Sex-dependent differences in ethanol inhibition of mouse lateral orbitofrontal cortex neurons.

Biological differences between males and females likely influence responses to alcohol and the propensity to engage in excessive drinking. In both humans and rodents, females escalate alcohol use and develop addiction-like behaviors faster than males, while males exhibit more severe withdrawal symptoms during abstinence. The mechanisms underlying these differences are not yet known but may reflect fundamental differences in the ethanol sensitivity of neurons in reward and control areas of the brain. To address this question, we recorded current-evoked spiking of lateral orbitofrontal cortex (lOFC) neurons in male and female C57BL/6J mice following acute and chronic exposure to ethanol. Ethanol (11-66 mM) reduced firing of lOFC neurons but produced less inhibition in neurons from female mice. As previously reported for male mice, the glycine receptor blocker strychnine blocked ethanol inhibition of spiking of lOFC neurons from female mice and prevented the ethanol-induced increase in tonic current. Following chronic intermittent ethanol (CIE) exposure, current-evoked spiking of lOFC neurons was significantly enhanced with a greater effect observed in males. After CIE treatment, acute ethanol had no effect on spiking in neurons from male mice, while it produced a slight but significant decrease in firing in females. Finally, like male mice, the inhibitory effect of the glycine transport inhibitor sarcosine was blunted in CIE-exposed female mice. Together, these results suggest that while lOFC neurons in male and female mice are similarly affected by ethanol, there are significant differences in sensitivity that may contribute to differences in alcohol actions between males and females.

Effects of monoamines on the intrinsic excitability of lateral orbitofrontal cortex neurons in alcohol-dependent and non-dependent female mice.

Changes in brain reward and control systems of frontal cortical areas including the orbitofrontal cortex (OFC) are associated with alcohol use disorders (AUD). The OFC is extensively innervated by monoamines, and drugs that target monoamine receptors have been used to treat a number of neuropsychiatric diseases, including AUDs. Recent findings from this laboratory demonstrate that D2, α2-adrenergic and 5HT1A receptors all decrease the intrinsic excitability of lateral OFC (lOFC) neurons in naïve male mice and that this effect is lost in mice exposed to repeated cycles of chronic intermittent ethanol (CIE) vapor. As biological sex differences may influence an individual's response to alcohol and contribute to the propensity to engage in addictive behaviors, we examined whether monoamines have similar effects on lOFC neurons in control and CIE exposed female mice. Dopamine, norepinephrine and serotonin all decreased spiking of lOFC neurons in naïve females via activation of Giα-coupled D2, α2-adrenergic and 5HT1A receptors, respectively. Firing was also inhibited by the direct GIRK channel activator ML297, while blocking these channels with barium eliminated the inhibitory actions of monoamines. Following CIE treatment, evoked spiking of lOFC neurons from female mice was significantly enhanced and monoamines and ML297 no longer inhibited firing. Unlike in male mice, the enhanced firing of neurons from CIE exposed female mice was not associated with changes in the after-hyperpolarization and the small-conductance potassium channel blocker apamin had no effect on current-evoked tail currents from either control or CIE exposed female mice. These results suggest that while CIE exposure alters monoamine regulation of OFC neuron firing similarly in males and female mice, there are sex-dependent differences in processes that regulate the intrinsic excitability of these neurons.

Chronic Alcohol, Intrinsic Excitability, and Potassium Channels: Neuroadaptations and Drinking Behavior.

