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.

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.

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.

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.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes.

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Stress-related synaptic plasticity in the hypothalamus.

Stress necessitates an immediate engagement of multiple neural and endocrine systems. However, exposure to a single stressor causes adaptive changes that modify responses to subsequent stressors. Recent studies examining synapses onto neuroendocrine cells in the paraventricular nucleus of the hypothalamus demonstrate that stressful experiences leave indelible marks that alter the ability of these synapses to undergo plasticity. These adaptations include a unique form of metaplasticity at glutamatergic synapses, bidirectional changes in endocannabinoid signalling and bidirectional changes in strength at GABAergic synapses that rely on distinct temporal windows following stress. This rich repertoire of plasticity is likely to represent an important building block for dynamic, experience-dependent modulation of neuroendocrine stress adaptation.

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.

Experience salience gates endocannabinoid signaling at hypothalamic synapses.

Alterations in synaptic endocannabinoid signaling are a widespread neurobiological consequence of many in vivo experiences, including stress. Here, we report that stressor salience is critical for bidirectionally modifying presynaptic CB-1 receptor (CB1R) function at hypothalamic GABA synapses controlling the neuroendocrine stress axis in male rats. While repetitive, predictable stressor exposure impairs presynaptic CB1R function, these changes are rapidly reversed upon exposure to a high salience experience such as novel stress or by manipulations that enhance neural activity levels in vivo or in vitro. Together these data demonstrate that experience salience, through alterations in afferent synaptic activity, induces rapid changes in endocannabinoid signaling.

Noradrenaline is a stress-associated metaplastic signal at GABA synapses.

Exposure to a stressor sensitizes behavioral and hormonal responses to future stressors. Stress-associated release of noradrenaline enhances the capacity of central synapses to show plasticity (metaplasticity). We found noradrenaline-dependent metaplasticity at GABA synapses in the paraventricular nucleus of the hypothalamus in rat and mouse that controls the hypothalamic-pituitary-adrenal axis. In vivo stress exposure was required for these synapses to undergo activity-dependent long-term potentiation (LTPGABA). The activation of ß-adrenergic receptors during stress functionally upregulated metabotropic glutamate receptor 1 (mGluR1), allowing for mGluR1-dependent LTPGABA during afferent bursts. LTPGABA was expressed postsynaptically and manifested as the emergence of new functional synapses. Our findings provide, to the best of our knowledge, the first demonstration that noradrenaline release during an in vivo challenge alters information storage capacity at GABA synapses. Because these GABA synapses become excitatory following acute stress, this metaplasticity may contribute to neuroendocrine sensitization to stress.

Glucocorticoid feedback uncovers retrograde opioid signaling at hypothalamic synapses.

Stressful experience initiates a neuroendocrine response culminating in the release of glucocorticoid hormones into the blood. Glucocorticoids feed back to the brain, causing adaptations that prevent excessive hormone responses to subsequent challenges. How these changes occur remains unknown. We found that glucocorticoid receptor activation in rodent hypothalamic neuroendocrine neurons following in vivo stress is a metaplastic signal that allows GABA synapses to undergo activity-dependent long-term depression (LTDGABA). LTDGABA was unmasked through glucocorticoid receptor-dependent inhibition of Regulator of G protein Signaling 4 (RGS4), which amplified signaling through postsynaptic metabotropic glutamate receptors. This drove somatodendritic opioid release, resulting in a persistent retrograde suppression of synaptic transmission through presynaptic µ receptors. Together, our data provide new evidence for retrograde opioid signaling at synapses in neuroendocrine circuits and represent a potential mechanism underlying glucocorticoid contributions to stress adaptation.

Cocaine potentiates excitatory drive in the perifornical/lateral hypothalamus.

The hypothalamus is a critical controller of homeostatic responses and plays a fundamental role in reward-seeking behaviour. Recently, hypothalamic neurones in the perifornical/lateral hypothalamic area (PF/LHA) have also been implicated in drug-seeking behaviour through projections to extra-hypothalamic sites such as the ventral tegmental area. For example, a population of neurones that expresses the peptide orexin has been strongly implicated in addiction-relevant behaviours. To date, the effect of addictive drugs on synaptic properties in the hypothalamus remains largely unexplored. Previous studies focusing on the PF/LHA neurones, however, have shown that the orexin system exhibits significant plasticity in response to food or sleep restriction. This neuroadaptive ability suggests that PF/LHA neurones could be highly susceptible to modifications by drug exposure. Here, we sought to determine whether cocaine produces synaptic plasticity in PF/LHA neurones. Whole-cell patch-clamp techniques were used to examine the effects of experimenter-administered (passive) or self-administered (SA) cocaine on glutamatergic synaptic transmission in PF/LHA neurones. These experiments demonstrate that both passive and SA cocaine exposure increases miniature excitatory postsynaptic current (mEPSC) frequency in PF/LHA neurones. In addition, SA cocaine reduced the paired-pulse ratio but the AMPA/NMDA ratio of evoked excitatory inputs was unchanged, indicative of a presynaptic locus for synaptic plasticity. Dual-labelling for orexin and excitatory inputs using the vesicular glutamate transporter (VGLUT2), showed that passive cocaine exposure increased VGLUT2-positive appositions onto orexin neurones. Further, a population of recorded neurones that were filled with neurobiotin and immunolabelled for orexin confirmed that increased excitatory drive occurs in this PF/LHA population. Given the importance of the PF/LHA and the orexin system in modulating drug addiction, we suggest that these cocaine-induced excitatory synapse-remodelling events within the hypothalamus may contribute to persistence in drug-seeking behaviour and relapse.

MAP kinases couple hindbrain-derived catecholamine signals to hypothalamic adrenocortical control mechanisms during glycemia-related challenges.

Physiological responses to hypoglycemia, hyperinsulinemia, and hyperglycemia include a critical adrenocortical component that is initiated by hypothalamic control of the anterior pituitary and adrenal cortex. These adrenocortical responses ensure appropriate long-term glucocorticoid-mediated modifications to metabolism. Despite the importance of these mechanisms to disease processes, how hypothalamic afferent pathways engage the intracellular mechanisms that initiate adrenocortical responses to glycemia-related challenges are unknown. This study explores these mechanisms using network- and cellular-level interventions in in vivo and ex vivo rat preparations. Results show that a hindbrain-originating catecholamine afferent system selectively engages a MAP kinase pathway in rat paraventricular hypothalamic CRH (corticotropin-releasing hormone) neuroendocrine neurons shortly after vascular insulin and 2-deoxyglucose challenges. In turn, this MAP kinase pathway can control both neuroendocrine neuronal firing rate and the state of CREB phosphorylation in a reduced ex vivo paraventricular hypothalamic preparation, making this signaling pathway an ideal candidate for coordinating CRH synthesis and release. These results establish the first clear structural and functional relationships linking neurons in known nutrient-sensing regions with intracellular mechanisms in hypothalamic CRH neuroendocrine neurons that initiate the adrenocortical response to various glycemia-related challenges.

Endocannabinoids gate state-dependent plasticity of synaptic inhibition in feeding circuits.

Changes in food availability alter the output of hypothalamic nuclei that underlie energy homeostasis. Here, we asked whether food deprivation impacts the ability of GABA synapses in the dorsomedial hypothalamus (DMH), an important integrator of satiety signals, to undergo activity-dependent changes. GABA synapses in DMH slices from satiated rats exhibit endocannabinoid-mediated long-term depression (LTD(GABA)) in response to high-frequency stimulation of afferents. When CB1Rs are blocked, however, the same stimulation elicits long-term potentiation (LTP(GABA)), which manifests presynaptically and requires heterosynaptic recruitment of NMDARs and nitric oxide (NO). Interestingly, NO signaling is required for eCB-mediated LTD(GABA). Twenty-four hour food deprivation results in a CORT-mediated loss of CB1R signaling and, consequently, GABA synapses only exhibit LTP(GABA). These observations indicate that CB1R signaling promotes LTD(GABA) and gates LTP(GABA). Furthermore, the satiety state of an animal, through regulation of eCB signaling, determines the polarity of activity-dependent plasticity at GABA synapses in the DMH.

