Serotonergic Fibers Research Articles

Aging is accompanied by a subfield-specific reduction of serotonergic fibers in the tree shrew hippocampal formation

The hippocampal formation is a crucial structure for learning and memory, and serotonin together with other neurotransmitters is essential in these processes. Although the effects of aging on various neurotransmitter systems in the hippocampus have been extensively investigated, it is not entirely clear whether or how the hippocampal serotonergic innervation changes during aging. Rat studies, which have mostly focused on aging-related changes in the dentate gyrus, have implied a loss of hippocampal serotonergic fibers. We used the tree shrew ( Tupaia belangeri), an intermediate between insectivores and primates, as a model of aging. We applied immunocytochemistry with an antibody against serotonin to assess serotonergic fiber densities in the various hippocampal subfields of adult (0.9–1.3 years) and old (5–7 years) tree shrews. Our results have revealed a reduction of serotonergic fiber densities in the stratum radiatum of CA1 and CA3, and in the stratum oriens of CA3. A partial depletion of serotonin in the hippocampal formation, as can be expected from our current observations, will probably have an impact on the functioning of hippocampal principal neurons. Our findings also indicate that the rat and the tree shrew hippocampal serotonergic innervation show some variations that seem to be differentially affected during aging.

5-Hydroxytryptamine 2C receptors on pudendal motoneurons innervating the external anal sphincter

The aim of this study was to determine the localization of 5-hydroxytryptamine 2C (5-HT 2C) receptors on the motoneurons innervating the external anal sphincter (EAS) of male rats. Motoneurons were retrogradely labeled after percutaneous intramuscular injection of Fluorogold (FG) into the EAS. Using fluorescent immunohistochemistry, FG-positive EAS motoneurons that were immunoreactive for the 5-HT 2C receptor (5-HT 2C-IR) were targeted for specific examination with widefield microscopy or confocal laser scanning microscopy with spectral separation. Widefield microscopy revealed distributions of FG-positive EAS motoneurons in the L5–S1 gray matter corresponding to the dorsomedial cell group. 5-HT 2C-IR positive cells were distributed in the intermediolateral cell column and the ventral horn. Ventral horn 5-HT 2C-IR labeling included the dorsomedial cell group as well as the dorsolateral, ventromedial and ventrolateral areas. Confocal analysis of FG-positive EAS motoneurons and 5-HT 2C-IR positive motoneuron profiles adjacent to EAS motoneurons that were not labeled with FG but presumably innervate the bulbospongiosus muscle confirmed that EAS motoneurons were immunopositive for the 5-HT 2C receptor. These data suggest that previously identified descending serotonergic immunopositive fibers observed terminating on EAS motoneurons might mediate their input through the activation of 5-HT 2C receptors.

105 Mdma (ECSTASY) Causes Neurodegeneration in the Developing Rat Brain

MDMA (3,4-methylenedioxymethamphetamine) causes damage to fine serotonergic fibers and apoptotic neurodegeneration in the adult rat brain. But little is known about the toxic effects of MDMA in the developing brain. The incidence of MDMA abuse is particularly high in adolescents. But the prevalence of MDMA use in woman of child-bearing age and the consequences on their offsprings are unknown. For this reason we administered MDMA (20 – 60 mg/kg) i.p. To 7-day-old rats. Rats treated with normal saline alone served as controls. Pups were sacrificed 24 hrs after injection and brain sections were examined histologically by means of De Olmos cupric silver staining to visualize degenerating cells. MDMA doses of 60 mg/kg induced significant apoptotic neurodegeneration. To explore pathogenetic mechanisms, we investigated the impact of MDMA on the neurotrophine BDNF (brain derived neurotrophic factor). Pups were sacrificed at defined time points (2, 6, 12 and 24 hrs) after injection and brains were processed for RT-PCR, real-time PCR and Western-Blot analysis. mRNA levels for BDNF were upregulated at 6 hrs in animals receiving MDMA compared to controls and even increased at 12 and 24 hours. real-time PCR analysis of the thalamus revealed similar pattern of upregulation of BDNF mRNA transcription. Determined by immunoblot protein expression of BDNF also showed an increase at 12 and 24 hours. These preliminary data suggest that a single injection of MDMA causes neurodegeneration in the neonatal rat brain and that higher doses are required for similar toxicity compared to adult rats. The upregulation in the expression of brain derived neurotrophic factor (BDNF) is may be an important mechanism that lead to survival of neuronal cells in the developing brain.

