Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Acetylcholine (ACh)-synthesizing neurons are major components of the enteric nervous system (ENS). They release ACh and peptidergic neurotransmitters onto enteric neurons and muscle. However, pharmacological interrogation has proven inadequate to demonstrate an essential role for ACh.

APEX2-enhanced electron microscopy distinguishes sigma-1 receptor localization in the nucleoplasmic reticulum.

The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization.

The Sigma-1 Receptor-A Therapeutic Target for the Treatment of ALS?

The membrane bound 223 amino acid Sigma-1 Receptor (S1R) serves as a molecular chaperone and functional regulator of many signaling proteins. Spinal cord motor neuron activation occurs, in part, via large ventral horn cholinergic synapses called C-boutons/C-terminals. Chronic excitation of motor neurons and alterations in C-terminals has been associated with Amyotrophic Lateral Sclerosis (ALS ).

Sigma-1 receptor expression in the dorsal root ganglion: Reexamination using a highly specific antibody.

Sigma-1 receptor (S1R) is a unique pluripotent modulator of living systems and has been reported to be associated with a number of neurological diseases including pathological pain. Intrathecal administration of S1R antagonists attenuates the pain behavior of rodents in both inflammatory and neuropathic pain models. However, the S1R localization in the spinal cord shows a selective ventral horn motor neuron distribution, suggesting the high likelihood of S1R in the dorsal root ganglion (DRG) mediating the pain relief by intrathecally administered drugs.

Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung's-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs.

Immunostaining to visualize murine enteric nervous system development.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine.

Role of the Sigma-1 receptor in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease affecting spinal cord motoneurons (MN) with an associative connection to Frontotemporal Lobar Dementia (FTLD). The endoplasmic reticulum (ER) bound Sigma-1 Receptor (S1R) chaperone protein localizes to specialized ER cisternae within 10 nm of the plasma membrane in spinal cord ventral horn cholinergic post synaptic C-terminals. Removal of the S1R gene in the Superoxide Dismutase-1 (SOD-1) mouse model of ALS exacerbated the neurodegenerative condition and resulted in a significantly reduced longevity when compared to the SOD-1/S1R wild type (WT) mouse.

Dopamine transporter gene susceptibility to methylation is associated with impulsivity in nonhuman primates.

Impulsivity, the predisposition to act without regard for negative consequences, is a characteristic of several psychiatric disorders and is thought to result in part from genetic variation in the untranslated region of the dopamine transporter (DAT) gene. As the exact link between genetic mutations and impulsivity has not been established, we used oculomotor behavior to characterize rhesus monkeys as impulsive or calm and genetic/epigenetic analysis and positron emission tomography (PET) to correlate phenotype to DAT genotype, DAT gene methylation, and DAT availability. We found three single nucleotide polymorphisms (SNPs) in the 3'-UTR of the DAT gene, one of which provided a potential site for methylation in the impulsive group.

Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung's disease.

Background/purpose: Hirschsprung's disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung's-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model.

Colonic enteric nervous system analysis during parenteral nutrition.

Introduction: Parenteral nutrition (PN) is a necessary therapy used to feed patients with gastrointestinal dysfunction. Unfortunately, PN results in intestinal atrophy and changes to host immune function. PN may also induce additional effects on gut motility that we hypothesized would result from changes in the enteric nervous system.

VEGF induces sensory and motor peripheral plasticity, alters bladder function, and promotes visceral sensitivity.

Background: This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.

Sacral neural crest-derived cells enter the aganglionic colon of Ednrb-/- mice along extrinsic nerve fibers.

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon.

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed.

Background: Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins.

Age-dependent changes in the gut environment restrict the invasion of the hindgut by enteric neural progenitors.

The enteric nervous system (ENS) develops from neural crest cells (NCCs) that enter the foregut and hindgut to become enteric neural-crest-derived cells (ENCCs). When these cells of neural crest origin fail to colonize the terminal hindgut, this aganglionic region becomes non-functional and results in a condition in humans known as Hirschsprung's disease (HSCR). One of the genes associated with HSCR is endothelin receptor type B (Ednrb).

Immunohistochemical analysis of neuron types in the mouse small intestine.

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons.

Targeting of endothelin receptor-B to the neural crest.

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development.

Behavior of enteric neural crest-derived cells varies with respect to the migratory wavefront.

Neural crest-derived cells colonize the entire gastrointestinal tract. The migration of these enteric neural crest-derived cells (ENCCs) occurs by their formation of cellular strands that extend into the intestinal mesenchyme. We have studied the behavior of crest cells that underlies the formation and extension of these strands by time-lapse microscopy.

The pattern of neural crest advance in the cecum and colon.

Neural crest cells leave the hindbrain, enter the gut mesenchyme at the pharynx, and migrate as strands of cells to the terminal bowel to form the enteric nervous system. We generated embryos containing fluorescent enteric neural crest-derived cells (ENCCs) by mating Wnt1-Cre mice with Rosa-floxed-YFP mice and investigated ENCC behavior in the intact gut of mouse embryos using time-lapse fluorescent microscopy. With respect to the entire gut, we have found that ENCCs in the cecum and proximal colon behave uniquely.

GDNF and insulin cooperate to enhance the proliferation and differentiation of enteric crest-derived cells.

Previously we have shown that glial derived neurotrophic factor (GDNF) stimulates modest increases in the proliferation of avian enteric crest-derived cells and similar increases in the phosphorylation of the phosphoinositide 3-kinase (PI3K) downstream substrate Akt (Akt-P). In the present study we tested whether GDNF-independent increases in PI3K activation would be sufficient to support proliferation. We found that insulin induces a large increase in the phosphorylation of Akt and can initiate DNA synthesis in avian enteric crest-derived cells, but is unable to maintain proliferation over time in culture, measured by BrdU incorporation.

Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells.

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections.