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Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia
Journal of the
Autonomic Nervous System
ELSEVIER
Journal of the Autonomic Nervous System 52 (1995) 107-116
Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia C . R . A n d e r s o n a,, J.B. F u r n e s s
a
H . L . W o o d m a n a S.L. E d w a r d s a p . j . C r a c k b A.I. Smith b
a Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3052, Australia b Baker Medical Research Institute, Commercial Road, Prahran, Victoria 3181, Australia
Received 6 July 1994; revision received 17 August 1994; accepted 30 August 1994
Abstract Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatinimmunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons. Keywords: Inferior mesenteric ganglion; Coeliac ganglion; Preganglionic neuron; Enteric neuron; Fast Blue; (Guinea
pig)
I. Introduction W i t h i n reflex p a t h w a y s t h a t c o n n e c t gastroint e s t i n a l r e g i o n s via p r e v e r t e b r a l g a n g l i a a r e n e u -
* Corresponding author. Tel.: (61-3) 344-5807; Fax: (61-3) 347-5219.
rons with cell b o d i e s in the gut wall a n d t e r m i n a l s in t h e coeliac, s u p e r i o r a n d inferior m e s e n t e r i c ganglia; t h e s e a r e k n o w n as i n t e s t i n o f u g a l neurons a n d a r e p a r t s o f t h e a f f e r e n t limbs of e n t e r o - e n t e r i c reflexes [16,26]. T r a n s m i s s i o n to t h e n e r v e cells of p r e v e r t e b r a l g a n g l i a f r o m intestinofugal n e u r o n s has b o t h c h o l i n e r g i c a n d nonc h o l i n e r g i c c o m p o n e n t s [9,15]. A m o n g s t t h e possible n o n - c h o l i n e r g i c t r a n s m i t t e r s a r e a n u m b e r
0165-1838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00150-2
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of neuropeptides, immunoreactivity for which has been found in intestinofugal neurons; these peptides include bombesin (gastrin-releasing peptide), cholecystokinin, dynorphin, enkephalin and vasoactive intestinal peptide [11,18,24]. Another possible participant in the transmission process from intestinofugal neurons has now been discovered, nitric oxide (NO). Immunoreactivity for the synthesising enzyme for NO, nitric oxide synthase (NOS) is found in nerve terminals forming dense baskets around nerve cells of the coeliac ganglion [13]. These immunoreactive terminals are lost if nerve trunks connecting the intestines with the coeliac ganglion are cut. However, the distribution of nerve cell bodies giving rise to these terminals has not been determined because sufficient quantities of antisera were not available at the time of the study to make surveys in whole mounts of intestine, which is necessary to determine this distribution [22]. For the same reason, the possible existence of NOS immunoreactivity in intestinofugal neurons projecting to the superior and inferior mesenteric ganglia was not studied. An important reason to examine the distribution of NOS immunoreactive neurons is the observation that the chemistries of neurons projecting to the coeliac ganglion differ depending on their origin [22] and NOS may mark a specific subgroup of such inputs. Sympathetic preganglionic neurons are a second source of nerve terminals containing NOS in the coeliac ganglion of the guinea pig [13], but it is not clear whether NOS-containing preganglionic neurons also project to the inferior ruesenteric ganglion. In order to determine the distribution of intestinofugal neurons with NOS immunoreactivity, the distributions of NOS-immunoreactive terminals in each of the prevertebral ganglia in the guinea pig and the possible contributions of NOS-immunoreactive preganglionic neurons to the innervation of these ganglia, we have raised and characterised a polyclonal antiserum specific to the neuronal form of NOS. We have labelled the cell bodies of both intestinofugal and preganglionic neurons by retrograde transport of Fast Blue from prevertebral ganglia and subsequently localised NOS immunoreactivity in the tissue.
2. Materials and methods
2.1. Production of antiserum The immunogen for antibody production was a polylysine tree to which was attached, through the N-terminal, multiple copies of a 21-amino-acid sequence from the neuronal form of NOS to create a large multivalent molecule for immunisation. The sequence chosen has previously been shown to generate antibodies to neuronal NOS [25] and was from amino acids 1409-1429 (R-SE-S-I-A-F-I-E-E-S-K-K-D-A-D-E-V-F-S-S) of the neuronal form of NOS isolated from the rat cerebellum [4]. This sequence is unique to the neuronal form of NOS, and does not occur in the endothelial or macrophage form [17,29]. The immunogen (20 /xg), dissolved in phosphate-buffered saline (PBS), was mixed with Freund's complete adjuvant (1:1) without being linked to a carrier molecule and injected subcutaneously into two rabbits. Booster injections, consisting of 20 ~g antigen in incomplete Freund's adjuvant were given six times at monthly intervals. Bleeds were taken 10 days after each boost. Both rabbits produced antisera, coded N73 and N74, with similar properties. Antiserum N74 from the fourth or fifth bleed has been used in the present study.
2.2. Western blotting Fresh rat brains were homogenised in 50 mM Tris-HC1 buffer (pH 7.4), containing 1 mM EDTA, 10 mg/1 leupeptin, 10 m g/ l pepstatin A and 100 m g / l phenylmethylsulphonyl fluoride. Following centrifugation at 20 000 x g for 30 min at 4°C, the supernatant was subjected to SDSPAGE and the samples then blotted onto polyvinylidene membranes. The membrane was exposed to antiserum followed by a peroxidase-conjugated anti-rabbit IgG (Silenus) and 4-chloronapthol as the chromogen.
2.3. Injection of Fast Blue Guinea pigs (250-350 g, of either sex) were anaesthetised by the subcutaneous injection of
C.R. Anderson et aL /Journal of the Autonomic Nervous System 52 (1995) 107-116
sodium pentobarbitone (15 mg/kg), followed by a mixture of fentanyl (0.6 m g / k g ) and fluanisone (5 mg/kg), injected intramuscularly. Following laparotomy, the coeliac or inferior mesenteric ganglion was exposed. Fast Blue (2% in 10% dimethylsulfoxide) was injected into one or other ganglion via a glass microelectrode that was bevelled to a tip diameter of 70-100 /zm. The left lobe of the coeliac ganglion was injected with 2-3 /xl of Fast Blue and the two lobes of the inferior mesenteric ganglion were injected with a total of 1-2 /xl. Following the injection of ganglia, the abdominal wall was closed and sutured and the animal allowed to recover from anaesthesia. Survival times were 7-10 days, before guinea pigs were killed and tissue taken. 2.4. Removal and processing o f tissue
Guinea pfgs were killed either by concussion and bleeding from the carotid arteries, or by perfusion under full anaesthesia, using the anaesthetic protocol described above. Perfusion was transcardiac with formaldehyde/picric acid (2% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer, p H 7.0). Segments of gastrointestinal tract that were taken from nonperfused animals were placed in PBS (0.9% NaC1 in 0.01 M sodium phosphate buffer, p H 7.0) containing 10 -5 M nicardipine, the contents were washed out and the segments were cut open along their anti-mesenteric borders and pinned tautly mucosa down on balsa board. The balsa was floated, tissue side down, in the formaldehyde/picric acid fixative for 24 h at 4°C. The regions of the gut taken for examination were the ileum, caecum, proximal and distal colon and the rectum. The gut distal from the junction with the caecum through to the anus was dissected out and the region between the pelvic brim and the anus removed as the rectum. The proximal and distal colon were separated at the colonic flexure. Coeliac, superior mesenteric and inferior mesenteric ganglia from non-perfused or perfused animals were immersion fixed in the same mixture. Spinal cords were dissected out only from perfused animals and were post-fixed overnight in the same fixative.
