and CY3-conjugated sheep anti-mouse Ž1:200. diluted in Triton-PBS buffer for 40 min at 378C, washed, rinsed and coverslipped. Sections were then photographed using a Zeiss axioplan microscope with a 488 and a 520 nm filter to visualize FITC and Cy3, respectively, in the same section, prior to processing for histochemical demonstration of NO synthase-related NADPH diaphorase activity.
submucous ganglia in sections of human colon triple labelled for GABA-T, Som and NOS-related NADPH dependent diaphorase activity or GABA-T, Enk and NOS-related NADPH dependent diaphorase activity. 2.3. Chemicals Reduced b-nicotinamide adenine dinucleotide phosphate ŽNADPH. and nitro blue tetrazolium ŽNBT. were purchased from Sigma Chemicals ŽToronto, Ont., Canada.. All of the remaining reagents utilized in this investigation were purchased from Aldrich Chemical Co. ŽMontreal, Que., Canada..
3. Results 2.1. NO synthase histochemistry Sections treated for GABA-T, GABA-TrSom, EnkrSom and GABA-TrEnk immunofluorescence were photographed, their microscope stage coordinates were recorded, the coverslips were subsequently removed and they were reacted for histochemical identification of NO synthase-related NADPH diaphorase activity after the method of Nichols et al. w42x. Slides were placed in a dark, moist chamber for 1 h at 378C covered with the reaction medium for NO synthase activity consisting of 1 mM NADPH, 0.5 mM nitro-blue tetrazolium ŽNBT. and 0.3% Triton X-100 in 10 mM sodium phosphate buffer, pH 8.0. Tissues were given an extensive final wash with PB Ž10 mM, pH 8.0. air dried, coverslipped and analyzed by light and fluorescence microscopy. 2.2. Cell counting procedure Counts were obtained in a single-blind fashion by two independent observers. Cells were counted while viewing ganglia at 20 = objective magnification and a 10 = eyepiece magnification as previously described w42x. Data were derived from counts of 200 myenteric and 150
Neurons positive for GABA-T immunoreactivity were abundant in the ganglionated nerve networks of the myenteric plexus ŽFig. 1a, Fig. 2a, Fig. 3a, Fig. 4a. and present in the submucosa within ganglia of Henle’s plexus ŽFig. 5a, Fig. 9a, Fig. 10a. which is located subjacent to the circular muscle layer, Meissner’s plexus ŽFig. 7a. which is located subjacent to the muscularis mucosae and within the ganglia of the intermediate nerve plexus Žunique to the human. which is located midway between the former two and close to the vascular plexus ŽFig. 6a.. In addition, GABA-T immunoreactivity was noted in fibres innervating the muscularis externa ŽFig. 1a, Fig. 3a and b, Fig. 9a, Fig. 10a.. Somatostatin ŽSom.-immunoreactive neurons were in ganglionated nerve networks of the wall of the colon, but were far less numerous than GABA-T immunoreactive cells, particularly within the myenteric plexus. Enkephalin ŽEnk.-immunoreactive neurons were found only in the myenteric plexus and there were varicose Enk-immunoreactive fibres were seen within the longitudinal and circular muscle layers. NADPH diaphorase staining, indicative of NO synthase activity ŽNOS activity., was present in nerve cells within all nerve layers of the colon wall and fibres within both nerve and muscle layers ŽFig. 2c, Fig. 9c, Fig. 10b..
Fig. 4. Micrographs of sections through a myenteric ganglion which has been double stained for GABA-T and Som immunoreactivity. Intensely GABA-T immunoreactive myenteric ganglion cells Ža. are often seen to colocalize Som immunoreactivity Žb, arrow.. Bar s 100 m m. Fig. 5. Micrographs of sections of the submucosa of the human colon double stained for GABA-T and Som immunoreactivity. Examples of GABA-T immunoreactive neurons Žarrow in a. colocalizing Som immunoreactivity Žarrow in b. in the ganglia of Henle’s plexus ŽHp.. Bar s 100 m m. Fig. 6. Micrographs of sections of the submucosa of the human colon double stained for GABA-T and Som immunoreactivity. Examples of GABA-T immunoreactive neurons Žarrow in a. colocalizing Som immunoreactivity Žarrow in b. in the ganglia of intermediate plexus ŽIp.. Bar s 100 m m. Fig. 7. Micrographs of sections of the submucosa of the human colon double stained for GABA-T and Som immunoreactivity. Examples of GABA-T immunoreactive neurons Žarrows in a. colocalizing Som immunoreactivity Žarrows in b. in the ganglia of Meissner’s plexus Žmp.. Bar s 100 m m. Fig. 8. Micrographs of fixed frozen cross sections of the mucosa of the human colon double stained for GABA-T Ža. and Som Žb. immunoreactivity. An endocrine-like cell in the mucosa Žmuc. shows clear evidence of colocalizing GABA-T and Som immunoreactivity.
A. Krantis et al.r Journal of the Autonomic NerÕous System 68 (1998) 33–42
It was possible to characterize cell shape and size to some extent. GABA-T immunopositive cells had elongated or round perikarya of moderate size, typical of Dogiel Type II neurons w33,46x, often with a single emergent process ŽFig. 10a.. Myenteric ganglia displayed a richly
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labelled neuropil within which single positive fibres were not discernible. Conversely, submucosal ganglia neuropil was less densely labelled and it was often possible to discern individual immunopositive fibres ŽFig. 9.. Although fibres could not be followed within the intercon-
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necting fasciculi of the nerve plexi, immunopositive fibres within nerve bundles of the muscularis could be seen ŽFig. 1a, Fig. 10a.. Some micrographs showed GABA-T positive fibres in the muscularis were varicose ŽFig. 3b..