The Key Role of Medullary 5-HT3 Receptors in the Serotonin-Mediated Neural Control of Cardiovascular Function.

The baroreceptor reflex plays a crucial role in the homeostatic control of cardiovascular parameters. In the central nervous system, the nucleus of the tractus solitarius (NTS) is critically involved in cardiovascular reflex control because it is both the first site of termination of glutamatergic baroreceptor afferent fibres and an important integrative area for the sensory afferent signals reaching the brainstem. In addition to glutamate, the NTS contains numerous neurotransmitters that could participate in the modulations of the baroreceptor reflex sensitivity which occur under various physiological conditions. In particular, a large body of evidence indicates that serotonin plays a modulatory role in the central control of blood pressure, especially at the level of the NTS, which is innervated by both central and peripheral serotonergic fibres. Indeed, serotonin exerts multiple cardiovascular influences through the activation of several receptors in the NTS. Actually, the NTS is the central area endowed with the highest density of serotonin3 (5-HT3) receptors whose stimulation triggers all the adaptive cardiovascular changes normally associated with behavioural responses to various stressful conditions. In this review, we first assess the current knowledge about the mechanisms underlying the cardiovascular effects of the specific activation of serotonergic receptors in the NTS. Secondly, we describe evidence that, in the NTS, 5-HT3 receptors play a key role in one of the crucial homeostatic responses that characterise the defence reaction: the inhibitory modulation of the parasympathetic cardiac component of the baroreceptor reflex. The possible functional interactions of 5-HT3 receptors with GABAA, NK1 and NMDA receptors within the NTS are also discussed.

Serotonergic hyperinnervation into the dopaminergic denervated striatum compensates for dopamine conversion from exogenously administered l-DOPA

The aim of this study was to determine whether raphe-striatal serotonergic neurons of adult rats with extensive nigro-striatal dopaminergic denervation are induced by injection of exogenous l-DOPA to contain dopamine. Double-labeling immunofluorescence study was conducted. In the lesioned striatum of rats that received l-DOPA, serotonergic hyperinnervation was observed, and dopamine was detected in serotonergic varicose fibers. These findings suggest that striatal serotonergic hyperinnervation can compensate for the lost function of dopaminergic neurons.

Diminished serotonergic innervation of adult medial prefrontal cortex after 6-OHDA lesions in the newborn rat

The development of the serotonergic (5HT) and dopaminergic (DA) systems may contribute to the onset of psychotic disorders during late adolescence and early adulthood. Previous studies in our laboratory have suggested that these systems may compete for functional territory on neurons during development, as lesions of the serotonergic system at postnatal day 5 (P5) result in an increase in the density of dopaminergic fibers in rat medial prefrontal cortex (mPFC). In the present study, the dopaminergic system of P5 rats was lesioned with intracisternal injections of 6-hydroxydopamine (6-OHDA). Quantification of serotonin-immunoreactivity (5HT-IR) in mPFC at adulthood (P70) revealed a significant decrease in fiber density within layers II and III of the Cg3 subdivision of mPFC in lesioned rats compared to sham controls. We propose that the decrease in serotonergic fibers in mPFC in response to a neonatal depletion of dopamine may be due to the loss of a trophic effect of this system on 5HT neurons and/or fibers during development. Taken together with previous findings, our data suggest that there may be an “inverse trophic” relationship between the cortical DA and 5HT systems whereby dopamine facilitates the ingrowth of 5HT fibers, while serotonin suppresses the ingrowth of DA fibers. We present a model based on neurotrophic interactions at the cortical and brainstem levels that could potentially explain these unexpected results.

964. Adenoviral-Mediated Expression of Brain-Derived Neuroptrophic Factor or Neurotrophin-3 Promotes Sprouting of Lesioned Serotonergic Axons in the Central Nervous System

