Multiple conductances are modulated by 5-HT receptor subtypes in rat subthalamic nucleus neurons

Firing patterns of subthalamic nucleus (STN) neurons influence normal and abnormal movements. The STN expresses multiple 5-HT receptor subtypes that may regulate neuronal excitability. We used whole-cell patch-clamp recordings to characterize 5-HT receptor-mediated effects on membrane currents in STN neurons in rat brain slices. In 80 STN neurons under voltage-clamp (-70 mV), 5-HT (30 microM) evoked inward currents in 64%, outward currents in 17%, and biphasic currents in 19%. 5-HT-induced outward current was caused by an increased K(+) conductance (1.4+/-0.2 nS) and was blocked by the 5-HT(1A) antagonist WAY 100135. The 5-HT-evoked inward current, which was blocked by antagonists at 5-HT(2C) and/or 5-HT(4) receptors, had two types of current-voltage (I-V) relations. Currents associated with the type 1 I-V relation showed negative slope conductance at potentials <-110 mV and were occluded by Ba(2+). In contrast, the type 2 I-V relation appeared linear and had positive slope conductance (0.64+/-0.11 nS). Type 2 inward currents were Ba(2+)-insensitive, and the reversal potential of -19 mV suggests a mixed cation conductance. In STN neurons in which 5-HT evoked inward currents, 5-HT potentiated burst firing induced by N-methyl-d-aspartate (NMDA). But in neurons in which 5-HT evoked outward current, 5-HT slowed NMDA-dependent burst firing. We conclude that 5-HT receptor subtypes can differentially regulate firing pattern by modulating multiple conductances in STN neurons.     

Diverse actions of 5-hydroxytryptamine on frog spinal dorsal horn neurons in vitro.

The effects of 5-hydroxytryptamine on the membrane potential and input resistance of 86 dorsal horn neurons were studied using intracellular recordings in isolated, hemisected spinal cords of adult frogs (Rana pipiens). Bath application of serotonin (5-100 microM) caused membrane depolarizations in 58 (67%) neurons, hyperpolarizations in 12 (14%) cells, biphasic responses in nine (11%) neurons, and no detectable change in seven (8%) cells. In some neurons depolarized by serotonin, the amine's responses could be mimicked by the selective 5-HT2 agonist (+/-)-1(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride and the 5-HT1C/2 agonist alpha-methyl-5-hydroxytryptamine, and blocked by the 5-HT1C/2 antagonists ketanserin and mianserin. In other neurons depolarized by serotonin, the 5-HT3 agonist 2-methyl-5-hydroxytryptamine mimicked, and the 5-HT3 antagonist, 3-tropanyl-3,5-dichlorobenzoate, blocked the serotonin-induced responses. Depolarizing responses due to activation of 5-HT1C/2 receptors were generally accompanied by increases in the membrane input resistance, whereas depolarizations mediated by 5-HT3 receptors were associated with a decreased membrane input resistance. Superfusion with tetrodotoxin or low-Ca2+/high-Mg(2+)-containing media abolished about half of the depolarizing responses. Hyperpolarizations caused by serotonin were associated with a decrease in membrane input resistance, and might have been due to activation of a potassium conductance. These responses persisted in bathing solutions containing tetrodotoxin or low-Ca2+/high-Mg2+. The 5-HT1A agonist 8-hydroxy-2-(di-N-propylamine)tetralin hydrobromide mimicked, whereas the 5-HT1A antagonist spiroxatrine blocked, these hyperpolarizing responses. Other antagonists selective for 5-HT1C/2 or 5-HT3 receptors were without effect. Serotonin-produced biphasic responses consisted of either an initial depolarization followed by a hyperpolarization or the reverse. The selective 5-HT2 agonist (+/-)-1(2,5-dimethyoxy-4-iodophenyl)-2-aminopropane hydrochloride could only mimic the depolarizations, whereas the 5-HT1A agonist 8-hydroxy-2-(di-N-propylamine)tetralin hydrobromide produced only the hyperpolarizations. Spiroxatrine, a 5-HT1A antagonist, blocked only the hyperpolarizations without affecting the depolarizations, and methysergide, a non-specific 5-HT receptor antagonist, depressed both the depolarizations and hyperpolarizations. Serotonin also appeared to affect spinal dorsal horn neurons indirectly because it produced excitatory postsynaptic potentials, inhibitory postsynaptic potentials, and a mixture of both.(ABSTRACT TRUNCATED AT 400 WORDS)     

