Acute stress causes sex-dependent changes to ventral subiculum synapses, circuitry, and anxiety-like behavior.

Experiencing a single severe stressor is sufficient to drive sexually dimorphic psychiatric disease development. The ventral subiculum (vSUB) emerges as a site where stress may induce sexually dimorphic adaptations due to its sex-specific organization and pivotal role in stress integration. Using a 1-hr acute restraint stress model, we uncover that stress causes a net decrease in vSUB activity in females that is potent, long-lasting, and driven by adrenergic receptor signaling. By contrast, males exhibit a net increase in vSUB activity that is transient and driven by corticosterone signaling. We further identified sex-dependent changes in vSUB output to the bed nucleus of the stria terminalis and in anxiety-like behavior in response to stress. These findings reveal striking changes in psychiatric disease-relevant brain regions and behavior following stress with sex-, cell-type, and synapse-specificity that contribute to our understanding of sex-dependent adaptations that may shape stress-related psychiatric disease risk.

Molecular and circuit determinants in the globus pallidus mediating control of cocaine-induced behavioral plasticity.

The globus pallidus externus (GPe) is a central component of the basal ganglia circuit that acts as a gatekeeper of cocaine-induced behavioral plasticity. However, the molecular and circuit mechanisms underlying this function are unknown. Here, we show that GPe parvalbumin-positive (GPePV) cells mediate cocaine responses by selectively modulating ventral tegmental area dopamine (VTADA) cells projecting to the dorsomedial striatum (DMS). Interestingly, GPePV cell activity in cocaine-naive mice is correlated with behavioral responses following cocaine, effectively predicting cocaine sensitivity. Expression of the voltage-gated potassium channels KCNQ3 and KCNQ5 that control intrinsic cellular excitability following cocaine was downregulated, contributing to the elevation in GPePV cell excitability. Acutely activating channels containing KCNQ3 and/or KCNQ5 using the small molecule carnosic acid, a key psychoactive component of Salvia rosmarinus (rosemary) extract, reduced GPePV cell excitability and impaired cocaine reward, sensitization, and volitional cocaine intake, indicating its therapeutic potential to counteract psychostimulant use disorder.

VTA µ-opioidergic neurons facilitate low sociability in protracted opioid withdrawal.

Opioids initiate dynamic maladaptation in brain reward and affect circuits that occur throughout chronic exposure and withdrawal that persist beyond cessation. Protracted withdrawal is characterized by negative affective behaviors such as heightened anxiety, irritability, dysphoria, and anhedonia, which pose a significant risk factor for relapse. While the ventral tegmental area (VTA) and mu-opioid receptors (MORs) are critical for opioid reinforcement, the specific contributions of VTAMOR neurons in mediating protracted withdrawal-induced negative affect is not fully understood. In our study, we elucidate the role of VTAMOR neurons in mediating negative affect and altered brain-wide neuronal activities following opioid exposure and withdrawal in male and female mice. Utilizing a chronic oral morphine administration model, we observe increased social deficit, anxiety-related, and despair-like behaviors during protracted withdrawal. VTAMOR neurons show heightened neuronal FOS activation at the onset of withdrawal and connect to an array of brain regions that mediate reward and affective processes. Viral re-expression of MORs selectively within the VTA of MOR knockout mice demonstrates that the disrupted social interaction observed during protracted withdrawal is facilitated by this neural population, without affecting other protracted withdrawal behaviors. Lastly, VTAMORs contribute to heightened neuronal FOS activation in the anterior cingulate cortex (ACC) in response to an acute morphine challenge, suggesting their unique role in modulating ACC-specific neuronal activity. These findings identify VTAMOR neurons as critical modulators of low sociability during protracted withdrawal and highlight their potential as a mechanistic target to alleviate negative affective behaviors associated with opioid withdrawal.

Molecular and circuit determinants in the globus pallidus mediating control of cocaine-induced behavioral plasticity.

