In vivo transplantation of fetal human gut‐derived enteric neural crest cells

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75^NTR were lentivirally labeled with either GFP or calcium‐sensitive GCaMP and transplanted into the hindgut of Rag2^?/γc^?/C5^?‐immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut‐derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.     

Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells

Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2^−/−) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2^−/− mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2^−/− mice exhibited smaller ganglia, fewer HuC/D^+ ve and nNOS^+ ve neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with those from WT-EGCs or WT-MΦ/DCs corrected the altered neuronal phenotype of TLR2^−/− mice. Supplementation of TLR2^−/− neuronal cultures with GDNF recapitulated the WT-SMC co-culture effect whereas the knockdown of GDNF expression in WT-SMCs using shRNA interference abolished the effect on TLR2^−/− neurons. These data revealed that by exploiting the repertoire of TLRs to decode gut-microbial signals, intestinal SMCs elaborate a cocktail of neurotrophic factors that in turn supports neuronal phenotype. In this view, the SMCs represent an attractive target for novel therapeutic strategies.     

Full‐field optical coherence microscopy is a novel technique for imaging enteric ganglia in the gastrointestinal tract

Background Noninvasive methods are needed to improve the diagnosis of enteric neuropathies. Full‐field optical coherence microscopy (FFOCM) is a novel optical microscopy modality that can acquire 1 μm resolution images of tissue. The objective of this research was to demonstrate FFOCM imaging for the characterization of the enteric nervous system (ENS). Methods Normal mice and EdnrB^‐/‐ mice, a model of Hirschsprung’s disease (HD), were imaged in three‐dimensions ex vivo using FFOCM through the entire thickness and length of the gut. Quantitative analysis of myenteric ganglia was performed on FFOCM images obtained from whole‐mount tissues and compared with immunohistochemistry imaged by confocal microscopy. Key Results Full‐field optical coherence microscopy enabled visualization of the full thickness gut wall from serosa to mucosa. Images of the myenteric plexus were successfully acquired from the stomach, duodenum, colon, and rectum. Quantification of ganglionic neuronal counts on FFOCM images revealed strong interobserver agreement and identical values to those obtained by immunofluorescence microscopy. In EdnrB^‐/‐ mice, FFOCM analysis revealed a significant decrease in ganglia density along the colorectum and a significantly lower density of ganglia in all colorectal segments compared with normal mice. Conclusions & Inferences Full‐field optical coherence microscopy enables optical microscopic imaging of the ENS within the bowel wall along the entire intestine. FFOCM is able to differentiate ganglionic from aganglionic colon in a mouse model of HD, and can provide quantitative assessment of ganglionic density. With further refinements that enable bowel wall imaging in vivo, this technology has the potential to revolutionize the characterization of the ENS and the diagnosis of enteric neuropathies.     

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models

Cannabinoid receptor 1 (CB₁ receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB₁ expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB₁ signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB₁ downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi‐quantitative immunohistochemistry, we confirmed previous studies showing that CB₁ expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB₁ is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)‐expressing interneurons while remaining unchanged in parvalbumin‐ and calretinin‐expressing interneurons. CB₁ downregulation in striatal NPY/nNOS‐expressing interneurons occurs in R6/2 mice, Hdh^Q150/Q150 mice and the caudate nucleus of patients with HD. In R6/2 mice, CB₁ downregulation in NPY/nNOS‐expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro‐survival signaling molecule cAMP response element‐binding protein in NPY/nNOS‐expressing interneurons. Loss of CB₁ signaling in NPY/nNOS‐expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.     

