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.

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.

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.

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.

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.