D2-like dopamine receptors modulate SKCa channel function in subthalamic nucleus neurons through inhibition of Cav2.2 channels

The activity patterns of subthalamic nucleus (STN) neurons are intimately related to motor function/dysfunction and modulated directly by dopaminergic neurons that degenerate in Parkinson's disease (PD). To understand how dopamine and dopamine depletion influence the activity of the STN, the functions/signaling pathways/substrates of D2-like dopamine receptors were studied using patch-clamp recording. In rat brain slices, D2-like dopamine receptor activation depolarized STN neurons, increased the frequency/irregularity of their autonomous activity, and linearized/enhanced their firing in response to current injection. Activation of D2-like receptors in acutely isolated neurons reduced transient outward currents evoked by suprathreshold voltage steps. Modulation was inhibited by a D2-like receptor antagonist and occluded by voltage-dependent Ca2+ (Cav) channel or small-conductance Ca2+-dependent K+ (SKCa) channel blockers or Ca2+-free media. Because Cav channels are targets of G(i/o)-linked receptors, actions on step- and action potential waveform-evoked Cav channel currents were studied. D2-like receptor activation reduced the conductance of Cav2.2 but not Cav1 channels. Modulation was mediated, in part, by direct binding of Gbetagamma subunits because it was attenuated by brief depolarization. D2 and/or D3 dopamine receptors may mediate modulation because a D4-selective agonist was ineffective and mRNA encoding D2 and D3 but not D4 dopamine receptors was detectable. Brain slice recordings confirmed that SKCa channel-mediated action potential afterhyperpolarization was attenuated by D2-like dopamine receptor activation. Together, these data suggest that D2-like dopamine receptors potently modulate the negative feedback control of firing that is mediated by the functional coupling of Cav2.2 and SKCa channels in STN neurons.     

5-HT excites globus pallidus neurons by multiple receptor mechanisms

Anatomical and neurochemical studies indicated that the globus pallidus receives serotonergic innervation from raphe nuclei but the membrane effects of 5-HT on globus pallidus neurons are not entirely clear. We address this question by applying whole-cell patch-clamp recordings on globus pallidus neurons in immature rat brain slices. Under current-clamp recording, 5-HT depolarized globus pallidus neurons and increased their firing rate, an action blocked by both 5-HT(4) and 5-HT(7) receptor antagonists and attributable to an increase in cation conductance(s). Further experiments indicated that 5-HT enhanced the hyperpolarization-activated inward conductance which is blocked by 5-HT(7) receptor antagonist. To determine if 5-HT exerts any presynaptic effects on GABAergic and glutamatergic inputs, the actions of 5-HT on synaptic currents were studied. At 10 microM, 5-HT increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but had no effect on both the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). However, 5-HT at a higher concentration (50 microM) decreased the frequency but not the amplitude of the mIPSCs, indicating an inhibition of GABA release from the presynaptic terminals. This effect was sensitive to 5-HT(1B) receptor antagonist. In addition to the presynaptic effects on GABAergic neurotransmission, 5-HT at 50 microM had no consistent effects on glutamatergic neurotransmission, significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in 4 of 11 neurons and decreased the frequency of mEPSCs in 3 of 11 neurons. In conclusion, we found that 5-HT could modulate the excitability of globus pallidus neurons by both pre- and post-synaptic mechanisms. In view of the extensive innervation by globus pallidus neurons on other basal ganglia nuclei, this action of 5-HT originated from the raphe may have a profound effect on the operation of the entire basal ganglia network.     