Reduced activity and protein expression of NOS in R6/2 HD transgenic mice: effects of -NAME on symptom progression

Previous work found that dietary l-arginine alters symptom progression in mice transgenic for Huntington’s disease (HD), and that cerebral blood flow (CBF) is abnormal in early stage HD patients. Both of these findings potentially implicate nitric oxide (NO) and its converting enzyme, nitric oxide synthase (NOS), in HD. The current experiment found that both NOS enzymatic activity and neuronal NOS (nNOS) protein expression were reduced (P<0.05) in R6/2 HD transgenic mice compared to non-HD controls (CON). Conversely, inducible NOS (iNOS) protein expression was not significantly different between groups. The changes in nNOS were accompanied by changes in protein expression of calmodulin kinase II (CaMKII) (P<0.05) and calmodulin kinase IV (CaMKIV) (P<0.05). Protein expression of 3-nitrotyrosine (3-NT), a marker for the neurotoxin peroxynitrite, was slightly increased in non-drug treated HD and was accompanied by increased immunostaining of 3-NT in cells adhering to the vasculature and choroid plexus. Mice that received the broad-spectrum NOS inhibitor N^g-nitro- -arginine methyl ester hydrochloride ( -NAME) via their drinking water had reduced NOS enzyme activity. NOS activity varied as a function of -NAME dose, was virtually eliminated in the 500-mg/l groups, and correlated (P<0.05) with the behavioral scores as revealed by regression and correlation analyses. High dose -NAME (500 mg/l) accelerated symptom onset in HD transgenics. These results support the hypothesis that nNOS activity and NO production are abnormal in HD, this in the setting of a more global dysregulation of calcium protein expression. Taken collectively with earlier data from our laboratory demonstrating abnormal CBF findings in early-stage HD patients, these results suggest that abnormalities in NOS function may significantly contribute to the neurodegeneration found in HD.     

Methylphenidate treatment increases Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in the cerebrum of young and adult rats

Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na⁺, K⁺-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na⁺, K⁺-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na⁺, K⁺-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na⁺, K⁺-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na⁺, K⁺-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na⁺, K⁺-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na⁺, K⁺-ATPase activity may be associated with neuronal excitability caused by methylphenidate.     

The effects of glucagon‐like peptide 2 on enteric neurons in intestinal inflammation

Background Intestinal inflammation alters the structure and function of the enteric nervous system (ENS). Glucagon‐like peptide 2 (GLP‐2) reduces intestinal inflammation and has trophic effects on isolated neurons. This study examined the effects of GLP‐2 treatment on the submucosal plexus of rat colon in the trinitrobenzene sulfonic acid (TNBS) model of colitis. Methods After administration of TNBS or saline/ethanol for controls, animals were allocated to treatment with GLP‐2 (50 μg kg^?1day^?1, s.c.) or sham injection of vehicle, twice daily. Animals were monitored, following clinical parameters, and killed on day 5. The number of neuronal cell bodies per ganglion was quantified using immunohistochemistry on submucosal whole mount preparations, with further characterization of specific subpopulations using antibodies against vasoactive intestinal polypeptide (VIP), neuronal nitric oxide synthase (nNOS), and enteric glial cells with glial fibrillary acid protein and S100. Key Results Glucagon‐like peptide 2 treatment was associated with a significant amelioration of weight loss, and reduced neutrophil infiltration and microscopic colitis scores in the TNBS animals. Inflammation resulted in a loss of enteric neurons in submucosal ganglia; GLP‐2 treatment restored the enteric neuronal populations to normal. In control, non‐inflamed animals, GLP‐2 treatment increased the number of VIP expressing neurons per ganglion; in TNBS‐treated animals, GLP‐2 prevented an inflammation‐induced reduction in the numbers of VIP expressing neurons per ganglion. Glucagon‐like peptide 2 did not change the numbers of nNOS neurons or enteric glial cells in either the control, or inflamed state. Conclusions & Inferences These findings show that GLP‐2 increased the number of VIP expressing neurons in normal animals, and prevents the inflammation‐induced loss of neurons in the colonic submucosal ganglia, with an increase in the proportion of VIP expressing neurons. They suggest that GLP‐2 may have a role in protecting or regulating the circuitry of the ENS under basal and inflamed states.     

