Neurochemical identification of enteric neurons expressing P2X(3) receptors in the guinea-pig ileum

It was hypothesised that P2X(3) receptors, predominantly labelling spinal and cranial sensory ganglionic neurons, are also expressed in intrinsic sensory enteric neurons, although direct evidence is lacking. The aim of this study was to localise P2X(3) receptors in the enteric nervous system of the guinea-pig ileum, and to neurochemically identify the P2X(3)-expressing neurons. In the submucous plexus, cholinergic neurons expressing calretinin (CRT), were immunostained for P2X(3). These neurons made up about 12% of the submucous neurons. In the myenteric plexus, approximately 36% of the neurons expressed P2X(3). Half of the latter neurons were immunoreactive for CRT, whereas about 20% were immunoreactive for nitric oxide synthase (NOS). Based on earlier neurochemical analysis of enteric neurons in the guinea-pig, the myenteric neurons exhibiting P2X(3)/CRT immunoreactivity were identified as longitudinal muscle motor neurons, and those expressing P2X(3)/NOS immunoreactivity as short inhibitory circular muscle motor neurons. In both plexuses, no colocalisation was observed between P2X(3) and calbindin, a marker for intrinsic sensory neurons. Multiple staining with antisera raised against somatostatin, neuropeptide Y, substance P or neurofilament protein did not reveal any costaining. It can be concluded that in the guinea-pig ileum, intrinsic sensory neurons do not express P2X(3) receptors. However, this does not negate the possibility that extrinsic sensory nerves expressing P2X(3) are involved in a purinergic mechanosensory transduction pathway as demonstrated in other organs.     

Calbindin immunoreactivity in the enteric nervous system of larval and adult zebrafish (Danio rerio)

Calbindin is a calcium-binding protein, commonly found in certain subpopulations of the enteric nervous system in mammals. Recently, calbindin-immunoreactive enteric neurons have also been demonstrated in shorthorn sculpin (Myoxocephalus scorpius). In the present study, calbindin immunoreactivity has been investigated in the gut of adult and larval zebrafish (Danio rerio) and differences and similarities between the two species are discussed. Calbindin immunoreactivity is present in 40%?C50% of all enteric neurons in adult zebrafish. It first appears at 3?days post-fertilisation (dpf) and is present in all regions of the gut by 13 dpf. Calbindin-immunoreactive nerve cell bodies do not differ in size from calbindin-negative cells. Zebrafish calbindin-immunoreactive neurons are serotonin-negative, with at least some being choline acetyltransferase (ChAT)-positive, in contrast to the sculpin in which cells are generally smaller than the average enteric neuron and are serotonin-positive and ChAT-negative. These findings further emphasise the importance of comparative studies for understanding the diversity of chemical coding in the enteric nervous system of fish and other vertebrates. Improved knowledge of the role of the enteric nervous system is also essential for future studies of gut activity with regard to zebrafish being used as a model organism.     

Neurochemical classification of enteric neurons in the guinea-pig distal colon

Previous studies have identified the chemistries, shapes, projections and electrophysiological characteristics of several populations of neurons in the distal colon of the guinea-pig but it is unknown how these characteristics correlate to define the classes of neurons present. We have used double-label immunohistochemical techniques to identify neurochemically distinct subgroups of enteric neurons in this region. On the basis of colocalisation of neurochemical markers and knowledge gained from previous studies of neural projections, 17 classes of neurons were identified. The myenteric plexus contained the cell bodies of 13 distinct types of neurons. Four classes of descending interneurons and three classes of ascending interneurons were identified, together with inhibitory and excitatory motor neurons to both the circular and longitudinal muscle layers. Dogiel type II neurons, which are presumed to be intrinsic primary afferent neurons, were located in myenteric and submucosal ganglia; they were all immunoreactive for choline acetyltransferase and often calbindin and tachykinins. Three classes of secretomotor neurons with cell bodies in submucosal ganglia were defined. Two of these classes were immunoreactive for choline acetyltransferase and the other class was immunoreactive for both vasoactive intestinal peptide and nitric oxide synthase. Some of the secretomotor neurons probably also have a vasomotor function. The neural subtypes defined in the present study are similar in many respects to those found in the small intestine, although differences are evident, especially in populations of interneurons. These differences presumably reflect the differing physiological roles of the two intestinal regions.     

