A Bone Morphogenetic Protein Signaling Inhibitor, LDN193189, Converts Ischemia-Induced Multipotent Stem Cells into Neural Stem/Progenitor Cell-Like Cells.

Stem cell therapy is used to restore neurological function in stroke patients. We have previously reported that ischemia-induced multipotent stem cells (iSCs), which are likely derived from brain pericytes, develop in poststroke human and mouse brains. Although we have demonstrated that iSCs can differentiate into neural lineage cells, the factors responsible for inducing this differentiation remain unclear. In this study, we found that LDN193189, a bone morphogenetic protein (BMP) inhibitor, caused irreversible changes in the shape of iSCs. In addition, compared with iSCs incubated without LDN193189, the iSCs incubated with LDN193189 (LDN-iSCs) showed upregulated expression of neural lineage-related genes and proteins, including those expressed in neural stem/progenitor cells (NSPCs), and downregulated expression of mesenchymal and pericytic-related genes and proteins. Moreover, microarray analysis revealed that LDN-iSCs and NSPCs had similar gene expression profiles. Furthermore, LDN-iSCs differentiated into electrophysiologically functional neurons. These results indicate that LDN193189 induces NSPC-like cells from iSCs, suggesting that bioactive molecules regulating BMP signaling are potential targets for promoting neurogenesis from iSCs in the pathological brain, such as during ischemic stroke. We believe that our findings will bring us one step closer to the clinical application of iSCs.

Morphology of Schwann Cell Processes Supports Renal Sympathetic Nerve Terminals With Local Distribution of Adrenoceptors.

Nerves in the renal parenchyma comprise sympathetic nerves that act on renal arteries and tubules to decrease blood flow and increase primary urine reabsorption, respectively. Synaptic vesicles release neurotransmitters that activate their effector tissues. However, the mechanisms by which neurotransmitters exert individual responses to renal effector cells remain unknown. Here, we investigated the spatial and molecular compositional associations of renal Schwann cells (SC) supporting the nerve terminals in male rats. The nerve terminals of vascular smooth muscle cells (SMCs) enclosed by renal SC processes were exposed through windows facing the effectors with presynaptic specializations. We found that the adrenergic receptors (ARs) α2A, α2C, and ß2 were localized in the SMC and the basal side of the tubules, where the nerve terminals were attached, whereas the other subtypes of ARs were distributed in the glomerular and luminal side, where the norepinephrine released from nerve endings may have indirect access to ARs. In addition, integrins α4 and ß1 were coexpressed in the nerve terminals. Thus, renal nerve terminals could contact their effectors via integrins and may have a structure, covered by SC processes, suitable for intensive and directional release of neurotransmitters into the blood, rather than specialized structures in the postsynaptic region.

Exploring Molecular Mechanisms Involved in the Development of the Depression-Like Phenotype in Interleukin-18-Deficient Mice.

Interleukin-18 (IL-18) is an inflammatory cytokine that has been linked to energy homeostasis and psychiatric symptoms such as depression and cognitive impairment. We previously revealed that deficiency in IL-18 led to hippocampal abnormalities and resulted in depression-like symptoms. However, the impact of IL-18 deficiency on other brain regions remains to be clarified. In this study, we first sought to confirm that IL-18 expression in neural cells can be found in human brain tissue. Subsequently, we examined the expression of genes in the prefrontal cortex of Il18 -/- mice and compared it with gene expression in mice subjected to a chronic mild stress model of depression. Extracted genes were further analyzed using Ingenuity® Pathway Analysis, in which 18 genes common to both the chronic mild stressed model and Il18 -/- mice were identified. Of those, 16 were significantly differentially expressed between Il18+/+ and Il18 -/- mice. We additionally measured protein expression of α-2-HS-glycoprotein (AHSG) and transthyretin (TTR) in serum and the brain. In the prefrontal cortex of Il18 -/- mice, TTR but not AHSG was significantly decreased. Conversely, in the serum of Il18 -/- mice, AHSG was significantly increased but not TTR. Therefore, our results suggest that in IL-18-deficit conditions, TTR in the brain is one of the mediators causally related to depression, and AHSG in peripheral organs is one of the regulators inducing energy imbalance. Moreover, this study suggests a possible "signpost" to clarify the molecular mechanisms commonly underlying the immune system, energy metabolism, neural function, and depressive disorders.

