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The basolateral amygdala (BLA) is reliably activated by psychological stress and hyperactive in conditions of pathological stress or trauma; however, subsets of BLA neurons are also readily activated by rewarding stimuli and can suppress fear and avoidance behaviours. The BLA is highly heterogeneous anatomically, exhibiting continuous molecular and connectivity gradients throughout the entire structure. A critical gap remains in understanding the anatomical specificity of amygdala subregions, circuits, and cell types explicitly activated by acute stress and how they are dynamically activated throughout stimulus exposure. Using a combination of topographical mapping for the activity-responsive protein FOS and fiber photometry to measure calcium transients in real-time, we sought to characterize the spatial and temporal patterns of BLA activation in response to a range of novel stressors (shock, swim, restraint, predator odour) and non-aversive, but novel stimuli (crackers, citral odour). We report four main findings: (1) the BLA exhibits clear spatial activation gradients in response to novel stimuli throughout the medial-lateral and dorsal-ventral axes, with aversive stimuli strongly biasing activation towards medial aspects of the BLA; (2) novel stimuli elicit distinct temporal activation patterns, with stressful stimuli exhibiting particularly enhanced or prolonged temporal activation patterns; (3) changes in BLA activity are associated with changes in behavioural state; and (4) norepinephrine enhances stress-induced activation of BLA neurons via the ß-noradrenergic receptor. Moving forward, it will be imperative to combine our understanding of activation gradients with molecular and circuit-specificity., (© 2024. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1 + sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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; PVN CRH ), 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 PVN CRH neurons and their input to other stress- and reward-related brain regions drives these behaviors. To further understand the direct involvement of PVN CRH 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 PVN CRH inputs in the LH produced a similar pattern of effects on sucrose motivation. Together, these data suggest that PVN CRH neuronal activity may be directly responsible for changes in motivational drive and that these behavioral changes may, in part, be driven by PVN CRH synaptic projections to the LH., (© 2024. The Author(s).)

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 CRH PVN 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 CRH PVN neurons. Finally, the aversive memory in CRH PVN 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., (© 2023. The Author(s).)

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., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/β-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

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., (© 2021. The Author(s).)

There has been a clear movement in recent years towards the adoption of more naturalistic experimental regimes for the study of behavior and its underlying neural architecture. Here we provide a protocol that allows experimenters working with mice, to mimic a looming and advancing predatory threat from the sky. This approach is easy to implement and can be combined with sophisticated neural recordings that allow access to real-time activity during behavior. This approach offers another option in a battery of tests that allow for a more comprehensive understanding of defensive behaviors., Competing Interests: Competing interestsThe authors declare no competing interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

In humans and rodents, the perception of control during stressful events has lasting behavioral consequences. These consequences are apparent even in situations that are distinct from the stress context, but how the brain links prior stressful experience to subsequent behaviors remains poorly understood. By assessing innate defensive behavior in a looming-shadow task, we show that the initiation of an escape response is preceded by an increase in the activity of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus (CRH PVN neurons). This anticipatory increase is sensitive to stressful stimuli that have high or low levels of outcome control. Specifically, experimental stress with high outcome control increases CRH PVN neuron anticipatory activity, which increases escape behavior in an unrelated context. By contrast, stress with no outcome control prevents the emergence of this anticipatory activity and decreases subsequent escape behavior. These observations indicate that CRH PVN neurons encode stress controllability and contribute to shifts between active and passive innate defensive strategies.

