Acute Ca(2+)-dependent desensitization of 5-HT(1A) receptors is mediated by activation of protein kinase A (PKA) in rat serotonergic neurons.

This report investigates acute changes in the sensitivity of 5-HT(1A) receptors in dorsal raphe (dr) neurons in response to elevated serotonin. DR neurons were isolated from adult rats and measurements of inhibition of Ca(2+) current by 5-HT were obtained using the whole cell patch clamp technique. During a 10-min application of 5-HT (with normal [Ca(2+)](i) approximately 100 nM) a desensitization occurred. The response to 20 nM 5-HT decreased by 66% relative to control and remained depressed for about 30 min. When the internal [Ca(2+)] was buffered to <1 nM only a weak transient desensitization occurred that was surmountable with higher [5-HT]. Adenylyl cyclase activation with forskolin mimicked the desensitization and selective inhibition of protein kinase A (PKA), but not protein kinase C (PKC), partially antagonized the desensitization induced by 5-HT. To measure the activity of PKA and phosphatase enzymes, dr slices were incubated with the selective agonist dipropyl-5-carboxamidotryptamine (DP-5-CT, 1 microM) for 10 min and the phosphorylation of the PKA substrate Kemptide was followed using ATP-gamma(32)P. DP-5-CT inhibited the cAMP stimulated maximal activity of PKA but raised basal PKA activity, thus increasing the percentage of PKA in the active state (activity ratio), an effect that was prevented by the selective 5-HT(1A) antagonist WAY100635. DP-5-CT also caused a significant inhibition of phosphatase activity. These data support a model in the dr where 5-HT(1A)-receptor stimulation of PKA promotes phosphorylation of a target and phosphatase inhibition leading to heterologous desensitization. The effect would be expected to have physiological consequences for 5-HT-mediated inhibitory post synaptic potentials and the Ca(2+) component of the action potentials of dr neurons.

Effect of pertussis toxin and N-ethylmaleimide on voltage-dependent and -independent calcium current modulation in serotonergic neurons.

Introduction of GTP-gamma-S into a neuronal cell spontaneously results in G-protein activation. A possible contribution to this mechanism is that some receptors have a constitutive activity that stimulates GDP/GTP exchange resulting in increased GTPase activity of G-protein alpha subunits, leading to a facilitation of GTP-gamma-S binding. It follows that partial or complete uncoupling of receptors and G-proteins could inhibit Ca(2+) current modulation by GTP-gamma-S. This possibility was tested in acutely isolated rat dorsal raphe neurons by uncoupling the receptor and G-protein using N-ethylmaleimide and pertussis toxin. Since these compounds have been suggested to differentially block voltage-dependent inhibition, relative to voltage-independent, we investigated whether the apparent voltage-independent component of Ca(2+) channel modulation by 5-hydroxytryptamine (5-HT) shares the same mechanism as the voltage-dependent component. N-ethylmaleimide inhibited the response to 5-HT by about 50% but had no effect on the response to GTP-gamma-S. In dorsal raphe neurons 28.9% of the total response to 5-HT was voltage-independent. N-ethylmaleimide had identical effects on the voltage-dependent and -independent components as measured by tail current inhibition. The response to 5-HT was completely sensitive to pertussis toxin, and completely uncoupling the receptors and G-proteins did not affect the maximal response to GTP-gamma-S. Our results suggest that the apparent voltage-independent component of Ca(2+) channel modulation by 5-HT in dorsal raphe neurons might share the same mechanism as does the voltage-dependent component. In addition, these experiments provided evidence that partial or even complete uncoupling of receptors and G-proteins did not affect Ca(2+) current modulation by direct activators of G-proteins.

A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels.

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.

ATP modulates Na+ channel gating and induces a non-selective cation current in a neuronal hippocampal cell line.

Extracellular ATP evoked two excitatory responses in hippocampal neuroblastoma cells (HN2). The first, an opening of a receptor-operated non-selective cation channel and the second was a leftward shift in Na+ channel activation. Both ATP (5-1000 microM) and 2',3'-(4-benzoyl)-benzoyl-ATP (Bb-ATP, 50 microM) activated a non-selective cation current reversing near 0 mV and shifted the Na+ activation and inactivation curves to the left. Based on a comparison of a series of agonists and antagonists, the inward current appeared to be partially mediated by activation of a P2X7 receptor, although hybrid channels cannot be ruled out. The shift in Na+ channel gating could be separated from the opening of the cation channel, as application of the P2Y antagonist Reactive Blue-2 and GTP shifted the Na+ current activation to the left but failed to elicit the inward cation current. Both responses to ATP and Bb-ATP were insensitive to block by the P2X antagonist suramin (300 microM) but were prevented by incubation in oxidized ATP (200 microM); a putative P2X7 receptor antagonist. Prior screening of the surface negative charge of the membrane with a high concentration of divalent cations prevented both responses. We suggest that ATP4- activates a P2X receptor and becomes trapped on a site, on or near the Na+ channel. Activation of the P2X receptor leads to the opening of a non-specific cation channel, while the binding of ATP4- leads to a modified charge sensed by the Na+ channel, similar to what occurs in the presence of charged amphiphiles as well as a number of beta-scorpion toxins.

