0306-4522/91 $3.00+ 0.00 PergamonPress plc © 1991IBRO

Neuroscience Vol. 42, No. 3, pp. 863-878, 1991

Printed in Great Britain

IDENTIFICATION A N D IMMUNOHISTOCHEMISTRY OF CHOLINERGIC A N D NON-CHOLINERGIC CIRCULAR MUSCLE MOTOR NEURONS IN THE GUINEA-PIG SMALL INTESTINE S. J. H. BROOKES,P. A. STEELEand M . COSTA Department of Physiology and Centre for Neuroscience, Flinders University of South Australia, GPO Box 2100, Adelaide 5001, Australia Abstract--Motor neurons which innervate the circular muscle layer of the guinea-pig small intestine were retrogradely labelled,/n vitro, with the carbocyanine dye, DiI, applied to the deep muscular plexus. By combining retrograde tracing and immunohistochemistry, the chemical coding of motor neurons was investigated. Five classes of neuron could be distinguished on the basis of the co-localization of immunoreactivity for the different antigens; the five classes were also characterized by different lengths and polarities of their axonal projections and by their cell body shapes. Two classes with local or orally directed axons were immunoreactive for choline acetyltransferase and substance P and are likely to be cholinergic excitatory motor neurons. Two other classes had anally directed axons; they were immunoreactive for vasoactive intestinal polypeptide and are likely to be inhibitory motor neurons. A small proportion of neurons with short projections to the circular muscle were immunoreactive for neither substance P nor for vasoactive intestinal polypeptide, but are likely to be cholinergie. The morphological and histochemicalidentification of excitatory and inhibitory motor neurons provides a neuroanatomical basis for the final motor pathways involved in the polarized reflex motor activity of the gut.

The role of the enteric nervous system in the control of gut function is now well established. Since the first studies of Bayliss and Starling7 the importance of the circular muscle layer in the propulsive activity of the intestine has been recognized. A large body of physiological and pharmacological evidence indicates that the circular muscle is supplied by both excitatory and inhibitory neurons intrinsic to the intestine)8 The reduction of circular muscle contractility by atropine during peristalsis7'54 indicates the predominance of cholinergic excitatory transmission to the circular muscle layer. 1 In the circular muscle of the guinea-pig small intestine, four types of physiological input have been identified. Firstly, cholinergic excitatory junction potentials have been recorded electrophysiologically.5,38,46 Secondly, in the presence of muscarinic antagonists, a non-cholinergic excitatory input has also been described 5'6'~4-~6 which may be largely mediated by substance P (SP) or similar tachykinins.23 The existence of intrinsic non-adrenergic, non-cholinergic enteric inhibitory neurons through-

Abbreviations: AMCA, 7-amino 4-methyl coumarin 3-acetic

acid; CHAT, choline acetyltransferase; DiI, I, l'-didodecyl-3,3,3,3"-tetramethylindocarbocyanine perchlorate; DMSO, dimethylsulphoxide; FITC, fluorescein isothiocyanate; NFP, neurofilament protein; NPY, neuropeptide Y; PB, phosphate buffer; PBS, phosphate-buffered saline; SP, substance P; VIP, vasoactive intestinal polypeptide.

out the gastrointestinal tract is now well established. 8,~s Fast non-cholinergic, non-adrenergic inhibitory junction potentials have been recorded from the circular muscle layer) '14'36'4° Fourthly, in the presence of apamin, the fast inhibitory junction potential is blocked and an additional slow inhibitory potential is revealed.46 A number of populations of enteric neurons have been characterized according to their combinations of immunohistochemical markers. 28'32 The main source of innervation of the circular muscle layer has been demonstrated ultrastructurally to be the non-ganglionated deep muscular plexus and several populations of nerve fibres, with different chemical coding, have been identified ultrastructurally.45 It has also been shown that the circular muscle motor neurons which project to the deep muscular plexus have their cell bodies exclusively in the myenteric plexus. ~ A recent study has revealed the distribution and morphological subtypes of circular muscle motor neurons retrogradely labelled from the deep muscular plexus.It The aim of the present study was to establish the chemical coding of the motor neurons to the circular muscle by combining retrograde tracing in organotypic culture, with immunohistochemistry. In addition, the neurochemically characterized classes have been related to the different types of physiological inputs, to reach a final identification of the morphology, neurochemistry and function of motor neurons to the circular muscle.