Short-term plasticity impacts information transfer at glutamate synapses onto parvocellular neuroendocrine cells in the paraventricular nucleus of the hypothalamus.

Glutamatergic synaptic inputs onto parvocellular neurosecretory cells (PNCs) in the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic-pituitary-adrenal (HPA) axis responses to stress and undergo stress-dependent changes in their capacity to transmit information. In spite of their pivotal role in regulating PNCs, relatively little is known about the fundamental rules that govern transmission at these synapses. Furthermore, since salient information in the nervous system is often transmitted in bursts, it is also important to understand the short-term dynamics of glutamate transmission under basal conditions. To characterize these properties, we obtained whole-cell patch clamp recordings from PNCs in brain slices from postnatal day 21-35 male Sprague-Dawley rats and examined EPSCs. EPSCs were elicited by electrically stimulating glutamatergic afferents along the periventricular aspect. In response to a paired-pulse stimulation protocol, EPSCs generally displayed a robust short-term depression that recovered within 5 s. Similarly, trains of synaptic stimuli (5-50 Hz) resulted in a frequency-dependent depression until a near steady state was achieved. Application of inhibitors of AMPA receptor (AMPAR) desensitization or the low-affinity, competitive AMPAR antagonist failed to affect the depression due to paired-pulse and trains of synaptic stimulation indicating that this use-dependent short-term synaptic depression has a presynaptic locus of expression. We used cumulative amplitude profiles during trains of stimulation and variance-mean analysis to estimate synaptic parameters. Finally, we report that these properties contribute to hamper the efficiency with which high frequency synaptic inputs generate spikes in PNCs, indicating that these synapses operate as effective low-pass filters in basal conditions.

Functional interactions between stress and the endocannabinoid system: from synaptic signaling to behavioral output.

Endocannabinoid signaling is distributed throughout the brain, regulating synaptic release of both excitatory and inhibitory neurotransmitters. The presence of endocannabinoid signaling within stress-sensitive nuclei of the hypothalamus, as well as upstream limbic structures such as the amygdala, suggests it may play an important role in regulating the neuroendocrine and behavioral effects of stress. The evidence reviewed here demonstrates that endocannabinoid signaling is involved in both activating and terminating the hypothalamic-pituitary-adrenal axis response to both acute and repeated stress. In addition to neuroendocrine function, however, endocannabinoid signaling is also recruited by stress and glucocorticoid hormones to modulate cognitive and emotional processes such as memory consolidation and extinction. Collectively, these data demonstrate the importance of endocannabinoid signaling at multiple levels as both a regulator and an effector of the stress response.

Metaplasticity of hypothalamic synapses following in vivo challenge.

Neural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic alpha2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge.

Can homeostatic circuits learn and remember?

Alterations in synaptic strength are thought to represent the cellular basis of learning and memory. While such processes appear to be fundamental to all synapses, until recently there has been a relative dearth of information regarding synaptic 'memory' processes in autonomic circuits. Here we examine recent advances in our understanding of plasticity at glutamatergic synapses onto magnocellular neurosecretory cells in the hypothalamus, paying particular attention to the contributions of noradrenaline in coding long-lasting pre- and postsynaptic changes in efficacy. We also highlight recent work demonstrating that glial cells play a crucial role in the induction of long-term potentiation. Based on the work reviewed here, we have a clearer picture of the synaptic and cellular mechanisms that allow autonomic pathways to learn and remember.