Inferior Mesenteric Ganglion Neurons Research Articles

Axotomy-Induced Changes in the Chemical Coding Pattern of Colon Projecting Calbindin-Positive Neurons in the Inferior Mesenteric Ganglia of the Pig

The present study examines the response of colon-projecting neurons localized in the inferior mesenteric ganglia (IMG) to axotomy in the pig animal model. In all animals (n = 8), a median laparotomy was performed under anesthesia and the retrograde tracer Fast Blue was injected into the descending colon wall. In experimental animals (n = 4), the descending colon was exposed and the bilateral caudal colonic nerves were identified and severed. All animals were euthanized and the inferior mesenteric ganglia were harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P, vesicular acetylcholine transporter, or galanin. Immunohistochemistry revealed significant changes in the chemical coding pattern of injured inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, NOS, LENK, and CB. In the experimental group, the numbers of TH-, NPY-, and SOM-expressing neurons were reduced, whereas the number of neurons immunoreactive to LENK was increased. Our data indicate that the colon-projecting neurons of the porcine IMG react to the axotomy in a similar, but not an identical manner in a comparison to other species, especially rodents. Further studies are needed to elucidate the detailed factors/mechanisms involved in the response to nerve injury.

Enhanced excitability of guinea pig inferior mesenteric ganglion neurons during and following recovery from chemical colitis

Postganglionic sympathetic neurons in the prevertebral ganglia (PVG) provide ongoing inhibitory tone to the gastrointestinal tract and receive innervation from mechanosensory intestinofugal afferent neurons primarily located in the colon and rectum. This study tests the hypothesis that colitis alters the excitability of PVG neurons. Intracellular recording techniques were used to evaluate changes in the electrical properties of inferior mesenteric ganglion (IMG) neurons in the trinitrobenzene sulfonic acid (TNBS) and acetic acid models of guinea pig colitis. Visceromotor IMG neurons were hyperexcitable 12 and 24 h, but not 6 h, post-TNBS during "acute" inflammation. Hyperexcitability persisted at 6 days post-TNBS during "chronic" inflammation, as well as at 56 days post-TNBS when colitis had resolved. In contrast, there was only a modest decrease in the current required to elicit an action potential at 24 h after acetic acid administration. Vasomotor neurons from inflamed preparations exhibited normal excitability. The excitatory effects of XE-991, a blocker of the channel that contributes to the M-type potassium current, and heteropodatoxin-2, a blocker of the channel that contributes to the A-type potassium current, were unchanged in TNBS-inflamed preparations, suggesting that these currents did not contribute to hyperexcitability. Riluzole, an inhibitor of persistent sodium currents, caused tonic visceromotor neurons to accommodate to sustained current pulses, regardless of the inflammatory state of the preparation, and restored a normal rheobase in neurons from TNBS-inflamed preparations but did not alter the rheobase of control preparations, suggesting that enhanced activity of voltage-gated sodium channels may contribute to colitis-induced hyperexcitability. Collectively, these data indicate that enhanced sympathetic drive as a result of hyperexcitable visceromotor neurons may contribute to small bowel dysfunction during colitis.

Su2000 Reduced Small Bowel Transit is Associated With Enhanced Excitability of Sympathetic Prevertebral Ganglion Neurons but Not Ileum Myenteric Neurons During Colitis in Guinea Pigs

