Publications using the RZ5P

Nicotine addiction remains a major cause of disease and premature death worldwide. Nicotine modulates neural pathways that underlie both rewarding and aversive behavioral effects, but persistent activation of brain reward circuitry drives nicotine consumption despite the negative consequences. When nicotine users attempt to quit, additional neural mechanisms are recruited to generate an aversive withdrawal state, which contributes to the remarkably high relapse rate among nicotine users. The interpeduncular nucleus (IPN) and its presynaptic inputs from the medial habenula are key mediators of aversive nicotine withdrawal symptoms, but the downstream neural targets mediating these effects are unknown. The aversive effects of acute exposure to high doses of nicotine require inhibitory IPN GABAergic projections to the laterodorsal tegmentum (LDTg), a key driver of reward-related dopamine signaling. Here we show that optogenetic inhibition of these IPN→LDTg projections reduces behavioral and physiological effects of nicotine withdrawal in male and female mice. Using fiber photometry, we found that nicotine withdrawal reduced reward-related signaling with decreases in both LDTg neuronal activity and nucleus accumbens dopamine release. These studies demonstrate a direct link between aversive and appetitive neural pathways that is active during nicotine withdrawal, providing novel targets for treating nicotine addiction.

Brain circuits in reward-associated behaviors are potent drivers of feeding behavior but also recently emerged as regulator of metabolism. Short or chronic exposures to caloric food alter brain structures and are associated with increased astrocytes reactivity and pro-inflammatory responses in both mice and humans. However, the role of striatal astrocytes in regulating adaptive and maladaptive behavioral and metabolic responses to energy-dense food remains elusive. In this study we reveal that chemogenetic manipulation of the astrocytes in striatal structures can exert a direct effect on peripheral metabolism in male mice, and that manipulation of astrocytes in the dorsal striatum can alter peripheral metabolism and is sufficient to restore cognitive deficit induced by chronic high fat high sucrose (HFHS) diet exposure in obese mice. Altogether, this work reveals a yet unappreciated role for striatal astrocytes as a direct operator of flexible behavior and metabolic control.

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 h acute restraint stress model in mice, we uncover that stress causes a net decrease in vSUB activity in females driven by adrenergic receptor signaling. By contrast, males exhibit a net increase in vSUB activity that is driven by corticosterone signaling. We further identified sexually dimorphic changes in vSUB output to the anterior bed nucleus of the stria terminalis and in anxiety-like behaviors 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 sexually dimorphic adaptations that may shape stress-related psychiatric disease risk.

The activity of neurotransmitter-based cell types in the cuneiform and pedunculopontine nuclei during locomotion, non-locomotor behaviors, and following sensory stimulation is not fully understood. Using fiber photometry in mice, we found cell-type specific responses to sensory stimuli. Glutamatergic and GABAergic cells responded to sound, visual looming, and air puffs, except for pedunculopontine GABAergic cells, which did not respond to visual looming. Cholinergic cells responded to air puffs. Air puffs triggered high-speed locomotion, whereas visual looming and sound stimuli evoked low-speed locomotion. During air puff–evoked locomotion, cuneiform glutamatergic neuron activity was higher than in trials without locomotion. In contrast, during locomotion evoked by visual looming or sound, activity in pedunculopontine glutamatergic neurons was higher than when no locomotion occurred. In the open-field arena, mice exhibited spontaneous low-speed locomotion during which activity increased in pedunculopontine glutamatergic cells. Activity also increased in a cell type-specific manner during grooming or rearing. Our study shows cell type-specific activity in the cuneiform or pedunculopontine nuclei during locomotion, non-locomotor behaviors, and following sensory stimulation. Sensory responsiveness likely has relevance in Parkinson’s disease, where sensory circuits are increasingly targeted to improve walking.

