Single cell tracing of Pomc neurons reveals recruitment of 'Ghost' subtypes with atypical identity in a mouse model of obesity.

The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of 'Ghost' neurons endowed with atypical molecular and functional identity. Compared to 'classical' Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are 'invisible' to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

Forgetting in obesity: The pregnenolone link.

Cognitive dysfunction is often diagnosed in people with obesity and associated metabolic disorders. In the latest issue of Cell Metabolism, Ramírez et al. highlight an impaired production of the neurosteroid pregnenolone in the hypothalamus as a mechanism for obesity-induced cognitive impairment in both rodent models and patients with obesity.

Hypothalamic bile acid-TGR5 signaling protects from obesity.

Bile acids (BAs) improve metabolism and exert anti-obesity effects through the activation of the Takeda G protein-coupled receptor 5 (TGR5) in peripheral tissues. TGR5 is also found in the brain hypothalamus, but whether hypothalamic BA signaling is implicated in body weight control and obesity pathophysiology remains unknown. Here we show that hypothalamic BA content is reduced in diet-induced obese mice. Central administration of BAs or a specific TGR5 agonist in these animals decreases body weight and fat mass by activating the sympathetic nervous system, thereby promoting negative energy balance. Conversely, genetic downregulation of hypothalamic TGR5 expression in the mediobasal hypothalamus favors the development of obesity and worsens established obesity by blunting sympathetic activity. Lastly, hypothalamic TGR5 signaling is required for the anti-obesity action of dietary BA supplementation. Together, these findings identify hypothalamic TGR5 signaling as a key mediator of a top-down neural mechanism that counteracts diet-induced obesity.

Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors.

The type 1 cannabinoid receptor (CB1) modulates numerous neurobehavioral processes and is therefore explored as a target for the treatment of several mental and neurological diseases. However, previous studies have investigated CB1 by targeting it globally, regardless of its two main neuronal localizations on glutamatergic and GABAergic neurons. In the context of cocaine addiction this lack of selectivity is critical since glutamatergic and GABAergic neuronal transmission is involved in different aspects of the disease. To determine whether CB1 exerts different control on cocaine seeking according to its two main neuronal localizations, we used mutant mice with deleted CB1 in cortical glutamatergic neurons (Glu-CB1) or in forebrain GABAergic neurons (GABA-CB1). In Glu-CB1, gene deletion concerns the dorsal telencephalon, including neocortex, paleocortex, archicortex, hippocampal formation and the cortical portions of the amygdala. In GABA-CB1, it concerns several cortical and non-cortical areas including the dorsal striatum, nucleus accumbens, thalamic, and hypothalamic nuclei. We tested complementary components of cocaine self-administration, separating the influence of primary and conditioned effects. Mechanisms underlying each phenotype were explored using in vivo microdialysis and ex vivo electrophysiology. We show that CB1 expression in forebrain GABAergic neurons controls mouse sensitivity to cocaine, while CB1 expression in cortical glutamatergic neurons controls associative learning processes. In accordance, in the nucleus accumbens, GABA-CB1 receptors control cocaine-induced dopamine release and Glu-CB1 receptors control AMPAR/NMDAR ratio; a marker of synaptic plasticity. Our findings demonstrate a critical distinction of the altered balance of Glu-CB1 and GABA-CB1 activity that could participate in the vulnerability to cocaine abuse and addiction. Moreover, these novel insights advance our understanding of CB1 neuropathophysiology.

Glycogen synthase kinase-3ß is involved in electroacupuncture pretreatment via the cannabinoid CB1 receptor in ischemic stroke.