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Fixative was removed by 3 washes in dimethylsulfoxide, followed by 3 changes of PBS. Tissue which was to be examined in whole mount was stored in PBS plus 0.1% sodium azide until it was dissected. Tissue to be examined in section was kept in the same solution to which 30% sucrose was added as a cryoprotectant. Whole mounts of the myenteric plexus plus longitudinal muscle were dissected from the gut wall. Ganglia and spinal cord were cut at 12-/xm thickness on a cryostat. The spinal cord was sectioned at 50/zm on a vibratome. 2.5. Immunohistochemistry
For immunohistochemistry of NOS in ganglia and gut, sections or whole mounts, respectively, were incubated in rabbit anti-NOS antiserum N74-5, diluted 1: 1000, at room temperature for 18-24 h. Dilution was with hypertonic PBS (NaCI concentration, 1.5%). The primary antiserum was washed from the tissue with PBS and replaced with fluorescein isothiocyanate (FITC) labelled sheep anti-rabbit antiserum, 1:160, (Wellcome Diagnostics), for 1 h at room temperature. Tissue was again washed in PBS and the sections or whole mounts coverslipped with phosphatebuffered glycerol (pH 8.6). For simultaneous localisation of NOS and somatostatin immunoreactivity, sections were incubated with a mixture of the anti-NOS antiserum with the monoclonal anti-somatostatin antibody $8 (1 : 20) [7]. This was followed by incubation in biotinylated horse antimouse IgG (Vecta Laboratories) for 2 h. After washing in PBS, the tissue was exposed to a mixture of FITC-labelled sheep anti-rabbit IgG (as above) and streptavidin-Texas Red complex, 1:50 (Amersham) for 90 min. Spinal cord sections were stained free floating in anti-NOS (1: 1000) containing 0.3% Triton X-100, then in Texas Red-labelled donkey anti-rabbit IgG (Jackson) for 1 h and then mounted in serial order. The immunofluorescence was examined in a Zeiss microscope fitted with filters to discriminate between Fast Blue, FITC and Texas Red fluorescence. For preabsorption studies of the antiserum against NOS, 500 /xl of the antiserum diluted
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1 : 1000 was incubated with 20/zg of the antigen or with 20 /.~g of a different but identically synthesised peptide of similar length whose sequence represented amino acids 251-270 of rat neuronal NOS [41•
3. Results
Serum N74 recognised a single band at an apparent molecular mass of 150 kDa on Western blots prepared by separating proteins in extracts from rat brain by SDS-PAGE (Fig. 1). Staining with antiserum N74 was restricted to neurons in the tissue examined. In the prevertebral ganglia and within the spinal cord both nerve terminals and nerve cells were stained. The staining was absent in tissue stained with antiserum preabsorbed with the immunogen but was not affected by preincubation with a similarly synthesised and sized peptide of different sequence. The antiserum has also been found to stain nerve cell bodies and processes in the central and peripheral nervous system of rat, cat, dog and sheep in a pattern consistent with it staining NOS-containing neurons (C.R. Anderson and J.B. Furness, unpublished observations).
A
B
C
205K---~ 116K----~ 80K--~ 50K---~
Fig. 1. Western blot of rat brain extract stained with NOS antiserum N74-5. A, Molecular mass markers; B, rat brain extract probed with NOS antiserum; C, rat brain extract probed with NOS antiserum preabsorbed with the immunogen. Note the single band around 150 kDa in B that is absent in C after preabsorbtion.
3.1• Distribution of immunoreactivity in prevertebral ganglia The distribution of NOS immunoreactivity (NOS-IR) in the superior and inferior mesenteric ganglia closely resembled that previously described for the coeliac ganglion [13]. Clumps of nerve cell bodies were surrounded by networks of strongly immunoreactive nerve fibres (Fig. 2).
Fig. 2. NOS immunoreactivity in cryostat sections of the inferior mesenteric ganglion (A and C) and in a wholemount preparation of a small ganglion of the hypogastric nerve (B), close to the inferior mesenteric ganglion. In the inferior mesenteric ganglion (A), strongly immunoreactive fibres surround groups of nerve cells (arrowheads), and in other regions less strongly reactive fibres are found amongst the nerve cells (arrows). Rare immunoreactive nerve cells are also found in these ganglia (C). A small proportion of nerve cells in hypogastric nerve ganglia close to the inferior mesenteric ganglion is reactive (B). Calibration, 50/zm.
C.R. Anderson et al. /Journal of the Autonomic Nervous System 52 (1995) 107-116
Faintly immunoreactive fibres were around most of the remaining nerve cells, as previously illustrated for the coeliac ganglion [13]. In both ganglia there were only very small numbers of strongly immunoreaetive nerve cell bodies (Fig. 2). NOSIR nerve cell bodies were more common in the small ganglia situated on the hypogastric nerve close to the IMG (Fig. 2). The intermesenteric and colonic nerves contained numerous NOS-immunoreactive, non-varicose nerve fibres. Chromaffin cells, which are prominent in the inferior mesenteric ganglia in particular, were not immunoreactive for NOS. The majority of somatostatin-IR nerve cells in the prevertebral ganglia were surrounded by fibres with strong immunoreactivity for NOS (Fig. 3); conversely, areas of ganglia without somatostatin-IR nerve cells lacked these strongly NOSimmunoreactive terminals. In samples of 100 successive somatostatin-IR nerve cells counted in ganglia from 3 animals, most somatostatin-IR cells were surrounded by baskets of nerve fibres with strong immunoreactivity for NOS (86.1 + 1.4 in the coeliac ganglion, 82.8 +_ 1.5 in the superior mesenteric ganglion and 83.2 + 2.1 in the inferior mesenteric ganglion. All values are means + SEM). Similarly, NOS baskets showing strong immunoreactivity were almost all around somatostatin-IR nerve cells (88.6 _+ 1.1 in the coeliac ganglion, 78.2 +_ 2.4 in the superior mesenteric ganglion and 85.8 +_ 1.7 in the inferior mesenteric ganglion. All values are means + SEM).