3.1. Colocalization Combined histochemical labelling experiments revealed that the GABAergic neurons of the myenteric and submu-
A. Krantis et al.r Journal of the Autonomic NerÕous System 68 (1998) 33–42 Table 1 Average number Ž"standard deviation. of neurons per ganglion labelled for GABA-T, somatostatin ŽSom., enkephalin ŽEnk. and NO synthase ŽNOS. within the different enteric nerve plexi
Myenteric Henle’s Intermediate Meissner’s
GABA-T
Som
Enk
NOS
5"1.0 2"0.2 3"0.4 3"0.2
2"0.3 2"0.4 2"0.2 2"0.3
1"0.2 0 0 0
3"1.0 2"0.2 2"0.1 3"0.1
Data were obtained from counts of 200 myenteric and 150 submucosal ganglia in sections of human colon triple labelled for Ž1. GABA-T, Som and NOS-related NADPH diaphorase activity or Ž2. GABA-T, Enk and NOS activity.
Table 2 Percentage of GABA-T immunoreactive cells which display colocalization of for somatostatin ŽSom., enkephalin immunoreactivity ŽEnk. or NO synthase activity ŽNOS. within the myenteric plexus and submucosa
Myenteric Submucous
NOS Ž%.
Som Ž%.
Enk Ž%.
23 17
40 37
9 0
Data were obtained from counts of 200 myenteric and 150 submucosal ganglia in sections of human colon triple labelled for Ž1. GABA-T, Som and NOS-related NADPH diaphorase activity or Ž2. GABA-T, Enk and NOS activity.
cosal ganglia were composed of distinct populations of neurons that could be further separated into those that contained either Som and GABA-T immunoreactivity, Enk and GABA-T immunoreactivity, GABA-T immunoreactivity and NOS activity, Enk immunoreactivity alone, GABA-T immunoreactivity alone and NOS activity alone. Som and Enk immunoreactivity were never seen to colocalize within the same cell Žimmunohistochemical protocol Žii., data not shown.. Similarly, NOS activity was never seen in either Som or Enk immunoreactive neurons Žcf. Fig. 2b and c, Fig. 9b and c.. Cell counts were made and the frequency distribution of labelled neurons is presented in Table 1. The proportion of GABAergic neurons colocalizing neuropeptide or NOS activity is shown in Table 2. Approximately 40% of GABAergic ŽGABA-T positive. neurons were also immunopositive for Som, 9% were immunopositive for Enk and 23% colocalize NOS activity. Conversely all of the Som immunoreactive neurons also contain GABA-T immunoreactivity and 81% of Enk immunoreactive neurons displayed GABA-T immunoreactivity ŽTable 3.. Of neu-
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Table 3 Percentage of somatostatin ŽSom., enkephalin immunoreactivity ŽEnk. or NO synthase activity ŽNOS. within the myenteric plexus and submucosa cells which display colocalization of for GABA-T immunoreactivity
Myenteric Submucous
NOS Ž%.
Som Ž%.
Enk Ž%.
46 60
100 69
81 0
Data were obtained from counts of 200 myenteric and 150 submucosal ganglia in sections of human colon triple labelled for Ž1. GABA-T, Som and NOS-related NADPH diaphorase activity or Ž2. GABA-T, Enk and NOS activity.
rons with NO synthase activity, 46% colocalize GABA-T immunoreactivity. Enkephalin immunoreactive neurons were not found within the submucosa. In contrast, GABA-T immunoreactive cells were evident within all of the networks ŽHenle’s, intermediate and Meissner’s plexuses. and Som positive cells were only slightly less numerous, Table 1. Within the submucosa, 69% of Som immunoreactive ganglion cells were GABA-T immunoreactive ŽFigs. 4–7 and 9., however only 37% of GABA-T positive cells showed Som immunoreactivity. Although NOS positive cells are abundant in the submucosal ganglia ŽFig. 2c, Fig. 9c, Fig. 10b., NOS was only found in 17% of submucosal GABAergic neurons ŽFig. 9c, Fig. 10b.. Conversely, 60% of the NOS positive cells were also GABA-T positive. These results are summarized in Tables 2 and 3. Many mucosal epithelial cells with the characteristics of endocrine cells were positive for either GABA-T or Som. Cells immunoreactive for Som were more numerous, in particular at the base of the crypts, whereas GABA-T immunoreactive epithelial cells were more often located on the villus. A small minority displayed both GABA-T and Som immunoreactivity ŽFig. 8.. These cells were always elongate with a long narrow process at the luminal aspect.
4. Discussion In this study, we demonstrate neurochemically distinct, non-overlapping subpopulations of GABAergic myenteric and submucosal neurons of the human colon, and provide additional evidence for the existence of GABAergic endocrine-like cells in the human colon mucosa. Using GABA-T-immunoreactivity in combination with neuropeptide, immunohistochemistry and NOS cytochemistry, the
Fig. 9. Micrographs of a single section through a ganglion of Henle’s plexus which is triple labelled for GABA-T, Som and NADPH-diaphorase. Ža. should be compared with Žb. and Žc.. Cells and circular muscle fibres colocalizing Som immunoreactivity Ž`. or NO synthase related NADPH-diaphorase staining Ž^. are shown. In addition, a GABA-T positive cell Žarrow. that is negative for Som or diaphorase staining is evident. Bar s 100 m m. Fig. 10. Micrographs of a single section through a ganglion of Henle’s plexus ŽHp. and adjacent circular muscle Žcm. which is double labelled for GABA-T and NADPH-diaphorase. Neural elements Žcells and nerve fibers. displaying colocalization of both GABA-T immunoreactivity and NO synthase related diaphorase are labelled Ž^.. A type II neuron, that is GABA-T positiverNADPH-diaphorase negative is shown ŽII.. Bar s 100 m m.
