Morphological characteristics and immunohistochemical detection of nicotinic acetylcholine receptors on intestinofugal afferent neurones in guinea-pig colon.
ABSTRACT Intestinofugal afferent neurones (IFANs) provide excitatory synaptic input to abdominal prevertebral ganglion neurones. Input is greatly reduced during blockade of nicotinic acetylcholine receptors (nAChRs) in the wall of the colon, suggesting two projection pathways: a direct pathway without synaptic interruption and an indirect pathway interrupted by at least one nicotinic cholinergic synapse. This study aimed to characterize the morphology of IFANs and examine the distribution of nAChRs on them. We identified IFANs in guinea-pig colon by retrograde labelling with fluorescent tracer DiI placed either on the lumbar colonic nerves in vitro or inferior mesenteric ganglion in vivo. Confocal laser scanning microscopy and computerized image-processing software were used for 3D image reconstruction. Approximately 70% of identified IFANs had Dogiel type I-like morphology, the remainder were Dogiel type II-like. In vivo labelled IFANs were injected with Lucifer Yellow and immunostained for nAChRs using monoclonal antibody MAb35. Approximately 3% of total plasma membrane surface of IFANs with Dogiel type I morphology had MAb35-IR. In contrast, <1% of membrane surface of IFANs with Dogiel type II morphology had MAb35-IR. The finding that IFANs displayed immunostaining for nAChRs suggests the presence of putative nicotinic synapses.
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ABSTRACT: The present investigation pertains to changes in substance P-like immunoreactive (SP-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically-induced inflammation and nerve injury (axotomy). The distribution pattern of SP-LI structures was studied using the double immunofluorescence technique in the myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses, as well as in the circular muscle and mucosal layers. Under physiological conditions, SP-LI neurons have been shown to constitute 4.13 ± 0.24%, 3.36 ± 0.26%, and 7.92 ± 0.16% in the MP, OSP, and ISP, respectively. Changes in SP-immunoreactivity depended on the pathological factor studied. The numbers of the SP-LI perikarya amounted to 7.89 ± 0.34, 5.56 ± 0.30, and 19.96 ± 0.57 in chemically-induced colitis, and 4.28 ± 0.13%, 7.18 ± 20%, and 11.62 ± 0.48% after axotomy in MP, OSP, and ISP, respectively. The both studied processes generally resulted in an increase in the number of SP-LI nerve fibers in the circular muscle and mucosal layers. The obtained results suggest that SP-LI nerve structures of the ENS may participate in various pathological processes in the porcine descending colon and exact functions of SP probably depend on the type of the pathological factor. © 2013 BioFactors 39(5):542-551, 2013.BioFactors 09/2013; 39(5):542-51. · 3.09 Impact Factor
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ABSTRACT: Intestinofugal neurons sense and receive information regarding mechanical distension of the bowel and transmit this information to postganglionic sympathetic neurons in the prevertebral ganglia. Previous studies have demonstrated that trinitrobenzene sulfonic acid (TNBS)-induced colitis is associated with a loss of myenteric neurons that occurs within the first twelve hours following the inflammatory insult. The purpose of this study was to test the hypothesis that intestinofugal neurons are among the myenteric neurons lost during TNBS-induced colitis. The retrograde tracing dye Fast Blue was used to label intestinofugal neurons and immunohistochemical staining for the RNA binding proteins HuC/D was used to count all myenteric neurons. Ongoing synaptic input to neurons in the guinea pig inferior mesenteric ganglion (IMG) was recorded via conventional intracellular electrophysiology. In control preparations, intestinofugal neurons account for 0.25% of myenteric neurons. In the distal colon of TNBS-treated animals, the proportion of intestinofugal neurons was reduced to 0.05% (an 80% reduction) within the region of inflammation where 20-25% of myenteric neurons were lost. Neither intestinofugal neurons specifically nor myenteric neurons were reduced in more proximal uninflamed regions. There is a reduction in the frequency of ongoing synaptic potentials in visceromotor neurons of the IMG at 12h, 24h, 6d and 56d after TNBS. Collectively, the results of this study suggest that intestinofugal neurons are among the myenteric neurons lost during inflammation and may be selectively targeted. Because intestinofugal neurons are a major driver of sympathetic output to the gut, the loss of intestinofugal neurons may have a profound pathophysiological significance.AJP Gastrointestinal and Liver Physiology 09/2012; · 3.65 Impact Factor
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ABSTRACT: Enteric viscerofugal neurons are mechanosensory interneurons that form the afferent limb of intestino-intestinal reflexes involving prevertebral sympathetic neurons. Fast synaptic inputs to viscerofugal neurons arise from other enteric neurons, but their sources are unknown. We aimed to describe the origins of synaptic inputs to viscerofugal neurons by mapping the locations of their cell bodies within the myenteric plexus. Viscerofugal neuron somata were retrogradely traced with DiI from colonic nerve trunks and impaled with microelectrodes, in longitudinal muscle/myenteric plexus preparations of guinea pig distal colon (39 impalements, n=14). Thirty eight viscerofugal neurons were uniaxonal and had the electrophysiological characteristics of myenteric S-neurons; one neuron was multipolar with AH-neuron electrophysiological characteristics. Depolarizing current pulses evoked either single or multiple action potentials in viscerofugal neurons (range 1-25 spikes, 500ms, 100-900pA, 21 cells). Electrical stimulation of internodal strands circumferential to viscerofugal neurons evoked fast EPSPs in 19/24 cells. Focal pressure-ejection of the nicotinic agonist DMPP (10μm) directly onto viscerofugal nerve cell bodies evoked large depolarizations and action potentials (23±10mV, latency 350±230ms, 21/22 cells). DMPP was then focally applied to multiple sites, up to 3mm from the recorded viscerofugal neuron, to activate other myenteric S-neurons. In a few sites in myenteric ganglia, DMPP evoked repeatable fast EPSPs in viscerofugal neurons (latency 300±316ms, 38/394 sites, 10 cells). The cellular sources of synaptic inputs to viscerofugal neurons were located both orally and aborally (19 oral, 19 aboral), but the amplitude of oral inputs was consistently greater than aboral inputs (13.1±4.3mV vs. 10.1±4.8mV, respectively, p<0.05, paired t-test, n=6). Most impaled viscerofugal neurons were NOS immunoreactive (20/27 cells tested). Thus, the synaptic connections onto viscerofugal neurons within the myenteric plexus suggest that multiple enteric neural pathways feed into intestino-intestinal reflexes, involving sympathetic prevertebral ganglia.Neuroscience 05/2014; · 3.12 Impact Factor