Food sensory perception has emerged as a potent regulator of specialized feeding circuits; yet, the consequences on feeding behaviour and the underlying neuronal basis remain poorly understood. Here, we reveal a sensory pathway that co-ordinately integrates food odours to control forthcoming nutrient intake in male mice. Unbiased whole-brain mapping of food odour-induced brain activity revealed a potent activation of the medial septum (MS), where food odours selectively activate MS glutamatergic neurons (MS VGLUT2 ). Activity dynamics of MS VGLUT2 neurons uncovered a biphasic modulation of their neuronal activity with a transient activation after detection of food odours and a long-lasting inhibition following food ingestion, independent of the caloric value and identity of the food. MS VGLUT2 neurons receive direct projections from the olfactory bulb (OB) and acute optogenetic stimulation of OB→MS projections selectively before food ingestion decreased feeding in lean mice. However, acute OB→MS optogenetic stimulation in diet-induced obese mice failed to reduce feeding, suggesting the involvement of this pathway in calorie-rich diet-induced hyperphagia and obesity development. Altogether, our study uncovered a sensory circuit by which the organism integrates olfactory food cues to prime satiety at the outset of a meal.

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.

Understanding how threats drive fear memory formation is crucial to understanding how organisms adapt to environments and treat threat-related disorders such as PTSD. While traditional Pavlovian conditioning studies have provided valuable insights, the exclusive reliance on electric shock as a threat stimulus has limited our understanding of diverse threats. To address this, we developed a conditioning paradigm using a looming visual stimulus as an unconditioned stimulus (US) in mice and identified a distinct neural circuit for visual threat conditioning. Parabrachial CGRP neurons were necessary for both conditioning and memory retrieval. Upstream neurons in the posterior insular cortex (pIC) responded to looming stimuli, and their projections to the parabrachial nucleus (PBN) induced aversive states and drove conditioning. However, this pIC-to-PBN pathway was not required for foot-shock conditioning. These findings reveal how non-nociceptive visual stimuli can drive aversive states and fear memory formation, expanding our understanding of aversive US processing beyond traditional models.

Phenylketonuria (PKU), an inborn error of phenylalanine (Phe) metabolism, is a common cause of intellectual disability. However, the mechanisms by which elevated Phe levels cause cognitive impairment remain unclear. Here, we show that submillimolar Phe perturbs synaptic plasticity through the hyperactivation of GluN2B-containing NMDARs. Pah Enu2 PKU model mice exhibited submillimolar and supramillimolar concentrations of Phe in the cerebrospinal fluid (CSF) and serum, respectively. l-Phe produced concentration-dependent bidirectional effects on NMDA-induced currents, without affecting synaptic NMDA receptors (NMDARs) in hippocampal CA1 neurons. l-Phe-induced hyperactivation of extrasynaptic GluN2B resulted in activity-dependent downregulation of AMPA receptors during burst or sustained synaptic activity. Administration of l-Phe in mice decreased neural activity and impaired memory, which were blocked by pretreatment with GluN2B inhibitors. Furthermore, pharmacological and virus-mediated suppression of GluN2B reversed the impaired learning in Pah Enu2 mice. Collectively, these results suggest the concentration of Phe in the CSF of patients with PKU perturbs extrasynaptic NMDARs and synaptic plasticity and that suppression of GluN2B may have the potential to improve cognitive function in patients with PKU.

Objective: This study aims to investigate how reproductive experience (RE) alters thermal preference and thermoregulation in female mice, with a focus on estrogen receptor alpha (ERα)-expressing neurons in the preoptic area (POA). Methods: Thermal preference and body temperature were measured in female mice with and without RE, and virgin female mice with selective deletion of ERα from the POA (ERαPOA-KO). The number and activity of ERα-expressing POA neurons (ERαPOA) were assessed using immunohistochemistry and in vitro electrophysiology in response to temperature changes and ERα agonist. Results: We showed that female mice prefer a cooler environment starting from late pregnancy and persisting long term postpartum. Female mice with RE (>4 weeks post-weaning) displayed lower body temperature and a lower thermal preferred temperature, and lost preference for warm environments (30 °C) but preserved avoidance of cold environments (15 °C). This was associated with a significant decrease in the number of ERαPOA neurons. Importantly, virgin female ERαPOA-KO mice displayed lower thermal preferred temperature and impaired warm preference, mimicking RE mice. We further found that distinct ERαPOA subpopulations can be regulated by temperature changes with or without presynaptic blockers, and by ERα agonist. More importantly, RE decreased the number of warm-activated ERαPOA neurons and reduced the excitatory effects of warmth and estrogen-ERα signaling, while cold-activated ERαPOA neurons were slightly enhanced in female mice with RE. Conclusion: Our results support that the thermosensing ability and estrogenic effects in ERαPOA neurons are regulated by reproductive experience, altering thermal preference.

