Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons

Catecholaminergic cells are transiently present during development of the fetal murine bowel. These transient catecholaminergic (TC) cells appear at Day E10, but by Day E13 can no longer be detected. In order to evaluate the hypothesis that these cells are the precursors of enteric neurons, we investigated the possibilities that TC cells coexpress neuronal and catecholaminergic markers, that they can be found along the presumed path followed by crest-derived cells migrating to the gut, and that they are proliferating. TC cells were identified immunocytochemically using polyclonal or monoclonal antibodies to tyrosine hydroxylase (TH). At Day E9.5, TH-immunoreactive cells were observed to be present along the wall of the primordial esophagus in lines that extended from the developing nodose ganglia down to the boundary of the stomach. At Day E9.5, TC cells were absent from the remaining foregut. These lines of esophageal TH-immunoreactive cells became continuous with similar cells in the wall of the stomach and duodenum on Day E10. Coincident expression of neurofilament immunoreactivity was seen in all of the esophageal TH-immunoreactive cells present at Day E9.5, as well as in the entire set of esophageal and lower enteric TH-immunoreactive cells present at Day E10 (or later); moreover, at Days E9.5 and E10, all of the neurofilament-immunoreactive cells in the esophagus, stomach, or duodenum were also TH-immunoreactive. In contrast, neurofilament immunoreactivity was not expressed by the endodermally derived pancreatic duct and islet cells, which were also TH-immunoreactive; nor could expression of neurofilament immunoreactivity be detected in the TH-immunoreactive cells of the nodose ganglia. It was not until Day E11 that neurofilament-immunoreactive cells, which did not coexpress TH immunoreactivity (the definitive phenotype of enteric neurons) began to appear in the gut. Vagal axons reached as far distally as the nodose ganglion on Day E9.5, the esophagogastric junction on Day E10, and did not enter the stomach until Day E11. When the vagus nerves reached their level, the TH-immunoreactive cells in the wall of the esophagus came to lie among the nerve fibers. TH-immunoreactive cells are thus present on the pathway ultimately followed by the vagus nerves, but they develop before vagal fibers reach their level. The vagal TH-immunoreactive cells, therefore, are probably not initially migrating on vagal fibers, but appear instead to be overtaken by the descending vagus nerves.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development and persistence of catecholaminergic neurons in cultured explants of fetal murine vagus nerves and bowel

Transient catecholaminergic (TC) cells have been found to appear in the vagal pathway and bowel of fetal mice and rats. It has been proposed that these cells are migrating vagal crest-derived precursors of enteric neurons that lose their catecholaminergic properties when they terminally differentiate. In the current experiments, segments of fetal mouse gut were explanted before (day E9) TC cells or any neural markers could be detected in situ. Tyrosine hydroxylase (TH)-immunoreactive neurons developed in vitro in 4/12 such explants; therefore, cells with a catecholaminergic potential are present in the gut of at least some animals prior to the in situ expression of this phenotype. The neurogenic potential of cells in the vagal pathway was similarly tested by studying cultures of explanted vagus nerves (day E11). These studies revealed that neural precursors were present in the vagi and gave rise in vitro to neurons that displayed acetylcholinesterase (AChE) activity and neuron-specific enolase (NSE) immunoreactivity. A subset of these neural precursors were capable of migrating and formed satellite ganglia at a distance from the explants. Coincident expression of NSE and TH immunoreactivities was observed, indicating that at least some of the neurons that developed in vitro were derived from TC cells. Vagal TC cells, therefore, are neurogenic. Catecholaminergic cells did not disappear from cultured explants of vagus nerves or gut provided that these tissues contained TC cells at the time of explantation. Instead, catecholaminergic neurons developed and persisted in vitro for as long as cultures were maintained. These neurons contained aromatic L-amino acid decarboxylase as well as TH, NSE and neurofilament immunoreactivities. In contrast, if the bowel was explanted after the in situ disappearance of TC cells, catecholaminergic cells did not arise in the cultures. These experiments indicate that the period of time during which a catecholaminergic phenotype is expressed by neural precursors in the fetal vagal pathway and gut is not fixed, but can be changed by altering the environment of the cells as occurs when the bowel is grown in vitro; moreover, contact with non-neuronal cells within the bowel is not by itself sufficient to inactivate catecholaminergic expression. The nature of the signal responsible for loss of the catecholaminergic phenotype in situ remains to be determined; however, the persistence of catecholaminergic expression in vitro should facilitate the investigation of this signal.     

