Hypothalamic vasopressin sex differentiation is observed by embryonic day 15 in mice and is disrupted by the xenoestrogen bisphenol A.

Arginine vasopressin (AVP) neurons of the hypothalamic paraventricular region (AVPPVN) mediate sex-biased social behaviors across most species, including mammals. In mice, neural sex differences are thought to be established during a critical window around birth ( embryonic (E) day 18 to postnatal (P) day 2) whereby circulating testosterone from the fetal testis is converted to estrogen in sex-dimorphic brain regions. Here, we found that AVPPVN neurons are sexually dimorphic by E15.5, prior to this critical window, and that gestational bisphenol A (BPA) exposure permanently masculinized female AVPPVN neuronal numbers, projections, and electrophysiological properties, causing them to display male-like phenotypes into adulthood. Moreover, we showed that nearly twice as many neurons that became AVP+ by P0 were born at E11 in males and BPA-exposed females compared to control females, suggesting that AVPPVN neuronal masculinization occurs between E11 and P0. We further narrowed this sensitive period to around the timing of neurogenesis by demonstrating that exogenous estrogen exposure from E14.5 to E15.5 masculinized female AVPPVN neuronal numbers, whereas a pan-estrogen receptor antagonist exposed from E13.5 to E15.5 blocked masculinization of males. Finally, we showed that restricting BPA exposure to E7.5-E15.5 caused adult females to display increased social dominance over control females, consistent with an acquisition of male-like behaviors. Our study reveals an E11.5 to E15.5 window of estrogen sensitivity impacting AVPPVN sex differentiation, which is impacted by prenatal BPA exposure.

Distinguishing motion artifacts during optical fiber-based in-vivo hemodynamics recordings from brain regions of freely moving rodents.

Significance: Motion artifacts in the signals recorded during optical fiber-based measurements can lead to misinterpretation of data. In this work, we address this problem during in-vivo rodent experiments and develop a motion artifacts correction (MAC) algorithm for single-fiber system (SFS) hemodynamics measurements from the brains of rodents. Aim: (i) To distinguish the effect of motion artifacts in the SFS signals. (ii) Develop a MAC algorithm by combining information from the experiments and simulations and validate it. Approach: Monte-Carlo (MC) simulations were performed across 450 to 790 nm to identify wavelengths where the reflectance is least sensitive to blood absorption-based changes. This wavelength region is then used to develop a quantitative metric to measure motion artifacts, termed the dissimilarity metric (DM). We used MC simulations to mimic artifacts seen during experiments. Further, we developed a mathematical model describing light intensity at various optical interfaces. Finally, an MAC algorithm was formulated and validated using simulation and experimental data. Results: We found that the 670 to 680 nm wavelength region is relatively less sensitive to blood absorption. The standard deviation of DM (σDM) can measure the relative magnitude of motion artifacts in the SFS signals. The artifacts cause rapid shifts in the reflectance data that can be modeled as transmission changes in the optical lightpath. The changes observed during the experiment were found to be in agreement to those obtained from MC simulations. The mathematical model developed to model transmission changes to represent motion artifacts was extended to an MAC algorithm. The MAC algorithm was validated using simulations and experimental data. Conclusions: We distinguished motion artifacts from SFS signals during in vivo hemodynamic monitoring experiments. From simulation and experimental data, we showed that motion artifacts can be modeled as transmission changes. The developed MAC algorithm was shown to minimize artifactual variations in both simulation and experimental data.

Optogenetic recruitment of hypothalamic corticotrophin-releasing-hormone (CRH) neurons reduces motivational drive.

Impaired motivational drive is a key feature of depression. Chronic stress is a known antecedent to the development of depression in humans and depressive-like states in animals. Whilst there is a clear relationship between stress and motivational drive, the mechanisms underpinning this association remain unclear. One hypothesis is that the endocrine system, via corticotropin-releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN; PVNCRH), initiates a hormonal cascade resulting in glucocorticoid release, and that excessive glucocorticoids change brain circuit function to produce depression-related symptoms. Another mostly unexplored hypothesis is that the direct activity of PVNCRH neurons and their input to other stress- and reward-related brain regions drives these behaviors. To further understand the direct involvement of PVNCRH neurons in motivation, we used optogenetic stimulation to activate these neurons 1 h/day for 5 consecutive days and showed increased acute stress-related behaviors and long-lasting deficits in the motivational drive for sucrose. This was associated with increased Fos-protein expression in the lateral hypothalamus (LH). Direct stimulation of the PVNCRH inputs in the LH produced a similar pattern of effects on sucrose motivation. Together, these data suggest that PVNCRH neuronal activity may be directly responsible for changes in motivational drive and that these behavioral changes may, in part, be driven by PVNCRH synaptic projections to the LH.

