Serotonin selectively attenuates glutamate-evoked activation of noradrenergic locus coeruleus neurons.

The effect of 5-HT on activity of noradrenergic locus coeruleus (LC) neurons was studied using microiontophoretic and micropressure drug application in anesthetized rats. 5-HT had no consistent effect on LC spontaneous discharge, eliciting a modest decrease overall. However, 5-HT reliably attenuated responses of LC neurons to excitatory amino acids (EAAs), one of the major classes of transmitters in afferents to these neurons. This effect was specific for EAA responses because it occurred for glutamate and kainate but not for ACh. In contrast, iontophoretic norepinephrine (NE) selectively attenuated spontaneous activity but not responses evoked by either glutamate or ACh. The responsiveness of LC neurons to EAAs as quantified by a response-contrast measure (evoked excitation/basal activity) was markedly reduced by 5-HT, but was increased by NE. For ACh, such responsiveness of LC cells was not changed by 5-HT, but was increased by NE. The effects of 5-HT were prevented and reversed by iontophoretically applied antagonists of 5-HT receptors, methysergide and methiothepin. Thus, 5-HT appears to selectively interact with EAA responses of LC neurons, acting as a filter to attenuate LC activity linked to its major EAA inputs while allowing other channels afferent to the LC (e.g., those utilizing ACh) to be expressed.     

Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock

Noxious stimuli, such as electrical shocks to the animal's tail, enhance Aplysia's gill- and siphon-withdrawal reflex. Previous experimental work has indicated that this behavioral enhancement, known as dishabituation (if the reflex has been habituated) or sensitization (if it has not been habituated), might be mediated, at least in part, by the endogenous monoaminergic transmitter serotonin (5-HT). To assess 5-HT's role in dishabituation and sensitization of Aplysia withdrawal reflex, we treated Aplysia with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). We found that 5,7-DHT treatment significantly reduced the dishabituation of the withdrawal reflex produced by tail shock. Treatment with the neurotoxin also blocked the heterosynaptic facilitation of monosynaptic connections between siphon sensory neurons and their follower cells, which contributes to the behavioral enhancement. Analysis by high-performance liquid chromatography indicated that 5,7-DHT treatment significantly reduced 5-HT levels in the Aplysia CNS. Moreover, the neurotoxic effects of 5,7-DHT appeared to be relatively specific for serotonergic pathways. Thus, 5,7-DHT treatment did not disrupt the ability of nonserotonergic facilitatory interneurons, the L29 cells, to facilitate the connections of siphon sensory neurons. Also, 5,7-DHT reduced 5-HT-dependent, but not dopamine-dependent, histofluorescence in Aplysia central ganglia. Finally, 5,7-DHT does not reduce the levels of the facilitatory peptides SCPA and SCPB within the Aplysia CNS. Our results, together with those of Mackey et al. (1989), indicate that 5-HT plays a major role in mediating dishabituation and sensitization of Aplysia's withdrawal reflex.     

Comparative localization of two serotonin receptors and sensorin in the central nervous system of Aplysia californica

Aplysia californica is a powerful model for understanding the cellular and molecular mechanisms underlying modulation of neuronal plasticity and learning. In the central nervous system of Aplysia, serotonin is associated with various behaviors. For example, it induces short-, intermediate-, and long-term synaptic changes in sensory neurons during learning and inhibits the afterdischarge of the bag cells that initiate egg-laying behavior. Little is known about the nature and contribution of serotonin receptors involved in the numerous serotonin-mediated physiological responses in Aplysia. Recently, two G(i)-coupled serotonin receptors (5-HT(ap1) and 5-HT(ap2)) were cloned. We now report that, by using in situ hybridization to express the profile of these receptors, we are able to gain critical insight into their roles in the behavior of Aplysia. We compared their distribution to that of sensorin-A, a peptide specifically found in sensory neurons. We wished to determine their involvement in some simple forms of behavioral modifications. 5-HT(ap1) and 5-HT(ap2) mRNAs are expressed in all ganglia of the Aplysia central nervous system. Stronger signal was observed with the 5-HT(ap2) antisense probe than with the 5-HT(ap1) antisense probe. Notably, mRNA coding for the receptors was found in several identified neurons, in the bag cells, in characterized serotonergic neurons, and in neurons of the mechanosensory clusters that expressed sensorin. We also observed heterogeneity of receptor expression between R2 and LPl1 and among neurons of a single cluster of sensory neurons. These results suggest that 5-HT(ap1) and 5-HT(ap2) receptors may regulate the response to serotonin and/or its release in several neurons.     