The globus pallidus externus (GPe) is a central component of the basal ganglia circuit, receiving strong input from the indirect pathway and regulating a variety of functions, including locomotor output and habit formation. We recently showed that it also acts as a gatekeeper of cocaine-induced behavioral plasticity, as inhibition of parvalbumin-positive cells in the GPe (GPe PV ) prevents the development of cocaine-induced reward and sensitization. However, the molecular and circuit mechanisms underlying this function are unknown. Here we show that GPe PV cells control cocaine reward and sensitization by inhibiting GABAergic neurons in the substantia nigra pars reticulata (SNr GABA ), and ultimately, selectively modulating the activity of ventral tegmental area dopamine (VTA DA ) cells projecting to the lateral shell of the nucleus accumbens (NAcLat). A major input to GPe PV cells is the indirect pathway of the dorsomedial striatum (DMS D 2 ), which receives DAergic innervation from collaterals of VTA DA →NAcLat cells, making this a closed-loop circuit. Cocaine likely facilitates reward and sensitization not directly through actions in the GPe, but rather in the upstream DMS, where the cocaine-induced elevation of DA triggers a depression in DMS D 2 cell activity. This cocaine-induced elevation in DA levels can be blocked by inhibition of GPe PV cells, closing the loop. Interestingly, the level of GPe PV cell activity prior to cocaine administration is correlated with the extent of reward and sensitization that animals experience in response to future administration of cocaine, indicating that GPe PV cell activity is a key predictor of future behavioral responses to cocaine. Single nucleus RNA-sequencing of GPe cells indicated that genes encoding voltage-gated potassium channels KCNQ3 and KCNQ5 that control intrinsic cellular excitability are downregulated in GPe PV cells following a single cocaine exposure, contributing to the elevation in GPe PV cell excitability. Acutely activating channels containing KCNQ3 and/or KCNQ5 using the small molecule carnosic acid, a key psychoactive component of Salvia rosmarinus (rosemary) extract, reduced GPe PV cell excitability and also impaired cocaine reward, sensitization, and volitional cocaine intake, indicating its potential as a therapeutic to counteract psychostimulant use disorder. Our findings illuminate the molecular and circuit mechanisms by which the GPe orchestrates brain-wide changes in response to cocaine that are required for reward, sensitization, and self-administration behaviors.

Neural circuit basis of adolescent THC-induced potentiation of opioid responses in adult mice.

Use of one drug of abuse typically influences the behavioral response to other drugs, either administered at the same time or a subsequent time point. The nature of the drugs being used, as well as the timing and dosing, also influence how these drugs interact. Here, we tested the effects of adolescent THC exposure on the development of morphine-induced behavioral adaptations following repeated morphine exposure during adulthood. We found that adolescent THC administration impacted morphine-induced behaviors across several dimensions, including potentiating reward and paradoxically impairing the development of morphine reward. We then mapped the whole-brain response to a reinstatement dose of morphine, finding that adolescent THC administration led to increased activity in the basal ganglia and increased functional connectivity between frontal cortical regions and the ventral tegmental area. Last, we show using rabies virus-based circuit mapping that adolescent THC exposure triggers a long-lasting elevation in connectivity from the frontal cortex regions onto ventral tegmental dopamine cells that has the potential to influence dopaminergic response to morphine administration during adulthood. Our study adds to the rich literature on the interaction between drugs of abuse and provides potential circuit substates by which adolescent THC exposure influences responses to morphine later in life.

Analysis of changes in inter-cellular communications during Alzheimer's Disease pathogenesis reveals conserved changes in glutamatergic transmission in mice and humans.

Analysis of system-wide cellular communication changes in Alzheimer's disease (AD) has recently been enabled by single nucleus RNA sequencing (snRNA-seq) and new computational methods. Here, we combined these to analyze data from postmortem human tissue from the entorhinal cortex of AD patients and compared our findings to those from multiomic data from the 5xFAD amyloidogenic mouse model at two different time points. Using the cellular communication inference tool CellChat we found that disease-related changes were largely related to neuronal excitability as well as synaptic communication, with specific signaling pathways including BMP, EGF, and EPHA, and relatively poor conservation of glial-related changes during disease. Further analysis using the neuron-specific NeuronChat revealed changes relating to metabotropic glutamate receptors as well as neuronal adhesion molecules including neurexins and neuroligins. Our results that cellular processes relating to excitotoxicity are the best conserved between 5xFAD mice and AD suggest that excitotoxicity is the main common feature between pathogenesis in 5xFAD mice and AD patients.

Mimicking opioid analgesia in cortical pain circuits.

