Behavioral Neuroscience publications

Many species display mating-related sleep suppression to maximize reproductive success, yet the underlying neural mechanisms in mammals remain unclear. Here, we report that male mice exhibit delayed sleep onset and reduced sleep duration in periods following successful mating, while females are unaffected. Single-cell transcriptomics identifies a group of sleep-promoting Rxfp1-expressing neurons in the preoptic area of hypothalamus (POA Rxfp1 neurons), and fiber photometry reveals selective inhibition of them during and after mating in male mice. Lifting this inhibition via chemogenetic activation reverses sleep suppression in mated males. Monosynaptic retrograde tracing from these POA Rxfp1 neurons identifies male-biased γ-aminobutyric acid–releasing inputs from the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr). Notably, optogenetic activation of these neurons induces immediate wakefulness specifically in male mice, and BNSTpr → POA Rxfp1 neurons are hyperactive after mating. Together, these findings establish a BNSTpr → POA Rxfp1 circuit that integrates reproductive signals for male-specific sleep suppression in mice.

The lateral habenula (LHb) integrates cortical and basal forebrain (BF) input and is implicated in negative affective states, but its involvement in negative affect associated with cannabis withdrawal is unknown. Here, effects of withdrawal from the psychoactive cannabis constituent, Δ 9 -tetrahydrocannabinol (Δ 9 -THC), on fear conditioning (FC) and LHb function were measured in rats using calcium photometry and electrophysiology. FC-associated LHb calcium signals, freezing behavior, and LHb excitatory-inhibitory (E-I) ratios were increased during Δ 9 -THC withdrawal, and local GABA release was impaired, indicating disinhibition of LHb output neurons. GABA release was also impaired at optogenetically activated BF-LHb axons, and not from ventral tegmental area-LHb inputs. The results indicate that Δ 9 -THC withdrawal increases LHb excitability through disinhibition, contributing to increased fear of primary and conditioned aversive stimuli. Our findings implicate the LHb in cannabis withdrawal-associated negative affect and offer insight into the neural mechanisms of cannabis use disorder.

Recent work shows that the brain’s arousal system remains active during sleep, with rhythmic locus coeruleus (LC) activity shaping sleep architecture and supporting memory consolidation. The LC releases norepinephrine (NE) in infraslow (~0.02 Hz) bouts that gate NREM sleep spindles. Here, we demonstrate that heart rate (HR) fluctuations during NREM are tightly phase-locked to these NE rhythms, identifying the LC as a key driver of very-low-frequency HR variability (VLF-HRV), an understudied autonomic signal. Using optogenetics, transient LC inhibition blunts HR slowing, whereas LC activation produces rapid HR acceleration, demonstrating a direct LC-HR relationship during sleep that is maintained across a defined range of LC activity levels but breaks down when LC activity becomes excessive. We further show that infraslow HR variability is a cross-species marker of spindle-dependent memory processing. In mice, the amplitude of HR decelerations during NREM correlates with spindle activity and subsequent memory performance. Remarkably, human sleepers show the same pattern: stronger VLF-HR fluctuations during NREM correspond to increased spindle expression and better overnight memory retention. These findings reveal a mechanistic pathway through which graded changes in LC activity, up to a critical level, modulates autonomic physiology during sleep and identify infraslow HR variability as a non-invasive marker of brainstem function and memory-promoting sleep. Because LC degeneration occurs early in neurodegenerative disease, sleep-derived HR metrics may provide a scalable indicator of emerging neuromodulatory dysfunction.