Vipoxin both activates and antagonizes three types of acetylcholine response inAplysia neurons

The effects of vipoxin, a 13,000 Dalton protein component of Russell's viper venom on responses of voltage-clamped Aplysia neurons to acetylcholine (ACh) and monoamines has been studied. At low doses vipoxin reversibly antagonizes all 3 types of ionic response to ACh or carbachol, the order of susceptibility to blockade being Na+ greater than K+ greater than Cl-. High doses of vipoxin directly evoke the same ionic response on a given cell as that evoked by ACh. Responses to vipoxin are reversibly antagonized by cholinergic antagonists (e.g. hexamethonium, tetraethylammonium), but not by monoamine antagonists (e.g. bufotenine, ergometrine, cimetidine). In addition to activation of cholinergic responses, high doses of vipoxin also produce a reversible potentiation of responses to dopamine and 5-hydroxytryptamine on some cells. In contrast to its effects on Aplysia neurons, vipoxin has neither agonist nor antagonist actions at the frog neuromuscular junction. These results suggest that this venom protein acts as a partial agonist at molluscan ACh receptors and provides evidence for some phylogenetic difference between molluscan and vertebrate ACh receptors.     

Protein kinase inhibitors selectively block phorbol ester- or forskolin-induced changes in excitability of Aplysia neurons

Exposure of the bag cell neurons of Aplysia to activators of protein kinase C, such as phorbol esters, enhances electrically evoked action potentials by increasing the voltage-dependent calcium current. We have hypothesized that this effect is mediated by the activation of protein kinase C (PKC). An important prediction of this hypothesis is that inhibitors of PKC should inhibit these phorbol ester-induced changes in bag cell neuronal excitability. We have now found that treatment of bag cell neurons with the protein kinase inhibitor 1-[5-isoquinolinesulfonyl]-2-methyl piperazine (H-7) inhibits the phorbol ester-induced enhancement of bag cell action potentials and prevents the enhancement of calcium current by phorbol esters. The height and width of electrically evoked action potentials in bag cell neurons can also be enhanced by cAMP analogs or agents that elevate cAMP. These agents do not influence the major voltage-dependent calcium current in the bag cell neurons but may act by modulating potassium currents and other voltage-dependent currents. We have found that microinjection of a protein inhibitor of cAMP-PK (PKA-I) into isolated bag cell neurons prevents and reverses the effect of the adenylate cyclase activator forskolin on action potentials of these cells. In contrast, H-7 does not inhibit the effects of forskolin on a variety of responses in these cells, including its effects on action potentials, granule movement, and 32P incorporation into phosphoproteins. This suggests that H-7 is selective for PKC relative to cAMP-PK in intact bag cell neurons.     

Localization of potential serotonergic facilitator neurons in Aplysia by glyoxylic acid histofluorescence combined with retrograde fluorescent labeling

A variety of evidence suggests that 5-HT participates in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) have shown that the 5-HT neurotoxin 5,7-DHT markedly reduces both the synaptic facilitation and behavioral dishabituation produced by tail shock. To provide more direct evidence for a role of 5-HT, I have used histological techniques to try to locate individual serotonergic facilitator neurons. I first used a modification of the glyoxylic acid histofluorescence technique to map serotonergic and dopaminergic neurons in the CNS of Aplysia. Intracellular fluorescent labeling combined with histofluorescence indicates that the previously identified L29 facilitator neurons are not serotonergic. Nerve transection experiments suggest that most of the perisomatic 5-HT histofluorescence in the abdominal ganglion (the location of the siphon sensory cells) comes from neurons whose cell bodies are located in the pedal or cerebral ganglia. As there are at least 500 serotonergic neurons in those ganglia, I combined retrograde fluorescent labeling with histofluorescence to identify a small subset of those neurons which send processes to the abdominal ganglion and are therefore potential serotonergic facilitators. In the following paper, Mackey et al. (1989) show that stimulation of 2 of those neurons in the cerebral ganglia (the CB1 cells) produces presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the withdrawal reflex.     

Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons

Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1989) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CB1 cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CB1 neurons are serotonergic. In a semiintact preparation, the CB1 neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CB1 neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CB1 neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia.     