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GABAergic myenteric interneurons in the human colon were categorized into three major subsets: Ži. neurons with somatostatin which accounted for about 40% of the GABA population, Žii. neurons with enkephalin which accounted for about 9% of the GABA neurons and Žiii. neurons with NADPH diaphorase-related NOS activity which accounted for about 23% of the GABA cells. Neither somatostatin nor enkephalin positive cells showed NOS activity. Within the submucosa and mucosa, GABAergic cells were seen to colocalize either Som or NOS but not both. The lack of enkephalin positive cells in the submucosa has been reported by others and corroborates the low levels of met-enkephalin in this layer compared to the muscularis externa. Enteric interneurons have been partially classified by their neurochemical content and projections. Whereas ascending interneurons are all cholinergic, forming chains of neurons utilizing the same neurotransmitter, descending interneurons are either cholinergic or non-cholinergic w6x. Cholinergic interneurons have been classified into three subpopulations, on the basis of colocalization with somatostatin, VIP or 5-HT. The somatostatin and serotoninimmunoreactive interneurons contact more anally situated interneurons of the same neurotransmitter type and as such they also form neurochemically homogeneous interneuronal chains. Non-cholinergic interneurons have been subcatagorized based on their being immunoreactive for VIP, NOS and gastrin-releasing peptide. In view of the identification of distinct populations of enteric GABAergic neurons, we propose that neurochemical classification of interneurons requires revision to take into account the presence of GABAergic interneurons w6,17,18x. Both GABA acting at GABA B receptors and enkephalin act prejunctionally to modulate cholinergic motor neuron output w9,31,40,45x. The myenteric GABAergic neurons colocalizing enkephalin documented in this study represent neurons providing this prejunctional input. This notion is supported by data with on the distribution of GABAergic neurons colocalizing somatostatin. As shown in the human intestine w51x, we observed that somatostatin neurons occur within the myenteric and submucous plexuses, but found no somatostatin-immunoreactive fibers in the muscularis where most if not all axon–axon interaction occurs. The fibers of somatostatin-containing neurons project caudally within the nerve plexuses w8,32x. Somatostatin has no direct effect on smooth muscle w19,21,52x, rather somatostatin causes either membrane depolarization or hyperpolarization of distinct populations of myenteric neurons w30x. On this basis, the subset of GABA-TrSom positive neurons are probably unrelated to any GABA-B modulation of motor function. It is more likely that these neurons are associated with GABA-A neural control of motor and secretomotor neurons. In the rat colon, half of all myenteric neuron perikarya have GABA-A receptors and hence are potential targets for GABAergic neurons w38x. In this study, somatostatin immunoreactive epithelial cells and GABA-T positive epithelial cells were found
throughout the colon mucosa. Double labelling experiments indicate that for the most part, these represent separate populations of cells however, a few endocrine-like cells show colocalization of GABA-T and Som. Somatostatin localization within human mucosal endocrine cells and it’s function as a gut hormonerparacrine agent have been well described w13x. GABA-T immunoreactivity and high affinity GABA uptake are also known to occur within endocrine-like cells of the mucosa throughout the rodent gastrointestinal tract w10,14,15,29,37x. In the rat stomach, GABA is colocalised in gastrin producing ŽG type. and somatostatin producing ŽD type. endocrine cells w10x. GABA synthesized by endocrine-like cells of the antrum has been shown to act locally at neural GABA A receptor sites to control antral gastrin and somatostatin secretion w26,43,44x. These enteric GABAergic endocrine-like cells, are also present to the rat intestine w13,34x, and display a granule morphology characteristic of D type endocrine cells w6x. In addition to affecting secretory function, local neuroendocrine factors are known to influence transepithelial water and electrolyte movement. In a study of the involvement of GABA in sensory afferent evoked epithelial secretion, researchers in our group found that neural GABA A sites are an important link in rat intestinal mucosal transport w39x. Taken together with the findings of GABA A evoked transport, the rat small intestine by Hardcastle et al. w25x, control of human epithelium by GABA originating from nerves or epithelial cells seems likely. The neurochemical division of GABAergic interneurons of the human colon into distinct subpopulations of neuropeptide or NOS containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and their pattern of release during reflex peristalsis. A large proportion of GABAergic neurons colocalize NOS activity, similar to what has been described on the spinal cord w50x. NO is a putative NANC inhibitory motor transmitter w23,44x and inhibition of NOS w3,24x increases gut motor activity. On this basis, NO is proposed to tonically modulate the release of mediators of contraction from local neuronal or intestinal cellular sources. There is now functional and morphological evidence for the existence of separate populations of enteric GABAergic neurons. The single models of the neural circuits regulating peristalsis proposed to date likely undervalue the neurochemically heterogeneous population of interneurons by not recognizing the GABAergic component. A useful modification of the neural circuit proposed by Grider w16x for control of the descending phase of peristalsis in light of present data would show that all somatostatin-immunoreactive neurons are true interneurons since they contain GABA-T. Enkephalin occurs within at least two populations of myenteric neurons, one of which colocalize GABA-T and, as such, are interneurons. The remaining enkephalin-immunoreactive neurons represent a neurochemically distinct population of interneurons. It has
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consistently proved useful for functional analysis of the enteric nervous system to neurochemically define the subpopulations of neurons within the different nerve layers of the gut wall. We are adding to previous studies showing GABA-T colocalization with NOS but not VIP in the human colon.
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Acknowledgements This study was funded by a Grant from the Medical Research Council of Canada to A.K. and W.S. K.N. was a recipient of an MRC Graduate Studentship. We thank Kim Wong for her expert technical assistance.
