Neuronal PAS domain 1 identifies a major subpopulation of wakefulness-promoting GABAergic neurons in basal forebrain.

Here we describe a novel group of basal forebrain (BF) neurons expressing neuronal PAS domain 1 (Npas1), a developmental transcription factor linked to neuropsychiatric disorders. Immunohistochemical staining in Npas1-cre-2A-TdTomato mice revealed BF Npas1 + neurons are distinct from well-studied parvalbumin or cholinergic neurons. Npas1 staining in GAD67-GFP knock-in mice confirmed that the vast majority of Npas1 + neurons are GABAergic, with minimal colocalization with glutamatergic neurons in vGlut1-cre-tdTomato or vGlut2-cre-tdTomato mice. The density of Npas1 + neurons was high, 5-6 times that of neighboring cholinergic, parvalbumin or glutamatergic neurons. Anterograde tracing identified prominent projections of BF Npas1 + neurons to brain regions involved in sleep-wake control, motivated behaviors and olfaction such as the lateral hypothalamus, lateral habenula, nucleus accumbens shell, ventral tegmental area and olfactory bulb. Chemogenetic activation of BF Npas1 + neurons in the light (inactive) period increased the amount of wakefulness and the latency to sleep for 2-3 hr, due to an increase in long wake bouts and short NREM sleep bouts. Non-REM slow-wave (0-1.5 Hz) and sigma (9-15 Hz) power, as well as sleep spindle density, amplitude and duration, were reduced, reminiscent of findings in several neuropsychiatric disorders. Together with previous findings implicating BF Npas1 + neurons in stress responsiveness, the anatomical projections of BF Npas1 + neurons and the effect of activating them suggest a possible role for BF Npas1 + neurons in motivationally-driven wakefulness and stress-induced insomnia. Identification of this major subpopulation of BF GABAergic neurons will facilitate studies of their role in sleep disorders, dementia and other neuropsychiatric conditions involving BF. SIGNIFICANCE STATEMENT: We characterize a group of basal forebrain (BF) neurons in the mouse expressing neuronal PAS domain 1 (Npas1), a developmental transcription factor linked to neuropsychiatric disorders. BF Npas1 + neurons are a major subset of GABAergic neurons distinct and more numerous than cholinergic, parvalbumin or glutamate neurons. BF Npas1 + neurons target brain areas involved in arousal, motivation and olfaction. Activation of BF Npas1 + neurons in the light (inactive) period increased wakefulness and the latency to sleep due to increased long wake bouts. Non-REM sleep slow waves and spindles were reduced reminiscent of findings in several neuropsychiatric disorders. Identification of this major subpopulation of BF GABAergic wake-promoting neurons will allow studies of their role in insomnia, dementia and other conditions involving BF.

Negative Allosteric Modulation of Gamma-Aminobutyric Acid A Receptors at α5 Subunit-Containing Benzodiazepine Sites Reverses Stress-Induced Anhedonia and Weakened Synaptic Function in Mice.

BACKGROUND: Abnormal reward processing, typically anhedonia, is a hallmark of human depression and is accompanied by altered functional connectivity in reward circuits. Negative allosteric modulators of GABAA (gamma-aminobutyric acid A) receptors (GABA-NAMs) have rapid antidepressant-like properties in rodents and exert few adverse effects, but molecular targets underlying their behavioral and synaptic effects remain undetermined. We hypothesized that GABA-NAMs act at the benzodiazepine site of GABAA receptors containing α5 subunits to increase gamma oscillatory activity, strengthen synapses in reward circuits, and reverse anhedonia. METHODS: Anhedonia was induced by chronic stress in male mice and assayed by preferences for sucrose and female urine (n = 5-7 mice/group). Hippocampal slices were then prepared for electrophysiological recording (n = 1-6 slices/mouse, 4-6 mice/group). Electroencephalography power was quantified in response to GABA-NAM and ketamine administration (n = 7-9 mice/group). RESULTS: Chronic stress reduced sucrose and female urine preferences and hippocampal temporoammonic-CA1 synaptic strength. A peripheral injection of the GABA-NAM MRK-016 restored hedonic behavior and AMPA-to-NMDA ratios in wild-type mice. These actions were prevented by pretreatment with the benzodiazepine site antagonist flumazenil. MRK-016 administration increased gamma power over the prefrontal cortex in wild-type mice but not α5 knockout mice, whereas ketamine promoted gamma power in both genotypes. Hedonic behavior and AMPA-to-NMDA ratios were only restored by MRK-016 in stressed wild-type mice but not α5 knockout mice. CONCLUSIONS: α5-Selective GABA-NAMs exert rapid anti-anhedonic actions and restore the strength of synapses in reward regions by acting at the benzodiazepine site of α5-containing GABAA receptors. These results encourage human studies using GABA-NAMs to treat depression by providing readily translatable measures of target engagement.

