Behavioral Neuroscience publications

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter ( MORp ) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene ( mMORp ) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp ( hMORp ) efficiently transduced macaque cortical OPRM1 + cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.

The medial prefrontal cortex (mPFC) plays a key role in learning, mood and decision making, including in how individuals respond to threats 1–6 . mPFC undergoes a uniquely protracted development, with changes in synapse density, cortical thickness, long-range connectivity, and neuronal encoding properties continuing into early adulthood 7–21 . Models suggest that before adulthood, the slow-developing mPFC cannot adequately regulate activity in faster-developing subcortical centers 22,23 . They propose that during development, the enhanced influence of subcortical systems underlies distinctive behavioural strategies of juveniles and adolescents and that increasing mPFC control over subcortical structures eventually allows adult behaviours to emerge. Yet it has remained unclear how a progressive strengthening of top-down control can lead to nonlinear changes in behaviour as individuals mature 24,25 . To address this discrepancy, here we monitored and manipulated activity in the developing brain as animals responded to threats, establishing direct causal links between frontolimbic circuit activity and the behavioural strategies of juvenile, adolescent and adult mice. Rather than a linear strengthening of mPFC synaptic connectivity progressively regulating behaviour, we uncovered multiple developmental switches in the behavioural roles of mPFC circuits targeting the basolateral amygdala (BLA) and nucleus accumbens (NAc). We show these changes are accompanied by axonal pruning coinciding with functional strengthening of synaptic connectivity in the mPFC-BLA and mPFC-NAc pathways, which mature at different rates. Our results reveal how developing mPFC circuits pass through distinct architectures that may make them optimally adapted to the demands of age-specific challenges.

Dopamine is broadly implicated in reinforcement learning, but how patterns of dopamine activity are generated is poorly resolved. Here, we demonstrate that two ion channels, Kv4.3 and BKCa1.1, regulate the pattern of dopamine neuron firing and dopamine release on different time scales to influence separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases ex vivo pacemaker activity and excitability that is associated with increased in vivo firing rate and ramping dynamics before lever press in a learned instrumental paradigm. Loss of Kv4.3 enhances performance of the learned response and facilitates extinction. In contrast, loss of BKCa1.1 increases burst firing and phasic dopamine release that enhances learning of an instrumental response and enhances extinction burst lever pressing in early extinction that is associated with a greater change in activity between reinforced and unreinforced actions. These data demonstrate that disruption of intrinsic regulators of neuronal activity differentially affects dopamine dynamics during reinforcement and extinction learning.

Head-fixed behavioral experiments in rodents permit unparalleled experimental control, precise measurement of behavior, and concurrent modulation and measurement of neural activity. Here, we present OHRBETS (Open-Source Head-fixed Rodent Behavioral Experimental Training System; pronounced ‘Orbitz’), a low-cost, open-source platform of hardware and software to flexibly pursue the neural basis of a variety of motivated behaviors. Head-fixed mice tested with OHRBETS displayed operant conditioning for caloric reward that replicates core behavioral phenotypes observed during freely moving conditions. OHRBETS also permits optogenetic intracranial self-stimulation under positive or negative operant conditioning procedures and real-time place preference behavior, like that observed in freely moving assays. In a multi-spout brief-access consumption task, mice displayed licking as a function of concentration of sucrose, quinine, and sodium chloride, with licking modulated by homeostatic or circadian influences. Finally, to highlight the functionality of OHRBETS, we measured mesolimbic dopamine signals during the multi-spout brief-access task that display strong correlations with relative solution value and magnitude of consumption. All designs, programs, and instructions are provided freely online. This customizable platform enables replicable operant and consummatory behaviors and can be incorporated with methods to perturb and record neural dynamics in vivo.