Relationship between lamellar sensory corpuscles distributed along the upper arm's deep arteries and pulsating sensation of blood vessels.

Vibration is detected by mechanoreceptors, including Pacinian corpuscles (PCs), which are widely distributed in the human body including the adventitia of large blood vessels. Although the distribution of PCs around large limb vessels has been previously reported, there remains no consensus on their distribution in the adventitia of the human deep blood vessels in the upper arm. In addition, the physiological functions of PCs located around the deep limb blood vessels remain largely unknown. This study aimed to elucidate detailed anatomical features and physiological function of lamellar sensory corpuscles structurally identified as PCs using the immunohistochemical methods around the deep vessels in the upper arm. We identified PCs in the connective tissue adjacent to the deep vessels in the upper arm using histological analysis and confirmed that PCs are located in the vascular sheath of the artery and its accompanying vein as well as in the connective tissue surrounding the vascular sheath and nerves. PCs were densely distributed on the distal side of deep vessels near the elbow. We also examined the relationship between vascular sound and pulsating sensation to evaluate the PCs functions around deep arteries and veins and found that the vascular sound made by pressing the brachial arteries in the upper arm was associated with the pulsating sensation of the examinee. Our results suggest that PCs, around deep vessels, function as bathyesthesia sensors by detecting vibration from blood vessels.

Interleukin-18-deficient mice develop hippocampal abnormalities related to possible depressive-like behaviors.

Interleukin-18 (IL-18) is an inflammatory cytokine linked to major depressive disorder (MDD). MDD is closely related to metabolic disorders, such as diabetes mellitus (DM) and obesity. Moreover, DM is associated with cognitive impairment and promotes apoptosis of hippocampal cells by activating pro-apoptotic and inhibiting anti-apoptotic factors. IL-18-deficient (Il18-/-) mice are obese and have DM. Therefore, we hypothesized a close relationship between IL-18 and death of hippocampal cells, affecting neurogenesis related to behavioral changes such as MDD. Il18-/- male mice were generated on the C57Bl/6 background and Il18+/+ mice were used as controls. Behavioral, histopathological, and molecular responses, as well as responses to intracerebral recombinant IL-18 administration, were examined. Compared with Il18+/+ mice, Il18-/- mice had impaired learning and memory and exhibited lower motivation. In the Il18-/- mice, degenerated mitochondria were detected in synaptic terminals in the molecular layer, the polymorphic layer, and in mossy fibers in the dentate gyrus, suggesting mitochondrial abnormalities. Because of the degeneration of mitochondria in the dentate gyrus, in which pro-apoptotic molecules were upregulated and anti-apoptotic factors were decreased, apoptosis inducers were not cleaved, indicating inhibition of apoptosis. In addition, neurogenesis in the dentate gyrus and the maturity of neuronal cells were decreased in the Il18-/- mice, while intracerebral administration of recombinant IL-18 promoted significant recovery of neurogenesis. Our findings suggested that IL-18 was indispensable for mitochondrial homeostasis, sustaining clearance of degenerative neural cells, and supporting neurogenesis, normal neuronal maturation and hippocampal function.

Platelet-derived growth factor receptor α gene is regulated by multiple first exons.