Neurotrophin-3 and neurotrophin receptor immunoreactivity in peptidergic enteric neurons

In the rat small intestine, neurotrophin-3 immunoreactivity was identified in ganglion cells and in processes mostly innervating the mucosa and occasionally the muscle layer and vasculature. The vast majority of neurotrophin-3 immunoreactive neurons contained vasoactive intestinal polypeptide (VIP), but not substance P or related tachykinin (SP/TK). Neurotrophin receptors visualized by pan-trk immunoreactivity were found in numerous ganglion cells of both plexuses and in nerve processes in the intestinal wall. Pan-trk submucosal neurons contained VIP (36%) or SP/TK-IR (47%). Pan-trk myenteric neurons contained VIP-IR (57%) or SP/TK (27%). Our data suggest that neurotrophin-3 and neurotrophin receptors may be involved in the maintenance of enteric neuronal circuits, transmission and phenotypic expression.     

Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

Summary The distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm² of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiel's type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.     

Neurokinin B- and substance P-like immunoreactivity are co-localized in enteric nerves of rat ileum

The tachykinins (TKs) substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) have conserved C-terminal sequences and mediate similar physiological responses by activating neurokinin receptors found on neural and smooth muscle cells. Many enteric nerves express preprotachykinin A (PPT A) mRNA and synthesize SP and NKA. However, it is unclear if NKB is synthesized in enteric neurons as many antibodies developed against NKB also recognize other TKs. Therefore, the cellular distribution of NKB-like-immunoreactivity (NKB-ir) in rat ileum was examined using selective antisera raised against either synthetic Cys10-NKB or peptide 2 (P2), a non-tachykinergic peptide sequence in NKB precursor protein. NKB-ir and P2-ir had a similar distribution in varicose nerve fibers in submucosal and myenteric ganglia and almost all ganglia contained immunoreactive nerves. Few submucosal or myenteric neuronal somata contained strong immunoreactivity. Preabsorption of NKB or P2 antisera with their respective cognate peptides, but not with other TK peptides, abolished specific immunostaining. Finally, co-localization of NKB-/P2-ir with SP-ir suggested that most NKB-/P2-ir nerve fibers contain SP-ir, but some SP-ir nerves do not contain detectable NKB-/P2-ir. These results indicate that PPT B products P2 and NKB are localized in a subpopulation of enteric nerves containing TKs encoded by PPT A. Stimulation of these nerves may release NKB to activate local neurokinin receptors.     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig

Summary Neuromedin U immunoreactivity was located histochemically in the guinea-pig small intestine. Projections of immunoreactive neurons were determined by analysing patterns of degeneration following nerve lesions. The co-localization of neuromedin U immunoreactivity with immunoreactivity for substance P, neuropeptide Y, vasoactive intestinal peptide and calbindin was also investigated. Neuromedin U immunoreactivity was found in nerve cells in the myenteric and submucous plexuses and in nerve fibres in these ganglionated plexuses, around submucous arterioles and in the mucosa. Reactive fibres did not supply the muscle layers. Most reactive nerve cells in the myenteric ganglia had Dogiel type-II morphology and in many there was co-localization of calbindin, although some Dogiel type-II neuromedin U neurons were calbindin negative. Lesion studies suggest that these myenteric neurons project circumferentially to local myenteric ganglia. Projections from myenteric neurons also run anally in the myenteric plexus, while other projections extend to submucous ganglia, and still further projections run from the intestine to provide terminals in the coeliac ganglia. In the submucous ganglia neuromedin U was co-localized in three populations of nerve cells: (i) those with vasoactive intestinal peptide immunoreactivity, (ii) neurons containing neuropeptide Y, and (iii) neurons containing substance P. Each of these populations sends nerve fibres to the mucosa. Neuromedin U immunoreactivity is thus located in a variety of neurons serving different functions in the intestine and therefore probably does not have a single role in intestinal physiology.     

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.     

Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep

We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20–25% of myenteric and 65–75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80±13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77±7% of CALB-IR cells were NeuN-positive. In mature sheep, 73±10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type.This work was supported by a grant from the Ministero dellIstruzione, dellUniversità e della Ricerca (MIUR)     

Choline acetyltransferase- and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig

Summary The peptides cholecystokinin (CCK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP), and the synthesizing enzyme for acetylcholine, choline acetyltransferase (ChAT) were localized immunohistochemically in nerve cell bodies of the submucous ganglia in the small intestine of the guinea-pig. VIP-like immunoreactivity was found in 45% of submucous neurons. ChAT immunoreactivity was observed in a separate group of nerve cells, which made up 54% of the total population. There were three subsets of neurons immunoreactive for ChAT: (1) ChAT neurons that also contained immunoreactivity for each of the peptides CCK, SOM and NPY, representing 29% of all submucous neurons; (2) ChAT neurons that also contained SP-like immunoreactivity, representing 11% of all submucous neurons, and (3) ChAT cells that did not contain any detectable amount of the peptides that were localized in this study.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Movement of villi induces endocytosis of NK1 receptors in myenteric neurons from guinea-pig ileum

Agitation of villi evokes reflexes that affect the motility of the guinea-pig small intestine. NK1 receptor endocytosis was used to investigate the possible involvement of tachykinins acting on neuronal NK1 receptors in these reflexes. Segments of guinea-pig ileum were incubated at 37°C in Krebs physiological saline containing 3×10^–6 M nicardipine, with or without agitation of the villi by gas bubbles. Gut segments were fixed after 0–75 min and processed for immunohistochemistry to reveal the NK1 receptors, following which cells were imaged by confocal microscopy. Initially, receptors were located on the surface and in the cytoplasm of myenteric neurons. In gut incubated without movement of the villi, NK1 receptors returned to the cell surface. After 45 and 60 min, NK1 receptors were detected almost exclusively at the cell surface of 83% and 97% (respectively) of nerve cells that were immunoreactive for NK1 receptors and only 12%–13% of the NK1 receptor fluorescence was located in the cytoplasm. Following the return of receptor to the cell surface, agitation of the villi caused a new wave of endocytosis of the NK1 receptors in 70%–80% of the NK1 receptor-immunoreactive neurons. The percentage of the NK1 receptor fluorescence that was in the cytoplasm increased more than 2-fold to 27±2% after 15 min villous agitation. Action potential blockade by tetrodotoxin (3×10^–7 M) prevented the internalisation of the NK1 receptor in response to villous agitation. The degree of internalisation caused by bubbling was similar to that caused by 2×10^–9 M substance P. These results indicate that, when enteric reflex circuits are activated by villous movement, tachykinins are released and cause endocytosis of the NK1 receptor in a subpopulation of myenteric neurons.     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

Summary Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with N^G-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

Multiple opiate receptors in guinea-pig ileum

Actions of the prototypic μ-, κ-, and σ-opiate receptor agonists, morphine (M) ketocyclazocine (K) and SKF-10,047. (S), respectively, were examined and differentiated using the guinea-pig ileum preparation. S, like M and K, depressed the electrically stimulated ileum. Naloxone antagonized the depressant actions of the prototypic drugs with different potencies. PA₂ values of naloxone for M, K, and S, respectively, were 8.81, 7.58 and 7.74. Relative cross tolerance of each prototypic drug to normorphine, a comparison standard, was also examined in morphine-pretreated ilea and quantitatively estimated as follows: (1) the median effective dose of each drug and of the standard drug normorphine were determined in the nontolerant ileum (IC50_NT) and in ilea with varying degrees of tolerance IC50_T); (2) cross-tolerance ratios (IC50_T/IC50_NT) of each drug and of normorphine were calculated for the varying degrees of tolerance; (3) cross-tolerance ratios of each drug were plotted against those of normorphine, the data were fit by a least squares straight line, and the slope of the line determined as the Relative Cross Tolerance Index (RCTI). RCTI for M was 2.21. K and S, however, had lower RCTI's of 0.44 and 0.64 respectively. In the morphine-pretreated tolerant ilea, slopes of the dose response curves of the prototypic drugs were found to differ: while M and K possessed steep and constant slopes for ilea with different degrees of tolerance, the slopes for S became shallower as ilea became more tolerant to morphine. A maximum ceiling effect of less than 50% depression was obtained for S in the most highly tolerant ilea. The above observations are consistent with possible existence of the three types of hypothesized opiate receptors in the guinea-pig ileum.