Serotonin augments the cationic current Ih in central neurons.

Serotonin (5-HT) induced a slow depolarization when superfused onto neurons of the rat brainstem nucleus prepositus hypoglossi (PH) in vitro. The depolarization was associated with a decrease in cell input resistance. In voltage clamp, 5-HT caused an inward current that activated at approximately -50 mV and was present only at potentials negative to this. With hyperpolarizing voltage-clamp steps, PH neurons exhibited a slow inward current relaxation. The properties of this conductance were consistent with the cationic, nonselective current, Ih. Bath-applied 5-HT augmented Ih. Extracellular CsCl blocked both Ih and the inward current produced by 5-HT. In addition, forskolin, isobutylmethylxanthine, and 8-bromo-cAMP mimicked the inward current seen with 5-HT. The 5-HT1 agonist 5-carboxamidotryptamine produced a similar inward current. We conclude that 5-HT excites PH neurons by augmenting Ih, probably through receptor-mediated stimulation of adenylate cyclase. As Ih is found in many types of neurons, this mechanism may be a common mode of regulating cell excitability.

Ion conductances affected by 5-HT receptor subtypes in mammalian neurons.

5-Hydroxytryptamine (5-HT) has both excitatory and inhibitory actions in the CNS and PNS. The development of new 5-HT ligands has led to the expansion of 5-HT receptor subtypes into three categories: 5-HT1, 5-HT2 and 5-HT3. Each category has further subdivisions. The literature concerning the biochemical basis of this division has been reviewed recently. While this approach has elucidated many of the pharmacological properties of 5-HT receptors, it has not addressed the question of how 5-HT modulates cell excitability. Physiological studies have confirmed the existence of a multiplicity of 5-HT receptors that act through a variety of ionic mechanisms. The purpose of this review is to summarize what is known of the ionic mechanisms associated with the activation of identified mammalian 5-HT receptor subtypes, as well as some effects of 5-HT where the receptor could not be defined.

Synaptic potentials in locus coeruleus neurons in brain slices.

Neurons of the locus coeruleus (LC) fire action potentials spontaneously in vitro in the absence of any stimulation. This spontaneous activity is thought to arise from intrinsic membrane properties that include a balance between at least two ion conductances. One is a persistent inward sodium current that is active near the threshold for action potential generation. The second is a calcium-dependent potassium current that is activated following the entry of calcium during the action potential, is responsible for the after-hyperpolarization following the action potential, and decays over a period of 1-2 sec following the action potential. The spontaneous activity of LC neurons can be altered by both excitatory and inhibitory synaptic inputs. One excitatory input has been described that is mediated by glutamate receptors of both the non-NMDA and NMDA subtypes. Inhibitory synaptic potentials include those mediated by GABA (acting on GABAA-receptors), glycine (acting on a strychnine-sensitive receptor) and noradrenaline (acting on alpha 2-adrenoceptors). The presence of synaptic potentials mediated by these transmitters, studied in vitro, correlate with studies made in vivo and with histochemical identification of synaptic inputs to the locus coeruleus.

A slow excitatory postsynaptic potential mediated by 5-HT2 receptors in nucleus prepositus hypoglossi.

Intracellular recordings were made from neurons of the nucleus prepositus hypoglossi in slices of guinea pig medulla. 5-HT (serotonin) caused a hyperpolarization followed by a late depolarization. The hyperpolarization was mediated by 5-HT1A receptor activation and could be selectively blocked by pindobind-5HT1A (PBD). 5-HT then caused a depolarization only. A selective 5-HT2 agonist, (+)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), also caused a depolarization. Ketanserin and spiperone, 5-HT2 antagonists, blocked the depolarization due to both 5-HT and DOI. Focal electrical stimulation caused an IPSP mediated by 5-HT acting upon 5-HT1A receptors and a slow EPSP (s-EPSP). PBD blocked the IPSP, leaving an isolated s-EPSP. Both spiperone and ketanserin antagonized the s-EPSP, while DOI occluded it. The s-EPSP was from 2 to 10 mV in amplitude and 35-50 sec in duration, and showed voltage dependence consistent with a decrease in potassium conductance. Both the IPSP and the s-EPSP were presynaptically inhibited by the 5-HT1D agonist sumatriptan. These data indicate that the s-EPSP is mediated by 5-HT acting upon 5-HT2 receptors. This represents strong support for the role of 5-HT as an excitatory neurotransmitter in the CNS. Further, it demonstrates that synaptic release of 5-HT can mediate opposing effects on the membrane potential of a single cell.

