Dose-dependent reduction in cocaine-induced locomotion by Clozapine-N-Oxide in rats with a history of cocaine self-administration

Abstract

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are novel tools for the dissection of circuitry mediating behavior and neural function. Designer receptors based on the muscarinic M3 and M4 subtypes were designed to be activated by clozapine-N-oxide (CNO), a ligand previously shown to be an inert metabolite of clozapine. However, recent work in rats has shown that CNO is reverse metabolized to its parent compound. Furthermore, CNO administration (5 mg/kg IP) attenuates amphetamine-induced locomotion and the evoked dopamine response that accompanies it. As these systems are routinely used to probe the neurocircuitry underlying cocaine-seeking behavior, here we sought to determine whether CNO would have similar effects on cocaine-induced locomotion in rats with a history of cocaine self-administration. In order for muscarinic-based DREADDs to be utilized for the dissection of circuitry underlying behavioral responses to cocaine, the doses of CNO administered to induce DREADD signaling must themselves have no effect on cocaine-induced behavior. Male Sprague-Dawley rats self-administered cocaine (0.35 mg/infusion) for 12 days, followed by 14-21 days of instrumental extinction training. Rats then underwent locomotor testing. CNO (0, 3, or 5 mg/kg) was injected (utilizing a within-subjects design), followed 20 min later by cocaine (10 mg/kg IP). Locomotion was monitored for the following 120 min. We found that the 5, but not the 3 mg/kg, dose of CNO reduced cocaine-induced locomotion. Thus, studies utilizing DREAADs to probe cocaine-induced behavior should consider these findings when choosing a dose of CNO and include non-DREADD CNO controls.

Keywords: Addiction; CNO; Circuitry; DREADDs; Reinstatement; Relapse.

Fig. 1. Rat self-administration and extinction data

Panel A. Mean number of infusions attained across the 12 day self-administration period. Panel B. Mean number of active and inactive lever presses during cocaine self-administration. Panel C. Mean number of active and inactive lever presses during the first 12 days of extinction training.

Fig. 2. CNO at 5 mg/kg but not 3 mg/kg (IP) attenuated cocaine-induced locomotion

Rats were administered CNO (0, 3, 5 mg/kg IP) utilizing a within-subjects, counterbalanced design. Each dose was tested on a different day, with at least two days separating tests. Rats were injected with CNO and placed into the locomotor chamber for 20 min prior to receiving cocaine (10 mg/kg IP). * = p<0.05 compared to both 0)0 and 3 mg/kg CNO doses. # = p<0.05 relative to 3 mg/kg CNO. & = p<0.05 relative to 0 mg/kg only. N=7.