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.

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.

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.

CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

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.

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.

Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers.

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.

Brain mechanisms involved in predatory aggression are activated in a laboratory model of violent intra-specific aggression.

Callous-unemotional violence associated with antisocial personality disorder is often called 'predatory' because it involves restricted intention signaling and low emotional/physiological arousal, including decreased glucocorticoid production. This epithet may be a mere metaphor, but may also cover a structural similarity at the level of the hypothalamus where the control of affective and predatory aggression diverges. We investigated this hypothesis in a laboratory model where glucocorticoid production is chronically limited by adrenalectomy with glucocorticoid replacement (ADXr). This procedure was proposed to model important aspects of antisocial violence. Sham and ADXr rats were submitted to resident/intruder conflicts, and the resulting neuronal activation patterns were investigated by c-Fos immunocytochemistry. In line with earlier findings, the share of attacks aimed at vulnerable targets (head, throat and belly) was dramatically increased by ADXr, while intention signaling by offensive threats was restricted. Aggressive encounters activated the mediobasal hypothalamus, a region involved in intra-specific aggression, but sham and ADXr rats did not differ in this respect. In contrast, the activation of the lateral hypothalamus that is tightly involved in predatory aggression was markedly larger in ADXr rats; moreover, c-Fos counts correlated positively with the share of vulnerable attacks and negatively with social signaling. Glucocorticoid deficiency increased c-Fos activation in the central amygdala, a region also involved in predatory aggression. In addition, activation patterns in the periaqueductal gray - involved in autonomic control - also resembled those seen in predatory aggression. These findings suggest that antisocial and predatory aggression are not only similar but are controlled by overlapping neural mechanisms.

Neural inputs of the hypothalamic "aggression area" in the rat.

A part of the mediobasal hypothalamus (known as hypothalamic attack area) plays a central role in the control of aggressive behavior as its electrical stimulation reliably and rapidly elicits biting attacks in cats and rodents. The efferent connections of this brain region were described in rats, but afferent pathways were not investigated so far. We injected the retrograde tracer cholera toxin B subunit into the mediobasal hypothalamus of male Wistar rats and studied the distribution of labeled cells by immunohistochemical method. The retrograde labeling outlined three continuous, distinct afferent cell populations: (i) a telencephalic midline "plate" containing the orbitofrontal - medial prefrontal - septal regions which ends in the bed nucleus of stria terminalis; (ii) a temporal column including the medial amygdala, amigdalohippocampal area and subiculum; (iii) a diffuse column along the medial hypothalamus which ends in the posterior hypothalamic nucleus. Sparse labeling was present in brainstem nuclei, except for the lateral parabrachial nucleus that provides a significant input. The projections of the medial prefrontal cortex to the hypothalamic attack area indicate a direct, earlier undescribed pathway with marked importance in the control of aggressive behavior. Similarly, we identified several brain regions which send very significant projections to the hypothalamic attack area but their importance in the control of aggressive behavior are nearly unknown. The comparison of the present and earlier findings shows that efferent and afferent connections overlap in many regions to a significant extent, suggesting that reverberating circuits are important in the control of aggressive behavior.