Multi-messenger innervation of the male sexual system of Lymnaea stagnalis

The male copulation behaviour of the hermaphrodite pond snail Lymnaea stagnalis is under the control of five groups of central neurons that produce a variety of neuropeptides and a classical transmitter, 5-hydroxy tryptamine (5HT). In this article, we describe how the male sexual organs of this snail are innervated by axons from these central neurons. We carried out immunocytochemistry with antisera against the tetra peptide Ala-Pro-Gly-TRP-NH₂ (APGWamide), the Lymnaea form of neuropeptide tyrosine (LNPY), conopressin, pedal peptide, the FRMFamide copeptide SEEPLY, the GDPFLRFamide co-peptide DEILSR, myomodulin, Lymnaea inhibitory peptide, and 5HT on tissue sections of the following male sexual organs that receive input from the penis nerve: the prostate gland, vas deferens, preputium, and penis. The results demonstrate that the axons of the separate muscle systems contain particular combinations of transmitters. In addition, two networks of peripheral neurons were revealed. In the tip of the everted preputium lies what appears to be a network of conopressin-containing sensory neurons, which is possibly involved in probing; probing is the part of copulation behaviour in which the male searches for the female genital pore. The other network of peripheral neurons surrounds the most proximal part of the vas deferens and is most likely involved in the pacemaker control of vas deferens motility. On the basis of the data obtained, we hypothesize how the preputium and penis are everted during copulation and which transmitters and central neurons might be involved. J. Comp. Neurol. 390:564–577, 1998. © 1998 Wiley-Liss, Inc.     

Neuropeptides myomodulin,small cardioactive peptide,and buccalin in the central nervous system of Lymnaea stagnalis: Purification,immunoreactivity, and artifacts

The neuropeptides myomodulin, small cardioactive peptide (SCP), and buccalin are widely distributed in the phylum Mollusca and have important physiological functions. Here, we describe the detailed distribution of each class of peptide in the central nervous system (CNS) of the snail Lymnaea stagnalis by the use of immunocytochemical techniques combined with dye-marking of electrophysiologically identified neurons. We report the isolation and structural characterization of a Lymnaea myomodulin, PMSMLRLamide, identical to myomodulin A of Aplysia californica. Myomodulin immunoreactivity was localized in all 11 ganglia, in their connectives, and in peripheral nerves. In many cases, myomodulin immunoreactivity appeared localized in neuronal clusters expressing FMRFamide-like peptides, but also in a large number of additional neurons. Double-labelling experiments demonstrated myomodulin immunoreactivity in the visceral white interneuron, involved in regulation of cardiorespiration. SCP-like immunoreactivity also appeared in all ganglia, and double-labelling experiments revealed that in many locations it was specifically associated with clusters expressing distinct exons of the FMRFamide gene that are differentially expressed in the CNS. Characterization of the two types of SCP-antisera used in this study, however, suggested that they cross-reacted with both FMRFamide and N-terminally extended FMRFamide-like peptides. Selective preadsorption with these cross-reacting peptides resulted in elimination of the widespread staining and retention of bona fide SCP immunoreactivity in the buccal and pedal ganglia only. Buccalin immunoreactivity was limited to the buccal and pedal ganglia. It did not coincide with the distribution of either myomodulin or SCP. Most immunoreactive clusters were found in the pedal ganglia. © 1994 Wiley-Liss, Inc.     

Organization of the serotonergic innervation of the feeding (buccal) musculature during the maturation of the pond snail Lymnaea stagnalis: a morphological and biochemical study

The serotonergic innervation of the buccal musculature responsible for feeding (radula protraction) was investigated during the maturation of the pond snail, Lymnaea stagnalis L., applying light and electron microscopic immunohistochemistry and biochemical approaches. According to epifluorescence and laser confocal microscopy, the first 5-HT-like-immunoreactive (5-HTLIR) processes appeared on the surface of the musculature at the postmetamorphic E80% embryonic stage. Until hatching, the innervation continued to increase in density, showing axon arborizations with projections into the deeper muscle levels. An adult-like pattern of 5-HTLIR innervation appeared at P2-P3 juvenile stages. At the ultrastructural level, close (16-20 nm) but mostly unspecialized neuromuscular contacts were formed by both unlabeled and 5-HTLIR axon profiles from the E80% embryonic stage. Labeled processes were also found located relatively far from the muscle cells. An HPLC assay showed a gradual increase of the 5-HT level in the buccal mass during development. The buccal mass was characterized by a single-component high-affinity 5-HT uptake system, and 5-HT release could be evoked by 100 mM K(+) and blocked in Ca(2+) -free medium. It is suggested that 5-HT plays a wide modulatory role in the peripheral feeding system and is also involved in the functional maturation of the muscle system.     

