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

Nimitvilai S, Arora DS, McElvain MA, Brodie MS - Front Cell Neurosci (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago Chicago, IL, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Change in baseline firing rate over 2 h of recording with micropipettes containing lentivirus and/or shRNA.


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

Nimitvilai S, Arora DS, McElvain MA, Brodie MS - Front Cell Neurosci (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569574&req=5

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.
Mentions: Change in baseline firing rate over 2 h of recording with micropipettes containing lentivirus and/or shRNA.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago Chicago, IL, USA.

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.

No MeSH data available.


Related in: MedlinePlus