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3.2. N O S immunoreactivity in intestinofugal neurons projecting to the coeliac ganglion
NOS-IR was examined in the myenteric ganglia of preparations of small intestine, caecum, proximal colon, distal colon and rectum that contained nerve cells that were retrogradely labelled following injection of the dye into the coeliac ganglion. The distributions and morphologies of NOS-IR nerve cell bodies and fibres were indistinguishable from those previously described in the guinea pig small and large intestine [8,19,20]. No retrogradely labelled nerve cells in the small intestine had NOS-IR, whereas 40-70% of retrogradely labelled nerve cells in the regions of large intestine that were examined had NOS-IR (Table 1; Fig. 4). The NOS-IR intestinofugal neurons were typically neither the smallest nor the largest neurons in the ganglia. The processes of the cells were not always distinct, but where these were apparent 3 - 8 tapering processes were observed (Fig. 4). 3.3. N O S immunoreactivity in intestinofugal neurons projecting to the inferior mesenteric ganglia
Two sample regions of distal colon, the middle (mid distal colon) and distal fifth (terminal distal colon) of the organ, and the rectum were examined for the presence of NOS-IR in retrogradely labelled nerve cells after injection of Fast Blue into the IMG. Neurons projecting to the inferior
Fig. 3. Simultaneous localisation of NOS and somatostatin-immunoreactivityin a thin (8 izm) section from the inferior mesenteric ganglion. NOS terminals (A) surround the somatostatin nerve cell bodies (A, arrows). Calibration, 50 izm.
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Table 1 Percentage of labelled enteric neurons showing NOS immunoreactivity after Fast Blue injection into the coeliac ganglion
Table 2 Percentage of labelled enteric neurons showing NOS immunoreactivity after Fast Blue injection into the inferior mesenteric ganglion
Ileum
Caecum
Proc. colon
Rectum
Mid distal colon
Terminal distal colon
Rectum
0 (n = 83)
70.2+7.8 (n = 32)
40.3_+11.6 61.7+13.3 (n = 209) (n = 38)
44.6+18.7 (n = 31)
60.9_+3.8 (n = 60)
68.3_+4.8 (n = 312)
66.2+_7.4 (n = 257)
Dist. colon
(Tissue from four animals, except for Ileum where eight animals were used). All values are means+SEM. Total cells counted are also indicated.
(Tissue from four animals). All values are means+SEM. Total cells counted are also indicated.
Fig. 4. Relation between retrograde labelling from prevertebral ganglia (A, B, C, D) and NOS immunoreactivity (/~, B', C', D') in the gut wall. (A, .~) A myenteric nerve cell in the small intestine that was retrogradely labelled from the coeliac ganglion is not immunoreactive (arrows). (B, B') Retrograde labelling of a NOS-immunoreactive nerve cell in the distal colon following Fast blue injection of the coeliac ganglion. (C, C') Retrograde labelling of a cell in the distal colon following injection of label into the inferior mesenteric ganglion. (D, D') Retrograde labelling of a cell in the rectum following injection of label into the inferior mesenteric ganglion. Calibrations, 50/~m (B, C and D are all same magnification).
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Table 3 Distribution of NOS-containing preganglionic neurons labelled with Fast Blue from the inferior mesenteric ganglion Level
T12 L1 L2
Distribution of FastBlue-labelled neurons (n = 2306)
Distribution of FastBlue-labelled neurons between I M L / D C N
Percentage of Fast-Bluelabelled neurons containing NOS-IR
NOS-IR in Fast-Blue-labelled neurons IML
DCN
23% 48% 28%
50%/50% 58%/42% 77%/23%
70% 69% 68%
56% 65% 52%
83% 74% 68%
mesenteric ganglia are absent from the small intestine and caecum and are very rare in the proximal colon [23] and so these regions were not examined. Labelled cells showing NOS-IR (Fig. 4) were equally common in both regions of distal colon and in the rectum that were investigated and comprised between 60% and 68% of the labelled cells (Table 2). The cells had tapering processes, similar to those observed for nerve cells labelled from the coeliac ganglion.
3.4. NOS immunoreactivity in preganglionic neurons projecting to the inferior mesenteric ganglia The spinal cord between T l l and L3 was examined in two animals where the IMG was injected with Fast Blue. Labelled preganglionic neurons were found in all five segments in both animals, in the intermediolateral column (IML) and in the dorsal eommissural nucleus (DCN), but were rare in T l l and in L3 and so only TI2-L2 were analysed in detail (Table 3). Similar numbers of cells were labelled in each animal and the results have been combined. A total of 2306 Fast-Blue-labelled cells were found bilaterally in both IMLs and in the DCN, with nearly half the cells in L1 and the rest approximately equally distributed between T12 and L2. In T12, labelled neurons were equally distributed between the IML and DCN, whereas in the more caudal segments more labelled neurons were progressively found in the IML. All Fast-Blue-labelled preganglionic neurons in each segment were examined in serial horizontal sections and assessed for expression of NOSIR. The majority (69%) of all labelled neurons were immunoreactive for NOS and these formed a similar proportion of the total labelled cells in
all three segments. In each segment, the DCN contained a higher proportion of retrogradelylabelled neurons containing NOS-IR than did the IML. There was no obvious difference in mediolateral distribution within each nucleus between labelled NOS-IR positive and NOS-IR negative neurons.
4. Discussion
The antiserum used in this study was raised against a peptide previously shown to produce antibodies suitable for immunohistochemistry [25]. In Western blots prepared from extracts of rat brain, the antiserum stained a single band at 150 kDa, the molecular mass of rat neuronal NOS [5]. The staining in both Western blots and tissue sections was completely suppressed by prior incubation of diluted antiserum with the peptide used to raise the antiserum but was not affected by other similarly sized peptides. The antiserum demonstrated patterns of staining of enteric and preganglionic neurons in the guinea pig identical to those seen with two other widely used NOS antisera [8,13]. In the present study this antiserum, in combination with retrograde tracing studies, has been used to identify the source of NOS-IR projections to the coeliac and inferior mesenteric ganglia of the guinea pig. Two sources of NOS-IR terminals were found: enteric neurons in the large intestine and sympathetic preganglionic neurons in the thoracolumbar spinal cord.