Background: Localized colitis causes a reduction in small bowel motor function in humans and non-human animals yet the mechanisms that contribute to this phenomenon is not completely understood. Aim: To determine the time course of changes in 1) small bowel motor function, 2) excitability of ileum myenteric plexus neurons and 3) excitability of inferior mesenteric ganglion (IMG) neurons following an inflammatory insult to the distal colon. Methods: Colitis was induced by a single enema of trinitrobenzene sulfonic acid (TNBS) dissolved in 30% EtOH. Small bowel transit was assessed by determining the location of the leading edge of charcoal 30min following gavage and reported as a percentage of the total length of the small intestine. Neuronal excitability was assessed by conventional intracellular electrophysiology. Results: Small bowel transit was reduced in guinea pigs 12h, and 24h post-TNBS, but returned to normal at 48h and 6d post-TNBS (control, 84±3;12h, 50±9; 24h, 44±3; 48h, 68±8; 6d, 75±2;P<0.05). AH neurons of the guinea pig ileum were hyperexcitable at 24h 48h and 6d post-TNBS, but were not different than controls at 12h post-TNBS. Evidences of excitability were a more depolarized membrane potential (mV: control, -76±3;12h, -73±2; 24h, -59±2; 48h, -63±2; 6d, -68±3;P<0.05), and increased number of action potentials (control, 1.2±0.2;12h, 1.1±0.7; 24h, 3.5±1.2; 48h, 4.5±1.3; 6d, 7.8±3.0;P<0.05). There was no change in the magnitude of the afterhyperpolarization or the threshold current. Tonic neurons of the IMG were hyperexcitable at 12h, 24h and 6d post-TNBS, the three time points tested. Evidences of excitability were a decreased threshold current (pA: control, 340±26;12h, 258±23; 24h, 192±17; 6d, 164±18;P<0.05), and increased number of action potentials (control, 3.1±0.1;12h, 5.7±0.7; 24h, 6.2±0.4; 6d, 5.1±0.3;P<0.05). There was no change in resting membrane potential. Conclusion: Reduced small bowel transit 12h following TNBS suggests rapid functional changes in response to the inflammatory insult. There is no evidence for excitability of myenteric AH neurons at this early time point so these cells may not directly contribute to the functional change. Conversely, PVG neurons are excitable at this early time point. This association supports the concept that PVG neurons contribute to small bowel motor dysfunction during colitis. The mechanisms contributing the later excitability of myenteric AH neurons and restoration of normal transit despite ongoing neuronal hyperexcitability remain unresolved. Supported by NIH Grant DK76665.

Proliferative enteropathy (PE)-induced changes in the calbindin-immunoreactive (CB-IR) neurons of inferior mesenteric ganglion supplying the descending colon in the pig.

A subpopulation of the pig inferior mesenteric ganglia (IMG) neurons projecting to the colon exhibit calbindin-like immunoreactivity. It is not known if there are any changes in the chemical coding patterns of these neurons during porcine proliferative enteropathy (PE). To answer this question, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PE; n = 3) and a group of uninfected controls (C; n = 3) were compared. The retrograde tracer fast blue (FB) was injected into the descending colons of all animals and then tissue comprising IMGs from both groups was processed for double-labeling immunofluorescence with calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase, Leu-enkephalin, substance P, vesicular acetylcholine transporter, galanin, or pituitary adenylate cyclase-activating polypeptide. Immunohistochemistry revealed changes in the chemical coding pattern of calbindin-immunoreactive neurons in the inferior mesenteric ganglia of the pig. In control animals, FB/CB-positive neurons were immunoreactive to TH, NPY, SOM, and VIP. In the experimental group, TH-expressing neurons were unaffected, NPY-expressing neurons were increased, whereas the number of neurons immunoreactive to SOM or VIP was reduced. Changes in chemical coding of CB neurons during PE may play an important role in adaptation of these IMG cells under pathological conditions.

Inflammation-Induced Changes in the Chemical Coding Pattern of Colon-Projecting Neurons in the Inferior Mesenteric Ganglia of the Pig

The present study examines the chemical coding of the inferior mesenteric ganglia after chemically induced colitis in the pig animal model. In all animals (n = 6), a median laparotomy was performed under anesthesia, and the Fast Blue retrograde tracer was injected into the descending colon wall. In experimental animals (n = 3), the thick descending colon were injected with formalin solution to induce inflammation. The animals were euthanized and the inferior mesenteric ganglion was harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P (SP), vesicular acetylcholine transporter (VAChT), or galanin (GAL). Immunohistochemistry revealed significant changes in the chemical coding pattern of inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, LENK, CB, and NOS. In the experimental group, TH, NPY, SOM, VIP, and LENK expressing neurons were reduced, whereas the number of neurons immunoreactive to CB, NOS, and GAL were increased. The increase of so-called neuroprotective neuropeptides suggests that the changes in the chemical coding of inferior mesenteric ganglion neurons reflect adaption under pathological conditions to promote their own survival.