Social interaction among conspecifics is essential for maintaining adaptive, cooperative, and social behaviors, along with survival among mammals. The 5-hydroxytryptamine (5-HT) neuronal system is an important neurotransmitter system for regulating social behaviors; however, the circadian role of 5-HT in social interaction behaviors is unclear. To investigate whether the circadian nuclear receptor REV-ERBα, a transcriptional repressor of the rate-limiting enzyme tryptophan hydroxylase 2 (Tph2) gene in 5-HT biosynthesis, may affect social interaction behaviors, we generated a conditional knockout (cKO) mouse by targeting Rev-Erbα in dorsal raphe (DR) 5-HT neurons (5-HT DR -specific REV-ERBα cKO) using the CRISPR/Cas9 gene editing system and assayed social behaviors, including social preference and social recognition, with a three-chamber social interaction test at two circadian time (CT) points, i.e., at dawn (CT00) and dusk (CT12). The genetic ablation of Rev-Erbα in DR 5-HTergic neurons caused impaired social interaction behaviors, particularly social preference but not social recognition, with no difference between the two CT points. This deficit of social preference induced by Rev-Erbα in 5-HT DR -specific mice is functionally associated with real-time elevated neuron activity and 5-HT levels at dusk, as determined by fiber-photometry imaging sensors. Moreover, optogenetic inhibition of DR to nucleus accumbens (NAc) 5-HTergic circuit restored the impairment of social preference in 5-HT DR -specific REV-ERBα cKO mice. These results suggest the significance of the circadian regulation of 5-HT levels by REV-ERBα in regulating social interaction behaviors.

Nicotine is an addictive drug whose popularity has recently increased, particularly among adolescents, because of the availability of electronic nicotine devices (i.e., “vaping”) and nicotine e-liquids containing additives with rich chemosensory properties. Some efforts to understand the role of these additives in nicotine reward suggest that they increase nicotine reward and reinforcement, but the sensory contributions of additives, especially in their vapor forms, are largely untested. Here, to better understand how a fruit-flavored (i.e., strawberry) additive influences nicotine reward and aversion, we used a conditioned place preference (CPP) procedure in which nicotine and a strawberry additive were delivered as a vapor to male and female adolescent mice. We found that nicotine vapor alone can lead to a dose-dependent CPP when using a biased design. The strawberry additive did not produce CPP on its own, and we did not observe an effect of the strawberry additive on nicotine vapor-induced reward. Nevertheless, mice exposed to nicotine plus strawberry additive vapor had higher plasma cotinine concentrations, which did not appear to reflect altered nicotine metabolism. Instead, by directly measuring vapor sampling through respiration monitoring, we uncovered an increase in the amount of sniffing toward strawberry-containing nicotine vapor compared with nicotine vapor alone. Together these data indicate that chemosensory-rich e-liquid additives may enhance the perceived sensory profile of nicotine vapors rather than the reward value per se, which leads to overall increased nicotine exposure.

Midbrain and striatal dopamine signals have been extremely well characterized over the past several decades, yet novel dopamine signals and functions in reward learning and motivation continue to emerge. A similar characterization of real-time sub-second dopamine signals in areas outside of the striatum has been limited. Recent advances in fluorescent sensor technology and fiber photometry permit the measurement of dopamine binding correlates, which can divulge basic functions of dopamine signaling in non-striatal dopamine terminal regions, like the dorsal bed nucleus of the stria terminalis (dBNST). Here, we record GRAB DA signals in the dBNST during a Pavlovian lever autoshaping task. We observe greater Pavlovian cue-evoked dBNST GRAB DA signals in sign-tracking (ST) compared to goal-tracking/intermediate (GT/INT) rats and the magnitude of cue-evoked dBNST GRAB DA signals decreases immediately following reinforcer-specific satiety. When we deliver unexpected rewards or omit expected rewards, we find that dBNST dopamine signals encode bidirectional reward prediction errors in GT/INT rats, but only positive prediction errors in ST rats. Since sign- and goal-tracking approach strategies are associated with distinct drug relapse vulnerabilities, we examined the effects of experimenter-administered fentanyl on dBNST dopamine associative encoding. Systemic fentanyl injections do not disrupt cue discrimination but generally potentiate dBNST dopamine signals. These results reveal multiple dBNST dopamine correlates of learning and motivation that depend on the Pavlovian approach strategy employed.

