Publications using the RZ10X

Aversion to opioid withdrawal is a significant barrier to achieving lasting opioid abstinence. The central amygdala (CeA), a key brain region for pain, threat-detection, autonomic engagement, and valence assignment, is active during opioid withdrawal. However, the role of molecularly distinct CeA neural populations in withdrawal remains underexplored. Here, we investigated the activity dynamics, brain-wide connectivity, and functional contribution of Protein Kinase C-delta (PKCδ)-expressing neurons in the CeA lateral capsule (CeLC PKCδ ) during fentanyl withdrawal in mice. Mapping activity-dependent gene expression in CeLC PKCδ neurons revealed a highly withdrawal-active subregion in the anterior half of the CeA. Fiber photometry calcium imaging showed that opioid-naïve CeLC PKCδ neurons respond to salient noxious and startling stimuli. In fentanyl-dependent mice, naloxone-precipitated withdrawal increased spontaneous neural activity and enhanced responses to noxious stimuli. Chronic inhibition of CeLC PKCδ neurons throughout fentanyl exposure, via viral overexpression of the potassium channel Kir2.1, attenuated withdrawal symptoms in fentanyl-dependent mice. Lastly, we identified putative opioid-sensitive inputs to CeLC PKCδ neurons using rabies-mediated monosynaptic circuit tracing and color-switching tracers to map mu-opioid receptor-expressing inputs to the CeLC. Collectively, these findings suggest that the hyperactivity of CeLC PKCδ neurons underlies the somatic signs of fentanyl withdrawal, offering new insights into the amygdala cell-types and circuits involved in opioid dependence.

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.

Background CLN3 disease, also known as juvenile Batten disease, is a recessively inherited neurodevelopmental disorder caused by mutations in the CLN3 gene. It represents the most common form of Neuronal Ceroid Lipofuscinoses (NCLs), a group of lysosomal storage disorders that impair brain function. Clinical features include progressive vision loss, language impairment, and cognitive decline. The early onset of visual deficits complicates the neurological assessment of cognitive dysfunction, while the rarity of CLN3 cases limits the study of sex-specific disease trajectories in humans. Therefore, there is a critical need for objective, translational biomarkers to monitor disease progression and support therapeutic development in preclinical animal models. Methods Building on our recent studies in individuals with CLN3 disease, we developed a parallel experimental paradigm using high-density electroencephalography (EEG) in Cln3 knockout ( Cln3-/-) mice to longitudinally assess auditory neurophysiological changes. We applied a duration-based mismatch negativity (MMN) paradigm, similar to that used in our human studies, to evaluate automatic detection of auditory pattern changes in male and female mice between 3 and 9 months of age. Results Wild-type (WT) mice of both sexes showed robust and stable duration MMN responses across this age range. In contrast, Cln3-/- mice showed marked sex- and age-dependent deficits: female mutants displayed persistent MMN deficits, whereas male mutants exhibited early MMN abnormalities that unexpectedly improved with age. Auditory brainstem responses confirmed intact peripheral hearing in Cln3-/- mice, indicating a central origin for the observed abnormalities. Further analyses revealed that MMN impairments were driven by age- and sex-specific alterations in auditory evoked potentials to both standard and deviant stimuli. Conclusions These findings demonstrate sex- and age-dependent disruptions in central auditory processing in Cln3-/- mice and support auditory duration MMN as a sensitive, translational biomarker of brain dysfunction in CLN3 disease. This approach offers a functional, cross-species measure for tracking disease progression and evaluating therapeutic interventions in Batten disease.

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.

Classical neuromodulators regulate arousal states, spanning deep sleep to vigilant wakefulness, primarily by activating cortical neurons. However, cortical astrocytes also express neuromodulatory G-protein-coupled receptors (GPCRs). While astrocytic noradrenergic receptors have been shown to modulate two critical regulators of arousal—cortical synchrony and extracellular adenosine levels—how other neuromodulatory signaling pathways similarly shape arousal remains unclear. Astrocytes in mammalian cortex express particularly high levels of the wake-promoting, Gq-coupled histamine-1-receptor (H1R), yet little is known about how astrocytic H1R contributes to regulation of arousal. To address this gap, we used pharmacological and genetic approaches in murine cortex to test how astrocyte-H1R signaling affects astrocyte calcium (Ca 2+ ), cortical neural activity across sleep/wake, and extracellular adenosine—an astrocytic output that regulates cortical arousal. Using ex vivo two-photon Ca 2+ imaging in acute cortical slices, we show that H1R mediates cell-autonomous astrocyte Ca 2+ responses to histamine (HA) and attenuates responses to norepinephrine (NE). Next, in vivo fiber photometry and electrophysiology results show that H1R deletion in cortical astrocytes disrupts local astrocyte Ca 2+ during wake and extracellular adenosine dynamics specifically around REM sleep transitions, when HA release is minimal. Further, astrocyte-specific H1R deletion in cortex promotes wakefulness and reduces REM sleep time. Our results indicate that H1R activity modulates astrocyte responses to non-histaminergic inputs by inducing lasting changes in astrocyte physiology that modulate extracellular adenosine and REM sleep. Our findings contribute to an emerging model in which neuromodulator GPCRs synergistically shape astrocyte physiology to regulate arousal behavior and adenosine signaling in the cortex.