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w36x A. Krantis, D.I.B. Kerr, B.J. Dennis, Autoradiographic study of the distribution of w3 Hxg-aminobutyrate-accumulating neural elements in guinea-pig intestine: Evidence for a transmitter function of g-aminobutyrate, Neuroscience 17 Ž1986. 1243–1255. w37x A. Krantis, K. Tufts, K. Nichols, G.P. Morris, w3 HxGABA uptake and GABA localization in mucosal endocrine cells of the rat stomach and colon, J. Auton. Nerv. Syst. 47 Ž1994. 225–232. w38x A. Krantis, L. Shabnavard, K. Nichols, A.L. deBlas, W. Staines, Localization of GABA A receptor immunoreactivity in NO synthase positive myenteric neurons, J. Auton. Nerv. Syst. 53 Ž1995. 157–165. w39x W.K. MacNaughton, B.C. Pineau, A. Krantis, g-Aminobutyric acid stimulates electrolyte transport in the guinea-pig ileum in vitro, Gastroenterology 110 Ž1996. 498–507. w40x K.S. Murthy, J.R. Grider, J.G. Jin, G.M. Makhlouf, Interplay of VIP and nitric oxide in the regulation of neuromuscular activity in the gut, Arch. Int. Pharmacodyn Ther. 329 Ž1. Ž1995. 27–38. w41x T. Nagai, P.L. McGeer, E.G. McGeer, Distribution of GABA-T-intensive neurons in the rat forebrain and midbrain, J. Comp. Neurol. 218 Ž1983. 220–238. w42x K. Nichols, W. Staines, A. Krantis, Neural sites of the human colon colocalize nitric oxide synthase-related NADPH diaphorase activity and neuropeptide Y, Gastroenterology 107 Ž1994. 968–975. w43x K. Nichols, W. Staines, J.-Y. Wu, A. Krantis, Immunopositive GABAergic neural sites display nitric oxide synthase-related NADPH diaphorase activity in the human colon, J. Auton. Nerv. Syst. 50 Ž1995. 253–262. w44x R.C. Spike, A.J. Todd, H.M. Johnston, Coexistence of NADPH diaphorase with GABA, glycine and acetylcholine in rat spinal cord, J. Comp. Neurol. 335 Ž1993. 320–333.
w45x K.M. Sanders, S.M. Ward, Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission, Am. J. Physiol. 262 Ž1992. G379–G392. w46x N. Saito, C. Tanaka, Immunohistochemical demonstration of GABA-containing neurons in the guinea-pig ileum using purified GABA antiserum, Brain Res. 376 Ž1986. 78–84. w47x W.A. Staines, W. Oertel, J.-Y. Wu, T. Hokfelt, Immunohistochemistry of amino acid transmitter enzymes, Acta Neurol. Scand. 69 Ž1984. 306–312. w48x C. Tanaka, g-Aminobutyric acid in peripheral tissues, Life Sci. 37 Ž1985. 2221–2235. w49x C. Tanaka, K. Taniyama, Substance P provoked gamma-aminobutyric acid release from the myenteric plexus of the guinea-pig small intestine, J. Physiol. London 362 Ž1985. 319–329. w50x L.H. Tsai, W.H. Rsai, J.-Y. Wu, Action of myenteric GABAergic neurons in guinea-pig stomach, Neurochem. Int. 23 Ž1993. 187–193. w51x S.R. Vincent, H. Kimura, E.G. McGeer, GABA-transaminase in the basal ganglia: A pharmacohistochemical study, Brain Res. 251 Ž1982. 93–104. w52x D.A. Wattchow, J.B. Furness, M. Costa, Distribution and coexistence of peptides in nerve fibres of the external muscle of the human gastrointestinal tract, Gastroenterology 95 Ž1988. 32–41. w53x J. Wiley, C. Owyang, Somatostatin inhibits cAMP-mediated cholinergic transmission in the myenteric plexus, Am. J. Physiol. ŽGastro. Liver Physiol.. 253 Ž16. Ž1987. G607–G612. w54x S. Williamson, S. Pompolo, J.B. Furness, GABA and nitric oxide synthase immunoreactivities are colocalized in a subset of inhibitory motor neurons of the guinea-pig small intestine, Cell Tissue Res. 284 Ž1996. 29–37.
Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon Anthony Krantis ) , Kim Nichols, William Staines DigestiÕe Diseases Research Group, Department of Cellular and Molecular Medicine, UniÕersity of Ottawa, 451 Smyth Road, Ottawa, Ont., Canada K1H 8M5 Received 11 April 1997; revised 15 September 1997; accepted 25 September 1997
Abstract GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide ŽNO. is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase ŽGABA-T. immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: Ži. neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, Žii. neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and Žiii. neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis. q 1998 Elsevier Science B.V. Keywords: GABAergic; Nitric oxide ŽNO.; NO synthase; Somatostatin; Enkephalin; GABA-transaminase ŽGABA-T. human colon; Immunohistochemistry; NADPH-diaphorase
1. Introduction The emerging neurochemical evidence for an extensive enteric neural and endocrine distribution of GABA, together with its well described actions in the mammalian gut, supports the proposal that the enteric GABAergic system is vast with respect to its distribution, types of communication and potential functions. GABAergic neurons and fibres have been identified in the myenteric and submucosal nerve layers of the guinea-pig and rat gut by 3 H-GABA high affinity autoradiography, as well as by ) Corresponding author. Present address: Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ont., Canada K1H 8M5. Tel.: q1 613 5625800 Ext. 8399; fax: q1 613 5625434; e-mail: [email protected].
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 7 . 0 0 1 1 3 - 6
immunohistochemical andror histochemical analysis of GABA and its synthetic enzymes w10,35,45,47,54x. Endocrine-like cells in the rat stomach and intestine have also been found to contain GABA and high affinity GABA uptake sites w10,29,31x and it is proposed that GABA in the gut may function as a neurotransmitter, hormone and paracrine agent. We have employed an immunofluorescence technique w29x used in the CNS for localizing the GABA-catabolizing enzyme 4-amino-butyrate:2-oxoglutarate transaminase ŽGABA-T., to identify GABAergic neurons w41,50x in the human colon w42x. We found GABAergic neurons in both the myenteric and submucosal ganglionated nerve networks and GABAergic nerve fibre projections within the smooth muscle layers. This distribution of GABAergic neural elements in human tissue is similar to that seen in the rodent gut w28,33,34,36,42,53x.