Markerless Mouse Tracking for Social Experiments.

Automated behavior quantification in socially interacting animals requires accurate tracking. While many methods have been very successful and highly generalizable to different settings, issues of mistaken identities and lost information on key anatomical features are common, although they can be alleviated by increased human effort in training or post-processing. We propose a markerless video-based tool to simultaneously track two interacting mice of the same appearance in controlled settings for quantifying behaviors such as different types of sniffing, touching, and locomotion to improve tracking accuracy under these settings without increased human effort. It incorporates conventional handcrafted tracking and deep-learning-based techniques. The tool is trained on a small number of manually annotated images from a basic experimental setup and outputs body masks and coordinates of the snout and tail-base for each mouse. The method was tested on several commonly used experimental conditions including bedding in the cage and fiberoptic or headstage implants on the mice. Results obtained without any human corrections after the automated analysis showed a near elimination of identities switches and a ∼15% improvement in tracking accuracy over pure deep-learning-based pose estimation tracking approaches. Our approach can be optionally ensembled with such techniques for further improvement. Finally, we demonstrated an application of this approach in studies of social behavior of mice by quantifying and comparing interactions between pairs of mice in which some lack olfaction. Together, these results suggest that our approach could be valuable for studying group behaviors in rodents, such as social interactions.

Hypothalamic CRH neurons represent physiological memory of positive and negative experience.

Recalling a salient experience provokes specific behaviors and changes in the physiology or internal state. Relatively little is known about how physiological memories are encoded. We examined the neural substrates of physiological memory by probing CRHPVN neurons of mice, which control the endocrine response to stress. Here we show these cells exhibit contextual memory following exposure to a stimulus with negative or positive valence. Specifically, a negative stimulus invokes a two-factor learning rule that favors an increase in the activity of weak cells during recall. In contrast, the contextual memory of positive valence relies on a one-factor rule to decrease activity of CRHPVN neurons. Finally, the aversive memory in CRHPVN neurons outlasts the behavioral response. These observations provide information about how specific physiological memories of aversive and appetitive experience are represented and demonstrate that behavioral readouts may not accurately reflect physiological changes invoked by the memory of salient experiences.

Impact and Role of Hypothalamic Corticotropin Releasing Hormone Neurons in Withdrawal from Chronic Alcohol Consumption in Female and Male Mice.

Worldwide, alcohol use and abuse are a leading risk of mortality, causing 5.3% of all deaths (World Health Organization, 2022). The endocrine stress system, initiated by the peripheral release of corticotropin releasing hormone (CRH) from primarily glutamatergic neurons in the paraventricular nucleus of the hypothalamus (PVN), is profoundly linked with alcohol use, abuse, and relapse (Blaine and Sinha, 2017). These PVN CRH-releasing (PVNCRH) neurons are essential for peripheral and central stress responses (Rasiah et al., 2023), but little is known about how alcohol affects these neurons. Here, we show that two-bottle choice alcohol consumption blunts the endocrine-mediated corticosterone response to stress during acute withdrawal in female mice. Conversely, using slice electrophysiology, we demonstrate that acute withdrawal engenders a hyperexcitable phenotype of PVNCRH neurons in females that is accompanied by increased glutamatergic transmission in both male and female mice. GABAergic synaptic transmission was unaffected by alcohol history. We then tested whether chemogenetic inhibition of PVNCRH neurons would restore stress response in female mice with a history of alcohol drinking in the looming disk test, which mimics an approaching predator threat. Accordingly, inhibition of PVNCRH neurons reduced active escape in hM4Di alcohol history mice only. This study indicates that stress-responsive PVNCRH neurons in females are particularly affected by a history of alcohol consumption. Interestingly, women have indicated an increase in heavy alcohol use to cope with stress (Rodriguez et al., 2020), perhaps pointing to a potential underlying mechanism in alcohol-mediated changes to PVNCRH neurons that alter stress response.SIGNIFICANCE STATEMENT Paraventricular nucleus of the hypothalamus neurons that release corticotropin releasing hormone (PVNCRH) are vital for stress response. These neurons have been understudied in relation to alcohol and withdrawal despite profound relations between stress, alcohol use disorders (AUD), and relapse. In this study, we use a variety of techniques to show that acute withdrawal from a history of alcohol impacts peripheral stress response, PVNCRH neurons, and behavior. Specifically, PVNCRH are in a hyperactive state during withdrawal, which drives an increase in active stress coping behaviors in female mice only. Understanding how alcohol use and withdrawal affects stress responding PVNCRH neurons may contribute to finding new potential targets for the treatment of alcohol use disorder.