Objective: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN., Methods: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis., Results: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure., Conclusion: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca 2+ -influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Limbic brain regions drive goal-directed behaviors. These behaviors often require dynamic motor responses, but the functional connectome of limbic structures in the diencephalon that control locomotion is not well known. The A11 region, within the posterior diencephalon has been postulated to contribute to motor function and control of pain. Here we show that the A11 region initiates movement. Photostimulation of channelrhodopsin 2 (ChR2) transfected neurons in A11 slice preparations showed that neurons could follow stimulation at frequencies of 20 Hz. Our data show that photostimulation of ChR2 transfected neurons in the A11 region enhances motor activity often leading to locomotion. Using vGluT2-reporter and vGAT-reporter mice we show that the A11 tyrosine hydroxylase positive (TH) dopaminergic neurons are vGluT2 and vGAT negative. We find that in addition to dopaminergic neurons within the A11 region, there is another neuronal subtype which expresses the monoenzymatic aromatic L-amino acid decarboxylase (AADC), but not TH, a key enzyme involved in the synthesis of catecholamines including dopamine. This monoaminergic-based motor circuit may be involved in the control of motor behavior as part of a broader diencephalic motor region.

Fiber photometry uses genetically encoded optical reporters to link specific cellular activity in stereotaxically targeted brain structures to specific behaviors. There are still a number of barriers that have hindered the widespread adoption of this approach. This includes cost, but also the high-levels of light required to excite the fluorophore, limiting commercial systems to the investigation of short-term transients in neuronal activity to avoid damage of tissue by light. Here, we present a cost-effective optoelectronic system for in vivo fiber photometry that achieves high-sensitivity to changes in fluorescence intensity, enabling detection of optical transients of a popular calcium reporter with excitation powers as low as 100 nW. By realizing a coherent detection scheme and by using a photomultiplier tube as a detector, the system demonstrates reliable study of in vivo neuronal activity, positioning it for future use in the experiments inquiring into learning and memory processes. The system was applied to study stress-evoked calcium transients in corticotropin-releasing hormone neurons in the mouse hypothalamus.

Two anorexigenic peptides, thyrotropin-releasing hormone (TRH) and urocortin 3 (UCN3), are co-expressed in a continuous neuronal group that extends from the perifornical area to the bed nucleus of stria terminalis, raising the possibility that this cell group may be involved in the regulation of energy homeostasis. In this study, therefore, we tested the hypothesis that the TRH/UCN3 neurons regulate food intake by influencing feeding-related neuropeptide Y (NPY) and/or proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC). Triple-labeled immunofluorescent preparations demonstrated that only very few NPY neurons (4.3 ± 1.3%) were contacted by double-labeled TRH/UCN3 axons in the ARC. In contrast, more than half of the POMC neurons (52.4 ± 8.5%) were contacted by double-labeled axons. Immuno-electron microscopy demonstrated that the UCN3 axons established asymmetric synapses with POMC neurons, indicating the excitatory nature of these synaptic specializations. Patch clamp electrophysiology revealed that TRH and UCN3 have antagonistic effects on the POMC neurons. While UCN3 depolarizes and increases the firing rate of POMC neurons, TRH prevents these effects of UCN3. These data demonstrate that TRH/UCN3 neurons in the perifornical/BNST region establish abundant synaptic associations with the POMC neurons in the ARC and suggest a potentially important role for these neurons in the regulation of food intake through an antagonistic interaction between TRH and UCN3 on the electrophysiological properties of POMC neurons.

Stress can trigger enduring changes in neural circuits and synapses. The behavioral and hormonal consequences of stress can also be transmitted to others, but whether this transmitted stress has similar effects on synapses is not known. We found that authentic stress and transmitted stress in mice primed paraventricular nucleus of the hypothalamus (PVN) corticotropin-releasing hormone (CRH) neurons, enabling the induction of metaplasticity at glutamate synapses. In female mice that were subjected to authentic stress, this metaplasticity was diminished following interactions with a naive partner. Transmission from the stressed subject to the naive partner required the activation of PVN CRH neurons in both subject and partner to drive and detect the release of a putative alarm pheromone from the stressed mouse. Finally, metaplasticity could be transmitted sequentially from the stressed subject to multiple partners. Our findings demonstrate that transmitted stress has the same lasting effects on glutamate synapses as authentic stress and reveal an unexpected role for PVN CRH neurons in transmitting distress signals among individuals.

