Clozapine-N-oxide impairs spatial memory independent of DREADDs

Chemogenetic studies using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) enable the precise manipulation of neuronal activity in specific brain regions and cell types. DREADDs are widely used to dissect neural circuits underlying animal behavior, including learning and memory. Clozapine-N-Oxide (CNO), a metabolite of clozapine and one of the earliest-developed ligands for muscarinic DREADDs, was initially considered pharmacologically inert. However, CNO is now known to undergo back-metabolism to clozapine, leading to undesired off-target behavioral effects, including alterations in locomotion and anxiety-related behaviors. New ligands such as Compound 21 (C21) have been developed to improve selectivity and reduce these effects. However, despite their widespread use, no studies to date have directly compared the effects of CNO and C21 themselves on specific cognitive processes such as hippocampus-dependent memory formation. Here, we measured acute effects of CNO and C21 on spatial memory encoding, using an object-location memory (OLM) paradigm in male and female mice. We also quantified encoding-associated hippocampal principal neuron and parvalbumin (PV⁺) interneuron activity by measuring cFos expression in these populations. Across dorsal hippocampal subregions, neither ligand altered overall neuronal activity nor PV⁺ interneuron activity during encoding. Nonetheless, we find that CNO administration impairs OLM encoding, while C21 does not. Together, these findings highlight a previously unrecognized behavioral effect of CNO administration on hippocampus-dependent memory formation-even in the absence of DREADD expression-and indicate that C21 may be a preferable ligand for chemogenetic studies examining memory and hippocampal function.