Rat Dorsal Raphe Neurons Research Articles

Distinct contribution of axonal and somatodendritic serotonin transporters in drosophila olfaction

The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. Here we introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting - otherwise fully functional - hSERT-R607A on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-R599A led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. We explored the possible physiological role of somatodendritic SERT by comparing flies harboring wild type SERT and dSERT-R599A in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R599A restored ethanol preference. In contrast, dSERT-R599A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. We conclude that, in CSD neurons, (i) somatodendritic SERT supports ethanol attraction, (ii) axonal SERT specifies ethanol aversion, (iii) the effect of axonal SERT can override that of somatodendritic SERT. These observations demonstrate a distinct biological role of somatodendritic and axonal serotonin transport.This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

Role of nitric oxide and WNK-SPAK/OSR1-KCC2 signaling in daily changes in GABAergic inhibition in the rat dorsal raphe neurons

Serotonergic neurons in the dorsal raphe nucleus (DRN) act as wake-inducing neurons in the sleep-wake cycle and are controlled by gamma-aminobutyric acid (GABA) synaptic inputs. We investigated daily changes in GABAergic inhibition of the rat DRN neurons and the role of nitric oxide (NO) and cation-chloride co-transporters in the GABAergic action. Neuronal NO synthase (nNOS) was co-expressed in 74% of serotonergic DRN neurons and nNOS expression was higher during daytime (the sleep cycle) than that during nighttime (the wake cycle). GABAergic hyperpolarization of DRN neurons produced by GABAA receptor agonist muscimol was greater and the equilibrium potential of muscimol showed a hyperpolarizing shift during daytime compared to that during nighttime. Expression levels of potassium-chloride co-transporter 2 (KCC2) were higher during daytime than that during nighttime, whereas there were no changes in sodium-potassium-chloride co-transporter 1 (NKCC1) expression. With-no-lysine kinase (WNK) isoform 1 was more highly expressed during daytime than that during nighttime. Total Ste20-related proline alanine rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) were also higher during daytime than during nighttime, while there were no changes in phosphorylated SPAK and OSR1. Consistent with the findings during the sleep-wake cycle, ex vivo treatment of DRN slices with a NO donor sodium nitroprusside (SNP) increased the expression of KCC2, WNK1, WNK2, WNK3, SPAK, and OSR1, whilst decreasing phosphorylated SPAK. These results suggest that GABAergic synaptic inhibition of DRN serotonergic neurons shows daily changes during the sleep-wake cycle, which might be regulated by daily changes in nNOS-derived NO and WNK-SPAK/OSR1-KCC2 signaling.

Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum.

Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes.

P.1.187 Chronic corticosterone attenuates 5-HT 1A-mediated but not GABA-mediated inhibition of rat dorsal raphe neurones

The effects of common antiepileptics, GABAmimetic drugs, excitatory amino acid antagonists as well as of clonidine, corynanthine, chlorpromazine and atropine were studied against clonic convulsion induced in mice by N-methyl-D,L-aspartic acid (NMDLA) after subcutaneous (340 mg/kg; ED97) administration. Among the antiepileptics studied, valproate (ED50: 340 mg/kg after subcutaneous injection of NMDLA) and diazepam (ED50: 0.78 mg/kg after intravenous and 14 mg/kg after subcutaneous injection of NMDLA) antagonized NMDLA-induced convulsions, whereas phenobarbital (up to 80 mgkg), diphenylhydantoin (up to 50 mg/kg) and ethosuximide (500 mg/kg) were totally ineffective. Moreover, 2-amino-5-phosphonopentanoic acid and 2-amino-7-phosphonoheptanoic acid (but not glutamic acid diethyl ester), aminooxyyacetic acid, cetyl GABA and clonidine protected strongly against the convulsant whereas progabide was only weakly active. THIP showed a higher activity against intravenous than against subcutaneous NMDLA. Baclofen and atropine even increased mortality and the remaining agents exerted no significant protective action. The data suggest that NMDLA-induced convulsions can be blocked effectively by direct antagonism of NMDLA-produced excitation, enhancement of GABA-mediated inhibition, and activation of central α2-adrenoceptors. The possible efficacy of valproate in cases of epilepsy with a distinct rise in plasma excitatory amino acid levels should be carefully considered.