Synaptic transmission in the inferior mesenteric ganglion of hypertensive and normotensive rats

The inferior mesenteric ganglion (IMG) is a prevertebral sympathetic ganglion that innervates the lower abdominal and pelvic viscera. While extensive studies have been performed on the IMG of several species, there is very little information available concerning the rat IMG. Because of the importance of the mesenteric circulation in the regulation of systemic blood pressure and in the genesis of salt‐sensitive hypertension, we compared the properties and response profiles to pre‐ganglionic input of IMG neurons in normotensive (NT) and hypertensive (HT) animals using intracellular recording in vitro. All rats were uninephrectomized 4 weeks prior to experiments. HT rats received 200mg/kg of deoxycorticosterone s.q. and 1%NaCl/0.2%KCl in drinking water. NT animals (mean arterial pressure &lt; 130mmHg) received tap water. Resting membrane potential, rheobasic current, action potential amplitude, input resistance, and membrane time constant show no significant differences between NT and HT animals. In contrast to the findings in the IMG of other species, nearly all neurons (94% in HT, 91% in NT rats) showed a phasic response to direct current injection. This is the first study of the IMG of the hypertensive rat. While there is a predominance of phasic neurons in this ganglion, changes in sympathetic outflow that occur with hypertension are not due to alterations in the basic properties of the ganglionic neurons. Supported by NIH grant HL70687

S8.4 Inferior mesenteric ganglion neurons of the guinea pig are hyperexcitable and exhibit non-cholinergic fast synaptic transmission during and following recovery from hapten-induced colitis

S8.4 Inferior mesenteric ganglion neurons of the guinea pig are hyperexcitable and exhibit non-cholinergic fast synaptic transmission during and following recovery from hapten-induced colitis

Identification of Rat Inferior Mesenteric Ganglion Neurons Differentially Innervating Splanchnic Veins and Arteries

The splanchnic circulation contains approximately 70% of the total body blood volume and the majority of this resides in the splanchnic veins. Splanchnic vascular resistance and capacitance can be differentially regulated by the sympathetic nervous system and in some species this is associated with separate arterial and venous innervation. We examined the innervation of mesenteric arteries and veins by neurons in the inferior mesenteric ganglion (IMG). An ex vivo preparation of the IMG attached to mesenteric vessels was placed in organotypic culture. After removing the endothelium fluorescent beads of different colors were injected separately into the arteries and veins of the mesentery. The preparation was cultured for 7 days. After 5 days a solution of fast blue was instilled around the ganglion to mark sympathetic ganglion neuron cell bodies. In 3 ganglia 90% of all neurons were filled when a single tracer was placed in both vessels. Of the neurons that innervate blood vessels 95% of them innervated either artery or vein and 5% innervated both artery and vein. These studies suggest that a majority of neurons in the IMG regulate mesenteric vascular tone and that there are separate neuronal pathways innervating artery or vein. Support:HL70687.

The β-subunit composition of nicotinic acetylcholine receptors in the neurons of the guinea pig inferior mesenteric ganglion

The antibodies against synthetic (183–192) fragments of β2- and β4-subunits of rat neuronal nicotinic acetylcholine receptor were used to study a β-subunit composition of nicotinic receptors in the inferior mesenteric ganglion of the guinea pig by both immunocytochemical staining and blocking of excitatory postsynaptic potentials induced by electric stimulation of the pre-ganglionic nerve (intermesenteric trunk). The β4-specific antibody stained 59.8±7.5% of neurons and inhibited the synaptic responses in all ( n=10) neurons studied by 25.5±1.8%. The β2-specific antibody did not stain ganglionic neurons and did not affect the synaptic transmission. Taking into account the previously obtained data on the α-subunits found in this ganglion, it is concluded that the neurons of inferior mesenteric ganglion contain nicotinic receptors of α3(α5)β4 subtypes involved in synaptic transmission through the intermesenteric tract.

β2- and β4-Subunit Composition of Nicotinic Acetylcholine Receptors in the Guinea Pig Inferior Mesenteric Ganglion: Immunohistochemical and Electrophysiological Studies