The physiological basis of transplantation of fetal catecholaminergic neurons in the transected spinal cord of the rat

1. Fetal (E.17) rat locus coeruleus and mesencephalic dopaminergic neurons when implanted into the transected spinal cord of the young adult rat survive for periods of longer than four months. Axons of up to 15 mm in length are observed growing from the cell bodies of the implanted neurons. 2. Fluorescent catecholaminergic (presumably dopaminergic) cell bodies are found in the caudal region of the transected, non-implanted spinal cord. 3. After transection of the spinal cord at the middle thoracic region in rats, at different postnatal ages (PN. 0, 7, 14, 21 and 28), there is substantial recovery of motor coordination involving all four limbs in the PN. 0 and PN. 7 groups. Recovery is best in the PN. 7 group. There is almost no recovery in the PN. 28 group, and very little recovery in the PN. 14 and PN. 21 groups. 4. Spinal locomotor generators in rat can, therefore, display a substantial degree of functional autonomy, if the spinal cord is cut before a certain critical stage of development (before PN. 14). These results have interesting implications with regard to current efforts to understand the mechanisms that regulate the spinal locomotor generators in experimental animals, and perhaps in man as well.     

Neurofilament expression in vagal neural crest-derived precursors of enteric neurons

In order to gain insight into the potential role of the enteric microenvironment in the neuronal determination of the neural crest-derived precursor cells of enteric neurons, an attempt was made to ascertain when and where along the migratory route of these cells that they first express neuronal properties. The immunocytochemical detection of the 160-kDa component of the triplet of the chick neurofilament peptides served as a neuronal marker. In addition, neurogenic potential was assessed by growing explants of tissue suspected of containing presumptive neuroblasts in culture or as grafts on the chorioallantoic membrane of chick embryonic hosts. Neurofilament immunoreactivity was first detected in the foregut by Day 4 of development and spread to the hindgut by Day 7. Within the hindgut, development was more advanced within the colorectum than within the more proximal terminal ileum and caecal appendages. This probably reflects the distal-proximal migration of sacral neural crest cells in the postumbilical bowel. The ability of enteric explants to show neuronal development in vitro correlated with whether or not cells containing neurofilament immunoreactivity had reached that segment of gut at the age of explantation. These data suggest that enteric neuronal precursors have already begun to differentiate as neurons by the time they colonize the gut. Prior to the appearance of fibrillar neurofilament immunoreactivity in the foregut, cells that express this marker were found transiently within the mesenchyme of branchial arches 3, 4, and 5. These cells had disappeared from this region by developmental Day 6. The neurogenic potential of branchial arches 3 and 4 was demonstrated by the correlation that was found between the ability of explants of these arches to show neuronal development in vitro and the presence within them of cells that display neurofilament immunoreactivity. No similar neurogenic potential was found in the more rostral branchial arches which lacked the masses of neurofilament-immunoreactive cells. The location of the caudal branchial arches below the migrating vagal neural crest, the transience of the neurofilament immunoreactivity in them, and the coincident transience of their neurogenic potential in vitro, suggested that the masses of neurofilament immunoreactive cells in the caudal branchial arches might be vagal neural crest-derived neuronal precursor cells en route to the pharynx and the rest of the gut. This possibility was supported by the observation of neurofilament immunoreactivity in a subset of cells of the premigratory and early migratory neural crest in the vagal, but not other, regions of the neuraxis prior to the appearance of neurofilament immunoreactivity in the branchial arches. Proliferative expansion of cells with neurofilament immunoreactivity was indicated by the observation of mitotic figures in them. It is suggested that the vagal neural crest cells that populate the ENS are already committed to the neuronal lineage while still in the vagal region of the neuraxis. It is therefore not likely that the enteric microenvironment plays a role in this process.     

