Different subpopulations of cholinergic and nitrergic myenteric neurones project to mucosa and circular muscle of the guinea-pig gastric fundus

Since the stomach lacks a well-developed ganglionated submucous plexus, the somata of enteric neurones innervating the muscle or the mucosa have to be localised within the myenteric plexus. The aim of this study was to determine the projection pathways and the neurochemical coding of myenteric neurones innervating these different targets in the gastric fundus. Myenteric cell bodies projecting to the mucosa or the circular muscle were retrogradely labelled by mucosa or muscle application of the fluorescent tracer DiI and subsequently characterised by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and/or neuropeptide Y (NPY). On average 143±91 and 89±49 myenteric neurones were labelled from the mucosa and the circular muscle, respectively. DiI-labelled neurones were either ChAT- or NOS-positive. DiI-labelled ChAT-positive neurones were mainly ascending and outnumbered NOS-positive neurones, which were mainly descending (79.3±6.2% vs 20.7±6.2% for mucosa neurones; 69.3±11.1% vs 30.7±11.1% for muscle neurones). Three ChAT-positive subpopulations (ChAT/–, ChAT/SP, ChAT/NPY) and two NOS-positive subpopulations (NOS/–, NOS/NPY) were found. ChAT/SP neurones projected mainly to the circular muscle (36.1±11.9% of the cholinergic muscle neurones; mucosa projection: 8.0±2.1%), whereas ChAT/NPY neurones projected mainly to the mucosa (38.1±9.2% of the cholinergic mucosa neurones; muscle projection: 5.7±2.4%). NOS/– cells projected predominantly to the muscle. This study demonstrates polarised pathways in the myenteric plexus consisting of ascending ChAT and descending NOS cells that innervate the circular muscle and the mucosa of the gastric fundus. The ChAT/SP neurones might function as circular muscle motor neurones, whereas ChAT/NPY neurones might represent secretomotor neurones.     

Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.     

Enkephalin-immunoreactive subpopulations in the myenteric plexus of the guinea-pig fundus project primarily to the muscle and not to the mucosa

Enkephalin (ENK) immunoreactivity was localised in different neuronal subpopulations of the myenteric plexus in the guinea-pig gastric fundus using immunohistochemistry for neurone-specific enolase (NSE), ENK, choline acetyltransferase (ChAT), substance P (SP), neuropeptide Y (NPY), calretinin (CALRET), and somatostatin (SOM). NADPH-diaphorase staining was used to label nitric oxide synthase (NOS)-containing neurones. ENK was observed in 44% of the myenteric neurones. The major ENK-positive subpopulations were ChAT/ENK (35% of ENK-positive neurones), ChAT/SP/ENK (26%), NOS/NPY/ENK (22%) and ChAT/SP/ENK/CALRET (9%). The projection pathways of these ENK-positive subpopulations to the circular muscle and the mucosa were determined using retrograde labelling with DiI in organ culture followed by immunohistochemistry. Of myenteric neurones retrogradely labelled from the mucosa and the circular muscle, 13% and 48% exhibited ENK immunoreactivity, respectively. Three major ENK-positive subpopulations innervating the mucosa or circular muscle were identified: ascending ChAT/SP/ENK (7% of all mucosa neurones; 24% of all circular muscle neurones), ascending ChAT/ENK (4%; 15%) and descending NOS/NPY/ENK (1%; 8%) neurones. Only very few CALRET- or SOM-positive neurones projected to the mucosa or circular muscle. ChAT/SP/ENK and ChAT/ENK neurones might function as ascending excitatory muscle motor neurones, whereas NOS/NPY/ENK neurones are most likely descending inhibitory muscle motor neurones. The relatively few ENK-positive mucosa neurones do not favour a major involvement of ENK-positive myenteric neurones in the control of gastric mucosa activity.     