Transcription of the platelet-derived growth factor receptor α (PDGFRA/Pdgfra) gene is considered to be precisely regulated. We have previously reported that the PDGFRA/Pdgfra gene is regulated by a dual promoter system in human and mouse, in which a novel PDGFRA/Pdgfra transcript has a first exon (exon 1ß) different from that of the canonical PDGFRA/Pdgfra transcript (exon 1α). To elucidate the function of each transcript, we first investigated the contribution of different PDGFRA transcripts to final protein levels. Notably, knockdown experiments suggested the existence of other PDGFRA transcripts, and we identified five additional first exons (exons 1γ, 1δ, 1ε, 1ζ, and 1η) in intron 1 in both the human and mouse genes. The first exons of the mouse Pdgfra gene showed unique expression patterns: exon 1α was broadly expressed; exon 1ß was highly expressed in embryos; exon 1γ was observed at relatively high levels in the adult central nervous system (CNS); and exon 1δ was expressed at relatively high levels in the developing CNS. Furthermore, in silico analysis of common putative transcription factor binding sites in the upstream regions of the first exons of both human and mouse PDGFRA/Pdgfra genes predicted common (such as Sry, Mzf1, and Cdx) and unique (such as Sox5, Lmo2, and GATA) transcription factors. Our findings show the diversity of the transcriptional regulation of the PDGFRA/Pdgfra gene.

Deficiency in interleukin-18 promotes differentiation of brown adipose tissue resulting in fat accumulation despite dyslipidemia.

BACKGROUND: The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis. METHODS: Il18-/- male mice were generated on the C57Bl/6 background, and littermate C57Bl/6 Il18+/+ male mice were used as controls. To reveal the direct effect of IL-18, primary cell cultures derived from both mice were established. Moreover, for molecular analysis, microarray, quantitative reverse transcription PCR and western blotting were performed using 6 and 12 weeks old mice. To evaluate the short- and long-term effects of IL-18 on BAT, recombinant IL-18 was administered for 2 and 12 weeks, respectively. RESULTS: Compared with Il18+/+ mice, BAT of Il18-/- mice showed earlier differentiation and lipid accumulation. To examine the direct effect of IL-18 on BAT, BA cell cultures were established. Myogenic factor 5-expressing adipose precursor cells were extracted from Il18+/+ and Il18-/- mice. PR domain containing 16 (PRDM16), a differentiation inducer, was strongly expressed in Il18-/- BAs, and uncoupling protein 1, a thermogenic and differentiation marker, was upregulated, resulting in the promotion of BA differentiation. Moreover, PRDM16-dependent and independent molecules related to BAT function, such as fibroblast growth factor 21, were activated. These findings were confirmed by comparing Il18+/+ and Il18-/- mice at 6 and 12 weeks of age. Additional analyses of the molecular mechanisms influencing the 'Quantity of adipocytes' identified three associated genes, apolipoprotein C3 (Apoc3), insulin-induced gene 1 (Insig1) and vitamin D (1,25-dihydroxyvitamin D3) receptor (Vdr). Intravenous administration of IL-18 not only significantly improved the expression of some of these genes, but it also significantly decreased the adipocytes' size. CONCLUSIONS: This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance.

Distribution of Pacini-Like Lamellar Corpuscles in the Vascular Sheath of the Femoral Artery.

Pacinian corpuscles are vibration-sensing mechanoreceptors that are densely distributed in the dermis of the human hand. Although they are also known to occur in various other regions/structures throughout the human body, including the adventitia of large vessels, their precise distribution and function in arteries remain unclear. In the present study, we identified Pacini-like lamellar corpuscles (LCs) adjacent to the femoral artery, and investigated their distribution with respect to that structure via a histological analysis. We identified nine LCs that were localized in the connective tissue surrounding the femoral artery and vein. We showed that although their distribution was heterogeneous, they were predominantly concentrated on the dorsal side of the femoral artery. Immunohistochemical analyses revealed that the identified femoral artery LCs exhibited features characteristic of typical LCs located in the dermis of the index finger. Thus, the results of the present study contribute to an improved understanding of the function of femoral artery LCs. Anat Rec, 301:1809-1814, 2018. © 2018 Wiley Periodicals, Inc.

Differential Distribution of Renal Nerves in the Sympathetic Ganglia of the Rat.