Reward-predictive cues acquire motivating and reinforcing properties that contribute to the escalation and relapse of drug use in addiction. The ventral pallidum (VP) and ventral tegmental area (VTA) are two key nodes in brain reward circuitry implicated in addiction and necessary for the performance of cue-driven behavior. Evidence suggests that VP neurons projecting to the VTA (VP→VTA) promote cue-induced reinstatement of drug-seeking, but the mechanisms by which these neurons do so are undefined. In addition, the role of these neurons in the pursuit of non-drug reward is not known. In the current study, we used in vivo fiber photometry and optogenetics to record from and manipulate VP→VTA in rats performing a discriminative stimulus task (DS task) with sucrose reward to determine the fundamental role these neurons play in invigoration and reinforcement by reward and associated discriminative cues. We find that VP→VTA neurons are selectively active during reward consumption, that optogenetic stimulation of these neurons paired with reward consumption biases choice, and that VP→VTA optogenetic stimulation is reinforcing. Critically, we found no significant encoding of cue-elicited reward-seeking vigor and acute optogenetic stimulation of these neurons paired with cue onset did not enhance the probability or vigor of reward-seeking. Our results suggest that VP→VTA neurons are active during the consumption of natural reward and that this activity reinforces seeking behavior.

Objective. Neuropixels (NP) probes are a significant advance in electrophysiological recording technology that enable monitoring of hundreds of neurons in the brain simultaneously at different depths. Application of this technology has been predominately in rodents, however widespread use in non-human primates (NHPs) such as rhesus macaques has been limited. In this study we sought to overcome two overarching challenges that impede acute NP implantation in NHPs: (1) traditional microdrive systems that mount to cephalic chambers are commonly used to access cortical areas for microelectrode recordings but are not designed to accommodate NP probes, and (2) NHPs have thick dura mater and tissue growth within the cephalic chambers which poses a challenge for insertion of the extremely fragile NP probe. Approach. In this study we present a novel NP guide tube system that can be adapted to commercial microdrive systems and demonstrate an implant method using the NP guide tube system. This system was developed using a combination of CAD design, 3D printing, and small part machining. Software programs, 3D Slicer and SolidWorks were used to target cortical areas, approximate recording depths and locations, and for in-silico implant testing. Main results. We performed in vivo testing to validate our methodology, successfully implanting, explanting, and reimplanting NP probes. We collected stable neurophysiological recordings in the premotor cortex of a rhesus macaque at rest and during performance of a reaching task. Significance. In this study we demonstrate a robust Neuropixels implant system that allows multiple penetrations with the same NP probe and share design files that will facilitate the adoption of this powerful recording technology for NHP studies.

Application of closed-loop approaches in systems neuroscience and brain-computer interfaces holds great promise for revolutionizing our understanding of the brain and for developing novel neuromodulation strategies to restore lost function. The anterior forebrain mesocircuit (AFM) of the mammalian brain is hypothesized to underlie arousal regulation of the cortex and striatum, and support cognitive functions during wakefulness. Dysfunction of arousal regulation is hypothesized to contribute to cognitive dysfunctions in various neurological disorders, and most prominently in patients following traumatic brain injury (TBI). Several clinical studies have explored the use of daily central thalamic deep brain stimulation (CT-DBS) within the AFM to restore consciousness and executive attention in TBI patients. In this study, we explored the use of closed-loop CT-DBS in order to episodically regulate arousal of the AFM of a healthy non-human primate (NHP) with the goal of restoring behavioral performance. We used pupillometry and near real-time analysis of ECoG signals to episodically initiate closed-loop CT-DBS and here we report on our ability to enhance arousal and restore the animal's performance. The initial computer based approach was then experimentally validated using a customized clinical-grade DBS device, the DyNeuMo-X, a bi-directional research platform used for rapidly testing closed-loop DBS. The successful implementation of the DyNeuMo-X in a healthy NHP supports ongoing clinical trials employing the internal DyNeuMo system (NCT05437393, NCT05197816) and our goal of developing and accelerating the deployment of novel neuromodulation approaches to treat cognitive dysfunction in patients with structural brain injuries and other etiologies.