Projections and pathways of submucous neurons to the mucosa of the guinea-pig small intestine

Summary Double-labelling immunohistochemistry and retrograde transport of the carbocyanine dye, DiI, were used to establish the pathways of submucous neurons to the mucosa of the guinea-pig small intestine. Following the application of DiI to a villus, DiI-labelled nerve cell bodies were found in the submucous plexus up to 8.3 mm circumferentially and 3.8 mm longitudinally. The size of each of the four characterised classes of submucous neurons was determined and their distributions and projections mapped. Cells characterised by vasoactive intestinal polypeptide immunoreactivity accounted for 52% of DiI-labelled cells and had the longest projections. Cells characterised by neuropeptide Y (19%) or by calretinin immunoreactivity (13% of all DiI-labelled neurons) had relatively short projections and cells with substance P immunoreactivity (20%) had intermediate lengths of projection. When DiI was applied directly to the submucous plexus, filled neurons of all classes had significantly shorter projections, indicating that they must run for considerable distances in other pathways to the mucosa, probably via the non-ganglionated plexus. On average, each villus is innervated by at least 70 submucous neurons. From quantitative estimates there are 9 submucous neurons per villus. Thus, each submucous neuron is likely to supply about 8 villi. This demonstrates a high degree of convergence and divergence in the innervation of the mucosa.     

Reciprocal activity of longitudinal and circular muscle during intestinal peristaltic reflex

A two-compartment, flat-sheet preparation of rat colon was devised, which enabled exclusive measurement of longitudinal muscle activity during the ascending and descending phases of the peristaltic reflex. A previous study using longitudinal muscle strips revealed the operation of an integrated neuronal circuit consisting of somatostatin, opioid, and VIP/pituitary adenylate cyclase-activating peptide (PACAP)/nitric oxide synthase (NOS) interneurons coupled to cholinergic/tachykinin motor neurons innervating longitudinal muscle strips that could lead to descending contraction and ascending relaxation of this muscle layer. Previous studies in peristaltic preparations have also shown that an increase in somatostatin release during the descending phase causes a decrease in Met-enkephalin release and suppression of the inhibitory effect of Met-enkephalin on VIP/PACAP/NOS motor neurons innervating circular muscle and a distinct set of VIP/PACAP/NOS interneurons. The present study showed that in contrast to circular muscle, longitudinal muscle contracted during the descending phase and relaxed during the ascending phase. Somatostatin antiserum inhibited descending contraction and augmented ascending relaxation of longitudinal muscle, whereas naloxone had the opposite effect. VIP and PACAP antagonists inhibited descending contraction of longitudinal muscle and augmented ascending relaxation. Atropine and tachykinin antagonists inhibited descending contraction of longitudinal muscle. As shown in earlier studies, the same antagonists and antisera produced opposite effects on circular muscle. We conclude that longitudinal muscle contracts and relaxes in reverse fashion to circular muscle during the peristaltic reflex. Longitudinal muscle activity is regulated by excitatory VIP/PACAP/NOS interneurons coupled to cholinergic/tachykinin motor neurons innervating longitudinal muscle.     

Characterization of excitatory and inhibitory motor neurons to the human gastric clasp and sling fibers

The aim of this study was to determine the morphology and position of the excitatory and inhibitory motor neurons to the human gastric sling and clasp fibers. Motor neurons were identified by retrograde staining with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (DiI), and choline acetyltransferase (ChAT) or nitric oxide synthase (NOS) immunoreactivity was then determined in these motor neurons. In the sling preparations, 46% of the DiI-stained cells were aboral motor neurons, 43% were local motor neurons, and only 10% were descending motor neurons. Overall, 58% were immunoreactive for ChAT, and 36% for NOS (P = 0.042). Sixty-two percent of local, and 66% of aboral DiI-stained motor neurons were immunoreactive for ChAT. In the clasp preparations, 52% of the DiI-stained cells were descending motor neurons, 45% were local motor neurons, and only 3% were aboral neurons. Overall, 31% were immunoreactive for ChAT and 65% for NOS (P = 0.039). Eighty-five percent of the DiI-stained descending motor neurons were immunoreactive for NOS. All of the cells that were labeled adequately had a single axon and a number of filamentous or flattened lobular dendrites, and fitted into the broad category of Dogiel type I neurons. In conclusion, the majority of the motor neurons to the sling fibers were ChAT-positive excitatory neurons from the myenteric plexus of the stomach and the local region, and to the clasp were predominantly NOS-positive inhibitory neurons from the esophagus.     