Disruption of tonic endocannabinoid signalling triggers cellular, behavioural and neuroendocrine responses consistent with a stress response.

BACKGROUND AND PURPOSE: Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is controlled by corticotropin releasing hormone (CRH) producing neurons in the paraventricular nucleus of the hypothalamus (PVN). Disruption of eCB signalling increases drive to the HPA axis, but the mechanisms subserving this process are poorly understood. EXPERIMENTAL APPROACH: Using an array of cellular, endocrine and behavioural readouts associated with activation of CRH neurons in the PVN, we evaluated the contributions of tonic eCB signalling to the generation of a stress response. KEY RESULTS: The CB1 receptor antagonist/inverse agonist AM251, neutral antagonist NESS243 and NAPE PLD inhibitor LEI401 all uniformly increased Fos in the PVN, unmasked stress-linked behaviours, such as grooming, and increased circulating CORT, recapitulating the effects of stress. Similar effects were also seen after direct administration of AM251 into the PVN, while optogenetic inhibition of PVN CRH neurons ameliorated stress-like behavioural changes produced by disruption of eCB signalling. CONCLUSIONS AND IMPLICATIONS: These data indicate that under resting conditions, constitutive eCB signalling restricts activation of the HPA axis through local regulation of CRH neurons in the PVN.

Particle-swarm based modelling reveals two distinct classes of CRHPVN neurons.

Electrophysiological recordings can provide detailed information of single neurons' dynamical features and shed light on their response to stimuli. Unfortunately, rapidly modelling electrophysiological data for inferring network-level behaviours remains challenging. Here, we investigate how modelled single neuron dynamics leads to network-level responses in the paraventricular nucleus of the hypothalamus (PVN), a critical nucleus for the mammalian stress response. Recordings of corticotropin releasing hormone neurons from the PVN (CRHPVN ) were performed using whole-cell current-clamp. These, neurons, which initiate the endocrine response to stress, were rapidly and automatically fit to a modified adaptive exponential integrate-and-fire model (AdEx) with particle swarm optimization (PSO). All CRHPVN neurons were accurately fit by the AdEx model with PSO. Multiple sets of parameters were found that reliably reproduced current-clamp traces for any single neuron. Despite multiple solutions, the dynamical features of the models such as the rheobase, fixed points, and bifurcations, were shown to be stable across fits. We found that CRHPVN neurons can be divided into two subtypes according to their bifurcation at the onset of firing: CRHPVN -integrators and CRHPVN -resonators. The existence of CRHPVN -resonators was then directly confirmed in a follow-up patch-clamp hyperpolarization protocol which readily induced post-inhibitory rebound spiking in 33% of patched neurons. We constructed networks of CRHPVN model neurons to investigate the network level responses of CRHPVN neurons. We found that CRHPVN -resonators maintain baseline firing in networks even when all inputs are inhibitory. The dynamics of a small subset of CRHPVN neurons may be critical to maintaining a baseline firing tone in the PVN. KEY POINTS: Corticotropin-releasing hormone neurons (CRHPVN ) in the paraventricular nucleus of the hypothalamus act as the final neural controllers of the stress response. We developed a computational modelling platform that uses particle swarm optimization to rapidly and accurately fit biophysical neuron models to patched CRHPVN neurons. A model was fitted to each patched neuron without the use of dynamic clamping, or other procedures requiring sophisticated inputs and fitting algorithms. Any neuron undergoing standard current clamp step protocols for a few minutes can be fitted by this procedure The dynamical analysis of the modelled neurons shows that CRHPVN neurons come in two specific 'flavours': CRHPVN -resonators and CRHPVN -integrators. We directly confirmed the existence of these two classes of CRHPVN neurons in subsequent experiments. Network simulations show that CRHPVN -resonators are critical to retaining the baseline firing rate of the entire network of CRHPVN neurons as these cells can fire rebound spikes and bursts in the presence of strong inhibitory synaptic input.