Glial cell line‐derived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system

Background The enteric nervous system (ENS) continues its structural and functional growth after birth, with formation of ganglia and the innervation of growing smooth muscle. However, little is known about factors in the postnatal intestine that influence these processes. Methods We examined the presence and potential role of glial cell line‐derived nerve growth factor (GDNF) in the rat postnatal ENS using neonatal tissue, primary co‐cultures of the myenteric plexus, smooth muscle, and glial cells as well as cell lines of smooth muscle or glial cells. Key Results Western blot analysis showed that GDNF and its co‐receptors rearranged during transfection (RET) and GDNF family receptor alpha‐1 were expressed in the muscle layer of the neonatal and adult rat intestine. Immunohistochemistry localized the receptors for GDNF to myenteric neurons, while GDNF was localized to smooth muscle cells. In a co‐culture model, GDNF but not nerve growth factor, brain derived neurotrophic factor or neurotrophin‐3 significantly increased neuronal survival and more than doubled the numbers of neurites in vitro. RT‐PCR, qPCR, Western blotting, ELISA, and immunocytochemistry as well as bioassays of neuronal survival and of RET phosphorylation all identified intestinal smooth muscle as the source of GDNF in vitro. GDNF also induced morphological changes in the structure and organization of neurons and axons, causing marked aggregation of neuronal cell bodies and collinear development of axons. As well, GDNF (50–150 ng mL^?1) significantly increased [³H]‐choline uptake and stimulated [³H]‐acetylcholine release. Conclusions & Inferences We conclude that GDNF derived from intestinal smooth muscle cells is a key factor influencing the structural and functional development of postnatal myenteric neurons.     

Endothelial nitric oxide synthase inhibition triggers inflammatory responses in the brain of male rats exposed to ischemia‐reperfusion injury

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a role in preserving and maintaining the brain's microcirculation, inhibiting platelet aggregation, leukocyte adhesion, and migration. Inhibition of eNOS activity results in exacerbation of neuronal injury after ischemia by triggering diverse cellular mechanisms, including inflammatory responses. To examine the relative contribution of eNOS in stroke‐induced neuroinflammation, we analyzed the effects of systemic treatment with l‐N‐(1‐iminoethyl)ornithine (L‐NIO), a relatively selective eNOS inhibitor, on the expression of MiR‐155‐5p, a key mediator of innate immunity regulation and endothelial dysfunction, in the cortex of male rats subjected to transient middle cerebral artery occlusion (tMCAo) followed by 24 hr of reperfusion. Inducible NO synthase (iNOS) and interleukin‐10 (IL‐10) mRNA expression were evaluated by real‐time polymerase chain reaction in cortical homogenates and in resident and infiltrating immune cells isolated from ischemic cortex. These latter cells were also analyzed for their expression of CD40, a marker of M1 polarization of microglia/macrophages.tMCAo produced a significant elevation of miR155‐5p and iNOS expression in the ischemic cortex as compared with sham surgery. eNOS inhibition by L‐NIO treatment further elevated the cortical expression of these inflammatory mediators, while not affecting IL‐10 mRNA levels. Interestingly, modulation of iNOS occurred in resident and infiltrating immune cells of the ischemic hemisphere. Accordingly, L‐NIO induced a significant increase in the percentage of CD40⁺ events in CD68⁺ microglia/macrophages of the ischemic cortex as compared with vehicle‐injected animals. These findings demonstrate that inflammatory responses may underlie the detrimental effects due to pharmacological inhibition of eNOS in cerebral ischemia.     