Synaptic transmission in simple motility reflex pathways excited by distension in guinea pig distal colon

We examined specific receptor/transmitter combinations used at functionally identified synapses in ascending and descending reflex pathways of guinea pig distal colon. Excitatory (EJPs) or inhibitory junction potentials (IJPs) were recorded intracellularly from nicardipine-paralyzed circular smooth muscle in either the oral or anal recording chamber of a three-chambered organ bath, respectively. Blockade of synaptic transmission in the central chamber with a 0.25 mM Ca2+/12 mM Mg2+ solution abolished EJPs evoked by distension applied either in the central or the far (anal) chamber. IJPs evoked by distension in the central or the far (oral) chamber were depressed to approximately 50% of control. Hexamethonium (nicotinic receptor antagonist, 200 microM) in the central chamber reduced IJPs evoked by far or central distension to 50%, whereas EJPs evoked by far distension were abolished and EJPs evoked by central distension were reduced to 70% of control. Hexamethonium in the recording chambers reduced both IJPs and EJPs evoked by central distension to approximately 50%. EJPs in the ascending pathway were unaffected by blockade of muscarinic receptors in the central chamber or blockade of neurokinin 3 tachykinin receptors in this or the recording chamber. In the descending pathway, blockade of P2 receptors in the same chambers had only a minor effect on distension-evoked IJPs. Thus some intrinsic sensory neurons of guinea pig colon have long descending projections (>30 mm), but ascending projections of <15 mm. In contrast to the ileum, transmission between ascending or descending interneurons and from sensory neurons to descending interneurons is predominantly via nicotinic receptors; but transmission to inhibitory or excitatory motoneurons and from sensory neurons to ascending interneurons involves nicotinic and other unidentified receptors.     

Functional coordination of motor activity in colonic smooth muscles in rat experimental model

Spontaneous and electrically-elicited motor activity was recorded by triple organ bath in rat segment-model preparation as display of excitation of local nerve networks and ascending or descending reflex pathways underlying contractile potency and functional coordination of colonic longitudinal and circular muscles. Spontaneous high-amplitude contractions, but not relaxations, appeared synchronously in both muscles. Electrical field stimulation applied to proximal or distal part of segments elicited both tetrodotoxin (0.1 microM)-sensitive local motor responses of the stimulated part and ascending or descending motor responses of the contralateral, nonstimulated part of the preparations. Contractions characterized the local response of longitudinal muscle. The circular muscle responded with relaxation followed by contraction. Synchronous ascending contractions and descending contraction of the longitudinal muscle and relaxation followed by contraction of the circular muscle were observed when the middle part of segments was stimulated, thus indicating that locally-induced nerve excitation propagated via intrinsic ascending or descending nerve pathways that could be synchronously coactivated by one and the same stimulus. The ascending motor responses were more pronounced and the motor responses of longitudinal muscle were expressed more than those of circular muscle suggesting an essential role of ascending reflex pathways and longitudinal muscle in the coordinated motor activity of colon.     