Disruption of raphé serotonergic neural projections to the cortex: a potential pathway contributing to remote loss of brainstem neurons following neonatal hypoxic–ischemic brain injury

Neuronal injury is a key feature of neonatal hypoxic–ischemic (HI) brain injury. However, the mechanisms underpinning neuronal losses, such as in the brainstem, are poorly understood. One possibility is that disrupted neural connections between the cortex and brainstem may compromise the survival of neuronal cell bodies in the brainstem. We investigated whether brainstem raphé serotonergic neurons that project to the cortex are lost after HI. We also tested if neuroinflammation has a role in disrupting brainstem raphé projections. Postnatal day 3 (P3) rats underwent unilateral carotid artery ligation followed by hypoxia (6% oxygen for 30 min). A retrograde tracer, choleratoxin b, was deposited in the motor cortex on P38. On P45 we found that retrogradely labelled neurons in the dorsal raphé dorsal, ventrolateral, interfascicular, caudal and ventral nuclei were lost after P3 HI. All retrogradely labelled neurons in the raphé nuclei were serotonergic. Numbers of retrogradely labelled neurons were also reduced in the ventromedial thalamus and basolateral amygdala. Minocycline treatment (45 mg/kg 2 h post‐HI, 22.5 mg/kg daily P4–P9) attenuated losses of retrogradely labelled neurons in the dorsal raphé ventrolateral, interfascicular and ventral raphé nuclei, and the ventromedial thalamus. These results indicate that raphé neurons projecting to the cortex constitute a population of serotonergic neurons that are lost after P3 HI. Furthermore, neuroinflammation has a role in the disruption of raphé and thalamic neural projections. Future studies investigating the cellular mechanisms of axonal degeneration may reveal new targets for interventions to prevent neuronal losses after neonatal HI.     

Central nervous system receptors involved in mediating the inhibitory action of neuropeptide Y on luteinizing hormone secretion in the male rhesus monkey (Macaca mulatta)

An earlier finding that gonadotropin-releasing hormone (GnRH) secretion may be triggered prematurely in the juvenile male monkey by central administration of 1229U91, a Y1 receptor antagonist, contributed to our current hypothesis that neuropeptide Y (NPY) is a major component of the brake that holds pulsatile GnRH release in check during prepubertal development in primates. However, 1229U91 is also a Y4 receptor agonist, and the present study was conducted to further examine the role of the Y1 receptor in mediating the putative inhibitory action of NPY on GnRH release. Agonadal juvenile and postpubertal male monkeys were implanted with i.v. and i.c.v. cannulae to gain continuous access to the venous and cerebroventricular circulations without sedation. Luteinizing hormone (LH) secretion was measured to provide an indirect index of GnRH release. The specific Y1 antagonists, VD-11 (476 microg; n = 4) and isopropyl 3-chloro-5-[1-((6-[2-(5-ethyl-4-methyl-1,3-thiazol-2-yl)ethyl]-4-morpholin-4-ylpyridin-2-yl)amino)ethyl]phenylcarbamate (Compound A, 300 microg; n = 4), did not mimic the stimulatory action of 1229U91 on GnRH secretion in the juvenile male monkey. Additionally, neither NPY (200 microg; n = 2), a general Y receptor agonist, nor rPP (100 microg; n = 4), a Y4 agonist, mimicked the action of 1229U91 in stimulating GnRH release. Moreover, previous exposure of the hypothalamus of juvenile monkeys (n = 5) to NPY (660 microg) failed to block 1229U91-induced (200 microg) GnRH release. However, the action of NPY (364 microg) in inhibiting GnRH release postpubertally was attenuated by 1229U91 (300 microg). We conclude that, although the action of exogenous NPY to suppress GnRH release from the postpubertal hypothalamus appears to be mediated, at least in part, by the Y1 receptor, the existence of a Y1 receptor pathway inhibitory to GnRH release in the prepubertal hypothalamus remains to be substantiated.     