The chemical code of porcine enteric neurons and the number of enteric glial cells are altered by dietary probiotics

Background The enteric nervous system (ENS) contains chemically coded populations of neurons that serve specific functions for the control of the gastrointestinal tract. The ability of neurons to modify their chemical code in response to luminal changes has recently been discovered. It is possible that enteric neuronal plasticity may sustain the adaptability of the gut to changes in intestinal activity or injury, and that gut neurons may respond to an altered intestinal environment by changing their neuropeptide expression. Methods We used immunohistochemical methods to investigate the presence and localization of several neuronal populations and enteric glia in both the small (ileum) and large (cecum) intestine of piglets. We assessed their abundance in submucosal and myenteric plexus from animals treated with the probiotic Pediococcus acidilactici compared with untreated controls. Key Results The treated piglets had a larger number of galanin‐ and calcitonin gene‐related peptide (CGRP)‐immunoreactive neurons than controls, but this was limited to the submucosal plexus ganglia of the ileum. Moreover, immunohistochemistry revealed that glial fibrillary acidic protein‐positive enteric glial cells were significantly higher in the inner and outer submucosal plexuses of treated animals. Conclusions & Inferences The neuronal and glial changes described here illustrate plasticity of the ENS in response to an altered luminal environment in the gastrointestinal tract.     

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS‐expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS‐immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα‐immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFP^Vglut2, EYFP^Vgat, and GFP^Gad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.     

CXCR4 enhances engraftment of muscle progenitor cells

Cell‐based therapy is a possible avenue for the treatment of Duchenne muscular dystrophy (DMD), an X‐linked skeletal muscle‐wasting disease. We have demonstrated that cultured myogenic progenitors derived from the adult skeletal muscle side population can engraft into dystrophic fibers of non‐irradiated, non–chemically injured mouse models of DMD (mdx^5cv) after intravenous and intraarterial transplantation, with engraftment rates approaching 10%. In an effort to elucidate the cell‐surface markers that promote progenitor cell extravasation and engraftment after systemic transplantation, we found that expression of the chemokine receptor CXCR4, whose ligand SDF‐1 is overexpressed in dystrophic muscle, enhances the extravasation of these cultured progenitor cells into skeletal muscle after intraarterial transplantation. At 1 day post‐transplantation, mice that received CXCR4‐positive enhanced green fluorescent protein (eGFP)‐positive cultured cells derived from the skeletal muscle side population displayed significantly higher amounts of eGFP‐positive mononuclear cells in quadriceps and tibialis anterior than mice that received CXCR4‐negative eGFP‐positive cells derived from the same cultured population. At 30 days posttransplantation, significantly higher engraftment rates of donor cells were observed in mice that received CXCR4‐positive cells compared with mice transplanted with CXCR4‐negative fractions. Our data suggest that CXCR4 expression by muscle progenitor cells increases their extravasation into skeletal muscle shortly after transplantation. Furthermore, this enhanced extravasation likely promotes higher donor cell engraftment rates over time. Muscle Nerve 40: 562–572, 2009     

Role of neuronal and inducible nitric oxide synthases in the guinea pig ileum myenteric plexus during in vitro ischemia and reperfusion

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation (in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Western‐immunoblotting and real time RT‐PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOS‐expressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS‐immunoreactive neurons, protein and mRNA levels were up‐regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by N^ω‐propyl‐l ‐arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R.     

Eph receptor expression defines midline boundaries for ephrin-positive migratory neurons in the enteric nervous system of Manduca sexta

Eph receptor tyrosine kinases and their ephrin ligands participate in the control of neuronal growth and migration in a variety of contexts, but the mechanisms by which they guide neuronal motility are still incompletely understood. By using the enteric nervous system (ENS) of the tobacco hornworm Manduca sexta as a model system, we have explored whether Manduca ephrin (MsEphrin; a GPI-linked ligand) and its Eph receptor (MsEph) might regulate the migration and outgrowth of enteric neurons. During formation of the Manduca ENS, an identified set of approximately 300 neurons (EP cells) populates the enteric plexus of the midgut by migrating along a specific set of muscle bands forming on the gut, but the neurons strictly avoid adjacent interband regions. By determining the mRNA and protein expression patterns for MsEphrin and the MsEph receptor and by examining their endogenous binding patterns within the ENS, we have demonstrated that the ligand and its receptor are distributed in a complementary manner: MsEphrin is expressed exclusively by the migratory EP cells, whereas the MsEph receptor is expressed by midline interband cells that are normally inhibitory to migration. Notably, MsEphrin could be detected on the filopodial processes of the EP cells that extended up to but not across the midline cells expressing the MsEph receptor. These results suggest a model whereby MsEphrin-dependent signaling regulates the response of migrating neurons to a midline inhibitory boundary, defined by the expression of MsEph receptors in the developing ENS.     