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.     

Opposing electrophysiological actions of 5-HT on noncholinergic and cholinergic neurons in the rat ventral pallidum in vitro

The ventral pallidum in rat is a basal forebrain structure that contains neurons that project in the limbic striatopallidal circuitry and magnocellular cholinergic corticopetal neurons. Because 5-hydroxytryptamine (5-HT) terminals on dorsal raphe projections form close appositions with these neurons, we made patch-clamp recordings in immature rat brain slices to determine whether they are modulated by postsynaptic 5-HT receptors. Inward currents were predominantly induced by 5-HT in noncholinergic neurons, which were distinguished from cholinergic neurons by immunohistochemical and electrophysiological criteria. The inward current induced by 5-HT was mimicked and occluded when adenylyl cyclase was stimulated with forskolin, and was almost abolished when h-currents in noncholinergic neurons were blocked with cesium. Consistent with 5-HT(7) receptor activation of h-curents by cAMP in other brain regions, we found inward currents were mimicked by the mixed 5-HT(1)/5-HT(7) agonists 5-methoxytryptamine, and by 5-carboxamidotryptamine (5-CT), which was more potent than 5-HT. In contrast, 5-HT(1) preferring 8-OH-DPAT was a weak partial agonist, and the 5-HT(1)-selective antagonist pindolol had no effect. However, despite this profile, antagonists that bind at the 5-HT(7) receptor only partly reduced the agonist inward current (SB-269970 and clozapine), or had no effect (mianserin and pimozide). We found in cholinergic neurons that 5-HT predominantly induced hyperpolarizing currents, which were carried by potassium channels, and were smaller than currents induced by 8-OH-DPAT and 5-CT. We conclude from this study that ascending 5-HT projections from the dorsal raphe could have direct and opposite effects on the activities of neurons within the limbic striatopallidal and cholinergic corticopetal circuitry in the ventral pallidum.     

Serotonergic modulation of respiratory motoneurons and interneurons in brainstem slices of perinatal rats

Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-B?tzinger complex (pre-B?tC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (approximately 11 mV) and tonic spike discharge in XII motoneurons, whereas pre-B?tC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-B?tC exclusively increased respiratory frequency by 30-220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-B?tC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT2 receptor agonist -methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-B?tC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT2 receptors of pre-B?tC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT1A receptors.     

Histamine H(2) receptor activated chloride conductance in myenteric neurons from guinea pig small intestine

Whole cell perforated patch-clamp methods were used to investigate ionic mechanisms underlying histamine-evoked excitatory responses in small intestinal AH-type myenteric neurons. When physiological concentrations of Na(+), Ca(2+), and Cl(-) were in the bathing medium, application of histamine significantly increased total conductance as determined by stepping to 50 mV from a holding potential of -30 mV. The current reversed at a membrane potential of -30 +/- 5 (SE) mV and current-voltage relations exhibited outward rectification. The reversal potential for the histamine-activated current was unchanged by removal of Na(+) and Ca(2+) from the bathing medium. Reduction of Cl(-) from 155 mM to 55 mM suppressed the current when the neurons were in solutions with depleted Na(+) and Ca(2+). Current-voltage curves in solutions with reduced Cl(-) were linear and the reversal potential was changed from -30 +/- 5 mV to 7 +/- 4 mV. Niflumic acid, but not anthracene-9-carboxylic acid (9-AC) nor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the histamine-activated current. A membrane permeable analogue of cAMP evoked currents similar to those activated by histamine. A selective histamine H(2) receptor agonist (dimaprit) mimicked the action of histamine and a selective histamine H(2) receptor antagonist (cimetidine) blocked the conductance increase evoked by histamine. A selective adenosine A(1) receptor agonist (CCPA) reduced the histamine-activated current and a selective adenosine A(1) receptor antagonist (CPT) reversed the inhibitory action. The results suggest that histamine acts at histamine H(2) receptors to increase Cl(-) conductance in AH-type enteric neurons. Cyclic AMP appears to be a second messenger in the signal transduction process. Results with a selective adenosine A(1) receptor agonist and antagonist add to existing evidence for co-coupling of inhibitory adenosine A(1) receptors and histamine H(2) receptors to adenylate cyclase in AH-type enteric neurons.     