Development of catecholaminergic neurons in the pond snail,Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120–140 fmol per animal by around E40. Dopamine content remained stable during mid-embryogenesis (E40–65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde- and glyoxylate-induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32–35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography. J. Comp. Neurol. 404:285–296, 1999. © 1999 Wiley-Liss, Inc.     

Sensorimotor-related discharge of simultaneously recorded, single neurons in the dorsal raphe nucleus of the awake, unrestrained rat

Multi-channel, multi-neuron recording procedures were used to monitor simultaneously the spike train activity of single neurons (n=7–16 cells/animal) in the dorsal raphe (DR) nucleus of the awake, freely moving rat. Putative serotonergic and non-serotonergic neurons were distinguished from one another on the basis of established criteria, i.e. waveform shape and duration, firing pattern and firing frequency. As a group, presumed serotonergic neurons exhibited low tonic discharge rates, depressed firing after serotonin (5HT)-1a agonist administration, and, except for the transition from sleep to waking, a general insensitivity to specific sensory or motor events. By contrast, non-serotonergic cells in midline and lateral wing sub-regions of the nucleus displayed responses to a variety of sensorimotor events including locomotion, grooming, head movement, chewing, auditory stimuli, and whisker movement (both passive and active). However, within this latter group, the sensorimotor response repertoire of individual cells was not uniform. Likewise, non-5HT cells with diverse response profiles were identified in both medial and lateral sub-regions of the nucleus. Cells categorized as non-serotonergic also had varied responses to 5HT1a agonist administration. These results emphasize the diverse input/output relationships of individual DR neurons and underscore the need for a more comprehensive analysis of such properties under waking conditions in order to obtain a better understanding of the role of the DR nucleus in brain function.     

Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis

The cerebral neurosecretory caudodorsal cells (CDCs) of the freshwater pulmonate snail Lymnaea stagnalis control egg laying, an event that involves a pattern of stereotyped behaviors. The CDCs synthesize and release multiple peptides, among which is the ovulation hormone (CDCH). It is thought that each peptide controls a specific aspect of the processes involved in egg laying. We isolated and characterized a CDC-specific cDNA clone that encodes the ovulation hormone (CDCH). RNA blot analysis and in situ hybridization experiments demonstrated that the CDCs are the major cell groups in the cerebral ganglia that transcribe the CDCH gene. In addition to CDCH, the 259-amino acid-long CDCH preprohormone contains 11 other predicted peptides. The overall homology of the CDCH preprohormone with the egg-laying hormone (ELH) preprohormones of the marine opisthobranch snails Aplysia californica and A. parvula is very low (29 and 26%, respectively). However, a more detailed comparison revealed a highly differential pattern of conservation of peptide regions. Significant homology was found between the regions containing (1) CDCH and ELH, (2) repeated pentapeptides, (3) alpha-caudodorsal cell peptide and alpha-bag cell peptide, and (4) 2 regions representing as yet unidentified peptides. Insignificant homology was found when comparing regions containing the other predicted peptides. The conserved peptides probably control similar aspects of the egg-laying fixed action patterns in these distantly related gastropod species. The pentapeptide region exhibits the highest level of homology (75%); in addition, an extra pentapeptide has been generated on the CDCH precursor. This indicates a vital function of these peptides in Aplysia, as well as in Lymnaea species.     