863

864

S . J . H . BROOKES et al. EXPERIMENTAL PROCEDURES

Adult male and female guinea-pigs were killed by a blow to the back of the head, followed by cutting of the carotid arteries. Methods of organotypic culture have been described previously.l~ Briefly, a short length of distal ileum was removed and placed in sterile Krebs' solution of the following composition (in mM): NaCI, 117; KC1, 5; CaC12, 2.5; MgSO4, 1.2; NaHCO3, 25; NaH2PO 4, 1.2; glucose, 10; bubbled with 95% 02, 5% CO 2, pH 7.4, containing 1/~M nicardipine. It was then cut open along the mesenteric border, pinned flat in a sylgard-lined Petri dish and the mucosa and submucosa were removed. A small crystal of the carbocyanine dye4~ 1,1'-didodecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, OR, U.S.A.) was then placed on the surface of the circular muscle layer adjacent to the submucous border where it adhered to the muscle and deep muscular plexus. The tissue was washed repeatedly in sterile Krebs' solution, pinned in a fresh sterile sylgard-lined Petri dish, covered with culture medium and incubated at 37°C with 5% CO2 for two to five days. The culture medium [Eagle's minimal essential medium (Flow Laboratories) or DME/F12 (Sigma)] contained 10% v/v fetal bovine serum, 100 IU/ml penicillin, 100 #g/ml streptomycin and 2.5/zg/ml fungizone (all from Flow Laboratories) and was changed daily. These techniques have been demonstrated to label selectively neurons which project to the deep muscular plexus, n For immunohistoehemical processing, tissue was fixed overnight in modified Zamboni's fixative [2% formaldehyde and 15% saturated pieric acid in 0.1 M phosphate buffer (laB), pH 7.0] 52 at 4°C, then cleared briefly in dimethylsulphoxide (DMSO), care being taken not to dissolve the DiI out of the tissue. For choline acetyltransferase (CHAT) immunohistochemistry, tissue was collected in PB (0.2 M sodium phosphate, pH 7.2) and fixed for 1 h at room temperature in PB with 4% paraformaldehyde and 15% saturated picric acid. It was then cleared with DMSO, washed with PB and stored in PB with 0.1% sodium azide. In order to enhance immunoreactivity for peptides in nerve cell bodies, some tissue was exposed to 250/~M colchicine for the final 24 h of the culture period. Indirect immunofluorescence techniques using primary antisera combined with fluorescent-labelled secondary antibodies or biotin-labelled secondary antibodies with streptavidin-labelled fluorophores, were used for single and double labelling of whole-mount preparations. Antisera were diluted with hypertonic phosphate-buffered saline (PBS) to reduce nonspeofic binding of immunoglobulins to tissue components. ~ The CHAT antiserum was diluted with 150 mM NaC1, 100 mM Tris (pH 7.4), containing 2% bovine serum albumin, 5% goat serum, and 0.1% sodium azide. The primary antisera used in the present study have all been histochemicaily characterized in the guinea-pig small intestine) 2 Following fixation and clearing, the circular muscle layer was removed and tissues were exposed overnight to combinations of primary antisera raised in different species for single or double labelling~°,55 in humidified chambers at room temperature, washed repeatedly in PBS and exposed to secondary antibodies for 2 h. Primary antisera used were as follows: mouse monoclonal anti-neurofilament protein triplet, NFIC8 (from M. Vitadello), 1:2000; rabbit polyclonal anti-vasoactive intestinal polypeptide (VIP), 7913 (from J. Walsh), 1:400; rat polyclonal anti-CHAT, Eck2 (from F. Eckenstein), 1:400, rat monoclonal anti-SP, NCI/34MC (Sera Lab.), 1:200; rabbit polyclonal anti-SP, RMSPI/II (from R. Murphy), 1 : 1000; rabbit polyclonal anti-caJretinin, AB-6B (from J. Rogers), 1 : 1000. Secondary antisera used were as follows. For rabbit primary antisera, fluorescein isothiocyanate (FITC)conjugated sheep anti-rabbit IgG (WeUcome Diagnostics,

Beckenham, U.K.; code 890520) was used at 1:160, or biotinylated goat anti-rabbit IgG (Vector, U.S.A.) used at 1 : 50. For rat primary antisera one of the following antisera was used: FITC-conjugated sheep anti-rat IgG (Wellcome; code 6759) at 1 : 80 or biotinylated goat anti-rat IgG (Jackson ImmunoResearch Labs Inc., PA, U.S.A.; code 10956) at 1 : 50. For mouse primary antisera, goat anti-mouse FITC, Cappel 31649 was used at I: 160, or biotinylated horse anti-mouse IgG (Vector Laboratories Inc., CA, U.S.A.) was used at 1:50. Biotinylated secondaries were followed by either streptavidin 7-amino 4-methyl coumarin 3-acetic acid (AMCA) S-875, (Molecular Probes, OR, U.S.A.) or fluorescein-streptavidin (RPN 1232; Amersham, U.K.). Specimens were given a final wash in PBS, mounted in bicarbonate-buffered glycerol, pH 8.6, and viewed on a Leitz microscope equipped with a fluorescence epi-illuminator. The red fluorescence of DiI was viewed with filter block N2, filter block L3 was used to view FITC fluorescence, and a filter block modified from an A2 filter block (with the excitation filter of an H3 filter used as a substitute emission filter together with an additional 430-nm long pass filter) was used for AMCA fluorescence. Kodak TMAX 400 film, rated at 800 ASA was used for black-and-white photography.

