Probes for CFOS

Autophagy represents a key regulator of aging and metabolism in sensing energy deprivation. We find that fasting in mice activates autophagy in the liver paralleled by activation of hypothalamic AgRP neurons. Optogenetic and chemogenetic activation of AgRP neurons induces autophagy, alters phosphorylation of autophagy regulators, and promotes ketogenesis. AgRP neuron-dependent induction of liver autophagy relies on NPY release in the paraventricular nucleus of the hypothalamus (PVH) via presynaptic inhibition of NPY1R-expressing neurons to activate PVHCRH neurons. Conversely, inhibiting AgRP neurons during energy deprivation abrogates induction of hepatic autophagy and rewiring of metabolism. AgRP neuron activation increases circulating corticosterone concentrations, and reduction of hepatic glucocorticoid receptor expression attenuates AgRP neuron-dependent activation of hepatic autophagy. Collectively, our study reveals a fundamental regulatory principle of liver autophagy in control of metabolic adaptation during nutrient deprivation.

The induction of nausea and emesis is a major barrier to maximizing the weight loss profile of obesity medications, and therefore, identifying mechanisms that improve tolerability could result in added therapeutic benefit. The development of Peptide YY (PYY)-based approaches to treat obesity are no exception, as PYY receptor agonism is often accompanied by nausea and vomiting. Here, we sought to determine whether glucose-dependent insulinotropic polypeptide (GIP) receptor agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist (GIPRA) reduced conditioned taste avoidance (CTA) without affecting hypophagia induced by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in the detection of aversive stimuli. Peripheral administration of a GIPRA induced neuronal activation (cFOS) in the AP. Further, whole-brain cFOS analyses indicated that PYY-induced CTA was associated with augmented neuronal activity in the parabrachial nucleus (PBN), an area of the brain that relays aversive/emetic stimuli to brain regions that control feeding behavior. Importantly, GIPR agonism reduced PYY-mediated neuronal activity in the PBN, providing a potential mechanistic explanation for how GIPRA treatment reduces PYY-induced nausea-like behavior. Together, our study provides a novel mechanism by which GIP-based therapeutics may benefit the tolerability of weight loss agents.

Trauma can cause dysfunctional fear regulation leading some people to develop disorders such as post-traumatic stress disorder (PTSD). The amygdala regulates fear, whereas PACAP (pituitary adenylate activating peptide) and PAC1 receptors are linked to PTSD symptom severity at genetic/epigenetic levels, with a strong link in females with PTSD. We discovered a PACAPergic projection from the basomedial amygdala (BMA) to the medial intercalated cells (mICCs) in adult mice. In vivo optogenetic stimulation of this pathway increased cfos expression in mICCs, decreased fear recall and increased fear extinction. Selective deletion of PAC1 receptors from the mICCs in females reduced fear acquisition, but enhanced fear generalization and reduced fear extinction in males. Optogenetic stimulation of the BMA-mICCs PACAPergic pathway produced excitatory postsynaptic currents (EPSCs) in mICC neurons, which were enhanced by the PAC1 receptor antagonist, PACAP 6-38. Our findings show that mICCs modulate contextual fear in a dynamic and sex-dependent manner via a microcircuit containing the BMA and mICCs, and in a manner that was dependent on behavioral state.SIGNIFICANCE STATEMENTTraumatic stress can affect different aspects of fear behaviors including fear learning, generalization of learned fear to novel contexts, how the fear of the original context is recalled, and how fear is reduced over time. While the amygdala has been studied for its role in regulation of different aspects of fear, the molecular circuitry of this structure is quite complex. In addition, aspects of fear can be modulated differently in males and females. Our findings show that a specific circuitry containing the neuropeptide PACAP and its receptor, PAC1, regulates various aspects of fear including acquisition, generalization, recall and extinction in a sexually dimorphic manner, characterizing a novel pathway that modulates traumatic fear.

Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated ion channel in primary sensory neurons of the dorsal root ganglia (DRG). Pharmacological and genetic studies implicated TRPM3 in various pain modalities, but TRPM3 inhibitors were not validated in TRPM3-/- mice. Here we tested two inhibitors of TRPM3 in male and female wild type and TRPM3-/- mice in nerve injury-induced neuropathic pain. We found that intraperitoneal injection of either isosakuranetin, or primidone reduced heat hypersensitivity induced by chronic constriction injury (CCI) of the sciatic nerve, in wild type, but not in TRPM3-/- mice. Primidone was also effective when injected locally in the hind paw, or intrathecally. Consistently, intrathecal injection of the TRPM3 agonist CIM0216 reduced paw withdrawal latency to radiant heat in wild type, but not in TRPM3-/- mice. Intraperitoneal injection of 2 mg/kg, but not 0.5 mg/kg isosakuranetin, inhibited cold and mechanical hypersensitivity in CCI, both in wild-type and TRPM3-/- mice, indicating a dose dependent off target effect. Primidone had no effect on cold sensitivity, and only a marginal effect on mechanical hypersensitivity. Genetic deletion or inhibitors of TRPM3 reduced the increase in the levels of the early genes cFos and pERK in the spinal cord and DRG in CCI mice, suggesting spontaneous activity of the channel. Intraperitoneal isosakuranetin also inhibited spontaneous pain related behavior in CCI in the conditioned place preference assay, and this effect was eliminated in TRPM3-/- mice. Overall our data indicate a role of TRPM3 in heat hypersensitivity and in spontaneous pain after nerve injury.SIGNIFICANCE STATEMENT:Neuropathic pain is a major unsolved medical problem. The heat-activated TRPM3 ion channel is a potential target for novel pain medications, but it is not clear what pain modalities it plays roles in. Here we used a combination of genetic and pharmacological tools to assess the role of this channel in spontaneous pain, heat-, cold- and mechanical hypersensitivity in a nerve injury model of neuropathic pain in mice. Our findings indicate a role for TRPM3 in heat hyperalgesia, and spontaneous pain, but not in cold, and mechanical hypersensitivity. We also find that not only TRPM3 located in the peripheral nerve termini, but also TRPM3 in the spinal cord, or proximal segments of DRG neurons is important for heat hypersensitivity.