Animals defend protein intake strongly, yet how protein status shapes moment-to-moment reward signalling in mesolimbic circuits remains unclear. Here, we examined how dietary protein restriction shapes nucleus accumbens (NAc) core dopamine signalling during operant feeding in the home-cage. C57BL/6 mice expressing the GRAB DA sensor in NAc core performed 1 h fixed-ratio 1 (FR1) and progressive ratio (ProgRatio) sessions to earn grain or sucrose pellets across three dietary phases; an initial non-restricted phase (NR1), a protein-restricted phase (PR), and a return to the non-restricted diet (NR2). Home-cage food intake remained stable across phases, whereas bodyweight gain was markedly reduced during PR and partially recovered in NR2. Behaviourally, operant responding increased during PR under both FR1 and ProgRatio schedules, with the most robust enhancement observed for grain pellets. Fibre photometry revealed a more selective neural effect. During FR1, pellet delivery evoked a larger NAc dopamine response for grain than for sucrose specifically during PR, whereas no pellet-type difference was detected during NR1 or NR2. By contrast, under progressive ratio, dopamine responses at pellet delivery and pellet retrieval did not differ between pellet types in any dietary phase. Together, these findings show that protein restriction does not globally amplify reward-related behaviour or dopamine signalling, but instead selectively biases NAc dopamine encoding in a manner that depends on nutritional status of the animal, nutrient context, and task demands.

Social behavior varies considerably across mammalian species, yet the neural basis for these variations remains unclear. We compared the behavior of three commonly used laboratory rodent strains (C57BL/6J and CD1 mice and SD rats) in social preference and free interaction tasks. Animals carried chronically implanted electrodes in brain regions associated with social motivation. The three strains exhibited distinctly structured social behavior: SD rats showed the strongest social motivation, CD1 mice were most active, and C57BL/6J mice displayed the most restrained behavior. Electrophysiology revealed strain- and task-specific theta and gamma power and coherence. Strikingly, SD rats displayed low baseline coherence followed by robust induction during interactions, while C57BL/6J mice exhibited opposite tendencies. Machine learning models allowed accurate strain separation based solely on brain electrophysiology, with theta and gamma coherence between the prelimbic cortex and nucleus accumbens shell playing a key role in defining distinct neuronal signatures of strain-specific social strategies.

The parabrachial nucleus (PBN) is an important hub located in the pons that relays sensory signals from peripheral regions. It is genetically diverse and contains many populations that modulate feeding and responses to threatening situations. A small Ntsr1 -expressing population of neurons was identified that projects selectively to the ventromedial hypothalamus (VMH). The Ntsr1 neurons are scattered throughout the lateral PBN with a cluster of cells along the border to the nucleus of the lateral lemniscus that overlap with Cck and Foxp2 expression. Chemogenetic activation of PBN Ntsr1 neurons results in Fos induction in Nr5a1 (SF1) and Bdnf neurons in the VMH. Activation of PBN Ntsr1 neurons or their terminals in the VMH reduces food intake after fasting and increases anxiety-like behaviors. In anxiogenic feeding assays, activation of PBN Ntsr1 neurons increases latency to feed as well as reducing food intake. Photometry showed that PBN Ntsr1 -neuronal activity increases during anxiogenic situations but is suppressed during food consumption, suggesting a role in threat-induced suppression of feeding. Silencing PBN Ntsr1 neurons with tetanus toxin light-chain increased food intake and reduced anxiety. These findings reveal a genetically defined PBN to VMH circuit that responds to threats and suppresses feeding behavior.

Exposure to stress can cause long-lasting enhancement of fear and other defensive responses that extend beyond the cues or contexts associated with the original traumatic event. These nonassociative consequences of stress, referred to as fear sensitization, are thought to underlie some symptoms of trauma-related disorders. Fear sensitization has been predominantly studied using the stress-enhanced fear learning (SEFL) paradigm, which models the stress-induced amplification of fear learning. Less is known about the mechanisms through which unlearned fear responses are sensitized by stress. Here, we investigated the neural mechanisms for sensitization of unlearned fear responses using a paradigm we termed stress-enhanced fear responding (SEFR). In this model, mice exposed to a single session of footshock stress exhibit enhanced freezing to a novel tone stimulus. To investigate brain regions that might mediate SEFR, we first used c-Fos mapping to identify neural activity changes associated with stress-induced enhancement of unlearned fear. Our c-Fos screen identified the posterior paraventricular thalamus (pPVT) as a region that was persistently hyperactive after footshock stress and whose activity correlated with behavioral expression of SEFR. Using fiber photometry, we observed that SEFR, but not SEFL, was associated with increased activity in the pPVT. Next, we found that chemogenetic inhibition of the pPVT blocked both the induction of SEFR during stress and its later expression, while artificial stimulation of pPVT in stress-naive mice was sufficient to recapitulate SEFR. Interestingly, pPVT inhibition or stimulation did not affect acquisition or expression of SEFL. In conclusion, our results indicate that sensitization of fear learning (SEFL) and sensitization of unlearned fear (SEFR) have distinct neural mechanisms. Our results identify pPVT hyperactivity as a mechanism for stress-induced sensitization of unlearned fear and highlight pPVT as a potential target for treating arousal and reactivity symptoms of trauma- and stressor-related disorders.

