Phylogeny of enteric serotonergic neurons

Serotonergic neurons have previously been identified in the enteric nervous systems of humans, subhuman primates, rodents, and rabbits. The distribution of enteric serotonergic neurons in lower vertebrates was examined in order to determine if these neurons are restricted to mammals and, if they are found more generally amongst vertebrates, when they first appear in vertebrate phylogeny. Since mammalian enteric serotonergic neurons take up ³H-serotonin by a highly specific mechanism, the radioautographic demonstration of axonal uptake of ³H-serotonin was used as the primary tool in looking for these neurons. As controls, conditions known to interfere with ³H-serotonin uptake by mammalian enteric neurons were also examined. These controls included incubation with 10 μ fluoxetine, a specific antagonist, incubation in Na⁺-free medium, and incubation in the presence of a 100-fold excess of nonradioactive serotonin. Radioautographic labeling had to be absent or greatly reduced under all three control conditions for labeling by ³H-serotonin to be considered specific. Labeled enteric axons were found in cyclostomes (hagfish), teleosts (goldfish), and amphibia (bullfrog) but not in tunicates (sea squirt; sea vase) or echinoderms (sea cucumber). In addition, the serotonin concentration was measured in the intestine of two vertebrates, hagfish and goldfish, that do not have serotonin-containing enterochromaffin cells. Serotonin was found in both; in hagfish, the amine concentration was highest in preparations of muscularis externa containing the myenteric plexus. It is concluded that enteric serotonergic neurons arose early in vertebrate evolution, possibly in an ancestral chordate resembling amphioxus, although probably not in more primitive prevertebrates, and that they are a general feature of the vertebrate bowel.     

Early postnatal administration of 5,7-DHT: Effects on serotonergic neurons and terminals

Serotonergic neurons throughout the brain were destroyed by early postnatal treatment of rats with an intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT), as demonstrated with biochemical measurements of serotonin and immunocytochemical localization of serotonin-containing neurons. Using these methods, it was shown that approximately 75–98% of serotonergic nuerons underwent cell death in rats which were treated on day 3. In contrast, intracisternal administration of 5,7-DHT in adult rats led to the loss of distal serotonergic terminals without apparent loss the cell bodies. Desipramine prevented significant effects of 5,7-DHT on noradrenergic neurons.     

Effects of intracerebral injections of quinolinic acid on serotonergic neurons in the rat brain

The effects of intrastriatal and intrahippocampal injections of the excitotoxic amino acid, quinolinic acid (QUIN), were examined in the rat using immunohistochemical and neurochemical techniques. Serotonin and 5-hydroxyindoleacetic acid measurements at 90 min, 6 h, 4 and 11 days following QUIN administration revealed highly elevated levels of the metabolite in the injected nuclei, with peak increases occurring after 4 days. Serotonin levels remained largely unchanged over the same time period. Direct visualization of hippocampal serotonergic fibers by immunohistochemistry demonstrated morphological changes (varicosities, swellings) in otherwise undamaged serotonin-positive afferents 4 days following a local QUIN injection. Hippocampal serotonin turnover was assessed at 4 days after an intrahippocampal QUIN-application: following inhibition of aromatic amino acid decarboxylase, the accumulation of 5-hydroxytryptophan was twice as rapid in QUIN-lesioned hippocampi as in controls. Dose-response relationships, examination of brain regions distant from the two injection sites and the temporal sequence of the changes described here suggest a close association between QUIN-induced neuronal degeneration and alterations in the serotonergic system.     

Effects of tobacco and cigarette smoke extracts on serotonergic raphe neurons in the rat

Tobacco components other than nicotine might participate in the behavioural effects of smoking. In this study, in-vivo recordings of serotonergic dorsal raphe neurons were performed in the anesthetized rat, whereas tobacco extracts, cigarette smoke extracts, nicotine, nornicotine or anabasine were intravenously injected. All substances inhibited the neurons, and all inhibitions were completely blocked by the nicotine receptor antagonist mecamylamine. The effects of the extracts were much more potent than those of individual substances. These results support the hypothesis that the acute inhibition of serotonin neurons by tobacco compounds is completely related to an effect on nicotine receptors. Tobacco extracts and tobacco smoke extracts may be useful tools for the study of the effects of central effects of smoking.     