All organisms possess innate behavioural and physiological programmes that ensure survival. In order to have maximum adaptive benefit, these programmes must be sufficiently flexible to account for changes in the environment. Here we show that hypothalamic CRH neurons orchestrate an environmentally flexible repertoire of behaviours that emerge after acute stress in mice. Optical silencing of CRH neurons disrupts the organization of individual behaviours after acute stress. These behavioural patterns shift according to the environment after stress, but this environmental sensitivity is blunted by activation of PVN CRH neurons. These findings provide evidence that PVN CRH cells are part of a previously unexplored circuit that matches precise behavioural patterns to environmental context following stress. Overactivity in this network in the absence of stress may contribute to environmental ambivalence, resulting in context-inappropriate behavioural strategies.

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.

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

Corticotropin-releasing hormone (CRH)-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) initiate and control neuroendocrine responses to psychogenic and physical stress. Investigations into the physiology of CRH neurons, however, have been hampered by the lack of tools for adequately targeting or visualizing this cell population. Here we characterize CRH neurons in the PVN of mice that express tdTomato fluorophore, generated by crosses of recently developed Crh-IRES-Cre driver and Ai14 Cre-reporter mouse strains. tdTomato containing PVN neurons in Crh-IRES-Cre;Ai14 mice are readily visualized without secondary-detection methods. These neurons are predominantly neuroendocrine and abundantly express CRH protein, but not other PVN phenotypic neuropeptides. After an acute stress, a large majority of tdTomato cells express neuronal activation marker c-Fos. Finally, tdTomato PVN neurons exhibit homogenous intrinsic biophysical and synaptic properties, and can be optogenetically manipulated by viral Cre-driven expression of channelrhodopsin. These observations highlight basic cell-type characteristics of CRH neurons in a mutant mouse, providing validation for its future use in probing neurophysiology of endocrine stress responses.

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 β-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.

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.

Recent studies indicate that the effect of thyrotropin-releasing hormone (TRH) on the regulation of food intake may be mediated by histaminergic neurons. To elucidate the anatomical basis for a functional relationship between TRH- and histamine-synthesizing neuronal systems, double-labeling immunocytochemistry was performed on the tuberomammillary nucleus (TMN) of rats, the exclusive location of histaminergic neurons. TRH-immunoreactive (IR) innervation of the histaminergic neurons were detected in all five subnuclei (E1-5) of the TMN, but was most prominent in the E4 and E5 subnuclei where 100% of the histamine-IR neurons were contacted. The number of TRH-IR varicosities in contact with histamine-IR neurons was also greatest in the E4 and E5 subnuclei, averaging 27.0±1.2 in E4 and 7.9±0.5 in E5. Somewhat fewer histamine-IR neurons were juxtaposed by TRH-IR varicosities in E2 and E3 and contacted by 6.3±0.2 and 6.8±0.2 varicosities/innervated cell, respectively. The number of juxtapositions of TRH-IR axon varicosities with histamine-IR neurons was the lowest in the E1 subnucleus (85.7±0.9%; 4.0±0.2 varicosities/innervated cell). Ultrastructural analysis demonstrated that TRH-IR axons established both asymmetric and symmetric type synapses on the perikaryon and dendrites of the histamine-IR neurons, although the majority of synapses were asymmetric type. These data demonstrate that TRH neurons heavily innervate histaminergic neurons in all subdivisions of the TMN, with the densest innervation in the E4 and E5 subdivisions, and are likely to exert activating effects., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions., (Copyright © 2011 Wiley-Liss, Inc.)

Hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central regulators of the hypothalamic-pituitary-thyroid axis, are located in the hypothalamic paraventricular nucleus (PVN) in a partly overlapping distribution with non-hypophysiotropic TRH neurons. The distribution of hypophysiotropic TRH neurons in the rat PVN is well understood, but the localization of these neurons is unknown in mice. To determine the distribution and phenotype of hypophysiotropic TRH neurons in mice, double- and triple-labeling experiments were performed on sections of intact mice, and mice treated intravenously and intraperitoneally with the retrograde tracer Fluoro-Gold. TRH neurons were located in all parts of the PVN except the periventricular zone. Hypophysiotropic TRH neurons were observed only at the mid-level of the PVN, primarily in the compact part. In this part of the PVN, TRH neurons were intermingled with oxytocin and vasopressin neurons, but based on their size, the TRH neurons were parvocellular and did not contain magnocellular neuropeptides. Co-localization of TRH and cocaine- and amphetamine-regulated transcript (CART) were observed only in areas where hypophysiotropic TRH neurons were located. In accordance with the morphological observations, hypothyroidism increased TRH mRNA content of neurons only at the mid-level of the PVN. These data demonstrate that the distribution of hypophysiotropic TRH neurons in mice is vastly different from the pattern in rats, with a dominant occurrence of these neurosecretory cells in the compact part and adjacent regions at the mid-level of the PVN. Furthermore, our data demonstrate that the organization of the PVN is markedly different in mice and rats.

Thyrotropin-releasing hormone (TRH) decreases food intake when administered intracerebroventricularly or into the ventromedial hypothalamus. However, it is unknown which population of TRH neurons exerts this anorexigenic function. In the rostral perifornical area, the pattern of TRH-expressing neurons is reminiscent of the distribution of neurons expressing urocortin3 (Ucn3) that also inhibits feeding when injected into the hypothalamic ventromedial nucleus (VMN). Since colocalization of TRH and Ucn3 may help to identify feeding-related TRH neurons, the putative coexpression of the two peptides was examined using fluorescent in situ hybridization combined with immunofluorescence. Almost all (95.5 +/- 0.2%) Ucn3-immunoreactive neurons in the perifornical area expressed pro-TRH mRNA, while 50.2 +/- 1.6% Ucn3 neurons were double-labeled in the bed nucleus of the stria terminalis (BNST). Only a few Ucn3/pro-TRH neurons were found outside these two areas. The distribution of axons containing both Ucn3 and TRH was examined by dual immunofluorescence. Ucn3/TRH fibers heavily innervated the VMN. In addition, high densities of double-labeled axons were observed in the lateral septal nucleus, posterior division of the BNST, medial amygdaloid nucleus, amygdalohippocampal area, and ventral hippocampus, forebrain areas associated with psychological stress and anxiety. We conclude that Ucn3 and TRH are coexpressed in a discrete, continuous population of neurons in the perifornical area and BNST, making Ucn3 a neurochemical marker to define a distinct subset of TRH neurons. The distribution of their axons suggests that Ucn3/TRH neurons may coordinate feeding and behavioral responses to stressful stimuli.

Hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, the central regulators of the hypothalamic-pituitary-thyroid axis, are located in the paraventricular nucleus of the hypothalamus (PVN). These neurons are well-known to be stimulated by cold exposure through activation of ascending brainstem pathways, and are heavily innervated by catecholaminergic axons that contain dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), enzymes that generate noradrenaline and adrenaline, respectively. However, whether noradrenergic cell groups that lack PNMT contribute to the innervation of TRH neurons is not known. Therefore, triple-labeling immunofluorescence was performed using antibodies against DBH, PNMT and proTRH to determine the relative involvement of adrenaline-synthesizing and noradrenergic neurons in the innervation of TRH neurons in the PVN of rats. Using confocal microscopy, the number of PNMT/DBH (adrenaline-synthesizing) and single-labeled DBH (noradrenergic) boutons juxtaposed to proTRH neurons was quantified. Both noradrenergic and PNMT-containing varicosities were observed in close apposition to virtually all proTRH neurons. An average of 11.8+/-0.6 PNMT-containing and 7.4+/-1.0 noradrenergic boutons was present on the surface of proTRH cell bodies and proximal dendrites. Of all catecholaminergic axon-varicosities juxtaposed to proTRH neurons, 63.5+/-1.2% contained PNMT while the remaining 36.5+/-1.2% were immunopositive for DBH only. We conclude that both adrenaline-synthesizing and noradrenergic axons innervate hypophysiotropic TRH neurons, although there is a predominance of adrenaline-synthesizing fibers. Since adrenaline-synthesizing and noradrenergic cell groups of the brainstem may respond differently to various physiological stimuli, we hypothesize that the two cell groups are likely to mediate the effects of distinct stimuli toward the hypophysiotropic TRH neurons.