Neuropeptide FF receptors 1 and 2 exert an anti-opioid activity in acutely dissociated rat dorsal raphe and periventricular hypothalamic neurones

We measured the reduction by nociceptin of the [Ca 2+] i transient triggered by depolarization in acutely dissociated neurones of the rat dorsal raphe and periventricular hypothalamic nuclei that express NPFF 2 and NPFF 1 receptors, respectively, in the absence and presence of 10 nM of NPA-NPFF or NPVF, two peptides selective for NPFF 2 and NPFF 1 receptors, respectively. In dorsal raphe neurones, NPA-NPFF reduces the inhibition of Ca 2+ conductances by nociceptin while NPVF is inactive. In periventricular hypothalamic neurones, both peptides reduce the inhibition of Ca 2+ transients by nociceptin, NPVF having a significantly larger effect than NPA-NPFF. These results demonstrate that activation of both NPFF 1 and NPFF 2 receptors has the same cellular anti-opioid effect.

Perospirone, a novel antipsychotic agent, hyperpolarizes rat dorsal raphe neurons via 5-HT1A receptor.

To investigate the effect of cis-N-[4-[4-(1,2-benz-isozole-3-yl)-1-piperazinyl]butyl] cyclohexane-1,2-dicarboximide hydrochloride (perospirone), a novel antipsychotic agent with high affinities for D(2)/5-HT(2) receptors, on the rat dorsal raphe (DR) neurons, an electrophysiological study was performed using the tight-seal whole-cell patch-clamp technique. Applications of perospirone at the concentration between 10(-)(9) and 10(-)(5) M hyperpolarized the membrane potential and inhibited spontaneous action potentials of the DR neurons in a concentration-dependent manner. This effect of perospirone on DR neurons is similar to that of typical 5HT(1A)-receptor agonists, including 8-OH-DPAT or tandospirone. In addition, WAY100635, a 5-HT(1A)-receptor antagonist, inhibited this perospirone-induced hyperpolarization of DR neurons, suggesting that perospirone physiologically acts on DR neurons as a 5HT(1A)-receptor agonist. These results provide new profiles of perospirone as an antipsychotic drug.

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

Introduction of GTP-γ-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 α subunits, leading to a facilitation of GTP-γ-S binding. It follows that partial or complete uncoupling of receptors and G-proteins could inhibit Ca 2+ current modulation by GTP-γ-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-γ-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-γ-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.

Structure-activity relationships of neuropeptide FF: role of C-terminal regions

A structure-activity study was carried out to determine the importance of the C-terminal amino acids of the octapeptide Neuropeptide FF (NPFF) in binding and agonistic activity. Affinities of NPFF analogues were tested toward NPFF receptors of the rat spinal cord and the human NPFF 2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF 2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine 8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine 7 by a lysine or D.Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe 8 and Arg 7 residues are also extremely critical for activation of anti-opioid effects on dorsal raphe neurons. The sequence of the C-terminal dipeptide seems also to be responsible for the high affinity and the activity on human NPFF 2 receptors. The results support the view that a code messaging the molecular interaction toward NPFF-receptors is expressed in the C-terminal region of these peptides but the N-terminal segment is important to gain very high affinity.