Basolateral amygdala (BLA) GABAergic interneurons (INs) are critical for associative learning; however, the comparative synaptic organization and learning-related activity of BLA IN types have not been systematically evaluated. Here, we show somatostatin (SOM) INs provide inhibition onto, and are excited by, local pyramidal cells (PCs), whereas vasoactive intestinal peptide (VIP) INs are driven by extrinsic afferents. Parvalbumin (PV) INs inhibit PCs and are activated by local and extrinsic inputs. Thus, SOM and VIP INs exhibit complementary roles in feedback and feedforward inhibition, respectively, while PV INs contribute to both microcircuit motifs. Functionally, each IN subtype exhibits unique activity patterns across fear and extinction learning, with SOM and VIP INs showing most divergent characteristics, and PV INs display an intermediate phenotype. Finally, SOM and PV INs dynamically track behavioral-state transitions across learning. These data provide insight into the synaptic microcircuit organization and activity of distinct BLA IN classes across aversive associative learning.

How Δ 9 -tetrahydrocannabinol (THC) impairs natural behaviors in mice remains unknown. We developed a video-monitored behavioral platform with machine learning classifiers to unravel discrete changes in natural mouse behaviors. THC infusion into the medial prefrontal cortex (mPFC) disrupted walking kinematic features characteristic of impairment responses. THC predominantly increased mPFC GABAergic activity preceding walk initiation shifting the mPFC excitatory/inhibitory (E/I) balance. Pose-defined closed loop photo-stimulation of mPFC GABAergic neurons demonstrated that THC exacerbates selected parameters of motor impairment. Surprisingly, THC also induced a time locked, movement-induced, transient potentiation of mPFC endocannabinoid (eCB) release and ensuing CB 1 R-mediated synaptic inhibition. Here we establish that THC-modifies mPFC E/I balance to excitation via dynamic changes in eCB release which acts to induce behavioral impairment.

In the hippocampal formation, cholinergic modulation from the medial septum/diagonal band of Broca is known to correlate with the speed of an animal's movements at subsecond timescales and also supports spatial memory formation. Yet, the extent to which subsecond cholinergic dynamics, if at all, align with transient behavioral and cognitive states supporting the encoding of novel spatial information remains unknown. In this study, we used fiber photometry to record the temporal dynamics in the population activity of septo-hippocampal cholinergic neurons at subsecond resolution during a hippocampus-dependent object location memory task using ChAT-Cre mice of both sexes. Using a linear mixed-effects model, we quantified the extent to which cholinergic dynamics were explained by changes in movement speed; behavioral states such as locomotion, grooming, and rearing; and hippocampus-dependent cognitive states such as recognizing a novel location of a familiar object. The data show that cholinergic dynamics contain a multiplexed code of fast and slow signals (1) coding for the logarithm of movement speed at subsecond timescales, (2) providing a phasic spatial novelty signal during the brief periods of exploring a novel object location, and (3) coding for recency of environmental change at a seconds-long timescale. Furthermore, behavioral event-related phasic cholinergic activity demonstrates that fast cholinergic transients correlate with a switch in cognitive and behavioral states. These findings enhance understanding of the mechanisms by which cholinergic modulation contributes to the coding of movement speed and encoding of novel spatial information.

Nicotine is one of the most widely used addictive substances, yet its primary reinforcing effects are relatively weak. Nicotine's ability to potentiate responding for conditioned reinforcers is thought to drive persistent drug use. Here, we show that nicotine failed to alter responding maintained by a primary sucrose reinforcer (under a variable-ratio [VR] 11 schedule) across a broad dose range (0.01-1.0 mg/kg). Yet, nicotine enhanced operant behavior under a second-order reinforcement schedule in which responses produced sucrose-associated cues and were only intermittently reinforced by sucrose itself. Together, we demonstrate that nicotine selectively augments behavior maintained by conditioned reinforcers (sucrose-associated cues) in a rate-dependent manner, increasing responding only in mice with low rates of reinforcement behavior at baseline. Using fiber photometry, we demonstrate that nicotine selectively amplified cue-evoked dopamine release in the dorsolateral striatum-but only in low baseline responders-while having no effect on dopamine signaling in the nucleus accumbens. These effects were blocked by the nicotinic receptor antagonist mecamylamine. Further, nicotine's influence on behavior was abolished when the contingency between action and conditioned stimuli was disrupted, indicating that nicotine strengthens cue control of behavior rather than increasing motivation generally. Collectively, these findings reveal that nicotine's behavioral actions emerge through an interaction between pharmacological mechanisms, behavioral contingencies, and individual differences in baseline behavioral control. SIGNIFICANCE STATEMENT: Nicotine strengthens the impact of environmental cues on behavior by amplifying dopamine signals in specific projection targets, but only in individuals with specific behavioral traits. This reveals how nicotine hijacks learning processes to promote persistent, cue-driven actions.

