Serotonin potentiates ethanol-induced excitation of ventral tegmental area neurons in brain slices from three different rat strains.

Neurons of the ventral tegmental area of Tsai (VTA) are important in mediation of the rewarding effects of drugs of abuse, including ethanol. We have demonstrated previously that ethanol excites neurons of the VTA recorded in a brain slice preparation. In the present study, we tested the interaction between serotonin and ethanol on putative dopamine neurons of the VTA. As reported previously, ethanol (20-160 mM) excited VTA neurons in a concentration-dependent manner. Serotonin (1-50 microM) produced only minor increases or decreases of the firing rate. In the presence of serotonin, the potency of ethanol was increased significantly. This potentiation of ethanol excitation by serotonin was seen in VTA slices taken from Sprague-Dawley, Fischer 344 and Lewis rats. The effect of 160 mM ethanol on VTA neurons from Fischer 344 rats, for example, was increased from 38.5 +/- 6.1% before serotonin to 77.4 +/- 16.7% after administration of 10 microM serotonin. Two serotonergic agonists, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride and alpha-methylserotonin, also potentiated the ethanol-induced excitation. The action of serotonin to increase the potency of ethanol to excite VTA neurons may be an important factor in the rewarding effects of ethanol, and might be exploited to develop effective pharmacotherapeutic agents for the treatment of alcohol craving.

Characterization of the adenosine A1 receptor-activated potassium current in rat locus ceruleus neurons.

The effect of adenosine on locus ceruleus neurons was investigated with intracellular recording in a totally submerged brain slice preparation. Bath application of adenosine (100 microM) hyperpolarized locus ceruleus neurons and inhibited their spontaneous firing; under voltage-clamp conditions, adenosine activated an inwardly rectifying, outward current (IAdo). The reversal potential of the IAdo was -110 mV and shifted by 59.2 mV per 10-fold change in external K+ concentration, very close to the shift predicted by the Nernst equation for a pure K+ current. The IAdo was due to a direct postsynaptic action, because it persisted in low Ca++/high Mg++ media that block Ca(++)-dependent neurotransmitter release. The IAdo was not blocked by glibenclamide, which indicates that it is not mediated by ATP-dependent K+ channels. The adenosine-activated current was concentration-dependent (10 microM-1 mM adenosine) and was blocked by the selective A1 antagonist 8-cyclopentyltheophylline in a competitive manner. Schild analysis in two neurons yielded estimates of the Kd value for 8-cyclopentyltheophylline of 1.4 and 4.6 nM, which indicates that the IAdo is mediated by A1 adenosine receptors. The adenosine-induced hyperpolarization, inhibition of firing and activation of outward current were blocked by external barium, but not by 4-aminopyridine. By contrast, we have previously shown that adenosine enhances A-current, thereby reducing action potential duration in locus ceruleus neurons, and these effects are blocked by 4-aminopyridine but not barium. These data indicate that the adenosine-induced hyperpolarization and inhibition of firing are mediated by the IAdo and that these effects are independent of adenosine's enhancement of A-current.

Ethanol enhances inward rectification in rat locus ceruleus neurons by increasing the extracellular potassium concentration.

The effect of ethanol on the current-voltage relationship of rat locus ceruleus (LC) neurons was examined under voltage-clamp conditions in a totally submerged brain slice preparation. LC neurons have the classic K(+)-specific type of inward rectification (IR), which is blocked by Ba++ and Cs+. Ethanol (40-200 mM) was found to enhance IR in LC neurons; it increased both the maximal slope conductance and shifted the voltage range for activation of IR in the depolarizing direction. These effects of ethanol were concentration dependent and fully reversible on washout. When IR was blocked with Ba++ or intracellular acetate, ethanol increased the slope conductance of the linear current-voltage relationship but did not restore IR. Ethanol also reduced the amplitude of afterhyperpolarizations that follow individual action potentials and trains of action potentials in LC neurons. Ethanol caused a depolarizing shift in the K+ equilibrium potential and its effects on IR could be mimicked by an increase in the K+ concentration in the medium. Specifically, 100 mM ethanol, on average, shifted the K+ equilibrium potential by 6.3 mV and its enhancement of IR was mimicked by an increase in the external K+ of 0.5-0.7 mM. From these data, it was concluded that ethanol enhances IR in LC neurons by increasing the extracellular K+ concentration.

