Immunohistochemical analysis of the mouse celiac ganglion: An integrative relay station of the peripheral nervous system

Abstract

Celiac ganglia are important sites of signal integration and transduction. Their complex neurochemical anatomy has been studied extensively in guinea pigs but not in mice. The goal of this study was to provide detailed neurochemical characterization of mouse celiac ganglia and noradrenergic nerves in two target tissues, spleen and stomach. A vast majority of mouse celiac neurons express a noradrenergic phenotype, which includes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, and the norepinephrine transporter. Over 80% of these neuron also express neuropeptide Y (NPY), and this coexpression is maintained by dissociated neurons in culture. Likewise, TH and NPY were colocalized in noradrenergic nerves throughout the spleen and in stomach blood vessels. Somatostatin was not detected in principal neurons but did occur in small, TH‐negative cells presumed to be interneurons and in a few varicose nerve fibers. Cholinergic nerves provided the most abundant input to the ganglia, and small percentages of these also contained nitric oxide synthase or vasoactive intestinal polypeptide. A low‐to‐moderate density of nerves also stained separately for the latter markers. Additionally, nerve bundles and varicose nerve fibers containing the sensory neuropeptides, calcitonin gene‐related polypeptide, and substance P, occurred at variable density throughout the ganglia. Collectively, these findings demonstrate that principal neurons of mouse celiac ganglia have less neurochemical diversity than reported for guinea pig and other species but receive input from nerves expressing an array of neurochemical markers. This profile suggests celiac neurons integrate input from many sources to influence target tissues by releasing primarily norepinephrine and NPY.

A Novel Mode of Sympathetic Reflex Activation Mediated by the Enteric Nervous System

“…In guinea pig, viscerofugal neurons selectively innervate sympathetic motor and secretomotor neurons but not vasoconstrictor neurons (Costa and Furness, 1984;Macrae et al, 1986;Meckler and McLachlan, 1988;Gibbins et al, 2003). The specificity of viscerofugal targets remain to be confirmed in mouse and there is currently no useful chemical coding scheme identified among sympathetic neurons in mouse to differentiate functional subtypes (Kaestner et al, 2019). However, we speculate the viscerofugal-sympathetic loop ensures CMCs cause inhibition of motility and secretion further oral along the gut (Messenger and Furness, 1993;Furness et al, 2000;Furness, 2003).…”

“…However, it may be suggested, at least in control preparations with intact spinal afferent neurons, that spinal afferent axons branching in prevertebral ganglia potentially activated postganglionic sympathetic neurons in a mechanism similar to the "Sokownin reflex" (Sokownin, 1877;Job and Lundberg, 1952;Bulygin, 1983). Spinal afferent collaterals contain peptide transmitters that can be released during gut distension causing slow depolarization of sympathetic neurons (Dun and Karczmar, 1979;Dun and Jiang, 1982;Peters and Kreulen, 1986;Webber and Heym, 1988;Dun and Mo, 1989;Ma and Szurszewski, 1996;Jobling and Gibbins, 1999;Kaestner et al, 2019). Slow depolarizations range seconds to minutes in duration, making this mode unlikely to account for the high-fidelity transmission of the ;2-Hz burst firing pattern from the gut to sympathetic neurons during CMCs.…”

“…Following biotinamide tracing, a subset of control and organ-cultured preparations were additionally assessed for immunoreactivity for calcitonin gene-related peptide (CGRP), expressed in the majority of colorectal spinal afferent neurons (Robinson et al, 2004;Christianson et al, 2006), and tyrosine hydroxylase (TH), a marker of sympathetic neurons (Jobling and Gibbins, 1999;Kaestner et al, 2019). Preparations were incubated with primary antisera for CGRP (rabbit; Peninsula; IHC 6006; RRID: AB_2314156; 1:2000 dilution) and TH (chicken; Abcam; AB76442; RRID: AB_ 1524535; 1:200 dilution) for 48 h at room temperature, followed by PBS washing (3 Â 10 min) and 4-h incubation in secondary antisera (Alexa Fluor 488; donkey anti-rabbit; Thermo Fisher Scientific; A21206; RRID: AB_2535792; 1:1000 dilution; and Alexa Fluor 647 donkey anti-chicken; Jackson ImmunoResearch; 703-605-155; RRID: AB_ 2340379; 1:1000 dilution).…”

A single cell transcriptomics map of paracrine networks in the intrinsic cardiac nervous system

“…Porcine RAGP fat pads were fixed in cold 4% paraformaldehyde in PBS for 24 h, cryoprotected in cold 20% sucrose in PBS, and sectioned at 30 mm thickness using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA). Sections were collected on charged slides and immunostained at room temperature using standard methods of fluorescence immunohistochemistry, as described previously (Hoard et al, 2008;Hoover et al, 2009;Kaestner et al, 2019). Antibodies to vesicular acetylcholine transporter ll OPEN ACCESS iScience 24, 102713, July 23, 2021 iScience Article (VAChT; Synaptic Systems, Cat.…”

A spatially-tracked single cell transcriptomics map of neuronal networks in the intrinsic cardiac nervous system

“…Porcine RAGP fat pads were fixed in cold 4% paraformaldehyde in PBS for 24 h, cryoprotected in cold 20% sucrose in PBS, and sectioned at 30 mm thickness using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA). Sections were collected on charged slides and immunostained at room temperature using standard methods of fluorescence immunohistochemistry, as described previously 23,62,63 .…”