Mark S. Brodie

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.

The effects of cholecystokinin (CCK-8) on dopamine-containing nerve terminals in the caudate nucleus and nucleus accumbens of the anesthetized rat: an in vivo electrochemical study

The action of cholecystokinin (CCK) on presynaptic function of dopaminergic nerve terminals has been the subject of much debate in the literature. In efforts to resolve some of the reported ambiguities, high speed in vivo electrochemical recordings were carried out in the caudate nucleus and nucleus accumbens of the urethane anesthetized rat, to determine effects of locally applied sulfated (CCK-8S) and unsulfated (CCK-8US) CCK octapeptide. Locally-applied CCK-8S and CCK-8US caused no increase in the baseline electrochemical signals recorded from either brain region. However, locally applied CCK-8S potentiated the potassium-evoked overflow of dopamine (DA) into the extracellular space in both the caudate and nucleus accumbens. In contrast, pressure ejection of CCK-8US produced no significant effects on the potassium-evoked overflow of DA in either structure. These data support a facilitatory effect of CCK-8S on potassium-evoked overflow from DA-containing nerve terminals in the urethane anesthetized rat that is likely mediated through a peripheral type CCK receptor.

Enduring Effects of Chronic Ethanol in the CNS: Basis for Alcoholism

This symposium focused on functional alterations in the mesolimbic dopamine system during the abstinence phase after chronic alcohol intake. Mark Brodie first described his recordings from midbrain slices prepared after chronic alcohol treatment in vivo by daily injection in C57BL/6J mice. No changes were found in the baseline firing frequency of dopaminergic neurones in the VTA (ventral tegmental area), but the excitation produced in these neurones by an acute ethanol challenge was significantly increased in neurons from ethanol-treated mice compared with those from the saline-treated controls. There was also a significant decrease in the inhibitory response to GABA by the dopamine neurones following the chronic ethanol treatment. These data suggest that the timing pattern and mode of ethanol administration may determine the types of changes observed in dopaminergic reward area neurons. Annalisa Muntoni lectured on the relationship between electrophysiological and biochemical in vivo evidence supporting a reduction in tonic activity of dopamine neurons projecting to the nucleus accumbens at various times after suspension of chronic ethanol treatment and morphological changes affecting dopamine neurons in rat VTA. Hilary J. Little then described changes in dopaminergic neurone function in the VTA during the abstinence phase. Decreases in baseline firing were seen at 6 days after withdrawal of mice from chronic ethanol treatment but were not apparent after 2 months abstinence. Increases in the affinity of D1 receptors in the striatum, but not in the cerebral cortex, were seen however up to 2 months after withdrawal. Scott Steffensen then described his studies recording in vivo from GABA containing neurones in the VTA in freely moving rats. Chronic ethanol administration enhanced the baseline activity of these neurones and resulted in tolerance to the inhibition by ethanol of these neurones. His results demonstrated selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA-GABA neurone activity.

Antinociception induced by local injections of carbachol into the nucleus raphe magnus in rats: Alteration by intrathecal injection of monoaminergic antagonists

Electrical stimulation of neurons located in the nucleus raphe magnus (NRM) produces antinociception which appears to result from inhibition of spinothalamic tract neurons located in the spinal cord dorsal horn. Iontophoretic application of acetylcholine also activates NRM neurons and microinjection of cholinergic agonists such as carbachol into the NRM produces a profound, long-lasting antinociception. Since the antinociception induced by electrical stimulation of NRM neurons is mediated, at least in part, by bulbospinal serotonergic and noradrenergic neurons, the role of these monoaminergic neurons in mediating the antinociception induced by microinjecting carbachol in the NRM was examined in the present study. To this end, various antagonists of serotonin and norepinephrine were injected into the spinal cord subarachnoid space following the induction of antinociception by the local injection of carbachol into the NRM. The serotonergic antagonist methysergide had no effect on carbachol-induced antinociception. However, the alpha 2-noradrenergic antagonist yohimbine attenuated, while the alpha 1-noradrenergic antagonists prazosin and WB4101 increased the effects of carbachol. The non-selective noradrenergic antagonist phentolamine also attenuated the effects of carbachol. These results lead to the suggestion that the antinociception induced by the local injection of carbachol into the NRM is mediated by selective activation of bulbospinal noradrenergic neurons. Furthermore, the antinociception resulting from the activation of these descending noradrenergic neurons appears to be mediated by alpha 2-noradrenergic receptors located in the spinal cord dorsal horn. Finally, the local injection of carbachol into the NRM also appears to activate another population of noradrenergic neurons which produces hyperalgesia mediated by alpha 1-noradrenergic receptors.

Ethanol Effects on Dopaminergic Ventral Tegmental Area Neurons During Block of Ih: Involvement of Barium-Sensitive Potassium Currents

The dopaminergic neurons of the ventral tegmental area (DA VTA neurons) are important for the rewarding and reinforcing properties of drugs of abuse, including ethanol. Ethanol increases the firing frequency of DA VTA neurons from rats and mice. Because of a recent report on block of ethanol excitation in mouse DA VTA neurons with ZD7288, a selective blocker of the hyperpolarization-activated cationic current Ih, we examined the effect of ZD7288 on ethanol excitation in DA VTA neurons from C57Bl/6J and DBA/2J mice and Fisher 344 rats. Ethanol (80 mM) caused only increases in firing rate in mouse DA VTA neurons in the absence of ZD7288, but in the presence of ZD7288 (30 microM), ethanol produced a more transient excitation followed by a decrease of firing. This same biphasic phenomenon was observed in DA VTA neurons from rats in the presence of ZD7288 only at very high ethanol concentrations (160-240 mM) but not at lower pharmacologically relevant concentrations. The longer latency ethanol-induced inhibition was not observed in DA VTA neurons from mice or rats in the presence of barium (100 microM), which blocks G protein-linked potassium channels (GIRKs) and other inwardly rectifying potassium channels. Ethanol may have a direct effect to increase an inhibitory potassium conductance, but this effect of ethanol can only decrease the firing rate if Ih is blocked.