The basolateral amygdala (BLA) is important for assigning emotional weight to environmental stimuli to guide learning, discrimination, and adaptive vigilance. The contribution of neuromodulator inputs such as dopamine to this process is unclear. Here, we recorded in vivo BLA dopamine signals across emotional learning paradigms. We show that BLA dopamine scales with a stimulus's emotional intensity, but not value or associative strength, and reward-predictive cues evoke BLA dopamine signals that diminish, rather than grow, with training. In a complex learning context, where rats discriminated between multiple valenced associations, the magnitude of cue-evoked BLA dopamine re-scaled to report the level of relative emotional saliency. Threat and safety cues prompted larger, sustained dopamine signals compared to reward and neutral cues. Together, our data reveal a pattern of dopamine encoding with a unique contribution to learning, for dynamic disambiguation of affective states.

Adaptive behavior requires that behaviorally relevant signals gain access to neural circuits guiding action. The thalamus has long been proposed to regulate information flow to cortical and subcortical systems, yet whether it also tracks internally generated goal signals remains unclear. Here, we show that neurons in the paraventricular thalamus (PVT) projecting to the nucleus accumbens (NAc) encode the motivational value of safety during active avoidance. As mice learn to avoid threat, PVT→NAc neurons develop a signal that emerges selectively at successful avoidance, is experience-dependent, and diminishes following outcome devaluation. Selective silencing of the PVT→NAc pathway at safety onset reduces the motivational value assigned to safety without impairing action–outcome learning. Mechanistically, PVT input engages cholinergic interneurons (CIN) in the NAc to regulate dopamine release via synaptic potentiation mediated by GluA2-lacking AMPA receptors at PVT–CIN synapses. Disrupting this plasticity reduces the motivational impact of safety. These findings identify a thalamostriatal mechanism through which learned goals gain stable access to motivational circuitry.

Many substances and drugs are administered intranasally. These include opioid overdose reversal drugs, anti-epileptic medications, migraine medications, hormone treatments and medicines to treat/prevent allergies, colds and flus, including nasally administered vaccines, corticosteroids, antihistamines and decongestants. In addition, intranasal (IN) administration is the preferred route of entry by users of illicit drugs. Despite the widespread use of the IN route of administration, there is no established paradigm to access this route of administration preclinically to yield precise and reliable control over delivery. This poses major gaps in therapeutic discovery/testing, establishing pharmacokinetic/pharmacodynamic relationships of therapeutics and understanding the mechanisms of actions of therapeutics. Here we developed an in-dwelling microfluidic device that, when implanted upon the nasal bone in mice, accesses the nasal cavity to allow reliable and precise IN fluid delivery during freely moving behavior. We validated this device, called the nasal access port (NAP), to confirm it allows rapid and precise control of fluids. We further exemplified the application of the NAP for studying outcomes of IN cocaine in mice, including its pharmacokinetic profile, and both the rapid release of dopamine and behavioral effects upon IN cocaine. By achieving precise and reliable access to the IN route of administration, the NAP represents a substantial methodological advance with broad applicability in the biomedical and life sciences, especially in the neuroscience, pharmacology, medicinal chemistry and physiology domains.