1 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to L. P. Voitenko (e-mail: lvoit@serv.biph. kiev.ua). Affinity-purified rabbit antibodies (Ab) against extracellular fragments 193-204 of nicotinic acetylcholine receptor (nAChR) β2 and β4 subunits were used in the experiments with immunoperoxidase labeling of guinea pig inferior mesenteric ganglion (IMG) neurons and intracellular recording of excitatory postsynaptic potentials (EPSP) from these units. Since the epitopes of these Ab are close to an acetylcholine-binding site located at the interface of α and β subunits, we expected that the Ab can block acetylcholine-mediated EPSP responses of IMG neurons, thus enabling us to determine the involvement of β2and β4-containing nAChR in synaptic transmission through intermesenteric pathways. Microscopic examination revealed the presence of two cell types in IMG preparations: principal ganglionic neurons (PGN), 48-60 μm in diameter, and small cells of unknown origin, 6-18 μm in diameter. The β4-specifi c Ab labeled 59.8 ± 7.5% of PGN, while the β2-specifi c Ab labeled less than 1% of these cells, the β4-positive label being much more intensive than the β2-positive one. The β4-specifi c labeling was similar to that revealed with the α3-specifi c Ab. It should be noted that the percentage of α5-immunoreactive IMG neurons differed insignifi cantly from that of α3-labeled PGN. The somata of small cells were labeled with the β4-specifi c Ab too, and a group of β2-positive small cells was also found. Both β4and β2-positive neurons were localized near the origins of the main nerve trunks branching from the IMG (mesenteric, colonic, and hypogastric nerves). In electrophysiological experiments, the β4-specific Ab decreased the EPSP amplitudes by 25.5 ± 1.8% in all 10 PGN tested. In contrast, the β2-specific Ab did not suppress the synaptic responses under study. Our findings showed that α3-, α5-, and β4-containing nAChR are present in the guinea pig IMG neurons and are involved in synaptic transmission through the intermesenteric pathways. Probably, the α3β4 nAChR subtype is more widespread that is consistent with the generally recognized nature of ganglionic nAChR.

α-Subunit Composition of Nicotinic Cholinoreceptors of Neurons of the Inferior Mesenteric Ganglion of the Guinea Pig: an Electrophysiological Study

We studied the subunit composition of nicotinic cholinoreceptors (nChR) of neurons of the inferior mesenteric ganglion (IMG) of the guinea pig using antibodies against the α3, α4, α5, and α7 nChR subunits and a standard technique of intracellular recording. Application of the α3 subunit-specific antibodies resulted in a decrease in the EPSP amplitude by 29.7 ± 1.8%, on average, in 17 of 20 studied neurons. The α4 subunit-specific antibodies evoked no changes in the amplitude of EPSP recorded from IMG neurons (n = 10). Effects of the α5 subunit-specific antibodies were studied in 20 neurons, where the EPSP amplitude dropped after application of these antibodies by 40.0 ± 1.8%, on average. Superfusion of the neurons with a solution containing the α3 and α5 subunit-specific antibodies completely suppressed synaptic responses (n = 3). The α7 subunit-specific antibodies provided an increase in the EPSP amplitude in 13 of 15 studied IMG neurons (by 37 ± 6%, on average); this fact allows us to suppose that α7-containing nChR are present in the IMG neurons and are indirectly involved in the processes of synaptic transmission. Application of the antibodies evoked no significant shifts in the membrane potential in IMG neurons. Our findings demonstrate that synaptic transmission through the guinea pig IMG is realized mostly with the involvement of the α3- and α5-containing nChR; the α4-containing receptors are not engaged in this process.

The three-dimensional structure of neurons in the guinea pig inferior mesenteric and pelvic hypogastric ganglia

The three-dimensional (3-D) morphology of sympathetic inferior mesenteric ganglion (IMG) neurons and sympathetic–parasympathetic pelvic hypogastric ganglion (PHG) neurons was studied using confocal laser scanning microscopy. Cell bodies of IMG neurons were disc-shaped and were arranged orderly in layers. The dendritic arbor of individual neurons was confined to a plane with a thickness that did not exceed the thickness of the parent cell body. The actual dendritic surface area (71,400 μm 2) and volume (81,500 μm 3) of the IMG neurons were up to 100-fold larger than previously reported for similar sympathetic neurons using data of 2-D measurements and estimations of the third dimension. PHG neurons had a much smaller dendritic surface area (4100 μm 2) and volume (2400 μm 3) compared to IMG neurons. The ratio dendritic/somal surface area for individual IMG and PHG neurons ranged from 5:1 to 14:1 and from 0.1:1 to 6:1, respectively. The total dendritic path-length was 8–42 times greater for IMG than for PHG neurons. Neurons in the IMG were either stellate with radiating dendrites or bipolar-shaped with dendrites emerging from the two poles of the cell body. Neurons in the PHG were of two morphological types. One type (nearly 2/3 of all the imaged PHG neurons) had two to seven relatively long dendrites and an axon; the other type had only one to three short unbranched dendrites and an axon. The spatial organization of neurons within the ganglia and the structural features of individual neurons are likely to have important implications regarding connectivity patterns between neurons within the ganglion as well as on how information is processed by the ganglion.