Regulation of L-Type Calcium Channel and Delayed Rectifier Potassium Channel Activity by p21-Activated Kinase-1 in Guinea Pig Sinoatrial Node Pacemaker Cells

Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p21-activated kinase-1 (Pak1), a serine–threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca2+ current (ICaL) and delayed rectifier potassium current (IK) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of ICaL and IK; (3) inhibition of protein phosphatase 2A increases the enhancement of IK and ICaL by isoproterenol in Ad-Pak1–infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.

Ethanol and acetaldehyde action on central dopamine systems: Mechanisms, modulation and relationship to stress

There has been a great deal of activity in recent years in the study of the direct effects of ethanol on the dopamine reward system originating in the ventral tegmental area (VTA). In addition, recent evidence suggests that acetaldehyde formed from ethanol in the brain or periphery may be a crucial factor in the central effects of ethanol. This critical review examines the actions of ethanol and acetaldehyde on neurons of the VTA and the possible interactions with stress, with a focus on electrophysiological studies in vivo and in vitro. Ethanol has specific effects on dopamine neurons and there is recent evidence that some of the in vivo and in vitro effects of ethanol are mediated by acetaldehyde. Stress has some analogous actions on neuronal activity in the VTA, and the interactions between the effects of stress and alcohol on VTA neurons may be a factor in ethanol-seeking behavior. Taken together, the evidence suggests that stress may contribute to the activating effects of ethanol on dopamine VTA neurons, that at least some actions of ethanol on dopamine VTA neurons are mediated by acetaldehyde, and that the interaction between stress and alcohol could play a role in susceptibility to alcoholism. The link between acetaldehyde and ethanol actions on brain reward pathways may provide a new avenue for the development of agents to reduce alcohol craving.

Hyposensitivity to Gamma-Aminobutyric Acid in the Ventral Tegmental Area During Alcohol Withdrawal: Reversal by Histone Deacetylase Inhibitors

Putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons have an important role in alcohol addiction. Acute ethanol increases the activity of pDAergic neurons, and withdrawal from repeated ethanol administration produces a decreased sensitivity of pDAergic VTA neurons to GABA. Recent studies show that behavioral changes induced by chronic alcohol are reversed by inhibitors of histone deacetylases (HDACs). Whether HDAC-induced histone modifications regulate changes in GABA sensitivity of VTA pDAergic neurons during withdrawal is unknown. Here, we investigated modulation of withdrawal-induced changes in GABA sensitivity of pDAergic VTA neurons by HDAC inhibitors (HDACi), and also measured the levels of HDAC2, histone (H3-K9) acetylation, and GABA-Aα1 receptor (GABA (A-α1) R) subunit in VTA during ethanol withdrawal. Mice were injected intraperitoneally (ip) with either ethanol (3.5 g/kg) or saline twice daily for 3 weeks. In recordings from pDAergic VTA neurons in brain slices from ethanol-withdrawn mice, sensitivity to GABA (50–500 μM) was reduced. In brain slices from ethanol-withdrawn mice incubated with the HDACi SAHA (vorinostat) or trichostatin A (TSA) for 2 h, the hyposensitivity of pDAergic VTA neurons to GABA was significantly attenuated. There was no effect of TSA or SAHA on GABA sensitivity of pDAergic VTA neurons from saline-treated mice. In addition, ethanol withdrawal was associated with an increase in levels of HDAC2 and a decrease in histone (H3-K9) acetylation and levels of GABA (A-α1) R subunits in the VTA. Therefore, blockade of upregulation of HDAC2 by HDACi normalizes GABA hyposensitivity of pDAergic neurons developed during withdrawal after chronic ethanol treatment, which suggests the possibility that inhibition of HDACs can reverse ethanol-induced neuroadaptational changes in reward circuitry.

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.

Reversal of Prolonged Dopamine Inhibition of Dopaminergic Neurons of the Ventral Tegmental Area

Drug abuse-induced plasticity of putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons may play an important role in changes in the mesocorticolimbic system that lead to the development of addiction. In the present study, extracellular recordings were used to examine time-dependent effects of dopamine (DA) on pDAergic VTA neurons in rat brain slices. Administration of DA (2.5–10 μM) for 40 min resulted in inhibition followed by partial or full reversal of that inhibition. The reduced sensitivity to DA inhibition lasted 30 to 90 min after washout of the long-term dopamine administration. The inhibition reversal was not observed with 40-min administration of the D2 agonist quinpirole (25–200 nM), so this phenomenon was not the result of desensitization induced solely by stimulation of D2 DA receptors. Inhibition reversal could be observed with the coapplication of quinpirole and the D1/D5 agonist SKF38393 [(±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide], suggesting a D1/D5 mechanism for the reversal. Furthermore, D1/D5 antagonists, given in the presence of prolonged DA exposure, prevented the inhibition reversal. Application of 3 μM quinpirole caused desensitization to low quinpirole concentrations that was blocked by a D1/D5 antagonist. These data suggest that coactivation of D1/D5 receptors and D2 receptors in the VTA results in desensitization of autoinhibitory D2 receptors. Prolonged increases in pDAergic tone in the VTA that may occur in vivo with drugs of abuse could reduce the regulation of firing by D2 dopamine receptor activation, producing long-term alteration in information processing related to reward and reinforcement.