The medial nucleus accumbens shell (medNAcSh) is a key regulator of hedonic feeding, controlling reward consumption through its projections to downstream structures. Recent studies showed that the primary cellular mediators of these effects are dopamine 1 receptor-positive striatal projection neurons (D1-SPNs). Specifically, D1-SPN activity gets inhibited during reward consumption, and such inhibition is necessary and sufficient to authorize consumption, independent of metabolic need. Anatomically, the medNAcSh spans 1–1.5 mm along the rostro-caudal axis in mice, and previous studies have reported functional gradients along this axis. For instance, pharmacological studies have suggested that rostral rather than caudal medNAcSh regulates appetitive behavior. However, the mechanisms underlying this topographical gradient remain unknown. Here, we hypothesized that D1-SPNs contribute to this gradient by regulating hedonic feeding via a specific hotspot in the rostral medNAcSh. Using calcium monitoring with fiber photometry in mice, we show that rostral medNAcSh D1-SPNs demonstrate inhibitory responses during reward consumption, while caudal D1-SPNs do not. Consistently, optogenetic stimulation of rostral D1-SPNs inhibits consumption, while stimulation of caudal D1-SPNs had minimal effects, confirming the existence of a functional rostro-caudal gradient. Importantly, we observed no differences between rostral and caudal D1-SPNs in their responses to aversive stimuli, suggesting that the D1-SPN gradient is specific to appetitive contexts. To investigate potential molecular correlates of this functional gradient, we leveraged open-source anatomy datasets and performed fluorescent in situ hybridization, identifying Stard5 and Peg10 as markers enriched in the rostral and caudal medNAcSh, respectively. Finally, we developed a novel Stard5-Flp driver line to selectively target the rostral hotspot and demonstrated that Stard5+ neurons recapitulate rostral D1-SPN activity patterns. Together, these findings establish a spatially confined rostral medNAcSh subregion as a critical regulator of reward consumption and introduce Stard5 as a molecular tool for its manipulation, offering new opportunities for intervention in dysregulated eating.

Quickly adapting behavior to changing environments is key for survival. Brain dopamine signals reinforce actions that procure rewards and avoid adverse outcomes. However, the molecular mechanisms within the circuits that actuate these signals at subsecond timescales remain unexplored. We show that midbrain endocannabinoid mobilization is the conditio sine qua non for behavioral invigoration evoked by external cues. Reward- and punishment-predictive signals require intact retrograde release of the endocannabinoid 2-arachidonoylglycerol (2-AG) from ventral tegmental area dopamine neurons. Moreover, we show that genetic deletion of presynaptic cannabinoid type-1 receptors (CB1Rs), the target for 2-AG, recapitulates behavioral and dopaminergic deficits. Exploiting causal inference tools to interpret continuous brain signals, we describe that 2-AG/CB1R communication is essential for striatal dopamine release to signal salient cues and propel conditioned responding. These findings reveal an endocannabinoid-mediated disinhibition mechanism exploited by dopamine neurons to orchestrate striatal dopamine release events, enabling adaptive action selection informed by cues.

Traumatic brain injuries (TBIs) are more than mere lesions and generate a persistent secondary pathology. This, combined with functional reorganization of circuits post-injury, may explain the increased risk for psychiatric disorders in patients with TBI. In the current studies, we demonstrate that frontal TBI changed the Pavlovian behavioral response to reinforcer-predicting cues and reduced the motivational value of cues. TBI also chronically impaired decision-making on a gambling-like task with reinforcer-paired cues. To investigate how these changes occur, we evaluated the nucleus accumbens (NAc) core. At a subacute time point (14 days), we confirmed reduced input to the NAc with optogenetics and evaluated electrophysiological and transcriptional changes. TBI increased neuronal excitability and the single nucleus RNA sequencing profile indicated a substantial stress and inflammatory response, but also high indicators of plasticity, particularly in D1- and D2-positive medium spiny neurons. To evaluate how these subacute changes transitioned to chronic NAc dysfunction, we measured immunohistochemical surrogates of activity post-mortem and recorded calcium activity from the NAc after TBI during Pavlovian conditioning. TBI reduced histological markers of activity and reduced cue-evoked calcium activity. Overall, these data indicate that substantial reorganization of the NAc occurs following frontal brain injury. A primary effect of this is to reduce the salience of environmental cues linked to outcomes. The inability to properly process outcomes could contribute to broader psychiatric symptoms after TBI, including impairments in decision-making, behavioral flexibility, and impulsivity but also presents a potential treatment target.