The serotonergic inhibitory postsynaptic potential in prepositus hypoglossi is mediated by two potassium currents.

Synaptic inhibition mediated by the activation of potassium channels has been reported from several types of neurons. In each case, despite mediation by different neurotransmitters, the K+ conductance underlying the synaptic potential is activated by a G protein and inwardly rectifies. We report here a second K+ current that contributes to synaptic inhibition. Intracellular recordings were made from guinea pig nucleus prepositus hypoglossi in vitro, where we have described a 5-HT-mediated IPSP. Voltage-clamp analysis of the current induced by applied 5-HT revealed two separate conductances: an inwardly rectifying, rapidly activating K+ current (IIR) and an outwardly rectifying, slowly activating K+ current (IOR). IIR was blocked by extracellular Ba2+ (200 microM) and TEA+ (126 mM). IOR was insensitive to this concentration of Ba2+ and TEA+, but was inhibited by Cd2+ and intracellular BAPTA, indicating Ca dependence. Single focal electrical stimuli evoked a 5-HT-mediated IPSP, or under voltage clamp, an inhibitory postsynaptic current (IPSC). Ba2+ blocked only a component of this IPSC, which corresponded to the current caused by IIR. When multiple stimuli were applied (to prolong the release of transmitter), the time-dependent current IOR was more fully activated, resulting in an augmentation of the IPSC. We conclude that the IPSC is caused by both currents and that its amplitude can be modulated by the degree to which IOR is activated. This represents a mechanism by which synaptic responses can be potentiated.

Cocaine and amphetamine interact at 5-HT synapses through distinct mechanisms in guinea-pig prepositus hypoglossi.

Intracellular recordings were made from guinea-pig prepositus hypoglossi (PH) neurons in vitro. Neurons within this nucleus are innervated by terminals that release 5-HT (serotonin) to mediate an IPSP. Cocaine caused a concentration-dependent prolongation of that IPSP, while having no effect on either membrane potential or firing rate. Cocaine also caused an increase in the IPSP amplitude at lower concentrations (less than or equal to 1 microM) and a decrease at higher concentrations. The selective 5-HT uptake inhibitor fluoxetine also prolonged the IPSP duration but depressed the amplitude at all concentrations tested. The effects of cocaine on IPSP duration can be completely accounted for by inhibition of 5-HT uptake. The depression of the IPSP is most likely due to a presynaptic effect, because cocaine augmented the response to applied 5-HT. Amphetamine (3-300 microM), unlike cocaine, changed the membrane potential. At lower concentrations, it caused a ketanserin-sensitive depolarization, while higher concentrations resulted in a spiperone-sensitive hyperpolarization. The hyperpolarization was most likely caused by the evoked release of 5-HT, while the depolarization may have been due to a direct effect of amphetamine on 5-HT2 receptors. Amphetamine also acted as a weak uptake inhibitor. The effects of cocaine, but not those of amphetamine, were observed at concentrations that are attained during self-administration.

Serotonin-mediated inhibitory postsynaptic potential in guinea-pig prepositus hypoglossi and feedback inhibition by serotonin.

1. Intracellular recordings were made from neurones of the nucleus prepositus hypoglossi (PH) in slices of guinea-pig brain. Focal stimulation evoked an inhibitory postsynaptic potential (IPSP) that was typically 10-25 mV in amplitude and 1 s in duration. The IPSP reversal potential showed a Nernstian dependence on the external potassium concentration ([K+]o). 2. Spiperone blocked the IPSP with an IC50 of 40 nM, while ketanserin and (-)sulpiride had no effect. Cocaine (1 microM) prolonged the IPSP half-duration by 157%, and increased the amplitude by 28%. 3. 5-Hydroxytryptamine (5-HT, serotonin) hyperpolarized PH cells with an EC50 of 8.5 microM in control, and 135 nM in cocaine (10 microM). 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) also hyperpolarized PH cells with an EC50 of 16 nM, although the maximal effect was only 81% of the maximum 5-HT hyperpolarization. Spiperone produced a parallel, right shift of the 5-HT concentration-response curve; Schild analysis gave a Kd of 10 nM. Application of 5-HT to neurones voltage-clamped near their resting potential (about -55 mV) caused an outward current and an increase in membrane conductance. 4. The amplitude of the IPSP was reversibly decreased by non-hyperpolarizing concentrations of 5-HT and by the 5-HT1 receptor agonists 1-(m-trifluoromethylphenyl)piperazine (TFMPP) and 1-(3-chlorophenyl)piperazine (mCPP). The IC50 values for the latter two compounds were 50 nM and 1.5 microM, respectively; the maximal effect was a 90% inhibition. Neither compound affected the membrane potential nor changed the hyperpolarization induced by 5-HT. Quipizine competitively antagonized TFMPP with an estimated Kd of 165 nM. 5. When trains of stimuli were applied, an inhibition of the IPSP was observed following the first stimulus. At a frequency of 1 Hz, the inhibition was approximately 75%. This frequency-dependent 'run-down' of the IPSP was markedly attenuated by pre-treatment with TFMPP (1 microM). 6. It is concluded that the IPSP in PH cells is caused by 5-HT acting on 5-HT1A receptors to activate a potassium conductance. The release of 5-HT can be inhibited by activation of a presynaptic 5-HT1D receptor. This presynaptic receptor appears to be at least partly responsible for the run-down phenomenon, and may be involved in the physiological regulation of 5-HT synaptic transmission.