Projections and actions of tachykininergic, cholinergic, and serotonergic neurones in the intestine of the Atlantic cod

The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine. Received: 6 March 1997 / Accepted: 15 September 1997     

The longitudinal smooth muscle layer of the pig small intestine is innervated by both myenteric and submucous neurons

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

The organization of exteroceptive information from the uropod to ascending interneurones of the crayfish

The organization of exteroceptive inputs to identified ascending interneurones of the crayfish, Procambarus clarkii (Girard), has been analyzed by stimulation of hairs on the uropod and simultaneous intracellular recordings from ascending interneurones. The spikes of single afferent neurones which innervated hairs on the distal ventral surface of the exopodite were consistently followed by a depolarizing synaptic potential in many identified ascending interneurones with a constant and short central delay of 0.7–1.5 ms. The amplitude of the potentials depended on the membrane potential of the ascending interneurones. Each afferent neurone made divergent outputs onto several ascending interneurones and each ascending interneurone received convergent inputs from several afferent neurones. Certain ascending interneurones made inhibitory or excitatory connections with other ascending interneurones. These central interactions were always one-way, and the spikes from one ascending interneurone consistently evoked excitatory or inhibitory post-synaptic potentials in other interneurones which followed with a constant and short latency of 0.7–1.0 ms. The inhibitory postsynaptic potential was reversed by injection of steady hyperpolarizing current.Abbreviations EPSP excitatory post-synaptic potential - IPSP inhibitory post-synaptic potential     

Vasoactive intestinal polypeptide and nitric oxide synthase distribution in the enteric plexuses of the human colon: an histochemical study and quantitative analysis

Vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) positive innervation patterns were immunohistochemically and statistically evaluated in the human colon. Specimens from the right colon (cecum, ascending and right transverse colon) and left colon (left transverse and descending colon) were obtained surgically, fixed either in paraformaldehyde or in Carnoy's or in Bouin's, and paraffin embedded. Sections were stained with hematoxylin-eosin, toluidine blue, cresyl violet, neuron-specific enolase, anti-VIP, and anti-NOS. The same results were obtained regardless of the fixative used. Enolase-positive, VIP-positive, and NOS-positive cells were occasionally found within the circular muscle and interpreted as neurons. VIP-positive nerve fibers were evenly distributed within the circular muscle while NOS-positive ones were lacking in its inner portion. The left colon was richer in neurons than the right colon, at both plexuses. VIP- and NOS-positive neuron densities were higher at the left than at the right colon, whereas at all colonic levels VIP-positive neuron percentages at both plexuses and NOS-positive ones at the myenteric plexus were simular. At the submucous plexus the NOS-positive neuron percentage was lower than that of the VIP-positive one. In conclusion: (a) the right colon contains a lower number of neurons and of VIP- and NOS-positive ones than the left colon, and (b) VIP- and NOS-positive fibers are differently distributed in the inner and outer portions of the circular muscle.     

The enteric nervous system: region and target specific projections and neurochemical codes.

The goal of this report is to summarise the current knowledge on the projection pathways of enteric neurones innervating the muscle and mucosa in different regions of the gut. Combination of neuronal tracing, immunohistochemical and electrophysiological methods has allowed researchers to gain insight into the enteric hardwiring of specific target tissue in the gut. A polarised innervation pattern of the circular muscle was demonstrated for the stomach fundus/corpus and the ileum with descending pathways being primarily nitrergic while ascending pathways were primarily cholinergic. This characteristic hardwiring is thought to set in part the functional basis for peristalsis. A similar polarised innervation pathway was found for the enteric innervation of the mucosa in the stomach and large intestine but not in the small intestine. In both the stomach (myenteric neurones) and in the proximal and distal colon (submucosal neurones), ascending pathways to the mucosa are primarily cholinergic while descending pathways are primarily non-cholinergic. In the colon, results suggest that activation of both pathways induces a cross potentiation of cholinergic and vasoactive intestinal polypeptidergic mediated secretion. Furthermore, a large population of myenteric neurone s projecting to the mucosa in the small and large intestine are probably intrinsic primary afferent neurones sensitive to mechanical as well as chemical stimuli.     

Motility regulating effects of galanin on smooth muscle of porcine ileum.