Adenosine decreases action potential duration by modulation of A-current in rat locus coeruleus neurons.

The possibility that adenosine modulates voltage-dependent conductances in locus coeruleus neurons was investigated in current-clamp and voltage-clamp experiments in a totally submerged rat brain slice preparation. Adenosine (100 microM) reduced the duration of control action potentials and action potentials prolonged by 1 mM barium. Adenosine (100 microM) also reduced the amplitude and slightly reduced the duration of TTX-resistant "calcium" action potentials. Action potential duration was also reduced by the adenosine receptor agonist 2-chloroadenosine in a concentration-dependent manner and the adenosine-induced reduction of action potential duration was blocked by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline, indicating that this action of adenosine is mediated by an adenosine receptor. The adenosine-induced reduction of action potential duration persisted in the presence of externally applied tetraethylammonium ion (6 mM) and cesium (3 mM). By contrast, adenosine did not reduce the duration of the action potential in the presence of 500 microM 4-aminopyridine (4-AP). Furthermore, 4-AP (30 microM) blocked the adenosine-induced reduction of action potential duration recorded in the presence of 1 mM barium. These data suggested that adenosine may be acting on the voltage-dependent, 4-AP-sensitive potassium current, IA. Single-electrode voltage clamp was used to study IA directly. IA was activated by depolarizing voltage pulses from a hyperpolarized holding potential and was blocked by 1 mM 4-AP. Adenosine (300 microM) enhanced IA by shifting the steady-state inactivation curve in the depolarizing direction.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-activated hyperpolarizations in rat locus coeruleus neurons in vitro.

1. Intracellular recordings were made from rat locus coeruleus (LC) neurons in completely submerged brain slices. Trains of action potentials in LC neurons were followed by a prolonged post-stimulus hyperpolarization (PSH). If trains were elicited with depolarizing current pulses of sufficient intensity, PSH was composed of a fast, early component (PSHE) and a slow, late component (PSHL). PSH which followed trains elicited with lower intensity depolarizing current pulses consisted only of PSHL. 2. Both PSHE and PSHL were augmented by increasing the number of action potentials in the train and both were associated with an increase in membrane conductance. The reversal potential for PSHE was -108 mV and for PSHL it was -114 mV. 3. When a hybrid voltage clamp protocol was used, the current underlying PSH (IPSH) was observed to consist of an early, rapidly decaying component, IE, followed by a late, slower decaying component, IL. The time course of decay of IPSH was biexponential with the time constant of decay of IL more than one order of magnitude larger than the time constant of decay of IE. An increase in the concentration of external K+ shifted the reversal potentials for IE and IL in the depolarizing direction; the mean value of shift per tenfold increase in external K+ concentration was 57.1 mV for IE and 57.6 mV for IL. 4. Both PSHE and PSHL were inhibited by lowering the external Ca2+ concentration or by application of the Ca2+ channel blockers Cd2+ (200-500 microM) or nifedipine (100 microM). Intracellular injection of EGTA abolished both components of PSH. Increasing the external Ca2+ concentration augmented both PSH components. 5. Superfusion of dantrolene (25 microM) or ryanodine (20 microM) decreased the amplitude and duration of PSHL with much less effect on PSHE. 6. d-Tubocurarine (20-200 microM) selectively blocked PSHE with no effect on PSHL; this effect is the same as that of apamin which we have previously described. Superfusion with charybdotoxin (40 nM) or TEA (400 microM-1 mM) did not reduce PSHE or PSHL. 7. Inhibition of IA by 4-aminopyridine or 2,4-diaminopyridine also did not reduce either component of PSH. In fact, these agents slightly augmented both components of PSH; this effect was probably secondary to the prolongation of action potential duration. Superfusion of TEA in concentrations of 2-10 mM increased the size and duration of PSHL and increased the duration but decreased the size of PSHE.(ABSTRACT TRUNCATED AT 400 WORDS)

GABAA and GABAB receptors and the ionic mechanisms mediating their effects on locus coeruleus neurons.