Serotonin agonists inhibit synaptic potentials in the rat locus ceruleus in vitro via 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors.

Intracellular recordings were made from rat locus ceruleus neurons in the slice preparation in vitro. Depolarizing synaptic potentials (DSP)2 elicited by electrical stimulation were typically 10 to 15 mV in amplitude and 200 msec in duration. Superfusion with 5-hydroxytryptamine (5-HT, serotonin) or the 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT), produced an inhibition of the DSP. The maximal inhibition was 55 +/- 2% (mean +/- S.E.M.). The EC50 for 5-CT was 60 nM, whereas for 5-HT it was 12 microM. Cocaine (10 microM) shifted the 5-HT concentration-response curve to the left and the EC50 to 320 nM. 8-Hydroxy-2-(di-n-propylamino)tetralin, a selective 5-HT1A receptor ligand, also inhibited the DSP, but only produced about 65% of the maximal 5-CT or 5-HT response (EC50 = 50 nM). A relatively selective 5-HT1B ligand (65-fold 5-HT1B greater than 5-HT1A), 1-(m-trifluoromethyl-phenyl)-piperazine, acted as a full agonist (EC50 = 110 nM). None of these compounds had any effects on the membrane properties of the cell at the doses tested. The response to 8-hydroxy-2-(di-n-propylamino) tetralin was antagonized by pretreatment with the 5-HT1A antagonist spiperone (1 microM). The estimated KD for spiperone was 16 nM. At this same concentration, however, there was no effect on the 5-CT-induced inhibition. The antagonist 4-(3-ter-butyl-amino-2-hydroxy-propoxyl)-indol-2-carbonic acid isopropyl ester (LM 21-009, 100 nM) was found to be a partial agonist producing a 26 +/- 4% inhibition of the DSP.(ABSTRACT TRUNCATED AT 250 WORDS)

DTG and (+)-3-PPP inhibit a ligand-activated hyperpolarization in mammalian neurons.

The effects of three compounds with high affinity for the haloperidol-sensitive alpha-binding site were studied with intracellular recordings in the vitro neuronal preparations of the rat locus ceruleus, rat dorsal raphe and the guinea pig submucous plexus. Both (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] and 1,3-di-o-tolylguanidine (DTG) inhibited the hyperpolarization induced by a ligand-activated potassium conductance. In the locus ceruleus, (+)-3-PPP and DTG produced a maximal 40 to 45% inhibition of the [Met5]enkephalin hyperpolarization, and had EC50 values of 6.6 and 2.2 microM, respectively. In the submucous plexus, the two compounds had a similar action on the alpha-2 adrenoceptor agonist UK14304 hyperpolarization, producing a maximal 50% inhibition with EC50 values of 140 and 32 nM, respectively. In addition, DTG inhibited the alpha-2-mediated inhibitory postsynaptic potential in both preparations. In contrast, (+)-3-PPP increased and prolonged the inhibitory postsynaptic potential. This action is qualitatively similar to the actions of cocaine on locus ceruleus and submucous plexus neurons. Haloperidol (1-10 microM) shared none of these actions. It is concluded that DTG and (+)-3-PPP are inhibitors of the opiate and alpha-2-mediated hyperpolarization at a postreceptor site, possibly the potassium channel. In addition, (+)-3-PPP, but not DTG, inhibits norepinephrine reuptake. None of these effects appear to be related to the sigma -binding site, because haloperidol acted as neither an agonist nor an antagonist.

Solubilization and characterization of sigma-receptors from guinea pig brain membranes.

The sigma-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for sigma-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective sigma-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 +/- 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]3-PPP] (KD = 36 +/- 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a sigma-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the sigma-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macromolecular complex.