Effects of GM1 ganglioside on developing and mature serotonin and noradrenaline neurons lesioned by selective neurotoxins

The effect of exogenous GM1 ganglioside on selective neurotoxin-induced lesions of serotonin (5-HT) and noradrenaline (NA) neurons in both the central and peripheral nervous systems has been investigated in developing and adult rats and mice by employing neuro- and histochemical techniques. 5,7-Dihydroxytryptamine (5,7-HT) was used to lesion 5-HT neurons, and 6-hydroxydopamine (6-OH-DA) was used to lesion NA neurons. In most lesion models investigated the neurotoxin causes primarily an axonal nerve terminal damage without notably affecting the perikarya. There was no evidence indicating that GM1 interferes with the primary and direct neurodegenerative actions of 5,7-HT or 6-OH-DA on 5-HT and NA nerve terminals, respectively. In all lesion models GM1 had in the chronic stage a counteracting effect on the neurotoxin-induced nerve terminal lesion or enhanced regrowth. The present results are compatible with the view that GM1 has a regrowth-stimulating effect and/or protective actions against secondary retrograde degeneration following the initial nerve terminal lesion induced by the neurotoxin.     

Ontogeny of serotonergic neurons in Aplysia californica

Although the identity, projection patterns, and functions of serotonergic neurons in juvenile and adult Aplysiaare relatively well understood, little is known about the development of these cells. We have used light and electron microscopic immunocytochemistry to investigate the genesis, differentiation, identity, and fate of the serotonergic cells in the embryonic, larval, and metamorphic stages of the life cycle of Aplysia. The results indicate that the first serotonergic cells emerge at midembryogenesis and that a total of five cells makes up the entire serotonergic system by hatching. These cells are part of a newly discovered ganglion in Aplysia, called the apical ganglion. This serotonergic system of five cells remains essentially intact throughout larval development. The apical ganglion, together with its serotonergic cells, is resorbed at metamorphosis. A distinct set of serotonergic cells, which begins to emerge by the end of the larval period, is rapidly elaborated during the metamorphic and early juvenile periods to form the adult serotonergic system. These results support the view that the larval and adult forms of the Aplysia nervous system consist of entirely distinct sets of serotonergic cells, each adapted to the stage-specific morphological and behavioral characteristics of the animal. J. Comp. Neurol. 386:477-490, 1997. © 1997 Wiley-Liss, Inc.     

Establishment and properties of separate cultures of enteric neurons and enteric glia

In this paper methods are described for the preparation of two types of culture derived from myenteric explants: (a) highly enriched neuronal cell cultures, and (b) purified glial cells (greater than 98%). Both procedures combine the technique of antibody complement-mediated cytolysis with the use of an antimitotic agent. Immunohistochemical methods were used to compare the purified cells to their counterparts in mixed cultures (see accompanying paper). Antibodies to the glycoprotein Thy-1 and the monoclonal antibody A2B5 which recognizes gangliosides, labelled the cell surface of all enteric neurons in enriched cultures while subpopulations of the neurons expressed the Leu 7 carbohydrate epitope, the neurotransmitter 5-hydroxytryptamine and the neuropeptides substance P, methionine-enkephalin and vasoactive intestinal polypeptide. Autoradiographic experiments show that a subpopulation of enriched neurons exhibit high-affinity uptake sites for gamma-[3H]aminobutyric acid (GABA). All purified enteric glia continue to express the calcium binding protein S100, the basement membrane glycoprotein laminin and the antigens recognized by the A2B5 antibody, and subpopulations of glia are labelled by the monoclonal antibodies LB1 which binds to GD3 gangliosides, and Leu 7. Thus enteric neurons and glia can survive independently of each other and express molecular properties which are present in cultures normally containing both cell types.     