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GABA exerts actions on motor neurons via two distinct populations of neural receptors, the excitatory GABA A and the prejunctional inhibitory GABA receptors respectively w4,5,11,12,48,49x. GABA A receptors may also modulate acid secretion w22,27x and epithelial H 2 O and electrolyte transport w25,39x. Centrally, the range of signalling across GABAergic synapses is often enhanced by co-storage and release of neuropeptides such as somatostatin and neuropeptide Y w1,2x. Similar insight with respect to enteric neuro-humoral signalling is limited. In the rat colon a circuit consisting of GABA- somatostatin- and enkephalin-containing noncholinergic interneurons is proposed to play a key role in the regulation of descending relaxation, i.e. activation of somatostatinergic neurons by muscle stretch, leads to inhibition of enkephalin neurons and, in turn, disinhibition of GABAergic interneurons and motor neurons containing both vasoactive intestinal peptide ŽVIP. and nitric oxide ŽNO. w16,20,40x. This latter detail is unlikely to be important in the human colon since VIP and NO synthase do not colocalize with single neurons w40x. Pharmacological data suggest that GABA, somatostatin and enkephalin interneurons function within neural circuits controlling NO inhibitory motor neurons, but the anatomical correlate to these findings is limited. Supportive data reveal that in the rat colon, almost half of the myenteric nerve cell perikarya, including 35% of the NO synthesizing neurons contain GABA A receptors w38x. A variety of enteric interneurons control the circuits underlying peristalsis, consisting of an ascending phase regulating motor neurons that release acetylcholine to cause contraction w6,17x and a descending relaxation phase that involves motor neurons releasing VIP andror NO, or adenosine triphosphate w6,20x. We recently reported w42x that GABAergic neurons within the myenteric and submucosal nerve layers of the human colon contain NO synthase ŽNOS. establishing that nitrergic interneurons exist in the human colon. Enteric neurons often colocalize a number of putative transmittersrmodulators and this provides an opportunity for physiologically and morphologically distinct subpopulation of neurons to be further categorized by the neuroactive substances they contain w7x. Using a triple-labelling strategy which involved double-labelling immunohistochemistry combined with the histochemical demonstration of NADPH diaphorase-related
NOS activity in the human colon we demonstrate that GABAergic neurons comprise non-overlapping populations of somatostatin immunoreactive, enkephalin immunoreactive and NOS containing neurons.
2. Materials and methods By consent Žunder approval of the Research Ethics Board of the Children’s Hospital of Eastern Ontario, CHEO. tissue specimens Žeach approximately 2 = 1 cm in dimension. were taken from the surgically resected normoganglionic segments of sigmoid colon obtained from four infants Žages 8 to 20 months. diagnosed with Hirschsprung’s disease, undergoing surgery for bowel obstruction at CHEO. Preoperative diagnosis included clinical, radiological and anorectal manometric assessment. Close cooperation and coordination between the pediatric pathologist at the time of diagnosis ensured the correct handling of tissue. The removed tissue examined in this study included only normal, ganglionated intestine. Tissue taken from full thickness resection margins were received at the time of surgery in a freshly prepared Kreb’s bicarbonate solution. These tissues were then processed for histochemistry: tissue segments were washed in ice cold 0.1 M sodium phosphate buffered saline ŽPBS, pH 7.4., pinned out on styrofoam and fixed for 2 h at 48C in 4% paraformaldehyde and 0.4% picric acid in 0.16 M sodium phosphate buffer ŽPB, pH 7.0.. The colon segments were kept at 48C in PB ŽpH 7.2. containing 10% sucrose and 0.1% sodium azide for a minimum of 24 h prior to sectioning. Serial frozen transverse or cross sections were taken at a 6 m m thickness and mounted onto glass slides. Three triple-labeling cytochemical strategies were employed in this study. Slide mounted frozen serial sections from each patient tissue sample Ž3 slidesrtissue sample. were reacted for immunofluorescent demonstration of GABA-T and Som, or GABA-T and Enk, or Som and Enk and then treated for NADPH diaphorase histochemistry. In some cases, sections were reacted singly for GABA-T immunofluorescence followed by NADPH diaphorase histochemistry. Three serial sections Ž1, 2 and 3. were treated in the respective order by incubating in Ži. rabbit antiGABA-T Ž1:400. and monoclonal mouse anti-Som Ž1:200;
Fig. 1. Micrographs of cross sections of the myenteric nerve layer and smooth muscle layers of the human colon. Ža. Intensely GABA-T immunoreactive nerve cells are present in a ganglion of the myenteric plexus ŽMp.. Within the circular muscle Žcm. intensely labelled fibre bundles Žlarge arrowheads. are distributed throughout the muscle. Žb. Micrograph of the same section showing intensely Enk immunoreactive cells in the ganglion. Enk immunoreactive fibres in the circular muscle layer were less intensely labelled. Bar s 100 m m. Fig. 2. Micrographs of myenteric plexus sections showing a single ganglion. Intensely GABA-T immunoreactive cells are shown with arrows Ža. Enk immunoreactivity Žb. and NO synthase related NADPH-diaphorase staining Žc. are presented alongside. GABA-T cells colocalizing Enk Ž`. and GABA-T cells stained with NO synthase related NADPH-diaphorase Ž^. are shown. Bar s 100 m m. Fig. 3. Micrographs of intensely GABA-T immunoreactive myenteric ganglion cells Ža. and nerve fibres Žarrows. in the longitudinal Žlm. and circular Žcm. muscle Žb.. In Žb., the varicose nature of these GABA-T positive nerve fibres is evident. Bar s 100 m m.