The renal nerve plexus comprises efferent and afferent fibers. It controls urine production and bodily fluid homeostasis. Efferent fibers to the kidney include sympathetic nerve fibers from their main ganglia, the prevertebral suprarenal ganglia (SrG), and the paravertebral sympathetic chain ganglia (ChG). In the present study, we examined topological innervation from these ganglia to the renal parenchymal segments of the left kidney of the rat. Fluoro-Gold was injected into the rostral or caudal poles of the left kidney. Approximately 50% of the cells in the SrG of rats injected in the rostral pole were labeled, while 60% of the cells in the ChG T13 of rats injected in the caudal pole were labeled. In addition, we performed dual-probe retrograde tracing of the nerves using two kinds of fluorescent-conjugated cholera toxins (f-CTbs) injected into the rostral and caudal poles of the left kidney. The cells labeled with each f-CTb were distributed differently in the left SrG and the lower ChGs; no dual-labeled cells were found in these ganglia. Anterograde tracing with pCAGGS-tdTomato vector transfected into the left SrG showed that tdTomato-labeled nerve varicosities extended to the cortical arterioles and urinary tubules. Immunohistochemistry revealed that they were positive to tyrosine hydroxylase and synaptophysin, suggesting that they possessed sympathetic nerve endings. Our results show that renal efferent nerves in the SrG may control the rostral part of the kidney and innervate the multiple effectors in the cortex. Anat Rec, 300:2263-2272, 2017. © 2017 Wiley Periodicals, Inc.

Possible Role of the Myelinated Neural Network in the Parietal Peritoneum in Rats as a Mechanoreceptor.

A network of myelinated nerve fibers in the peritoneum covers the abdominal wall. We studied the topographic distribution of this network, explored the fibers' destination in the central nervous system, and examined the markers in these fibers in order to identify the nature of the sensation conveyed by the network of nerve fibers in rats. We used Sihler's method, which stains myelinated fibers in whole mount materials, and observed a dense nerve network and endings toward the peritoneal cavity in the peritoneum that covers the abdomen's lateral bulge. We studied the axonal transport of cholera toxin subunit B to investigate the central projections of this network in order to identify its function. After applying the tracer in the peritoneum, we observed many labeled terminals in the medial part of laminae 3-5 of the spinal cord. A small number of labeled terminals was observed in the dorsal nucleus of Clarke and gracile nucleus. Labeled somata were observed in the dorsal root ganglia (DRG). Most (96%) were larger than 35 µm. We performed immunohistochemistry of the abdominal wall, using antiserum against the 200-kD neurofilament (a marker for mechanosensory neurons). We observed many positive nerve fibers in the peritoneum. Because cell bodies in the DRG were large, their nerve terminals ended in the base of the dorsal horn, which is known to transmit proprioceptive information, and the network possesses the marker for mechanosensitive fibers; therefore, it appears that the myelinated nerve network conveys information about distension and/or contraction of the abdominal wall. Anat Rec, 300:1662-1669, 2017. © 2017 Wiley Periodicals, Inc.

Interleukin-18 and its receptor are expressed in gonadotropin-releasing hormone neurons of mouse and rat forebrain.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling ß chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

Interleukin-18-deficient mice develop dyslipidemia resulting in nonalcoholic fatty liver disease and steatohepatitis.

We investigated potential pathophysiological relationships between interleukin 18 (IL-18) and dyslipidemia, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). Compared with Il18(+/+) mice, IL-18 knockout (Il18(-/-)) mice developed hypercholesterolemia and hyper-high-density-lipoprotein-cholesterolemia as well as hypertriglyceridemia as they aged, and these disorders occurred before the manifestation of obesity and might cause secondary NASH. The analyses of molecular mechanisms involved in the onset of dyslipidemia, NAFLD, and NASH in Il18(-/-) mice identified a number of genes associated with these metabolic diseases. In addition, molecules related to circadian rhythm might affect these extracted genes. The intravenous administration of recombinant IL-18 significantly improved dyslipidemia, inhibited the body weight gain of Il18(+/+) mice, and prevented the onset of NASH. The expression of genes related to these dysfunctions was also affected by recombinant IL-18 administration. In conclusion, this study demonstrated the critical function of IL-18 in lipid metabolism and these findings might contribute to the progress of novel treatments for NAFLD or NASH.