Windows into stress: a glimpse at emerging roles for CRHPVN neurons.

The corticotropin-releasing hormone cells in the paraventricular nucleus of the hypothalamus (CRHPVN) control the slow endocrine response to stress. The synapses on these cells are exquisitely sensitive to acute stress, leveraging local signals to leave a lasting imprint on this system. Additionally, recent work indicates that these cells also play key roles in the control of distinct stress and survival behaviors. Here we review these observations and provide a perspective on the role of CRHPVN neurons as integrative and malleable hubs for behavioral, physiological, and endocrine responses to stress.

MicroSweat: A Wearable Microfluidic Patch for Noninvasive and Reliable Sweat Collection Enables Human Stress Monitoring.

Stress affects cognition, behavior, and physiology, leading to lasting physical and mental illness. The ability to detect and measure stress, however, is poor. Increased circulating cortisol during stress is mirrored by cortisol release from sweat glands, providing an opportunity to use it as an external biomarker for monitoring internal emotional state. Despite the attempts at using wearable sensors for monitoring sweat cortisol, there is a lack of reliable wearable sweat collection devices that preserve the concentration and integrity of sweat biomolecules corresponding to stress levels. Here, a flexible, self-powered, evaporation-free, bubble-free, surfactant-free, and scalable capillary microfluidic device, MicroSweat, is fabricated to reliably collect human sweat from different body locations. Cortisol levels are detected corresponding to severe stress ranging from 25 to 125 ng mL-1 averaged across multiple body regions and 100-1000 ng mL-1 from the axilla. A positive nonlinear correlation exists between cortisol concentration and stress levels quantified using the perceived stress scale (PSS). Moreover, owing to the sweat variation in response to environmental effects and physiological differences, the longitudinal and personalized profile of sweat cortisol is acquired, for the first time, for various body locations. The obtained sweat cortisol data is crucial for analyzing human stress in personalized and clinical healthcare sectors.

Chronic alcohol consumption alters home-cage behaviors and responses to ethologically relevant predator tasks in mice.

BACKGROUND: Alcohol withdrawal is a key component of severe alcohol use disorder. Animal models of alcohol withdrawal tend to focus on traditional anxiety/stress tests. While these have been essential to advancing our understanding of the biology of alcohol withdrawal, abrupt cessation of drinking following heavy alcohol consumption can also trigger withdrawal-related affective states that impact responses to a variety of life events and stressors. To this end, we show that behaviors in a variety of tasks that differ in task demand and intensity are altered during withdrawal in male and female mice after voluntary alcohol access. METHODS: Male and female miceunderwent six weeks of intermittent two-bottle choice alcohol exposure followed by behavioral tests. The tests included-Home cage: low-stress baseline environment to measure spontaneous natural behaviors; Open field: anxiety-inducing bright novel environment; Looming disc: arena with a protective hut where mice are exposed to a series of discs that mimic an overhead advancing predator, and Robogator-simulated predator task: forced foraging behavioral choice in the presence of an advancing robot predator that "attacks" when mice are near a food pellet in a large open arena. RESULTS: A history of alcohol exposure impacted behaviors in these tasks in a sex-dependent manner. In the home cage, alcohol induced reductions in digging and heightened stress coping through an increase in grooming time. In males, increased rearing yielded greater vigilance/exploration in a familiar environment. The open-field test revealed an anxiety phenotype in both male and female mice exposed to alcohol. Male mice showed no behavioral alterations to the looming disc task, while females exposed to alcohol showed greater escape responses than water controls, indicative of active stress-response behaviors. In males, the Robogator task revealed a hesitant/avoidant phenotype in alcohol-exposed mice under greater task demands. CONCLUSIONS: Few drugs show robust evidence of efficacy in clinical trials for alcohol withdrawal. Understanding how withdrawal alters a variety of behaviors in both males and females that are linked to stress coping can increase our understanding of alcohol misuse and aid in developing better medications for treating individuals with AUD.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

A versatile computational algorithm for time-series data analysis and machine-learning models.

Here we introduce Local Topological Recurrence Analysis (LoTRA), a simple computational approach for analyzing time-series data. Its versatility is elucidated using simulated data, Parkinsonian gait, and in vivo brain dynamics. We also show that this algorithm can be used to build a remarkably simple machine-learning model capable of outperforming deep-learning models in detecting Parkinson's disease from a single digital handwriting test.