Anatomical, neurochemical, and electrophysiological studies have provided evidence that gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the locus coeruleus (LC) nucleus. We have used intracellular recording to study the actions of GABA on putative noradrenergic neurons of the rat LC, in a brain slice preparation. GABA application in the bath, or more locally by micropressure ejection inhibited spontaneous firing and increased the conductance of LC neurons. In addition, GABA could hyperpolarize or depolarize LC neurons; the size of these responses depended on the Cl- gradient across the membrane. GABA responses were antagonized by bicuculline. These data indicate that the actions of GABA on LC neurons are primarily mediated by activation of GABAA receptors which increases the Cl- conductance. When GABA is applied to LC neurons after blockade of GABAA receptors with bicuculline, a residual action mediated by GABAB type receptors can be seen. Similar responses can be obtained with the GABAB-selective agonist baclofen. GABAB activation inhibits spontaneous firing and causes membrane hyperpolarization due to an increase in K+ conductance. Single-electrode voltage clamp experiments were used to study the voltage dependency of GABA responses in LC neurons. GABA-induced current showed outward rectification. The conductance increase caused by a given amount of GABA decreased with membrane hyperpolarization. The time constant of decay of the GABA current also decreased with membrane hyperpolarization. Due to the voltage dependency of GABA responses, GABA exerts a stronger inhibitory effect on LC neurons at depolarized potentials than at hyperpolarized potentials, which could serve as a negative feedback mechanism to control excitability of these neurons.

Functional significance of the apamin-sensitive conductance in rat locus coeruleus neurons.

The effects of apamin on rat locus coeruleus (LC) neurons were studied in a brain slice preparation with intracellular recording. Bath application of apamin (2-500 nM) reduced the amplitude of an intermediate component of the afterhyperpolarization (AHP) following single spontaneous action potentials, but did not change the size or time-course of fast and slow components of the AHP, spike amplitude or duration. Apamin blocked the early component of the post-stimulus hyperpolarization (PSH) which follows a train of action potentials. The size of the late component of PSH was sometimes augmented by apamin. Apamin increased the number of spikes evoked by a depolarizing current pulse and increased the slope of the spike frequency-current intensity relation. Accommodation of firing during long depolarizing pulses showed a biexponential time-course indicating 2 distinct components. Apamin specifically reduced the contribution of the fast component of accommodation and increased its time constant. These data indicate that the apamin-sensitive conductance is functionally important in accommodation at faster firing rates such as those seen during evoked spike trains in the present experiments, and which may occur in vivo during behavioral arousal and in anxiety or drug withdrawal syndromes.

Enhancement of current induced by superfusion of GABA in locus coeruleus neurons by pentobarbital, but not ethanol.

gamma-Aminobutyric acid (GABA), pentobarbital and ethanol were applied by bath superfusion to rat locus coeruleus (LC) neurons in a brain slice preparation. The GABA-induced current and conductance increase was measured with single-electrode voltage clamp. Pentobarbital potentiated the GABA-induced current and conductance increase in all LC neurons tested. In contrast, ethanol did not alter the current and conductance increase induced by bath application of GABA to LC neurons.

gamma-Aminobutyric acid responses in rat locus coeruleus neurones in vitro: a current-clamp and voltage-clamp study.