Electron-microscopic cytochemistry of the catecholaminergic innervation of TRH neurons in the rat hypothalamus

Summary The catecholaminergic innervation of thyrotropin-releasing hormone (TRH) neurons was examined by use of a combined method of 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline (³H-NA) and immunocytochemistry for TRH in the same tissue sections at the electron-microscopic level.TRH-like immunoreactive nerve cell bodies were distributed abundantly in the parvocellular part of the paraventricular nucleus (PVN), in the suprachiasmatic preoptic nucleus and in the dorsomedial nucleus of the rat hypothalamus. In the PVN, a large number of immunonegative axon terminals were found to make synaptic contact with TRH-like immunoreactive cell bodies and fibers. In the combined autoradiography or 5-OHDA labeling with immunocytochemistry, axon terminals labeled with ³H-NA or 5-OHDA were found to form synaptic contacts with the TRH immunoreactive nerve cell bodies and fibers. These findings suggest that catecholamine-containing neurons, probably noradrenergic, may innervate TRH neurons to regulate TRH secretion via synapses with other unknown neurons in the rat PVN.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Cholecystokinin activates specific enteric neurons in the rat small intestine

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.     

Neuroplasticity and neuroprotection in enteric neurons: Role of epithelial cells

Neurons of enteric nervous system (ENS) regulate intestinal epithelial cells (IEC) functions but whether IEC can impact upon the neurochemical coding and survival of enteric neurons remain unknown. Neuro-epithelial interactions were studied using a coculture model composed of IEC lines and primary culture of rat ENS or human neuroblastoma cells (SH-SY5Y). Neurochemical coding of enteric neurons was analysed by immunohistochemistry and quantitative PCR. Neuroprotective effects of IEC were tested by measuring neuron specific enolase (NSE) release or cell permeability to 7-amino-actinomycin D (7-AAD). Following coculture with IEC, the percentage of VIP-immunoreactive (IR) neurons but not NOS-IR and VIP mRNA expression were significantly increased. IEC significantly reduced dopamine-induced NSE release and 7-AAD permeability in culture of ENS and SH-SY5Y, respectively. Finally, we showed that NGF had neuroprotective effects but reduced VIP expression in enteric neurons. In conclusion, our study identified a novel role for IEC in the regulation of enteric neuronal properties.     

Prospective identification and culture of rat enteric neural stem cells (ENSCs)

Hirschprung’s disease (HD), a very common congenital abnormality in children, occurs mainly due to the congenital developmental defect of the enteric nervous system. The absence of enteric ganglia from the distal gut due to deletion in gut colonization by neural crest progenitor cells may lead to HD. The capacity to identify and isolate the enteric neuronal precursor cells from developing and mature tissues would enable the development of cell replacement therapies for HD. However, a mature method to culture these cells is a challenge. The present study aimed to propose a method to culture enteric neural stem cells (ENSCs) from the DsRed transgenic fetal rat gut. The culture medium used contained 15 % chicken embryo extract, basic fibroblast growth factor, and epidermal growth factor. ENSCs were cultured from embryonic day 18 in DsRed transgenic rat. Under inverted microscope and fluorescence staining, ENSCs proliferated to form small cell clusters on the second day of culture. The neurospheres-like structure were suspended in the medium, and there were some filaments between the adherent cells from day 3 to day 6 of the culture. The neurospheres were formed by ENSCs on day 8 of the culture. Network-like connections were formed between the adherent cells and differentiated cells after adding 10 % FBS. The differentiated cells were positive for neurofilament and glial fibrillary acidic protein antibodies. The present study established a method to isolate and culture ENSCs from E18 DsRed transgenic rats in the terminal stage of embryonic development. This study would offer a way to obtain plenty of cells for the future research on the transplantation of HD.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Electron-microscopic cytochemistry of the catecholaminergic innervation of ACTH-containing neurons in the rat hypothalamic arcuate nucleus