We tested whether adenoviral-mediated expression of the neurotrophins Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), or Nerve Growth Factor (NGF) is sufficient to induce regeneration of lesioned serotonergic axons in the cerebral cortex. A single, subcutaneous injection of the neurotoxin p-chloroamphetamine (PCA, 9mg/kg) selectively degenerates the majority of serotonergic axons in the rat cortex while sparing the cell bodies located in the Raphe Nuclei of the brain stem. At this concentration of PCA a small number of axons remain in the cortex, providing potential targets for inducing axon growth. To test whether cortical expression of neurotrophins would elicit a regenerative sprouting response from the remaining cortical serotonergic axons, 107 pfu of adenoviral vectors carrying the genes for BDNF, NT-3, NGF, or eGFP were directly injected into rat frontal cortex 5 days after a subcutaneous injection of PCA. Injections of 107pfu of adenovirus only elicited an acute immune response that was tightly confined to the site of needle injection, similar to PBS controls. The adenoviral constructs were designed under the control of the human elongation factor promoter, EF|[alpha]|, and contained the NGF signal sequence to target the neurotrophins for secretion into the extracellular space. Local concentrations of BDNF, NT-3 or NGF were significantly higher near the injection sites of Adv.BDNF, Adv.NT-3 or Adv.NGF, respectively. Nine days after vector injections, serotonin concentrations were over 50% higher surrounding the injection sites of Adv.BDNF and Adv.NT-3 versus Adv.eGFP control, indicating serotonergic reinnervation of the lesioned cortex. Confocal micrographs of immunohistochemical localization of serotonin in the cortex revealed a four-fold increase in the density of serotonergic fibers surrounding the injection sites of Adv.BDNF and Adv.NT-3 compared to the control vector, Adv.eGFP. In contrast, there was no difference in serotonergic fiber density between Adv.NGF and control. These data demonstrate that localized adenoviral-mediated expression of BDNF or NT-3, but not NGF, is sufficient to promote compensatory sprouting of serotonergic axons to reinnervate the cortex following an experimental neurotoxic injury.

Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb

The centrifugal systems innervating the olfactory bulb are important elements in the functional regulation of the olfactory pathway. In this study, the selective innervation of specific glomeruli by serotonergic, noradrenergic and cholinergic centrifugal axons was analyzed. Thus, the morphology, distribution and density of positive axons were studied in the glomerular layer of the main olfactory bulb of the rat, using serotonin-, serotonin transporter- and dopamine- β-hydroxylase-immunohistochemistry and acetylcholinesterase histochemistry in serial sections. Serotonin-, serotonin transporter-immunostaining and acetylcholinesterase-staining revealed a higher heterogeneity in the glomerular layer of the main olfactory bulb than previously reported. In this sense, four types of glomeruli could be identified according to their serotonergic innervation. The main distinctive feature of these four types of glomeruli was their serotonergic fibre density, although they also differed in their size, morphology and relative position throughout the rostro-caudal main olfactory bulb. In this sense, some specific regions of the glomerular layer were occupied by glomeruli with a particular morphology and a characteristic serotonergic innervation pattern that was consistent from animal to animal. Regarding the cholinergic system, we offer a new subclassification of glomeruli based on the distribution of cholinergic fibres in the glomerular structure. Finally, the serotonergic and cholinergic innervation patterns were compared in the glomerular layer. Sexual differences concerning the density of serotonergic fibres were observed in the atypical glomeruli (characterized by their strong cholinergic innervation). The present report provides new data on the heterogeneity of the centrifugal innervation of the glomerular layer that constitutes the morphological substrate supporting the existence of differential modulatory levels among the entire glomerular population.

Serotonergic fiber sprouting to external anal sphincter motoneurons after spinal cord contusion

The present study analyzed the anatomical plasticity of serotonergic immunoreactive projections to external anal sphincter (EAS) motoneurons, and the behavioral plasticity of EAS reflexes, penile erection, and locomotion in rats with spinal contusion injury (SCI) or complete spinal cord transection (TX). Electromyographic activity of the EAS, penile erection latency, and BBB locomotor score exhibited parallel recovery over the 6-week recovery period after contusion SCI. This pattern of recovery was not observed in TX animals. While locomotor scores demonstrated a small increase after TX, erectile and anorectal function remained at abnormal levels established immediately after injury. Serotonergic immunofluorescent (5-HT-IF) staining at the lesion site identified a small number of fibers spared after SCI that may provide a substrate for functional recovery. Pixel density measurements of 5-HT-IF in the vicinity of retrogradely labeled EAS and unlabeled pudendal motoneurons necessary for penile erection provide indirect evidence of serotonergic sprouting that parallels the observed functional recovery in animals with SCI. No 5-HT-IF was detected caudal to the injury site in TX animals. These studies indicate: (1) lumbosacral eliminative and reproductive reflexes provide a valid means of studying the mechanisms of post-SCI plasticity; (2) the similar recovery curves suggest similar return of descending control, perhaps through sprouting of descending serotonergic fibers; (3) the observed deficits after TX likely represent the permanent removal of descending inhibition and reflect reorganization of segmental circuitry.