Brain-derived neurotrophic factor immunoreactive vagal sensory neurons innervating the gastrointestinal tract of the rat.

We have determined whether brain-derived neurotrophic factor immunoreactive (BDNF-ir) neurons in the vagal ganglia innervate the gastrointestinal tract. Many BDNF-ir neurons were medium in size and located throughout the jugular and nodose ganglia. When Fluorogold was injected into the wall of the cervical esophagus, many retrogradely Fluorogold-labeled neurons were found in both the jugular ganglion and the nodose ganglion. When Fluorogold was injected into the body of the stomach or applied to the cut end of the subdiaphragmatic vagus nerve, numerous Fluorogold-labeled neurons were found mostly in the nodose ganglion. Double-labeling combining immunohistochemistry for BDNF and retrograde tracing with Fluorogold showed that more than 90% of the neurons in the jugular ganglion and the nodose ganglion projecting to the cervical esophagus contained BDNF-like immunoreactivity. In the cases of both Fluorogold injection into the stomach and Fluorogold application to the subdiaphragmatic vagus nerve, almost all Fluorogold-labeled neurons in the nodose ganglion contained BDNF-like immunoreactivity. These results indicated that almost all vagal sensory neurons located in either the jugular ganglion or the nodose ganglion that innervate the gastrointestinal tract are BDNF-ir neurons.

Expression of adrenergic and cholinergic receptors in murine renal intercalated cells.

Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems.

Origin of efferent fibers of the renal plexus in the rat autonomic nervous system.

To clarify the origin of efferent nerves containing renal plexus, the retrograde neuronal tracing was utilized with a new exact closed injection system with microcapsules. The microcapsule was positioned in the rat left renal plexus, and the capsule was filled with fluoro-gold. Retrograde labeled cells were observed in the ipsilateral sympathetic trunk, especially T12 and T13, and the ipsilateral suprarenal ganglia (SrG). There were no labeled cells in the parasympathetic nuclei in medulla oblongata and sacral cords. These results indicated that the origins of efferent nerves in the rat renal plexus are almost all sympathetic ganglia, such as sympathetic trunk and SrG, and cells in other ganglia may be secondary or accessory innervations.

Calcitonin gene-related peptide immunoreactive sensory neurons in the vagal and glossopharyngeal ganglia innervating the larynx of the rat.

We have examined whether calcitonin gene-related peptide-immunoreactive (CGRP-ir) neurons in the vagal and glossopharyngeal ganglia innervate the larynx. Many CGRP-ir neurons were located mostly in the superior glossopharyngeal-jugular ganglion complex that was fused the superior glossopharyngeal ganglion and the jugular ganglion in the cranial cavity. When Fluorogold was applied to the cut end of the superior laryngeal nerve (SLN) or the recurrent laryngeal nerve (RLN), many Fluorogold-labeled neurons were found in the superior glossopharyngeal-jugular ganglion complex and the nodose ganglion. Double-labeling for CGRP and Fluorogold showed that about 80% of Fluorogold-labeled neurons in the superior glossopharyngeal-jugular ganglion complex expressed CGRP-like immunoreactivity in the case of application to the SLN, and about 50% of Fluorogold-labeled neurons expressed CGRP-like immunoreactivity in the case of the RLN. Only a few double-labeled neurons were found in the nodose ganglion. The number of the Fluorogold-labeled neurons and double-labeled neurons in the superior glossopharyngeal-jugular ganglion complex in the case of the SLN was larger than that in the case of the RLN. These results indicate that sensory information from the larynx might be conveyed by many CGRP-ir neurons located in the superior glossopharyngeal-jugular ganglion complex by way of the SLN and the RLN.