Fructose consumption impairs serotonergic signaling in the murine enteric nervous system

The intake of free fructose has increased substantially since the development of high‐fructose corn syrup. This has not only been associated with metabolic disorders but recent evidence also indicates that chronic fructose consumption can affect neuronal and cognitive function. In this study we investigated the effects of fructose consumption on serotonergic signaling and neuronal activity in the mouse submucous plexus. Male mice were put on a control or fructose (23% solution) diet for 6 weeks or were assigned to a recovery group that received normal water (2 weeks) after 4 weeks of fructose. At the end of the diet, gene expressions and enteric neuronal activity, after depolarization with high K⁺ and 5‐HT, were measured using Ca²⁺ imaging and RT‐qPCR, respectively. Even in the lack of gain weight and the absence of changes in duodenal permeability, the total number of 5‐HT‐responding neurons and the depolarization and 5‐HT‐evoked Ca²⁺ amplitudes were significantly lower after fructose consumption. Expression of synaptobrevin Ca_V2.1 and Ca_V2.2 mRNA did not differ after fructose intake; however, Ca_V2.1 mRNA levels were significantly higher in the recovery animals. SERT mRNA concentration, isolated from submucosal plexus containing mucosal epithelium, was significantly decreased after fructose consumption. Chronic fructose consumption impairs serotonergic signaling in the mouse submucous plexus, prior to weight gain and detectable intestinal permeability problems.     

Aquaporin-4 water channels in enteric neurons

Aquaporin-4 is a water channel predominantly found in astrocytes in the central nervous system and is believed to play a critical role in the formation and maintenance of the blood-brain barrier and in water secretion from the brain. As enteric glial cells were found to share several similarities with astrocytes, we hypothesized that enteric glia might also contain aquaporin-4. We used immunohistochemistry to identify aquaporin-4 in the myenteric and submucosal plexuses of the mouse and the rat colon. We found that subpopulations of neurons in both enteric plexuses were positively labeled for human aquaporin-4. Double staining of the enteric ganglia with antibodies to the neuronal marker neurofilament-heavy chain 100 and to aquaporin-4 showed that a minority of myenteric neurons were aquaporin-4 positive (about 12% in the mouse and 13% in the rat). In contrast, in the submucosal plexus significant numbers of neurons were positive for aquaporin-4 (about 79% in both the mouse and the rat). Double labeling for aquaporin-4 and for the glial marker glial fibrillary acidic protein verified that glial cells were not immunoreactive to aquaporin-4. We further confirmed our findings with additional aquaporin-4 antibodies and Western blot analysis. We found that, in addition to expressing aquaporin-4, the myenteric plexus and, to a greater extent, the submucosal plexus both expressed aquaporin-1. We conclude that neurons rather than glial cells contain aquaporin-4 in the colonic enteric plexuses. It is known that submucosal neurons control transport processes in the intestinal mucosa, and the high percentage of aquaporin-4-positive submucosal neurons suggests that aquaporin-4 contributes to this function.     

Neuropeptides mark functionally distinguishable cholinergic enteric neurons

Subpopulations of physiologically identified cholinergic enteric neurons in cell culture contain somatostatin (SOM)- or vasoactive intestinal peptide (VIP)- like immunoreactivity (LIR). These subpopulations differ in their synaptic effects on other neurons: cholinergic neurons that contain SOM-LIR cause fast nicotinic excitatory postsynaptic potentials (EPSPs) that have significantly larger amplitudes than do EPSPs caused by cholinergic neurons that lack SOM-LIR. Cholinergic neurons containing VIP-LIR cause slow non-cholinergic depolarizations in addition to fast nicotinic EPSPs. These findings are the first correlation between neuropeptide content and functional differences in the synaptic effects of subpopulations of cholinergic enteric neurons.     