The basolateral amygdala (BLA) assigns valence to sensory stimuli, with a dedicated nociceptive ensemble encoding the negative valence of pain. However, the effects of chronic pain on the transcriptomic signatures and projection architecture of this BLA nociceptive ensemble are not well understood. Here, we show that optogenetic inhibition of the nociceptive BLA ensemble reduces affective-motivational behaviors in chronic neuropathic pain. Single-nucleus RNA sequencing revealed peripheral injury–induced changes in genetic pathways involved in axonal and presynaptic organization in nociceptive BLA neurons. Next, we identified a previously uncharacterized nociceptive hotspot in the nucleus accumbens shell that is innervated by BLA nociceptive neurons. Axonal calcium imaging of BLA projections to the accumbens and chemogenetic inhibition of this pathway revealed pain-related transmission from the amygdala to the medial nucleus accumbens, facilitating both acute and chronic pain affective-motivational behaviors. Together, this work defines a critical nociceptive amygdala-striatal circuit underlying pain unpleasantness across pain states.

The neurotransmitter histamine is involved in control of food intake, yet its dynamics during individual feeding episodes remain unexplored. Therefore, we used the novel genetically‐encoded histamine sensor, HisLightG, combined with fiber photometry to measure histamine release in two hypothalamic regions critical for the food‐suppressive effects of histamine, the paraventricular nucleus of the hypothalamus (PVH), and the ventromedial hypothalamus (VMH). Male mice were tested under different conditions to assess whether hunger, time of day, or the caloric content of the solution they were given affected histamine fluctuations. We found that histamine levels changed rapidly in response to eating. These histamine fluctuations were influenced by experimental conditions, with slightly smaller responses when the test solution was sucralose (both regions) or during the light cycle (PVH only). Notable regional differences were identified, such that in the PVH histamine rebounded to above baseline levels, whereas in the VMH histamine remained lower than baseline for at least 10 s after licking ceased. In a separate cohort of male and female mice, enhancing histamine tone via administration of a histamine precursor (L‐histidine) reduced the number of licks across multiple sucrose concentrations. Together, these findings indicate that histaminergic activity is modulated rapidly during ingestive episodes, and that understanding these release patterns will give insight into histamine's role in appetite suppression. image

Dopamine (DA) neurons of the substantia nigra (SN) are essential for motor control and selectively degenerate in Parkinson’s disease (PD). However, DA neurons are molecularly heterogeneous, with some showing greater vulnerability and others resilience. Here, we show that the DA subtype marker Anxa1 , identified in mice, labels PD-vulnerable DA neurons in human SN. Using mice, we found that excitatory inputs from subthalamic (STN) and pedunculopontine (PPN) nuclei evoked frequency-dependent excitation in SN GABA neurons, but complex multiphasic DA neuron responses, suggesting heterogeneous DA subtype responses. Indeed, excitatory inputs evoked differential DA responses in striatal subregions, an increase in caudal striatum, but inhibition followed by rebound in dorsolateral striatum. Additionally, PD-resilient Vglut2+ DA neurons were excited by STN/PPN input, while vulnerable Anxa1+ DA neurons were inhibited. These findings demonstrate that DA subtypes are embedded in distinct functional networks, suggesting that some therapeutic interventions may differentially impact vulnerable and resilient DA subtypes.

Post-traumatic stress disorder (PTSD) develops in a sub-population of people exposed to trauma and is highly comorbid with opioid use disorder (OUD). The neurobiology of comorbid PTSD+OUD, especially with respect to sex differences, is poorly understood. Here we investigated the sex-specific effects of predator scent stress on intravenous (IV) oxycodone self-administration (SA), cue-primed reinstatement, and pre- and post-stress corticosterone (CORT) concentrations. Upon detecting effects of stress on oxycodone IVSA, the nucleus accumbens (NA) dopamine response to oxycodone was assessed. Male and female rats received a single 10-minute exposure to the predator scent TMT or the control condition. One week later, rats were tested for anxiety-like behavior on the elevated plus maze and underwent oxycodone IVSA, instrumental extinction, and a cue-primed reinstatement test. In a separate cohort of only female rats, the NA core dopamine response to IV oxycodone (0, 0.25, 0.5 mg/kg) was assessed using fiber photometry. TMT increased anxiety-like behavior one week later in male and female rats. Baseline CORT was negatively correlated with anxiety-like behavior in males. TMT exposure reduced oxycodone intake exclusively in females, with no effects on instrumental extinction or cue-primed reinstatement in either sex. Female rats demonstrated a dose-dependent increase in NA core dopamine signal following oxycodone administration that was blunted by a history of TMT exposure. These results indicate that TMT exposure diminishes the NA core dopamine response to oxycodone in females, reducing oxycodone IVSA. These results are consistent with stress-induced reward insensitivity that has been observed following stress in both rodents and humans.