4.1. lntestinofugal projections to the coeliac and inferior mesenteric ganglia Although nerve cells in both the small and large intestines project to the coeliac ganglion, we
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found that NOS-IR occurred only in neurons projecting from the large intestine. Of the neurons projecting from the large intestine, 40-68%, depending on the region of intestine, were NOSIR. A number of interpretations might be placed on these data. On the one hand, NOS-IR could be a marker of a functional group of intestinofugal neurons that is present in the large, but not in the small intestine. It is also feasible that NOS-IR is a region-specific feature of intestinofugal neurons of the large intestine that do have functional equivalents in the small intestine; region-specific chemical coding of apparently equivalent neurons has been suggested by previous studies of the chemical coding of neurons [14,21]. In the coeliac ganglion, the dominant connection made by the NOS-IR intestinofugal neurons was with somatostatin-IR nerve cells [13]. In the SMG and IMG the intensely stained baskets of NOS-IR terminals also surround SOM-IR cells and it is likely that these fibres are the terminals of the NOS-IR intestinofugal neurons. Furthermore, the characteristic pericellular baskets formed around somatostatin-IR neurons by NOS-IR terminals suggest the input is predominantly axo-somatic. Somatostatin-IR neurons in the coeliac ganglion have been deduced to supply inhibitory innervation of secretomotor neurons in the small intestine. The NOS-IR intestinofugal neurons may, therefore, contribute to the reflex inhibition, from the large intestine, of fluid secretion in the small intestine. Such a reflex could reduce fluid loading of the large intestine and limit, for example, the effect of diarrhoea. The somatostatin-IR neurons in the IMG, whose function remains to be determined, are likely to be involved in colon to colon reflexes as the colon is the major gastrointestinal target of postganglionic neurons in the IMG [27].
4.2. Preganglionic projections to the IMG NOS is found in preganglionic neurons in rat, guinea pig, mouse and cat [1,6,13,28]. It is present in a majority of the preganglionic neuron somata [1] and can also be visualised in nerve terminals in all autonomic ganglia examined to date as well as in preganglionic terminals in the adrenal medulla [2,3]. If in the SMG and IMG the in-
tensely stained pericellular baskets of terminals around SOM-IR neurons represent the intestinofugal projections as they do in the coeliac ganglion [13], then the more weakly stained NOSIR terminals throughout the ganglion are likely to represent the preganglionic terminals. In contrast to the inputs by the presumed intestinofugal NOS-IR neurons, preganglionic inputs did not form distinct pericellular baskets and so the preganglionic inputs may well be axo-dendritic. In this study, preganglionic neurons projecting to the 1MG were found predominantly in L1 with significant numbers in T12 and L2. Preganglionic neurons projecting to the guinea-pig IMG have previously been identified in L2 and L3, with very few ceils in L1 [10,12]. This one segment difference in distribution between the two studies may simply reflect how the segmental boundaries were defined. However, the earlier study also incorrectly identified a thirteenth thoracic segment, which does not exist in the guinea pig. It is unclear whether this may also have affected the recorded distribution of preganglionic neurons. When preganglionic neurons projecting to the coeliac ganglion of the guinea pig were identified by retrograde tracing [13], there was found to be a lower total proportion of labelled neurons containing NOS-IR (42%) than were found in the present study when the IMG was injected (69%). In addition, there was a striking tendency for the NOS-IR neurons labelled from the coeliac ganglion to lie laterally in the IML. This was not seen in the present study where NOS-IR neurons projecting to the IMG were spread evenly across both the IML and DCN. The differences may reflect the different targets of the IMG relative to the coeliac ganglion and also the absence of the DCN at the level of the coeliac preganglionic neurons. Enkephalin has been identified previously in 40% of the preganglionic neurons labelled by Fast Blue injected into the IMG [12]. In contrast to NOS, enkephalin is found predominantly in preganglionic neurons in the IML. As NOS-IR neurons never accounted for more than 65% of the labelled neurons in the IML it is impossible to predict whether enkephalin and NOS coexist in preganglionic neurons. However, the distinctly
C.R. Anderson et al. /Journal of the Autonomic Nervous System 52 (1995) 107-116
different distribution in the spinal cord of NOS and enkephalin in preganglionic neurons projecting to the IMG raises the possibility that the two substances mark neurons with different functions.
Acknowledgements This work was supported by grants from the National Health and Medical Research Council.
References [1] Anderson, C.R., NADPH diaphorase-positive neurons in the rat spinal cord include a sub-population of autonomic preganglionic neurons, Neurosci. Lett., 139 (1992) 280284. [2] Anderson, C.R., Edwards, S.L., Furness, J.B., Bredt, D.S. and Snyder, S.H., The distribution of nitric oxide synthase-cont~fining autonomic preganglionic terminals in the rat, Brain Res., 614 (1993) 78-85. [3] Blottner, D. and Baumgarten, H.-G., Nitric oxide synthetase (NOS)-containing sympathoadrenal cholinergic neurons of the rat IML-cell column: evidence from histochemistry, immunohistochemistry and retrograde labelling, J. Comp. Neurol., 316 (1992) 45-55. [4] Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R. and Snyder, S.H., Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature, 351 (1991) 714-718. [5] Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. USA, 87 (1990) 682-685. [6] BriJning, G., Localization of NADPH diaphorase, a histochemical marker for nitric oxide synthase, in the mouse spinal cord, Acta Histochem., 93 (1992) 397-401. [7] Buchan, A.M.J., Sikora, B.K.J., Levy, J.G., Mclntosh, C.H.S., Dyck, I. and Brown, J.C., An immunocytochemical investigation with monoclonal antibodies to somatostatin, Histochemistry, 83 (1985) 175-180. [8] Costa, M., Furness, J.B., Pompolo, S., Brookes, S.J.H., Bornstein, J.C., Bredt, D.S. and Snyder, S.H., Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine, Neurosci. Lett., 148 (1992) 121-125. [9] Crowcroft, PJ., Holman, M.E. and Szurszewski, J.H., Excitatory input from the distal colon to the inferior mesenteric ganglion in the guinea-pig, J. Physiol., 219 (1971) 443-461. [10] Dalsgaard, C.-J. and Elfvin, L.-G., Spinal origin of preganglionic fibers projecting onto the superior cervical ganglion and inferior mesenteric ganglion of the guinea pig, as demonstrated by the horseradish peroxidase technique, Brain Res., 172 (1979) 139-143.