We have previously shown that electroacupuncture (EA) pretreatment produces neuroprotective effects, which were mediated through an endocannabinoid signal transduction mechanism. Herein, we have studied the possible contribution of the phosphorylated form of glycogen synthase kinase-3ß (GSK-3ß) in EA pretreatment-induced neuroprotection via the cannabinoid CB1 receptor (CB1R). Focal transient cerebral ischemia was induced by middle cerebral artery occlusion in rats. Phosphorylation of GSK-3ß at Ser-9 [p-GSK-3ß (Ser-9)] was evaluated in the penumbra tissue following reperfusion. Infarct size and neurological score were assessed in the presence of either PI3K inhibitors or a GSK-3ß inhibitor 72 h after reperfusion. Cellular apoptosis was evidenced by TUNEL staining and determination of the Bax/Bcl-2 ratio 24 h after reperfusion. The present study showed that EA pretreatment increased p-GSK-3ß(Ser-9) 2 h after reperfusion in the ipsilateral penumbra. Augmented phosphorylation of GSK-3ß induced similar neuroprotective effects as did EA pretreatment. By contrast, inhibition of PI3K dampened the levels of p-GSK-3ß(Ser-9), and reversed not only the neuroprotective effect but also the anti-apoptotic effect following EA pretreatment. Regulation of GSK-3ß by EA pretreatment was abolished following treatment with a CB1R antagonist and CB1R knockdown, whereas two CB1R agonists enhanced the phosphorylation of GSK-3ß. Therefore we conclude that EA pretreatment protects against cerebral ischemia/reperfusion injury through CB1R-mediated phosphorylation of GSK-3ß.

Activation of STAT3 is involved in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptors in rats.

Pretreatment with electroacupuncture (EA) attenuates cerebral ischemic injury through the endocannabinoid system, although the molecular mechanisms mediate this neuroprotection are unknown. It is well-known that signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell survival and proliferation. Therefore, we investigated whether STAT3 is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors (CB1R) after transient focal cerebral ischemia in rats. Two hours after EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MACO) for 120 min. The expression of pSTAT3(Ser727), which is necessary for STAT3 activation, was examined in the ipsilateral ischemic penumbra. Infarct volumes and neurological scores were evaluated at 72 h after MACO in the presence or absence of the STAT3 inhibitor peptide (PpYLKTK). Neuronal apoptosis and the Bax/Bcl-2 ratio were also evaluated 24h after reperfusion. Our results showed that EA pretreatment significantly enhanced neuronal expression of pSTAT3(Ser727) in the ischemic penumbra 6h after reperfusion. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and decreased the Bax/Bcl-2 ratio following reperfusion. The beneficial effects of EA were attenuated by PpYLKTK administered 30 min before MACO, and PpYLKTK effectively reversed the increase in pSTAT3(Ser727) expression. Furthermore, CB1R antagonist or CB1R knockdown with siRNA blocked the elevation of pSTAT3(Ser727) expression by EA pretreatment, whereas the two CB1R agonists increased STAT3 activation. In conclusion, EA pretreatment enhances STAT3 activation via CB1R to protect against cerebral ischemia, suggesting that STAT3 activation may be a novel target for stroke intervention.

Striatal GABAergic and cortical glutamatergic neurons mediate contrasting effects of cannabinoids on cortical network synchrony.

Activation of type 1 cannabinoid receptors (CB1R) decreases GABA and glutamate release in cortical and subcortical regions, with complex outcomes on cortical network activity. To date there have been few attempts to disentangle the region- and cell-specific mechanisms underlying the effects of cannabinoids on cortical network activity in vivo. Here we addressed this issue by combining in vivo electrophysiological recordings with local and systemic pharmacological manipulations in conditional mutant mice lacking CB1R expression in different neuronal populations. First we report that cannabinoids induce hypersynchronous thalamocortical oscillations while decreasing the amplitude of faster cortical oscillations. Then we demonstrate that CB1R at striatonigral synapses (basal ganglia direct pathway) mediate the thalamocortical hypersynchrony, whereas activation of CB1R expressed in cortical glutamatergic neurons decreases cortical synchrony. Finally we show that activation of CB1 expressed in cortical glutamatergic neurons limits the cannabinoid-induced thalamocortical hypersynchrony. By reporting that CB1R activations in cortical and subcortical regions have contrasting effects on cortical synchrony, our study bridges the gap between cellular and in vivo network effects of cannabinoids. Incidentally, the thalamocortical hypersynchrony we report suggests a potential mechanism to explain the sensory "high" experienced during recreational consumption of marijuana.