The posterior medial (POm) thalamus is heavily interconnected with sensory and motor circuitry and is likely involved in behavioral modulation and sensorimotor integration. POm provides axonal projections to the dorsal striatum, a hotspot of sensorimotor processing, yet the role of POm-striatal projections has remained undetermined. Using optogenetics with mouse brain slice electrophysiology, we found that POm provides robust synaptic input to direct and indirect pathway striatal spiny projection neurons (D1- and D2-SPNs, respectively) and parvalbumin-expressing fast spiking interneurons (PVs). During the performance of a whisker-based tactile discrimination task in head-restrained mice, POm-striatal projections displayed learning-related activation correlating with anticipatory, but not reward-related, pupil dilation. Inhibition of POm-striatal axons across learning caused slower reaction times and an increase in the number of training sessions for expert performance. Our data indicate that POm-striatal inputs provide a behaviorally relevant arousal-related signal, which may prime striatal circuitry for efficient integration of subsequent choice-related inputs.

The ability to evaluate valence of a social agent based on social experience is essential for an animal's survival in its social group1. Although hippocampal circuits have been implicated in distinguishing novel and familiar conspecifics2-7, it remains unclear how social valence is constructed on the basis of social history and what mechanisms underlie the heightened valence versatility in dynamic relationships. Here we demonstrate that the ventral (v)CA1 integrates serotonin (5-HT) inputs from the dorsal raphe and neurotensin inputs from the paraventricular nucleus of the thalamus (PVT) to determine positive or negative valence of conspecific representations. Specifically, during an appetitive social interaction 5-HT is released into the vCA1 and disinhibits pyramidal neurons through 5-HT1B receptors, whereas neurotensin is released during an aversive social interaction and potentiates vCA1 neurons directly through NTR1s. Optogenetic silencing of dorsal raphe 5-HT and PVT neurotensin inputs into the vCA1 impairs positive and negative social valence, respectively, and excitation flexibly switches valence assignment. These results show how aversive and rewarding social experiences are linked to conspecific identity through converging dorsal raphe 5-HT and PVT neurotensin signals in the vCA1 that instruct opposing valence, and represent a synaptic switch for flexible social valence computation.

Dopamine release in the nucleus accumbens (NAc) plays a critical role in the motivation to perform actions that promote survival. However, the NAc dopamine response to innately threatening visual stimuli, such as predators descending from above, and the innate behaviors they promote has not been fully characterized. Using the genetically encoded sensor dLight1, we investigated looming visual threat–evoked dopamine release in the lateral (LNAc) and medial NAc shell (NAcS) regions in freely moving mice during performance of a looming stimulus assay. We found that dopamine release related to visual threat perception in the NAcS, but not in the LNAc, predicts the timing and vigor of a future defensive action, yet dopamine released during the performance of the action itself does not. Optogenetic inhibition of dopaminergic terminals in the NAcS at visual stimulus onset prevented escape, confirming a role for ventral striatal dopamine in promoting threat-related behaviors.

Social novelty preference is an important aspect of social interaction for evaluating new threats and opportunities for survival, but the underlying neuronal mechanism remains unclear. Here, we identify a molecularly defined medial prefrontal cortex (mPFC) excitatory neuron subtype, located in layer 5 expressing Il1rapl2 , which is highly associated with social deficit disorders in genome-wide association studies and might be responsible for regulating social novelty preference. Using an Il1rapl2 -Cre mouse line, we show that chemogenetic activation of the mPFC Il1rapl2 -expressing neurons impairs social novelty preference but with little effect on sociability. In addition, fiber photometry recording indicates that this neuron subtype is inhibited when mice interact with novel but not with familiar mice. Furthermore, viral tracing and terminal manipulation reveal that basolateral amygdala (BLA)–projecting Il1rapl2 + neurons mediate the social novelty preference. Thus, our study uncovers a molecularly defined mPFC-BLA circuit that specifically regulates social novelty preference, highlighting that specific neuron subtypes and circuits could modulate distinct aspects of social behaviors.