The anterior cingulate cortex plays a pivotal role in the cognitive and affective aspects of pain perception. Both endogenous and exogenous opioid signaling within the cingulate mitigate cortical nociception, reducing pain unpleasantness. However, the specific functional and molecular identities of cells mediating opioid analgesia in the cingulate remain elusive. Given the complexity of pain as a sensory and emotional experience, and the richness of ethological pain-related behaviors, we developed a standardized, deep-learning platform for deconstructing the behavior dynamics associated with the affective component of pain in mice-LUPE (Light aUtomated Pain Evaluator). LUPE removes human bias in behavior quantification and accelerated analysis from weeks to hours, which we leveraged to discover that morphine altered attentional and motivational pain behaviors akin to affective analgesia in humans. Through activity-dependent genetics and single-nuclei RNA sequencing, we identified specific ensembles of nociceptive cingulate neuron-types expressing mu-opioid receptors. Tuning receptor expression in these cells bidirectionally modulated morphine analgesia. Moreover, we employed a synthetic opioid receptor promoter-driven approach for cell-type specific optical and chemical genetic viral therapies to mimic morphine's pain-relieving effects in the cingulate, without reinforcement. This approach offers a novel strategy for precision pain management by targeting a key nociceptive cortical circuit with on-demand, non-addictive, and effective analgesia.

Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Regulation of stress-induced sleep fragmentation by preoptic glutamatergic neurons.

Sleep disturbances are detrimental to our behavioral and emotional well-being. Stressful events disrupt sleep, in particular by inducing brief awakenings (microarousals, MAs), resulting in sleep fragmentation. The preoptic area of the hypothalamus (POA) is crucial for sleep control. However, how POA neurons contribute to the regulation of MAs and thereby impact sleep quality is unknown. Using fiber photometry in mice, we examine the activity of genetically defined POA subpopulations during sleep. We find that POA glutamatergic neurons are rhythmically activated in synchrony with an infraslow rhythm in the spindle band of the electroencephalogram during non-rapid eye movement sleep (NREMs) and are transiently activated during MAs. Optogenetic stimulation of these neurons promotes MAs and wakefulness. Exposure to acute social defeat stress fragments NREMs and significantly increases the number of transients in the calcium activity of POA glutamatergic neurons during NREMs. By reducing MAs, optogenetic inhibition during spontaneous sleep and after stress consolidates NREMs. Monosynaptically restricted rabies tracing reveals that POA glutamatergic neurons are innervated by brain regions regulating stress and sleep. In particular, presynaptic glutamatergic neurons in the lateral hypothalamus become activated after stress, and stimulating their projections to the POA promotes MAs and wakefulness. Our findings uncover a novel circuit mechanism by which POA excitatory neurons regulate sleep quality after stress.

Cationic peptides erase memories by removing synaptic AMPA receptors through endophilin-mediated endocytosis.

Administration of the Zeta Inhibitory Peptide (ZIP) interferes with memory maintenance and long-term potentiation (LTP). However, mice lacking its putative target, the protein kinase PKMζ, exhibit normal learning and memory as well as LTP, making ZIP's mechanism unclear. Here, we show that ZIP disrupts LTP by removing surface AMPA receptors through its cationic charge alone. This effect was fully blocked by drugs that block macropinocytosis and is dependent on endophilin A2 (endoA2)-mediated endocytosis. ZIP and other cationic peptides selectively removed newly inserted AMPAR nanoclusters, providing a mechanism by which these peptides erase memories without effects on basal synaptic function. Lastly, cationic peptides can be administered locally and/or systemically and can be combined with local microinjection of macropinocytosis inhibitors to modulate memories on local and brain-wide scales. Our findings have critical implications for an entire field of memory mechanisms and highlight a previously unappreciated mechanism by which memories can be lost.

Prefrontal Cortical Regulation of REM Sleep.

Rapid-eye-movement (REM) sleep is accompanied by intense cortical activity, underlying its wake-like electroencephalogram (EEG). The neural activity inducing REM sleep is thought to originate from subcortical circuits in brainstem and hypothalamus. However, whether cortical neurons can also trigger REM sleep has remained unknown. Here, we show in mice that the medial prefrontal cortex (mPFC) strongly promotes REM sleep. Bidirectional optogenetic manipulations demonstrate that excitatory mPFC neurons promote REM sleep through their projections to the lateral hypothalamus (LH) and regulate phasic events, reflected in accelerated EEG theta oscillations and increased eye-movement density during REM sleep. Calcium imaging reveals that the majority of LH-projecting mPFC neurons are maximally activated during REM sleep and a subpopulation is recruited during phasic theta accelerations. Our results delineate a cortico-hypothalamic circuit for the top-down control of REM sleep and identify a critical role of the mPFC in regulating phasic events during REM sleep.

Reanalysis of primate brain circadian transcriptomics reveals connectivity-related oscillations.

