{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:27:22Z","timestamp":1785414442080,"version":"3.56.0"},"posted":{"date-parts":[[2026,1,12]]},"group-title":"Neuroscience","reference-count":27,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T00:00:00Z","timestamp":1768176000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R01NS118440"],"award-info":[{"award-number":["R01NS118440"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01MH135565"],"award-info":[{"award-number":["R01MH135565"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["K99AG088300"],"award-info":[{"award-number":["K99AG088300"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014989","name":"Chan Zuckerberg Initiative","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100014989","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/00jmfr291","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,1,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  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\n                  <jats:sup>+<\/jats:sup>\n                  ) interneuron activity by measuring cFos expression in these populations. Across dorsal hippocampal subregions, neither ligand altered overall neuronal activity nor PV\n                  <jats:sup>+<\/jats:sup>\n                  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\u2014even in the absence of DREADD expression\u2014and indicate that C21 may be a preferable ligand for chemogenetic studies examining memory and hippocampal function.\n                <\/jats:p>","DOI":"10.64898\/2026.01.11.698821","type":"posted-content","created":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T05:10:14Z","timestamp":1768281014000},"source":"Crossref","is-referenced-by-count":0,"title":["DREADD agonist Clozapine-N-oxide, but not Compound 21, impairs spatial memory encoding"],"prefix":"10.64898","author":[{"given":"Brayden","family":"Bunce","sequence":"first","affiliation":[{"name":"Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, Ann Arbor, Michigan, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna A.","family":"VanKampen","sequence":"additional","affiliation":[{"name":"Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, Ann Arbor, Michigan, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annie","family":"He","sequence":"additional","affiliation":[{"name":"Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, Ann Arbor, Michigan, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2921-0509","authenticated-orcid":false,"given":"Sara J.","family":"Aton","sequence":"additional","affiliation":[{"name":"Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, Ann Arbor, Michigan, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8287-0496","authenticated-orcid":false,"given":"Frank","family":"Raven","sequence":"additional","affiliation":[{"name":"Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, Ann Arbor, Michigan, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020420531399000_2026.01.11.698821v1.1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0700293104"},{"key":"2026020420531399000_2026.01.11.698821v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2016.01.040"},{"key":"2026020420531399000_2026.01.11.698821v1.3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-pharmtox-010814-124803"},{"key":"2026020420531399000_2026.01.11.698821v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2009.06.014"},{"key":"2026020420531399000_2026.01.11.698821v1.5","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.neuroscience.2025.08.015","article-title":"Sleep modulation via Nucleus Accumbens A2AR neurons to probe cognition and behavior in mice","volume":"584","year":"2025","journal-title":"Neuroscience"},{"key":"2026020420531399000_2026.01.11.698821v1.6","doi-asserted-by":"publisher","DOI":"10.1126\/science.aan2475"},{"key":"2026020420531399000_2026.01.11.698821v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-22116-z"},{"key":"2026020420531399000_2026.01.11.698821v1.8","doi-asserted-by":"publisher","DOI":"10.1021\/acschemneuro.7b00079"},{"key":"2026020420531399000_2026.01.11.698821v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/0893-133X(94)00129-N"},{"key":"2026020420531399000_2026.01.11.698821v1.10","doi-asserted-by":"publisher","DOI":"10.1007\/BF02245606"},{"issue":"Suppl B","key":"2026020420531399000_2026.01.11.698821v1.11","first-page":"47","article-title":"An overview of the mechanism of action of clozapine","volume":"55","year":"1994","journal-title":"J Clin Psychiatry"},{"key":"2026020420531399000_2026.01.11.698821v1.12","doi-asserted-by":"crossref","unstructured":"MacLaren DA , Browne RW , Shaw JK , Krishnan Radhakrishnan S , Khare P , Espa\u00f1a RA , et al. Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments. eNeuro. 2016;3(5).","DOI":"10.1523\/ENEURO.0219-16.2016"},{"key":"2026020420531399000_2026.01.11.698821v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.neulet.2020.135432"},{"issue":"1","key":"2026020420531399000_2026.01.11.698821v1.14","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1021\/acsptsci.8b00012","article-title":"DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo","volume":"1","year":"2018","journal-title":"ACS Pharmacol Transl Sci"},{"key":"2026020420531399000_2026.01.11.698821v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-41088-2"},{"key":"2026020420531399000_2026.01.11.698821v1.16","doi-asserted-by":"publisher","DOI":"10.1021\/cn500325v"},{"key":"2026020420531399000_2026.01.11.698821v1.17","doi-asserted-by":"publisher","DOI":"10.1007\/s00213-023-06465-w"},{"key":"2026020420531399000_2026.01.11.698821v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms15039"},{"key":"2026020420531399000_2026.01.11.698821v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.05.027"},{"key":"2026020420531399000_2026.01.11.698821v1.20","doi-asserted-by":"publisher","DOI":"10.1111\/jsr.12972"},{"key":"2026020420531399000_2026.01.11.698821v1.21","doi-asserted-by":"crossref","unstructured":"Raven F , Vankampen AA , He A , Aton SJ . Dentate gyrus network regulation by somatostatin- and parvalbumin-expressing interneurons differentially impacts hippocampal spatial memory processing. iScience. 2026; In press.","DOI":"10.1101\/2025.07.23.666335"},{"key":"2026020420531399000_2026.01.11.698821v1.22","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2019318118"},{"issue":"4","key":"2026020420531399000_2026.01.11.698821v1.23","doi-asserted-by":"crossref","first-page":"109408","DOI":"10.1016\/j.isci.2024.109408","article-title":"Sleep-dependent engram reactivation during hippocampal memory consolidation associated with subregion-specific biosynthetic changes","volume":"27","year":"2024","journal-title":"iScience"},{"key":"2026020420531399000_2026.01.11.698821v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/j.conb.2006.09.002"},{"key":"2026020420531399000_2026.01.11.698821v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/nature10360"},{"key":"2026020420531399000_2026.01.11.698821v1.26","doi-asserted-by":"publisher","DOI":"10.1093\/cercor\/bhy174"},{"key":"2026020420531399000_2026.01.11.698821v1.27","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.27868"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.01.11.698821","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T04:53:23Z","timestamp":1770267203000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.01.11.698821"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,12]]},"references-count":27,"URL":"https:\/\/doi.org\/10.64898\/2026.01.11.698821","relation":{},"subject":[],"published":{"date-parts":[[2026,1,12]]},"subtype":"preprint"}}