Mammillary body (MB) is traditionally viewed as a structural node of an anatomic circuit for emotion and memory. However, little is known about its molecular and cellular organizations. Here, a discovery that MB contains four subtypes of neurons that occupy different spatial subregions is reported. Of these, two subtypes of neurons are tagged by parvalbumin (PV) and dopamine receptor‐D2 (Drd2) markers. PV neurons are spontaneously active, whereas Drd2 neurons are inactive at rest and generate rebound bursts. These two distinct electrophysiological properties are encoded by Kcnn4 and Cacna1h . PV and Drd2 neurons generate two distinct cell‐type specific circuits by receiving inputs from two discrete subiculum neuronal classes. Gain‐ and loss‐of‐function studies on these cortical‐subcortical circuits demonstrate their differential roles for place and object recognition memory. This finding provides a comprehensive molecular and structural atlas of MB neurons at single‐cell resolution and reveals that MB contains molecularly, structurally, and functionally dissociable streams within its serial architecture.

The endogenous dynorphin/kappa opioid receptor (KOR) system in the brain mediates the dysphoric effects of stress, and KOR antagonists may have therapeutic potential for the treatment of drug addiction, depression, and psychosis. One class of KOR antagonists, the long-acting norBNI-like antagonists, have been suggested to act by causing KOR inactivation through a cJun-kinase mechanism rather than by competitive inhibition. In this study, we screened for other opioid ligands that might produce norBNI-like KOR inactivation and found that nalfurafine (a G-biased KOR agonist) and nalmefene (a KOR partial agonist) also produce long-lasting KOR inactivation. Neither nalfurafine nor nalmefene are completely selective KOR ligands, but KOR inactivation was observed at doses 10-100 fold lower than necessary for mu opioid receptor actions. Daily microdosing with nalfurafine or nalmefene blocked KORs responsible for antinociceptive effects, blocked KORs mediating stress-induced aversion, and mitigated the aversion during acute and protracted withdrawal in fentanyl-dependent mice. Both nalfurafine and nalmefene have long histories of safety and use in humans and could potentially be repurposed for the treatment of dynorphin-mediated stress disorders.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD). 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 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. In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine consolidate sleep. Furthermore, inhibiting LC-NE neurons restores memory. Finally, rabies-mediated screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory-regulatory regions in 16p11.2 deletion mice. Our findings identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Inhibitory neuronal circuits within the lateral septum (LS) play a key role in regulating mood and stress responses. Even though glial cells can modulate these circuits, the impact of astrocytes on LS neural circuits and their functional interactions remains largely unexplored. Here, we demonstrate that astrocytes exhibit increased intracellular Ca²⁺ levels in response to aversive sensory and social stimuli in both male and female mice. This astrocytic Ca²⁺ elevation inhibits neighboring LS neurons by reducing excitatory synaptic transmissions through A1R-mediated signaling in both the dorsal (LSd) and intermediate LS (LSi) and enhancing inhibitory synaptic transmission via A2AR-mediated signaling in the LSi. At the same time, astrocytes reduce inhibitory tone on distant LS neurons. In the LSd, astrocytes promote social avoidance and anxiety, as well as increased heart rate in socially stressed male mice. In contrast, astrocytes in the LSi contribute to elevated heart rate and heightened blood corticosterone levels in unstressed male mice. These results suggest that the dynamic interactions between astrocytes and neurons within the LS modulate physiological and behavioral responses to stressful experiences.

