Viral Induction of Cholinergic Neuron Expression

Following behavioral testing, one cohort of the Chat::Cre+ transgenic rats (N = 4 males, N = 4 females) were injected with an adeno‐associated viral vector (AAV) into the basal forebrain (BF) to induce enhanced yellow fluorescent protein (EYFP) expression in cholinergic neurons. Rats were anesthetized with isoflurane (5% for induction, 2.5% for maintenance, E‐Z Systems Palmer, PA) in oxygen, placed in a Kopf stereotaxic device (David Kopf Instruments, Tujunga CA), and body temperature was maintained using a homeothermic blanket (Harvard Apparatus, Holliston, MA). After administration of a local anesthetic (2% carbocaine, s.c.) at the incision site, the basal forebrain was targeted by drilling two holes through the skull using the following coordinates measured from Bregma with skull flat: A/P‐0.8, L/M+/− 2.4, DV ‐8.6‐8.8.⁴⁶ Rats were injected bilaterally with 2 μl of rAAV5/Ef1a‐DIO‐EYFP (UNC Viral Vector Core; LOT AV4310L) using a 33‐gauge needle on a Neuros Hamilton syringe at a rate of 0.2 μl/min using a motorized injector (Stoelting QSI Stereotaxic injector Wood Dale, IL). Following injections, the viral vector was allowed to diffuse for 10 min before the needle was withdrawn. Nalbuphine (2 mg/kg, s.c.) was administered postoperatively for pain management, the diet was supplemented with bacon softies (Bio‐serve, Frenchtown, NJ) to maintain postoperative weight, and topical nitrofurazone powder (NFZ puffer, Neogen Corporation) was used for prevention of infection at the incision site. Animals were allowed 3 weeks of recovery prior to perfusion and euthanasia to determine the number of BF cholinergic neurons expressing eYFP using immunofluorescence for choline acetyltransferase (ChAT; described below).