Neuropeptide FF receptors couple to a cholera toxin-sensitive G-protein in rat dorsal raphe neurones

In rat dorsal raphe neurones, nociceptin (300 nM) reduced the peak [Ca 2+] i transient, triggered by depolarization, by 36.7±1.8% ( n=46). This effect of nociceptin decreased to 16.7±2.9% ( n=18) after pre-treatment of the neurones with pertussis toxin (5 μg/ml, 2–6 h) but was unchanged (37.4±2.1%, n=44) after pre-incubation with cholera toxin (5 μg/ml, 2–6 h). This suggests that, in dorsal raphe neurones, the ORL1 receptor couples to inhibitory (G i/o) G-proteins. The neuropeptide FF analogue, [ d-Tyr 1, (N-Me)Phe 3]neuropeptide FF (10, 100, 1000 nM), acted as an anti-opioid and reduced the effect of nociceptin (300 nM, 30 s) by 62.0±3.3% ( n=28). Following pre-incubation with cholera toxin (5 μg/ml, 2–6 h) [ d-Tyr 1, (N-Me)Phe 3] neuropeptide FF was unable, at the three concentrations tested, to block nociceptin activity. We conclude that, in rat dorsal raphe neurones, neuropeptide FF receptors couple to stimulatory G-proteins (G s).

Inhibition of the 5-HT 1A receptor-mediated inwardly rectifying K + current by dextromethorphan in rat dorsal raphe neurones

The effect of dextromethorphan (DM) on the inwardly rectifying K + currents mediated by 5-HT 1A receptors in acutely dissociated dorsal raphe (DR) neurones of rats was studied using nystatin-perforated patch and conventional whole-cell patch recording configurations under voltage-clamp conditions. DM rapidly and reversibly inhibited the K + currents induced by 10 −7 M 5-HT in a concentration-dependent manner with a half-maximum inhibitory concentration of 1.43×10 −5 M. The inhibitory effect of DM was neither voltage- nor use-dependent. DM caused a suppression of the maximum response of the 5-HT concentration–response curve, thus suggesting a non-competitive type of inhibition. In neurones perfused intracellularly with a pipette-solution containing the nonhydrolyzable GTP analog GTPγS, 5-HT activated K + currents in an irreversible manner. DM suppressed the current irreversibly activated by intracellular GTPγS even in the absence of the agonist. DM also inhibited the inwardly rectifying K + currents regulated by α 2-adrenoceptors in freshly isolated rat locus coeruleus neurones. These results suggest that DM may inhibit the G-protein coupled inwardly rectifying K + channels, but not the neurotransmitter receptors, in the central nervous system.

Are Neuropeptides FF and SF Neurotransmitters in the Rat?

We have compared the affinities and anti-opioid activities of the different peptides putatively produced by the rat NPFF precursor, NPAFLFQPQRF-NH2 (NPA-NPFF) and EFWSLAAPQRF-NH2 (EFW-NPSF), with those already identified in nervous tissue, FLFQPQRF-NH2 (NPFF) and SLAAPQRF-NH2 (NPSF). NPFF and NPA-NPFF exhibit a high affinity (0.34 and 0.14 nM, respectively) for [125I]1DMe binding sites of the rat spinal cord. In contrast, EFW-NPSF displays an affinity 13 times higher than NPSF (1.99 and 9.5 nM, respectively). In rat dorsal raphe neurones, EFW-NPSF, NPFF, and NPA-NPFF maximally reduce the inhibitory effect of nociceptin on the [Ca2+]i transients triggered by depolarization by 39, 31, and 58%, respectively. NPSF is inactive in the same test. We conclude that NPA-NPFF and EFW-NPSF are likely to be the physiologically active neurotransmitters in rat brain.

Discharge modulation of rat dorsal raphe neurons during sleep and waking: effects of preoptic/basal forebrain warming