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[11] Dalsgaard, C,J., H6kfelt, T., Schultzberg, M., Lundberg, J.M., Terenius, I., Dockray, G.J. and Goldstein, M., Origin of peptide-containing fibers in the inferior mesenteric ganglion of the guinea-pig immunohistochemical studies with antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin, Neuroscience, 9 (1983) 191-211. [12] Dalsgaard, D.-L., H6kfelt, T., Elfvin, L.-G. and Terenius, L., Enkephalin-containing sympathetic preganglionic neurons projecting to the inferior mesenteric ganglion: evidence from combined retrograde tracing and immunohistochemistry, Neuroscience, 7 (1982) 2039-2050. [13] Furness, J.B. and Anderson, C.R., Origins of nerve terminals containing nitric oxide synthase in the guinea-pig coeliac ganglion, J. Autonom. Nerv. Syst., 46 (1994) 4754. [14] Gibbins, I.L., Morris, J.L., Furness, J.B. and Costa, M., Chemical coding of autonomic neurons, Exp. Brain Res., 16 (1987) 23-27. [15] Kreulen, D.L. and Peters, S., Non-cholinergic transmission in a sympathetic ganglion of the guinea-pig elicited by colon distention, J. Physiol., 374 (1986) 315-334. [16] Kuntz, A. and Saccomanno, G., Reflex inhibition of intestinal motility mediated through decentralized prevertebral ganglia, J. Neurophysiol., 7 (1944) 163-170. [17] Lamas, S., Marsden, P.A., Li, G.K., Tempst, P. and Michel, T., Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constituitive enzyme isoform, Proc. Natl. Acad. Sci. USA, 89 (1992) 6348-6352. [18] Macrae, I.M., Furness, J.B. and Costa, M., Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig, Cell Tissue Res., 244 (1986) [19] McConalogue, K. and Furness. J.B., Projections of nitric oxide synthesizing neurons in the guinea-pig colon, Cell Tissue Res., 271 (1993) 545-553. [20] Messenger, J.P., Immunohistochemical analysis of neurons and their projections in the proximal colon of the guinea pig, Arch. Histol. Cytol., 56 (1993) 459-473. [21] Messenger, J.P. and Furness, J.B., Projections of chemically-specified neurons in guinea-pig colon, Arch. Histol. Cytol., 53 (1990) 467-495. [22] Messenger, J.P. and Furness, J.B., Distribution of enteric nerve cells that project to the coeliac ganglion of the guinea pig, Cell Tissue Res., 269 (1992) 119-132. [23] Messenger, J.P. and Furness, J.B., Distribution of enteric nerve cells projecting to the superior and inferior mesenteric ganglia of the guinea-pig, Cell Tissue Res.. 271 (1993) 333-339. [24] Schultzberg, M. and Dalsgaard, C.-J., Enteric origin of bombesin fibres in the rat coeliac-superior mesenteric ganglion, Brain Res., 269 (1983) 190-195. [25] Springall, D.R., Riveros-Moreno, V., Buttery, L., Suburo, A., Bishop, A.E., Merrett, M., Moncada, S. and Polak, J.M., Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms, Histochemistry, 98 (1992) 259-266. [26] Szurszewski, J.H. and King, B.F., Physiology of preverte-
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bral ganglia in mammals with special reference to inferior mesenteric ganglion. In: S.G. Schultz, J.D. Wood and B.B. Rauners (Eds.), Handbook of Physiology, Section 6, The Gastrointestinal Tract, American Physiological Society, Washington, DC, 1989, pp. 519-592. [27] Trudrung, P., Furness, J.B., Pompolo, S. and Messenger, J.P., Locations and chemistries of sympathetic nerve cells that project to the gastrointestinal tract and spleen, Arch. Histol. Cytol., 57 (1994) 139-150. [28] Vizzard, M.A., Erdman, S.L., Erickson, V.L., Stewart,
R.J., Roppolo, J.R. and de Groat, W.C., Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat, J, Comp. Neurol., 339 (1994) 62-75. [29] Xie, Q.-W., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., Ding, A., Trosco, T. and Nathan, C., Cloning and characterization of inducible nitric oxide synthase from mouse macrophages, Science, 256 (1992) 225-228.
Autonomic Nervous System
ELSEVIER
Journal of the Autonomic Nervous System 52 (1995) 107-116
Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia C . R . A n d e r s o n a,, J.B. F u r n e s s
a
H . L . W o o d m a n a S.L. E d w a r d s a p . j . C r a c k b A.I. Smith b
a Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3052, Australia b Baker Medical Research Institute, Commercial Road, Prahran, Victoria 3181, Australia
Received 6 July 1994; revision received 17 August 1994; accepted 30 August 1994
Abstract Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatinimmunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons. Keywords: Inferior mesenteric ganglion; Coeliac ganglion; Preganglionic neuron; Enteric neuron; Fast Blue; (Guinea
pig)
I. Introduction W i t h i n reflex p a t h w a y s t h a t c o n n e c t gastroint e s t i n a l r e g i o n s via p r e v e r t e b r a l g a n g l i a a r e n e u -
* Corresponding author. Tel.: (61-3) 344-5807; Fax: (61-3) 347-5219.
rons with cell b o d i e s in the gut wall a n d t e r m i n a l s in t h e coeliac, s u p e r i o r a n d inferior m e s e n t e r i c ganglia; t h e s e a r e k n o w n as i n t e s t i n o f u g a l neurons a n d a r e p a r t s o f t h e a f f e r e n t limbs of e n t e r o - e n t e r i c reflexes [16,26]. T r a n s m i s s i o n to t h e n e r v e cells of p r e v e r t e b r a l g a n g l i a f r o m intestinofugal n e u r o n s has b o t h c h o l i n e r g i c a n d nonc h o l i n e r g i c c o m p o n e n t s [9,15]. A m o n g s t t h e possible n o n - c h o l i n e r g i c t r a n s m i t t e r s a r e a n u m b e r
0165-1838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00150-2
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of neuropeptides, immunoreactivity for which has been found in intestinofugal neurons; these peptides include bombesin (gastrin-releasing peptide), cholecystokinin, dynorphin, enkephalin and vasoactive intestinal peptide [11,18,24]. Another possible participant in the transmission process from intestinofugal neurons has now been discovered, nitric oxide (NO). Immunoreactivity for the synthesising enzyme for NO, nitric oxide synthase (NOS) is found in nerve terminals forming dense baskets around nerve cells of the coeliac ganglion [13]. These immunoreactive terminals are lost if nerve trunks connecting the intestines with the coeliac ganglion are cut. However, the distribution of nerve cell bodies giving rise to these terminals has not been determined because sufficient quantities of antisera were not available at the time of the study to make surveys in whole mounts of intestine, which is necessary to determine this distribution [22]. For the same reason, the possible existence of NOS immunoreactivity in intestinofugal neurons projecting to the superior and inferior mesenteric ganglia was not studied. An important reason to examine the distribution of NOS immunoreactive neurons is the observation that the chemistries of neurons projecting to the coeliac ganglion differ depending on their origin [22] and NOS may mark a specific subgroup of such inputs. Sympathetic preganglionic neurons are a second source of nerve terminals containing NOS in the coeliac ganglion of the guinea pig [13], but it is not clear whether NOS-containing preganglionic neurons also project to the inferior ruesenteric ganglion. In order to determine the distribution of intestinofugal neurons with NOS immunoreactivity, the distributions of NOS-immunoreactive terminals in each of the prevertebral ganglia in the guinea pig and the possible contributions of NOS-immunoreactive preganglionic neurons to the innervation of these ganglia, we have raised and characterised a polyclonal antiserum specific to the neuronal form of NOS. We have labelled the cell bodies of both intestinofugal and preganglionic neurons by retrograde transport of Fast Blue from prevertebral ganglia and subsequently localised NOS immunoreactivity in the tissue.
