Conditional inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function

摘要

Conditional and inducible gene silencing in midbrain DA neurons. a Strategy for conditional and inducible Rit2 silencing in DA neurons. Conditional and inducible Rit2-KD in DA neurons was achieved using the Tet-Off system, in which tTA expression was driven downstream of the DAergic transcription factor, Pitx3. Following injection with an AAV encoding TRE-shRit2-GFP, tTA binds the Tet-response element (TRE) and drives both shRit2 and GFP reporter expression under (−)dox conditions. When mice are maintained on a (+)dox diet, doxycycline binds the tTA and prevents TRE-driven shRit2 and GFP expression. bPitx3IRES-tTA/+ mouse VTA was bilaterally injected with AAV9-TRE-GFP and mice were maintained ± dox diet ≥6 weeks. c–f Immunohistochemistry: Brains were sectioned 6 weeks post-injection, stained for TH (red) and GFP (green), and imaged as described in “Methods” section. GFP was selectively expressed in TH + midbrain neurons, and (+)dox diet suppressed GFP expression in both the VTA (c, d) and SNc (e, f). g–i RT-qPCR studies. The indicated brain regions were harvested by tissue punch and/or laser capture microscopy, and Rit2 mRNA levels were quantified by RT-qPCR, as described in “Methods”. Data are presented as 2−ΔCt values, ± S.E.M. g, h Female data: AAV9-TRE-shRit2 did not affect Rit2 expression in non-DAergic, SNr (g; p = 0.92), but significantly decreased Rit2 expression in SNc (h; *p < 0.02). i Male data from midbrain punches: AAV9-TRE-shRit2 significantly decreased midbrain Rit2 (p = 0.021), one-tailed Student’s t test, n = 7–12