Objective: Exposure to 60% high fat diet (HFD) leads to a robust consummatory preference over well-balanced chow standard diet (SD) when mice are presented with a choice. This passive HFD-induced SD devaluation following HFD challenge and withdrawal is highlighted by the significant reduction in SD food intake even in states of caloric deprivation. The elements of HFD that lead to this SD depreciation remains unclear. Possibly important factors include the amount and type of fat contained in a diet as well as past eating experiences dependent on sensory properties including taste and post ingestive feedback. We aimed to explore the role of these components to HFD-induced SD devaluation. Methods: Wildtype mice were longitudinally presented discrete HFDs in conjunction with SD and feeding and metabolic parameters were analyzed. A separate cohort of animals were assessed for acute HFD preference in 3 conditions: 1) ad libitum fed (sated), 2) overnight fasted (physiologically hungry), and 3) ad libitum fed (artificially hungry), elicited through chemogenetic Agouti-related peptide (AgRP) neuron activation. Population dynamics of AgRP neurons were recorded to distinct inaccessible and accessible diets both before and after consummatory experience. Transient receptor potential channel type M5 (TRPM5) knockout mice were used to investigate the role of fat taste perception and preference to HFD-induced SD devaluation. The clinically approved lipase inhibitor orlistat was used to test the contribution of fat absorption to HFD-induced SD devaluation. Results: HFD-induced SD devaluation is dependent on fat content, composition, and preference. This effect scaled both in strength and latency with higher percentages of animal fat. 60% HFD was preferred and almost exclusively consumed in preference to other diets across hours and days, but this was not as evident upon initial introduction over seconds and minutes, suggesting ingestive experience is critical. Optical fiber photometry recordings of AgRP activity supported this notion as neuronal suppression by the different diets was contingent on prior intake. While taste transduced via TRPM5 influenced HFD-evoked weight gain, it failed to impact either HFD preference or HFD-induced SD devaluation. Perturbation of post ingestive feedback through orlistat-mediated diminishment of fat absorption prevented HFD-evoked weight gain and abolished HFD-induced SD devaluation. Conclusions: Post ingestive feedback via fat digestion is vital for expression of HFD-induced SD devaluation.

Leptin is an adipose tissue hormone that maintains homeostatic control of adipose tissue mass by regulating the activity of specific neural populations controlling appetite and metabolism 1 . Leptin regulates food intake by inhibiting orexigenic agouti-related protein (AGRP) neurons and activating anorexigenic pro-opiomelanocortin (POMC) neurons 2 . However, whereas AGRP neurons regulate food intake on a rapid time scale, acute activation of POMC neurons has only a minimal effect 3–5 . This has raised the possibility that there is a heretofore unidentified leptin-regulated neural population that rapidly suppresses appetite. Here we report the discovery of a new population of leptin-target neurons expressing basonuclin 2 ( Bnc2 ) in the arcuate nucleus that acutely suppress appetite by directly inhibiting AGRP neurons. Opposite to the effect of AGRP activation, BNC2 neuronal activation elicited a place preference indicative of positive valence in hungry but not fed mice. The activity of BNC2 neurons is modulated by leptin, sensory food cues and nutritional status. Finally, deleting leptin receptors in BNC2 neurons caused marked hyperphagia and obesity, similar to that observed in a leptin receptor knockout in AGRP neurons. These data indicate that BNC2-expressing neurons are a key component of the neural circuit that maintains energy balance, thus filling an important gap in our understanding of the regulation of food intake and leptin action.

The brain processes an array of stimuli, enabling the selection of appropriate behavioural responses, but the neural pathways linking interoceptive inputs to outputs for feeding are poorly understood 1–3 . Here we delineate a subcortical circuit in which brain-derived neurotrophic factor (BDNF)-expressing neurons in the ventromedial hypothalamus (VMH) directly connect interoceptive inputs to motor centres, controlling food consumption and jaw movements. VMH BDNF neuron inhibition increases food intake by gating motor sequences of feeding through projections to premotor areas of the jaw. When food is unavailable, VMH BDNF inhibition elicits consummatory behaviours directed at inanimate objects such as wooden blocks, and inhibition of perimesencephalic trigeminal area (pMe5) projections evokes rhythmic jaw movements. The activity of these neurons is decreased during food consumption and increases when food is in proximity but not consumed. Activity is also increased in obese animals and after leptin treatment. VMH BDNF neurons receive monosynaptic inputs from both agouti-related peptide (AgRP) and proopiomelanocortin neurons in the arcuate nucleus (Arc), and constitutive VMH BDNF activation blocks the orexigenic effect of AgRP activation. These data indicate an Arc → VMH BDNF → pMe5 circuit that senses the energy state of an animal and regulates consummatory behaviours in a state-dependent manner.

