Paav hsyn hm3d gq mcherry

Plasmids for transfection were purified by using Qiagen's endotoxin-free HiSpeed Gigaprep kit (Qiagen) and quantified by spectrophotometry. The integrity of the cis plasmids and ITRs were confirmed by restriction digest and NGS. Complete reagent information is listed in Supplementary Table S5.
The following plasmids were used to prepare the rAAV described in this study; pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA (Addgene 20298, a gift of Karl Deisseroth, unpublished) AAV-Cre-GFP (Addgene 68544, a gift of Eric Nestler), AAV-EF1a-DIO-GCaMP6s-P2A-nls-dTomato (Addgene 51082, a gift of Jonathan Ting), AAV-FLEX-rev-ChR2(H134R)-mCherry (Addgene 18916, a gift from Scott Sternson), rAAV2-retro helper (Addgene 81070, a gift of Alla Karpova), pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene 44361, a gift of Bryan Roth), pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362, a gift of Bryan Roth), pAAV-hSyn-hM3D(Gq)-mCherry (Addgene 50474, a gift of Bryan Roth, unpublished), pAAV-hSyn-EGFP(Addgene 50465, a gift of Bryan Roth, unpublished), pAAV-CAG-GFP (Addgene 37825, a gift from Edward Boyden, unpublished), pAAV-CAG-tdTomato (Addgene 59462, a gift of Edward Boyden, unpublished), and pUCmini-iCAP-PHP.eB (Addgene 103005, a gift of Viviana Gradinaru).^{15–20 (link)}

For some experiments, an AAV retrograde virus expressing pAAV-hSyn-hM4D(Gi)-mCherry (Addgene #50475) or pAAV-hSyn-hM3D(Gq)-mCherry (Addgene #50474) was injected into either the ipsilateral pons (relative to lambda, 1.00 mm lateral, 0.20 mm posterior, depth of 4.55 mm) or contralateral mPFC (relative to bregma, 0.48 mm lateral, 2.1 mm anterior, depth of 1.6 mm) of 6- to 9-week-old mice to express DREADD (“designer receptor exclusively activated by designer drugs”) receptors selectively in PT (ipsilateral pons injections) or IT (contralateral mPFC injections) neurons in the prefrontal cortex. Under sterile surgical conditions, animals were anesthetized with continuous isoflurane (~2%) and a craniotomy made at the above coordinates. A 33 gauge microsyringe (Hamilton) containing undiluted virus was slowly lowered into place over a period of ~5 minutes. After waiting anther 5 minutes, the virus was injected at a rate of 50 nL per minute for a total volume of 450 nL (pons) or 300 nL (mPFC). Five minutes after the injection was completed, the microsyringe was slowly removed and the wound sutured. Mice were allowed to recover from surgery for at least 21 days before use in experiments.