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Žii. rabbit anti-Enk Ž1:400. and monoclonal mouse anti-Som Ž1:200. and Žiii. rabbit anti-GABA-T Ž1:400. and mouse anti-met-Enk Ž1:200. in PBS Žphosphate buffered saline, pH 7.2. containing 0.3% Triton X-100 for 18 h at 48C. Following a 15 min wash in PBS, each of the three serial tissue sections were incubated in fluorescein isothiocyanate ŽFITC.-conjugated donkey anti-rabbit Ž1:20. ŽAmersham, Ont., Canada. and CY3-conjugated sheep anti-mouse Ž1:200. diluted in Triton-PBS buffer for 40 min at 378C, washed, rinsed and coverslipped. Sections were then photographed using a Zeiss axioplan microscope with a 488 and a 520 nm filter to visualize FITC and Cy3, respectively, in the same section, prior to processing for histochemical demonstration of NO synthase-related NADPH diaphorase activity.
submucous ganglia in sections of human colon triple labelled for GABA-T, Som and NOS-related NADPH dependent diaphorase activity or GABA-T, Enk and NOS-related NADPH dependent diaphorase activity. 2.3. Chemicals Reduced b-nicotinamide adenine dinucleotide phosphate ŽNADPH. and nitro blue tetrazolium ŽNBT. were purchased from Sigma Chemicals ŽToronto, Ont., Canada.. All of the remaining reagents utilized in this investigation were purchased from Aldrich Chemical Co. ŽMontreal, Que., Canada..
3. Results 2.1. NO synthase histochemistry Sections treated for GABA-T, GABA-TrSom, EnkrSom and GABA-TrEnk immunofluorescence were photographed, their microscope stage coordinates were recorded, the coverslips were subsequently removed and they were reacted for histochemical identification of NO synthase-related NADPH diaphorase activity after the method of Nichols et al. w42x. Slides were placed in a dark, moist chamber for 1 h at 378C covered with the reaction medium for NO synthase activity consisting of 1 mM NADPH, 0.5 mM nitro-blue tetrazolium ŽNBT. and 0.3% Triton X-100 in 10 mM sodium phosphate buffer, pH 8.0. Tissues were given an extensive final wash with PB Ž10 mM, pH 8.0. air dried, coverslipped and analyzed by light and fluorescence microscopy. 2.2. Cell counting procedure Counts were obtained in a single-blind fashion by two independent observers. Cells were counted while viewing ganglia at 20 = objective magnification and a 10 = eyepiece magnification as previously described w42x. Data were derived from counts of 200 myenteric and 150
Neurons positive for GABA-T immunoreactivity were abundant in the ganglionated nerve networks of the myenteric plexus ŽFig. 1a, Fig. 2a, Fig. 3a, Fig. 4a. and present in the submucosa within ganglia of Henle’s plexus ŽFig. 5a, Fig. 9a, Fig. 10a. which is located subjacent to the circular muscle layer, Meissner’s plexus ŽFig. 7a. which is located subjacent to the muscularis mucosae and within the ganglia of the intermediate nerve plexus Žunique to the human. which is located midway between the former two and close to the vascular plexus ŽFig. 6a.. In addition, GABA-T immunoreactivity was noted in fibres innervating the muscularis externa ŽFig. 1a, Fig. 3a and b, Fig. 9a, Fig. 10a.. Somatostatin ŽSom.-immunoreactive neurons were in ganglionated nerve networks of the wall of the colon, but were far less numerous than GABA-T immunoreactive cells, particularly within the myenteric plexus. Enkephalin ŽEnk.-immunoreactive neurons were found only in the myenteric plexus and there were varicose Enk-immunoreactive fibres were seen within the longitudinal and circular muscle layers. NADPH diaphorase staining, indicative of NO synthase activity ŽNOS activity., was present in nerve cells within all nerve layers of the colon wall and fibres within both nerve and muscle layers ŽFig. 2c, Fig. 9c, Fig. 10b..
Fig. 4. Micrographs of sections through a myenteric ganglion which has been double stained for GABA-T and Som immunoreactivity. Intensely GABA-T immunoreactive myenteric ganglion cells Ža. are often seen to colocalize Som immunoreactivity Žb, arrow.. Bar s 100 m m. Fig. 5. Micrographs of sections of the submucosa of the human colon double stained for GABA-T and Som immunoreactivity. Examples of GABA-T immunoreactive neurons Žarrow in a. colocalizing Som immunoreactivity Žarrow in b. in the ganglia of Henle’s plexus ŽHp.. Bar s 100 m m. Fig. 6. Micrographs of sections of the submucosa of the human colon double stained for GABA-T and Som immunoreactivity. Examples of GABA-T immunoreactive neurons Žarrow in a. colocalizing Som immunoreactivity Žarrow in b. in the ganglia of intermediate plexus ŽIp.. Bar s 100 m m. Fig. 7. Micrographs of sections of the submucosa of the human colon double stained for GABA-T and Som immunoreactivity. Examples of GABA-T immunoreactive neurons Žarrows in a. colocalizing Som immunoreactivity Žarrows in b. in the ganglia of Meissner’s plexus Žmp.. Bar s 100 m m. Fig. 8. Micrographs of fixed frozen cross sections of the mucosa of the human colon double stained for GABA-T Ža. and Som Žb. immunoreactivity. An endocrine-like cell in the mucosa Žmuc. shows clear evidence of colocalizing GABA-T and Som immunoreactivity.
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It was possible to characterize cell shape and size to some extent. GABA-T immunopositive cells had elongated or round perikarya of moderate size, typical of Dogiel Type II neurons w33,46x, often with a single emergent process ŽFig. 10a.. Myenteric ganglia displayed a richly
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labelled neuropil within which single positive fibres were not discernible. Conversely, submucosal ganglia neuropil was less densely labelled and it was often possible to discern individual immunopositive fibres ŽFig. 9.. Although fibres could not be followed within the intercon-
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necting fasciculi of the nerve plexi, immunopositive fibres within nerve bundles of the muscularis could be seen ŽFig. 1a, Fig. 10a.. Some micrographs showed GABA-T positive fibres in the muscularis were varicose ŽFig. 3b..
