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Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems


The designer receptor exclusively activated by designer drugs (DREADD) system is one of the most widely used chemogenetic techniques to modulate the activity of cell populations in the brains of behaving animals. DREADDs are activated by acute or chronic administration of their ligand, clozapine-N-oxide (CNO). There is, however, a current lack of a non-invasive CNO administration technique that can control for drug timing and dosing without inducing substantial distress for the animals. Here, we evaluated whether the recently developed micropipette-guided drug administration (MDA) method, which has been used as a non-invasive and minimally stressful alternative to oral gavages, may be applied to administer CNO orally to activate DREADDs in a dosing- and timing-controlled manner. Unlike standard intraperitoneal injections, administration of vehicle substances via MDA did not elevate plasma levels of the major stress hormone, corticosterone, and did not attenuate exploratory activity in the open field test. At the same time, however, administration of CNO via MDA or intraperitoneally was equally efficient in activating hM3DGq-expressing neurons in the medial prefrontal cortex, as evident by time-dependent increases in mRNA levels of neuronal immediate early genes (cFos, Arc and Zif268) and cFos-immunoreactive neurons. Compared to vehicle given via MDA, oral administration of CNO via MDA was also found to potently increase locomotor activity in mice that express hM3DGq in prefrontal neurons. Taken together, our study confirms the effectiveness of CNO given orally via MDA and provides a novel method for non-stressful, yet well controllable CNO treatments in mouse DREADD systems.

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Fig. 1: The three procedural steps of the MDA method in C57BL6/N mice.
Fig. 2: Effects of VEH administration via intraperitoneal injection or MDA on acute stress response and locomotor activity in the open field test.
Fig. 3: Neuronal activation in the mPFC after CNO administration via IP injection or MDA.
Fig. 4: Effect of chemogenetically induced neuronal activation in the mPFC using the MDA method on spontaneous locomotor activity.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.


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This study was supported by a Postdoc Mobility grant (grant no. P2ZHP3_174868) awarded to T.N. by the Swiss National Science Foundation. Additional financial support was received from the Swiss National Science Foundation (grant no. 310030_188524 awarded to U.M.; grant no. PZ00P3_18009/1 awarded to J.R.). We would like to thank the Hodge Foundation UK, as well as the Viral Vector Facility (VVF) of the Neuroscience Center Zurich (ZNZ). Imaging was performed with equipment maintained by the Center for Microscopy and Image Analysis, University of Zurich.

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Authors and Affiliations



S.M.S. designed and performed research, analyzed data and contributed to the preparation of the manuscript. F.S.M., J.S., J.R. and U.W.-S. performed research. U.M. designed research and contributed to the preparation of the manuscript. T.N. designed and performed research, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Tina Notter.

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Competing interests

Unrelated to the present study, U.M. has received financial support from Boehringer Ingelheim Pharma GmbH & Co. and from Wren Therapeutics Ltd. All authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Immediate early gene expression in the mPFC after CNO (1 mg/kg) or VEH administration via IP injection or MDA in the absence of the modified human muscarinic M3 G-protein-coupled receptor (DREADD).

The scatter bar blots represent mRNA levels of cFos, Arc and Zif268 in the mPFC of adult (12-week-old) C57Bl6/N mice 60 min after treatment with VEH or CNO given via MDA or IP injections; n(VEH/IP) = 7, n(CNO/IP) = 7, n(VEH/MDA) = 7, n(CNO/MDA) = 7. Each dot in the scatter bar plot represents an individual animal, and error bars represent s.e.m.

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Schalbetter, S.M., Mueller, F.S., Scarborough, J. et al. Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems. Lab Anim 50, 69–75 (2021).

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