Serotonergic modulation of neurotransmission in the rat basolateral amygdala.

Whole cell patch-clamp recordings were obtained from projection neurons and interneurons of the rat basolateral amygdala (BLA) to understand local network interactions in morphologically identified neurons and their modulation by serotonin. Projection neurons and interneurons were characterized morphologically and electrophysiologically according to their intrinsic membrane properties and synaptic characteristics. Synaptic activity in projection neurons was dominated by spontaneous inhibitory postsynaptic currents (IPSCs) that were multiphasic, reached 181 +/- 38 pA in amplitude, lasted 296 +/- 27 mS, and were blocked by the GABAA receptor antagonist, bicuculline methiodide (30 microM). In interneurons, spontaneous synaptic activity was characterized by a burst-firing discharge patterns (200 +/- 40 Hz) that correlated with the occurrence of 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive, high-amplitude (260 +/- 42 pA), long-duration (139 +/- 19 mS) inward excitatory postsynaptic currents (EPSCs). The interevent interval of 831 +/- 344 mS for compound inhibitory postsynaptic potentials (IPSPs), and 916 +/- 270 mS for EPSC bursts, suggested that spontaneous IPSP/Cs in projection neurons are driven by burst of action potentials in interneurons. Hence, BLA interneurons may regulate the excitability of projection neurons and thus determine the degree of synchrony within ensembles of BLA neurons. In interneurons 5-hydroxytryptamine oxalate (5-HT) evoked a direct, dose-dependent, membrane depolarization mediated by a 45 +/- 6.9 pA inward current, which had a reversal potential of -90 mV. The effect of 5-HT was mimicked by the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT), but not by the 5-HT1A receptor agonist, (+/-) 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), or the 5-HT1B agonist, CGS 12066A. In projection neurons, 5-HT evoked an indirect membrane hyperpolarization ( approximately 2 mV) that was associated with a 75 +/- 42 pA outward current and had a reversal potential of -70 mV. The response was independent of 5-HT concentration, blocked by TTX, mimicked by alpha-methyl-5-HT but not by 8-OH-DPAT. In interneurons, 5-HT reduced the amplitude of the evoked EPSC and in the presence of TTX (0.6 microM) reduced the frequency of miniature EPSCs but not their quantal content. In projection neurons, 5-HT also caused a dose-dependent reduction in the amplitude of stimulus evoked EPSCs and IPSCs. These results suggest that acute serotonin release would directly activate GABAergic interneurons of the BLA, via an activation of 5-HT2 receptors, and increase the frequency of inhibitory synaptic events in projection neurons. Chronic serotonin release, or high levels of serotonin, would reduce the excitatory drive onto interneurons and may act as a feedback mechanism to prevent excess inhibition within the nucleus.     

5-HT inhibits synaptic transmission in rat subthalamic nucleus neurons in vitro

The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 microM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35+/-4%. However, a much higher concentration of 5-HT (100 microM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration-response curves showed that the 5-HT inhibitory concentration 50% (IC50) for inhibition of IPSCs (20.2 microM) was more than fivefold greater than the IC50 for inhibition of EPSCs (3.4 microM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT1B receptors.     