In cats, putative serotonergic neurons (PSNs) recorded from the dorsal raphe nucleus (DRN) across the sleep-wake cycle exhibit the so-called rapid eye movement sleep-off (REM-off) discharge pattern. Since, the sleep-wake discharge patterns of DRN neurons in behaving rats is poorly known, the present study examined this neuronal populations. The PSNs recorded in this study exhibited: (1) progressive decrease in discharge rate from waking to NREM to REM sleep; (2) long action potential duration, and (3) reduction of discharge rate after systemic administration of a selective 5-HT 1A agonist, (±)-8-hydroxy-2-(di- n-propylamino) tetralin hydrobromide (8-OH-DPAT). Evidence supports the hypothesis that NREM sleep is modulated by thermoregulatory mechanisms localized in the preoptic area and adjacent basal forebrain (POA/BF). We previously reported that POA/BF warming suppresses the discharge of wake-promoting neurons in the posterior hypothalamus and the basal forebrain. Since the DRN is one component of the brainstem arousal system and receives projections from POA/BF, we examined the effects of local POA/BF warming by 1.5–2.0°C during waking on the discharge of DRN neurons. POA/BF warming reduced the discharge in 14 of 19 PSNs and in 12 of 17 other wake-related neurons in the DRN. DRN neuronal discharge reduction occurred without accompanying EEG frequency or behavioral changes. These results suggest that PSNs recorded in DRN in unrestrained and unanesthetized rats exhibit a “wake-active REM-off” discharge pattern and further support the hypothesis that the POA/BF warm-sensitive hypnogenic system induces sleep by a coordinated inhibition of multiple arousal systems including that modulated by the DRN.

O-284 - Antitussives inhibit the 5-HT1A receptor-mediated inwardly rectifying K+ current in rat dorsal raphe neurons

O-284 - Antitussives inhibit the 5-HT1A receptor-mediated inwardly rectifying K+ current in rat dorsal raphe neurons

Effects of nicotine and mecamylamine on rat dorsal raphe neurons

This study investigates the hypothesis that serotonin mediates certain nicotine effects, such as mood improvement and the suppression of the ponto-geniculo-occipital spikes of rapid eye movement sleep. The influence of nicotine (10–300 μM) on the firing rate of dorsal raphe neurons and on serotonin release was therefore, studied in rat midbrain slices. Nicotine increased the firing rate, 10–90%, in 67.5% recorded neurons and decreased it, 8–100%, in the remaining 32.5%. Serotonin release increased 2–7 times after nicotine administration, regardless of firing frequency, but the absolute value of serotonin release was 3 times higher during the decreases than during the increases in firing rate. Mecamylamine (1–20 μM) transiently stimulated the dorsal raphe neurons and competitively antagonized the nicotine-induced serotonin release. The results support the working hypothesis and additionally show that mecamylamine also stimulates dorsal raphe neurons.

Tandospirone-induced K+ current in acutely dissociated rat dorsal raphe neurones.

1. The effects of tandospirone (TDS) on dissociated rat dorsal raphe neurones were investigated using the patch-clamp method. 2. Under current-clamp conditions, TDS hyperpolarized the cell membrane, resulting in the reduction of firing rates. 3. Under voltage-clamp conditions, TDS induced an inward rectifying K+ current in a concentration-dependent manner. 4. The TDS-induced K+ currents (I(TDS)) were mimicked by 8-OH-DPAT, a 5-HT1A agonist. The I(TDS) was blocked by spiperone, a 5-HT1A receptor antagonist, in a concentration-dependent manner. 5. N-Ethylmaleimide, an agent which uncouples between the receptor and the G-protein, irreversibly blocked the I(TDS). 6. In neurones perfused intracellularly with a pipette-solution containing GTP using the conventional whole-cell patch recording, the I(TDS) showed a gradual rundown. When the neurones were perfused with GTPgammaS, TDS activated the inwardly rectifying K+ current in an irreversible manner. 7. In the inside-out patch recording mode, TDS-activated single K+ channel currents (i(TDS)) which also showed an inward rectification. When the GDP in cytosolic side was completely replaced with GTP, the open probability of i(TDS) significantly increased. 8. These results indicate that the activation of 5-HT1A receptors by TDS directly opens the inward rectifying K+ channels via a G-protein mediated process.