As a key oscillatory activity in the brain, thalamic spindle activities are long believed to support memory consolidation. However, their propagation characteristics and causal actions at systems level remain unclear. Using functional MRI (fMRI) and electrophysiology recordings in male rats, we found that optogenetically-evoked somatosensory thalamic spindle-like activities targeted numerous sensorimotor (cortex, thalamus, brainstem and basal ganglia) and non-sensorimotor limbic regions (cortex, amygdala, and hippocampus) in a stimulation frequency- and length-dependent manner. Thalamic stimulation at slow spindle frequency (8 Hz) and long spindle length (3 s) evoked the most robust brain-wide cross-modal activities. Behaviorally, evoking these global cross-modal activities during memory consolidation improved visual-somatosensory associative memory performance. More importantly, parallel visual fMRI experiments uncovered response potentiation in brain-wide sensorimotor and limbic integrative regions, especially superior colliculus, periaqueductal gray, and insular, retrosplenial and frontal cortices. Our study directly reveals that thalamic spindle activities propagate in a spatiotemporally specific manner and that they consolidate associative memory by strengthening multi-target memory representation.

Fear extinction is a form of inhibitory learning that suppresses the expression of aversive memories and plays a key role in the recovery of anxiety and trauma-related disorders. Here, using male mice, we identify a cerebello-thalamo-cortical pathway regulating fear extinction. The cerebellar fastigial nucleus (FN) projects to the lateral subregion of the mediodorsal thalamic nucleus (MD), which is reciprocally connected with the dorsomedial prefrontal cortex (dmPFC). The inhibition of FN inputs to MD in male mice impairs fear extinction in animals with high fear responses and increases the bursting of MD neurons, a firing pattern known to prevent extinction learning. Indeed, this MD bursting is followed by high levels of the dmPFC 4 Hz oscillations causally associated with fear responses during fear extinction, and the inhibition of FN-MD neurons increases the coherence of MD bursts and oscillations with dmPFC 4 Hz oscillations. Overall, these findings reveal a regulation of fear-related thalamo-cortical dynamics by the cerebellum and its contribution to fear extinction.

Multiple strategies have evolved to compensate for masking noise, leading to changes in call features. One call adjustment is the Lombard effect, an increase in call amplitude in response to noise. Another strategy involves call production in periods where noise is absent. While mechanisms underlying vocal adjustments have been well studied, mechanisms underlying noise avoidance strategies remain largely unclear. We systematically perturbed ongoing phee calls of marmosets to investigate noise avoidance strategies. Marmosets canceled their calls after noise onset and produced longer calls after noise-phases ended. Additionally, the number of uttered syllables decreased during noise perturbation. This behavior persisted beyond the noise-phase. Using machine learning techniques, we found that a fraction of single phees were initially planned as double phees and became interrupted after the first syllable. Our findings indicate that marmosets use different noise avoidance strategies and suggest vocal flexibility at different complexity levels in the marmoset brain.