We studied the effect of synthetic porcine galanin on circular and longitudinal oriented strips of pig ileal muscle. Galanin 10(-11)-10(-6) M had no effect on resting tension in the two layers. In circular muscle precontracted with carbachol 10(-6) M, galanin dose-dependently inhibited the amplitude of contractions to a maximum of 33 +/- 8% at 10(-6) M. In longitudinal muscle the amplitude of contractions induced by carbachol 10(-7) M or transmural field stimulation increased after addition of galanin 10(-9)-10(-7) M to a maximum of 21 +/- 6%, while at higher concentrations inhibition occurred. Maximal inhibition was 36 +/- 14% at galanin 10(-6) M. Tetrodotoxin did not influence the effects of galanin in the preparations. The results indicate that in the homologous species galanin inhibits the circular muscle layer, possibly by a direct action on the smooth muscle. In the longitudinal muscle the effect of galanin is apparently excitatory. The inhibition observed with high concentration of galanin could be due to tachyphylaxis and desensitization. Alternatively, an additional population of low affinity, inhibitory receptors may exist.     

Firing of 5-HT neurones in the dorsal and median raphe nucleus in vitro shows differential alpha1-adrenoceptor and 5-HT1A receptor modulation

The median raphe nucleus and dorsal raphe nucleus together are the major source of ascending 5-HT projections. Here, using in vitro extracellular single unit electrophysiology we examined the responses of individual neurones in the rat median raphe nucleus and dorsal raphe nucleus to alpha(1)-adrenoceptor and 5-HT(1A) receptor activation and made comparisons between the two nuclei. In the presence of the alpha(1)-adrenoceptor agonist phenylephrine (1microM) all spontaneously active neurones recorded in the median and dorsal raphe nuclei fired slowly (<5Hz) and regularly. Most were inhibited by 5-HT (10-50microM), although a few were excited by 5-HT. 5-HT-induced inhibition was attenuated by the 5-HT(1A) receptor antagonist, WAY100635 (100nM). Compared to those in the dorsal raphe nucleus, the neurones in the median raphe nucleus which were inhibited by 5-HT had: (1) lower basal firing rates in the continuous presence of phenylephrine (1microM), (2) smaller excitatory responses to higher concentrations of phenylephrine (3-10microM), (3) smaller excitatory responses to brief application of norepinephrine (10-100microM) and (4) smaller inhibitory responses to 5-HT (10-50microM). The lower sensitivity of median raphe neurones to alpha(1)-adrenoceptor excitation and 5-HT(1A) receptor inhibition will have consequences for 5-HT neurotransmission in forebrain regions innervated by the two nuclei.     

Reflex modulation of motoneurone activity in the cheliped of the crayfish Astacus leptodactylus.

1. The reflex activity elicited by movement of the mero-carpopodite (M-C) joint in the cheliped of the crayfish Astacus leptodactylus is investigated and the role of the different proprioceptors (chordotonal and myochordotonal organs) separately studied. 2. The reflex discharge involves mainly the tonic motoneurones of the extensor (E), the flexor (F) and the accessory flexor (AF) muscles. 3. M-C joint posture is also regulated by the cuticular stress detector (CSD2) afferents: they increase mainly the F discharge and secondarily the AF command. 4. The activity of the motor axons supplying the muscles of the meropodite can be also influenced by a variety of natural stimuli applied to other appendages. The effect usually produced is a general flexion reaction which is characterized by a reciprocity between E and F involving both central and peripheral mechanisms. 5. The AF muscle is innervated by two antagonistic motoneurones, an excitatory neurone functionally linked in its discharge with one of the four excitors supplying F and an inhibitory motoneurone, common with E. The resulting competitive effect between these two neurones has been recorded intracellularly in AF muscle fibres. 6. The role of the myochordotonal organ (MCO) in the crayfish is discussed. In particular the modulation of the AF command in relation to the discharges of the motor nerves to the main muscle E and F is studied.     

