The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice

Manvich, Daniel F.; Webster, Kevin A.; Foster, Stephanie L.; Farrell, Martilias S.; Ritchie, James C.; Porter, Joseph H.; Weinshenker, David

doi:10.1038/s41598-018-22116-z

Cited by 286 publications

(267 citation statements)

References 23 publications

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“…17,18 Therefore novel ligands, such as compound 21, perlapine, JHU37160/152, and clozapine have been suggested for direct DREADD activation. Moreover, DREADD ligands did not modulate epileptic activity in non-DREADD animals.…”

Section: Discussionmentioning

confidence: 99%

“…Although the designer receptors were initially designed to be sensitive to the inert molecule CNO, it recently became clear that in vivo effects of CNO are mediated by its back-conversion to clozapine, which crosses the BBB and activates DREADD receptors. 17,18 Therefore novel ligands, such as compound 21, perlapine, JHU37160/152, and clozapine have been suggested for direct DREADD activation. 5,19 We now confirm that low doses of clozapine can effectively be used as hM4Di activators in vivo.…”

Section: Discussionmentioning

confidence: 99%

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Long‐term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy

et al. 2019

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Objective: More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. Methods: Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. Results: Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. Significance: This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects. | 2315 DESLOOVERE Et aL.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Long‐term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy

et al. 2019

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“…There is no evidence that CNO alters hunger or thirst, but a recent study demonstrated that CNO can, in fact, produce a detectable state in rats that may be related to the metabolism of CNO into the psychoactive compound Clozapine (Gomez et al, 2017;Manvich et al, 2018). Specifically, Manvich et al, (2018) first trained systemic Clozapine (1.25 mg/kg) versus saline injection as a discriminative stimulus during an instrumental task, such that one lever was reinforced under Saline conditions, whereas a different lever was reinforced under Clozapine conditions. After establishing this discrimination, rats were challenged with CNO (1.0, 3.2, 10 mg/kg, i.p.)…”

Section: Discussionmentioning

confidence: 99%

“…Sections were then visualized using an upright epifluorescence manual system microscope (Olympus, BX43) with an XM10 camera; images were taken at 2x, 10x, and 20x (cellSens). Assessment of viral expression location was performed visually, using standard anatomical landmarks to identify the BLA (Paxinos Watson, 2007). For hM4Di transduced rats, only data from subjects with bilateral hM4Di-mCherry expression localized to the BLA were included for analysis (n= 20 included, 15 rejected).…”

Section: Pit Testingmentioning

confidence: 99%

Effects of hM4Di activation in CamKII basolateral amygdala neurons and CNO treatment on Sensory-Specific vs. General-PIT; refining PIT circuits and considerations for using CNO

Derman¹,

Bass

Ferrario

2019

Preprint

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Pavlovian stimuli can influence instrumental behaviors, a phenomenon known as Pavlovian-to-instrumental transfer (PIT). PIT arises via psychologically and neurobiologically independent processes as Sensory-Specific-PIT (SS-PIT) and General-PIT. SS-, but not General-PIT, relies on the basolateral amygdala (BLA), however the specific BLA neuronal populations involved are unknown. Therefore, here we determined the contribution of glutamatergic BLA neurons to SS-PIT. The BLA was transduced with virus containing either GFP or hM4Di, driven by the CamKII promoter. Rats were then tested for SS- and General-PIT following Vehicle or Clozapine-n-oxide (CNO, the hM4Di-activating ligand) injection. CNO had no effect on SS-PIT in the GFP control group, but selectively blocked its expression in the hM4Di-expressing group. Furthermore, CNO did not alter the expression of Pavlovian outcome devaluation effects in GFP or hM4Di expressing groups, indicating that the hM4Di-mediated loss of SS-PIT did not result from an inability to recall the sensory-specific details of the Pavlovian stimulus-outcome associations. Unexpectedly, CNO disrupted General-PIT in both GFP and hM4Di expressing groups, indicating that CNO alone is sufficient to disrupt affective, but not sensory-specific processes. Disruption of General-PIT by CNO was not due to generalized motor effects, but instead may be related to shifts in internal state produced by CNO. Together these data identify BLA CamKII neurons as critical for the expression of SS-PIT, and reveal important considerations for using CNO to study general affective motivation.

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“…The seemingly negative effect of CNO might have adversely affected motor performance in comparison with Saline group(Gomez JL et al 2017;Mahler SV and G Aston-Jones 2018;Manvich DF et al 2018). There are mixed findings about whether selective ACh reduction affects motor performance(Conner JM et al 2003;Gharbawie OA and …”

mentioning

confidence: 99%

Low acetylcholine during early sleep is important for motor memory consolidation

Qandeel

2018

Preprint

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The synaptic homeostasis theory of sleep proposes that low neurotransmitter activity in sleep is optimal for memory consolidation. We tested this theory by asking whether increasing acetylcholine levels during early sleep would disrupt motor memory consolidation. We trained separate groups of adult mice on the rotarod walking and skilled reaching for food tasks, and after training, administered physostigmine, an acetylcholinesterase inhibitor, to increase cholinergic tone in subsequent sleep. Post-sleep testing suggested that physostigmine impaired motor skill acquisition. Home-cage video monitoring and electrophysiology revealed that physostigmine disrupted sleep structure, delayed non-rapid-eyemovement sleep onset, and reduced slow-wave power in the hippocampus and cortex. The impaired motor performance with physostigmine, however, was not solely due to its effects on sleep structure, as one hour of sleep deprivation after training did not impair rotarod performance. A reduction in cholinergic tone by inactivation of cholinergic neurons during early sleep also affected rotarod performance. Administration of agonists and antagonists of muscarinic and nicotinic acetylcholine receptors revealed that activation of muscarinic receptors during early sleep impaired rotarod performance. The experiments suggest that the increased slow wave activity and inactivation of muscarinic receptors during early sleep due to reduced acetylcholine contribute to motor memory consolidation.

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The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice

Cited by 286 publications

References 23 publications

Long‐term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy

Long‐term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy

Effects of hM4Di activation in CamKII basolateral amygdala neurons and CNO treatment on Sensory-Specific vs. General-PIT; refining PIT circuits and considerations for using CNO

Low acetylcholine during early sleep is important for motor memory consolidation

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