3.1. Colocalization Combined histochemical labelling experiments revealed that the GABAergic neurons of the myenteric and submu-
A. Krantis et al.r Journal of the Autonomic NerÕous System 68 (1998) 33–42 Table 1 Average number Ž"standard deviation. of neurons per ganglion labelled for GABA-T, somatostatin ŽSom., enkephalin ŽEnk. and NO synthase ŽNOS. within the different enteric nerve plexi
Myenteric Henle’s Intermediate Meissner’s
GABA-T
Som
Enk
NOS
5"1.0 2"0.2 3"0.4 3"0.2
2"0.3 2"0.4 2"0.2 2"0.3
1"0.2 0 0 0
3"1.0 2"0.2 2"0.1 3"0.1
Data were obtained from counts of 200 myenteric and 150 submucosal ganglia in sections of human colon triple labelled for Ž1. GABA-T, Som and NOS-related NADPH diaphorase activity or Ž2. GABA-T, Enk and NOS activity.
Table 2 Percentage of GABA-T immunoreactive cells which display colocalization of for somatostatin ŽSom., enkephalin immunoreactivity ŽEnk. or NO synthase activity ŽNOS. within the myenteric plexus and submucosa
Myenteric Submucous
NOS Ž%.
Som Ž%.
Enk Ž%.
23 17
40 37
9 0
Data were obtained from counts of 200 myenteric and 150 submucosal ganglia in sections of human colon triple labelled for Ž1. GABA-T, Som and NOS-related NADPH diaphorase activity or Ž2. GABA-T, Enk and NOS activity.
cosal ganglia were composed of distinct populations of neurons that could be further separated into those that contained either Som and GABA-T immunoreactivity, Enk and GABA-T immunoreactivity, GABA-T immunoreactivity and NOS activity, Enk immunoreactivity alone, GABA-T immunoreactivity alone and NOS activity alone. Som and Enk immunoreactivity were never seen to colocalize within the same cell Žimmunohistochemical protocol Žii., data not shown.. Similarly, NOS activity was never seen in either Som or Enk immunoreactive neurons Žcf. Fig. 2b and c, Fig. 9b and c.. Cell counts were made and the frequency distribution of labelled neurons is presented in Table 1. The proportion of GABAergic neurons colocalizing neuropeptide or NOS activity is shown in Table 2. Approximately 40% of GABAergic ŽGABA-T positive. neurons were also immunopositive for Som, 9% were immunopositive for Enk and 23% colocalize NOS activity. Conversely all of the Som immunoreactive neurons also contain GABA-T immunoreactivity and 81% of Enk immunoreactive neurons displayed GABA-T immunoreactivity ŽTable 3.. Of neu-
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Table 3 Percentage of somatostatin ŽSom., enkephalin immunoreactivity ŽEnk. or NO synthase activity ŽNOS. within the myenteric plexus and submucosa cells which display colocalization of for GABA-T immunoreactivity
Myenteric Submucous
NOS Ž%.
Som Ž%.
Enk Ž%.
46 60
100 69
81 0
Data were obtained from counts of 200 myenteric and 150 submucosal ganglia in sections of human colon triple labelled for Ž1. GABA-T, Som and NOS-related NADPH diaphorase activity or Ž2. GABA-T, Enk and NOS activity.
rons with NO synthase activity, 46% colocalize GABA-T immunoreactivity. Enkephalin immunoreactive neurons were not found within the submucosa. In contrast, GABA-T immunoreactive cells were evident within all of the networks ŽHenle’s, intermediate and Meissner’s plexuses. and Som positive cells were only slightly less numerous, Table 1. Within the submucosa, 69% of Som immunoreactive ganglion cells were GABA-T immunoreactive ŽFigs. 4–7 and 9., however only 37% of GABA-T positive cells showed Som immunoreactivity. Although NOS positive cells are abundant in the submucosal ganglia ŽFig. 2c, Fig. 9c, Fig. 10b., NOS was only found in 17% of submucosal GABAergic neurons ŽFig. 9c, Fig. 10b.. Conversely, 60% of the NOS positive cells were also GABA-T positive. These results are summarized in Tables 2 and 3. Many mucosal epithelial cells with the characteristics of endocrine cells were positive for either GABA-T or Som. Cells immunoreactive for Som were more numerous, in particular at the base of the crypts, whereas GABA-T immunoreactive epithelial cells were more often located on the villus. A small minority displayed both GABA-T and Som immunoreactivity ŽFig. 8.. These cells were always elongate with a long narrow process at the luminal aspect.
4. Discussion In this study, we demonstrate neurochemically distinct, non-overlapping subpopulations of GABAergic myenteric and submucosal neurons of the human colon, and provide additional evidence for the existence of GABAergic endocrine-like cells in the human colon mucosa. Using GABA-T-immunoreactivity in combination with neuropeptide, immunohistochemistry and NOS cytochemistry, the
Fig. 9. Micrographs of a single section through a ganglion of Henle’s plexus which is triple labelled for GABA-T, Som and NADPH-diaphorase. Ža. should be compared with Žb. and Žc.. Cells and circular muscle fibres colocalizing Som immunoreactivity Ž`. or NO synthase related NADPH-diaphorase staining Ž^. are shown. In addition, a GABA-T positive cell Žarrow. that is negative for Som or diaphorase staining is evident. Bar s 100 m m. Fig. 10. Micrographs of a single section through a ganglion of Henle’s plexus ŽHp. and adjacent circular muscle Žcm. which is double labelled for GABA-T and NADPH-diaphorase. Neural elements Žcells and nerve fibers. displaying colocalization of both GABA-T immunoreactivity and NO synthase related diaphorase are labelled Ž^.. A type II neuron, that is GABA-T positiverNADPH-diaphorase negative is shown ŽII.. Bar s 100 m m.