RESULTS

The distribution of DiI-labeiled neurons was recorded in each row of myenteric ganglia, in order to obtain distributions which were independent of the degree of stretch of the tissue at the time of fixation. Myenteric ganglia are arranged in rows which are regularly spaced along the longitudinal axis of the gut, with approximately two rows per millimetre in maximally stretched fixed tissue (in unfixed, moderately stretched tissue, 10 rows of ganglia occupy approximately 4 m m of tissue). The distributions of cells were quantified in units of rows of myenteric ganglia in order to minimize variation due to different degrees of stretch of the preparations. The distribution of labelled cell bodies in the present study was similar to that previously described ~' with about three-quarters of cell bodies being located within four rows of myenteric ganglia oral and anal to the application site, but with some nerve cell bodies located up to 18 rows anal, or up to 40 rows oral to the dye application site. Immunoreactivity of DiIlabelled neurons for CHAT, SP, VIP and neurofilament protein (NFP)-triplet will be considered in turn. The present study includes data on 2798 cells retrogradely labelled from the deep muscular plexus of 36 tissue specimens from 28 animals.

Choline acetyltransferase labelled motor neurons

immunoreactivity o f DiI-

C h A T immunoreactivity can be used to identify cholinergic neurons. 262° The distribution of C h A T in the myenteric plexus of the guinea-pig small intestine has been described 3°,5~ and was present in about half of the DiI-labelled m o t o r neurons to the circular muscle in the present study. In order to conserve limited stocks of the antiserum it was necessary to limit the size and number of preparations. It

Figs 1-4. Figs 1-4. ChAT immunohistochemistry of circular muscle motor neurons rctrogradely labelled with DiI from the deep muscular plexus. Scale bar (bottom right)= 50/~m. Fig. I. DiI-labelled neuron (arrows) located 19 rows of ganglia oral to the application site in the deep muscular plexus (A), not immunorcactive for ChAT (B). Fig. 2. DiI-labelled lamellar neuron (arrows) three rows of ganglia oral to the applicaton site (A), not immunoreactive for ChAT (B). Fig. 3. Two DiI-labeUed motor neurons (arrows) one row of ganglia anal to the DiI application site (A), both clearly immunorcactive for ChAT (B). Fig. 4. Two DiI-labeUed motor neurons (arrows) l0 rows of ganglia anal to the application site (A), both immunoreactive for ChAT (B). Scale bar = 50 #m. 865

866

S.J.H. BROOKESet al. Distribution of Dil labelled cells - 5 preparations Choline acetyltransferase (CHAT) immunohistochemistry

o~ o. oa

18-1 16,

r~l

Total No cells

II

ChAT +re ceils

14.

6

Z

12. 10. 8. 6. 4-

2. 0

-20

-15

Oral

-10

-5

0

Rows of ganglia

5

10

15

20 Anal

Application site at row 0

Fig. 5. Histogram summarizing the distribution of ChAT immunoreactivity in DiI-labelled cells in each row of myenteric ganglia oral and anal to the application site from five preparations. ChAT immunoreactivity was not detectable in motor neurons located more than one row of ganglia oral to the appplication site, but was present in nearly all neurons close to, or anal to, the application site. was readily apparent that most DiI-labelled neurons located more than one row of myenteric ganglia oral to the DiI application site, most of which had lamellar dendrites, were not immunoreactive for C h A T (Figs 1, 2). However, over half of the cells in the first row of ganglia oral to the application site were C h A T immunoreactive, as were virtually all of the cells level with (row 0) and anal to the application site (Figs 3, 4). F r o m the morphology of the cells revealed by DiI it was clear that neurons with filamentous dendrites located from one row of ganglia oral to four rows of ganglia anal to the application site were nearly all C h A T positive, as were all of the lamellar type neurons located from 5-18 rows anally. The distribution of C h A T immunoreactivity in rows of myenteric ganglia oral and anal to the DiI application site in the deep muscular plexus is summarized in the histogram in Fig. 5. Only in the first two rows of ganglia oral to the DiI application site were substantially mixed populations of ChAT-immunoreactive and non-immunoreactive m o t o r neuron cell bodies present.