Combining Schwann Cell Bridges and Olfactory-Ensheathing Glia Grafts with Chondroitinase Promotes Locomotor Recovery after Complete Transection of the Spinal Cord

Numerous obstacles to successful regeneration of injured axons in the adult mammalian spinal cord exist. Consequently, a treatment strategy inducing axonal regeneration and significant functional recovery after spinal cord injury has to overcome these obstacles. The current study attempted to address multiple impediments to regeneration by using a combinatory strategy after complete spinal cord transection in adult rats: (1) to reduce inhibitory cues in the glial scar (chondroitinase ABC), (2) to provide a growth-supportive substrate for axonal regeneration [Schwann cells (SCs)], and (3) to enable regenerated axons to exit the bridge to re-enter the spinal cord (olfactory ensheathing glia). The combination of SC bridge, olfactory ensheathing glia, and chondroitinase ABC provided significant benefit compared with grafts only or the untreated group. Significant improvements were observed in the Basso, Beattie, and Bresnahan score and in forelimb/hindlimb coupling. This recovery was accompanied by increased numbers of both myelinated axons in the SC bridge and serotonergic fibers that grew through the bridge and into the caudal spinal cord. Although prominent descending tracts such as the corticospinal and reticulospinal tracts did not successfully regenerate through the bridge, it appeared that other populations of regenerated fibers were the driving force for the observed recovery; there was a significant correlation between numbers of myelinated fibers in the bridge and improved coupling of forelimb and hindlimb as well as open-field locomotion. Our study tests how proven experimental treatments interact in a well-established animal model, thus providing needed direction for the development of future combinatory treatment regimens.

Anti-CD11d Integrin Antibody Treatment Restores Normal Serotonergic Projections to the Dorsal, Intermediate, and Ventral Horns of the Injured Spinal Cord

Spinal serotonergic pathways provide inhibitory and excitatory modulation of sensory, autonomic, and motor processing. After spinal cord injury (SCI), the acute inflammatory response is one process that damages descending pathways. Increases in serotonergic fiber density in spinal segments rostral and decreases caudal to the lesion have been observed previously and may contribute to neuropathic pain and motor dysfunction associated with SCI. We investigated the effect of an acute anti-inflammatory treatment on the density of serotonergic fibers rostral and caudal to a thoracic SCI lesion. This treatment, a monoclonal antibody to the CD11d subunit of the leukocyte CD11d/CD18 integrin, limits the trafficking of neutrophils and macrophages into the SCI site. In the dorsal horn, after treatment, the typically increased serotonin immunoreactivity rostral to injury was reduced, whereas that caudal to the lesion increased toward normal. Coincidently, mechanical allodynia in the dorsal trunk and hindpaws was significantly reduced. Increased serotonergic fiber density below the lesion also occurred in the intermediolateral cell column and ventral horn of treated rats, relative to controls. Improved locomotor recovery paralleled this increased serotonin. The treatment increased compact myelin in and near the lesion epicenter and increased serotonergic fiber bundles coursing around part of the lesion but had no consistent effect on the number of raphe-spinal neurons retrogradely labeled by tracer injection below the injury. In conclusion, this anti-CD11d integrin antibody treatment is neuroprotective after SCI, corresponding with improved patterns of intraspinal serotonergic innervation. The improvement in serotonergic fiber projections paralleled reduced mechanical allodynia and enhanced locomotor recovery.

A serotonergic system in the brain of the Antarctic fish, Trematomus bernacchii

The immunohistochemical distribution of serotonin-containing nerve fibres and cells has been described in the brain of the Antarctic fish, Trematomus bernacchii. The largest serotonergic system was associated with the diencephalic and rhombencephalic ventricles. In particular, serotonin-positive cells have been found in the lateral recess and neuropile zone of the diencephalic ventricle, where we have identified the serotonergic portion of the paraventricular organ. Numerous serotonin cells were localized in the dorsal nucleus of the raphe, the dorsal tegmental nucleus and the central gray. Two large cell groups, arranged in a pair of well-defined columns and connecting the central gray with the dorsal reticular formation, were immunostained in the region of the trigeminal nuclei. In addition, few positive cells have been found in the preoptic area and the cerebellar valvula, and few serotonergic nerve fibres, probably belonging to the lateral lemniscus, have been identified. The distribution of serotonin elements in the brain of T. bernacchii has been compared with that described in other fish, where it showed some modifications in the immunoreactive pattern. Finally, the lack of a serotonergic system at the level of the reticular superior formation has been reported; however, it was not possible to rule out a phylogenetic or environmental explanation.