The synaptic relationship between catecholamine terminals and adrenocorticotropic hormone (ACTH)-containing neurons in the arcuate nucleus (AN) of the rat hypothalamus was investigated by electron microscopy, using ACTH immunocytochemistry combined with autoradiography after 3H-dopamine (3H-DA) injection or 5-hydroxydopamine (5-OHDA) uptake in the same tissue section. ACTH-like (ACTH-LI) immunoreactive nerve cell bodies and fibers received synaptic inputs by axon terminals labeled with 3H-DA or 5-OHDA in the AN. This suggests that catecholaminergic neurons, at least DA- and 5-OHDA-containing neurons, may play an important role in the regulation of ACTH secretion or other functions of ACTH neurons via synapses in the AN of the rat hypothalamus.     

Immunocytochemical study of catecholaminergic neurons in grafted mesencephalon transplanted into the hypothalamus of the rat

Mesencephalic fragments from 14 day old embryonic rat brain were transplanted into the third ventricle of adult rats neonatally treated with monosodium glutamate. From two to twelve months after grafting, the implanted tissue was still present in the ventricle and contained TH immunoreactive neurons which displayed a normal appearance at ultrastructural level. While endogenous TH containing neurons were still present in dopaminergic regions of the recipient hypothalamus, grafted mesencephalic fragments could survive and develop. They contained TH immunopositive most probably dopaminergic neurons which are able, in some cases, to innervate the host brain. This model should be of interest in the study of neuroendocrine functions of dopaminergic neurons.     

Bee venom acupoint stimulation increases Fos expression in catecholaminergic neurons in the rat brain

Fos immunocytochemistry was combined with tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH) immunolabeling to examine brainstem catecholaminergic neuronal activation resulting from bee venom (BV) stimulation of the Zusanli acupoint (ST36) in Sprague-Dawley rats. BV injection into the Zusanli acupoint caused increased Fos expression in catecholaminergic neurons located in the hypothalamic arcuate nucleus (Arc), the dorsal raphe (DR), the A5 cell group (A5) and the locus coeruleus (LC). BV acupoint stimulation significantly increased Fos-TH double-labeled neurons in the Arc, LC and DR. Fos-DBH positive neurons were also increased by BV acupoint stimulation in the LC and A5. In contrast BV stimulation of a non-acupoint only increased Fos expression and Fos-TH double-labeled neurons in the Arc. These data indicate that BV acupoint stimulation activates brainstem catecholaminergic neurons and that this activation underlies BV acupoint-induced antinociception.     

Development of catecholaminergic neurons in the human medulla oblongata

Distribution and maturation of catecholaminergic (CA) neurons have been studied by tyrosine hydroxylase immunohistochemistry in the medulla oblongata of human fetuses aged 14.5-25 weeks of gestation. Already at 14.5 weeks, CA neurons were observed in two longitudinally oriented cell clusters, one located ventrolaterally in the area of the lateral reticular and ambiguous nuclei, the other one dorsomedially forming 4 groups related to the dorsal vagal nucleus, the commissural nucleus of the vagus, the nucleus of the tractus solitarius and the area postrema. CA neurons in the area postrema were often found close to blood vessels. Scattered intermediate CA neurons were seen between these two larger clusters. CA neurons still appeared immature exhibiting bipolar morphology with only one or two short stout processes, which hardly branched. At 21 weeks, CA neurons occupied essentially the same location, but had a more mature morphology. Though still bipolar in shape, they had thinner and much longer processes which frequently branched. Both in the ventrolateral and the dorsomedial cell clusters, these processes were frequently lying close to blood vessels. At 25 weeks, CA cells had matured into multipolar neurons with long thin processes forming fine fiber networks in the ventrolateral medulla as well as around and within the dorsal vagal and solitarius nuclei. Only at this stage, a distinct CA fiber tract was seen located in the region of the tractus solitarius. Our results indicate that CA neurons in the human medulla, which are presumably involved in the control of ventilation and blood pressure, though generated rather early during development, mature relatively late.     