Heterogeneous actions of serotonin on interneurons in rat visual cortex

The effects of serotonin (5-HT) on excitability of two cortical interneuronal subtypes, fast-spiking (FS) and low threshold spike (LTS) cells, and on spontaneous inhibitory postsynaptic currents (sIPSCs) in layer V pyramidal cells were studied in rat visual cortical slices using whole-cell recording techniques. Twenty-two of 28 FS and 26 of 35 LTS interneurons responded to local application of 5-HT. In the group of responsive neurons, 5-HT elicited an inward current in 50% of FS cells and 15% of LTS cells, an outward current was evoked in 41% of FS cells and 81% of LTS cells, and an inward current followed by an outward current in 9% of FS cells and 4% LTS cells. The inward and outward currents were blocked by a 5-HT(3) receptor antagonist, tropisetron, and a 5-HT(1A) receptor antagonist, NAN-190, respectively. The 5-HT-induced inward and outward currents were both associated with an increase in membrane conductance. The estimated reversal potential was more positive than -40 mV for the inward current and close to the calculated K(+) equilibrium potential for the outward current. The 5-HT application caused an increase, a decrease, or an increase followed by a decrease in the frequency of sIPSCs in pyramidal cells. The 5-HT(3) receptor agonist 1-(m-chlorophenyl) biguanide increased the frequency of larger and fast-rising sIPSCs, whereas the 5-HT(1A) receptor agonist (+/-)8-hydroxydipropylaminotetralin hydrobromide elicited opposite effects and decreased the frequency of large events. These data indicate that serotonergic activation imposes complex actions on cortical inhibitory networks, which may lead to changes in cortical information processing.     

5-HT(2) receptor subtypes mediate different long-term changes in GABAergic activity to parasympathetic cardiac vagal neurons in the nucleus ambiguus

Serotonin (5-HT), and in particular 5-HT(2) receptors, play an important role in cardiorespiratory function within the brainstem. In addition, abnormalities in the 5-HT system have been implicated in many cardiorespiratory disorders, including sudden infant death syndrome. However, little is known about the mechanisms of action of 5-HT(2) receptors in altering the activity of parasympathetic cardiac neurons in the brainstem. In this study we examined the effects of activation of different subtypes of 5-HT(2) receptors on spontaneous and respiratory-evoked GABAergic neurotransmission to cardioinhibitory vagal neurons within the nucleus ambiguus as well as rhythmic fictive inspiratory-related activity in rats. A single application of alpha-Me-5-hydroxytryptamine maleate (alpha-Me-5-HT), a 5-HT(2) receptor agonist, did not significantly alter the frequency of spontaneous or respiratory-evoked GABAergic inhibitory postsynaptic currents (IPSCs) in cardiac vagal neurons. However, repetitive successive applications of alpha-Me-5-HT elicited a long-lasting (>/=1 h) decrease in the frequency of spontaneous as well as inspiratory-related GABAergic IPSCs to cardiac vagal neurons. This study demonstrates multiple, but not single applications of the 5-HT(2) receptor agonist alpha-Me-5-HT caused a long-lasting inhibition of both spontaneous and fictive inspiratory-related GABAergic neurotransmission to CVNs, which can be prevented by the 5-HT(2B) receptor antagonist SB204741, but persisted with the 5-HT(2A/2C) receptor antagonist ketanserin. The 5-HT(2) receptor agonist alpha-Me-5-HT also reversibly and transiently excited central fictive inspiratory activity, which was abolished by ketanserin, but was unaffected by the 5-HT(2B) receptor antagonist SB204741.     

Excitatory effects of serotonin on rat striatal cholinergic interneurones

We investigated the effects of 5-hydroxytryptamine (5-HT, serotonin) in striatal cholinergic interneurones with gramicidin-perforated whole-cell patch recordings. Bath-application of serotonin (30 μ m ) significantly and reversibly increased the spontaneous firing rate of 37/45 cholinergic interneurones tested. On average, in the presence of serotonin, firing rate was 273 ± 193% of control. Selective agonists of 5-HT_1A, 5-HT₃, 5-HT₄ and 5-HT₇ receptors did not affect cholinergic interneurone firing, while the 5-HT₂ receptor agonist α-methyl-5-HT (30 μ m ) mimicked the excitatory effects of serotonin. Consistently, the 5-HT₂ receptor antagonist ketanserin (10 μ m ) fully blocked the excitatory effects of serotonin. Two prominent after-hyperpolarizations (AHPs), one of medium duration that was apamin-sensitive and followed individual spikes, and one that was slower and followed trains of spikes, were both strongly and reversibly reduced by serotonin; these effects were fully blocked by ketanserin. Conversely, the depolarizing sags observed during negative current injections and mediated by hyperpolarization-activated cationic currents were not affected. In the presence of apamin and tetrodotoxin, the slow AHP was strongly reduced by 5-HT, and fully abolished by the calcium channel blocker nickel. These results show that 5-HT exerts a powerful excitatory control on cholinergic interneurones via 5-HT₂ receptors, by suppressing the AHPs associated with two distinct calcium-activated potassium currents.     