Neural mechanisms underlying alcohol use disorder remain elusive, and this lack of understanding has slowed the development of efficacious treatment strategies for reducing relapse rates and prolonging abstinence. While synaptic adaptations produced by chronic alcohol exposure have been extensively characterized in a variety of brain regions, changes in intrinsic excitability of critical projection neurons are understudied. Accumulating evidence suggests that prolonged alcohol drinking and alcohol dependence produce plasticity of intrinsic excitability as measured by changes in evoked action potential firing and after-hyperpolarization amplitude. In this chapter, we describe functional changes in cell firing of projection neurons after long-term alcohol exposure that occur across species and in multiple brain regions. Adaptations in calcium-activated (KCa2), voltage-dependent (KV7), and G protein-coupled inwardly rectifying (Kir3 or GIRK) potassium channels that regulate the evoked firing and after-hyperpolarization parallel functional changes in intrinsic excitability induced by chronic alcohol. Moreover, there are strong genetic links between alcohol-related behaviors and genes encoding KCa2, KV7, and GIRK channels, and pharmacologically targeting these channels reduces alcohol consumption and alcohol-related behaviors. Together, these studies demonstrate that chronic alcohol drinking produces adaptations in KCa2, KV7, and GIRK channels leading to impaired regulation of the after-hyperpolarization and aberrant cell firing. Correcting the deficit in the after-hyperpolarization with positive modulators of KCa2 and KV7 channels and altering the GIRK channel binding pocket to block the access of alcohol represent a potentially highly effective pharmacological approach that can restore changes in intrinsic excitability and reduce alcohol consumption in affected individuals.

Correction to: Chronic Alcohol, Intrinsic Excitability, and Potassium Channels: Neuroadaptations and Drinking Behavior.

In section 8.1 on the 10th line in first paragraph the reference citation Mateos-Aparicio et al. 2014 is incorrect.

Orbitofrontal Neuroadaptations and Cross-Species Synaptic Biomarkers in Heavy-Drinking Macaques.

Cognitive impairments, uncontrolled drinking, and neuropathological cortical changes characterize alcohol use disorder. Dysfunction of the orbitofrontal cortex (OFC), a critical cortical subregion that controls learning, decision-making, and prediction of reward outcomes, contributes to executive cognitive function deficits in alcoholic individuals. Electrophysiological and quantitative synaptomics techniques were used to test the hypothesis that heavy drinking produces neuroadaptations in the macaque OFC. Integrative bioinformatics and reverse genetic approaches were used to identify and validate synaptic proteins with novel links to heavy drinking in BXD mice. In drinking monkeys, evoked firing of OFC pyramidal neurons was reduced, whereas the amplitude and frequency of postsynaptic currents were enhanced compared with controls. Bath application of alcohol reduced evoked firing in neurons from control monkeys, but not drinking monkeys. Profiling of the OFC synaptome identified alcohol-sensitive proteins that control glutamate release (e.g., SV2A, synaptogyrin-1) and postsynaptic signaling (e.g., GluA1, PRRT2) with no changes in synaptic GABAergic proteins. Western blot analysis confirmed the increase in GluA1 expression in drinking monkeys. An exploratory analysis of the OFC synaptome found cross-species genetic links to alcohol intake in discrete proteins (e.g., C2CD2L, DIRAS2) that discriminated between low- and heavy-drinking monkeys. Validation studies revealed that BXD mouse strains with the D allele at the C2cd2l interval drank less alcohol than B allele strains. Thus, by profiling of the OFC synaptome, we identified changes in proteins controlling glutamate release and postsynaptic signaling and discovered several proteins related to heavy drinking that have potential as novel targets for treating alcohol use disorder.SIGNIFICANCE STATEMENT Clinical research identified cognitive deficits in alcoholic individuals as a risk factor for relapse, and alcoholic individuals display deficits on cognitive tasks that are dependent upon the orbitofrontal cortex (OFC). To identify neurobiological mechanisms that underpin OFC dysfunction, this study used electrophysiology and integrative synaptomics in a translational nonhuman primate model of heavy alcohol consumption. We found adaptations in synaptic proteins that control glutamatergic signaling in chronically drinking monkeys. Our functional genomic exploratory analyses identified proteins with genetic links to alcohol and cocaine intake across mice, monkeys, and humans. Future work is necessary to determine whether targeting these novel targets reduces excessive and harmful levels of alcohol drinking.

Ethanol Dependence Abolishes Monoamine and GIRK (Kir3) Channel Inhibition of Orbitofrontal Cortex Excitability.

