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

Abstract

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.

Keywords: Gq; desensitization; dopamine D2 receptor; lentivirus; neurotensin; shRNA; ventral tegmental area.

Figure 1

Reduction of neurotensin-induced excitation of VTA neurons by Gq shRNA. (A,B) Ratemeter diagrams. Firing rate over 5 s intervals is represented by the height of the vertical bars; duration of drug application is shown by the horizontal bars. Neurotensin (10 nM, 5 min applications) was tested at 30 min intervals while recording from DA VTA neurons with micropipettes containing 0.9% NaCl to which either lentivirus containing Gq shRNA (A) or lentivirus without shRNA (B) was added. The magnitude of neurotensin excitation over time in the recording shown in (A) was 25.99, 19.33, 8.3, and 2.7%, respectively; the magnitude of neurotensin excitation over time in the recording shown in (B) was 58.5, 62.8, 61.3, and 56.4%, respectively. (C) Bars representing the mean responses to neurotensin in recordings similar to those shown in (A,B). Mean excitatory effect of neurotensin is shown at each time period for cells recorded with micropipettes to which Gq shRNA (filled bars) or lentiviral control (open bars) was added. There was a significant change in the excitatory effect of neurotensin in recordings in which Gq shRNA was present in the micropipettes.

Figure 2

Blockade of dopamine inhibition reversal by Gq shRNA. (A–C) Ratemeter diagrams. Firing rate over 5 s intervals is represented by the height of the vertical bars; duration of drug application is shown by the horizontal bars. Recordings were maintained for 2 h before dopamine was applied for 40 min. Recordings were made from DA VTA neurons with micropipettes containing 0.9% NaCl to which either lentivirus containing Gq shRNA (A), lentivirus without shRNA (B), or lentivirus containing G_olf shRNA (C) was added. The inhibition produced by dopamine subsided over time when lentivirus without shRNA or lentivirus containing G_olf shRNA was present in the pipette. No reversal of dopamine-induced inhibition was observed when lentivirus containing Gq shRNA was in the micropipette. (D) Relative change in firing rate (mean ± SEM) is plotted as a function of time. In experiments similar to those shown in parts (A–C) above, the effect of dopamine at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. There was a significant reversal in dopamine-induced inhibition when either Golf shRNA (▼) or lentiviral control (■) was present in the micropipettes. A significant inhibition produced by dopamine was observed with the inclusion of Gq shRNA (•) in the micropipettes.

Figure 3

Effect of cycloheximide on neurotensin excitation and DIR. (A–D) Ratemeter diagrams. Firing rate over 5 s intervals is represented by the height of the vertical bars; duration of drug application is shown by the horizontal bars. (A,B) Neurotensin (100 nM) was applied for 5 min at 30 min intervals while recording from DA VTA neurons with micropipettes containing 0.9% NaCl to which either cycloheximide (A) or vehicle (B) was added. The magnitude of neurotensin excitation over time in the recording shown in (A) was 78, 17, 11.7, and 15%, respectively; the magnitude of neurotensin excitation over time in the recording shown in (B) was 55.4, 40.6, 43.2, and 52.8%, respectively. (C,D) Recordings were maintained for 2 h before dopamine was applied for 40 min. Recordings were made from DA VTA neurons with micropipettes containing 0.9% NaCl to which either cycloheximide (C) or vehicle (D) were added. The inhibition produced by dopamine subsided over time when vehicle was present in the pipette. No reversal of dopamine-induced inhibition was observed when cycloheximide was included in the micropipette. (E) Bars representing the mean responses to neurotensin in recordings similar to those shown in (A,B). Mean excitatory effect of neurotensin is shown at each time period for cells recorded with micropipettes to which cycloheximide (filled bars) or vehicle (open bars) was added. There was a significant reduction in the excitatory effect of neurotensin in recordings in which cycloheximide was present in the micropipettes. (F) Relative change in firing rate (mean ± SEM) is plotted as a function of time. In experiments similar to those shown in (C,D) above, the effect of dopamine [after 2 h application of either cycloheximide (▼) or vehicle (■)] at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. No reversal of dopamine-induced inhibition was observed when cycloheximide was present in the micropipettes.