S u b s t a n c e P i m m u n o r e a c t i v i t y in D i I - l a b e l l e d m o t o r neurons

SP immunoreactivity is present in many fibres in the deep muscular plexus and in the bulk of the circular muscle layer of the guinea-pig small intestine. 22'45 Immunohistochemical localization of SP was combined with retrograde labelling of circular muscle motor neurons in order to determine the projections of different types of motor neurons. In order to visualize SP immunoreactivity in the cell bodies of myenteric neurons, it was necessary to expose the tissue to 2 5 0 / t M colchicine for the final 24 h of organotypic culture. 22 Colchicine did not appear to affect the distribution of DiI-labelled cells but did noticeably distort the morphology of nerve cell bodies compared to control tissue. SP immunoreactivity in the cell bodies of neurons retrogradely labelled from the deep muscular plexus, had a similar distribution to that of CHAT. Very few DiI-labelled nerve cell bodies located more than one row of ganglia oral to the application site were immunoreactive for SP (Figs 6--8). Thus two rows

Figs 6-1 I. Triple labelling of myenteric ganglia with motor neurons retrogradely labelled with DiI (A), VIP immunoreactivity (B) and SP immunoreactivity (C); all micrographs are from the same preparation. Scale bar (bottom right)= 50/am. Rows of myenteric ganglia are on average 400-500/am apart. Fig. 6. DiI-labelled motor neuron, 28 rows of ganglia oral to the application site (A), showing immunoreactivity for VIP (B) but not SP (C). Fig. 7. DiI-labelled motor neuron (arrow), two rows of ganglia oral to the application site (A), showing immunoreactivity for VIP (B), but not for SP (C). Fig. 8. DiI-labelled motor neuron (arrow), two rows of ganglia oral to the application site (A), immunoreactive for neither VIP (B) nor SP (C). Such neurons were always found within three rows of ganglia either side of the application site. Scale bar = 50/am.

Cholinergic and non-cholinergic circular muscle motor neurons

Figs 6-8.

867

868

S.J.H. BROOKESet al.

oral to the application site approximately 12% of labelled neurons were immunoreactive for SP; one row oral, 50% of DiI-labelled neurons were SP immunoreactive whereas in the rows of ganglia level with the application site and up to four rows anally approximately 80% of cells were SP immunoreactive. However, unlike ChAT immunoreactivity, a proportion of cells with short local projections to the circular muscle (i.e. within 1 mm oral and 2 mm anal to the application site) in every preparation were not immunoreactive for SP. All of the cells located more than four rows anal to the application site had SP immunoreactivity and it was notable that the intensity of SP immunoreactivity correlated with the length of the projection to the circular muscle (neurons with long projections were usually more intensely labelled than neurons with shorter projections; Fig. 11). The distribution of SP immunoreactivity in DiI-labelled neurons in each row of ganglia relative to the DiI application site is summarized in the histogram in Fig. 12.

VIP. These observations lead to the prediction that circular muscle motor neuron cell bodies would be either immunoreactive for SP or for VIP but not for both peptides. This was directly studied in five preparations double labelled for both SP and VIP. The majority of cells did conform with these predictions; over 80% of neurons were immunoreactive for either SP or for VIP. However, in every preparation studied there were some DiI-labelled nerve cell bodies which were not immunoreactive for either SP or VIP (Fig. 8). Such cells were always located within three rows of myenteric ganglia oral or anal to the DiI application site. Very rare DiI-labelled nerve cell bodies (three out of 386 DiI-labelled neurons, n = 5) were also found which were immunoreactive for both SP and VIP. Interestingly, all three cells were found in the rows of ganglia (one to two rows oral to the application site) where there was a transition from VIP- to SP-immunoreactive nerve cell bodies (Fig. 9). Neurofilament protein triplet immunoreactivity o f

Vasoactive intestinal polypeptide immunoreactivity in DiI-labelled motor neurons

Dil-labelled motor neurons

VIP immunoreactivity is present in many nerve fibres in the circular muscle, ~9but probably in separate axons from SP. 45 VIP-immunoreactive nerve cell bodies had a very different distribution from both ChAT- and SP-immunoreactive nerve cell bodies labelled by DiI applied to the deep muscular plexus. Virtually all neurons more than two rows of ganglia oral to the application site were immunoreactive for VIP (Figs 6, 7). However, neurons level with and anal to the application site were rarely immunoreactive for VIP (Figs 10, 11). The transition from neurons with VIP immunoreactivity to neurons with ChAT or SP immunoreactivity was restricted to the first two rows of ganglia oral to the DiI application site. In fact, only in the first row of ganglia oral to the application site were substantial numbers of both VIP- and SP-immunoreactive neurons present in the same row. The distribution of VIP immunoreactivity amongst circular muscle motor neurons is summarized in Fig. 13. It has previously been reported in ultrastructural studies that 95% of varicose axons within the circular muscle layer are either immunoreactive for SP or VIP. 4s In the myenterie plexus extremely few nerve cell bodies are immunoreactive for both SP and