Neurogastroenterology of tegaserod (HTF 919) in the submucosal division of the guinea-pig and human enteric nervous system.

Actions of the 5-HT(4) serotonergic receptor partial agonist, tegaserod, were investigated on mucosal secretion in the guinea-pig and human small intestine and on electrophysiological behaviour of secretomotor neurons in the guinea-pig small intestinal submucosal plexus. Expression of 5-HT(4) receptor protein and immunohistochemical localization of the 5-HT(4) receptor in the submucosal plexus in relation to expression and localization of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter were determined for the enteric nervous system of human and guinea-pig small intestine. Immunoreactivity for the 5-HT(4) receptor was expressed as ring-like fluorescence surrounding the perimeter of the neuronal cell bodies and co-localized with the vesicular ACh transporter. Exposure of mucosal/submucosal preparations to tegaserod in Ussing chambers evoked increases in mucosal secretion reflected by stimulation of short-circuit current. Stimulation of secretion had a relative high EC(50) of 28.1 +/- 1.3 mumol L(-1), was resistant to neural blockade and appeared to be a direct action on the secretory epithelium. Tegaserod acted at presynaptic 5-HT(4) receptors to facilitate the release of ACh at nicotinic synapses on secretomotor neurons in the submucosal plexus. The 5-HT(2B) receptor subtype was not involved in actions at nicotinic synapses or stimulation of secretion.     

Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA

The molecular mechanism underlying the signal transduction from muscarinic and serotonergic receptors to Cl- channels were investigated in Xenopus oocyte microinjected with rat brain poly(A)+ mRNA. Transient Cl- current responses of the mRNA-injected oocytes to acetylcholine (ACh) and serotonin (5-HT) were similar in amplitude and onset. Although pharmacological characterization indicated that distinct M1-like and S1-like receptors of rat brain are involved in the ACh and 5-HT responses, respectively, these responses cross-desensitized each other completely. A common involvement of the GTP-binding proteins coupled to phosphoinositide breakdown was suggested by the findings that intracellular application of guanosine 5'-O-(2-thio)bisphosphate (GDP beta S) or neomycin greatly suppressed both ACh and 5-HT responses. These responses were not affected by exposure of the mRNA-injected cells to cholera toxin, but they were inhibited by pertussis toxin. The increase in inositol trisphosphate (IP3) responsive both to ACh and 5-HT coincided with the expression of Cl- current responses. However, only 5-HT but not ACh slightly increased the cyclic AMP (cAMP) content of the mRNA-injected cells. Intracellular injection of either IP3 or Ca2+ produced a transient Cl- current in the mRNA-injected cells as well as in non-injected cells, while 1-oleoyl-2-acetylglycerol (OAG), cAMP or cyclic GMP (cGMP) never elicited chloride current responses. It was proposed that muscarinic and serotonergic receptors are commonly linked to phosphoinositide breakdown through the mediation of GTP-binding proteins Ni and/or No in mRNA-injected oocytes.     

Uptake of [3H]serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex in Aplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [3H]serotonin ([3H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure of Aplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10(-6) M [3H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [3H]5-HT. In addition, [3H]-5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [3H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [3H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the uptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Effects of intermittent hypoxia on cat petrosal ganglion responses induced by acetylcholine, adenosine 5'-triphosphate and NaCN

Exposure to chronic intermittent hypoxia (CIH) for 4 days enhances the cat carotid body (CB) chemosensory responses to acute hypoxia. However, it is not known if CIH enhances the responses of the petrosal ganglion (PG) neurons that innervate the CB chemoreceptor cells. Accordingly, we studied the effects of the CB putative excitatory transmitter acetylcholine (ACh) and adenosine 5 -triphosphate (ATP), and the effects of citotoxic hypoxia (NaCN) applied to the isolated PG from cats exposed to CIH for 4 days. The dose-dependent curve parameters of the frequency of discharges evoked in the carotid sinus nerve by the application of ACh, ATP and NaCN to the isolated PG in control condition were not significantly modified in the CIH-treated cats. Present results suggest that CIH enhances the chemosensory responses to acute hypoxia acting primarily at the chemoreceptor cells, without major changes in the response of PG neurons evoked by the application of putative CB excitatory transmitters to their somata.     