Research shows that brain circuits controlling vital physiological processes are closely linked with endogenous time-keeping systems. In this study, we aimed to examine oscillatory gene expression patterns of well-characterized neuronal circuits by reanalyzing publicly available transcriptomic data from a spatiotemporal gene expression atlas of a non-human primate. Unexpectedly, brain structures known for regulating circadian processes (e.g., hypothalamic nuclei) did not exhibit robust cycling expression. In contrast, basal ganglia nuclei, not typically associated with circadian physiology, displayed the most dynamic cycling behavior of its genes marked by sharp temporally defined expression peaks. Intriguingly, the mammillary bodies, considered hypothalamic nuclei, exhibited gene expression patterns resembling the basal ganglia, prompting reevaluation of their classification. Our results emphasize the potential for high throughput circadian gene expression analysis to deepen our understanding of the functional synchronization across brain structures that influence physiological processes and resulting complex behaviors.

Prefrontal cortical regulation of REM sleep.

Rapid eye movement (REM) sleep is accompanied by intense cortical activity, underlying its wake-like electroencephalogram. The neural activity inducing REM sleep is thought to originate from subcortical circuits in brainstem and hypothalamus. However, whether cortical neurons can also trigger REM sleep has remained unknown. Here we show in mice that the medial prefrontal cortex (mPFC) strongly promotes REM sleep. Bidirectional optogenetic manipulations demonstrate that excitatory mPFC neurons promote REM sleep through their projections to the lateral hypothalamus and regulate phasic events, reflected in accelerated electroencephalogram theta oscillations and increased eye movement density during REM sleep. Calcium imaging reveals that the majority of lateral hypothalamus-projecting mPFC neurons are maximally activated during REM sleep and a subpopulation is recruited during phasic theta accelerations. Our results delineate a cortico-hypothalamic circuit for the top-down control of REM sleep and identify a critical role of the mPFC in regulating phasic events during REM sleep.

Spatiotemporal Mapping of Brain Cilia Reveals Region-Specific Oscillation of Length and Orientation.

In this study, we conducted high-throughput spatiotemporal analysis of primary cilia length and orientation across 22 mouse brain regions. We developed automated image analysis algorithms, which enabled us to examine over 10 million individual cilia, generating the largest spatiotemporal atlas of cilia. We found that cilia length and orientation display substantial variations across different brain regions and exhibit fluctuations over a 24-hour period, with region-specific peaks during light-dark phases. Our analysis revealed unique orientation patterns of cilia at 45 degree intervals, suggesting that cilia orientation within the brain is not random but follows specific patterns. Using BioCycle, we identified circadian rhythms of cilia length in five brain regions: nucleus accumbens core, somatosensory cortex, and three hypothalamic nuclei. Our findings present novel insights into the complex relationship between cilia dynamics, circadian rhythms, and brain function, highlighting cilia crucial role in the brain's response to environmental changes and regulation of time-dependent physiological processes.

Challenges and future trends in wearable closed-loop neuromodulation to efficiently treat methamphetamine addiction.

Achieving abstinence from drugs is a long journey and can be particularly challenging in the case of methamphetamine, which has a higher relapse rate than other drugs. Therefore, real-time monitoring of patients' physiological conditions before and when cravings arise to reduce the chance of relapse might help to improve clinical outcomes. Conventional treatments, such as behavior therapy and peer support, often cannot provide timely intervention, reducing the efficiency of these therapies. To more effectively treat methamphetamine addiction in real-time, we propose an intelligent closed-loop transcranial magnetic stimulation (TMS) neuromodulation system based on multimodal electroencephalogram-functional near-infrared spectroscopy (EEG-fNIRS) measurements. This review summarizes the essential modules required for a wearable system to treat addiction efficiently. First, the advantages of neuroimaging over conventional techniques such as analysis of sweat, saliva, or urine for addiction detection are discussed. The knowledge to implement wearable, compact, and user-friendly closed-loop systems with EEG and fNIRS are reviewed. The features of EEG and fNIRS signals in patients with methamphetamine use disorder are summarized. EEG biomarkers are categorized into frequency and time domain and topography-related parameters, whereas for fNIRS, hemoglobin concentration variation and functional connectivity of cortices are described. Following this, the applications of two commonly used neuromodulation technologies, transcranial direct current stimulation and TMS, in patients with methamphetamine use disorder are introduced. The challenges of implementing intelligent closed-loop TMS modulation based on multimodal EEG-fNIRS are summarized, followed by a discussion of potential research directions and the promising future of this approach, including potential applications to other substance use disorders.

A noradrenergic-hypothalamic neural substrate for stress-induced sleep disturbances.