The distribution of serotonergic fibers in the macaque monkey amygdala: An immunohistochemical study using antisera to 5-hydroxytryptamine

Though both the amygdala and the serotonin system appear to play critical roles in regulating fear and anxiety, little is known regarding the organization of serotonergic inputs to the primate amygdala. The present study employed immunohistochemistry to determine the distribution of serotonin fibers in the macaque amygdala. The brains of three adult male Macaca fascicularis monkeys were prepared for histological analysis using a polyclonal antibody to serotonin. The macaque amygdala is densely innervated by serotonergic fibers and demonstrates a distinctive pattern of fiber distribution and density among the 13 nuclei and cortical areas. The highest density of 5-hydroxytryptamine immunoreactive fibers is observed in the central nucleus, the nucleus of the lateral olfactory tract, the paralaminar nucleus, the anterior amygdaloid area and a small region of the amygdalohippocampal area. Moderate fiber densities are found in portions of the basal, lateral, and intercalated nuclei. The lowest fiber densities are observed in the accessory basal, posterior cortical, anterior cortical and medial nuclei, and in subregions of the periamygdaloid cortex. The present study provides evidence that the serotonergic system can have substantial influence on the ongoing activity of the amygdaloid complex.

Slow excitatory post-synaptic potentials in myenteric AH neurons of the guinea-pig ileum are reduced by the 5-hydroxytrytamine 7 receptor antagonist SB 269970

Serotonin (5-HT) is a key modulator of neuronal excitability in the central and peripheral nervous system. In the enteric nervous system, 5-HT causes a slow depolarization in the intrinsic sensory neurons, but the receptor responsible for this has not been correlated with known gene products. The aim of this study was to determine whether the newly characterized 5-HT 7 receptor may participate in the 5-HT-mediated depolarization of, and synaptic transmission to, the intrinsic sensory neurons of the guinea-pig ileum. Intracellular electrophysiological recordings were made from intrinsic sensory neurons identified as myenteric AH neurons from guinea-pig ileum. 5-HT (5μM) applied to the cell body evoked both a fast depolarization (5-HT 3 mediated) and/or a slow depolarization (5-HT 1P-like). The 5-HT 1/5/7 receptor agonist 5-carboxamidotryptamine (5-CT) (5μM) evoked only a slow depolarization. When the fast depolarization evoked by 5-HT was blocked with granisetron (1μM, 5-HT 3 receptor antagonist), only a slow depolarization remained; this was abolished by the 5-HT 7 receptor antagonist SB 269970 (1μM, control: 14±2 mV, granisetron+SB 269970: −1±2 mV). The slow depolarization evoked by 5-CT was also significantly reduced by SB 269970 (control: 14±1 mV, SB 269970: 5±2 mV) suggesting a 5-HT 7 receptor was activated by exogenous application of 5-CT and 5-HT. Slow excitatory postsynaptic potentials evoked by stimulating descending neural pathways (containing serotonergic fibers) were reduced by SB 269970 (control: 8±3 mV, SB 269970: 3±1 mV). However, SB 269970 had no effect on slow excitatory postsynaptic potentials evoked by stimulation of circumferential (tachykinergic) pathways (control: 7±1 mV, SB 269970: 6±1 mV). These data are consistent with the presence on enteric AH neurons of functional 5-HT 7 receptors that participate in slow synaptic transmission.