Competition between internal AlF(4)(-) and receptor-mediated stimulation of dorsal raphe neuron G-proteins coupled to calcium current inhibition.

Intracellular aluminum fluoride (AlF(4)(-)), placed in a patch pipette, activated a G-protein, resulting in a "tonic" inhibition of the Ca(2+) current of isolated serotonergic neurons of the rat dorsal raphe nucleus. Serotonin (5-HT) also inhibits the Ca(2+) current of these cells. After external bath application and quick removal of 5-HT to an AlF(4)(-) containing cell, there was a reversal or transient disinhibition (TD) of the inhibitory effect of AlF(4)(-) on Ca(2+) current. A short predepolarization of the membrane potential to +70 mV, a condition that is known to reverse G-protein-mediated inhibition, reversed the inhibitory effect of AlF(4)(-) on Ca(2+) current and brought the Ca(2+) current to the same level as that seen at the peak of the TD current. With AlF(4)(-) in the pipette, the TD phenomenon could be eliminated by lowering pipette MgATP, or by totally chelating pipette Al(3+). In the presence of AlF(4)(-), but with either lowered MgATP or extreme efforts to eliminate pipette Al(3+), the rate of recovery from 5-HT on wash was slowed, a condition opposite to that where a TD occurred. The putative complex of AlF(4)(-)-bound G-protein (Galpha.GDP. AlF(4)(-)) appeared to free G-betagamma-subunits, mimicking the effect on Ca(2+) channels of the G.GTP complex. The ON-rate of the inhibition of Ca(2+) current, after a depolarizing pulse, by betagamma-subunits released by AlF(4)(-) in the pipette was significantly slower than that of the agonist-activated G-protein. The OFF-rate of the AlF(4)(-)-mediated inhibition in response to a depolarizing pulse, a measure of the affinity of the free G-betagamma-subunit for the Ca(2+) channel, was slightly slower than that of the agonist stimulated G-protein. In summary, AlF(4)(-) modified the OFF-rate kinetics of G-protein activation by agonists, but had little effect on the kinetics of the interaction of the betagamma-subunit with Ca(2+) channels. Agonist application temporarily reversed the effects of AlF(4)(-), making it a complementary tool to GTP-gamma-S for the study of G-protein interactions.

Agonist stimulation of the serotonin1A receptor causes suppression of anoxia-induced apoptosis via mitogen-activated protein kinase in neuronal HN2-5 cells.

Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin1A receptor (5-HT1A-R), could cause attenuation of the activity of both N-type Ca2+ channels and N-methyl-D-aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5-HT1A-R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca2+ channels. Using a hippocampal neuron-derived cell line (HN2-5) that is Ca2+ channel-deficient, we demonstrate here that an alternative pathway is responsible for 5-HT1A-R-mediated protection of these cells from anoxia-triggered apoptosis, assessed by deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL). The 5-HT1A-R agonist-evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation-mediated activation of mitogen-activated protein kinase (MAPK), as evidenced by the elimination of the agonist-elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI-3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5-HT1A-R caused a 60% inhibition of anoxia-stimulated caspase 3-like activity in the HN2-5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5-HT1A-R caused an activation of PI-3Kgamma in HN2-5 cells, our results showed that this PI-3Kgamma activity was not linked to the 5-HT1A-R-promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2-5 cells, agonist binding to the 5-HT1A-R results in MAPK-mediated inhibition of a caspase 3-like enzyme and a 60-70% suppression of anoxia-induced apoptosis through a Ca2+ channel-independent pathway.

Order of application determines the interaction between phorbol esters and GTP-gamma-S in dorsal raphe neurons: evidence that the effect of 5-HT is modified upstream of the G protein Ca channel interaction.

