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 2h. 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 2h after initiation of recording. As the time interval (2h) 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

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 play 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 pDA 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 pDA VTA neurons by HDAC inhibitors and also measured the levels of HDAC2, histone (H3-K9) acetylation, and GABA-A1 receptor subunit in VTA during ethanol withdrawal. Mice were injected intraperitoneally with either ethanol (3.5 g/kg) or saline twice daily for 3 weeks. In recordings from pDA 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 HDAC inhibitor SAHA (vorinostat) or trichostatin A (TSA) for two hours, the hyposensitivity of pDA VTA neurons to GABA was significantly attenuated. There was no effect of TSA or SAHA on GABA sensitivity of pDA 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- 1) R subunits in the VTA. Therefore, blockade of upregulation of HDAC2 by HDAC inhibitors 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