Alcohol abuse disorders are associated with dysfunction of frontal cortical areas including the orbitofrontal cortex (OFC). The OFC is extensively innervated by monoamines, and drugs that target monoamine receptors have been used to treat a number of neuropsychiatric diseases, including alcoholism. However, little is known regarding how monoamines affect OFC neuron excitability or whether this modulation is altered by chronic exposure to ethanol. In this study, we examined the effect of dopamine, norepinephrine, and serotonin on lOFC neuronal excitability in naive mice and in those exposed to chronic intermittent ethanol (CIE) treatment. All three monoamines decreased current-evoked spike firing of lOFC neurons and this action required Giα-coupled D2, α2-adrenergic, and 5HT1A receptors, respectively. Inhibition of firing by dopamine or the D2 agonist quinpirole, but not norepinephrine or serotonin, was prevented by the GABAA receptor antagonist picrotoxin. GABA-mediated tonic current was enhanced by dopamine or the D1 agonist SKF81297 but not quinpirole, whereas the amplitude of spontaneous IPSCs was increased by quinpirole but not dopamine. Spiking was also inhibited by the direct GIRK channel activator ML297, whereas blocking these channels with barium increased firing and eliminated the inhibitory actions of monoamines. In the presence of ML297 or the G-protein blocker GDP-ß-S, DA induced a further decrease in spike firing, suggesting the involvement of a non-GIRK channel mechanism. In neurons from CIE-treated mice, spike frequency was nearly doubled and inhibition of firing by monoamines or ML297 was lost. These effects occurred in the absence of significant changes in expression of Gi/o or GIRK channel proteins. Together, these findings show that monoamines are important modulators of lOFC excitability and suggest that disruption of this process could contribute to various deficits associated with alcohol dependence.

Differential Effects of Toluene and Ethanol on Dopaminergic Neurons of the Ventral Tegmental Area.

Drugs of abuse increase the activity of dopaminergic neurons of the ventral tegmental area (VTA), and output from the VTA is critical for both natural and drug-induced reward and reinforcement. Ethanol and the abused inhalant toluene both enhance VTA neuronal firing, but the mechanisms of this effect is not fully known. In this study, we used extracellular recordings to compare the actions of toluene and ethanol on DA VTA neurons. Both ethanol and toluene increased the firing rate of DA neurons, although toluene was ~100 times more potent than ethanol. The mixed ion channel blocker quinine (100 µM) blocked the increases in firing produced by ethanol and toluene, indicating some similarity in mechanisms of excitation. A mixture of antagonists of GABA and cholinergic receptors did not prevent toluene-induced or ethanol-induced excitation, and toluene-induced excitation was not altered by co-administration of ethanol, suggesting independent mechanisms of excitation for ethanol and toluene. Concurrent blockade of NMDA, AMPA, and metabotropic glutamate receptors enhanced the excitatory effect of toluene while having no significant effect on ethanol excitation. Nicotine increased firing of DA VTA neurons, and this was blocked by the nicotinic antagonist mecamylamine (1 µM). Mecamylamine did not alter ethanol or toluene excitation of firing but the muscarinic antagonist atropine (5 µM) or a combination of GABA antagonists (bicuculline and CGP35348, 10 µM each) reduced toluene-induced excitation without affecting ethanol excitation. The Ih current blocker ZD7288 abolished the excitatory effect of toluene but unlike the block of ethanol excitation, the effect of ZD7288 was not reversed by the GIRK channel blocker barium, but was reversed by GABA antagonists. These results demonstrate that the excitatory effects of ethanol and toluene have some similarity, such as block by quinine and ZD7288, but also indicate that there are important differences between these two drugs in their modulation by glutamatergic, cholinergic, and GABAergic receptors. These findings provide important information regarding the actions of abused inhalants on central reward pathways, and suggest that regulation of the activation of central dopamine pathways by ethanol and toluene partially overlap.

Inactivation of the lateral orbitofrontal cortex increases drinking in ethanol-dependent but not non-dependent mice.