Phorbol esters activating protein kinase C (PKC) partially uncouple the inhibitory effect of serotonin (5-HT) from serotonergic neuron Ca2+ current. Presently the site of action of PKC is not known and may be the receptor, G protein, or ion channel. We recorded Ca2+ current from acutely isolated neurons with the use of the patch-clamp technique to study the site of action of PKC. Activation of the G protein with internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) occluded the response to 5-HT, but unexpectedly this effect was not reversed by the addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) despite the voltage-dependent reversal of the effect of GTP-gamma-S by long depolarizing steps to +80 mV. PMA was, however, able to partially reverse 5-HT-induced inhibition of Ca2+ current. The rate of reinhibition of the Ca2+ current (related to the concentration of activated G proteins) by GTP-gamma-S after the addition of PMA at -50 mV was identical to the rate when only GTP-gamma-S was present. By contrast, when cells were exposed first to PMA, and then GTP-gamma-S was perfused into the cell, GTP-gamma-S lost about half of its ability to activate the G protein. The rate of reinhibition of the Ca2+ current by internal GTP-gamma-S was also reduced in cells pretreated with PMA. The original result in which PMA did not reverse the action of GTP-gamma-S suggested that the channel was not the functional site of action of PMA, nor was the site on the G protein that binds to the channel, but it did not rule out the receptor. When the receptor was bypassed, after prior PKC activation, it was found that direct activation of the G protein by a nonhydrolyzable analogue of GTP was reduced; taken as a whole, this indicates that in dorsal raphe, and perhaps other neurons, the site of the critical phosphorylation may be on the G protein and possibly at the GTP binding site.

QEHA27, a peptide that binds to G-protein beta gamma-subunits, reduces the inhibitory effect of 5-HT on the Ca2+ current of rat dorsal raphe neurons.

We studied a 27 amino acid peptide (QEHA27) containing the G beta gamma binding motif QXXER, found in adenylyl cyclase 2 and several effector proteins of the G beta gamma-subunit. The patch-clamp technique was utilized to record Ca2+ current from serotonergic dorsal raphe neurons and to introduce the peptide QEHA27 into the cell. We investigated whether it antagonized the inhibitory modulation of Ca2+ current. Compared to the control group, 5-HT was 41% less effective at inhibiting Ca2+ current with 500 microM QEHA27 in the pipette. The QEHA27 peptide did not simply occlude the response to 5-HT because there was no stimulating effect of QEHA27 on the G-protein mediated response. This appears to be the first report of an interference between this peptide and the modulation of Ca2+ currents; it suggests that at least part of the action of the G-protein may be mediated by a beta gamma -subunit.

Differential effects of protein kinase C activation on 5-HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurones.

1. Activation of the enzyme protein kinase C (PKC) partially uncouples receptors from the inhibition of Ca2+ current. We have studied the effect of PKC activation on 5-HT1A receptor coupling of Ca2+ currents and 5-HT-induced K+ current (IK,5-HT) in acutely isolated adult rat dorsal raphe neurones. 2. The phorbol ester 4 beta-phorbol 12-myristate, 13-acetate (PMA; 1 microM) did not significantly alter the peak Ca2+ current. A maximal dose of 5-HT inhibited Ca2+ current on average by 52%; after application of PMA, the inhibition was only 30% and the effect was irreversible for the duration of the experiment. 3. The inactive phorbol ester 4 alpha-phorbol (1 microM) did not reduce the effectiveness of 5-HT. When the kinase inhibitor staurosporine (ST; 200 nM) was added, PMA reduced the effect of 5-HT by only 13.9%. ST partially prevented or reversed the effect of PMA, depending on the order of addition. 4. The voltage-dependent rate or re-inhibition by 5-HT was reduced by PMA, suggesting that fewer activated G-protein subunits are available to interact with Ca2+ channel after the action of PMA. 5. In contrast, PMA (1 microM) did not have a significant effect on IK,5-HT. 6. PKC activation has an inhibitory effect on one branch of the 5-HT1A receptor transduction fork, namely inhibition of Ca2+ influx, but not on the activation of IK,5-HT.

Actions of methoxylated amphetamine hallucinogens on serotonergic neurons of the brain.