Hypothalamic CB1 cannabinoid receptors regulate energy balance in mice.

Cannabinoid type 1 (CB(1)) receptor activation is generally considered a powerful orexigenic signal and inhibition of the endocannabinoid system is beneficial for the treatment of obesity and related metabolic diseases. The hypothalamus plays a critical role in regulating energy balance by modulating both food intake and energy expenditure. Although CB(1) receptor signaling has been implicated in the modulation of both these mechanisms, a complete understanding of its role in the hypothalamus is still lacking. Here we combined a genetic approach with the use of adeno-associated viral vectors to delete the CB(1) receptor gene in the adult mouse hypothalamus and assessed the impact of such manipulation on the regulation of energy balance. Viral-mediated deletion of the CB(1) receptor gene in the hypothalamus led to the generation of Hyp-CB(1)-KO mice, which displayed an approximately 60% decrease in hypothalamic CB(1) receptor mRNA levels. Hyp-CB(1)-KO mice maintained on a normocaloric, standard diet showed decreased body weight gain over time, which was associated with increased energy expenditure and elevated ß(3)-adrenergic receptor and uncoupling protein-1 mRNA levels in the brown adipose tissue but, surprisingly, not to changes in food intake. Additionally, Hyp-CB(1)-KO mice were insensitive to the anorectic action of the hormone leptin (5 mg/kg) and displayed a time-dependent hypophagic response to the CB(1) inverse agonist rimonabant (3 mg/kg). Altogether these findings suggest that hypothalamic CB(1) receptor signaling is a key determinant of energy expenditure under basal conditions and reveal its specific role in conveying the effects of leptin and pharmacological CB1 receptor antagonism on food intake.

Requirement of cannabinoid receptor type 1 for the basal modulation of hypothalamic-pituitary-adrenal axis function.

The endocannabinoid system affects the neuroendocrine regulation of hormone secretion, including the activity of the hypothalamus-pituitary-adrenal (HPA) axis. However, the mechanisms by which endocannabinoids regulate HPA axis function have remained unclear. Here we demonstrate that mice lacking cannabinoid receptor type 1 (CB1-/-) display a significant dysregulation of the HPA axis. Although circadian HPA axis responsiveness is preserved, CB1-/- mice are characterized by an enhanced circadian drive on the HPA axis, resulting in elevated plasma corticosterone concentrations at the onset of the dark as compared with wild-type (CB1+/+) littermates. Moreover, CB1-/--derived pituitary cells respond with a significantly higher ACTH secretion to CRH and forskolin challenges as compared with pituitary cells derived from CB1+/+ mice. Both CBL-/- and CB1+/+ mice properly respond to a high-dose dexamethasone test, but response to low-dose dexamethasone is influenced by genotype. In addition, CB1-/- mice show increased CRH mRNA levels in the paraventricular nucleus of the hypothalamus but not in other extrahypothalamic areas, such as the amygdala and piriform cortex, in which CB1 and CRH mRNA have been colocalized. Finally, CB1-/- mice have selective glucocorticoid receptor mRNA down-regulation in the CA1 region of the hippocampus but not in the dentate gyrus or paraventricular nucleus. Conversely, mineralocorticoid receptor mRNA expression levels were found unchanged in these brain areas. In conclusion, our findings indicate that CB1 deficiency enhances the circadian HPA axis activity peak and leads to central impairment of glucocorticoid feedback, thus further outlining the essential role of the endocannabinoid system in the modulation of neuroendocrine functions.

The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis.

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1-/-) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1-/- mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1-/- mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and preproorexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1-/- mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.