1. Intracellular recordings were made from locus coeruleus (LC) neurones in a totally submerged brain slice preparation from adult rats. The effect of gamma-aminobutyric acid (GABA) on LC neurones was studied under current-clamp and voltage-clamp conditions. GABA caused inhibition of spontaneous firing and a large conductance increase in LC neurones. These effects could be accompanied by depolarization, hyperpolarization or little change in membrane potential depending on the presence or absence of Cl- in the recording microelectrode. 2. The reversal potential for GABA-induced changes in membrane potential (EGABA) was -71.3 +/- 1.1 mV (S.E.M., n = 21) in cells impaled with potassium acetate electrodes and -47.5 +/- 1.4 mV (S.E.M., n = 15) in cells impaled with KCl electrodes. When the external Cl- concentration was reduced EGABA was shifted in the depolarizing direction by 51.5 mV per tenfold change in external Cl- which is close to the shift predicted by the Nernst equation for a selective increase in CL- conductance. 3. GABA effects on LC neurones result from a direct action since they persist in low-Ca2+ and high-Mg2+ media which block synaptic transmission. 4. The effects of GABA were concentration dependent and antagonized by bicuculline (10 microM) and bicuculline methiodide (80-100 microM) indicating that they were mediated predominantly by an action on GABAA receptors. In the presence of bicuculline, EGABA was shifted towards the K+ equilibrium potential which indicated a residual bicuculline-resistant action at GABAB receptors. 5. GABA-induced responses were membrane potential dependent. GABA conductance was observed to decrease with membrane hyperpolarization in a linear manner. GABA-induced current showed outward rectification. In the voltage range studied (rest to -110 mV) the extent of this rectification was predicted by the Goldman-Hodgkin-Katz equation, suggesting that it was due to the unequal distribution of Cl- across the membrane. In addition, the time constant of decay of GABA current was decreased by membrane hyperpolarization; this could be due to a voltage-dependent change in receptor or channel kinetics. 6. These data suggest that the primary action of GABA on LC neurones is to increase Cl- conductance by activation of bicuculline-sensitive GABAA receptors. Due to the voltage dependence of GABA responses, GABA will exert a stronger inhibitory effect on LC neurones at depolarized than at hyperpolarized membrane potentials. This could serve as a negative feedback mechanism to control excitability of these neurones.

Ethanol increases the firing rate of dopamine neurons of the rat ventral tegmental area in vitro.

The ventral tegmental area (VTA) is a brain region rich in dopamine-containing neurons. Since most agents which act as substrates for self-administration increase dopaminergic outflow in the mesolimbic or mesocortical areas, the VTA slice preparation may be useful for identifying drugs with potential for abuse. While ethanol (EtOH) is a drug of abuse which has been widely studied, the properties of ethanol which contribute to its abuse potential are not known. We have developed a brain slice preparation of the VTA in order to study the action of EtOH on putative dopamine neurons. Concentrations of EtOH from 20 to 320 mM produce a dose-dependent excitation of the dopamine-type neurons of the VTA. About 89% of neurons which have electrophysiological characteristics established for presumed dopamine-containing neurons were excited by ethanol in the pharmacologically relevant concentration range. This excitation persists in low-calcium, high-magnesium medium, which suggests a direct excitatory action of EtOH on dopamine-type cells in the VTA slice.

Baclofen increases the potassium conductance of rat locus coeruleus neurons recorded in brain slices.

Baclofen causes a concentration-dependent inhibition of spontaneous firing, hyperpolarization and resistance decrease in locus coeruleus (LC) neurons recorded intracellularly in a brain slice preparation. The (-) isomer is active while the (+) isomer has little or no activity which indicates that the baclofen effect is stereoselective. Baclofen action on LC neurons is a direct postsynaptic effect since it remains in low Ca2+, high Mg2+ media. Baclofen actions on LC neurons are resistant to the GABAA antagonist bicuculline. The baclofen-induced hyperpolarization reverses at the K+ equilibrium potential, as estimated by the reversal potential of the post-stimulus hyperpolarization which follows an evoked train of action potentials. When the K+ concentration in the superfusion media is increased, the reversal potential for the baclofen-induced hyperpolarization shifts linearly with a slope of 61 mV per 10-fold change as predicted by the Nernst equation for a pure K+ conductance. The baclofen-induced K+ conductance increase is prevented by addition of the K+-channel blocker Ba2+ to the external media. Taken together, these data suggest that baclofen directly hyperpolarizes LC neurons by activation of GABAB receptors which leads to an increase in K+ conductance.

Anomalous rectification in rat locus coeruleus neurons.