SignificanceGenetically encoded optical probes to image calcium levels in neurons in vivo are used widely as a real-time measure of neuronal activity in the brain. Mesoscale calcium imaging through a cranial window provides a method of studying the interaction of circuit activity between cortical areas but lacks access to subcortical regions.AimWe have developed an optical and surgical preparation that preserves wide-field imaging of the cortical surface while also permitting access to specific subcortical networks.ApproachThis was achieved using an optical fiber implanted in the striatum, along with a bilateral widefield cranial window, enabling simultaneous mesoscale cortical imaging and subcortical fiber photometry recording of calcium signals in a transgenic animal expressing GCaMP. Subcortical signals were collected from the dorsal regions of the striatum. We combined this approach with multiple sensory-motor tasks, including specific auditory and visual stimulation, and video monitoring of animal movements and pupillometry during head-fixed behaviors.ResultsWe found high correlations between cortical and striatal activity in response to sensory stimulation or movement. Furthermore, spontaneous activity recordings revealed that specific motifs of cortical activity are correlated with presynaptic activity recorded in the striatum, enabling us to select for corticostriatal activity motifs.ConclusionWe believe that this method can be utilized to reveal not only global patterns but also cell-specific connectivity that provides insight into corticobasal ganglia circuit organization.

We hypothesized that auditory stimulation could reduce the progression of Alzheimer’s disease (AD), and that audiovisual stimulation could have additional effects through multisensory integration. We exposed 12 month old Apoetm1.1(APOE*4)Adiuj mice (a mouse model of sporadic AD) to auditory (A) or audiovisual stimulation (AV) at 40 Hz for 14 days in a soundproof chamber system (no stimulation, N). Behavioral tests were performed before and after each session, and their brain tissues were assessed for amyloid-beta expression and apoptotic cell death, after 14 days. Furthermore, brain levels of acetylcholine and apoptosis-related proteins were analyzed. In the Y-maze test, the percentage relative alternation was significantly higher in group A than in group N mice. Amyloid-beta and TUNEL positivity in the hippocampal CA3 region was significantly lower in group A and group AV mice than in group N mice (p < 0.05). Acetylcholine levels were significantly higher in group A and group AV mice than in group N mice (p < 0.05). Compared to group N mice, expression of the proapoptotic proteins Bax and caspase-3 was lower in group A, and expression of the antiapoptotic protein Bcl-2 was higher in group AV. In a mouse model of early-stage sporadic AD, auditory or audiovisual stimulation improved cognitive performance and neuropathology.

Extended wakefulness is associated with reduced performance and the build-up of sleep pressure. In the cortex, this manifests as changes in network activity. These changes show local variation depending on the waking experience, and their underlying mechanisms represent targets for overcoming the effects of tiredness. Here, we reveal a central role for intracellular chloride regulation, which sets the strength of postsynaptic inhibition via GABAA receptors in cortical pyramidal neurons. Wakefulness results in depolarizing shifts in the equilibrium potential for GABAA receptors, reflecting local activity-dependent processes during waking and involving changes in chloride cotransporter activity. These changes underlie electrophysiological and behavioral markers of local sleep pressure within the cortex, including the levels of slow-wave activity during non-rapid eye movement sleep and low-frequency oscillatory activity and reduced performance levels in the sleep-deprived awake state. These findings identify chloride regulation as a crucial link between sleep-wake history, cortical activity and behavior.

Breathing is dynamically modulated by metabolic needs as well as by emotional states. Even though rodents are invaluable models for investigating the neural control of respiration, current literature lacks systematic characterization of breathing dynamics across a broad spectrum of rodent behaviors. Here we uncover a wide diversity in breathing patterns across spontaneous, attractive odor-, stress-, and fear-induced behaviors in mice. Direct recordings of intranasal pressure afford more detailed respiratory information than more traditional whole-body plethysmography. K-means clustering groups 11 well-defined behavioral states into four clusters with distinct key respiratory features. Furthermore, we implement RUSBoost (random undersampling boost) classification, a supervised machine learning model, and find that breathing patterns can separate these behaviors with an accuracy of 80%. Taken together, our findings highlight the tight relationship between breathing and behavior and the potential use of breathing patterns to aid in distinguishing similar behaviors and inform about their internal states.