Developing safe and effective pain medications is an ongoing challenge for human health. Agonists for the µ-opioid receptor (MOR) are essential pain medications, but their high intrinsic efficacy also induces adverse side effects, including respiratory depression, constipation, tolerance, dependence, withdrawal and addiction 1–7 . Strategies to limit adverse effects traditionally include developing MOR agonists that have low intrinsic efficacy or that preferentially activate G-protein signalling over β-arrestin signalling 8 . Here we identify a novel MOR agonist with supramaximal intrinsic efficacy and a unique pharmacological profile that produced effective analgesia in rodents with minimal adverse effects. N -desethyl-fluornitrazene (DFNZ) was derived from a class of synthetic benzimidazole opioids called nitazenes. DFNZ has impaired brain penetrance, a unique spatiotemporal MOR cellular signalling profile, and diminished efficacy at the MOR–galanin 1 receptor (GAL1) heteromer. DFNZ does not induce respiratory depression, tolerance or MOR downregulation after repeated exposure. Compared with other MOR agonists, DFNZ has limited effects on dopamine neurotransmission in nucleus accumbens and weaker reinforcing effects in the drug self-administration procedure. These results provide novel insights about MOR and nitazene pharmacology, have important implications for pain and addiction treatment, and challenge the prevailing dogma that high-efficacy MOR agonists cannot constitute safe and effective therapeutic agents.

Glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone, and pharmacologic modulation of central GIP receptors (GIPRs) improves energy homeostasis and prevents conditioned taste avoidance (CTA). However, the mechanisms by which GIPR signaling impact food intake and aversion are incompletely understood. Here, we show that GIPR agonism abrogates the aversive and enhances the anorexigenic effects of the pro-inflammatory cytokine interleukin-1β (IL-1β). Aversion-encoding parabrachial calcitonin gene-related peptide (CGRP) neurons were required for IL-1β-induced CTA but not anorexia. Moreover, systemic IL-1β increased CGRP neural activity in vivo, and this was significantly attenuated by co-administration of a GIPR agonist. By contrast, GIPR in the dorsal vagal complex was required for the acute anorectic effect of GIPR agonism but not its anti-aversive effect. Taken together, our data suggest that GIPR agonism reduces food intake and prevents aversion via distinct circuits and that GIPR agonism may represent an effective approach to alleviate inflammation-induced aversion.

The ability to associate environmental stimuli with positive outcomes is a fundamental form of learning. While extensive research has focused on midbrain dopamine neurons during associative learning, less is known about learning-mediated changes in the afferents that shape dopamine neuron responses. We demonstrate in rats that during critical phases of learning, anion homeostasis in midbrain inhibitory GABA neurons - a primary source of input to dopamine neurons - is disrupted due to downregulation of the potassium chloride cotransporter KCC2. This alteration in GABA neurons preferentially impacted lateral mesoaccumbal dopamine pathways and was not observed after learning was established. At the network level, learning-mediated KCC2 downregulation was associated with enhanced synchronization between individual GABA neurons and increased dopamine responses to rewards and reward-related stimuli. Conversely, enhancing KCC2 function during learning reduced GABA synchronization, diminished relevant dopamine signaling, and prevented cue-reward associations. Thus, circuit-specific adaptations in midbrain GABA neurons are crucial for forming new reward-related behaviors.

Drugs of abuse in adolescence impact brain maturation and increase psychiatric risk, with differences in sensitivity between males and females. Amphetamine in early adolescence (postnatal day; PND 21 ± 1–32 ± 1) in male, but not female mice, causes dopamine axons intended to innervate the nucleus accumbens and to grow ectopically to the prefrontal cortex (PFC). This is mediated by drug-induced downregulation of the Netrin-1 receptor DCC. How off-target dopamine axons function in the adult PFC remains to be determined. Here we report that males and females show place preference for amphetamine in early adolescence. However, only in males, amphetamine increases PFC dopamine transporter expression in adulthood (PND 101 ± 15): leading to aberrant baseline dopamine transients, faster dopamine release, and exaggerated responses to acute methylphenidate. Upregulation of DCC in adolescence, using CRISPRa, prevents all these changes. Mesolimbic dopamine axons rerouted to the PFC in adolescence retain anatomical and functional phenotypes of their intended target, rendering males enduringly vulnerable to the harmful effects of drugs of abuse.