Long-term consumption of ethanol affects cortical areas that are important for learning and memory, cognition, and decision-making. Deficits in cortical function may contribute to alcohol-abuse disorders by impeding an individual's ability to control drinking. Previous studies from this laboratory show that acute ethanol reduces activity of lateral orbitofrontal cortex (LOFC) neurons while chronic exposure impairs LOFC-dependent reversal learning and induces changes in LOFC excitability. Despite these findings, the role of LOFC neurons in ethanol consumption is unknown. To address this issue, we examined ethanol drinking in adult C57Bl/6J mice that received an excitotoxic lesion or viral injection of the inhibitory DREADD (designer receptor exclusively activated by designer drug) into the LOFC. No differences in ethanol consumption were observed between sham and lesioned mice during access to increasing concentrations of ethanol (3-40%) every other day for 7 weeks. Adulterating the ethanol solution with saccharin (0.2%) or quinine (0.06 mM) enhanced or inhibited, respectively, consumption of the 40% ethanol solution similarly in both groups. Using a chronic intermittent ethanol (CIE) vapor exposure model that produces dependence, we found no difference in baseline drinking between sham and lesioned mice prior to vapor treatments. CIE enhanced drinking in both groups as compared to air-treated animals and CIE treated lesioned mice showed an additional increase in ethanol drinking as compared to CIE sham controls. This effect persisted during the first week when quinine was added to the ethanol solution but consumption decreased to control levels in CIE lesioned mice in the following 2 weeks. In viral injected mice, baseline drinking was not altered by expression of the inhibitory DREADD receptor and repeated cycles of CIE exposure enhanced drinking in DREADD and virus control groups. Consistent with the lesion study, treatment with clozapine-N-oxide (CNO) further enhanced consumption only in CIE exposed DREADD mice with no change in air-treated mice. These results suggest that the LOFC is not critical for the initiation and maintenance of ethanol drinking in non-dependent mice, but may regulate the escalated drinking observed during dependence.

Chronic Intermittent Ethanol Exposure Enhances the Excitability and Synaptic Plasticity of Lateral Orbitofrontal Cortex Neurons and Induces a Tolerance to the Acute Inhibitory Actions of Ethanol.

Alcoholism is associated with changes in brain reward and control systems, including the prefrontal cortex. In prefrontal areas, the orbitofrontal cortex (OFC) has been suggested to have an important role in the development of alcohol-abuse disorders and studies from this laboratory demonstrate that OFC-mediated behaviors are impaired in alcohol-dependent animals. However, it is not known whether chronic alcohol (ethanol) exposure alters the fundamental properties of OFC neurons. In this study, mice were exposed to repeated cycles of chronic intermittent ethanol (CIE) exposure to induce dependence and whole-cell patch-clamp electrophysiology was used to examine the effects of CIE treatment on lateral OFC (lOFC) neuron excitability, synaptic transmission, and plasticity. Repeated cycles of CIE exposure and withdrawal enhanced current-evoked action potential (AP) spiking and this was accompanied by a reduction in the after-hyperpolarization and a decrease in the functional activity of SK channels. CIE mice also showed an increase in the AMPA/NMDA ratio, and this was associated with an increase in GluA1/GluA2 AMPA receptor expression and a decrease in GluN2B NMDA receptor subunits. Following CIE treatment, lOFC neurons displayed a persistent long-term potentiation of glutamatergic synaptic transmission following a spike-timing-dependent protocol. Lastly, CIE treatment diminished the inhibitory effect of acute ethanol on AP spiking of lOFC neurons and reduced expression of the GlyT1 transporter. Taken together, these results suggest that chronic exposure to ethanol leads to enhanced intrinsic excitability and glutamatergic synaptic signaling of lOFC neurons. These alterations may contribute to the impairment of OFC-dependent behaviors in alcohol-dependent individuals.

KCNN Genes that Encode Small-Conductance Ca2+-Activated K+ Channels Influence Alcohol and Drug Addiction.

