Does chronic systemic injection of the DREADD agonists clozapine-N-oxide or compound 21 change behavior relevant to locomotion, exploration, anxiety, and depression in male non-DREADD-expressing mice?

Abstract

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a chemogenetic tool commonly-used to manipulate rodent brain circuit activity. The most widely-used synthetic ligand for DREADDs is Clozapine-N-oxide (CNO). However, CNO is back-metabolized to clozapine, which itself activates numerous endogenous receptors and therefore may influence rodent behavior. To eliminate potential off-target effects of CNO, a new DREADD agonist, Compound 21 (C21), has been proposed as an alternative as it lacks active metabolites. The literature is mixed on whether acute administration of CNO or C21 changes mouse behavior. In contrast, there is no substantial literature on whether chronic administration of CNO or C21 changes mouse behavior. Here we tested whether chronic injections of these two distinct DREADD agonists change key behaviors in non-designer receptor-expressing mice relative to Vehicle (Veh)-injected control mice. Mice (CamKIIα-icre males) were injected i.p. with Veh (0.5% dimethyl sulfoxide in 0.9% saline, 5ml/kg), CNO (0.2mg/ml, 1mg/kg), or C21 (0.2mg/ml, 1mg/kg) 5 days a week for 16 weeks. All three groups had similar weight gain over the 16 week-experiment, and showed similar measures in behaviors assessed during week 3 (beam breaks in a 30-min locomotion task, time in center of open field or open arms of elevated plus maze) and week 14-16 (ambulatory distance during 240-min activity monitoring, percent marbles buried, grooming time during the sucrose splash test). These data show chronic injections of CNO or C21 do not affect key behaviors as compared to chronic injections of Veh, and may be helpful for behavioral neuroscientists when study design requires repeated injection of these DREADD agonists. Highlights Acute injection of CNO changes behavior of non-DREADD-expressing mice It’s not known if chronic CNO or alternative agonist C21 also changes mouse behavior DREADD agonists or Veh were given chronically to non-DREADD-expressing mice Mice given Veh, CNO, or C21 were similar in regard to locomotion and other behaviors Thus CNO and C21 can be injected repeatedly without non-specific behavior effects

Chronic Behavioral Manipulation via Orally Delivered Chemogenetic Actuator in Macaques

“…It is also beneficial for neuroscience research aimed at long-term control of neuronal activity in freely-moving animals without restraint or stress. However, recent studies have reported that CNO has low permeability into the brain, and that its metabolite, clozapine, activates not only DREADDs but also endogenous receptors and transporters, leading to potential off-target effects (Gomez et al, 2017;Tran et al, 2020). To circumvent this issue, alternative selective DREADDs agonists have been explored and validated (Bonaventura et al, 2019;Weston et al, 2019;Nagai et al, 2020).…”

Oligodendrocyte lineage cells driven by neuronal activity in selected brain regions are required for episodic memory formation

“…one hour before IA training to transiently silence neuronal activity in the ACC (Fig. 7A) (Jendryka et al, 2019; Luo et al, 2021; Tran et al, 2020). In addition to hM4Di, the AAV-hSyn-hM4D(Gi)-mCherry viral construct expresses the fluorescent protein mCherry in neurons.…”

Seizing Control of Neuronal Activity: Chemogenetic Applications in Epilepsy

“…Moreover, it has been suggested that the in vivo activation of DREADDs after CNO administration is not due to CNO, which is poorly penetrant to the brain, but rather a metabolic production of clozapine. 21 -24 Deciphering which effects are DREADD-mediated, and which are due to off target effects of DREADD agonists is an important consideration …”