2. Materials and methods
2.1. Production of antiserum The immunogen for antibody production was a polylysine tree to which was attached, through the N-terminal, multiple copies of a 21-amino-acid sequence from the neuronal form of NOS to create a large multivalent molecule for immunisation. The sequence chosen has previously been shown to generate antibodies to neuronal NOS [25] and was from amino acids 1409-1429 (R-SE-S-I-A-F-I-E-E-S-K-K-D-A-D-E-V-F-S-S) of the neuronal form of NOS isolated from the rat cerebellum [4]. This sequence is unique to the neuronal form of NOS, and does not occur in the endothelial or macrophage form [17,29]. The immunogen (20 /xg), dissolved in phosphate-buffered saline (PBS), was mixed with Freund's complete adjuvant (1:1) without being linked to a carrier molecule and injected subcutaneously into two rabbits. Booster injections, consisting of 20 ~g antigen in incomplete Freund's adjuvant were given six times at monthly intervals. Bleeds were taken 10 days after each boost. Both rabbits produced antisera, coded N73 and N74, with similar properties. Antiserum N74 from the fourth or fifth bleed has been used in the present study.
2.2. Western blotting Fresh rat brains were homogenised in 50 mM Tris-HC1 buffer (pH 7.4), containing 1 mM EDTA, 10 mg/1 leupeptin, 10 m g/ l pepstatin A and 100 m g / l phenylmethylsulphonyl fluoride. Following centrifugation at 20 000 x g for 30 min at 4°C, the supernatant was subjected to SDSPAGE and the samples then blotted onto polyvinylidene membranes. The membrane was exposed to antiserum followed by a peroxidase-conjugated anti-rabbit IgG (Silenus) and 4-chloronapthol as the chromogen.
2.3. Injection of Fast Blue Guinea pigs (250-350 g, of either sex) were anaesthetised by the subcutaneous injection of
C.R. Anderson et aL /Journal of the Autonomic Nervous System 52 (1995) 107-116
sodium pentobarbitone (15 mg/kg), followed by a mixture of fentanyl (0.6 m g / k g ) and fluanisone (5 mg/kg), injected intramuscularly. Following laparotomy, the coeliac or inferior mesenteric ganglion was exposed. Fast Blue (2% in 10% dimethylsulfoxide) was injected into one or other ganglion via a glass microelectrode that was bevelled to a tip diameter of 70-100 /zm. The left lobe of the coeliac ganglion was injected with 2-3 /xl of Fast Blue and the two lobes of the inferior mesenteric ganglion were injected with a total of 1-2 /xl. Following the injection of ganglia, the abdominal wall was closed and sutured and the animal allowed to recover from anaesthesia. Survival times were 7-10 days, before guinea pigs were killed and tissue taken. 2.4. Removal and processing o f tissue
Guinea pfgs were killed either by concussion and bleeding from the carotid arteries, or by perfusion under full anaesthesia, using the anaesthetic protocol described above. Perfusion was transcardiac with formaldehyde/picric acid (2% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer, p H 7.0). Segments of gastrointestinal tract that were taken from nonperfused animals were placed in PBS (0.9% NaC1 in 0.01 M sodium phosphate buffer, p H 7.0) containing 10 -5 M nicardipine, the contents were washed out and the segments were cut open along their anti-mesenteric borders and pinned tautly mucosa down on balsa board. The balsa was floated, tissue side down, in the formaldehyde/picric acid fixative for 24 h at 4°C. The regions of the gut taken for examination were the ileum, caecum, proximal and distal colon and the rectum. The gut distal from the junction with the caecum through to the anus was dissected out and the region between the pelvic brim and the anus removed as the rectum. The proximal and distal colon were separated at the colonic flexure. Coeliac, superior mesenteric and inferior mesenteric ganglia from non-perfused or perfused animals were immersion fixed in the same mixture. Spinal cords were dissected out only from perfused animals and were post-fixed overnight in the same fixative.
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Fixative was removed by 3 washes in dimethylsulfoxide, followed by 3 changes of PBS. Tissue which was to be examined in whole mount was stored in PBS plus 0.1% sodium azide until it was dissected. Tissue to be examined in section was kept in the same solution to which 30% sucrose was added as a cryoprotectant. Whole mounts of the myenteric plexus plus longitudinal muscle were dissected from the gut wall. Ganglia and spinal cord were cut at 12-/xm thickness on a cryostat. The spinal cord was sectioned at 50/zm on a vibratome. 2.5. Immunohistochemistry
For immunohistochemistry of NOS in ganglia and gut, sections or whole mounts, respectively, were incubated in rabbit anti-NOS antiserum N74-5, diluted 1: 1000, at room temperature for 18-24 h. Dilution was with hypertonic PBS (NaCI concentration, 1.5%). The primary antiserum was washed from the tissue with PBS and replaced with fluorescein isothiocyanate (FITC) labelled sheep anti-rabbit antiserum, 1:160, (Wellcome Diagnostics), for 1 h at room temperature. Tissue was again washed in PBS and the sections or whole mounts coverslipped with phosphatebuffered glycerol (pH 8.6). For simultaneous localisation of NOS and somatostatin immunoreactivity, sections were incubated with a mixture of the anti-NOS antiserum with the monoclonal anti-somatostatin antibody $8 (1 : 20) [7]. This was followed by incubation in biotinylated horse antimouse IgG (Vecta Laboratories) for 2 h. After washing in PBS, the tissue was exposed to a mixture of FITC-labelled sheep anti-rabbit IgG (as above) and streptavidin-Texas Red complex, 1:50 (Amersham) for 90 min. Spinal cord sections were stained free floating in anti-NOS (1: 1000) containing 0.3% Triton X-100, then in Texas Red-labelled donkey anti-rabbit IgG (Jackson) for 1 h and then mounted in serial order. The immunofluorescence was examined in a Zeiss microscope fitted with filters to discriminate between Fast Blue, FITC and Texas Red fluorescence. For preabsorption studies of the antiserum against NOS, 500 /xl of the antiserum diluted
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1 : 1000 was incubated with 20/zg of the antigen or with 20 /.~g of a different but identically synthesised peptide of similar length whose sequence represented amino acids 251-270 of rat neuronal NOS [41•
3. Results
Serum N74 recognised a single band at an apparent molecular mass of 150 kDa on Western blots prepared by separating proteins in extracts from rat brain by SDS-PAGE (Fig. 1). Staining with antiserum N74 was restricted to neurons in the tissue examined. In the prevertebral ganglia and within the spinal cord both nerve terminals and nerve cells were stained. The staining was absent in tissue stained with antiserum preabsorbed with the immunogen but was not affected by preincubation with a similarly synthesised and sized peptide of different sequence. The antiserum has also been found to stain nerve cell bodies and processes in the central and peripheral nervous system of rat, cat, dog and sheep in a pattern consistent with it staining NOS-containing neurons (C.R. Anderson and J.B. Furness, unpublished observations).