Harnessing psilocybin: antidepressant-like behavioral and synaptic actions of psilocybin are independent of 5-HT2R activation in mice.

Depression is a widespread and devastating mental illness and the search for rapid-acting antidepressants remains critical. There is now exciting evidence that the psychedelic compound psilocybin produces not only powerful alterations of consciousness, but also rapid and persistent antidepressant effects. How psilocybin exerts its therapeutic actions is not known, but it is widely presumed that these actions require altered consciousness, which is known to be dependent on serotonin 2A receptor (5-HT2AR) activation. This hypothesis has never been tested, however. We therefore asked whether psilocybin would exert antidepressant-like responses in mice and, if so, whether these responses required 5-HT2AR activation. Using chronically stressed male mice, we observed that a single injection of psilocybin reversed anhedonic responses assessed with the sucrose preference and female urine preference tests. The antianhedonic response to psilocybin was accompanied by a strengthening of excitatory synapses in the hippocampus-a characteristic of traditional and fast-acting antidepressants. Neither behavioral nor electrophysiological responses to psilocybin were prevented by pretreatment with the 5-HT2A/2C antagonist ketanserin, despite positive evidence of ketanserin's efficacy. We conclude that psilocybin's mechanism of antidepressant action can be studied in animal models and suggest that altered perception may not be required for its antidepressant effects. We further suggest that a 5-HT2AR-independent restoration of synaptic strength in cortico-mesolimbic reward circuits may contribute to its antidepressant action. The possibility of combining psychedelic compounds and a 5-HT2AR antagonist offers a potential means to increase their acceptance and clinical utility and should be studied in human depression.

(R)-Ketamine exerts antidepressant actions partly via conversion to (2R,6R)-hydroxynorketamine, while causing adverse effects at sub-anaesthetic doses.

BACKGROUND AND PURPOSE: (R)-Ketamine (arketamine) may have utility as a rapidly acting antidepressant. While (R)-ketamine has lower potency than (R,S)-ketamine to inhibit NMDA receptors in vitro, the extent to which (R)-ketamine shares the NMDA receptor-mediated adverse effects of (R,S)-ketamine in vivo has not been fully characterised. Furthermore, (R)-ketamine is metabolised to (2R,6R)-hydroxynorketamine (HNK), which may contribute to its antidepressant-relevant actions. EXPERIMENTAL APPROACH: Using mice, we compared (R)-ketamine with a deuterated form of the drug (6,6-dideutero-(R)-ketamine, (R)-d2 -ketamine), which hinders its metabolism to (2R,6R)-HNK, in behavioural tests predicting antidepressant responses. We also examined the actions of intracerebroventricularly infused (2R,6R)-HNK. Further, we quantified putative NMDA receptor inhibition-mediated adverse effects of (R)-ketamine. KEY RESULTS: (R)-d2 -Ketamine was identical to (R)-ketamine in binding to and functionally inhibiting NMDA receptors but hindered (R)-ketamine's metabolism to (2R,6R)-HNK. (R)-Ketamine exerted greater potency than (R)-d2 -ketamine in several antidepressant-sensitive behavioural measures, consistent with a role of (2R,6R)-HNK in the actions of (R)-ketamine. There were dose-dependent sustained antidepressant-relevant actions of (2R,6R)-HNK following intracerebroventricular administration. (R)-Ketamine exerted NMDA receptor inhibition-mediated behaviours similar to (R,S)-ketamine, including locomotor stimulation, conditioned-place preference, prepulse inhibition deficits, and motor incoordination, with approximately half the potency of the racemic drug. CONCLUSIONS AND IMPLICATIONS: Metabolism of (R)-ketamine to (2R,6R)-HNK increases the potency of (R)-ketamine to exert antidepressant-relevant actions in mice. Adverse effects of (R)-ketamine require higher doses than those necessary for antidepressant-sensitive behavioural changes in mice. However, our data revealing that (R)-ketamine's adverse effects are elicited at sub-anaesthetic doses indicate a potential risk for sensory dissociation and abuse liability.

Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.