Collateral projections of the dorsal motor nucleus of the vagus nerve to the stomach and the intestines in the rat.

The vagal motor neurons project to the gastrointestinal tract by way of the gastric, celiac and hepatic branches of the vagus trunk. We have examined whether single neurons in the dorsal motor nucleus of the vagus nerve (DMV) have collateral projections to the stomach, the duodenum and the intestines using a double-labeling tracing method. Following application of Fluorogold to the cut end of the accessory celiac branch and injection of cholera toxin subunit b (CTb) into the body of stomach, many Fluorogold- and CTb-labeled neurons were found throughout the DMV. Most CTb-labeled neurons (about 90%) were also labeled with Fluorogold. When Fluorogold was applied to the cut end of the accessory celiac or the gastric branch and CTb was injected into the duodenum, many Fluorogold-labeled neurons and CTb-labeled neurons were found in the DMV. About 20% of CTb-labeled neurons were also labeled with Fluorogold. These results indicate that many neurons in the DMV send collateral projections to both the stomach and the intestines innervated by way of the celiac branch. However, many neurons in the DMV projecting to the duodenum do not project to the stomach or the intestines caudal to the duodenum.

Systemic administration of lipopolysaccharide increases the expression of aquaporin-4 in the rat anterior pituitary gland.

We investigated the effects of lipopolysaccharide (LPS)-induced endotoxemia on the expression of aquaporin-4 (AQP4) in the rat anterior pituitary gland, using the real-time polymerase chain reaction and immunohistochemistry. After intraperitoneal injection of LPS, the level of AQP4 mRNA doubled at 2, 4 and 8 hr. Immunohistochemical analysis showed an increase with time in AQP4 immunostaining in folliculo-stellate cells following LPS injection; the intensity of immunoreactivity peaked at 8 hr. At the same time, some cyst-like structures, formed by AQP4-positive cells, were observed. These findings indicate that LPS induces the expression of AQP4 in the anterior pituitary gland. The present results should provide an important key to elucidate the pathogenesis of the anterior pituitary gland during endotoxemia.

Localization in the vagal ganglia of calcitonin gene-related peptide- and calretinin-immunoreactive neurons that innervate the cervical and the subdiaphragmatic esophagus of the rat.

We have determined the localization of calcitonin gene-related peptide-immunoreactive (CGRP-ir) and calretinin-ir neurons in the vagal ganglia that innervate the cervical or subdiaphragmatic esophagus. Many CGRP-ir neurons were found exclusively in the jugular ganglion located in the cranial cavity. Calretinin-ir neurons were distributed throughout the vagal ganglia. Injection of Fluorogold into the cervical esophagus resulted in many Fluorogold-labeled neurons in the jugular and nodose ganglia. Injection of Fluorogold into the subdiaphragmatic esophagus resulted in many Fluorogold-labeled neurons, with most in the nodose ganglion. In the case of Fluorogold injection into the cervical esophagus, double-labeling combining immunohistochemistry and retrograde tracing showed that about 40% of the Fluorogold-labeled neurons in the jugular ganglion express CGRP-like immunoreactivity, and about 20% of the Fluorogold-labeled neurons in both the jugular and nodose ganglia express calretinin-like immunoreactivity. In the case of injection into the subdiaphragmatic esophagus, only a few Fluorogold-labeled neurons express CGRP-like immunoreactivity or calretinin-like immunoreactivity in the vagal ganglia. These results indicate that the cervical esophagus receives projections from many CGRP-ir neurons in the jugular ganglion and from calretinin-ir neurons in the jugular and nodose ganglia, while the subdiaphragmatic esophagus receives projections from only a few CGRP-ir and calretinin-ir neurons in the vagal ganglia.

Cyclical and patch-like GDNF distribution along the basal surface of Sertoli cells in mouse and hamster testes.

BACKGROUND AND AIMS: In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.