A proportion of both SP- and VIP-immunoreactive neurons in the myenteric plexus of the guinea-pig small intestine has been demonstrated to be immunoreactive for NFP-triplet24 and immunoreactive nerve fibres are present in the deep muscular plexus. DiI-labelled nerve cell bodies with short projections (i.e. within three rows of ganglia from the DiI application site) were rarely immunoreactive for N F P triplet (e.g. Fig. 17) whereas the majority of cells with longer projections (i.e. located more orally or anally than three rows) were consistently immunoreactive for N F P triplet (Figs 14-16, 18, 19). Within the two populations of NFP-triplet-immunoreactive motor neurons to the circular muscle (located oral and a n a l to the DiI application site) there was a consistent variation in the intensity of staining. Neurons in either population with long projections (Figs 14, 19) had more intense immunoreactivity than immunoreactive neurons with shorter projections (e.g. Fig. 16). Neurons with intermediate length projections had intermediate intensity of immunoreactivity (Figs 15, 18). The distribution of NFP-triplet-immunoreactive motor neurons relative to the DiI applications site in the deep muscular plexus is summarized in Fig. 20.

Fig. 9. Two DiI-labeUed motor neurons (closed arrow and open arrow), one row of ganglia oral to the application site (A), one cell is immunorcactive for VIP only (B; closed arrow) the other is immunoreactive for both VIP and SP (B, C; open arrow). Only three such double-labelled neurons were detected in the present study. Fig. 10. DiI-labelled motor neuron (arrow), in the row of ganglia level with the application site (0 rows) (A), showing immunoreactivity for SP (C) but not VIP (B). Fig. 11. DiI-labelled motor neuron (arrow), 11 rows of ganglia anal to the application site (A), intensely immunoreactive for SP (C) but not for VIP (B). Scale bar = 50 #m.

Cholinergic and non-cholinergic circular muscle motor neurons

Figs 9-11. NSC 42[~--I

869

870

S . J . H . BROOKESet al. Distribution of Dil labelled cells - 6 preparations Substance P (SP) immunohistochemistry

Total No cells SP +ve cells

181 16, 8

d

z

14 12, lO. 8, 6. 4,

24 o~ -35

-30

-25

-20

-15

Oral

-10

-5

0

5

10

15

Rows of ganglia ~

20 Anal

site at row0

Fig. 12. Summary histogram of the distribution of SP immunoreactivity in neurons retrogradely labelled from the deep muscular plexus. SP immunoreactivity was absent from neurons more than one row of ganglia oral to the application site, but was present in the majority of cells with short local projections and was invariably present in DiI-labelled nerve cell bodies located more than four rows of ganglia anal to the application site. Calretinin immunoreactivity o f DiI-labelled motor neurons Calretinin, a novel calcium-binding protein f o u n d in some n e u r o n s in the C N S 47 can be detected with i m m u n o h i s t o c h e m i c a l m e t h o d s in two p o p u l a t i o n s o f myenteric neurons. 12 Calretinin i m m u n o r e a c t i v i t y is f o u n d in a substantial p o p u l a t i o n of n e u r o n s with

processes in the tertiary plexus, which are likely to be m o t o r n e u r o n s to the longitudinal muscle. A second p o p u l a t i o n o f calretinin-immunoreactive neurons with cell bodies in the myenteric plexus supplies varicose fibres to o t h e r ganglia which pass t h r o u g h the internodal strands. However, there are very few calretinin-immunoreactive nerve fibres in the circular muscle layer. Thus it would be predicted from the

Distribution of Dil labelled cells - 8 p r e p a r a t i o n s 24

VIP immunohistochemistry

22 20 18 O.

~a

r-I

Total No. cells

BB

V I P + v e cells

16 14

d z ¢1

12" 108" 6 4 2"

-35 Or~

-30

-25

-20

-15

-10

-5

0

Rowsof ganglia

5

10

15

20 An~

Application site at row 0

Fig. 13. Summary histogram of VIP immunoreactivity in circular muscle motor neurons. Virtually all neurons oral to the application site were immunoreactive for VIP whereas none of the cells located anal to the application site were immunoreactive for VIP.

Chotinergic and non-cholinergic circular muscle mot or neurons distribution of calretinin-immunoreactive nerve fibres that very few neurons labelled with DiI applied to the deep muscular plexus would be immunoreactive for calretinin. Only 2% of labelled cells were immunoreactive for calretinin (eight out of 396 cells, n = 5). The small proportion of neurons labelled from the deep muscular plexus with calretinin immunoreactivity suggests a high degree of specificity of DiI labelling for circular muscle motor neurons, as reported previously. 11 DISCUSSION

