VTA DA Research Articles

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.

Parthenolide Blocks Cocaines Effect on Spontaneous Firing Activity of Dopaminergic Neurons in the Ventral Tegmental Area

Chronic cocaine administration leads to catecholamine reuptake inhibition which enhances reward and motivational behaviors. Ventral Tegmental Area dopaminergic (VTA DA) neuronal firing is associated with changes in reward predictive signals. Acute cocaine injections inhibit putative VTA DA cell firing in vertebrates. Parthenolide, a compound isolated from the feverfew plant (Tanacetum parthenium), has been shown to substantially inhibit cocaine’s locomotion effects in a planarian animal model (Pagán et al., 2008). Here we investigated the effects of parthenolide on the spontaneous firing activity of putative VTA DA neurons in anesthetized male rats (250-300g). Single-unit recordings were analyzed after intravenous (i.v.) parthenolide administration followed by 1mg/kg i.v. cocaine injection. Results showed that parthenolide at 0.125 mg/kg and 0.250mg/kg significantly blocked cocaine’s inhibitory effect on DA neuronal firing rate and bursting activity (p< 0.05, two way ANOVA). We propose that parthenolide might inhibit cocaine’s effects on VTA DA neurons via its interaction with a common binding site at monoamine transporters. It is suggested that parthenolide could have a potential use as an overdose antidote or therapeutic agent to cocaine intoxication.

P.1.c.059 Electrophysiological properties of the triple reuptake inhibitor SEP 225289 on monoaminergic neurons

SEP 225289 reduced the firing rate of NE, DA and 5-HT neurons through the activation of 2, D2 and 5-HT1A autoreceptors, respectively. The predominant inhibitory effect of SEP 225289 was detected in the LC while it produces only a partial decrease in VTA DA and DRN 5-HT neuronal activities. The unexpected moderate inhibitory effect of SEP 225289 in the DRN is not due to a lesser degree of reuptake blockade of 5-HT than for NE because it is similarly effective in prolonging the recovery of firing of pyramidal neurons following 5-HT and NE applications. It is thus possible that noradrenergic and/or dopaminergic inputs innervating the DRN produce excitatory influence on 5-HT neurons thereby limiting the inhibitory effect of SEP 225289 in the DRN. The observation that SEP 225289 activates the firing of 5-HT neurons in the presence of the 5-HT1A receptor antagonist WAY 100635 is consistent with this hypothesis. Supported by funding from SEPRACOR Inc.

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.

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.

The Effects of Long Chain-Lengthn-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.

Dopamine and serotonin: Integrating current affective engagement with longer-term goals

Interpreting VTA dopamine activity as a facilitator of affective engagement fits Depue &amp; Collins's agency dimension of extraverted personality and also Watson's and Tellegen's (1985) engagement dimension of state mood. Serotonin, by turning down the gain on dopaminergic affective engagement, would permit already prepotent responses or habits to prevail against the behavior-switching incentive-simulation-driven temptations of the moment facilitated by fickle VTA DA. Intelligent switching between openly responsive affective engagement and constraint by long-term plans, goals, or values presumably involves environment-sensitive balancing of these neuromodulators, such as socially dominant primates may show.