Serotonin receptor subtypes that depolarize guinea pig inferior mesenteric ganglion neurons

Our previous studies indicated that serotonin (5-HT) depolarized a majority of guinea pig inferior mesenteric ganglion (IMG) neurons and may be another transmitter for the noncholinergic late slow excitatory postsynaptic potential (ls-EPSP) in the IMG. However, the subtypes of 5-HT receptor mediating these responses have not yet been identified. Using intracellular recording, we examined the effect of 5-HT receptor antagonists with specificity to various 5-HT receptor subtypes on the 5-HT-mediated depolarization and ls-EPSP in IMG neurons in vitro. Cyproheptadine, a 5-HT 1/2 receptor antagonist, reversibly inhibited the slow, but not the fast, depolarization and ls-EPSP in the 5-HT-sensitive neurons. Both mianserin and spiperone, 5-HT 2 and 5-HT 1A receptor antagonists, did not significantly alter either the fast or slow depolarizing responses or the ls-EPSP. The 5-HT 3 receptor antagonist MDL 72222 (Bemesetron) completely inhibited the fast depolarization with little diminution of the slow depolarization and ls-EPSP. Superfusion of putative 5-HT 1P receptor antagonist, BRL 24924 (Renzapride), reversibly attenuated both the depolarization and ls-EPSP. However, 5-HT-insensitive neurons with ls-EPSP were found to be insensitive to both cyproheptadine and BRL 24924. In most 5-HT-sensitive neurons, the 5-HT 3 receptor agonist, 2-methyl-5-HT, and the selective 5-HT 1P agonist, MCPP or 5-OHIP, evoked a fast and a slow depolarization in 55.6 and 71.4% of the neurons, respectively, without a significant effect on the membrane potential in 85.7 and 100% of the 5-HT-insensitive neurons. In 5-HT-sensitive neurons, MDL 72222 reversibly abolished the fast depolarization induced by 2-methyl-5-HT; BRL 24924 significantly inhibited the slow depolarization induced by MCPP or 5-OHIP, but not by SP. Prolonged superfusion of 5-HT-sensitive neurons with MCPP abolished the evoked ls-EPSP without inhibition of action potential. These results suggest that the fast and slow depolarizations in these neurons are mediated by 5-HT 3 and 5-HT 1P receptor subtypes, respectively. The latter may also mediate the ls-EPSP in 5-HT-sensitive neurons.

Immediate-Early Gene Expression in the Inferior Mesenteric Ganglion and Colonic Myenteric Plexus of the Guinea Pig

Activation of neurons in the inferior mesenteric ganglion (IMG) was assessed using c-fos, JunB, and c-Jun expression in the guinea pig IMG and colonic myenteric plexus during mechanosensory stimulation and acute colitis in normal and capsaicin-treated animals. Intracolonic saline or 2% acetic acid was administered, and mechanosensory stimulation was performed by passage of a small (0.5 cm) balloon either 4 or 24 hr later. Lower doses of capsaicin or vehicle were used to activate primary afferent fibers during balloon passage. c-Jun did not respond to any of the stimuli in the study. c-fos and JunB were absent from the IMG and myenteric plexus of untreated and saline-treated animals. Acetic acid induced acute colitis by 4 hr, which persisted for 24 hr, but c-fos was found only in enteric glia in the myenteric plexus and was absent from the IMG. Balloon passage induced c-fos and JunB in only a small subset of IMG neurons and no myenteric neurons. However, balloon passage induced c-fos and JunB in IMG neurons (notably those containing somatostatin) and the myenteric plexus of acetic acid-treated animals. After capsaicin treatment, c-fos and JunB induction by balloon passage was inhibited in the IMG, but there was enhanced c-fos expression in the myenteric plexus. c-fos and JunB induction by balloon stimulation was also mimicked by acute activation of capsaicin-sensitive nerves. These data suggest that colitis enhances reflex activity of the IMG by a mechanism that involves activation of both primary afferent fibers and the myenteric plexus.

Two populations of sympathetic neurons project selectively to mesenteric artery or vein.

