Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord.

The motor programme executed by the spinal cord to generate locomotion involves glutamate-mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5-hydroxytryptamine (5-HT), we investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5-HT (2.5-30 microM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5-HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min-1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5-HT doses were increased (range 20-40 microM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non-NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5-HT, albeit with doses larger than in the control solution (15-40 microM). When NMDA and non-NMDA receptors were simultaneously blocked, 5-HT (5-120 microM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non-NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.     

Divergent actions of serotonin receptor activation during fictive swimming in frog embryos

We have investigated the pharmacology underlying locomotor system responses to serotonin (5-HT) in embryos of the frog, Rana temporaria, to provide a comparison to studies in embryos of its close relative, Xenopus laevis. Our findings suggest that two divergent mechanisms underlie the modulation of locomotion by 5-HT in Rana. Bath-applied 5-HT or 5-carboxamidotyptamine, a 5-HT_1,5A,7 receptor agonist, can modulate fictive swimming in a dose-dependent manner, increasing burst durations and cycle periods. However, activation of 5-HT_1,7 receptors with R8-OHDPAT or 8-OHDPAT fails to mimic 5-HT, and in some cases exerts exactly the opposite response; decreasing burst durations and cycle periods. Elevating endogenous 5-HT levels by blocking re-uptake with clomipramine transiently increases burst durations. The receptors involved in this endogenous response include 5-HT_1A receptors, as in Xenopus, but also 5-HT₇ receptors. However, like the 8-OHDPAT enantiomers, prolonged re-uptake inhibition can result in a motor response in the opposite direction to exogenous 5-HT. This effect is not reversed by 5-HT_1A and/or 5-HT₇ receptor antagonism, implicating 5-HT_1B/1D receptors. Remarkably, antagonism of these receptors using methiothepin unmasks a dose-dependent response to clomipramine, reminiscent of exogenous 5-HT. Our data suggest that 5-HT_1A,7 and 5-HT_1B/1D receptors act as gain-setters of burst durations, whilst 5-HT_5A receptors are involved in the effects of bath-applied 5-HT on locomotion.     

Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal dorsal horn in vitro

ABSTRACT: BACKGROUND: 5-hydroxytryptamine (5-HT) is one of the major neurotransmitters widely distributed in the CNS. Several 5-HT receptor subtypes have been identified in the spinal dorsal horn which act on both pre- and postsynaptic sites of excitatory and inhibitory neurons. However, the receptor subtypes and sites of actions as well as underlying mechanism are not clarified rigorously. Several electrophysiological studies have been performed to investigate the effects of 5-HT on excitatory transmission in substantia gelatinosa (SG) of the spinal cord. In the present study, to understand the effects of 5-HT on the inhibitory synaptic transmission and to identify receptor subtypes, the blind whole cell recordings were performed from SG neurons of rat spinal cord slices. RESULTS: Bath applied 5-HT (50 microM) increased the frequency but not amplitudes of spontaneous inhibitory postsynaptic currents (sIPSCs) in 58% of neurons, and both amplitude and frequency in 23 % of neurons. The frequencies of GABAergic and glycinergic mIPSCs were both enhanced. TTX (0.5 microM) had no effect on the increasing frequency, while the enhancement of amplitude of IPSCs was eliminated. Evoked-IPSCs (eIPSCs) induced by focal stimulation near the recording neurons in the presence of CNQX and APV were enhanced in both amplitude by 5-HT. In the presence of Ba2+ (1 mM), a potassium channel blocker, 5-HT had no effect on both frequency and amplitude. A 5-HT2Areceptor agonist, TCB-2 mimicked the 5-HT effect, and ketanserin, an antagonist of 5-HT2A receptor, inhibited the effect of 5-HT partially and TCB-2 almost completely. A 5-HT2C receptor agonist WAY 161503 mimicked the 5-HT effect and this effect was blocked by a 5-HT2C receptor antagonist, N-desmethylclozapine. The amplitude of sIPSCs were unaffected by both agonists. A 5-HT3 receptor agonist mCPBG enhanced both amplitude and frequency of sIPSCs. This effect was blocked by a 5-HT3 receptor antagonist ICS-205,930. The perfusion of 5-HT2B receptor agonist had no effect on sIPSCs. CONCLUSIONS: Our results demonstrated that 5-HT modulated the inhibitory transmission in SG by the activation of 5-HT2A and 5-HT2C receptors subtypes located predominantly at inhibitory interneuron terminals, and 5-HT3 receptors located at inhibitory interneuron terminals and soma-dendrites, consequently enhanced both frequency and amplitude.     