On the basis of morphology, polarity and chemical coding, the present study has provided evidence that the circular muscle layer is innervated by five classes of motor neuron. These are discussed in turn, starting with the cells which project anally from myenteric ganglia to the circular muscle. The first class of DiI-labelled cells was located from five to 40 rows of ganglia oral to the DiI application site in the deep muscular plexus and were immunoreactive for VIP and NFP-triplet, but not for ChAT or SP. These cells typically had large somata with lamellar dendrites and a single axon that could be traced for long distances running anally in internodal strands in the myenteric plexus. Previous studies have shown that this class of neuron is also immunoreactive for gastrin-releasing peptide and dynorphin. 24 The second class of cell was located from one to 10 rows of ganglia oral to the DiI application site and also had lamellar dendrites and a single axon. Such cells were immunoreactive for VIP, but NFPtriplet immunoreactivity was either very faint or absent. Cells of this type were not immunoreactive for SP or CHAT. Previous studies have demonstrated that this class of cell is also immunoreactive for neuropeptide Y (NPY), enkephalin and dynorphin.29, 32 Most DiI-iabelled cell bodies located from one row of myenteric ganglia oral to the application site, to five rows anally, had small oval or round nerve cell bodies with short filamentous dendrites. Such cells were consistently immunoreactive for CHAT, and for SP. However, this class of cell was not immunoreactive for either VIP or for NFP-triplet. Preliminary experiments indicate that a proportion of these cells is also immunoreactive for enkephalin. A small proportion of cells located within three rows of the DiI application site, with either lamellar or oval cell bodies, was not immunoreactive for either SP or VIP. In an ultrastructural study, LlewellynSmith et al. 45 demonstrated that approximately 95% of varicose fibres in the circular muscle layer was immunoreactive for either SP or VIP. In the present study, up to 20% of nerve cell bodies retrogradely labelled from circular muscle lacked immunoreactivity for both SP and VIP; however, from comparison of Figs 5 and 13 it is likely that these cells are immunoreactive for CHAT. The ChAT-immuno-

871

reactive motor neurons with short projections to the circular muscle that lack SP immunoreactivity appear to fall into two groups. Some of these cells had lamellar dendrites and short anally directed axons and are similar to the class of neuron that is immunoreactive for GABA. 33 However, a proportion of cholinergic neurons located level with, or anal to, the application site, also lacked both SP and VIP immunoreactivity and hence is unlikely to be immunoreactive for GABA. 33 This class of cell is likely to comprise only a relatively small proportion of the total number of circular muscle motor neurons. The last neurocbemical class of motor neurons was located from four to 18 rows of ganglia anal to the DiI application site. Cells of this class showed some variability but usually had relatively short lamellar dendrites. Such cells were intensely immunoreactive for SP, and were immunoreactive for both NFPtriplet and CHAT. None of these cells was immunoreactive for VIP. Previous studies have shown that cells with this chemical coding are immunoreactive for enkephalin and often for dynorphin. 24 Correlation o f neurochemical and functional classes o f motor neuron Excitatory motor neurons. The three classes of motor neuron that are immunoreactive for ChAT are likely to mediate excitatory transmission to the circular muscle layer. The antibody used to localize ChAT in the present study identifies known or suspected populations of cholinergic neurons in the CNS 26 and in the gut. 3° Physiological studies have demonstrated the major role of cholinergic transmission in excitation of the circular muscle layer during peristalsis7'44'54 and pharmacological studies have confirmed that acetylcholine and muscarinic agonists cause contraction of circular muscle in the guinea-pig small intestine) TM SP immunoreactivity is co-localized in two of the three classes of cholinergic motor neuron: i.e. in neurons located well anal to the application site (co-localized with CHAT, enkephalin, dynorphin, NFP-triplet) and in short, local motor neurons. SP evokes powerful contractions of the circular muscle layer and it, or a similar tachykinin, is likely to be the major non-cholinergic excitatory transmitter in the guinea-pig small intestine circular muscle layer. 23 An atropine-resistant component of peristalsis has been reported, 53 which may, in part, be mediated by SP. 3 Thus the presence of SP immunoreactivity in two of the classes of ChAT-immunoreactive DiI-labelled cells supports the proposal that they function as excitatory motor neurons. This study provides the first direct anatomical evidence that the noncholinergic excitatory input to the circular muscle (mediated by SP) is likely to arise from a subset of the cholinergic neurons. Coexistence of SP and ChAT in enteric neurons has previously been demonstrated in a proportion of submucous neurons. 3~ As in the longitudinal muscle layer2 it has been reported that different stimulation parameters preferentially evoke

872

S.J.H. BROOKESet al.