Immunohistological characterization of striatal and amygdalar structures in the telencephalon of the fire-bellied toad bombina orientalis

The subpallium of the fire-bellied toad Bombina orientalis was studied by means of enzyme-histological detection of NADPH-diaphorase and immunohistological demonstration of aspartate, GABA, calretinin, choline-acetyl transferase, Leu-and Met-enkephalin, neuropeptide Y, 5-hydroxy-tryptamine (serotonin), somatostatin, substance P and tyrosine-hydroxylase. As in other vertebrates, the striato-pallidum is characterized by GABA-, substance P- and enkephalin-immunoreactivity. Neurons and fibers differing in immunoreactivity are arranged in layers. Choline-acetyl transferase-immunoreactive neurons were found in a position corresponding to the mammalian cholinergic cell-group (Ch4-group), which therefore may be homologous to the nucleus basalis of Meynert. Within the amygdaloid complex, the cortical and lateral (vomeronasal) nuclei are similar in calretinin-, GABA-, NADPH-diaphorase-, enkephalin, substance P- and neuropeptide Y-(immuno)histology. The medial and central amygdaloid nuclei reveal a dense peptidergic innervation, and the medial amygdala additionally exhibits serotonergic fibers and cell bodies staining for neuropeptides and tyrosine-hydroxylase. Differences between Bombina and other anuran species exist, such as the absence of cholinergic neurons in the striatum. Our findings corroborate the view based on recent studies on the hodology and cytoarchitecture of the anuran telencephalon that the anuran ventral telencephalon contains most of the structures found in the mammalian brain. This concerns a septal region, a dorsal and ventral striato-pallidum including a nucleus accumbens and an amygdaloid complex consisting of a central, cortical and vomeronasal amygdala. The only major difference appears to concern the lack of a basolateral amygdala.

The effects of dopamine receptor agonists and antagonists on the secretory rate of cockroach ( Periplaneta americana) salivary glands

The acinar salivary glands of the cockroach, Periplaneta americana, are innervated by dopaminergic and serotonergic nerve fibers. Serotonin stimulates the secretion of protein-rich saliva, whereas dopamine causes the production of protein-free saliva. This suggests that dopamine acts selectively on ion-transporting peripheral cells within the acini and the duct cells, and that serotonin acts on the protein-producing central cells of the acini. We have investigated the pharmacology of the dopamine-induced secretory activity of the salivary gland of Periplaneta americana by testing several dopamine receptor agonists and antagonists. The effects of dopamine can be mimicked by the non-selective dopamine receptor agonist 6,7-ADTN and, less effectively, by the vertebrate D1 receptor-selective agonist chloro-APB. The vertebrate D1 receptor-selective agonist SKF 38393 and vertebrate D2 receptor-selective agonist R(−)-TNPA were ineffective. R(+)-Lisuride induces a secretory response with a slower onset and a lower maximal response compared with dopamine-induced secretion. However, lisuride-stimulated glands continue secreting saliva, even after lisuride-washout. Dopamine-induced secretions can be blocked by the vertebrate dopamine receptor antagonists cis( Z)-flupenthixol, chlorpromazine, and S(+)-butaclamol. Our pharmacological data do not unequivocally indicate whether the dopamine receptors on the Periplaneta salivary glands belong to the D1 or D2 subfamily of dopamine receptors, but we can confirm that the pharmacology of invertebrate dopamine receptors is remarkably different from that of their vertebrate counterparts.

Estrogen reduces the neurite growth of serotonergic cells expressing estrogen receptors.

Serotonergic innervation of the central nervous system has a sexual dimorphism. The serotonin level in the hypothalamus was modulated by estrogen, and the formation of sexual dimorphism of serotonergic fiber innervation in the hypothalamus has been shown by the effect of sexual hormone during the critical perinatal period. In this study, we examined the direct effect of estrogen on the neurite growth of serotonergic neurons in primary culture from embryonic day 14 (E14) of rat mesencephalon. The total neurite length of serotonin-immunoreactive (IR) cells was significantly decreased by estradiol benzoate (E2, 10(-8)M) treatment for 7 days, compared with the case of no treatment. Moreover, the presence of estrogen receptor (ER) alpha and ERbeta mRNA in the E14 mesencephalon with reverse transcription-polymerase chain reaction (RT-PCR), and the ERalpha or ERbeta protein in the cultured serotonin-IR cells with double fluorescence immunohistochemistry were also demonstrated. Our results suggest that the inhibitory effects of E2 on the neurite growth of serotonergic cells expressing ERalpha or ERbeta might be involved in the formation of the sexual dimorphic distribution of serotonergic innervation.