Effect of the acute and chronic administration of the selective 5-HT6 receptor antagonist SB-271046 on the activity of midbrain dopamine neurons in rats: an in vivo electrophysiological study

This study examined the effect of the acute and repeated per os (p.o.) administration of the selective 5-HT(6) receptor antagonist SB-271046, on the number, as well as the firing pattern of spontaneously active dopamine (DA) neurons in the rat substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized male Sprague-Dawley rats. This was accomplished using the technique of extracellular in vivo electrophysiology. A single p.o. administration of either 1, 3, or 10 mg/kg of SB-271046 did not significantly alter the number of spontaneously active SNC DA neurons per stereotaxic electrode tract compared to vehicle-treated animals. The acute administration of either 1 or 3 mg/kg of SB-271046 did not significantly alter the number of spontaneously active VTA DA neurons. In contrast, a significant decrease in the number of spontaneously active VTA DA neurons was observed after a single administration of 10 mg/kg of SB-271046 compared to vehicle-treated animals. The acute p.o. administration of SB-271046 significantly altered the firing pattern parameters of all (bursting + nonbursting DA neurons) DA neurons, particularly those in the VTA, compared to vehicle-treated animals. The repeated p.o. administration (once per day for 21 days) of 1, 3, or 10 mg/kg of SB-271046 did not significantly alter the number of spontaneously active VTA DA neurons compared to vehicle-treated animals. The repeated administration of 3 or 10 mg/kg of SB-271046 significantly increased the number of spontaneously active SNC DA neurons compared to vehicle controls. Overall, the repeated administration of SB-271046 had relatively little effect on the firing pattern of midbrain DA neurons. The results obtained following the chronic administration of SB-271046 show that this compound has a profile different from that of typical or atypical antipsychotic drugs in this model. Clinical studies are required to understand what role 5-HT(6) receptor blockade might eventually play in the treatment of schizophrenia.     

Effect of glial cell line‐derived neurotrophic factor on behavior and key members of the brain serotonin system in mouse strains genetically predisposed to behavioral disorders

The effect of glial cell line‐derived neurotrophic factor (GDNF) on behavior and on the serotonin (5‐HT) system of a mouse strain predisposed to depressive‐like behavior, ASC/Icg (Antidepressant Sensitive Cataleptics), in comparison with the parental “nondepressive” CBA/Lac mice was studied. Within 7 days after acute administration, GDNF (800 ng, i.c.v.) decreased cataleptic immobility but increased depressive‐like behavioral traits in both investigated mouse strains and produced anxiolytic effects in ASC mice. The expression of the gene encoding the key enzyme for 5‐HT biosynthesis in the brain, tryptophan hydroxylase‐2 (Tph‐2), and 5‐HT_1A receptor gene in the midbrain as well as 5‐HT_2A receptor gene in the frontal cortex were increased in GDNF‐treated ASC mice. At the same time, GDNF decreased 5‐HT_1A and 5‐HT_2A receptor gene expression in the hippocampus of ASC mice. GDNF failed to change Tph2, 5‐HT_1A, or 5‐HT_2A receptor mRNA levels in CBA mice as well as 5‐HT transporter gene expression and 5‐HT_1A and 5‐HT_2A receptor functional activity in both investigated mouse strains. The results show 1) a GDNF‐induced increase in the expression of key genes of the brain 5‐HT system, Tph2, 5‐HT_1A, and 5‐HT_2A receptors, and 2) significant genotype‐dependent differences in the 5‐HT system response to GDNF treatment. The data suggest that genetically defined cross‐talk between neurotrophic factors and the brain 5‐HT system underlies the variability in behavioral response to GDNF. © 2013 Wiley Periodicals, Inc.     

Transmitter localization and vesicle turnover at a serotoninergic synapse between identified leech neurons in culture