Rat locus coeruleus neurons recorded under current clamp conditions show anomalous rectification (AR) as indicated by a progressive decrease in slope resistance measured with hyperpolarizing current pulses of increasing amplitude. AR was most prominent at potentials more negative than the K+ equilibrium potential. AR was strongly dependent on external K+ concentration and was blocked by external Ba2+ and Cs+, and intracellular injection of acetate.

Serotonin decreases the duration of action potentials recorded from tetraethylammonium-treated bullfrog dorsal root ganglion cells.

Neurotransmitter effects on calcium currents activated by sensory neuron action potentials have been previously studied in embryonic or neonatal dorsal root ganglion (DRG) cells in culture. In the present study we examined the effects of serotonin (5-HT) on the shape of action potentials recorded from fully differentiated primary afferent neurons in isolated DRG of adult bullfrogs. Intracellular recordings were obtained from cell bodies of type A and C neurons. Concentrations of 5-HT that had no effect on membrane potential or input resistance had little or no effect on action potential shape. Treatment with 5-20 mM tetraethylammonium ion (TEA) led to the appearance of a plateau phase on the falling limb of the spike. This plateau phase appears to result from calcium influx, as it was dramatically reduced in amplitude and duration by solutions containing low concentrations of calcium or the calcium channel blocker, manganese. In preparations treated with 7.5 mM TEA, low concentrations of 5-HT (10 nM-1 microM) produced a dose-dependent narrowing of the calcium-dependent plateau phase of the mixed sodium/calcium spike. A decrease in spike afterhyperpolarization was also noted. The decrease in spike duration was recorded from 74% of type A neurons and 57% of type C neurons, and was not secondary to a change in resting potential or input resistance. The 5-HT receptor antagonists methysergide and metergoline did not block the response to 5-HT. Instead, they exhibited weak agonist-like actions. Serotonin also reduced the rate of rise and peak amplitude of calcium spikes recorded in the presence of tetrodotoxin and TEA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine inhibits locus coeruleus neurons: an intracellular study in a rat brain slice preparation.

Intracellular recording was used to study the effect of adenosine (3-100 microM) on rat locus coeruleus (LC) neurons in a brain slice preparation. Bath application of adenosine (100 microM) reduced the rate of spontaneous firing in 88% of LC neurons. In some LC neurons adenosine also caused membrane hyperpolarization (2-10 mV) and reductions in input resistance of 9-24%. Adenosine effects were dose-dependent and antagonized by the adenosine receptor antagonist theophylline.

Basal firing rate of rat locus coeruleus neurons affects sensitivity to ethanol.

Intracellular recordings were made from spontaneously active rat locus coeruleus (LC) neurons in a totally submerged brain slice preparation. Bath application of ethanol (ETOH) (1-60 mM) inhibited the spontaneous firing of LC neurons. These ETOH concentrations are equal to or below ETOH concentrations found in the brain during mild to moderate intoxication. The basal frequency of spontaneous firing of LC neurons ranged from 0.4-7 Hz. For 9 LC neurons which showed complete block of firing by ETOH, the latency to block was found to be directly related to the logarithm of the firing rate (correlation coefficient 0.94). This relationship was not secondary to a relationship between membrane potential and latency to block since for the same 9 neurons, membrane potential and latency to block were not significantly correlated. We conclude that the basal firing rate of a neuron can affect its sensitivity to the inhibitory effects of ETOH.

Serotonin depolarizes type A and C primary afferents: an intracellular study in bullfrog dorsal root ganglion.

Intracellular recordings were obtained from the somata of type A and C primary afferents in the isolated bullfrog dorsal root ganglion (DRG) preparation. Bath application of serotonin (5-HT) in concentrations of 0.25-1.0 mM led to slow and fast depolarizing responses. Slow, maintained 5-HT depolarizations were observed in 47% of type A and 70% of type C neurons. These slow depolarizations were associated with an underlying increase in input resistance (Rin). In some type A neurons, the Rin increase was masked by a decrease in Rin due to depolarization-induced rectification. The slow 5-HT depolarization of type A, but not type C neurons showed pronounced tachyphylaxis to repeated 5-HT applications. In type C afferents, serotonin's slow action was often accompanied by spontaneous firing. Manganese decreased slow 5-HT depolarizations of both cell types. A slow depolarization and excitation of type C afferents by methysergide and cinanserin was also observed. Fast transient 5-HT depolarizations accompanied by a rapid decrease in Rin were observed in 7% of type A and 24% of type C neurons. In some DRG cells the fast and slow depolarizations combined to form a biphasic response. The actions of 5-HT reported here resemble in some ways 5-HT responses recorded extracellularly from the spinal terminations of primary afferents.