cholinergic or non-cholinergic inputs to the circular muscle.23 From the present study, SP is only present in a subset of cholinergic motor neurons, thus it seems probable that this is caused by different release mechanisms from the same neurons, rather than by stimulation of separate populations of neurons. The third class of ChAT-immunoreactive neurons which lacks immunoreactivity for SP is also likely to have a predominantly excitatory input to the circular muscle and may represent a population of purely cholinergic neurons to the circular muscle layer. The identification of three classes of excitatory motor neurons to the circular muscle layer, located either local to, or anal to, the point of innervation, is compatible with a study using physiological techniques to trace neuronal pathways in which Smith et aL, 5° demonstrated that non-cholinergic excitatory junction potentials are mediated by neurons with either short local projections, or with orally directed axons up to 10mm in length. The projections of SP-immunoreactive neurons in the present study are entirely compatible with such a pattern of projection. More recently, it has been reported that cholinergic transmission to the circular muscle is dominated by neurons with local projections ( < 2 mm) to the circular muscle layer. ~7

are likely to be the inhibitory motor neurons in the guinea-pig small intestine circular muscle.28 The present study supports this speculation; two classes of VIP-immunoreactive neuron were characterized: neurons with long anally directed projections and neurons with short anally directed projections. The projections of neurons mediating the fast inhibitory junction potential to the circular muscle layer have been traced using physiological techniques;9 two classes of inhibitory motor neurons were distinguished. One class had short anally directed projections, the other had long anally directed projections up to 30 mm in length. It is likely that the two classes of VIP-immunoreactive neuron described in the present study underlie fast inhibitory transmission to the circular muscle, as well as the slow inhibition which may be mediated by VIP. The absence of ChAT immunoreactivity in inhibitory motor neurons could be anticipated as electrophysiological studies have shown that excitatory and inhibitory inputs to the circular muscle of the guinea-pig small intestine can be separately activated by physiological stimuli.48,49 The possibility that two transmitters are released from the same inhibitory motor neurons is comparable to the release of both acetylcholine and SP from most excitatory motor neurons.

Inhibitory motor neurons

Physiological significance o f motor neuron projections

The two classes of neurons located oral to the DiI application site, both of which are immunoreactive for VIP, are likely to be inhibitory motor neurons to the circular muscle. Two types of inhibitory transmission to the circular muscle layer of the guinea-pig small intestine have been characterized physiologically. The identity of the transmitter of the fast inhibitory junction potential remains uncertain; however, a small, slow inhibitory junction potential has been demonstrated in the presence of apamin and a SP antagonist.~ An apamin-resistant relaxation in guinea-pig circular muscle is mimicked by exogenous VIP. 2~ From their projections it has been suggested that a proportion of VIP-immunoreactive neurons

DiI applied to any point in the circular muscle layer reliably labels five classes of neuron with characteristic chemical coding, morphology and polarity (summarized diagrammatically in Fig. 21). This confirms previous immunohistochemical studies which showed that nerve fibres with a variety of chemical coding could be identified at every point in the circular muscle layer. 45 The different types of input are likely to converge onto any single region of circular muscle from inhibitory and excitatory final motor neurons with cell bodies located in different rows of myenteric ganglia orally and anally. Thus it is likely that these excitatory and inhibitory motor neurons belong to separate motor pathways and hence are capable of

Figs 14-19. DiI-labelled motor neurons (A) in myenteric ganglia stained for NFP-triplet (B) and VIP immunoreactivity (C). All micrographs were from the same preparation. Similar exposure times were used in all micrographs of NFP-triplet immunoreactivity to demonstrate the correlation of the intensity of NFP-triplet immunoreactivity with the length of the axonal projections. Scale bar (bottom fight) = 50 # m. Fig. 14. DiI-labeUedneuron (arrow), 17 rows of myenteric ganglia oral to the application site (A), showing immunoreactivity for VIP (C) and intense immunoreactivity for NFP-triplet (B). Fig. 15. DiI-labelled neuron (arrow), 10 rows of ganglia oral to the DiI application site (A), showing immunoreactivity for VIP (C) and moderately intense NFP-triplet immunoreactivity (B). Fig. 16. DiI-labelled neuron (arrow), five rows of ganglia oral to the application site (A), showing immunoreactivity for VIP (C), and very faint immunoreactivity for NFP-triplet (B). Scale bar = 50 #m. Figs 17. Two DiI-labeUed neurons (closed arrow and open arrow), one row of ganglia oral to the application site (A). One cell (open arrow) is immunoreactive for VIP (C), the other lacks VIP immunoreactivity (closed arrow). Both cells had no detectable NFP-triplet immunoreactivity (B). Fig. 18. DiI-labelled motor neuron (arrow), six rows of ganglia anal to the application site, lacking VIP immunoreactivity (C) but with moderately intense NFP-triplet immunoreactivity (B). Fig. 19. DiI-labelled motor neuron (arrow), 14 rows of ganglia anal to the application site (A), lacking VIP immunoreactivity (C) but with intense NFP-triplet immunoreactivity (B). Scale bar = 50 #m.

Cholinergic and non-cholinergic circular muscle motor neurons

Figs 14-16.