Evidence for a spinal serotonergic control of the peripheral inflammation in the rat

We investigated the effect of serotonergic agonists and antagonists injected intrathecally by direct punction of the spinal cord at the lumbar level (between L5-L6) on peripheral inflammatory edema. Edema was induced by carrageenan injected subcutaneously in one hindpaw 30 min after spinal treatments. Serotonin (0.1, 1, 10 pmol) caused a graded-inhibition of the inflammatory paw edema. The corticosteroid inhibitor aminoglutethimide (100 mg/kg, p.o. 1.5 h before spinal treatment) did not modify this effect. The 5-HT1A agonist buspirone and the 5-HT1B/1D agonist sumatriptan (0.1, 1.0 and 10 nmol) also inhibited paw edema. The 5-HT1,2 antagonist methysergide (10 and 100 pmol) enhanced edema, but higher doses ( 4 and 8 nmol) diminished edema. NAN-190 (5-HT1 antagonist; 1 and 10 nmol) increased paw edema, while ritanserin (5-HT2 antagonist; 1 nmol) inhibited paw edema. Ondansetron (5-HT3 antagonist; up to 10 nmol) did not affect edema, but metoclopramide (5-HT3 antagonist / 5-HT4 agonist; 5, 10 and 30 pmol) inhibited edema. These data suggest that a tonic release of serotonin in the spinal cord may occurs during ongoing peripheral inflammation, modulating the neurogenic component of edema either by an inhibitory action on 5-HT1 receptors or by a stimulatory action on 5-HT2 receptors. A disfunction in such mechanism may be involved in the pathophysiology of certain types of headaches or migraine, which seem to depend on neurogenic vasodilation, and may also help to explain the therapeuthic effectiveness of some serotonergic agents in these conditions.     

Functional organization of locomotor interneurons in the ventral lumbar spinal cord of the newborn rat

Although the mammalian locomotor CPG has been localized to the lumbar spinal cord, the functional-anatomical organization of flexor and extensor interneurons has not been characterized. Here, we tested the hypothesis that flexor and extensor interneuronal networks for walking are physically segregated in the lumbar spinal cord. For this purpose, we performed optical recordings and lesion experiments from a horizontally sectioned lumbar spinal cord isolated from neonate rats. This ventral hemi spinal cord preparation produces well-organized fictive locomotion when superfused with 5-HT/NMDA. The dorsal surface of the preparation was visualized using the Ca(2+) indicator fluo-4 AM, while simultaneously monitoring motor output at ventral roots L2 and L5. Using calcium imaging, we provided a general mapping view of the interneurons that maintained a stable phase relationship with motor output. We showed that the dorsal surface of L1 segment contains a higher density of locomotor rhythmic cells than the other segments. Moreover, L1 segment lesioning induced the most important changes in the locomotor activity in comparison with lesions at the T13 or L2 segments. However, no lesions led to selective disruption of either flexor or extensor output. In addition, this study found no evidence of functional parcellation of locomotor interneurons into flexor and extensor pools at the dorsal-ventral midline of the lumbar spinal cord of the rat.     

Cell-specific signaling of the 5-HT1A receptor. Modulation by protein kinases C and A.