Sensory neurotoxins: Chemically induced selective destruction of primary sensory neurons

Neonatal capsaicin treatment has been shown to cause selective degeneration of chemosensitive primary sensory neurons involved in the mediation of chemogenic pain and in neurogenic inflammatory responses. In the present study the neurotoxic effect of capsaicin congeners was investigated in the newborn rat. Some quantitative data on the selective neurotoxic action of capsaicin are also reported. Electron microscopy indicates that some pungent congeners of capsaicin also induce the selective degeneration of type ‘B’ sensory ganglion cells. At high doses the distribution pattern of axon terminal degeneration within the spinal cord and brain stem was equivalent to that observed after neonatal capsaicin treatment. The neurotoxic potency of capsaicin congeners, unlike desensitizing activity, is closely related to the sensory irritant property of these compounds. It is concluded that primary sensory neurons degenerating after the administration of these capsaicin congeners may correspond to substance P-containing chemosensitive primary sensory neurons involved in the transmission of nociceptive impulses.     

Types of neurons in the enteric nervous system

This paper, written for the symposium in honour of more than 40 years' contribution to autonomic research by Professor Geoffrey Burnstock, highlights the progress made in understanding the organisation of the enteric nervous system over this time. Forty years ago, the prevailing view was that the neurons within the gut wall were post-ganglionic neurons of parasympathetic pathways. This view was replaced as evidence accrued that the neurons are part of the enteric nervous system and are involved in reflex and integrative activities that can occur even in the absence of neuronal influence from extrinsic sources. Work in Burnstock's laboratory led to the discovery of intrinsic inhibitory neurons with then novel pharmacology of transmission, and precipitated investigation of neuron types in the enteric nervous system. All the types of neurons in the enteric nervous system of the small intestine of the guinea-pig have now been identified in terms of their morphologies, projections, primary neurotransmitters and physiological identification. In this region there are 14 functionally defined neuron types, each with a characteristic combination of morphological, neurochemical and biophysical properties. The nerve circuits underlying effects on motility, blood flow and secretion that are mediated through the enteric nervous system are constructed from these neurons. The circuits for simple motility reflexes are now known, and progress has been made in analysing those involved in local control of blood flow and transmucosal fluid movement in the small intestine.     

Intraspinal transplants of serotonergic neurons in the adult rat

Adult male Sprague-Dawley rats were made paraplegic by a complete transection of the spinal cord at lower thoracic level. One week later they were transplanted, below the level of the section, with a cell suspension prepared from the raphe region of 14-day embryos. After survival periods of 10 days to 1 year, the animals were sacrificed and the spinal cord processed for the immunocytochemical detection of 5-HT. Axons from grafted cells grew extensively into the grey matter of the host, and established axosomatic and axodendritic synapses in the anterior horn and intermediolateral column, similar to those of the intact animal. In addition, a group of transplanted animals was tested for sexual reflexes which are under the control of serotonin. It was found that ejaculation reflexes, which are absent in paraplegic rats, are restored in transplanted animals.     

Activity of medullary serotonergic neurons in freely moving animals

In the mammalian brain, serotonergic neurons in the medulla (n. raphe magnus, obscurus, and pallidus) send dense projections into the spinal cord, especially to the dorsal horn, intermediolateral column, and ventral horn. We have conducted a series of studies examining the single unit activity of these neurons in behaving cats. The experiments were directed at determining whether changes in unit activity were related to pain (n. raphe magnus), autonomic activity (n. raphe obscurus and pallidus), or motor activity (n. raphe obscurus and pallidus). The strongest relationship was between neuronal activity and motor output, especially tonic and repetitive motor activity. We hypothesize that the primary functions of this motor-related activity are to facilitate motor output, suppress processing of some forms of afferent activity, and to coordinate autonomic functioning with the current motor demand.     

