Cno dihydrochloride

Most drugs were dissolved in water to make stock solutions (typically at 10,000× concentration) that were frozen as aliquots and used as needed during experiments. ACh (Fisher Scientific) was bath-applied with physostigmine hemisulfate (eserine; Tocris Bioscience), a blocker of acetylcholinesterase (AChE). Atropine and pirenzepine dihydrochloride were purchased from Sigma-Aldrich. TTX citrate and CGP 52432 were purchased from HelloBio, Inc. CNO dihydrochloride, DNQX, and D-AP5 were purchased from Tocris Bioscience. SR-95531 (gabazine) was purchased from MedChemExpress and dissolved in DMSO for stock solutions.

To avoid the use of the toxic solvent dimethyl sulfoxide (DMSO), which is typically used for the preparation of CNO products in concentrations of up to 15% (Campbell and Marchant, 2018 (link)), we opted for the use of a water-soluble salt preparation of CNO, CNO dihydrochloride (Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: 6329) dissolved in sterile saline. The dihydrochloride preparation of CNO undergoes less back-metabolism to clozapine but has a higher bioavailability compared to CNO-DMSO as indicated by pharmacokinetic work in rhesus macaques (Allen et al., 2019 (link)). This product has previously been used in sleep studies on mice at concentrations between 1 and 5 mg/kg (Fernandez et al., 2018 (link); Stucynski et al., 2021 (link)). For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124). We chose a dose of 3 mg/kg because a detailed pharmacokinetic assessment of this product at this specific concentration as well as behavioural testing in a five-choice serial-reaction-time task did not reveal any behavioural effects at this dose (Jendryka et al., 2019 (link)).

In order to minimize the mechanical effects of subsequent drug infusions and habituate the animals to the infusion procedure, animals underwent an initial saline infusion at least 24 h before the conflict test. On critical test days, animals received 0.3 μl of CNO (1 mM, CNO-dihydrochloride, BioTechne, MN) or 0.9% saline vehicle (only for homecage feeding tests and EPM/c-Fos) into the LS target site. The substance was infused at a rate of 0.3 μl/min for 1 min via 30-gauge microinjectors (33 gauge; Plastics One) projecting 1.5 mm below the indwelling guide cannulae using an infusion pump (Harvard Apparatus, Hollison, MA) mounted with 10 μl Hamilton syringes. The microinjectors were left in place for a further 1 min to allow the drug to diffuse away from the injector tip. During microinfusion, animals were held and carefully handled to minimize stress and avoid equipment damage due to movement. The relevant test was administered a minimum of 10 min after the end of each infusion. Rats received six separate microinfusions following infusion habituation, which occurred on separate days prior to the conflict test (followed by locomotor test), cue preference/avoidance tests, novelty detection (followed by NESF), both free feeding tests, and EPM (Fig. 1).