Ethanol inhibition of m-current and ethanol-induced direct excitation of ventral tegmental area dopamine neurons.

Ethanol-induced excitation of ventral tegmental area dopamine (DA VTA) neurons is thought to be critical for the reinforcing effects of ethanol. Although ligand-gated ion channels are known to be the targets of ethanol, ethanol modulation of voltage-dependent ion channels of central neurons has not been well studied. We have demonstrated that ethanol excites DA VTA neurons by the reduction of sustained K(+) currents and recently reported that M-current (I(M)) regulates action potential generation through fast and slow afterhyperpolarization phases. In the present study we thus examined whether ethanol inhibition of I(M) contributes to the excitation of DA VTA neurons using nystatin-perforated patch current- and voltage-clamp recordings. Ethanol (20-120 mM) reduced I(M) in a concentration-dependent manner and increased the spontaneous firing frequency of DA VTA neurons. Ethanol-induced increase in spontaneous firing frequency correlated positively with ethanol inhibition of I(M) with a slope value of 1.3. Specific I(M) inhibition by XE991 (0.3-10 microM) increased spontaneous firing frequency which correlated positively with I(M) inhibition with a slope value of 0.5. In the presence of 10 muM XE991, a concentration that produced maximal inhibition of I(M), ethanol still increased the spontaneous firing frequency of DA VTA neurons in a concentration-dependent manner. Thus we conclude that, although ethanol causes inhibition of I(M) and this results in some increase in the firing frequency of DA VTA neurons, another effect of ethanol is primarily responsible for the ethanol-induced increase in firing rate in these neurons.

Characterization of M-current in ventral tegmental area dopamine neurons.

M-current (I(M)) is a voltage-gated potassium current (KCNQ type) that affects neuronal excitability and is modulated by some drugs of abuse. Ventral tegmental area (VTA) dopamine (DA) neurons are important for the reinforcing effects of drugs of abuse. Therefore we studied I(M) in acutely dissociated rat DA VTA neurons with nystatin-perforated patch recording. The standard deactivation protocol was used to measure I(M) during voltage-clamp recording with hyperpolarizing voltage steps to -65 mV (in 10-mV increments) from a holding potential of -25 mV. I(M) amplitude was voltage dependent and maximal current amplitude was detected at -45 mV. The deactivation time constant of I(M) was voltage dependent and became shorter at more negative voltages. The I(M)/KCNQ antagonist XE991 (0.3-30 microM) caused a concentration-dependent reduction in I(M) amplitude with an IC(50) of 0.71 microM. Tetraethylammonium (TEA, 0.3-10 mM) caused a concentration-dependent inhibition of I(M) with an IC(50) of 1.56 mM. In current-clamp recordings, all DA VTA neurons were spontaneously active. Analysis of evoked action potential shape indicated that XE991 (1-10 microM) reduced the fast and slow components of the spike afterhyperpolarization (AHP) without affecting the middle component of the AHP. Action potential amplitude, duration, and threshold were not affected by XE991. In addition, 10 microM XE991 significantly shortened the interspike intervals in evoked spike trains. In conclusion, I(M) is active near threshold in DA VTA neurons, is blocked by XE991 (10 microM) and TEA (10 mM), may contribute to the shape of the AHP, and may decrease excitability of these neurons.

A-type K+ current of dopamine and GABA neurons in the ventral tegmental area.

A-type K(+) current (I(A)) is a rapidly inactivating voltage-dependent potassium current which can regulate the frequency of action potential (AP) generation. Increased firing frequency of ventral tegmental area (VTA) neurons is associated with the reinforcing effects of some drugs of abuse like nicotine and ethanol. In the present study, we classified dopamine (DA) and GABA VTA neurons, and investigated I(A) properties and the physiological role of I(A) in these neurons using conventional whole cell current- and voltage-clamp recording. DA VTA neurons had a mean firing frequency of 3.5 Hz with a long AP duration. GABA VTA neurons had a mean firing frequency of 16.7 Hz with a short AP duration. For I(A) properties, the voltage-dependence of steady-state I(A) activation and inactivation was similar in DA and GABA VTA neurons. I(A) inactivation was significantly faster and became faster at positive voltages in GABA neurons than DA neurons. Recovery from inactivation was significantly faster in DA neurons than GABA neurons. I(A) current density at full recovery was significantly larger in DA neurons than GABA neurons. In DA and GABA VTA neurons, latency to the first AP after the recovery from membrane hyperpolarization (repolarization latency) was measured. Longer repolarization latency was accompanied by larger I(A) current density in DA VTA neurons, compared with GABA VTA neurons. We suggest that I(A) contributes more to the regulation of AP generation in DA VTA neurons than in GABA VTA neurons.