Selective modulation of spike duration by serotonin and the neuropeptides, FMRFamide, SCPB, buccalin and myomodulin in different classes of mechanoafferent neurons in the cerebral ganglion of Aplysia

An examination of the cellular properties and synaptic outputs of mechanoafferent neurons found on the ventrocaudal surface of the cerebral ganglion of Aplysia indicated that the cerebral mechanoafferent (CM) neurons are a heterogeneous population of cells. Based on changes in action potential duration in response to bath applications of 5-HT in the presence of TEA, CM neurons could be divided into 2 broad classes: mechanoafferents whose spikes broaden in response to 5-HT (CM-SB neurons) and mechanoafferents whose spikes narrow in response to 5-HT (CM-SN neurons). Morphological and electrophysiological studies of the CM-SN neurons indicated that they were comprised of previously identified interganglionic cerebral-buccal mechanoafferent (ICBM) neurons and a novel set of sensory neurons that send an axon into the LLAB cerebral nerve and have perioral zone receptive fields that are similar to those of ICBM neurons. Changes in spike width due to 5-HT were correlated with changes in synaptic output as indicated by the magnitudes of EPSPs evoked in postsynaptic neurons. Electrical stimulation of cerebral nerves and connectives also produced spike narrowing or broadening, and the sign of the effect was a function of the parameters of stimulation. Both heterosynaptic facilitation and heterosynaptic depression of EPSPs evoked in follower cells could be demonstrated. A variety of putative neuromodulators other than 5-HT were also found to affect the duration of action potentials in both classes of CM neurons. FMRFamide had effects opposite to that of 5-HT. SCPB and a recently characterized Aplysia neuropeptide, buccalin, broadened the spikes of both CM classes. Another neuropeptide, myomodulin, decreased the duration of CM-SB neuron spikes but had no effect on CM-SN spikes. Since the CM neurons appear to mediate a variety of competing behaviors, including feeding, locomotion, and defensive withdrawal, the various neuromodulator actions may contribute to the mechanisms whereby behaviors are selected and modified.     

Reciprocal modulation of calcium current by serotonin and dopamine in the identified Aplysia neuron R15

Voltage-clamp methods were employed to study the effects of serotonin (5-HT) and dopamine on the pharmacologically isolated calcium current in the identified Aplysia neuron R15 grown in cell culture. Neurons were obtained from juvenile animals and had not yet developed the bursting pacemaker pattern of activity characteristic of R15 in mature animals. In R15 5-HT elicits a biphasic response consisting of excitatory depolarization followed by an inhibitory hyperpolarization and dopamine elicits an inhibitory hyperpolarization. 5-HT increased the Ca2+ current without affecting its voltage dependence. The 5-HT effect persisted when Ba2+ was employed to carry current through Ca2+ channels. 5-HT did not affect the rate of Ca2+-dependent Ca2+ current inactivation other than through its effect on the magnitude of the Ca2+ current. The adenylate cyclase activator forskolin, in the presence of a phosphodiesterase inhibitor, also increased the magnitude of the Ca2+ or Ba2+ current. This result suggested that the 5-HT-induced enhancement of Ca2+ current was mediated by cAMP. Dopamine inhibited Ca2+ current when either Ca2+ or Ba2+ was employed as the current carrier. Dopamine did not affect the rate of Ca2+-dependent inactivation of Ca2+ current other than through its effect on the magnitude of the Ca2+ current. Intracellular injection of the Ca2+ chelator EGTA inhibited serotonergic modulation of the Ca2+ current but not dopaminergic modulation. These results indicated that the putative neurotransmitters 5-HT and dopamine may regulate bursting activity in mature R15 neurons through modulation of Ca2+ current.     

Serotonin modulation of Hermissenda type B photoreceptor light responses and ionic currents: implications for mechanisms underlying associative learning