Small-conductance Ca(2+)-activated K(+) (KCa2) channels control neuronal excitability and synaptic plasticity, and have been implicated in substance abuse. However, it is unknown if genes that encode KCa2 channels (KCNN1-3) influence alcohol and drug addiction. In the present study, an integrative functional genomics approach shows that genetic datasets for alcohol, nicotine, and illicit drugs contain the family of KCNN genes. Alcohol preference and dependence QTLs contain KCNN2 and KCNN3, and Kcnn3 transcript levels in the nucleus accumbens (NAc) of genetically diverse BXD strains of mice predicted voluntary alcohol consumption. Transcript levels of Kcnn3 in the NAc negatively correlated with alcohol intake levels in BXD strains, and alcohol dependence enhanced the strength of this association. Microinjections of the KCa2 channel inhibitor apamin into the NAc increased alcohol intake in control C57BL/6J mice, while spontaneous seizures developed in alcohol-dependent mice following apamin injection. Consistent with this finding, alcohol dependence enhanced the intrinsic excitability of medium spiny neurons in the NAc core and reduced the function and protein expression of KCa2 channels in the NAc. Altogether, these data implicate the family of KCNN genes in alcohol, nicotine, and drug addiction, and identify KCNN3 as a mediator of voluntary and excessive alcohol consumption. KCa2.3 channels represent a promising novel target in the pharmacogenetic treatment of alcohol and drug addiction.

Dopamine D2 receptor desensitization by dopamine or corticotropin releasing factor in ventral tegmental area neurons is associated with increased glutamate release.

Neurons of the ventral tegmental area (VTA) are the source of dopaminergic (DAergic) input to important brain regions related to addiction. Prolonged exposure of these VTA neurons to moderate concentrations of dopamine (DA) causes a time-dependent decrease in DA-induced inhibition, a complex desensitization called DA inhibition reversal (DIR). DIR is mediated by conventional protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of some Gq-linked receptors. Corticotropin releasing factor (CRF) acts via Gq, and can modulate glutamater neurotransmission in the VTA. In the present study, we used brain slice electrophysiology to characterize the interaction of DA, glutamate antagonists, and CRF agonists in the induction and maintenance of DIR in the VTA. Glutamate receptor antagonists blocked induction but not maintenance of DIR. Putative blockers of neurotransmitter release and store-operated calcium channels blocked and reversed DIR. CRF and the CRF agonist urocortin reversed inhibition produced by the D2 agonist quinpirole, consistent with our earlier work indicating that Gq activation reverses quinpirole-mediated inhibition. In whole cell recordings, the combination of urocortin and quinpirole, but not either agent alone, increased spontaneous excitatory postsynaptic currents (sEPSCs) in VTA neurons. Likewise, the combination of a D1-like receptor agonist and quinpirole, but not either agent alone, increased sEPSCs in VTA neurons. In summary, desensitization of D2 receptors induced by dopamine or CRF on DAergic VTA neurons is associated with increased glutamatergic signaling in the VTA.

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.

Suppression of Gq Function Using Intra-Pipette Delivery of shRNA during Extracellular Recording in the Ventral Tegmental Area.

Selective suppression of protein function in the brain can be achieved using specific silencing RNAs administered in vivo. A viral delivery system is often employed to transfect neurons with small hairpin RNA (shRNA) directed against specific proteins, and intervals of several days are allowed between microinjection of the shRNA-containing virus into the brain and experiments to assess suppression of gene function. Here we report studies using extracellular recording of dopaminergic neurons of the ventral tegmental area (DA VTA neurons) recorded in brain slices in which lentivirus containing shRNA directed against Gq was included in the recording pipette, and suppression of Gq-related function was observed within the time frame of the recording. The action of neurotensin (NT) is associated with activation of Gq, and the firing rate of DA VTA neurons is increased by NT. With shRNA directed against Gq in the pipette, there was a significant reduction of NT excitation within 2 h. Likewise, time-dependent dopamine desensitization, which we have hypothesized to be Gq-dependent, was not observed when shRNA directed against Gq was present in the pipette and dopamine was tested 2 h after initiation of recording. As the time interval (2 h) is relatively short, we tested whether blockade of protein synthesis with cycloheximide delivered via the recording pipette would alter Gq-linked responses similarly. Both NT-induced excitation and dopamine desensitization were inhibited in the presence of cycloheximide. Inclusion of shRNA in the recording pipette may be an efficient and selective way to dampen responses linked to Gq, and, more generally, the use of lentiviral-packaged shRNA in the recording pipette is a means to produce selective inhibition of the function of specific proteins in experiments.