Characterization of outward currents induced by 5-HT in neurons of rat dorsolateral septal nucleus

Properties of the 5-hydroxytryptamine (5-HT)-induced current (I(5-HT)) were examined in neurons of rat dorsolateral septal nucleus (DLSN) by using whole cell patch-clamp techniques. I(5-HT) was associated with an increase in the membrane conductance of DLSN neurons. The reversal potential of I(5-HT) was -93 +/- 6 (SE) mV (n = 7) in the artificial cerebrospinal fluid (ACSF) and was changed by 54 mV per decade change in the external K(+) concentration, indicating that I(5-HT) is carried exclusively by K(+). Voltage dependency of the K(+) conductance underlying I(5-HT) was investigated by using current-voltage relationship. I(5-HT) showed a linear I-V relation in 63%, inward rectification in 21%, and outward rectification in 16% of DLSN neurons. (+/-)-8-Hydroxy-dipropylaminotetralin hydrobromide (30 microM), a selective 5-HT(1A) receptor agonist, also produced outward currents with three types of voltage dependency. Ba(2+) (100 microM) blocked the inward rectifier I(5-HT) but not the outward rectifier I(5-HT). In I(5-HT) with linear I-V relation, blockade of the inward rectifier K(+) current by Ba(2+) (100 microM) unmasked the outward rectifier current in DLSN neurons. These results suggest that I(5-HT) with linear I-V relation is the sum of inward rectifier and outward rectifier K(+) currents in DLSN neurons. Intracellular application of guanosine-5'-O-(3-thiotriphosphate) (300 microM) and guanosine-5'-O-(2-thiodiphosphate) (5 mM), blockers of G protein, irreversibly depressed I(5-HT). Protein kinase C (PKC) 19-36 (20 microM), a specific PKC inhibitor, depressed the outward rectifier I(5-HT) but not the inward rectifier I(5-HT). I(5-HT) was depressed by N-ethylmaleimide, which uncouples the G-protein-coupled receptor from pertussis-toxin-sensitive G proteins. H-89 (10 microM) and adenosine 3',5'-cyclic monophosphothioate Rp-isomer (300 microM), protein kinase A inhibitors, did not depress I(5-HT). Phorbol 12-myristate 13-acetate (10 microM), an activator of PKC, produced an outward rectifying K(+) current. These results suggest that both 5-HT-induced inward and outward rectifying currents are mediated by a G protein and that PKC is probably involved in the transduction pathway of the outward rectifying I(5-HT) in DLSN neurons.     