The anterior parvocellular subdivision of the PVN (aPVN) contains nonhypophysiotropic thyrotropin-releasing hormone (TRH) neurons that are densely innervated by feeding-related neuronal groups of the hypothalamic arcuate nucleus. To determine how these TRH neurons are integrated within the brain, the major projection fields of this cell group were studied by anterograde and retrograde tract-tracing methods. Projection sites were identified by injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the aPVN, and subsequent double immunofluorescent staining was used to visualize axons containing both PHAL and pro-TRH. To distinguish between the projection sites of TRH neurons residing in the aPVN and the closely situated perifornical area, the retrograde tracer cholera toxin B subunit (CTB) was injected into regions where PHAL/pro-TRH-containing axons were densely accumulated. TRH neurons in the aPVN were found to project to the hypothalamic arcuate, dorsomedial and ventral premammillary nuclei, medial preoptic region, tuber cinereum area, paraventricular thalamic nucleus, bed nucleus of the stria terminalis, lateral septal nucleus, and central amygdaloid nucleus. Projection fields of perifornical TRH neurons were in partial overlap with those of the aPVN TRH cells. In addition, these neurons also innervated the hypothalamic ventromedial nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area. The data suggest that, through its efferent connections, aPVN TRH neurons may be involved in the regulation of energy homeostasis coordinately with effects on behavior, locomotor activity, and thermogenesis. In addition, the major differences in the projection fields of aPVN and perifornical TRH neurons suggest that these two TRH-synthesizing neuronal groups are functionally different., (Copyright 2009 Wiley-Liss, Inc.)

CRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN) integrate neuronal and hormonal inputs and serve as a final common pathway to regulate the hypothalamic-pituitary-adrenal axis. One of the neuronal regulators of CRH neurons is neuropeptide Y (NPY) contained in axons that densely innervate CRH neurons. The three main sources of NPY innervation of the PVN are the hypothalamic arcuate nucleus and the noradrenergic and adrenergic neurons of the brainstem. To elucidate the origin of the NPY-immunoreactive (NPY-IR) innervation to hypophysiotropic CRH neurons, quadruple-labeling immunocytochemistry for CRH, NPY, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase was performed. Approximately 63% of NPY-IR varicosities on the surface of CRH neurons were catecholaminergic (22% noradrenergic and 41% adrenergic), and 37% of NPY-IR boutons were noncatecholaminergic. By triple-labeling immunofluorescence detection of NPY, CRH, and agouti-related protein, a marker of NPY axons projecting from the arcuate nucleus, the noncatecholaminergic, NPY-ergic axon population was shown to arise primarily from the arcuate nucleus. When NPY was administered chronically into the cerebral ventricle of fed animals, a dramatic reduction of CRH mRNA was observed in the PVN (NPY vs. control integrated density units, 23.9 +/- 2.7 vs. 77.09 +/- 15.9). We conclude that approximately two thirds of NPY-IR innervation to hypophysiotropic CRH neurons originates from catecholaminergic neurons of the brainstem, whereas the remaining one third arises from the arcuate nucleus. The catecholaminergic NPY innervation seems to modulate the activation of CRH neurons in association with glucoprivation and infection, whereas the NPY input from the arcuate nucleus may contribute to inhibition of CRH neurons during fasting.