Cholinergic Agents

For analysis of cholinergic markers, rats were deeply anesthetized (5% isoflurane) followed by intracardiac perfusion with clearing solution (0.1 M phosphate buffer, 0.5 mM EDTA, 0.05% NaNO₂) followed by ice cold 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde. Brains were removed and placed in 4% paraformaldehyde (0.1 M PB, pH 7.4) to postfix at 4°C. Coronal sections containing the basal forebrain and the amygdala plus hippocampus were cut at 50 μm on a vibratome (VT1000S, Leica, Nussloch, Germany) and stored at 4°C in 0.1 M phosphate buffer until staining. For longer term storage, sections were transferred to Anti‐freezing solution (30% Sucrose in 0.1 M phosphate buffer with 30% ethylene glycol), then stored at −20°C.
Immunofluorescence labeling for ChAT and VAChT used methods described in Tryon et al., (2022).⁴⁷ For ChAT immunolabeling, sections were washed three times in Tris buffer (TBS) for 10 min, blocked in TBS containing 0.5% Triton X‐100 and 10% normal donkey serum for 30 min at room temperature, washed in TBS then incubated with goat polyclonal anti‐choline acetyltransferase antibodies (1:1000; Millipore Cat# AB144P, RRID:AB_2079751;used previously in⁴⁷) in TBS containing 0.5% Triton X‐100 and 2% normal donkey serum for 2 days at room temperature. Sections were then incubated in donkey‐anti goat conjugated to Alexa Fluor 647 (1:400; Jackson ImmunoResearch Labs Cat# 705‐605‐147, RRID:AB_2340437) in TBS with 0.5% Triton X‐100 and 2% normal donkey serum for 3 h at room temperature. To detect labeling of vesicular acetylcholine transporter (VAChT), separate sections were washed three times in TBS then blocked for 30 min at room temperature in TBS containing 0.5% Triton X‐100 and 10% normal donkey serum, after which they were washed three times in TBS. Sections were incubated in TBS containing goat anti‐vesicular acetylcholine transporter antibodies (1:1000; Millipore Cat# ABN100, RRID:AB_2630394), 0.5% Triton X‐100 and 2% normal donkey serum overnight at room temperature. Sections were washed three times in TBS then incubated in TBS containing donkey‐anti goat conjugated to Alexa Fluor 555 (1:500; Molecular Probes Cat# A‐21432, RRID:AB_141788), 0.5% Triton X‐100 and 2% normal donkey serum. Sections labeled for ChAT were coverslipped in Vectashield Vibrance Antifade Mounting Media (Vector Laboratories Cat#H‐1700, Burlingame, CA) and sections labeled for VAChT were coverslipped in Prolong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA) and stored at 4°C until imaging.
Coronal sections including the BF, BLA and hippocampus were also stained for acetylcholinesterase (ACHE) activity. As described previously, sections were incubated in a solution of 0.2 M Tris maleate buffer (pH 5.7), 0.1 M sodium citrate, 0.03 M cupric sulfate, 5 mM potassium ferricyanide, and 1.7 mM acetylthiocholine iodide for ~60 min at room temperature followed by a 70% ethanol rinse and coverslipping.⁴⁸, ⁴⁹

All animal treatments and procedures were in compliance with the American Association for Laboratory Animal Science (AALAS) and experimental procedures were approved by both the University of South Carolina and Columbia VA Health Care System Institutional Care and Use Committees. Upon arrival, rats were maintained in the vivarium on a 12‐hour light/dark cycle with ad libitum access to food and water. As Long Evans Tg (ChAT::Cre) rats originally developed by the Deisseroth group have genetically‐restricted expression of Cre recombinase in cholinergic (ChAT expressing) neurons,²⁶ four ChAT:Cre hemizygous male rats were purchased from the Rat Resource and Research Center (RRRC# 00658: Long Evans‐Tg(ChAT‐Cre)5.1Deis; University of Missouri, Columbia MO; RRID:RRRC_00658) and were ~ 2 months of age upon arrival. They were bred with four adult female wildtype Long Evans rats (~2 months old on arrival; Envigo RRID:RGD_5508398). The day before weaning, rat pups were color coded by tail marking and each given a unique number. At 21 days, the four resulting litters (N = 41 pups) were weaned. At weaning, tail snips were taken (~1 mm) with a sterile scalpel and placed into a labeled 96 well plate (provided by TransnetYX). The plate was sealed and sent to TransnetYX (Cordova, TN) for genotyping using polymerase chain reaction (PCR) for detection of the Cre recombinase gene.
Following weaning, animals were housed in groups of 2–3 with same‐sex littermates, but were single housed starting ~2 weeks prior to behavioral testing per our previous studies.¹⁷, ⁴², ⁴³ Breeding of the transgenic ChAT::Cre+ males with wildtype females yielded 41 offspring that were tested in a cue‐conditioned fear and extinction protocol during early light phase of the light: dark cycle (see Figure 1; as described in¹⁷, ⁴², ⁴³). The groups tested were Cre+ males (N = 12), Cre‐ males (N = 8), Cre+ females (N = 15), and Cre‐ females (N = 6). Unfortunately, due to the COVID‐19 pandemic we were unable to add additional litters to create similar sample sizes in each group.
Males and females were handled for 2 weeks prior to behavioral testing, and daily vaginal lavage was performed in the females to determine estrous cycle stage; males were handled similarly. Vaginal lavage was also performed after behavioral testing each day to assess the stage of the cycle during each behavioral test while avoiding additional handling stress.⁴³, ⁴⁴, ⁴⁵ Vaginal cytology was determined using the fresh cytology samples under a microscope, and slides were then fixed using 95% ethanol for additional staining using hematoxylin and eosin for additional verification (see Refs. 43 (link), 44 (link) for detailed methods).