Neuromodulatory role of serotonin in the ferret thalamus

Serotonergic fibers broadly innervate the thalamus and may influence the sleep wake cycle, attention, and other processes through modulation of neurons in this structure. However, the actions of serotonin in the dorsal thalamus have been investigated in detail only in the dorsal lateral geniculate nucleus. In the present study, we examined the action of serotonin in several different regions of the ferret dorsal thalamus, including the associative nuclei, using the in vitro slice preparation and intracellular recording techniques. In nearly all nuclei examined, the predominant action of serotonin was one of hyperpolarization and inhibition of the tonic firing mode. The magnitude of the hyperpolarizing response decreased with age and varied greatly across and somewhat within nuclei maintaining the following relationship (in descending order of magnitude): lateral posterior, lateral dorsal, pulvinar, mediodorsal, center median, anteroventral, central lateral, ventral basal, and medial geniculate. This hyperpolarization is elicited through two mechanisms: one direct and the other via local interneurons. The direct action occurs through an increase in potassium conductance mediated through the 5-HT(1A) receptor. This conclusion is supported by the findings that it persists in the presence of tetrodotoxin and block of GABAergic synaptic transmission, the reversal potential shifts in a Nernstian fashion with changes in extracellular potassium concentration, and the response is antagonized by the 5-HT(1A) antagonist WAY100635 and mimicked by the application of the 5-HT(1A)-selective agonist 8-OH DPAT. The second mechanism by which 5-HT evoked a hyperpolarization was through the activation of local interneurons. In slices in which GABA receptors were not blocked, 5-HT application increased the frequency and amplitude of spontaneous inhibitory postsynaptic potentials (IPSPs) occurring in thalamocortical neurons. Application of 5-HT to physiologically or morphologically identified interneurons evoked a prolonged suprathreshold depolarization. Our results suggest that serotonergic inputs act differentially across the thalamus in a complex manner involving direct and indirect mechanisms. It appears that 5-HT has a greater direct postsynaptic inhibitory influence in the posterior, medial, and intralaminar nuclei than in the primary sensory nuclei.     

大鼠三叉神经节神经元5-羟色胺激活电流及其特征

采用全细胞膜片钳技术观察大鼠三叉神经节神经元5-羟色胺激活电流(I5-HT)及其特征。在大多数受检细胞(64／87,73.6％)特别是中、小型细胞,外加5-HT可引起一快去敏感的内向电流,此内向电流可被5-HT3受体特异性激动剂2-甲基-5-羟色胺所模拟,被5-HT3受体拮抗剂ICS 250-930可逆性阻断。I5-HT浓度-反应曲线的阈浓度为3×10-7mol／L,饱和浓度约为3×10-3mol／L,EC50为21.4+2.2μmol／L,Hill系数为0.96。I5-HT的翻转电位在-2.5 mV左右。胞内透析GDP-β-S证明I5-HT不依赖于G蛋白而存在。本研究结果为了解5-HT3受体介导电流及其特征提供了有关的资料。     

Mechanisms of dopamine activation of fast-spiking interneurons that exert inhibition in rat prefrontal cortex

Prefrontal cortical dopamine (DA) modulates pyramidal cell excitability directly and indirectly by way of its actions on local circuit GABAergic interneurons. DA modulation of interneuronal functions is implicated in the computational properties of prefrontal networks during cognitive processes and in schizophrenia. Morphologically and electrophysiologically distinct classes of putative GABAergic interneurons are found in layers II-V of rat prefrontal cortex. Our whole cell patch-clamp study shows that DA induced a direct, TTX-insensitive, reversible membrane depolarization, and increased the excitability of fast-spiking (FS) interneurons. The DA-induced membrane depolarization was reduced significantly by D1/D5 receptor antagonist SCH 23390, but not by the D2 receptor antagonist (-)sulpiride, D4 receptor antagonists U101958 or L-745870, alpha1-adrenoreceptor antagonist prazosin, or serotoninergic receptor antagonist mianserin. The D1/5 agonists SKF81297 or dihydrexidine, but not D2 agonist quinpirole, also induced a prolonged membrane depolarization. Voltage-clamp analyses of the voltage-dependence of DA-sensitive currents, and the effects of changing [K(+)](O) on reversal potentials of DA responses, revealed that DA suppressed a Cs(+)-sensitive inward rectifier K(+) current and a resting leak K(+) current. D1/D5, but not D2 agonists mimicked the suppressive effects of DA on the leak current, but the DA effects on the inward rectifier K(+) current were not mimicked by either agonist. In a subgroup of FS interneurons, the slowly inactivating membrane outward rectification evoked by depolarizing voltage steps was also attenuated by DA. Collectively, these data showed that DA depolarizes FS interneurons by suppressing a voltage-independent 'leak' K(+) current (via D1/D5 receptor mechanism) and an inwardly rectifying K(+) current (via unknown DA mechanisms). Additional suppression of a slowly inactivating K(+) current led to increase in repetitive firing in response to depolarizing inputs. This D1-induced increase in interneuron excitability enhances GABAergic transmission to PFC pyramidal neurons and could represent a mechanism via which DA suppresses persistent firing of pyramidal neurons in vivo.     