Spatial memories guide navigation efficiently toward desired destinations. However, the neuronal and circuit mechanisms underlying the encoding of goal locations and its translation into goal-directed navigation remain unclear. Here we demonstrate that mice rapidly form a spatial memory of a shelter during shelter experiences, guiding escape behavior toward the goal location-a shelter-when under threat. Dopaminergic neurons in the ventral tegmental area and their projection to the nucleus accumbens (NAc) encode safety signals associated with the shelter. Optogenetically induced phasic dopamine signals are sufficient to create a place memory that directs escape navigation. Converging dopaminergic and hippocampal glutamatergic inputs to the NAc mediate the formation of a goal-related memory within a subpopulation of NAc neurons during shelter experiences. Artificial co-activation of this goal-related NAc ensemble with neurons in the dorsal periaqueductal gray was sufficient to trigger memory-guided, rather than random, escape behavior. These findings provide causal evidence of cognitive circuit modules linking memory with goal-directed action.

Anatomical connectivity and lesion-deficit studies have shown that the dorsal and ventral hippocampi contribute to cognitive and emotional processes, respectively. However, the role of the dorsal hippocampus (dHP) in emotional or stress-related behaviors remains unclear. Here, we showed that neuronal activity in the dHP affects stress-coping behaviors in mice via excitatory projections to the medial prefrontal cortex (mPFC). The antidepressant ketamine rapidly induced c-Fos expression in both the dorsal and ventral hippocampi. The suppression of GABAergic transmission in the dHP-induced molecular changes similar to those induced by ketamine administration, including eukaryotic elongation factor 2 (eEF2) dephosphorylation, brain-derived neurotrophic factor (BDNF) elevation, and extracellular signal-regulated kinase (ERK) phosphorylation. These synaptic and molecular changes in the dHP induced a reduction in the immobility time of the mice in the tail-suspension and forced swim tests without affecting anxiety-related behavior. Conversely, pharmacological and chemogenetic potentiation of inhibitory neurotransmission in the dHP CA1 region induced passive coping behaviors during the tests. Transneuronal tracing and electrophysiology revealed monosynaptic excitatory connections between dHP CA1 neurons and mPFC neurons. Optogenetic stimulation of dHP CA1 neurons in freely behaving mice produced c-Fos induction and spike firing in the mPFC neurons. Chemogenetic activation of the dHP-recipient mPFC neurons reversed the passive coping behaviors induced by suppression of dHP CA1 neuronal activity. Collectively, these results indicate that neuronal activity in the dHP modulates stress-coping strategies to inescapable stress and contributes to the antidepressant effects of ketamine via the dHP-mPFC circuit.

Females exhibit complex, dynamic behaviours during mating with variable sexual receptivity depending on hormonal status 1–4 . However, how their brains encode the dynamics of mating and receptivity remains largely unknown. The ventromedial hypothalamus, ventrolateral subdivision contains oestrogen receptor type 1-positive neurons that control mating receptivity in female mice 5,6 . Here, unsupervised dynamical system analysis of calcium imaging data from these neurons during mating uncovered a dimension with slow ramping activity, generating a line attractor in neural state space. Neural perturbations in behaving females demonstrated relaxation of population activity back into the attractor. During mating, population activity integrated male cues to ramp up along this attractor, peaking just before ejaculation. Activity in the attractor dimension was positively correlated with the degree of receptivity. Longitudinal imaging revealed that attractor dynamics appear and disappear across the oestrus cycle and are hormone dependent. These observations suggest that a hypothalamic line attractor encodes a persistent, escalating state of female sexual arousal or drive during mating. They also demonstrate that attractors can be reversibly modulated by hormonal status, on a timescale of days.

Current concepts of corticothalamic organization in the mammalian brain are mainly based on sensory systems, with less focus on circuits for higher-order cognitive functions. In sensory systems, first-order thalamic relays are driven by subcortical inputs and modulated by cortical feedback, while higher-order relays receive strong excitatory cortical inputs. The applicability of these principles beyond sensory systems is uncertain. We investigated mouse prefronto-thalamic projections to the midline thalamus, revealing distinct top-down control. Unlike sensory systems, this pathway relies on indirect modulation via the thalamic reticular nucleus (TRN). Specifically, the prelimbic area, which influences emotional and motivated behaviors, impacts instrumental avoidance responses through direct and indirect projections to the paraventricular thalamus. Both pathways promote defensive states, but the indirect pathway via the TRN is essential for organizing avoidance decisions through disinhibition. Our findings highlight intra-thalamic circuit dynamics that integrate cortical cognitive signals and their role in shaping complex behaviors.