Spinal and supraspinal contributions to central sensitization in peripheral neuropathy

We will focus on spinal cord dorsal horn lamina I projection neurones, their supraspinal targets and involvement in pain processing. These spinal cord neurons respond to tonic peripheral inputs by wind-up and other intrinsic mechanisms that cause central hyper-excitability, which in turn can further enhance afferent inputs. We describe here another hierarchy of excitation - as inputs arrive in lamina I, neurones rapidly inform the parabrachial area (PBA) and periaqueductal grey (PAG), areas associated with the affective and autonomic responses to pain. In addition, PBA can connect to areas of the brainstem that send descending projections down to the spinal cord - establishing a loop. The serotonin receptor, 5HT3, in the spinal cord mediates excitatory descending inputs from the brainstem. These descending excitatory inputs are needed for the full coding of polymodal peripheral inputs from spinal neurons and are enhanced after nerve injury. Furthermore, activity in this serotonergic system can determine the actions of gabapentin (GBP) that is widely used in the treatment of neuropathic pain. Thus, a hierarchy of separate, but interacting excitatory systems exist at peripheral, spinal and supraspinal sites that all converge on spinal neurones. The reciprocal relations between pain, fear, anxiety and autonomic responses are likely to be subserved by these spinal-brainstem-spinal pathways we describe here. Understanding these pain pathways is a first step toward elucidating the complex links between pain and emotions.     

Mucosal acid challenge activates nitrergic neurons in myenteric plexus of rat stomach

We tested the hypothesis that intrinsic neurons of the rat gastric myenteric plexus can be activated by an acid (HCl) challenge of the mucosa. Activated neurons were visualized by immunohistochemical detection of c-Fos, a marker for neuronal excitation. The neurochemical identity of the neurons activated by the HCl challenge was determined by colocalizing c-Fos with a marker for excitatory pathways, choline acetyltransferase (ChAT), and a marker for inhibitory pathways, nitric oxide synthase (NOS). Two hours after intragastric administration of HCl or saline, stomachs were removed and immunofluorescence triple labeling of myenteric neurons was carried out on whole mount preparations. Treatment with 0.35, 0.5, and 0.7 M HCl induced c-Fos in 8%, 56%, and 64%, respectively, of NOS-positive but not ChAT-positive neurons. c-Fos was also seen in glial cells of HCl-treated rats, whereas in saline-treated animals c-Fos was absent from the myenteric plexus. HCl treatment did not change the proportion of ChAT- and NOS-immunoreactive neurons in the myenteric ganglia. It is concluded that gastric acid challenge concentration-dependently stimulates a subpopulation of nitrergic, but not cholinergic, myenteric plexus neurons, which may play a role in muscle relaxation, vasodilatation, and/or secretion.     

Morphological and neurochemical identification of enteric neurones with mucosal projections in the human small intestine

Data on the axonal projections of enteric neurones in the human intestine are still scarce. The present study aimed to identify the morphology and neurochemical coding of enteric neurones in the human small intestine, which are involved in the innervation of the mucosa. The lipophilic neuronal tracer DiI was applied to one mucosal villus of small intestinal resection specimens. The tissue was kept in organotypic culture and subsequently processed for immunohistochemistry. Neurones labelled from the mucosa were located in all ganglionated nerve networks, including the myenteric plexus. In all plexuses, at least five neurochemical types of neurones could be observed, i.e. SOM-IR neurones, SP-IR neurones, SOM/SP-IR neurones, VIP-IR neurones and neurones lacking immunoreactivity for any of these markers. Most of the DiI-labelled neurones were multidendritic; a minority of neurones could be identified as Dogiel type II cells, suggesting the existence of a subgroup of primary afferent neurones in the DiI-filled cell population. The ratio of labelled multidendritic neurones (assumed to be secretomotor) to labelled Dogiel type II neurones (assumed to be primary afferent) in the myenteric plexus is higher in large mammals (pig and human) than in small mammals (guinea pig). This might point to the existence of a different topographical distribution of subsets of primary afferent neurones and/or topographically distinct intrinsic mucosal reflex circuits in large mammals, including humans.