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GABAergic myenteric interneurons in the human colon were categorized into three major subsets: Ži. neurons with somatostatin which accounted for about 40% of the GABA population, Žii. neurons with enkephalin which accounted for about 9% of the GABA neurons and Žiii. neurons with NADPH diaphorase-related NOS activity which accounted for about 23% of the GABA cells. Neither somatostatin nor enkephalin positive cells showed NOS activity. Within the submucosa and mucosa, GABAergic cells were seen to colocalize either Som or NOS but not both. The lack of enkephalin positive cells in the submucosa has been reported by others and corroborates the low levels of met-enkephalin in this layer compared to the muscularis externa. Enteric interneurons have been partially classified by their neurochemical content and projections. Whereas ascending interneurons are all cholinergic, forming chains of neurons utilizing the same neurotransmitter, descending interneurons are either cholinergic or non-cholinergic w6x. Cholinergic interneurons have been classified into three subpopulations, on the basis of colocalization with somatostatin, VIP or 5-HT. The somatostatin and serotoninimmunoreactive interneurons contact more anally situated interneurons of the same neurotransmitter type and as such they also form neurochemically homogeneous interneuronal chains. Non-cholinergic interneurons have been subcatagorized based on their being immunoreactive for VIP, NOS and gastrin-releasing peptide. In view of the identification of distinct populations of enteric GABAergic neurons, we propose that neurochemical classification of interneurons requires revision to take into account the presence of GABAergic interneurons w6,17,18x. Both GABA acting at GABA B receptors and enkephalin act prejunctionally to modulate cholinergic motor neuron output w9,31,40,45x. The myenteric GABAergic neurons colocalizing enkephalin documented in this study represent neurons providing this prejunctional input. This notion is supported by data with on the distribution of GABAergic neurons colocalizing somatostatin. As shown in the human intestine w51x, we observed that somatostatin neurons occur within the myenteric and submucous plexuses, but found no somatostatin-immunoreactive fibers in the muscularis where most if not all axon–axon interaction occurs. The fibers of somatostatin-containing neurons project caudally within the nerve plexuses w8,32x. Somatostatin has no direct effect on smooth muscle w19,21,52x, rather somatostatin causes either membrane depolarization or hyperpolarization of distinct populations of myenteric neurons w30x. On this basis, the subset of GABA-TrSom positive neurons are probably unrelated to any GABA-B modulation of motor function. It is more likely that these neurons are associated with GABA-A neural control of motor and secretomotor neurons. In the rat colon, half of all myenteric neuron perikarya have GABA-A receptors and hence are potential targets for GABAergic neurons w38x. In this study, somatostatin immunoreactive epithelial cells and GABA-T positive epithelial cells were found
throughout the colon mucosa. Double labelling experiments indicate that for the most part, these represent separate populations of cells however, a few endocrine-like cells show colocalization of GABA-T and Som. Somatostatin localization within human mucosal endocrine cells and it’s function as a gut hormonerparacrine agent have been well described w13x. GABA-T immunoreactivity and high affinity GABA uptake are also known to occur within endocrine-like cells of the mucosa throughout the rodent gastrointestinal tract w10,14,15,29,37x. In the rat stomach, GABA is colocalised in gastrin producing ŽG type. and somatostatin producing ŽD type. endocrine cells w10x. GABA synthesized by endocrine-like cells of the antrum has been shown to act locally at neural GABA A receptor sites to control antral gastrin and somatostatin secretion w26,43,44x. These enteric GABAergic endocrine-like cells, are also present to the rat intestine w13,34x, and display a granule morphology characteristic of D type endocrine cells w6x. In addition to affecting secretory function, local neuroendocrine factors are known to influence transepithelial water and electrolyte movement. In a study of the involvement of GABA in sensory afferent evoked epithelial secretion, researchers in our group found that neural GABA A sites are an important link in rat intestinal mucosal transport w39x. Taken together with the findings of GABA A evoked transport, the rat small intestine by Hardcastle et al. w25x, control of human epithelium by GABA originating from nerves or epithelial cells seems likely. The neurochemical division of GABAergic interneurons of the human colon into distinct subpopulations of neuropeptide or NOS containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and their pattern of release during reflex peristalsis. A large proportion of GABAergic neurons colocalize NOS activity, similar to what has been described on the spinal cord w50x. NO is a putative NANC inhibitory motor transmitter w23,44x and inhibition of NOS w3,24x increases gut motor activity. On this basis, NO is proposed to tonically modulate the release of mediators of contraction from local neuronal or intestinal cellular sources. There is now functional and morphological evidence for the existence of separate populations of enteric GABAergic neurons. The single models of the neural circuits regulating peristalsis proposed to date likely undervalue the neurochemically heterogeneous population of interneurons by not recognizing the GABAergic component. A useful modification of the neural circuit proposed by Grider w16x for control of the descending phase of peristalsis in light of present data would show that all somatostatin-immunoreactive neurons are true interneurons since they contain GABA-T. Enkephalin occurs within at least two populations of myenteric neurons, one of which colocalize GABA-T and, as such, are interneurons. The remaining enkephalin-immunoreactive neurons represent a neurochemically distinct population of interneurons. It has
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consistently proved useful for functional analysis of the enteric nervous system to neurochemically define the subpopulations of neurons within the different nerve layers of the gut wall. We are adding to previous studies showing GABA-T colocalization with NOS but not VIP in the human colon.
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Acknowledgements This study was funded by a Grant from the Medical Research Council of Canada to A.K. and W.S. K.N. was a recipient of an MRC Graduate Studentship. We thank Kim Wong for her expert technical assistance.
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