An electron microscopic study has been made of chemical synapses that develop between identified nerve cells isolated from the CNS of the leech and maintained in culture. Structures resembling synapses were observed in pairs of Retzius cells and P sensory cells at which chemical transmission had been demonstrated by recording with microelectrodes. Vesicle recycling was shown by following the uptake of extracellular markers after stimulation. The membrane separation between the presynaptic Retzius cell (which is known to liberate serotonin) and the postsynaptic P cell was wider in synaptic than in extrasynaptic regions. The Retzius cell contained clusters of clear vesicles apposed to thickenings of the presynaptic membrane. These clear vesicle clusters were capped by a layer of dense core vesicles that did not contact the presynaptic membrane thickenings. Subsynaptic cisternae were found in the postsynaptic cell opposite the presynaptic membrane thickenings. Occasional slight postsynaptic membrane thickenings were seen. Extracellular material was observed within the synaptic cleft. Similar synaptic structures developed between pairs of Retzius cells in culture; even a single Retzius cell was able to form autapses upon itself. Structures resembling transmitter release sites were found in Retzius cells at a distance from any postsynaptic membranes. These are presumed to be locations for the diffuse release of transmitter. Presynaptic structures resembling release sites were never observed in P cells apposed to Retzius cells. Antibody to serotonin (5-HT) labelled with colloidal gold showed serotonin to be localized in the dense core vesicles in Retzius cells. Stimulation of pairs of Retzius and P cells by raised concentrations of K+ resulted in uptake of extracellular markers. Only Retzius cells became labelled. Ferritin was found in cisternae, in dense core vesicles, and in clear vesicles. HRP was found in cisternae and in clear vesicles. Colloidal gold was taken up by coated vesicles and was occasionally found in both clear and dense core vesicles. The uptake of extracellular markers following stimulation was blocked by high Mg++. These results show that structures develop between pairs of cells at which chemical transmission develops and that transmitter release leads to turnover of dense core and clear vesicles.     

Anatomical identification of neurons in selected brain regions associated with maternal behavior deficits induced by knife cuts of the lateral hypothalamus in rats

The present experiment identified neurons associated with maternal behavior deficits induced by damage to the lateral hypothalamus (LH) in rats. Fully maternal lactating rats received bilateral coronal knife cuts through either the dorsal or ventral LH at the level of the ventromedial nucleus. The blade of the wire knife used to make the cuts was coated with horseradish peroxidase (HRP). The maternal behavior of all females was studied for 4 days postoperatively and then the brains were processed for the localization of neurons retrogradely filled with HRP. The analysis focused on those brain regions thought to be important for maternal behavior. The dorsal LH cuts severely disrupted maternal behavior while the ventral LH cuts did not. The ventral cuts labeled more medial preoptic area (MPOA) and septal-diagonal band neurons with HRP than did the dorsal cuts. The dorsal LH cuts labeled more neurons with HRP in the lateral preoptic area (LPOA), magnocellular preoptic area, bed nucleus of the stria terminalis, ventral tegmental area (VTA), substantia nigra, and central gray than did the ventral cuts. Previous research has suggested that the lateral efferents of the MPOA interact with the VTA in the control of maternal behavior. The results of the present experiment suggest that MPOA axons which descend directly to the brainstem via the ventral LH are not essential for maternal behavior. Our results are consistent with the view that an MPOA-to-LPOA-to-VTA circuit may be important for maternal behavior.     

Serotonin may stimulate granule cell proliferation in the adult hippocampus, as observed in rats grafted with foetal raphe neurons

The long-term effects of hippocampal serotonergic denervation and reinnervation by foetal raphe tissue were examined in the dentate gyrus where neurons are continously born in the adult. Complete lesion of serotonin neurons following injections of 5, 7-dihydroxytryptamine in the dorsal and medial raphe nuclei produced long-term decreases in the number of newly generated granule cells identified with 5-Bromo-2'-deoxyuridine (BrdU) and the polysialylated form of neural cell adhesion molecule (PSA-NCAM) immunostaining, as observed in 2-month-survival rats. The raphe grafts, but not the control grafts of embryonic spinal tissue, reversed the postlesion-induced decreases in the density of BrdU- and PSA-NCAM-labelled cells detected in the granule layer. Inhibition of serotonin synthesis in animals with raphe grafts reversed back to lesion-induced changes in granule cell proliferation. Furthermore, extensive serotonergic reinnervation of the dentate gyrus in the area proximal to the raphe graft could be associated with supranormal density of BrdU-labelled cells. These results indicate that serotonin may be considered a positive regulatory factor of adult granule cell proliferation. Finally, the lack of effect of embryonic nonserotonergic tissue grafted to serotonin-deprived rats suggests that neurotrophic factors may not be involved in the effects of serotonin on adult neurogenesis.     