Membrane properties of rat locus coeruleus neurones.

Intracellular recordings were made from neurones in the locus coeruleus contained within a slice cut from rat pons and maintained in vitro. Most neurones fired action potentials spontaneously at frequencies of between 1 and 5 Hz; this did not arise from spontaneous synaptic input but appeared to result from endogenous properties of the membrane conductances. Under voltage clamp at potentials near threshold for action potential generation (-55 mV) there was a persistent inward calcium current. This current became less with membrane hyperpolarization and was abolished at about -70 mV. Two potassium currents were observed. The first had properties similar to that generally described as the "fast" potassium current (IK,A); it flowed transiently (for about 200 ms) when the membrane potential passed from about -65 to -45 mV, and was blocked by 4-aminopyridine. The second was a calcium-activated potassium current (IK,Ca); it flowed for several seconds following a burst of calcium action potentials. Spontaneous and evoked action potentials had both tetrodotoxin-sensitive and tetrodotoxin-resistant components. The latter was apparently due to calcium entry. The potential changes occurring during the spontaneous firing of locus coeruleus neurones could be reconstructed qualitatively from the ionic conductances observed. The membrane properties of the locus coeruleus neurones were remarkably uniform; however, about 5% of cells impaled within the region of the locus coeruleus were electrophysiologically distinct. These atypical cells had short duration action potentials, did not fire spontaneously and had large spontaneous depolarizing synaptic potentials.

Electrophysiological properties of neurons contained in the locus coeruleus and mesencephalic nucleus of the trigeminal nerve in vitro.

Intracellular recordings were made from neurones contained in the locus coeruleus and mesencephalic nucleus of the trigeminal nerve (MNV), in tissue slices cut from guinea-pig pons and maintained in vitro. Locus coeruleus neurones were of -52.7 +/- 2.7 mV resting membrane potential; had an input resistance of 58.0 +/- 7.6 M omega and a membrane time constant of 7.3 +/- 1.0 ms. These neurones fired action potentials in response to depolarizing current pulses. Depolarizing synaptic potentials (DSPs) were recorded in locus coeruleus neurones in response to focal stimulation of the surface of the slice. MNV neurones were of -51.9 +/- 3.6 mV resting membrane potential; had an input resistance of 15.0 +/- 1.8 M omega and a membrane time constant of 1.35 +/- 0.16 ms. These neurones were also excitable but differed from locus coeruleus neurones in that they showed accommodation to depolarizing current pulses and time-dependent anomalous rectification with hyperpolarizing current pulses. In MNV neurones focal stimulation did not give rise to DSPs. Intracellular injection of Lucifer yellow revealed that the cell bodies of locus coeruleus neurones were small and multipolar whereas MNV neurones had larger, monopolar cell bodies.

Opiate effects on rabbit vagus nerve: electrophysiology and radioligand binding.

Intracellular recordings were made from rabbit nodose ganglion cells in vitro. Morphine (up to 100 micro M), normorphine (up to 10 micro M) and D-Ala2, Leu5-enkephalin (DADLE) (up to 5 micro M) each had no detectable effect on the electrical properties of the cell membrane, except for local anesthetic-like actions at the highest concentrations which were not reversed by naloxone. Extracellular recordings were made from the infranodose vagus nerve in vitro using a sucrose gap method. No effects of morphine, normorphine or DADLE were detected on the resting potential, compound action potential or compound action potential enhanced by barium or tetraethylammonium. Moderate levels of stereospecific binding of tritiated dihydromorphine and DADLE were detected in both the nodose ganglion and vagus nerve. It is surmised that the radioligand binding sites on the nodose ganglion and vagus nerve are not functionally linked to detectable electrophysiological effects.