Estrogen enhances light-induced activation of dorsal raphe serotonergic neurons

The serotonergic system has been implicated in the modulation of physiological processes including circadian rhythms, learning, memory, mood and food intake. In females, cessation of ovarian function produces deleterious changes in all of these processes and estrogen treatment often ameliorates these conditions. Estrogen may produce these effects by acting on the midbrain raphe, an estrogen-sensitive region that receives direct projections from sensory systems. Here we examined the ability of estradiol to modulate neuronal responses of neurons within raphe nuclei to photic stimulation. Ovariectomized rats treated with estradiol or cholesterol were killed 1 h after the normal onset of light (Zeitgeber time 0) or after a 2-h phase advance (Zeitgeber time 22). In a second study, estradiol-treated ovariectomized rats under constant dark conditions were exposed to light 2 h before the subjective onset of circadian time [(CT)22] and killed 1 h later (CT23). The brains from all animals were processed for Fos and/or serotonin (5-HT) immunocytochemistry. Comparisons showed that the phase shift increased Fos immunoreactivity in all dorsal raphe nucleus (DRN) regions. Although estradiol did not alter the overall number of Fos-positive nuclei, it significantly increased the number of Fos/5-HT double-labelled cells in the medial and lateral DRN. In contrast, neither a phase shift nor estradiol altered the number of Fos-immunoreactive cells or the proportion of Fos-positive 5-HT cells in the median raphe nucleus. Results reveal that the DRN 5-HT system responds to changes in the light : dark cycle and that these responses are modulated by estrogen.     

Denervation of serotonergic fibers in the hippocampus induced a trophic factor which enhances the maturation of transplanted serotonergic neurons but not norepinephrine neurons

The trophic effects of specific denervation on the growth and survival of fetal serotonergic (5-HT) or norepinephrinergic (NE) neurons grafted into the hippocampus were assessed by means of two transplantation paradigms. In the first, fetal raphe cells (containing 5-HT neurons) were transplanted into the control hippocampus. In the second, the transplantation was performed 2 weeks after the specific removal of 5-HT afferents to the hippocampus with 5,7-dihydroxytryptamine (5,7-DHT). We found that a month after transplantation, the number of 5-HT immunoreactive neurons was not significantly different between the two experimental paradigms. However, transplanted raphe neurons had 400% more 5-HT synaptosomal high-affinity uptake and 380% higher content of 5-HT in the hippocampus with prior 5,7-DHT lesion than in control hippocampus. Furthermore, immunocytochemistry showed that the transplanted 5-HT neurons had denser processes and varicosities in the hippocampus with lesion than in control hippocampus. The somatic area of the neurons with these denser processes and varicosities was 42% larger than that of control group. A greater 5-HT level could be achieved if transplanted neurons in the control hippocampus were treated with the supernatant extracted from the hippocampus with 5,7-DHT lesion. In contrast, the NE level of the implanted fetal locus ceruleus (containing NE neurons) was not significantly higher in the 5-HT denervated hippocampus than in control hippocampus a month after transplantation. These results suggest that 5-HT denervation in the hippocampus induces a trophic substance which promotes the maturation rather than survival of 5-HT neurons but not NE neurons.     

The experimental study of the damage of environmental neurotoxins on the cultured rat dopaminergic neurons

Objective To establish the culture system of rat dopaminergic neurons. and to determine whether Paraquat and Dieldrin selectively destroy cultured rat dopaminergic neurons respectively. Methods The cultured rat dopaminergic neurons were treated for 24h with Paraquat and Dieldrin(0.001 to 100 μ mol/L) respectively, Data were expressed as percentage of surviving TH-positive(TH+) cells and other cells per culture dish. Results Paraquat was not effective in selectively destroying TH+ neurons. Dieldrin (1 μ mol/L) selectively decreased the number of TH+ neurons without affecting other cells. The EC50 of Dieldrin on TH+ neurons was 27.6 l mol/L. Conclusion: Paraquat can not selectively destroy dopaminergic neurons in culture. Dieldrin (1 μ mol/L) can selectively destroy the dopaminergic neurons in culture, which make it a potential etiological agent for PD. The possible parkinsonogenic effect of Dieldrin is deserved for further investigation.     

Alpha-methylserotonin, a substitute transmitter for serotonergic neurons.

1. Administration to rats of alpha-methyltryptophan (AMTP) gives rise to alpha-(AM5HT) in the brain along with a decrease of cerebral 5HT. 2. Analysis of fractions prepared from brains of AMTP-injected rats shows that AM5HT occurs mainly in the synaptosomes. 3. The synaptosomal content of AM5HT in proportion to the total AM5HT in the brain represents the same ratio as for the corresponding fractions of 5HT.