The objective of this study was to determine whether sympathetic neurons of the inferior mesenteric ganglion (IMG) projecting to mesenteric arteries could be distinguished by their localization, neurochemical phenotype, and electrophysiological properties from neurons projecting to mesenteric veins. In an in vitro intact vasculature-IMG preparation, neurons were labeled following intraluminal injection of Fluoro-Gold or rhodamine beads into the inferior mesenteric artery (IMA) or vein (IMV). The somata of neurons projecting to IMA were localized in the central part of the IMG, whereas those projecting to IMV were localized more peripherally. None of the labeled neurons was doubly labeled. Neuropeptide Y immunoreactivity was found in 18.9% of neurons innervating the IMA, but not in neurons innervating the IMV. Identified neurons were dissociated and characterized using whole cell patch-clamp recording. After direct soma depolarization, all of the labeled arterial and venous neurons were classified as tonic firing, compared with only 40% of unlabeled neurons; the remaining 60% of unlabeled neurons were phasic firing. The results indicate that IMG neurons projecting to mesenteric arteries are distinct from neurons projecting to mesenteric veins.

Protein kinase-mediated reciprocal modulatory changes in anesthetic sensitivity of (BK)-K +- and GABA-A receptor-gated conductances in guinea-pig sympathetic neurons

(1) The interaction of substance P (SP)-mediated synaptic transmission with general anesthetics remains unknown. (2) Intracellular recordings were obtained from guinea-pig inferior mesenteric ganglion neurons to study monosynaptic responses to exogenous SP and GABA. (3) Propofol (1–100 μM) caused an increase in SP-evoked inward current responses and a concurrent decrease in peak amplitude of the afterspike hyperpolarization of intermittently evoked action potentials. These effects were occluded by the (BK)-K +-channel-selective blocker charybdotoxin (10 nM), and prevented by the protein kinase inhibitor staurosporine (100 nM). (4) Propofol also increased GABA-evoked current ( I GABA) responses. (5) When elicited during a SP response, I GABA was significantly diminished compared to control. In the presence of staurosporine (100 nM), the inhibitory effect of SP upon I GABA was abolished, and the propofol-induced augmentation of I GABA was significantly increased. (6) Thus, SP-evoked protein kinase activity produced reciprocal changes in anesthetic sensitivity of (BK)-K +- and GABA A-receptor-gated currents of these sympathetic neurons.

Neuronal circuitry between the inferior mesenteric ganglion and lower intestine of the dog.

Neuronal circuitry between the inferior mesenteric ganglion (IMG) and the distal colon as well as the rectum, forming the intestino-intestinal reflex pathway, was investigated in the dog using immunohistochemistry combined with retrograde tract tracing and denervation experiments. Virtually all IMG neurons were tyrosine hydroxylase (TH)-immunoreactive. Of these ganglionic neurons, about 64% were also immunoreactive for calbindin (Calb), some 35% for neuropeptide Y (NPY), and 2% for vasoactive intestinal peptide (VIP). The retrograde tracer experiments revealed that both Calb/TH neurons and NPY/TH neurons projected to the distal colon and the rectum. In these intestinal walls, Calb/TH positive varicose fibers were found in the myenteric and submucous ganglia as well as in the longitudinal muscle layer, while NPY/TH positive fibers were mainly distributed around the vascular walls. Around Calb/TH neurons of the IMG, abundant varicose nerve fibers immunoreactive for VIP, dynorphin (DYN), calcitonin gene-related peptide (CGRP), enkephalin (ENK), substance P (SP) and bombesin (BOM) were distributed. These immunoreactive fibers disappeared after the total denervation of the IMG. After the application of Fast Blue into the IMG or distal stumps of transected lumbar colonic and hypogastric nerves, retrogradely labeled neurons occurred in the myenteric plexus with increasing density along the distal colon and rectum, and were immunoreactive for VIP, DYN, CGRP, ENK, SP or BOM. Double immunostaining of nerve fibers in the distal stumps of the ligated colonic and hypogastric nerves revealed the presence of viscerofugal fibers containing VIP with DYN and/or CGRP and those containing ENK with SP and/or BOM. These results demonstrate for the first time that the efferent limb of the canine intestino-intestinal reflex arch via the IMG consists of Calb-immunoreactive ganglion neurons projecting to the longitudinal muscles in addition to the enteric plexus of the lower intestine and also of NPY-immunoreactive ganglion neurons projecting to the intestinal blood vessels, and that the afferent limb is composed of at least two discrete groups with different peptide contents, i.e., myenteric neurons containing VIP with DYN and/or CGRP and those containing ENK with SP and/or BOM.