Increasing extracellular levels of serotonin (5-HT) in the brain ameliorates symptoms of depression and anxiety-related disorders, e.g., social phobias and post-traumatic stress disorder. Recent evidence from preclinical and clinical studies established the therapeutic potential of drugs inducing the release of 5-HT via the 5-HT-transporter. Nevertheless, current 5-HT releasing compounds under clinical investigation carry the risk for abuse and deleterious side effects. Here, we demonstrate that S -enantiomers of certain ring-substituted cathinones show preference for the release of 5-HT ex vivo and in vivo, and exert 5-HT-associated effects in preclinical behavioral models. Importantly, the lead cathinone compounds (1) do not induce substantial dopamine release and (2) display reduced off-target activity at vesicular monoamine transporters and 5-HT 2B -receptors, indicative of low abuse-liability and low potential for adverse events. Taken together, our findings identify these agents as lead compounds that may prove useful for the treatment of disorders where elevation of 5-HT has proven beneficial.

Drug addiction is characterized by a sustained vulnerability to relapse even after long periods of abstinence. A deeper understanding of the brain systems underlying this state could inform therapeutic strategies with novel prognostic biomarkers aimed at preventing renewed drug seeking. Most drugs of abuse, in particular psychostimulants such as cocaine, lead to long-lasting mesolimbic dopamine system adaptations, that ultimately facilitate drug seeking following exposure to drug-paired cues. This “dopaminergic hypothesis” of relapse has been previously addressed, but technical limitations in measuring in vivo dopamine release have precluded the assessment of its sufficiency without introducing pharmacological, electrical, or optogenetic confounds. Using a dopamine receptor-based fluorescent sensor in freely moving mice, we show that long-lasting dopamine recordings in the nucleus accumbens (NAc), throughout the animal’s entire history of cocaine self-administration, are strong predictors of relapse as well as the time it takes an animal to extinguish its drug seeking behavior. Moreover, we reveal previously unseen sex-specific trajectories of cocaine-related phasic dopamine responses from acquisition to relapse. We show that males exhibit higher-amplitude phasic dopamine responses, a trait accompanied by a greater resistance to extinguish their cocaine seeking, compared to females. Furthermore, we show that a semi-parametric model of the transition to extinction – using only multivariate patterns of dopamine release and sex as covariates – faithfully recapitulates male-specific vulnerability to persistent cocaine seeking. In conclusion, we present a predictive model of reinstatement behavior that uses information exclusively conveyed by NAc phasic dopamine responses, thus confirming, and actuating the sufficiency of the dopaminergic hypothesis of relapse.

The question of how the brain links behavioral and biological features of defensive responses has remained elusive. The importance of this problem is underscored by the observation that behavioral passivity in stress coping is associated with elevations in glucocorticoid hormones, and each may carry risks for susceptibility to a host of stress-related diseases. Past work implicates the medial prefrontal cortex (mPFC) in the top-down regulation of stress-related behaviors; however, it is unknown whether such changes have the capacity to buffer against the longer-lasting biological consequences associated with aversive experiences. Using the shock probe defensive burying test in rats to naturalistically measure behavioral and endocrine features of coping, we observed that the active behavioral component of stress coping is associated with increases in activity along a circuit involving the caudal mPFC and midbrain dorsolateral periaqueductal gray (PAG). Optogenetic manipulations of the caudal mPFC-to-dorsolateral PAG pathway bidirectionally modulated active (escape and defensive burying) behaviors, distinct from a rostral mPFC–ventrolateral PAG circuit that instead limited passive (immobility) behavior. Strikingly, under conditions that biased rats toward a passive coping response set, including exaggerated stress hormonal output and increased immobility, excitation of the caudal mPFC–dorsolateral PAG projection significantly attenuated each of these features. These results lend insight into how the brain coordinates response features to overcome passive coping and may be of importance for understanding how activated neural systems promote stress resilience.