5-HT1B receptor-mediated presynaptic inhibition of GABA release in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) receives a dense serotonergic innervation that modulates photic input to the SCN via serotonin 1B (5-HT1B) presynaptic receptors on retinal glutamatergic terminals. However, the majority of 5-HT1B binding sites in the SCN are located on nonretinal terminals and most axonal terminals in the SCN are GABAergic. We therefore tested the hypothesis that 5-HT1B receptors might also be located on SCN GABAergic terminals by examining the effects of the highly selective 5-HT1B receptor agonist CP-93,129 on SCN miniature inhibitory postsynaptic currents (mIPSCs). Whole cell patch-clamp recordings of mIPSCs were obtained from rat and mouse SCN neurons in hypothalamic slices. Using CsCl-containing microelectrodes with QX314, we isolated mPSCs that were sensitive to the GABAA receptor antagonist, bicuculline. Bath application of CP-93,129 (1 microM) decreased the frequency of mIPSCs by an average of 22% (n = 7) in rat SCN neurons and by an average of 30% (n = 8) in mouse SCN neurons with no clear effect on mIPSC amplitude. In mice lacking functional 5-HT1B receptors, CP-93,129 (1 microM) had no clear effect on the frequency or the amplitude of mIPSCs recorded in any of the cells tested (n = 4). The decrease in the frequency of mIPSCs of SCN neurons produced by the selective 5-HT1B receptor agonist CP-93,129 is consistent with the interpretation that 5-HT1B receptors are located on GABA terminals in the SCN and that 5-HT inhibits GABA release via a 5-HT1B presynaptic receptor-mediated mechanism.     

P2Y1 receptor modulation of Ca2+-activated K+ currents in medium-sized neurons from neonatal rat striatal slices

ATP signaling to neurons and glia in the nervous system occurs via activation of both P2Y and P2X receptors. Here, we investigated the effects of P2Y(1) receptor stimulation in developing striatal medium-sized neurons using patch-clamp recordings from acute brain slices of 7- and 28-day-old rats. Application of the selective P2Y(1) receptor agonist 2-(Methylthio) ADP trisodium salt (2-MeSADP; 250 nM) increased outward K(+) currents evoked by a ramp depolarization protocol in voltage-clamp recordings. This effect was observed in 59 out of 82 cells (72%) and was blocked completely by the P2Y(1) antagonist, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate. The averaged 2-MeSADP-sensitive conductance was fitted by the sum of a linear conductance and a Boltzmann relation, giving one-half activation voltage of -14.2 mV and an equivalent charge of 2.91. The 2MeSADP-mediated effect was sensitive to submillimolar concentrations of tetraethylammonium (TEA; 200 μM), to 200 nM iberiotoxin and to 100 nM apamin, suggesting the involvement of both big and small potassium (BK and SK, respectively) calcium-activated channels. In current-clamp experiments, 2-MeSADP decreased depolarization-evoked action potential (AP) firing in all 26 cells investigated, and this effect was reversed by TEA and by apamin but not by iberiotoxin. We conclude that the stimulation of P2Y(1) receptors in developing striatal neurons leads to activation of calcium-activated potassium channels [I(K(Ca))] of both BK and SK subtypes, the latter responsible for decreasing the frequency of AP firing in response to current injection. Therefore, P2Y(1) signaling leading to activation of I(K(Ca)) may be important in regulating the activity of medium-sized neurons in the striatum.