873

875

Cholinergic and non-cholinergic circular muscle motor neurons Distribution of Dil labelled cells - 14 p r e p a r a t i o n s N e u r o f i l a m e n t (NF) i m m u n o h i s t o c h e m i s t r y

24 22

I--'] Total No cells

2o

P=

NF +ve cells

I

16

w

14 12, 10,

< 6, 4, 2 o

-35

-30

-25

-20

Oral

-15

-10

-5

0

5

10

15

20

Rows of ganglia

Anal

Applicationsite at row 0

Fig. 20. Histogram summarizing the distribution of NFP-triplet immunoreactivity in circular muscle motor neurons retrogradely labelled from the deep muscular plexus. No attempt was made to quantify the intensity of NFP-triplet immunoreactivity: cells were either scored as positive or negative. Note that cells with short projections to the circular muscle were not immunorcactive whereas cells with longer projections (>five rows of ganglia either orally or anally) were consistently immunorcactive for the NFP-triplet. being activated independently by physiological stimuli. 38,48,49It is apparent that the contractile activity of any area of circular muscle at a particular m o m e n t will reflect the temporal summation of the different types of physiological input. Neurons with the coding of the five classes of circular muscle motor neurons can be identified immunohistochemically in every myenteric ganglion, suggesting that there is no functional specialization of ganglia, at least with regard to their innervation of the circular muscle layer. Since every point in the deep muscular plexus receives similar inputs, the arrangement of circular muscle motor neurons (Fig. 21) can be redrawn to show the projections

of the five classes from any single row of ganglia (Fig. 22). The collection of inhibitory and excitatory motor neurons in a single row of ganglia could be considered as a functional module, which comprises the repeating neuronal units that control intestinal motility. Simultaneous activation of inhibitory and excitatory motor neurons in such a module would lead to relaxation of the muscle anal to the site of stimulation, and contraction orally. Thus the polarized nature of enteric reflexes reported by Bayliss and Starling 7 is reflected in the projections of the classes of circular muscle motor neuron. The co-ordination between these modules, presumably through ascending and descending interneuron

Oral

Anal

VlP/NF/GRP/DYN ~-

~

-

-

_ |

VIP/ENK /NPY/DY1M -1-

ChAT C h' A T t K /SP/+ENK

ChAT/SP/NF /ENK/+DYN ~

-

~

(HI appUcation site

Fig. 21. Summary diagram showing projections of five classes of circular muscle motor neuron in the myenteric ganglia to the DiI application site in the deep muscular plexus. The two classes of proposed inhibitory motor neurons are immunorcactive for VIP and arc oral to the application site. Three classes of proposed excitatory motor neuron are all immunorcactive for ChAT and are located either local, or anal, to the application site. NF, neurofilament protein triplet; GRP, gastrin-releasing peptide; DYN, dynorphin; ENK, enkephalin.

876

S.J.H. BROOKESet al. Oral

Myenterlc plexus J

Anal

~ .......

",,

:

J

:

/

.-- "-~

"

CKI~T SP NF ENK :k DYN +

VIP ENK NPY DYN

T CHATS1:1: ENK

VlP NF GRP DYN

++

Fig. 22. Diagram of projections of the five classes of circular muscle motor neuron from a single ganglion to the circular muscle. Synchronous excitation of the five types of neuron would lead to excitation (+) oral to the stimulus, and inhibition ( - ) anal to the stimulus, thus providing a basis for polarized motor reflex activity in the gut.

pathways, and perhaps through feedback through sensory elements, is likely to determine the macroscopic behaviour of longer segments of small intestine during complex behaviours such as peristalsis and migrating m o t o r complexes. It is likely that all of the classes of circular muscle m o t o r neurons do receive predominantly excitatory synaptic inputs since it has been shown 11 that they all have Dogiel type I soma morphology, 25'29 which correlates with S-cell electrophysiological activity. 1°,27,42,43 Thus all of the classes of circular muscle m o t o r neurons are likely to receive prominent nicotinic excitatory inputs. 37'39 Such nicotinic inputs to circular muscle m o t o r neurons are likely to be physiologically important since it has been demonstrated that nicotinic antagonists strongly inhibit co-ordinated m o t o r activity during peristalsis. 4'44

CONCLUSION The present study provides for the first time a full account of the morphology, neurochemistry and functions of the classes of motor neurons that innervate the circular muscle of the guinea-pig ileum. The results can account for, and are compatible with, a wide range of pharmacological, physiological and immunohistochemical observations. The ability to distinguish excitatory and inhibitory m o t o r neurons in the myenteric plexus will permit more detailed and directed studies of the m o t o r control of the gastrointestinal tract, than have hitherto been possible. Acknowledgements--We would like to thank Tania Neville, Rachel Short, Zan Min Song and Leisel Wion for valuable technical assistance throughout this project. Work was supported by a grant from the NH and MRC (Australia).

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