The effects of long chain-length n-alcohols on the firing frequency of dopaminergic neurons of the ventral tegmental area.

The dopaminergic neurons of the ventral tegmental area (DA VTA neurons) have been implicated in the reinforcing properties of drugs of abuse, including ethanol (ethyl alcohol). Ethanol increases the spontaneous firing frequency of DA VTA neurons in vitro, in both brain slices and acutely dissociated neurons, and also in vivo. In many systems, longer n-alkyl alcohols have a more potent effect than ethanol, and the potency is a function of the number of carbons in the alkyl chain. We studied n-alcohols of chain length 1 (methanol) to 5 (pentanol) on the firing rate of DA VTA neurons in brain slice preparations. All of the alcohols studied produced increases in the spontaneous firing frequency in DA VTA neurons; as the chain length increased, lower concentrations of the alcohols were needed to produce the same percentage increase in firing. With very high concentrations of all the alcohols except methanol, we observed apparent depolarization block of firing. In addition, trichloroethanol (TCE), the active metabolite of chloral hydrate, increased the firing frequency of DA VTA neurons, and the EC(40) (concentration to produce a 40% increase in firing rate) of TCE was below that of ethanol. These studies indicate that excitation of VTA dopamine neurons by n-alcohols is related to the chain length of the carbons. This is likely to be a characteristic of the ethanol-sensitive element of DA VTA neurons and may be useful in identifying the element of the membrane that is responsible for ethanol-induced excitation.

Ethanol excitation of dopaminergic ventral tegmental area neurons is blocked by quinidine.

The dopaminergic (DA) neurons in the ventral tegmental area (VTA) are important for the reinforcing effects of ethanol. We have shown that ethanol directly excites DA VTA neurons and reduces the afterhyperpolarization (AHP) that follows spontaneous action potentials in these neurons. These data suggested that ethanol may be increasing the firing rate of DA VTA neurons by modulating currents that contribute to the AHP, either by reducing a K+ current or by increasing the inward current Ih. In the present study, different blockers of K+ channels and Ih were tested to determine whether any could prevent the ethanol excitation of DA VTA neurons. Extracellular single-unit recordings and whole-cell patch-clamp recordings were made from DA VTA neurons in brain slices from Fischer-344 rats and ethanol (40-120 mM) and channel blockers were applied in the bath. Ethanol excitation was not reduced by blockade of Ih with cesium (5 mM) or ZD7288 (30 microM), or by block of G-protein-coupled inwardly rectifying K+ channels with barium (500 microM). Tetraethylammonium (TEA) ion (2-10 mM), which blocks the large conductance calcium-dependent potassium K+ current and some types of delayed rectifier currents, had no effect on the ethanol-induced excitation. Interestingly, ethanol excitation of DA VTA neurons was blocked by quinidine (20-80 microM), a drug that blocks many types of delayed rectifier K+ channels, including some insensitive to TEA. This effect of quinidine was concentration-dependent and reversible. These results suggest that ethanol excites DA VTA neurons by reducing a quinidine-sensitive K+ current.

Serotonin reduces the hyperpolarization-activated current (Ih) in ventral tegmental area dopamine neurons: involvement of 5-HT2 receptors and protein kinase C.

Dopaminergic neurons of the ventral tegmental area (VTA) have been implicated in the rewarding properties of drugs of abuse and in the etiology of schizophrenia; serotonin modulation of these neurons may play a role in these phenomena. Whole cell patch-in-the-slice recording in rat brain slices was used to investigate modulation of the hyperpolarization-activated cationic current Ih by serotonin in these neurons. Serotonin (50-500 microM) reduced the amplitude of Ih in a concentration-dependent manner; this effect was reversible after prolonged washout of serotonin. This effect was mimicked by the 5-HT2 agonist alpha-methylserotonin (25 microM) and reversed by the 5-HT2 antagonist ketanserin (25 microM). Serotonin reduced the maximal Ih current and conductance (measured at -130 mV) and caused a negative shift in the voltage dependence of Ih activation. The serotonin-induced reduction in Ih amplitude was antagonized by intracellular administration of the nonspecific protein kinase inhibitor H-7 (75 microM) and the selective protein kinase C inhibitor chelerythrine (25 microM). The protein kinase C activator phorbol 12, 13 diacetate (PDA, 2 microM) reduced Ih amplitude; when PDA and serotonin were applied together, the effect on Ih was less than additive. These data support the conclusion that serotonin reduces Ih in dopaminergic VTA neurons by acting at serotonin 5-HT2 receptors, which activate protein kinase C. This reduction of Ih may be physiologically important, as the selective inhibitor of Ih, ZD7288, significantly increased dopamine inhibition of firing rate of dopaminergic VTA neurons, an effect that we previously demonstrated with serotonin.