Heterologous expression of the rat 5-HT1A receptor in stably transfected GH4C1 rat pituitary cells (clone GH4ZD10) and mouse Ltk- fibroblast cells (clone LZD-7) (Albert, P.R., Zhou, Q.-Y., VanTol, H.H.M., Bunzow, J.R., and Civelli, O. (1990) J. Biol. Chem. 265, 5825-5832) was used to characterize the cellular specificity of signal transduction by the 5-HT1A receptor. We demonstrate that the 5-HT1A receptor, acting via pertussis toxin-sensitive G proteins, can change its inhibitory signaling phenotype and become a stimulatory receptor, depending on the cell type, differentiation state, or intracellular milieu of the cell in which it is expressed. When expressed in pituitary GH4ZD10 cells, activation of 5-HT1A receptors decreased both basal and vasoactive intestinal peptide-enhanced cAMP accumulation and blocked (+/-)-Bay K8644-induced influx of calcium, inhibitory responses which are typical of neurons which endogenously express this receptor. Similarly, 5-hydroxytryptamine (5-HT) also inhibited adenylyl cyclase in fibroblast LZD-7 cells, reducing the forskolin-induced enhancement of cAMP levels by 50%, but did not alter basal cAMP levels. In contrast to GH4ZD10 cells, where 5-HT had no effect on basal or thyrotropin-releasing hormone-induced phosphatidylinositol turnover, 5-HT enhanced the accumulation of inositol phosphates and induced a biphasic increase in [Ca2+]i in LZD-7 cells. These dominant stimulatory actions of 5-HT, as well as the inhibitory effects, were absent in untransfected cells and displayed the potency and pharmacological specificity of the 5-HT1A receptor, indicating that the 5-HT1A subtype coupled to both inhibitory and stimulatory pathways in the fibroblast cell. The actions of 5-HT in GH and L cells were blocked by 24-h pretreatment with pertussis toxin, suggesting that inhibitory G proteins (Gi/G(o)) mediate both inhibitory and stimulatory signal transduction of the 5-HT1A receptor. However, the 5-HT-induced stimulatory pathway in fibroblasts was blocked selectively by acute (2-min) pretreatment with TPA, an activator of protein kinase C. This action of protein kinase C was potentiated by activation of protein kinase A, indicating that the expression of the stimulatory pathway of the 5-HT1A receptor in LZD-7 cells is modulated by second messengers.     

Anatomical study of serotonergic innervation and 5-HT1A receptor in the human spinal cord

Serotonergic innervation of the spinal cord in mammals has multiple roles in the control of motor, sensory and visceral functions. In rats, functional consequences of spinal cord injury at thoracic level can be improved by a substitutive transplantation of serotonin (5-HT) neurons or regeneration under the trophic influence of grafted stem cells. Translation to either pharmacological and/or cellular therapies in humans requires the mapping of the spinal cord 5-HT innervation and its receptors to determine their involvement in specific functions. Here, we have performed a preliminary mapping of serotonergic processes and serotonin-lA (5-HT_1A) receptors in thoracic and lumbar segments of the human spinal cord. As in rodents and non-human primates, 5-HT profiles in human spinal cord are present in the ventral horn, surrounding motoneurons, and also contact their presumptive dendrites at lumbar level. 5-HT_1A receptors are present in the same area, but are more densely expressed at lumbar level. 5-HT profiles are also present in the intermediolateral region, where 5-HT_1A receptors are absent. Finally, we observed numerous serotonergic profiles in the superficial part (equivalent of Rexed lamina II) of the dorsal horn, which also displayed high levels of 5-HT_1A receptors. These findings pave the way for local specific therapies involving cellular and/or pharmacological tools targeting the serotonergic system.     