A
B
C
205K---~ 116K----~ 80K--~ 50K---~
Fig. 1. Western blot of rat brain extract stained with NOS antiserum N74-5. A, Molecular mass markers; B, rat brain extract probed with NOS antiserum; C, rat brain extract probed with NOS antiserum preabsorbed with the immunogen. Note the single band around 150 kDa in B that is absent in C after preabsorbtion.
3.1• Distribution of immunoreactivity in prevertebral ganglia The distribution of NOS immunoreactivity (NOS-IR) in the superior and inferior mesenteric ganglia closely resembled that previously described for the coeliac ganglion [13]. Clumps of nerve cell bodies were surrounded by networks of strongly immunoreactive nerve fibres (Fig. 2).
Fig. 2. NOS immunoreactivity in cryostat sections of the inferior mesenteric ganglion (A and C) and in a wholemount preparation of a small ganglion of the hypogastric nerve (B), close to the inferior mesenteric ganglion. In the inferior mesenteric ganglion (A), strongly immunoreactive fibres surround groups of nerve cells (arrowheads), and in other regions less strongly reactive fibres are found amongst the nerve cells (arrows). Rare immunoreactive nerve cells are also found in these ganglia (C). A small proportion of nerve cells in hypogastric nerve ganglia close to the inferior mesenteric ganglion is reactive (B). Calibration, 50/zm.
C.R. Anderson et al. /Journal of the Autonomic Nervous System 52 (1995) 107-116
Faintly immunoreactive fibres were around most of the remaining nerve cells, as previously illustrated for the coeliac ganglion [13]. In both ganglia there were only very small numbers of strongly immunoreaetive nerve cell bodies (Fig. 2). NOSIR nerve cell bodies were more common in the small ganglia situated on the hypogastric nerve close to the IMG (Fig. 2). The intermesenteric and colonic nerves contained numerous NOS-immunoreactive, non-varicose nerve fibres. Chromaffin cells, which are prominent in the inferior mesenteric ganglia in particular, were not immunoreactive for NOS. The majority of somatostatin-IR nerve cells in the prevertebral ganglia were surrounded by fibres with strong immunoreactivity for NOS (Fig. 3); conversely, areas of ganglia without somatostatin-IR nerve cells lacked these strongly NOSimmunoreactive terminals. In samples of 100 successive somatostatin-IR nerve cells counted in ganglia from 3 animals, most somatostatin-IR cells were surrounded by baskets of nerve fibres with strong immunoreactivity for NOS (86.1 + 1.4 in the coeliac ganglion, 82.8 +_ 1.5 in the superior mesenteric ganglion and 83.2 + 2.1 in the inferior mesenteric ganglion. All values are means + SEM). Similarly, NOS baskets showing strong immunoreactivity were almost all around somatostatin-IR nerve cells (88.6 _+ 1.1 in the coeliac ganglion, 78.2 +_ 2.4 in the superior mesenteric ganglion and 85.8 +_ 1.7 in the inferior mesenteric ganglion. All values are means + SEM).
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3.2. N O S immunoreactivity in intestinofugal neurons projecting to the coeliac ganglion
NOS-IR was examined in the myenteric ganglia of preparations of small intestine, caecum, proximal colon, distal colon and rectum that contained nerve cells that were retrogradely labelled following injection of the dye into the coeliac ganglion. The distributions and morphologies of NOS-IR nerve cell bodies and fibres were indistinguishable from those previously described in the guinea pig small and large intestine [8,19,20]. No retrogradely labelled nerve cells in the small intestine had NOS-IR, whereas 40-70% of retrogradely labelled nerve cells in the regions of large intestine that were examined had NOS-IR (Table 1; Fig. 4). The NOS-IR intestinofugal neurons were typically neither the smallest nor the largest neurons in the ganglia. The processes of the cells were not always distinct, but where these were apparent 3 - 8 tapering processes were observed (Fig. 4). 3.3. N O S immunoreactivity in intestinofugal neurons projecting to the inferior mesenteric ganglia
Two sample regions of distal colon, the middle (mid distal colon) and distal fifth (terminal distal colon) of the organ, and the rectum were examined for the presence of NOS-IR in retrogradely labelled nerve cells after injection of Fast Blue into the IMG. Neurons projecting to the inferior
Fig. 3. Simultaneous localisation of NOS and somatostatin-immunoreactivityin a thin (8 izm) section from the inferior mesenteric ganglion. NOS terminals (A) surround the somatostatin nerve cell bodies (A, arrows). Calibration, 50 izm.
C.R. Anderson et al. /Journal of the Autonomic NerL,ous System 52 (1995) 107-116
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Table 1 Percentage of labelled enteric neurons showing NOS immunoreactivity after Fast Blue injection into the coeliac ganglion
Table 2 Percentage of labelled enteric neurons showing NOS immunoreactivity after Fast Blue injection into the inferior mesenteric ganglion
Ileum
Caecum
Proc. colon
Rectum
Mid distal colon
Terminal distal colon
Rectum
0 (n = 83)
70.2+7.8 (n = 32)
40.3_+11.6 61.7+13.3 (n = 209) (n = 38)
44.6+18.7 (n = 31)
60.9_+3.8 (n = 60)
68.3_+4.8 (n = 312)
66.2+_7.4 (n = 257)
Dist. colon
(Tissue from four animals, except for Ileum where eight animals were used). All values are means+SEM. Total cells counted are also indicated.
(Tissue from four animals). All values are means+SEM. Total cells counted are also indicated.
Fig. 4. Relation between retrograde labelling from prevertebral ganglia (A, B, C, D) and NOS immunoreactivity (/~, B', C', D') in the gut wall. (A, .~) A myenteric nerve cell in the small intestine that was retrogradely labelled from the coeliac ganglion is not immunoreactive (arrows). (B, B') Retrograde labelling of a NOS-immunoreactive nerve cell in the distal colon following Fast blue injection of the coeliac ganglion. (C, C') Retrograde labelling of a cell in the distal colon following injection of label into the inferior mesenteric ganglion. (D, D') Retrograde labelling of a cell in the rectum following injection of label into the inferior mesenteric ganglion. Calibrations, 50/~m (B, C and D are all same magnification).
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Table 3 Distribution of NOS-containing preganglionic neurons labelled with Fast Blue from the inferior mesenteric ganglion Level
T12 L1 L2
Distribution of FastBlue-labelled neurons (n = 2306)
Distribution of FastBlue-labelled neurons between I M L / D C N
Percentage of Fast-Bluelabelled neurons containing NOS-IR
NOS-IR in Fast-Blue-labelled neurons IML
DCN
23% 48% 28%
50%/50% 58%/42% 77%/23%
70% 69% 68%
56% 65% 52%
83% 74% 68%
mesenteric ganglia are absent from the small intestine and caecum and are very rare in the proximal colon [23] and so these regions were not examined. Labelled cells showing NOS-IR (Fig. 4) were equally common in both regions of distal colon and in the rectum that were investigated and comprised between 60% and 68% of the labelled cells (Table 2). The cells had tapering processes, similar to those observed for nerve cells labelled from the coeliac ganglion.
