Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0–20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.

The locus coeruleus (LC) is the major loci of noradrenergic innervation to the forebrain. Due to the extensive central nervous system innervation of the LC noradrenergic system, a reduction in the number of LC neurons could result in significant changes in noradrenergic function in many forebrain regions. LC noradrenergic neurons were lesioned in adult male C57Bl/6 mice with the unilateral administration of 6-hydroxydopamine (6OHDA) (vehicle on the alternate side). Noradrenergic markers were measured 3 weeks later to determine the consequence of LC loss in the forebrain. Direct administration of 6OHDA into the LC results in the specific reduction of noradrenergic neurons in the LC (as measured by electrophysiology, immunoreactivity and in situ hybridization), the lateral tegmental neurons and dopaminergic neurons in the substantia nigra (SN) and ventral tegmental region were unaffected. The loss of LC noradrenergic neurons did not result in compensatory changes in the expression of mRNA for norepinephrine (NE) synthesizing enzymes. The loss of LC noradrenergic neurons is associated with reduced NE tissue concentration and NE transporter (NET) binding sites in the frontal cortex and hippocampus, as well as other forebrain regions such as the amygdala and SN. Adrenoreceptor (AR) binding sites (α1- and α2-AR) were not significantly affected on the 6OHDA-treated side compared to the vehicle-treated side, although there is a reduction of AR binding sites on both the vehicle- and 6OHDA-treated side in specific forebrain regions. These studies indicate that unilateral stereotaxic injection of 6OHDA into mice reduces noradrenergic LC neurons and reduces noradrenergic innervation to many forebrain regions, including the contralateral side.

The dopamine D 3 receptor (D 3 R) has emerged as a promising pharmacotherapeutic target for the treatment of several diseases including schizophrenia, Parkinson's disease, and substance use disorders. However, studies investigating the D 3 R's precise role in dopamine neurotransmission or how it may be exploited to modulate responses to drugs of abuse have produced contrasting results, in part because most D 3 R-targeted compounds often also interact with D 2 receptors (D 2 R). To resolve this issue, we set out to systematically characterize and compare the consequences of selective D 2 R or D 3 R antagonists on the behavioral-stimulant properties of cocaine in mice, and to identify putative neurobiological mechanisms underlying their behavior-modifying effects. Pretreatment with the selective D 2 R antagonist L-741,626 attenuated, while pretreatment with the selective D 3 R antagonist PG01037 enhanced, the locomotor-activating effects of both acute cocaine administration as well as sensitization following repeated cocaine dosing. While both antagonists potentiated cocaine-induced increases in presynaptic dopamine release, we report for the first time that D 3 R blockade uniquely facilitated dopamine-mediated excitation of D 1-expressing medium spiny neurons in the nucleus accumbens. Collectively, our results demonstrate that selective D 3 R antagonism potentiates the behavioralstimulant effects of cocaine in mice, an effect that is in direct opposition to that produced by selective D 2 R antagonism or nonselective D 2-like receptor antagonists, and is likely mediated by facilitating D 1-mediated excitation in the nucleus accumbens. These findings provide novel insights into the neuropharmacological actions of D 3 R antagonists on mesolimbic dopamine neurotransmission and their potential utility as pharmacotherapeutics.

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