Upregulation of mucosal 5-HT3 receptors is involved in restoration of colonic transit after pelvic nerve transection

Background Colonic dysfunction occurs after pelvic autonomic nerve damage. The enteric nervous system can compensate. We investigated the role of mucosal serotonin receptors, 5‐HT₃ and 5‐HT₄, in the colonic motility restoration over 2 weeks after parasympathetic pelvic nerve transection in a rat model. Methods Male Sprague‐Dawley rats underwent pelvic nerve transection or sham operation. Colonic transit was expressed as the geometric center of ⁵¹Cr distribution. Mucosal 5‐HT₃ and 5‐HT₄ receptor expression was evaluated by Western blot. Intraluminal pressure increase was measured after 5‐HT₃ (ondansetron) or 5‐HT₄ receptor antagonist (GR125487) administration in vitro in sham and denervated distal colons. Key Results At 2 weeks, colonic transit in the denervated group was 30% slower compared to the sham group (P < 0.01). At 1 and 2 weeks, 5‐HT₃ receptor expression was increased two‐fold in the denervated group, compared to shams (P < 0.05). A three‐fold smaller dose of ondansetron was required in denervated tissues to inhibit intraluminal pressure rise than in sham colons (P < 0.01). There was no difference in the expression of 5‐HT₄ receptor or the response to GR125487 in denervated vs sham colons. Conclusions & Inferences Colonic motility was restored to approximately 70% normal over 1 week without further improvement at 2 weeks. Enteric nervous system compensated by upregulating mucosal 5‐HT_3, but not 5‐HT_4, receptors.     

Embryonic differentiation of serotonin-containing neurons in the enteric nervous system of the locust (Locusta migratoria)

The enteric nervous system (ENS) of the locust consists of four ganglia (frontal and hypocerebral ganglion, and the paired ingluvial ganglia) located on the foregut, and nerve plexus innervating fore- and midgut. One of the major neurotransmitters of the ENS, serotonin, is known to play a vital role in gut motility and feeding. We followed the anatomy of the serotonergic system throughout embryonic development. Serotonergic neurons are generated in the anterior neurogenic zones of the foregut and migrate rostrally along the developing recurrent nerve to contribute to the frontal ganglion. They grow descending neurites, which arborize in all enteric ganglia and both nerve plexus. On the midgut, the neurites closely follow the leading migrating midgut neurons. The onset of serotonin synthesis occurs around halfway through development-the time of the beginning of midgut closure. Cells developing to serotonergic phenotype express the serotonin uptake transporter (SERT) significantly earlier, beginning at 40% of development. The neurons begin SERT expression during migration along the recurrent nerve, indicating that they are committed to a serotonergic phenotype before reaching their final destination. After completion of the layout of the enteric ganglia (at 60%) a maturational phase follows, during which serotonin-immunoreactive cell bodies increase in size and the fine arborizations in the nerve plexus develop varicosities, putative sites of serotonin release (at 80%). This study provides the initial step for future investigation of potential morphoregulatory functions of serotonin during ENS development.     

Dopamine neurotransmission is involved in the attenuating effects of 5-HT3 receptor antagonist MDL 72222 on acute methamphetamine-induced locomotor hyperactivity in mice

We have previously shown that 5-HT3 receptors are involved in the development and expression of methamphetamine (MAP)-induced locomotor sensitization in mice. In the present study, we further examined whether the dopaminergic system is involved in the attenuating effects of MDL 72222, a 5-HT3 receptor antagonist, on acute MAP-induced locomotor hyperactivity. For this, we examined alterations of dopamine (DA) in the form of D1 receptor, D2 receptor, and dopamine transporter (DAT) binding labeled with [3H]SCH23390 for D1, [3H]raclopride for D2, and [3H]mazindol for DAT binding in the mouse brains with acute MAP exposure or pretreatment of MDL 72222 with MAP. No significant differences were detected in the D1 receptor, D2 receptor, or DAT binding between any of the groups studied. Interestingly, we found increased DA levels in the striatum following acute MAP exposure; these increased levels were reversed by pretreatment with MDL 72222, but did not affect 5-HT levels in the dorsal raphe. Overall, our results suggest that dopamine neurotransmission plays an important role in the attenuating effects of 5-HT3 receptor antagonist MDL 72222 on acute MAP-induced locomotor hyperactivity in mice.     