1. Previous studies of phenethylamine hallucinogenic drugs have reported that intravenous injection of DOI or similar compounds produce an inhibition of the firing rate of serotonergic dorsal raphe neurons of the rat; potentially lowering levels of 5-HT throughout the brain. 2. Direct application by iontophoresis demonstrated only weak non-specific effects, but injection into the nucleus produced slowing of cell firing possibly due to the reduction of on-going synaptic excitation. 3. The literature concerning phenethylamine hallucinogens is discussed in the context of new results obtained from acutely isolated raphe neurons. The effect of DOI (10 microM) was tested on isolated DR neurons that responded to 5-HT with the activation of an inwardly rectifying K+ current but it was without effect. Under conditions that isolated inward Ca2+ current in raphe neurons that was inhibited by 5-HT, DOI was similarly without effect, neither blocking nor mimicking the action of 5-HT. Brief applications of glutamate produced an inward current similar to those produced by synaptic excitation in a slice preparation, but DOI failed to influence the size or duration of these responses. 4. These results suggest that DOI does not directly influence the cell bodies of DR neurons. 5. Further studies using raphe slices subject to synaptic influences will be required to reveal any indirectly mediated mechanism that may underlie the effects of DOI on DR neurons. Alternative indirect mechanisms of DR neuronal inhibition by i.v. DOI and DOM are suggested.

Toxin-insensitive Ca current in dorsal raphe neurons.

About 54% of the whole-cell Ca current recorded in dorsal raphe neurons cannot be categorized as N-, L-, or P-type Ca current. This current, ICa-Raphe, was not blocked by a combination of nimodipine, omega-CgTx-GVIA, and omega-AGA-IVA. Differences in toxin sensitivity and voltage dependence suggest that ICa-Raphe is distinct from Q- or R-type Ca currents. In raphe neurons activation of 5-HT1A receptors by 5-HT inhibits approximately 50% of the Ca current and slows activation. 5-HT inhibits both N-type Ca channels and ICa-Raphe channels by approximately 50% and slows the activation of both currents to a similar extent. Other similarities between ICa-Raphe and N-type Ca current were observed; they are both blocked to a similar extent by Ni2+, their activation properties, their current kinetics and channel availability as a function of holding potential are almost identical. Thus, ICa-Raphe represents a current that is not sensitive to known antagonists, but which is similar to N-type Ca current. Although it is possible that ICa-Raphe belongs to a heretofore undiscovered family of Ca channels it is also possible that it represents an omega-CgTx GVIA-insensitive isoform of the N-type Ca channel family.

Effects of LSD on Ca++ currents in central 5-HT-containing neurons: 5-HT1A receptors may play a role in hallucinogenesis.

Drugs that influence the activity of central serotonergic neurons by activating a 5-hydroxytryptamine subtype of receptor (5-HT1A) alter mood and perception. Previously, we demonstrated with whole-cell recordings from acutely isolated 5-HT-containing dorsal raphe (DR) neurons from the adult rat that 5-HT inhibited Ca++ current and activated K+ current in DR neurons. We now show that D-lysergic acid diethylamide (LSD) mimics the actions of 5-HT; it dramatically suppresses Ca++ current in a dose-dependent manner and activates an inwardly rectifying K+ conductance. Spiperone (0.2 microM), a 5-HT1A/5-HT2 antagonist, blocks the effect of both LSD and 5-HT. The nonhallucinogenic structural analog 2-bromo-LSD (2-Bol) at 10 microM has no effect on either Ca++ or K+ current by itself, but it competitively antagonizes both effects of LSD. Inhibition of 5-HT release resulting from 5-HT1A receptor activation may play an integral role in the hallucinogenic actions of LSD by reducing competition between 5-HT and LSD for the postsynaptic 5-HT receptors.

Unitary properties of potassium channels activated by 5-HT in acutely isolated rat dorsal raphe neurones.

1. Single inwardly rectifying K+ channel currents were recorded from acutely isolated adult serotonergic dorsal raphe (DR) neurones using the cell-attached and outside-out patch clamp configuration. 2. Four equally spaced conductance levels were observed in both outside-out and cell-attached patch recordings with conductance levels averaging 11, 21, 30 and 40 pS. Larger conductance openings (50-120 pS) were seen less frequently. 3. When using 136 [K+]0 the single channel I-V relation was linear in the range 0 mV to -100 mV in all cases. 4. Transitions between the various conductance levels were observed, as were apparent direct opening and closing to each individual conductance level. Furthermore openings of 11, 21 and 30 pS were observed in almost all the patches. These results suggest that the different-sized events result from substrates of a single channel rather than several different channels with different conductances. 5. Unitary K+ channel current probability of opening, recorded in cell-attached patch, was unchanged after 5-hydroxytryptamine (5-HT) was added to the bath outside the patch pipette which suggests that no easily diffusible second messenger was involved. 6. The single K+ channel activity, however, was increased on average by 670% following the addition of 5-HT to the bath when recording channel activity in the outside-out configuration. Usually all K+ channel subconductance levels increased in activity but the largest increases occurred in the events with 30 and 40 pS conductance. 7. These results suggest that 5-HT enhances the probability of opening of the resting K+ channel activity, which can open to several levels of conductance, and that no new channel or freely diffusible second messenger is involved in the response.