Stimulation of the catecholaminergic and serotoninergic neurons in the rat brain by R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane, (-)-BPAP

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane HCl, (-)-BPAP, the recently developed selective and much more potent catecholaminergic/serotoninergic enhancer (CAE/SAE) substance than (-)-deprenyl enhances the performance of midbrain neurons, both in vivo and ex vivo, in a characteristic complex manner, presenting one bell shape dose/concentration effect curve in the low nanomolar range and another at higher micromolar range. For example, 4.7 +/- 0.10 nmol/g wet weight noradrenaline was released within 20 min from the quickly removed locus coeruleus of saline treated rats. This amount was increased 30 min after the subcutaneous administration of 0.0005 mg/kg (-)-BPAP to 15.4 +/- 0.55 nmol/g (P < 0.001). However, following the injection of a hundred times higher, 0.05 mg/kg, dose of (-)-BPAP, the amount of noradrenaline (4.3 +/- 0.25 nmol/g) released from the locus coeruleus did not differ from the control value. In ex vivo experiments, when the isolated locus coeruleus was soaked in an organ bath containing (-)-BPAP, the release of noradrenaline was significantly enhanced from 10(-16) M concentration, reached a peak effect at 10(-13) M concentration, but 10(-10) M (-)-BPAP was ineffective. A significant enhancer effect was detected also in the high concentration range from 10(-8) M, the peak effect was reached at 10(-6) M concentration and 10(-5) M (-)-BPAP was ineffective. (-)-BPAP enhanced in the low concentration range the performance of dopaminergic and serotoninergic neurons with a peak effect at 10(-13) and 10(-12) M concentration, respectively. The results with (-)-BPAP, the highly specific artificial enhancer substance, suggest that (i) high and low affinity "enhancer" receptors may exist in the brain, and (ii) that they may be identified with the recently cloned family of the "trace amine" receptors, activated by beta-phenylethylamine and tryptamine, the prototypes of the endogenous enhancer substances.     

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X₂ and P2X₇ receptors (P2X₂R, P2X₇R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X₂R, P2X₇R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X₂R and P2X₇R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X₂R and P2X₇R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding.     

Cell lineage analysis of pancreatic islet development: glucagon and insulin cells arise from catecholaminergic precursors present in the pancreatic duct

We have previously reported that cells transiently expressing tyrosine hydroxylase (TH), the first enzyme of the catecholamine biosynthetic pathway, are present in the pancreas of mouse embryos from prenatal Day 11 (E11) and that, at E12, some TH cells contain glucagon. Cells containing TH were also found in adults which, unlike the TH cells of embryos, did not contain glucagon (G. Teitelman, T. H. Joh, and D. J. Reis (1981). Proc. Natl. Acad. Sci. 78, 5225). These findings suggested to us that the TH cells of embryonic pancreas were the precursors of glucagon cells of adults. In this study we used immunocytochemical and autoradiographic techniques to determine whether cells containing TH (a) were present in pancreas throughout pre- and postnatal development, (b) were localized to a specific region of the gland, (c) contained insulin at any time, and (d) proliferated. We found that TH cells were present in pancreas throughout life. In embryos, cells containing TH localized only along the pancreatic duct, also contained either glucagon or insulin, and were able to proliferate. In contrast, after birth, the pancreatic duct contained no TH cells. Cells containing TH in postnatal and adult mice also differed from embryonic TH cells in that they were found in all islets, contained insulin but not glucagon, and did not synthesize DNA, and hence did not proliferate. These findings suggest that progenitor cells that contain catecholamines and are present in the pancreatic duct give rise to glucagon and insulin cells of adult islets. They also indicate that the TH-insulin cells of postnatal and adult mice are not stem cells but are postmitotic cells that appear in the islets after birth.     

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.