Evidence for a complex influence of nicotinic acetylcholine receptors on hippocampal serotonin release

The effects of nicotine on 5-hydroxytryptamine (5-HT) release from serotonergic nerve endings in rat dorsal hippocampal slices were studied. Nicotine (50-500 microM:) caused a concentration-dependent increase in 5-HT release. This effect was antagonised by mecamylamine (0.5 microM:), indicating an action at nicotinic receptors. Nicotine-evoked 5-HT release was not affected by tetrodotoxin (3 microM:), cadmium chloride (0.1 mM:), or the absence of Ca(2+) or Na(+) in the superfusion medium. Unexpectedly, higher concentrations of mecamylamine alone (1-50 microM:) increased 5-HT release. This suggested the presence of inhibitory input to 5-HT neurones and that these inhibitory neurones possess tonically active nicotinic receptors. The effect of mecamylamine (50 microM:) on 5-HT release was reduced by the muscarinic M(1) receptor agonist, McN-A-343 (100 microM:), but pirenzepine (0.005-1 microM:), which blocks M(1) receptors, alone increased 5-HT release. Hippocampal serotonergic neurones are known to possess both excitatory nicotinic receptors and inhibitory M(1) receptors. Although there may be several explanations for our results, one possible explanation is that nicotine stimulates 5-HT release by activating nicotinic heteroreceptors on 5-HT terminals. Mecamylamine (0.5 microM:) antagonises this effect, but higher concentrations increase 5-HT release indirectly by blocking the action of endogenous acetylcholine on nicotinic receptors situated on cholinergic neurones that provide muscarinic inhibitory input to 5-HT neurones.     

Stimulation of cAMP synthesis by Gi-coupled receptors upon ablation of distinct Galphai protein expression. Gi subtype specificity of the 5-HT1A receptor.

The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level.     

Pharmacological analysis of locomotion and heart contraction during the development of Helisoma (Mollusca: Gastropoda). Short communication

We investigated involvement of different 5-HT receptors in regulation of ciliary rotation, gliding locomotion and heartbeat of Helisoma embryo at pre- and post-metamorphic stages. Pharmacological analysis suggested that activation of 5-HT1 receptor enhance ciliary rotation but do not affect gliding locomotion. Activation of 5-HT4 receptor depresses both types of locomotion. Before metamorphosis heart contraction is depressed by activation of 5-HT4 and enhanced by activation of 5-HT7 receptor. However, the heart became insensitive to all agonists by hatching. We hypothesized that alterations in affinity or expression of particular 5-HT receptors can underlie the well-coordinated character of serotonin-dependent larval behavior.     

Different receptor subtypes are involved in the serotonin-induced modulation of epileptiform activity in rat frontal cortex in vitro.

The frontal cortex is innervated by serotonergic terminals from the raphe nuclei and it expresses diverse 5-HT receptor subtypes. We investigated the effects of 5-HT and different 5-HT receptor subtype-selective agonists on spontaneous discharges which had developed in rat cortical slices perfused with a Mg2+-free medium and the GABA(A) receptor antagonist picrotoxin. The frequency of synchronous discharges, recorded extracellularly in superficial layers (II/III) of the frontal cortex, was dose-dependently enhanced by 5-HT (2.5-40 microM). That excitatory effect was blocked by the 5-HT2 receptor selective antagonist ketanserin. The 5-HT2A/2C receptor-selective agonist DOI and the 5-HT4 receptor agonist zacopride also increased the frequency of spontaneous discharges. In the presence of ketanserin, 5-HT decreased the discharge rate; a similar effect was observed when the 5-HT1A receptor agonist 8-OH-DPAT or the 5-HT1B receptor agonist CGS-12066B was applied. The 5-HT3 receptor agonist m-CPBG was ineffective. In conclusion, 5-HT produces multiple effects on epileptiform activity in the frontal cortex via activation of various 5-HT receptor subtypes. The excitatory action of 5-HT, which predominates, is mediated mainly by 5-HT2 receptors. The inhibitory effects can be attributed to activation of 5-HT1A and 5-HT1B receptors.     