Type B photoreceptors in the eyes of the nudibranch mollusc Hermissenda have previously been shown to exhibit enhanced steady-state depolarizing light responses, increases in steady-state light-induced impulse frequency and a decrease in resting membrane conductance on days following exposure to repeated pairings of light and rotation. These training-produced changes in the electrophysiological properties of B cells have been attributed to reductions in a voltage-dependent K+ current (IA), a calcium-activated K+ current (IK-Ca), and enhancement of a voltage-dependent Ca2+ current (ICa). Current- and voltage-clamp analysis of the effects of serotonin (5-HT) upon B cells revealed that 5-HT mimicked many of the effects of associative training. 5-HT enhanced the steady-state light response and decreased resting membrane conductance of B cells. Voltage-clamp analysis revealed five distinct effects of 5-HT upon the voltage-, calcium-, and light-dependent ionic conductances of B photoreceptors. In the dark-adapted cell, 5-HT enhanced but then suppressed IA, IK-Ca, and enhanced ICa. The presentation of 5-HT in combination with light accelerated the reduction of K+ currents. 5-HT also transiently reduced the fast component of light-induced inward current (INa-light), and enhanced a slower component of light-induced inward current. In an accompanying paper, 5-HT is shown to be localized within the cerebropleural neuropil where terminals presynaptic to B photoreceptors are located. Disruption of serotonergic neurotransmission by a variety of drugs is also shown to reduce the pairing-specific differences in Type B photoreceptor cumulative depolarization and resting membrane conductance decreases that are produced by in vitro conditioning. Collectively, the results indicate that 5-HT plays an important role in mediating the conductance changes produced in Type B photoreceptors by associative training.     

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT₃ agonist-induced inhibition of electrically evoked [³H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT₂, 5-HT₃ and D₂ receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT₃ agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 μM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 μM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 μM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT₂ receptor antagonist, ICS-205,930, a 5-HT₃ receptor antagonist or sulpiride, a D₂ receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 μM) and was completely reversed by sulpiride (1 μM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT₂, 5-HT₃ and D₂ receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D₂ receptors and high concentrations of the 5-HT₃ receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.     

Serotonin modifies the neuronal inhibitory responses to gamma-aminobutyric acid in the red nucleus: a microiontophoretic study in the rat

The effects of 5-hydroxytryptamine (5-HT) on the inhibitory responses evoked by gamma-aminobutyric acid (GABA) in neurons of the red nucleus (RN) were studied using a microiontophoretic technique. Extracellular unitary recordings performed in anesthetized rats demonstrated that 5-HT ejection influenced GABA-evoked inhibition in 94% of RN neurons, enhancing them in 52% and depressing them in 46% of cases. Both effects were specific and dose-dependent,although enhancements or depressions of the GABA responses were respectively inversely and directly related to the doses of 5-HT applied. The type of modulation exerted by 5-HT on the GABA responses was independent of the action of the amine on background firing. In fact, 5-HT induced an enhancement of the GABA responses in neurons mostly located in the rostral RN and a depression in those in the caudal RN. The application of 8-hydroxy-2(di-n-propylamino)tetralin, a specific 5-HT(1A) receptor agonist, enhanced GABA responses, whereas alpha-methyl-5-hydroxytryptamine, a 5-HT(2A) receptor agonist, depressed them. Both the 5-HT(2) antagonist methysergide and the 5-HT(2A) selective antagonist ketanserin were able to block partially or totally the depressive action of 5-HT on GABA responses. In contrast, the same 5-HT antagonists mimicked the enhancing action of 5-HT on the GABA responses or were ineffective. Application of bicuculline, a GABA(A) receptor antagonist, enhanced the excitatory action of 5-HT on the background firing and slightly reduced the inhibitory action. It is concluded that 5-HT is able to modulate GABA-evoked responses in RN neurons by acting on both 5-HT(1A) and 5-HT(2A) receptors. The functional significance of a serotonergic control on GABAergic inhibitory effects in RN is discussed.     

Modulation of acetylcholine and 5-hydroxytryptamine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts containing cholinergic and/or serotonergic neurons