Obesity-induced astrocyte dysfunction impairs heterosynaptic plasticity in the orbitofrontal cortex.

Overconsumption of highly palatable, energy-dense food is considered a key driver of the obesity pandemic. The orbitofrontal cortex (OFC) is critical for reward valuation of gustatory signals, yet how the OFC adapts to obesogenic diets is poorly understood. Here, we show that extended access to a cafeteria diet impairs astrocyte glutamate clearance, which leads to a heterosynaptic depression of GABA transmission onto pyramidal neurons of the OFC. This decrease in GABA tone is due to an increase in extrasynaptic glutamate, which acts via metabotropic glutamate receptors to liberate endocannabinoids. This impairs the induction of endocannabinoid-mediated long-term plasticity. The nutritional supplement, N-acetylcysteine rescues this cascade of synaptic impairments by restoring astrocytic glutamate transport. Together, our findings indicate that obesity targets astrocytes to disrupt the delicate balance between excitatory and inhibitory transmission in the OFC.

Subcellular specificity of cannabinoid effects in striatonigral circuits.

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.

Should I Stay or Should I Go? CRHPVN Neurons Gate State Transitions in Stress-Related Behaviors.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus are the canonical controllers of the endocrine response to stress. Here we propose a new role for these cells as a gate for state transitions that allow the organism to engage in stress-related behaviors. Specifically, we review evidence indicating that activation of these cells at critical times allows organisms to move to a state that is permissive for motor action. This is evident when the organism is under duress (defensive behavior), when the organism has successfully vanquished a threat (coping behavior), and when an organism initiates approach to a conspecific (social behavior). The motor behavior that follows from the activation of CRH neurons is not necessarily under the control of these cells but is determined by higher order circuits that discriminate more refined features of environmental context to execute the appropriate behavior.

Social communication of affective states.

Social interactions promote the communication of explicit and implicit information between individuals. Implicit or subconscious sharing of cues can be useful in conveying affective states. Knowing the affective state of others can guide future interactions, while an inability to decipher another's affective state is a core feature of autism spectrum disorder. The precise neural circuitry and mechanisms involved in communicating affective states are not well understood. Over the past few years, a number of important observations in rodent models have increased our knowledge of the neural processes for social communication of affective state. Here we highlight these contributions by first describing the rodent models used to investigate social communication of affect and then summarising the neural circuitry and processes implicated by these rodent models. We relate these findings to humans as well as to the current global context where social interactions have been modified by the Covid-19 pandemic.

Cage-lid hanging behavior as a translationally relevant measure of pain in mice.

ABSTRACT: The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

Sex-Specific Vasopressin Signaling Buffers Stress-Dependent Synaptic Changes in Female Mice.

In many species, social networks provide benefit for both the individual and the collective. In addition to transmitting information to others, social networks provide an emotional buffer for distressed individuals. Our understanding about the cellular mechanisms that contribute to buffering is poor. Stress has consequences for the entire organism, including a robust change in synaptic plasticity at glutamate synapses onto corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN). In females, however, this stress-induced metaplasticity is buffered by the presence of a naive partner. This buffering may be because of discrete behavioral interactions, signals in the context in which the interaction occurs (i.e., olfactory cues), or it may be influenced by local signaling events in the PVN. Here, we show that local vasopressin (VP) signaling in PVN buffers the short-term potentiation (STP) at glutamate synapses after stress. This social buffering of metaplasticity, which requires the presence of another individual, was prevented by pharmacological inhibition of the VP 1a receptor (V1aR) in female mice. Exogenous VP mimicked the effects of social buffering and reduced STP in CRHPVN neurons from females but not males. These findings implicate VP as a potential mediator of social buffering in female mice.SIGNIFICANCE STATEMENT In many organisms, including rodents and humans, social groups are beneficial to overall health and well-being. Moreover, it is through these social interactions that the harmful effects of stress can be mitigated, a phenomenon known as social buffering. In the present study, we describe a critical role for the neuropeptide vasopressin (VP) in social buffering of synaptic metaplasticity in stress-responsive corticotropin-releasing hormone (CRH) neurons in female mice. These effects of VP do not extend to social buffering of stress behaviors, suggesting this is a very precise and local form of sex-specific neuropeptide signaling.