Implication of serotonin in the control of vigilance states as revealed by knockout-mouse studies

Genetic manipulation of the 5-HT system leads to alterations of 5-HT neurotransmission and provides new opportunities to investigate the role of 5-HT in sleep regulations. Indeed, it represents an alternative to the use of pharmacological tools and, to some extent, of localized lesions of the 5-HT system, which have been, from the 1960s until recently, the main approaches to investigate this question. Homologous recombination knocking-out genes encoding various proteins involved in 5-HT neurotransmission in the mouse has recently allowed further assesment of the role of the serotonin transporter (5-HTT), the monoamine oxidase A (MAO-A), and the 5-HT1A, 5-HT1B and 5-HT2A receptors in the regulation of sleep. In 5-HT1A -/- and 5-HT1B -/- knock-out mice, Rapid Eye Movement sleep (REMs) was enhanced. Pharmacological blockade of these receptors had the same effects in wild-types. Thus, both receptor types exert a tonic inhibitory influence on REMs. In addition, 5-HT1A -/- and 5-HT1B -/- mutants were hypersensitive to 5-HT1B and 5-HT1A receptor agonists, respectively, which suggests that adaptive changes at 5-HT neurotransmission develop in knock-out animals. In the same manner, 5-HTT-/- knock-out mice exhibited increased REMs. This may be accounted for by a decrease in 5-HT1A and 5-HT1B receptor-mediated sleep regulations. In contrast, decreased REMs was observed in MAOA -/- knock-outs, a phenomenon that mimics the effect of pharmacological MAO inhibition. Finally, 5-HT2A -/- and 5-HT2C -/- mice exhibited more wakefulness and less slow wave sleep (SWS) than wild-types. These effects could not be reproduced by 5-HT2A or 5-HT2c receptor blockade in wild-types. To conclude, constitutive knock-outs undergo adaptive processes involving other proteins than those encoded by the invalidated gene, which renders interpretation of the corresponding sleep phenotype difficult. Inducible knock-outs will probably help to overcome this difficulty. Finally, combination of genetic manipulations with relevant pharmacological ones should allow further progress in the understanding of sleep mechanisms.     

Pharmacology of the serotonergic inhibition of steroid-induced reinitiation of oocyte meiosis in the teleost Fundulus heteroclitus

Serotonin (5-HT) was found to inhibit steroid (17α,20β-dihydroxy-4-pregnen-3-one; 17,20βP)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > α-methyl-5-HT > 2-methyl-5-HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20βP-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT_1A, 5-HT₂, and 5-HT₃ receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs. Mol. Reprod. Dev. 48:282–291, 1997. © 1997 Wiley-Liss, Inc.     

Motor effects of serotonin in the central nervous system

Serotoneric pathways in the CNS affect posture and movement, as well as behavioral responses to arousing stimuli. Pharmacologic analysis of these effects has led to an increasingly complex and confusing literature. Increased availability of serotonin (5-HT) by administration of precursors, or by its direct intracranial infusion can induce inhibitory or excitatory behavioral effects depending on the conditions of the experiment, although generally motor inhibition has been found. Availability of 5-HT has been decreased by electrolytic lesions of the raphe nuclei, inhibition of tryptophan hydroxylase, or use of selective neurotoxins. These treatments have generally increased motor activity, especially spontaneous locomotion in a familiar environment, as well as sexual or aggressive behaviors; other behaviors, such as responses in a novel environment, presumably associated with curiosity or fear, have been paradoxically decreased after loss of 5-HT. This differentiation may occur to an important extent through 5-HT projections to hippocampus and limbic structures, notably from the median raphe. Increases or decreases of brain 5-HT, respectively, generally tend to decrease and increase responses to catecholamine agonists such as amphetamines, and some effects of 5-HT may be mediated through catecholaminergic systems. Increased availability of 5-HT in the presence of MAO inhibitors, or challenge with 5-HT-agonists after selective 5-HT-denervation in the CNS has led to a behavioral syndrome marked by hyperactivity, autonomic arousal and myoclonic seizures. The mechanisms underlying this complex response may be mediated by descending 5-HT systems that result in motor excitation at the spinal cord level, in contrast to rostral projections to forebrain that may mediate many behaviorally inhibitory responses.     

Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods

Developing neural networks follow common trends such as expression of spontaneous, recurring activity patterns, and appearance of neuromodulation. How these processes integrate to yield mature, behaviorally relevant activity patterns is largely unknown. We examined the integration of serotonergic neuromodulation and its role in the functional organization of the accessible locomotor network in developing zebrafish at behavioral and cellular levels. Locally restricted populations of serotonergic neurons and their projections appeared in the hindbrain and spinal cord of larvae after hatching (approximately day 2). However, 5-HT affected the swimming pattern only from day 4 on, when sustained spontaneous swimming appeared. 5-HT and its agonist quipazine increased motor output by reducing intervals of inactivity, observed behaviorally (by high-speed video) and in recordings from spinal neurons during fictive swimming (by whole-cell current clamp). 5-HT and quipazine had little effect on the properties of the activity periods, such as the duration of swim episodes and swim frequency. Further, neuronal input resistance, rheobasic current, and resting potential were not affected significantly. The 5-HT antagonists methysergide and ketanserin decreased motor output by prolonging the periods of inactivity with little effect on the active swim episode or neuronal properties. Our results suggest that 5-HT neuromodulation is integrated early in development of the locomotor network to increase its output by reducing periods of inactivity with little effect on the activity periods, which in contrast are the main targets of 5-HT neuromodulation in neonatal and adult preparations.     

Effect of antagonists of serotonin receptors on modulation with serotonin of synaptic activity of projectional neurons of rat amygdala dorsolateral nucleus

The work studies role of different receptor types of serotonin (5-hydroxytryptamine; 5-HT) in the process of synaptic activity modulation with 5-HT of rat dorsolateral amygdala projection neurons. The selective antagonist of 5-HT_1,2 receptors methylsergid maleate was shown to suppress the 5-HT inhibitory action on amplitude of the postsynaptic currents evoked by glutamate and GABA, whereas the antagonist of 5-HT_3,4 receptors SDZ202-557 produced no effect on the above-mentioned 5-HT action. The obtained action indicates that the 5-HT modulatory effect on the projectional neuron synaptic inputs is mediated by 5-HT receptors of the 1 and 2 types.     

Concerted action of antiepileptic and antidepressant agents to depress spinal neurotransmission: Possible use in the therapy of spasticity and chronic pain

Chronic pain states and epilepsies are common therapeutic targets of voltage-gated sodium channel blockers. Inhibition of sodium channels results in central muscle relaxant activity as well. Selective serotonin reuptake inhibitors are also applied in the treatment of pain syndromes. Here, we investigate the pharmacodynamic interaction between these two types of drugs on spinal neurotransmission in vitro and in vivo. Furthermore, the ability of serotonin reuptake inhibitors to modulate the anticonvulsant and windup inhibitory actions and motor side effect of the sodium channel blocker lamotrigine was investigated. In the hemisected spinal cord model, we found that serotonin reuptake inhibitors increased the reflex inhibitory action of sodium channel blockers. The interaction was clearly more than additive. The potentiation was prevented by blocking 5-HT(2) receptors and PKC, and mimicked by activation of these targets by selective pharmacological tools, suggesting the involvement of 5-HT(2) receptors and PKC in the modulation of sodium channel function. The increase of sodium current blocking potency of lamotrigine by PKC activation was also demonstrated at cellular level, using the whole-cell patch clamp method. Similar synergism was found in vivo, in spinal reflex, windup, and maximal electroshock seizure models, but not in the rotarod test, which indicate enhanced muscle relaxant, anticonvulsant and analgesic activities with improved side effect profile. Our findings are in agreement with clinical observations suggesting that sodium channel blocking drugs, such as lamotrigine, can be advantageously combined with selective serotonin reuptake inhibitors in some therapeutic fields, and may help to understand the molecular mechanisms underlying the interaction.     

