Robert C. Malenka

Studies reporting the effects of psychedelic drugs on mouse behavior are rapidly emerging. However, the reproducibility and reliability of these effects remain uncertain. Here we implemented a multi-institutional collaborative approach to assess which effects of psilocybin on mouse behavior are robust and replicable. Five independent laboratories performed the same experiments to test the acute and persistent effects of psilocybin (2 mg kg⁻¹, intraperitoneally) on various behaviors. Psilocybin acutely increased anxiety- and avoidance-related behaviors and decreased fear expression in a robust and replicable manner. Surprisingly, however, psilocybin did not have replicable effects 24 h after administration in terms of reducing anxiety- and depression-like behaviors or facilitating fear extinction learning. Additionally, we did not observe psilocybin-induced alterations in social preference or social reward learning. Our characterization of psilocybin’s behavioral effects, using ~200 male and female mice per experiment across five laboratories, demonstrates several robust acute effects and suggests that some of psilocybin’s reported persistent effects in mice may be modest and/or inconsistent.

Synaptic NMDA-type glutamate receptors (NMDARs) are central components of all neural computations. NMDAR levels critically vary between synapses but how the synaptic localization of NMDARs is controlled remains unclear. Here we show that SIK3, a serine–threonine protein kinase, localizes to synapses where it phosphorylates the obligatory NMDAR subunit GluN1 at a single site. This single phosphorylation event controls NMDAR recruitment to synapses. Genetic deletion of SIK3 severely and selectively decreases synaptic GluN1 levels by ~60% without altering AMPA-type glutamate receptor levels or inducing major transcriptional changes. Pharmacological inhibition of SIK3 impairs synaptic NMDAR responses within 45 minutes. Mutating the SIK3-phosphorylation site inhibits GluN1 synaptic recruitment. ‘Sleepy’, a gain-of-function mutation in SIK3 that increases sleep, selectively enhances synaptic NMDAR levels by increasing GluN1 phosphorylation. Thus, SIK3 phosphorylation of GluN1 provides a key signal for recruiting NMDARs to synapses.

Nucleus accumbens (NAc) deep brain stimulation (DBS) has been increasingly explored as a treatment modality for refractory neuropsychiatric disorders. Uncovering the accumbens network that is engaged by DBS is a critical step forward in understanding how modulating this important node impacts the broader mesocorticolimbic circuit. Using whole-brain clearing and unbiased, brain-wide neural activity mapping, we found that NAc DBS increases neural activity in a coordinated mesocorticolimbic network in mice. Simultaneous intracranial electrophysiology recordings from the human NAc and brief stimulation epochs of analogous mesocorticolimbic nodes revealed patterns of connectivity between these regions and the NAc. Altogether, these results link brain-wide network mapping in mice with a hypothesis-driven test of human NAc connectivity, consistent with a coordinated mesocorticolimbic network that may be relevant to the mechanisms of NAc DBS.

The opioid crisis has emphasized the need for more effective treatments for opioid use disorder (OUD) ^1–3 , which is characterized by habitual opioid use to avoid withdrawal symptoms ^4,5 . Both physical and affective symptoms contribute to opioid withdrawal yet whether different neural mechanisms mediate these different symptom domains and contribute distinctly to opioid relapse is unknown. While neurons expressing mu opioid receptors (MORs) gate opioids’ reinforcing effects ^6–8 by increasing dopamine (DA) release in nucleus accumbens (NAc), sharp decreases in NAc DA release are associated with withdrawal ^9–11 , the cellular and circuit mechanisms of which are unknown. Here we describe an unusual population of evolutionarily-conserved MOR+ neurons in the NAc expressing the transcription factor Tshz1 . Increased activity in these neurons is required for withdrawal aversion learning. Deletion of MORs in Tshz1 neurons prevented withdrawal-induced decreases in DA release and affective aversion, but not physical symptoms associated with withdrawal. Pharmacological activation of mGluR8, which is preferentially expressed in Tshz1 neurons, reduced withdrawal aversion. Thus, by dissociating the circuit mechanisms contributing to the physical and affective components of opioid withdrawal focusing on the critical role of Tshz1 neurons, we have identified a novel druggable target with therapeutic potential for treating key OUD withdrawal symptoms.