Serotonin (5‐HT) regulates neurite outgrowth through 5‐HT1A and 5‐HT7 receptors in cultured hippocampal neurons

Serotonin (5‐HT) production and expression of 5‐HT receptors (5‐HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5‐HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5‐HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G‐protein‐coupled 5‐HT_1AR and 5‐HT₇R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5‐HT promotes neurite outgrowth through 5‐HT_1AR and 5‐HT₇R. The involvement of 5‐HT_1AR and 5‐HT₇R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5‐HT and specific antagonists for 5‐HT_1AR and 5‐HT₇R (WAY‐100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5‐HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT₇R was blocked, the addition of 5‐HT increased the number of primary neurites, suggesting that 5HT₇R could inhibit neuritogenesis. In contrast, 5‐HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY‐100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5‐HT_1AR and 5‐HT₇R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth. © 2014 Wiley Periodicals, Inc.     

Mapping study of serotoninergic input to diencephalic-projecting dorsal column neurons in the rat

Several lines of evidence indicate that the processing of somatosensory information in the dorsal column nuclei (DCN) is subject to descending controls. Anatomical experiments have demonstrated projections to the DCN from the sensorimotor cerebral cortex and the reticular formation. Physiological studies have shown that the activity of DCN neurons can be altered following stimulation of the cerebral cortex, reticular formation, periaqueductal gray, or raphe nuclei. Recent biochemical and electrophysiological evidence suggests a serotoninergic modulation of DCN neurons. The present study identifies serotonin-containing contacts on cells in the DCN that project to the thalamus in the rat. Retrograde labeling of brainstem neurons by horseradish peroxidase demonstrated projections to the DCN from the nucleus reticularis paragigantocellularis lateralis and from several raphe nuclei, including nuclei raphe obscurus (RO), pallidus (RP), and magnus (RM). Double labeling with horseradish peroxidase and antibody for serotonin indicated that the RO, RP and RM are likely to be the sources of the serotoninergic projections to the DCN. Thus, the role of the serotoninergic output from the raphe nuclei includes modulation of activity in the DCN.     

Effect of acute and chronic administration of the selective 5-HT2C receptor antagonist SB-243213 on midbrain dopamine neurons in the rat: an in vivo extracellular single cell study

In this study, we examined the effect of the acute and chronic administration of the selective 5-HT2C receptor antagonist SB-243213 (SB) on the activity of spontaneously active dopamine (DA) cells in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized, albino, male Sprague-Dawley rats. This was accomplished using the technique of in vivo extracellular single cell recording. The acute i.v. administration of SB-243213 (0.025-3.2 mg/kg) did not significantly alter the basal firing rate or pattern of either spontaneously active SNC or VTA DA neurons compared to vehicle-treated controls. The acute i.p. administration of either 1 or 10 mg/kg of SB-243213 did not significantly alter the number of spontaneously active DA cells in the SNC or VTA compared to vehicle-treated controls, whereas the 3 mg/kg dose only significantly decreased the number of spontaneously active VTA DA neurons. Overall, the 1 mg/kg dose of SB-243213 did not significantly alter the firing pattern of either SNC or VTA DA neurons compared to vehicle-treated controls. In contrast, the 3 mg/kg dose significantly altered the firing pattern of SNC DA neurons, whereas the 10 mg/kg dose altered the firing pattern of DA neurons in both the SNC and VTA. The repeated i.p. administration (21 days) of 1, 3, and 10 mg/kg of SB-243213 or 20 mg/kg of clozapine produced a significant decrease in the number of spontaneously active DA cells in the VTA compared to vehicle-treated controls. The decrease in the number of spontaneously active VTA DA cells was not reversed by the i.v. administration of (+)-apomorphine (50 microg/kg). The repeated administration of either 1 or 3 mg/kg of SB-243213 had minimal effects on the firing pattern of either SNC or VTA DA neurons. In contrast, the firing pattern of VTA DA neurons was significantly altered by 10 mg/kg dose of SB-243213. Overall, our results indicate that antagonism of the 5-HT2C receptor alters the activity of midbrain DA neurons in anesthetized rats and suggest that SB-243213 has an atypical antipsychotic profile following chronic administration.