Serotonergic modulation of murine fundic tone

Fundic tone is maintained through a balance of excitatory and inhibitory input to fundic smooth muscle. The aim of this study was to determine the role of serotonin (5-HT) and 5-HT receptors in modulating murine fundic tone. Muscle strips were prepared from the murine fundus. Intracellular recordings were made from circular smooth muscle cells, and the effects of 5-HT on tone and excitatory and inhibitory junction potentials evoked by electrical field stimulation (EFS) were determined. 5-HT induced a concentration-dependent contraction and smooth muscle depolarization that was tetrodotoxin resistant. The 5-HT(1B/D) receptor antagonists GR-127935 and BRL-155172 significantly inhibited 5-HT-induced contractions. The 5-HT(1B/D) agonist sumatriptan contracted murine fundic muscle. The 5-HT(1A) receptor agonist buspirone relaxed fundic smooth muscle, and the relaxation was inhibited by WAY-100135 but not by N(omega)-nitro-l-arginine or tetrodotoxin. 5-HT enhanced both the excitatory and inhibitory responses to EFS. The 5-HT(3) receptor antagonist MDL-72222 partly inhibited both the excitatory and inhibitory response elicited by EFS, whereas the 5-HT(4) receptor antagonist GR-113808 partly inhibited the EFS-evoked inhibitory response. The 5-HT reuptake inhibitor fluoxetine contracted smooth muscle strips, a contraction that was partially inhibited by GR-127935 and abolished by tetrodotoxin. In conclusion, the data suggest that 5-HT modulates murine fundic contractile activity through several different receptor subtypes. Sustained release of 5-HT maintains fundic tone through postjunctional 5-HT(1B/D) receptors. 5-HT(3) receptors modulate excitatory neural input to murine fundic smooth muscle, and both 5-HT(3) and 5-HT(4) receptors modulate inhibitory neural input to murine fundic smooth muscle.     

Peptidergic neuromodulation of the lumbar locomotor network in the neonatal rat spinal cord

It is now well established that a dynamic balance of neurotransmitters and neuromodulators finely influence the output of neuronal networks and subsequent behaviors. In the present study, to further understand the modulatory processes that control locomotor behavior, we investigated the action of 11 neuropeptides, chosen among the various peptide subfamilies, on the lumbar neuronal network in the in vitro neonatal rat spinal cord preparation. Peptides were bath-applied alone, in combination with N-methyl-D,L-aspartate (NMA) or with the classical 'locomotor cocktail' of NMA and serotonin. Using these different experimental paradigms, we show that each peptide can neuromodulate the lumbar locomotor network and that peptides exhibit different neuromodulatory profiles and potencies even within the same family. Only vasopressin, oxytocin, bombesin and thyrotropin releasing hormone triggered tonic or non-organized rhythmic activities when bath-applied alone. All the neuropeptides modulated NMA induced activity and/ or ongoing sequences of fictive locomotion to varying degrees. These results suggest that neuropeptides play an important role in the control of the neural network for locomotion in the neonatal rat. Their various profiles of action may account in part for the great flexibility of motor behaviors.     

Effects of ginsenoside Rg2 on the 5-HT3A receptor-mediated ion current in Xenopus oocytes

Treatment with ginsenosides, major active ingredients of Panax ginseng, produces a variety of pharmacological or physiological responses with effects on the central and peripheral nervous systems. Recent reports showed that ginsenoside Rg2 inhibits nicotinic acetylcholine receptor-mediated Na+ influx and channel activity. In the present study, we investigated the effect of ginsenoside Rg2 on human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity, which is also one of the ligand-gated ion channel families. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. The ginsenoside Rg2 itself had no effect on the oocytes that were injected with H2O as well as on the oocytes that were injected with the 5-HT3A receptor cRNA. In the oocytes that were injected with the 5-HT3A receptor cRNA, the pretreatment of ginsenoside Rg2 inhibited the 5-HT-induced inward peak current (I5-HT) The inhibitory effect of ginsenoside Rg2 on I5-HT was dose dependent and reversible. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 was 22.3 +/- 4.6 microM. The inhibition of I5-HT by ginsenoside Rg2 was non-competitive and voltage-independent. These results indicate that ginsenoside Rg2 might regulate the 5-HT3A receptors that are expressed in Xenopus oocytes. Further, this regulation on the ligand-gated ion channel activity by ginsenosides might be one of the pharmacological actions of Panax ginseng.