Classic theories propose opponent functions for striatal dopamine (DA) and serotonin (5-hydroxytryptamine; 5HT), with DA promoting approach and 5HT promoting patience or avoidance. How these neuromodulators regulate downstream circuits to achieve such antagonistic effects remains mysterious. Here, we mapped striatal 5HT receptor expression in mice to reveal preferential enrichment of putatively excitatory, G_q-coupled 5HT receptors on medium spiny neurons expressing inhibitory D2 DA receptors (D2-MSNs). Acute slice recordings from genetically identified striatal neurons showed that DA and 5HT inversely regulate D2-MSN firing and confirmed that 5HT’s excitatory effect on these cells is blocked by 5HT2a and 5HT2c receptor antagonists. Pharmacologically upregulating striatal 5HT release preferentially induced cfos expression in D2-MSNs, validating that 5HT’s excitatory effect on these cells also occurs in vivo. Finally, 5HT2c receptor loss-of-function in D2-MSNs, but not D1-MSNs, enhanced animals’ sensitivity to the behavioral effects of cocaine – a potent releaser of DA and 5HT – showing that 5HTergic excitation of D2-MSNs counteracts the reinforcing effects of striatal DA release. Altogether these results demonstrate that DA and 5HT inversely modulate D2-MSN activity to regulate cocaine reward, identifying a key circuit mechanism underlying the opponent behavioral effects of these important neuromodulators.

Serotonin (5-hydroxytryptamine; 5HT) has long been considered anti-aggressive, but the mechanisms by which 5HT regulates downstream circuits to control aggression remain unclear. Combining fiber photometry, optogenetics, and miniaturized microscope recordings in double-transgenic male mice, we find that 5HT levels ramp up in the nucleus accumbens during aggression, inhibiting a subset of D1 medium spiny neurons to suppress attacks. Our results reveal a novel 5HT-mediated neuromodulatory mechanism for limiting aggressive behavior.

Effort is costly: given a choice, we tend to avoid it¹. However, in many cases, effort adds value to the ensuing rewards². From ants³ to humans⁴, individuals prefer rewards that had been harder to achieve. This counterintuitive process may promote reward seeking even in resource-poor environments, thus enhancing evolutionary fitness⁵. Despite its ubiquity, the neural mechanisms supporting this behavioural effect are poorly understood. Here we show that effort amplifies the dopamine response to an otherwise identical reward, and this amplification depends on local modulation of dopamine axons by acetylcholine. High-effort rewards evoke rapid acetylcholine release from local interneurons in the nucleus accumbens. Acetylcholine then binds to nicotinic receptors on dopamine axon terminals to augment dopamine release when reward is delivered. Blocking the cholinergic modulation blunts dopamine release selectively in high-effort contexts, impairing effortful behaviour while leaving low-effort reward consumption intact. These results reconcile in vitro studies, which have long demonstrated that acetylcholine can trigger dopamine release directly through dopamine axons^{6,7,8,9,10,11}, with in vivo studies that failed to observe such modulation^12,13,14, but did not examine high-effort contexts. Our findings uncover a mechanism that drives effortful behaviour through context-dependent local interactions between acetylcholine and dopamine axons.

The psychedelic psilocybin may have lasting therapeutic effects for patients with chronic pain syndromes. Some preclinical data suggest these putative benefits derive from direct analgesic effects; however, this possibility has not been comprehensively tested in preclinical models. Here, we evaluated the analgesic properties of a single exposure to psilocybin at acute and chronic time points in Complete Freund’s Adjuvant-induced inflammatory pain, spared nerve injury model of neuropathic pain, and acid-induced muscle pain. Across these models, we tested a range of doses (0.3, 2, and 10 mg/kg i.p.) in male and female mice using multiple behavioral assays evaluating sensory aspects (von Frey, cold plate, Hargreaves, thermal place preference, and muscle withdrawal threshold) and functional aspects of pain (marble burying). We further tested the effects of psilocybin on the affective dimension of pain in a surgical model of acute pain (mouse grimace scale). Except for cold sensitivity, we found no effect of psilocybin across pain models, behavioral assays, drug doses, or sex. The apparent reduction in cold sensitivity may be explained by profound hypothermia induced by psilocybin rather than true analgesia.