In our daily life, we are exposed to uncontrollable and stressful events that disrupt our sleep. However, the underlying neural mechanisms deteriorating the quality of non-rapid eye movement sleep (NREMs) and REM sleep are largely unknown. Here, we show in mice that acute psychosocial stress disrupts sleep by increasing brief arousals (microarousals [MAs]), reducing sleep spindles, and impairing infraslow oscillations in the spindle band of the electroencephalogram during NREMs, while reducing REMs. This poor sleep quality was reflected in an increased number of calcium transients in the activity of noradrenergic (NE) neurons in the locus coeruleus (LC) during NREMs. Opto- and chemogenetic LC-NE activation in naïve mice is sufficient to change the sleep microarchitecture similar to stress. Conversely, chemogenetically inhibiting LC-NE neurons reduced MAs during NREMs and normalized their number after stress. Specifically inhibiting LC-NE neurons projecting to the preoptic area of the hypothalamus (POA) decreased MAs and enhanced spindles and REMs after stress. Optrode recordings revealed that stimulating LC-NE fibers in the POA indeed suppressed the spiking activity of POA neurons that are activated during sleep spindles and REMs and inactivated during MAs. Our findings reveal that changes in the dynamics of the stress-regulatory LC-NE neurons during sleep negatively affect sleep quality, partially through their interaction with the POA.

Defining the interconnectivity of the medial prefrontal cortex and ventral midbrain.

Dysfunction in dopamine (DA) signaling contributes to neurological disorders ranging from drug addiction and schizophrenia to depression and Parkinson's Disease. How might impairment of one neurotransmitter come to effect these seemingly disparate diseases? One potential explanation is that unique populations of DA-releasing cells project to separate brain regions that contribute to different sets of behaviors. Though dopaminergic cells themselves are spatially restricted to the midbrain and constitute a relatively small proportion of all neurons, their projections influence many brain regions. DA is particularly critical for the activity and function of medial prefrontal cortical (mPFC) ensembles. The midbrain and mPFC exhibit reciprocal connectivity - the former innervates the mPFC, and in turn, the mPFC projects back to the midbrain. Viral mapping studies have helped elucidate the connectivity within and between these regions, which likely have broad implications for DA-dependent behaviors. In this review, we discuss advancements in our understanding of the connectivity between the mPFC and midbrain DA system, focusing primarily on rodent models.

An inhibitory brainstem input to dopamine neurons encodes nicotine aversion.

Nicotine stimulates the dopamine (DA) system, which is essential for its rewarding effect. Nicotine is also aversive at high doses; yet, our knowledge about nicotine's dose-dependent effects on DA circuits remains limited. Here, we demonstrate that high doses of nicotine, which induce aversion-related behavior in mice, cause biphasic inhibitory and excitatory responses in VTA DA neurons that can be dissociated by distinct projections to lateral and medial nucleus accumben subregions, respectively. Guided by computational modeling, we performed a pharmacological investigation to establish that inhibitory effects of aversive nicotine involve desensitization of α4ß2 and activation of α7 nicotinic acetylcholine receptors. We identify α7-dependent activation of upstream GABA neurons in the laterodorsal tegmentum (LDT) as a key regulator of heterogeneous DA release following aversive nicotine. Finally, inhibition of LDT GABA terminals in VTA prevents nicotine aversion. Together, our findings provide a mechanistic circuit-level understanding of nicotine's dose-dependent effects on reward and aversion.

An extended amygdala-midbrain circuit controlling cocaine withdrawal-induced anxiety and reinstatement.

Although midbrain dopamine (DA) circuits are central to motivated behaviors, our knowledge of how experience modifies these circuits to facilitate subsequent behavioral adaptations is limited. Here we demonstrate the selective role of a ventral tegmental area DA projection to the amygdala (VTADA→amygdala) for cocaine-induced anxiety but not cocaine reward or sensitization. Our rabies virus-mediated circuit mapping approach reveals a persistent elevation in spontaneous and task-related activity of inhibitory GABAergic cells from the bed nucleus of the stria terminalis (BNST) and downstream VTADA→amygdala cells that can be detected even after a single cocaine exposure. Activity in BNSTGABA→midbrain cells is related to cocaine-induced anxiety but not reward or sensitization, and silencing this projection prevents development of anxiety during protracted withdrawal after cocaine administration. Finally, we observe that VTADA→amygdala cells are strongly activated after a challenge exposure to cocaine and that activity in these cells is necessary and sufficient for reinstatement of cocaine place preference.