GFAP-Positive Progenitor Cell Production is Concentrated in Specific Encephalic Regions in Young Adult Mice

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP⁺) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP⁺ cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP⁺ cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP⁺ cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP⁺ cells mainly generated NeuN⁺ neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP⁺ cells at the young adult stage is significantly different from that at the adult stage.     

Leptin excites enteric neurons of guinea‐pig submucous and myenteric plexus

Background Leptin, one of the most prominent mediators released from adipocytes, influences neuronal activity in the central nervous system. The enteric nervous system (ENS) expresses leptin receptors but consequence of activation of these receptors on enteric neuron activity has not been systematically studied. An adipocyte‐ENS axis is suggested by close apposition between enteric nerves and adipocytes. The aim of this study was to investigate the effects of leptin on guinea‐pig submucous and myenteric neurons. Methods Using voltage sensitive dye imaging, we recorded neural responses to application of leptin (0.0625 nmol L^?1) in myenteric and submucous neurons, nicotine (10 μmol L^?1) served as a reference for neuronal excitation. Mucosal ion secretion and muscle activity were measured in vitro with Ussing and organ bath techniques, respectively. Key Results Leptin induced spike discharge in 13.6% of submucous neurons and in 8.2% of myenteric neurons (1.1 ± 0.9 and 1.2 ± 1.0 Hz, respectively). Although there was an overlap of nicotine and leptin responses, 38.5% of submucous and 25% of myenteric neurons activated by leptin did not respond to nicotine. Leptin did not inhibit ongoing spike discharge or fast excitatory postsynaptic potentials. Leptin (0.0625 nmol L^?1) did not affect mucosal secretion or muscle activity suggesting a subtle modulatory action of leptin at the level of the ENS. Conclusions & Inferences Leptin activates submucous and myenteric neurons indicating relevance for adipocyte‐ENS signaling. These results set the basis for further studies to reveal the functional correlate of the neural action of leptin in the ENS.     

Development of myenteric cholinergic neurons in ChAT‐Cre;R26R‐YFP mice

Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT‐Cre;R26R‐YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre‐ and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time. J. Comp. Neurol. 521:3358–3370, 2013. © 2013 Wiley Periodicals, Inc.     

Differentiation of Ionic Currents in CNS Progenitor Cells: Dependence upon Substrate Attachment and Epidermal Growth Factor

Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na⁺and K⁺currents (I_Na, I_K) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative “spheres,” and differentiated cells were studied 1–25 days after plating spheres onto polylysine/laminin-treated coverslips.I_NaandI_Kwere detected together in 58%,I_Naalone in 11%, andI_Kalone in 19% of differentiated cells recorded with K⁺-containing pipettes. With internal Cs⁺(to isolateI_Na),I_Naup to 45 pA/pF was observed in some cells within 1 day after plating.I_Naranged up to 150 pA/pF subsequently. Overall, 84% of cells expressedI_Na, with an average of 38 pA/pF.I_Nahad fast kinetics, as in neurons, but steady-state inactivation curves were strongly negative, resembling those of glialI_Na. Inward tail currents sensitive to [K⁺]_outwere observed upon repolarization after the 10-ms test pulse with internal Cs⁺, indicating the expression of K⁺channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or noI_NaorI_K-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF.I_Nawas observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.