Identification of uterine-related sympathetic neurons in the rat inferior mesenteric ganglion: neurotransmitter content and afferent input

The rat uterus is innervated by sensory and autonomic nerves. Sensory and sympathetic fibers travel in the hypogastric nerves and are associated with the thoracolumbar spinal cord levels T 13-L 3. The inferior mesenteric ganglion (IMG) contains the somata of sympathetic postganglionic neurons and some of these may project axons to the uterus. Sensory and parasympathetic fibers travel in the pelvic nerve and are associated with the lumbosacral cord levels L 6-S 1 and pelvic ganglion (PG). We previously reported data concerning the neurochemical anatomy of the PG with regard to the uterine innervation; the present study was undertaken to characterize the neurochemical anatomy of the IMG with regard to its involvement in uterine innervation. A retrograde axonal tracer was used to verify projections of axons of IMG neurons to the uterus. Immunostaining of cryostat sections of the IMG revealed neurons immunoreactive for neuropeptide Y (NPY) and for tyrosine hydroxylase (TH). Immunostaining for the synaptic terminal protein synapsin I (SYN) revealed numerous fine terminals immediately surrounding the principal neurons and in the interneuronal spaces. Varicosities immunoreactive for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), substance P (SP) and galanin (GAL) appeared to be associated with principal neurons. Additional varicosities stained for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and nitric oxide synthase (NOS), thus indicating sites of neuronal nitric oxide synthesis. This study revealed that the IMG contains uterine-related neurons and that some of the retrogradely labeled uterine-related neurons contain NPY, TH or both NPY/TH. In addition, uterine-related neurons received abundant afferent inputs indicated by SYN-immunoreactive (-ir) terminals and some of these varicosities labeled for GAL, CGRP, VIP, ENK, or NADPH-d/NOS.

A physiologically-evoked M 1-muscarinic depolarization in guinea-pig inferior mesenteric ganglion neurons

The possibility of physiologically-evoked muscarinic excitatory synaptic potentials was examined in the inferior mesenteric ganglion (IMG) with intracellular microelectrodes in vitro. Three types of depolarizing responses were evoked concurrently by colonic distension: (1) fast nicotinic excitatory postsynaptic potentials (EPSPs); (2) an ‘intermediate’ time course depolarization and (3) a long time course potential that persisted throughout the period of distension. After hexamethonium was superfused over the ganglia the long time course potential was observed in 91% of IMG neurons. Intermediate time course depolarizations were observed in 100% of IMG neurons and correlated with each propulsive contraction of the distal colon. The intermediate depolarizations had an average amplitude of 1.8 ± 0.1 mV ( n = 175 individual events; 27 preparations) with an average duration of 11.9 ± 0.8 sec ( n = 28 individual events). The intermediate time course synaptic potentials were accompanied by an increase in input resistance of 15% ( n = 6). Superfusion of atropine (1 μM; n = 6) on the IMG or the M 1-selective antagonist pirenzepine (1 μM; n = 5) abolished the intermediate time course synaptic potentials during distension. Superfusion of the M 2-selective antagonist AF-DX 116 (1 μM; n = 4) had no effect. In all preparations examined, distension-induced intermediate time course depolarizations were blocked by tetrodotoxin (TTX) (3 μM). Pressure ejection of carbachol (1 mM, 60–100 ms pulses) evoked depolarizations of similar amplitude (6.5 ± 0.7 mV; n = 18) and duration to the intermediate depolarizations observed during propulsive contractions. In all preparations examined, carbachol-induced depolarizations were antagonized by atropine (1 μM) and pirenzepine (1 μM) but not by AF-DX 116 (1 μM). These experiments demonstrate the existence of a physiologically-evoked muscarinic excitatory synaptic potential in IMG neurons of the guinea pig which is mediated through the M 1-receptor subtype.

Muscarinic depolarizations evoked in inferior mesenteric ganglion neurons during colonie distension are voltage-sensitive

Muscarinic depolarizations evoked in inferior mesenteric ganglion neurons during colonie distension are voltage-sensitive