Galanin–5-hydroxytryptamine interactions: electrophysiological, immunohistochemical and in situ hybridization studies on rat dorsal raphe neurons with a note on galanin R1 and R2 receptors

Galaninergic mechanisms related to 5-hydroxytryptamine neurons in the dorsal raphe nucleus of the rat were analysed using electrophysiology, immunohistochemistry and in situ hybridization. Galanin caused a dose-dependent hyperpolarization accompanied by a decrease in membrane resistance in most 5-hydroxytryptamine-sensitive dorsal raphe neurons. The galanin-induced outward current reversed at about −105 mV and shifted to a more positive potential with increasing extracellular potassium concentrations. The 5-hydroxytryptamine-induced outward current was enhanced and prolonged by preincubation with a low concentration of galanin (1–10 nM). The immunohistochemical analysis showed (i) generally low levels of galanin in the 5-hydroxytryptamine cell bodies, (ii) moderate numbers of galanin-positive nerve endings around the 5-hydroxytryptamine cell bodies, (iii) presence of galanin-like immunoreactivity in 5-hydroxytryptamine-positive dendrites and (iv) galanin-positive, 5-hydroxytryptamine-negative boutons making synaptic contact with 5-hydroxytryptamine-positive dendrites. The in situ hybridization results suggest that the galanin receptor present in the galanin/5-hydroxytryptamine neurons is not of the recently cloned galanin-R1 type. Taken together these results indicate that galanin exerts an inhibitory effect via an increase in K + conductance in 5-hydroxytryptamine neurons by acting on a postsynaptic receptor. In addition, galanin at low, possibly physiological concentrations enhances the inhibitory effect of 5-hydroxytryptamine at the cell soma level. We propose that galanin primarily is released from adjacent galaninboutons lacking 5-hydroxytryptamine and also from soma and dendrites of galanin/5-hydroxytryptamine dorsal raphe neurons. Galanin may thus be involved in the manifold functions hitherto ascribed to ascending 5-hydroxytryptamine neurons, for example in mood regulation.

QEHA 27, a peptide that binds to G-protein βγ-subunits, reduces the inhibitory effect of 5-HT on the Ca 2+ current of rat dorsal raphe neurons

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

O-270 - Ionic mechanisms underlying excitation by direct activation of prostaglandin E receptor EP3 subtype in rat dorsal raphe neurons

O-270 - Ionic mechanisms underlying excitation by direct activation of prostaglandin E receptor EP3 subtype in rat dorsal raphe neurons

Characterization of the K + current mediated by 5-HT 1A receptor in the acutely dissociated rat dorsal raphe neurons

The action of 5-hydroxytryptamine (5-HT) via the 5-HT 1A receptor on dissociated rat dorsal raphe neurons was characterized under the whole-cell mode by using the nystatin-perforated patch-clamp technique. Under voltage-clamp conditions, 5-HT induced an inwardly rectifying K + current ( I 5-HT) in a concentration-dependent manner. I 5-HT was mimicked by 8-OH-DPAT and buspirone, which are both 5-HT 1A receptor agonists. I 5-HT was reversibly blocked by such 5-HT 1A receptor antagonists as ( S)-UH-301 and spiperone but not by ketanserin, a 5-HT 2 receptor antagonist, granisetron, a 5-HT 3 receptor antagonist, and GR-113808, a 5-HT 4 receptor antagonist. I 5-HT was antagonized concentration-dependently by such K + channel blockers as quinine, Ba 2+ and 4-aminopyridine but was relatively insensitive to both Cs + and tetraethylammonium. When the neurons were loaded with guanosine 5′- O-3-thiotriphosphate through a patch pipette, the K + current induced by 5-HT became irreversible. N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, irreversibly blocked I 5-HT. The intracellular perfusion with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA), a Ca 2+ chelator, or neomycine, a phospholipase C inhibitor, never significantly affected the 5-HT-induced response. 12-Myristate 13-acetate diester (PMA), a protein kinase C (PKC) activator, had only a weak inhibitory effect on I 5-HT, and staurosporine, a PKC inhibitor, failed to significantly occlude I 5-HT. Therefore, the K + conductance activated via the 5-HT 1A receptor of dorsal raphe neurons was thus characterized by the sensitivity to such K + channel blockers as quinine, Ba 2+ and 4-aminopyridine. Moreover, G protein, which is NEM-sensitive and can couple to the 5-HT 1A receptor, is thus considered to activate the inwardly rectifying K + conductance without being mediated by such second messengers as Ca 2+ and PKC.

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.