Serotonergic collateralized projections from Barrington's nucleus to the medial preoptic area and lumbo-sacral spinal cord

In this study, we employed triple fluorescent labelling to reveal the distribution of the direct serotonergic neurons within Barrington's nucleus (BN) that supply branching collateral input to the medial preoptic area (MPA) and to the lumbo-sacral spinal cord (LSC). Immunocytochemical detection of the monoclonal antibody raised against serotonin was used for identification of the neurons. The projections were defined by injections of two retrograde tracers: fluoro gold and rhodamine in the MPA and LSC, respectively. The aim of this study is to identify the direct projections to BN and MPA and/or LSC. The present study confirms findings of others describing BN–LSC projections and extends previous findings by demonstrating an single or collateralized fibers with MPA, and serotonergic immunoreactive fibers.

The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres

Previous work has shown there is an increase in motoneurone excitability produced by hyperpolarization of the threshold potential at which an action potential is elicited (Vth) at the onset, and throughout brainstem-induced fictive locomotion in the decerebrate cat. This represents a transient facilitation in the membrane potential for activation dependent on the presence of fictive locomotion. The present study tests the hypothesis that a similar neuromodulatory mechanism facilitating neuronal recruitment also exists in the neonatal rat, and the endogenous pathway mediating the Vth hyperpolarization can be activated by electrical stimulation of the neonatal brainstem. Isolated brainstem-spinal cord preparations from 1- to 5-day-old neonatal rats, and whole-cell recording techniques were used to examine the patterns of ventral root (VR) activity produced, and the effect of electrical stimulation of the ventromedial medulla on lumbar spinal neurones. Hyperpolarization of Vth was seen in 10/11 (range -2 to -18 mV) neurones recorded during locomotor-like VR activity, and appeared analogous to the locomotor-dependent Vth hyperpolarization previously described in the cat. However, in the present study, Vth hyperpolarization was also seen during electrical brainstem stimulation that evoked alternating, rhythmic, or tonic VR activity, or failed to evoke VR activity. Thirty-six of 71 neurones were antidromically identified as lumbar motoneurones and 33/36 showed a hyperpolarization of Vth (-2 to -14 mV) during electrical brainstem stimulation. Of the unidentified lumbar ventral horn neurones, 31/35 also showed hyperpolarization of Vth (-2 to -20 mV) during brainstem stimulation. The hyperpolarization of Vth and VR activity induced by brainstem stimulation was reversibly blocked by cooling of the cervical cord, indicating it is mediated by descending fibres, and application of the serotonergic antagonist ketanserin to the spinal cord was effectively able to block the brainstem-evoked hyperpolarization of Vth. These results demonstrate a previously unknown action of the endogenous descending serotonergic system to facilitate spinal motoneuronal recruitment and firing by inducing a hyperpolarization of Vth. This modulatory process can be examined in the neonatal rat brainstem-spinal cord preparation without the requirement for ongoing locomotor activity.

Monoamines in the pedal plexus of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata).

In molluscs, the number of peripheral neurons far exceeds those found in the central nervous system. Although previous studies on the morphology of the peripheral nervous system exist, details of its organization remain unknown. Moreover, the foot of the terrestrial species has been studied less than that of the aquatic species. As this knowledge is essential for our experimental model, the pulmonate gastropod Megalobulimus oblongus, the aim of the present study was to investigate monoamines in the pedal plexus of this snail using two procedures: glyoxylic acid histofluorescence to identify monoaminergic structures, and the unlabeled antibody peroxidase anti-peroxidase method using antiserum to detect the serotonergic component of the plexus. Adult land snails weighing 48-80 g, obtained from the counties of Barra do Ribeiro and Charqueadas (RS, Brazil), were utilized. Monoaminergic fibers were detected throughout the pedal musculature. Blue fluorescence (catecholamines, probably dopamine) was observed in nerve branches, pedal and subepithelial plexuses, and in the pedal muscle cells. Yellow fluorescence (serotonin) was only observed in thick nerves and in muscle cells. However, when immunohistochemical methods were used, serotonergic fibers were detected in the pedal nerve branches, the pedal and subepithelial plexuses, the basal and lateral zones of the ventral integument epithelial cells, in the pedal ganglion neurons and beneath the ventral epithelium. These findings suggest catecholaminergic and serotonergic involvement in locomotion and modulation of both the pedal ganglion interneurons and sensory information. Knowledge of monoaminergic distribution in this snail s foot is important for understanding the pharmacological control of reflexive responses and locomotive behavior.