3.4. NOS immunoreactivity in preganglionic neurons projecting to the inferior mesenteric ganglia The spinal cord between T l l and L3 was examined in two animals where the IMG was injected with Fast Blue. Labelled preganglionic neurons were found in all five segments in both animals, in the intermediolateral column (IML) and in the dorsal eommissural nucleus (DCN), but were rare in T l l and in L3 and so only TI2-L2 were analysed in detail (Table 3). Similar numbers of cells were labelled in each animal and the results have been combined. A total of 2306 Fast-Blue-labelled cells were found bilaterally in both IMLs and in the DCN, with nearly half the cells in L1 and the rest approximately equally distributed between T12 and L2. In T12, labelled neurons were equally distributed between the IML and DCN, whereas in the more caudal segments more labelled neurons were progressively found in the IML. All Fast-Blue-labelled preganglionic neurons in each segment were examined in serial horizontal sections and assessed for expression of NOSIR. The majority (69%) of all labelled neurons were immunoreactive for NOS and these formed a similar proportion of the total labelled cells in
all three segments. In each segment, the DCN contained a higher proportion of retrogradelylabelled neurons containing NOS-IR than did the IML. There was no obvious difference in mediolateral distribution within each nucleus between labelled NOS-IR positive and NOS-IR negative neurons.
4. Discussion
The antiserum used in this study was raised against a peptide previously shown to produce antibodies suitable for immunohistochemistry [25]. In Western blots prepared from extracts of rat brain, the antiserum stained a single band at 150 kDa, the molecular mass of rat neuronal NOS [5]. The staining in both Western blots and tissue sections was completely suppressed by prior incubation of diluted antiserum with the peptide used to raise the antiserum but was not affected by other similarly sized peptides. The antiserum demonstrated patterns of staining of enteric and preganglionic neurons in the guinea pig identical to those seen with two other widely used NOS antisera [8,13]. In the present study this antiserum, in combination with retrograde tracing studies, has been used to identify the source of NOS-IR projections to the coeliac and inferior mesenteric ganglia of the guinea pig. Two sources of NOS-IR terminals were found: enteric neurons in the large intestine and sympathetic preganglionic neurons in the thoracolumbar spinal cord.
4.1. lntestinofugal projections to the coeliac and inferior mesenteric ganglia Although nerve cells in both the small and large intestines project to the coeliac ganglion, we
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found that NOS-IR occurred only in neurons projecting from the large intestine. Of the neurons projecting from the large intestine, 40-68%, depending on the region of intestine, were NOSIR. A number of interpretations might be placed on these data. On the one hand, NOS-IR could be a marker of a functional group of intestinofugal neurons that is present in the large, but not in the small intestine. It is also feasible that NOS-IR is a region-specific feature of intestinofugal neurons of the large intestine that do have functional equivalents in the small intestine; region-specific chemical coding of apparently equivalent neurons has been suggested by previous studies of the chemical coding of neurons [14,21]. In the coeliac ganglion, the dominant connection made by the NOS-IR intestinofugal neurons was with somatostatin-IR nerve cells [13]. In the SMG and IMG the intensely stained baskets of NOS-IR terminals also surround SOM-IR cells and it is likely that these fibres are the terminals of the NOS-IR intestinofugal neurons. Furthermore, the characteristic pericellular baskets formed around somatostatin-IR neurons by NOS-IR terminals suggest the input is predominantly axo-somatic. Somatostatin-IR neurons in the coeliac ganglion have been deduced to supply inhibitory innervation of secretomotor neurons in the small intestine. The NOS-IR intestinofugal neurons may, therefore, contribute to the reflex inhibition, from the large intestine, of fluid secretion in the small intestine. Such a reflex could reduce fluid loading of the large intestine and limit, for example, the effect of diarrhoea. The somatostatin-IR neurons in the IMG, whose function remains to be determined, are likely to be involved in colon to colon reflexes as the colon is the major gastrointestinal target of postganglionic neurons in the IMG [27].
4.2. Preganglionic projections to the IMG NOS is found in preganglionic neurons in rat, guinea pig, mouse and cat [1,6,13,28]. It is present in a majority of the preganglionic neuron somata [1] and can also be visualised in nerve terminals in all autonomic ganglia examined to date as well as in preganglionic terminals in the adrenal medulla [2,3]. If in the SMG and IMG the in-
tensely stained pericellular baskets of terminals around SOM-IR neurons represent the intestinofugal projections as they do in the coeliac ganglion [13], then the more weakly stained NOSIR terminals throughout the ganglion are likely to represent the preganglionic terminals. In contrast to the inputs by the presumed intestinofugal NOS-IR neurons, preganglionic inputs did not form distinct pericellular baskets and so the preganglionic inputs may well be axo-dendritic. In this study, preganglionic neurons projecting to the 1MG were found predominantly in L1 with significant numbers in T12 and L2. Preganglionic neurons projecting to the guinea-pig IMG have previously been identified in L2 and L3, with very few ceils in L1 [10,12]. This one segment difference in distribution between the two studies may simply reflect how the segmental boundaries were defined. However, the earlier study also incorrectly identified a thirteenth thoracic segment, which does not exist in the guinea pig. It is unclear whether this may also have affected the recorded distribution of preganglionic neurons. When preganglionic neurons projecting to the coeliac ganglion of the guinea pig were identified by retrograde tracing [13], there was found to be a lower total proportion of labelled neurons containing NOS-IR (42%) than were found in the present study when the IMG was injected (69%). In addition, there was a striking tendency for the NOS-IR neurons labelled from the coeliac ganglion to lie laterally in the IML. This was not seen in the present study where NOS-IR neurons projecting to the IMG were spread evenly across both the IML and DCN. The differences may reflect the different targets of the IMG relative to the coeliac ganglion and also the absence of the DCN at the level of the coeliac preganglionic neurons. Enkephalin has been identified previously in 40% of the preganglionic neurons labelled by Fast Blue injected into the IMG [12]. In contrast to NOS, enkephalin is found predominantly in preganglionic neurons in the IML. As NOS-IR neurons never accounted for more than 65% of the labelled neurons in the IML it is impossible to predict whether enkephalin and NOS coexist in preganglionic neurons. However, the distinctly
C.R. Anderson et al. /Journal of the Autonomic Nervous System 52 (1995) 107-116
different distribution in the spinal cord of NOS and enkephalin in preganglionic neurons projecting to the IMG raises the possibility that the two substances mark neurons with different functions.
Acknowledgements This work was supported by grants from the National Health and Medical Research Council.
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