Reversal of dopamine D2 agonist-induced inhibition of ventral tegmental area neurons by Gq-linked neurotransmitters is dependent on protein kinase C, G protein-coupled receptor kinase, and dynamin.

Dopaminergic neurons of the ventral tegmental area are important components of brain pathways related to addiction. Prolonged exposure of these neurons to moderate concentrations of dopamine (DA) decreases their sensitivity to inhibition by DA, a process called DA-inhibition reversal (DIR). DIR is mediated by phospholipase C and conventional subtype of protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of 5-HT(2) or neurotensin receptors. In the present study, we further characterized this phenomenon by use of extracellular recordings in brain slices to examine whether DIR is linked to G protein-coupled receptor kinase-2 (GRK2) or dynamin by assessing DIR in the presence of antagonists of these enzymes. DIR was blocked by ß-ARK1 inhibitor, which inhibits GRK2, and by dynasore, which blocks dynamin. Reversal of inhibition by D2 agonist quinpirole was produced by serotonin (50 µM) and by neurotensin (5-10 nM). Serotonin-induced or neurotensin-induced reversal was blocked by ß-ARK1 inhibitor, dynasore, or cPKC antagonist 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4c]carbazole-12-propanenitrile (Gö6976). This further characterization of DIR indicates that cPKC, GRK2, and dynamin play important roles in the desensitization of D2 receptors. As drugs of abuse produce persistent increases in DA concentration in the ventral tegmental area, reduction of D2 receptor sensitivity as a result of drug abuse may be a critical factor in the processes of addiction.

Phorbol ester reduces ethanol excitation of dopaminergic neurons of the ventral tegmental area: involvement of protein kinase C theta.

Neurons of the ventral tegmental area (VTA) play a key role in the rewarding and reinforcing effects of drugs of abuse, including alcohol. Ethanol directly increases the firing rate of dopaminergic (DAergic) VTA neurons, but modulation of the firing rate of DAergic VTA neurons can be controlled by a number of factors, including some that are under the control of protein kinase C (PKC). Application of phorbol esters activates PKC and the present study assessed the effect of a phorbol ester, phorbol 12-myristate 13-acetate (PMA), on ethanol-induced excitation of DA VTA neurons. Ethanol-induced excitation of DAergic VTA neurons was reduced significantly in the presence of PMA. This action of PMA was antagonized by chelerythrine chloride, a non-selective antagonist of PKC, but not by moderate concentrations of antagonists of conventional PKC isoforms (Gö6976 and Gö6983). A PKC δ/θ inhibitor antagonized PMA-induced reduction of ethanol excitation. Since PKCδ antagonist Gö6983 did not antagonize the effect of PMA on ethanol excitation, the PMA reduction of ethanol excitation is most likely to be mediated by PKCθ. Antagonists of intracellular calcium pathways were ineffective in antagonizing PMA action on ethanol excitation, consistent with the lack of calcium dependence of PKCθ. In summary, ethanol-induced excitation of VTA neurons is attenuated in the presence of PMA, and this attenuation appears to be mediated by PKCθ. This novel mechanism for interfering with ethanol activation of reward-related neurons could provide a new target for pharmacotherapy to ameliorate alcoholism.

Ethanol blocks the reversal of prolonged dopamine inhibition of dopaminergic neurons of the ventral tegmental area.