Whole-cell recordings of inwardly rectifying K+ currents activated by 5-HT1A receptors on dorsal raphe neurones of the adult rat.

1. An inwardly rectifying K+ current activated by serotonin (5-HT) was recorded from acutely isolated adult dorsal raphe (DR) neurones using the whole-cell recording mode of the patch clamp technique. 2. The 5-HT-induced K+ current (I5-HT) was only visible at an [K+]0 > 5 mM and it was observed in 69% of the cells. 3. The reversal potential for I5-HT was close to the potassium equilibrium potential and was shifted by 51 mV per 10-fold change in [K+]0 indicating that I5-HT was carried predominantly by K+. The chord conductance of I5-HT at -90 mV was proportional to the external [K+] raised to a fractional power. 4. A dose-response relationship revealed that I5-HT was activated with an ED50 of 30 nM. Ba2+ (0.1 mM) blocked I5-HT completely. Spiperone reversibly antagonized the response to 5-HT and 8-OHDPAT (8-hydroxy-2-(di-n-propylamino)tetralin) mimicked the response indicating that the receptor activated was of the 5-HT1A subtype. 5. The response to 5-HT was largely prevented by in vitro pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX-sensitive G-protein in the transduction mechanism. 6. cAMP and lipoxygenase metabolites, both implicated in the modulation of similar currents in other preparations, were found not to alter the effectiveness of 5-HT. 7. Glibenclamide and tolbutamide, blockers of the ATP-regulated K+ channel, did not reduce the effect of 5-HT in DR neurones. 8. These results show that in acutely isolated adult DR neurones 5-HT activates an inwardly rectifying K+ current and this involves a PTX-sensitive G-protein in the transduction pathway which may interact with the K+ channel directly.

Ionic dependence of a slow inward tail current in rat dorsal raphe neurones.

1. The mechanism underlying a large slow inward tail current was studied in serotonergic dorsal raphe (DR) neurones. The tail current is most easily observed under conditions of suppressed K+ channel outward currents and follows the activation of a calcium current. This current may underlie a slow after-depolarizing potential (ADP) which follows action potentials observed in acutely isolated DR neurones. 2. The after-hyperpolarizing potential (AHP) following action potentials which should reverse at EK (the reversal potential for potassium) becomes an ADP at less negative potentials than expected due to contamination by the slow inward tail current. 3. DR neurones were acutely isolated enzymatically; the ADP in current clamp and the tail current underlying it in voltage clamp were studied using the patch clamp method. When the external Na+ was replaced with TEA or choline the slow inward tail current was completely abolished. Blocking K+ channels from the inside of the cell membrane with 40 mM TEACl or large concentrations of internal Cs+ also blocked the slow inward tail current. 4. The tail current proved to be independent of calcium influx or intracellular calcium release as it was not affected by inorganic calcium channel blockers or caffeine. 5. The tail grew exponentially upon lengthening the depolarizing test pulse and appeared to reverse close to 0 mV indicating that the current was carried by a nonselective cation conductance. Removal of external Na+ and replacement with Li+ ions reversibly blocked the tail current by 77%. 6. The data rule out several mechanisms for the generation of the current, namely: a calcium-activated chloride conductance, a calcium-activated non-selective cation conductance, a Na(+)-Ca2+ exchange pump current or a sodium-activated K+ conductance. 7. The slow tail current may be explained by postulating an inward movement of Na+ through a channel which is blocked by high concentrations of external TEA and Li+ or internal Cs+ or 40 mM TEA.

Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons.

Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.

A study of the mechanism of Ca2+ current inhibition produced by serotonin in rat dorsal raphe neurons.