The lateral habenula (LHb) integrates cortical and basal forebrain (BF) input to control monoaminergic outflow and is implicated in depression, anxiety, impulsivity, and aversion. Although cannabis withdrawal is associated with negative affect and heightened anxiety, LHb involvement is not established. Here, effects of withdrawal from the psychoactive cannabis constituent Δ 9 -tetrahydrocannabinol (Δ 9 -THC) on LHb neurons were assessed with photometric calcium measurement during fear conditioning and in vitro electrophysiology. LHb calcium signals were larger during footshock, and presentation of a tone paired with footshock in Δ 9 -THC withdrawn rats. Fear-induced freezing to the tone was also larger during Δ 9 -THC withdrawal. Electrophysiology revealed larger LHb excitatory-inhibitory (E-I) ratios during Δ 9 -THC withdrawal, resulting from impaired synaptic GABA release. Moreover, GABA release via optogenetic activation of BF-LHb inputs was impaired during Δ 9 -THC withdrawal, whereas no changes occurred at ventral tegmental area-LHb inputs. Recovery of BF-LHb GABA release and cannabinoid CB1 receptor desensitization were incomplete at 30 days of Δ 9 -THC withdrawal. The data show that LHb responsivity to primary aversive and conditioned stimuli are increased during Δ 9 -THC withdrawal, and that this is likely mediated by altered E-I balance. We propose negative affect experienced during cannabis withdrawal is related to LHb hypersensitivity to aversive stimuli and this facilitates encoding of associated environmental cues.

Anxiety disorders impose a substantial global burden, yet current pharmacotherapies often lack sustained efficacy and carry risks of dependence. Dexmedetomidine (Dex), a selective α2 adrenergic receptor agonist primarily used for sedation, demonstrates promising anxiolytic properties, but its long-term neural mechanisms remain unclear. Here, using a chronic restraint stress (CRS) mouse model, we reveal that tyrosine hydroxylase (TH)-positive neurons in the locus coeruleus (LC) are persistently hyperactivated during chronic anxiety states, driving elevated norepinephrine (NE) release in the medial prefrontal cortex (mPFC). A single low dose of Dex (50 µg/kg) produced anxiolytic effects lasting ≥3 days, evidenced by improved performance in open-field, light-dark box, and elevated plus maze tests. In vivo fiber photometry and chemogenetic approaches demonstrated that Dex suppresses LC-NE neuronal hyperactivity and normalizes mPFC NE levels. Crucially, neuron-specific knockdown of α2 receptors in the LC abolished Dex's anxiolytic effects, confirming their essential role. These findings elucidate a neural mechanism wherein Dex sustains anxiety relief via α2 receptor-mediated inhibition of the LC-NE-mPFC circuit, highlighting its potential as a novel long-acting therapeutic strategy for anxiety disorders.

Long-term sustained pain following acute physical injury is a prominent feature of chronic pain conditions1. Populations of neurons that rapidly respond to noxious stimuli or tissue damage have been identified in the spinal cord and several nuclei in the brain2-4. Understanding the central mechanisms that signal ongoing sustained pain, including after tissue healing, remains a challenge5. Here we use spatial transcriptomics, neural manipulations, activity recordings and computational modelling to demonstrate that activity in an ensemble of anatomically and molecularly diverse parabrachial neurons that express the neuropeptide Y (NPY) receptor Y1 (Y1R neurons) is increased following injury and predicts functional coping behaviour. Hunger, thirst or predator cues suppressed sustained pain, regardless of the injury type, by inhibiting parabrachial Y1R neurons via the release of NPY. Together, our results demonstrate an endogenous analgesic hub at pain-responsive parabrachial Y1R neurons.