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing fetal cells from the mesencephalic raphe (rich in serotonergic neurons; RAPHE), the medial septum and the diagonal band of Broca (rich in cholinergic neurons; SEPT), or a mixture of both (COTR). Lesion-only (LES) and sham-operated rats (SHAM) were used as controls. Hippocampal slices of these rats (5-9 month after surgery) were preincubated with [3H]choline or [3H]5-HT, superfused continuously (in the presence of hemicholinium-3 or fluvoxamine) and stimulated electrically (360 pulses, 2 ms, 3 Hz, 26-28 mA) in order to study the presynaptic modulation of acetylcholine (ACh) and serotonin (5-HT) release. The accumulation of [3H]choline and the evoked overflow of [3H]ACh were significantly reduced in slices from LES and RAPHE rats, but reached a close-to-normal level in SEPT and COTR rats. As to accumulation and overflow of [3H]5-HT, the lesion-induced reduction was compensated for only in RAPHE and COTR rats. The relative amount of evoked [3H]5-HT release (in % of tissue-3H) was significantly increased in LES and SEPT rats. Only slight differences (group LES) were found in the sensitivity of muscarinic and serotonergic autoreceptors towards oxotremorine and CP 93,129, respectively. Moreover, CP 93,129 induced a significantly weaker inhibition of ACh release in slices of COTR rats than in all other groups. Using the 5-HT1A receptor agonist 8-OH-DPAT and antagonist Way 100,635, no evidence for a modulatory influence of 5-HT1A receptors was found in RAPHE and COTR rats. It is concluded that despite substantial lesion- and graft-induced changes in the amount of ACh and 5-HT released by hippocampal slices of lesion-only or grafted rats, the presynaptic modulation of these transmitters is only slightly affected by changes in the neuronal environment.     

Regulation of the circadian clock in the Aplysia eye: mimicry of neural action by serotonin

In Aplysia, activity of efferent fibers in the optic nerve can influence the circadian clock in the eye. In addition, serotonin (5-HT) is present in the eye and influences the function of the clock. Accordingly, we hypothesized that 5-HT is the transmitter of the optic efferents, and tested the prediction that exogenous 5-HT would mimic the action of the optic efferents on the clock. We also tested the prediction that the action of the efferents would be prevented by blocking synaptic secretions with high Mg2+, low Ca2+ (HMLC). Activity of the optic efferents enhances clock resetting in response to the onset of darkness. We used this neural enhancement as a measure of neural action on the eye clock. We found that HMLC blocked neural enhancement. Serotonin enhanced resetting to the same extent as efferent activity. Enhancement by 5-HT did not summate with neural enhancement. High concentrations of 5-HT ([5-HT] greater than or equal to 6 X 10(-4) did not cause enhancement. High concentrations of 5-HT also blocked neural enhancement. Enhancement by 5-HT depended on the phase of drug application, much as neural enhancement depends on the phase of neural activity. Enhancement by 5-HT depended on day length in a manner similar to the enhancement by neural activity. HMLC did not block the facilitatory action of 5-HT. Several other transmitters that may be active in the eye did not enhance resetting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of serotonin-immunoreactive cell bodies and processes in the abdominal ganglion of mature Aplysia

Sensitization of the gill withdrawal reflex in Aplysia californica is an elementary form of learning, in part resulting from presynaptic facilitation of the LE mechanoreceptor neurons of the abdominal ganglion. It has previously been established that either application of serotonin or direct stimulation of a group of facilitatory neurons, the L29 cells of the abdominal ganglion, can simulate the effect of physiological stimulation in producing presynaptic facilitation. Because the evidence that serotonin serves as a facilitatory transmitter was indirect, we examined the distribution of serotonin-immunoreactive fibers and cell bodies in the abdominal ganglion in order to answer two questions: (1) do the sensory neurons receive serotonergic innervation and (2) are the L29 cells serotonergic? We observed two distinctive patterns of serotonergic innervation within the ganglion, sparse and dense. The sparse pattern is correlated with a serotonin-stimulated increase in cAMP in identified target cells, while the dense innervation is not. We found a sparse distribution of serotonin-immunoreactive fibers with varicosities close to both cell bodies and processes of identified LE sensory cells. It therefore is likely that the sensory neurons do receive serotonergic innervation. We also mapped the population of serotonergic neuronal cell bodies in the ganglion, and found five clusters of neurons. Cells in one of these clusters, the identified RB neurons, had previously been shown to synthesize serotonin from tryptophan and to contain the neurotransmitter in high concentration. Identified L29 facilitator cells marked by injection with Lucifer Yellow do not contain serotonin immunoreactivity and therefore evidently are not a source of serotonergic input onto sensory cells.     

G proteins are involved in the regulation of transmitter release at an Aplysia cholinergic synapse

At an identified cholinergic synapse of the Aplysia buccal ganglion, presynaptic injections of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) depressed the amplitude of evoked postsynaptic responses. This reduction of acetylcholine (ACh) release by GTP-gamma-S, prevented by pre-injection of guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) in the presynaptic neuron, was due to a reduction of the number of ACh quanta released. The mean amplitude of the evoked miniature postsynaptic current (MPSC) was unchanged. The presynaptic Ca2+ influx was lowered.