Pulled glass micropipette

Stereotaxic coordinates are in millimeters relative to bregma.¹¹ Animals were deeply anesthetized with ketamine/xylazine (90/15 mg/kg, intramuscular). For anterograde tracing (Figure 1A), we used a recombinant adeno-associated virus (rAAV5) vector with strong affinity for neurons.^{12 (link)} The vector carried archaerhodopsin (ArchT) conjugated with tdTomato under the CAG promoter (AAV-CAG-ArchT-tdTomato; #29778, Addgene)^{3 (link)}; 0.8 μl was injected into the left LS (anteroposterior [AP], +0.6; mediolateral [ML], +0.7; superior-inferior [SI], −5.0) at 0.04 μl/min using a Quintessential Stereotaxic Injector (53 311, Stoelting) via a pulled glass micropipette (21–175B, Fisher Scientific), held in place for 10 minutes wait time after injection. After 2 weeks,^{3 (link)} animals were perfused with 4% paraformaldehyde and the brain was removed.
For retrograde tracing (Figure 1A), identical procedures were utilized, with minor modifications. We used cholera toxin B (CTB) conjugated with Alexa Fluor 488 (C34775, Thermo Fisher Scientific)^{13 (link)}; 1 μl was injected into BF (AP, −1; ML, +2.7; SI, −6.7) at 0.04 μl/min followed by a 5-minute wait time, and the brain was harvested after 1 week.^{13 (link)}

To generate cell-type specific expression of ChR2 in cholinergic neurons, we used a Cre-lox approach, by injecting a Cre-inducible recombinant AAV vector containing ChR2 (pAAV-Ef1α-DIO-hChR2(H134R)-EYFP-WPRE-pA) into rats expressing Cre-recombinase in cholineacetyltransferase (ChAT)-positive neurons (Figure 1A) ^{5 (link)}. This viral construct carries an inverted ChR2 fused to the fluorescent marker EYFP, flanked by a pair of canonical loxP sites (loxP) and a pair of mutated loxP sites (lox2272). In the presence of Cre, ChR2-EYFP is inverted into the sense direction and expressed from the EF1-α promoter ^{6 (link); 7 (link)}. AAV particles of serotype 5 were produced by the Vector Core Facility at The University of North Carolina at Chapel Hill.
Rats were deeply anesthetized using ketamine/xylazine (90/15 mg/kg, i.m.) and virus was delivered with a Quintessential Stereotaxic Injector (53311, Stoelting) via a pulled glass micropipette (21-175B, Fisher Scientific) lowered through a small durotomy to 6.2mm below the cortical surface. A bolus of 0.8μl of virus (4×10¹² viral molecules per ml) was injected into the PPT (coordinates below) at 0.1μl/min. The pipette was held in place for 5min after the injection before being removed. Animals were maintained for 2-4 weeks to allow ChR2 expression prior to experiments.