Calcium currents and their modulation by 5-HT were studied using both whole-cell and single-channel patch-clamp techniques in acutely isolated adult rat dorsal raphe neurons. Evidence for three types of Ca channels (T, N, L) was obtained in both whole-cell and single-channel experiments. Approximately 4% of the total high-threshold Ca current (L-type) was sensitive to dihydropyridines (DHPs) while approximately 40% of the Ca current (N-type) was sensitive to omega-conotoxin (omega-CgTx). About 56% of the whole-cell current was insensitive to either DHPs or omega-CgTx and may thus represent a different kind of Ca current. 5-HT reduced raphe neuron Ca currents by approximately 50%, while slowing activation. 5-HT inhibited both omega-CgTx-sensitive and -insensitive Ca current. Inhibition by 5-HT was voltage dependent; prepulses to +80 mV lasting for 20 msec almost completely abolished the 5-HT-mediated inhibition. The voltage dependence of the response to 5-HT suggested that trains of action potentials might overcome the inhibition due to 5-HT. Trains of brief depolarizations were used to simulate action potentials; only about 5% of the 5-HT-induced inhibition was relieved by the trains. These results suggest that while large depolarizations could restore the Ca current inhibited by 5-HT, physiological stimuli, such as trains of action potentials, could not. The action of 5-HT was made irreversible by inclusion of GTP-gamma-S in the patch pipette, suggesting a G-protein mediation of the response to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin receptor heterogeneity and the role of potassium channels in neuronal excitability.

Intracellular recordings in vitro from a variety of central neuronal types have shown both inhibition and excitation to be modulatory consequences of serotonin (5-HT) receptor activation. These responses can be seen in isolation or in some cases (e.g. hippocampal pyramidal cells) as a complex biphasic combination of hyperpolarisation followed by depolarisation, suggesting overall control of neuronal excitability may be dependent on the interaction between activation of more than one post-synaptic receptor and/or mechanism. Our studies have confirmed the 5-HT evoked depolarisation of rat facial motorneurones (FM's) and the hyperpolarisation seen in presumed serotonergic neurones of the dorsal raphe nucleus (DRN) to be the result of opposite effects on K+ ion permeability. Suppression of a resting K+ conductance leads to depolarisation while activation leads to hyperpolarisation. The same mechanisms appear to be responsible for the 5-HT evoked responses in hippocampal pyramidal cells but in addition there is also a suppression of a Ca++ dependent K+ conductance responsible for the long spike after hyperpolarisation (AHP). Data from the hippocampus and DRN indicate the 5-HT induced hyperpolarisation to be sensitive to Pertussis Toxin (PTX) and irreversibly mimicked by GTP gamma S, a non-hydrolysable analogue of GTP, suggesting the involvement of a G protein in K+ channel activation. The mechanism of K+ channel closure is less clear as it is unaffected by PTX or activation of adenylate cyclase, however there is indirect evidence that the phosphoinositide pathway may be involved from the cloned 5-HT1C receptor which also closes a K+ channel in cell lines. The results show that hyperpolarisation evoked by 5-HT in the hippocampus and DRN to be mimicked and blocked by 5-HT1A agonists and antagonists. However, the depolarisations in the hippocampus and FM's are mediated by site-dependent receptors with profiles which do not fit into the current 5-HT receptor subtype classification.

Serotonin receptor activation reduces calcium current in an acutely dissociated adult central neuron.

The release of serotonin (5-HT) from the terminals of serotonergic (raphe) neurons is under inhibitory feed-back control. 5-HT, acting on raphe cell body autoreceptors, also mediates inhibitory postsynaptic potentials as a result of release from collaterals from neighboring raphe neurons. This may involve a ligand (5-HT)-gated increase in the membrane potassium conductance, leading to a decrease in action potential frequency, which could indirectly reduce calcium influx into nerve terminals. In this report we demonstrate that 5-HT can also directly reduce calcium influx at potentials including and bracketing the peak of calcium current activation. Using acutely isolated, patch-clamped dorsal raphe neurons, we found that low concentrations of 5-HT and the 5-HT1A-selective agonist 8-OH-DPAT reversibly decrease whole-cell calcium current. This effect is antagonized by the putative 5-HT1A-selective antagonist NAN 190. Hence, the inhibition of calcium current may serve a physiological role in these cells and elsewhere in the brain.

Electrophysiology of adult rat facial motoneurones: the effects of serotonin (5-HT) in a novel in vitro brainstem slice.

Studies of adult rat motoneurones using in vitro slice preparations are rare. We here describe a novel brainstem slice of the adult rat containing the facial motor nucleus (FMN). Data obtained for facial motoneurones (FM) by intracellular recording indicate that they display several passive and active properties seen in other rat cranial and spinal motoneurones. Bath application of serotonin (5-HT) evokes a reversible depolarization of FMs which is associated with an increase in input resistance due to a reduction in potassium permeability. This effect is unaffected by tetrodotoxin indicating a postsynaptic site of action.