BACKGROUND: Dopaminergic (DAergic) neurons of the ventral tegmental area (VTA) are important for the rewarding and reinforcing properties of alcohol and other drugs of abuse. Regulation of the firing of DAergic VTA neurons is controlled by a number of factors, including autoregulation of firing by D2 dopamine (DA) receptors. The inhibitory effects of DA on these neurons exhibit concentration- and time-dependent desensitization, which we have termed dopamine inhibition reversal (DIR), as it requires concurrent stimulation of D1/D5 and D2 receptors. METHODS: Extracellular recording of DAergic VTA neurons in brain slices was used to test the effects of ethanol (EtOH) (10 to 80 mM) on DIR. RESULTS: DIR was reduced by concentrations of EtOH as low as 10 mM and was blocked by higher EtOH concentrations. In addition, as we have shown that reversal of inhibition by the selective D2 agonist quinpirole can be observed in the presence of an activator of protein kinase C (PKC), we tested whether EtOH could antagonize the reversal of quinpirole inhibition in the presence of phorbol 12-myristate 13-acetate (PMA). EtOH (80 mM) blocked the reversal of quinpirole seen in the presence of PMA, suggesting that the antagonism of DIR by EtOH is owing to an action at a stage in the mechanism at or distal to PKC. Once achieved, DIR is not antagonized by EtOH. CONCLUSIONS: The blockade by relatively low concentrations of EtOH of DIR may play an important role in the spectrum of action of EtOH on DAergic neurons of the VTA and may be important in the acute and chronic actions of EtOH on the excitability of these brain reward/reinforcement neurons.

Reversal of quinpirole inhibition of ventral tegmental area neurons is linked to the phosphatidylinositol system and is induced by agonists linked to G(q).

Putative dopaminergic (pDAergic) ventral tegmental area neurons play an important role in brain pathways related to addiction. Extended exposure of pDAergic neurons to moderate concentrations of dopamine (DA) results in a time-dependent decrease in sensitivity of pDAergic neurons to DA inhibition, a process called dopamine inhibition reversal (DIR). We have shown that DIR is mediated by phospholipase C and conventional protein kinase C through concurrent stimulation of D2 and D1-like receptors. In the present study, we further characterized this phenomenon by using extracellular recordings in brain slices to examine whether DIR is linked to phosphatidylinositol (PI) or adenylate cyclase (AC) second-messenger pathways. A D1-like dopaminergic agonist associated with PI turnover (SKF83959), but not one linked to AC (SKF83822), promoted reversal of inhibition produced by quinpirole, a dopamine D2-selective agonist. Other neurotransmitter receptors linked to PI turnover include serotonin 5-HT(2), α(1)-adrenergic, neurotensin, and group I metabotropic glutamate (mGlu) receptors. Both serotonin and neurotensin produced significant reversal of quinpirole inhibition, but agonists of α(1)-adrenergic and group I mGlu receptors failed to significantly reverse quinpirole inhibition. These results indicate that some agonists that stimulate PI turnover can facilitate desensitization of D2 receptors but that there may be other factors in addition to PI that control that interaction.

Reversal of dopamine inhibition of dopaminergic neurons of the ventral tegmental area is mediated by protein kinase C.

Adaptation of putative dopaminergic (pDA) neurons in the ventral tegmental area (VTA) to drugs of abuse may alter information processing related to reward and reinforcement and is an important factor in the development of addiction. We have demonstrated that prolonged increases in the concentration of dopamine (DA) result in a time-dependent decrease in sensitivity of pDA neurons to DA, which we termed DA inhibition reversal (DIR). In this study, we used extracellular recordings to examine factors mediating DIR. A 40 min administration of DA (2.5-10 µM), but not the DA D2 receptor agonist quinpirole (50-200 nM), resulted in inhibition of neuronal firing followed by DIR. In the presence of 100 nM cocaine, inhibition followed by DIR was seen with much lower DA concentrations. Reversal of quinpirole inhibition could be induced by an activator of protein kinase C, but not of protein kinase A. Inhibitors of protein kinase C or phospholipase C blocked the development of DIR. Disruption of intracellular calcium release also prevented DIR. Reduction of extracellular calcium or inhibition of store-operated calcium entry blocked DIR, but the L-type calcium channel blocker nifedipine did not. DIR was age-dependent and not seen in pDA VTA neurons from rat pups younger than 15 days postnatally. Our data indicate that DIR is mediated by protein kinase C, and implicate a conventional protein kinase C. This characterization of DIR gives insight into the regulation of autoinhibition of pDA VTA neurons, and the resulting long-term alteration in information processing related to reward and reinforcement.

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 microM) 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 microM 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.