Probes for C-FOS

AIMS: Many patients taking risperidone for the treatment of psychiatric disorders experience substantial body weight gain. Researchers have speculated that risperidone induces obesity by modulating central signals; however, the precise central mechanisms involved remain to be fully elucidated. METHODS: Twenty-four C57BL/6J mice were divided into four groups: a control group; a risperidone-treated group; a lorcaserin-treated group; and a combined risperidone + lorcaserin-treated group. The mice were received the corresponding treatments for 4 weeks, and their brains were collected for in situ hybridization analysis. A subset of C57BL/6J mice was administrated with risperidone or placebo, and brains were collected 60 minutes post-treatment for determination of c-fos activity. In addition, brains of NPY-GFP mice treated with or without risperidone were collected to perform colocalization of NPY and c-fos, as well as NPY and 5-HT2c receptor using immunohistochemistry. RESULTS: There was significantly elevated c-fos expression in the hypothalamic arcuate nucleus (Arc) of risperidone-treated mice. More than 68% c-fos-positive neurons were NPY-expressing neurons. Furthermore, in situ hybridization revealed that Arc NPY mRNA expression was significantly increased in the risperidone-treated group compared with control group. Moreover, we identified that 95% 5-HT2c receptors were colocalized with NPY positive neurons, and increased Arc NPY mRNA expression induced by risperidone was markedly reduced by cotreatment with lorcaserin, a specific 5-HT2c receptor agonist. CONCLUSION: Our findings provide critical insight into the mechanisms underlying antipsychotic-induced obesity, which may assist the development of therapeutic strategies to address metabolic side effects of risperidone.

Background The neuropeptide PACAP is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. Methods We used AAV neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57Bl6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. Results PACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex (mPFC) to hypothalamus, impairs c-fos activation and CRH mRNA elevation in PVN after 2 hr of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in non-hypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala (EA), on the other hand, attenuates ARS-induced hypophagia, along with EA fos induction, without affecting ARS-induced CRH mRNA elevation in PVN. PACAP projections to EA terminate at PKCδ neurons in both central amygdala (CeA) and oval nuclei of bed nucleus of stria terminalis (BNSTov). Silencing of PKCδ neurons in CeA, but not in BNSTov, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n>5 per group. Conclusions A frontocortical descending PACAP projection controls PVN CRH mRNA production, to maintain hypothalamo-pituitary adrenal (HPA) axis activation, and regulate the endocrine response to stress. An ascending PACAPergic projection from eLPBn to PKCδ neurons in central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.

Spreading depolarization (SD), the underlying mechanism of migraine aura, may trigger the opening of the Pannexin-1 (Panx1) pore to sustain the cortical neuroinflammatory cascades involved in the genesis of headache. Yet, the mechanism underlying SD-evoked neuroinflammation and trigeminovascular activation remains incompletely understood. We characterized the identity of inflammasome activated following SD-evoked Panx1 opening. Pharmacological inhibitors targeting Panx1 or NLRP3 as well as genetic ablation of Nlrp3 and Il1b were applied to investigate the molecular mechanism of the downstream neuroinflammatory cascades. In addition, we examined whether SDs-triggered microglial activation facilitates neuronal NLRP3-mediated inflammatory cascades. Pharmacological inhibition of toll-like receptors TLR2/4, the potential receptors of the damage-associated molecular pattern HMGB1, was further employed to interrogate the neuron-microglia interplay in SD-induced neuroinflammation. We found that NLRP3 but not NLRP1 or NLRP2 inflammasome was activated following Panx1 opening after single or multiple SDs evoked by either KCl topical application or noninvasively with optogenetics. The SD-evoked NLRP3 inflammasome activation was observed exclusively in neurons but not microglia or astrocytes. Proximity ligation assay demonstrated that the assembly of NLRP3 inflammasome was as early as 15 mins after SD. Genetic ablation of Nlrp3 or Il1b or pharmacological inhibition of Panx1 or NLRP3 ameliorated SD-induced neuronal inflammation, middle meningeal artery dilatation, calcitonin gene-related peptide expression in trigeminal ganglion, and c-Fos expression in trigeminal nucleus caudalis. Moreover, multiple SDs induced microglial activation subsequent to neuronal NLRP3 inflammasome activation, which in turn orchestrated with neurons to mediate cortical neuroinflammation, as demonstrated by decreased neuronal inflammation after pharmacological inhibition of microglia activation or blockade of the TLR2/4 receptors. To conclude, single or multiple SDs evoked activation of neuronal NLRP3 inflammasomes and its downstream inflammatory cascades to mediate cortical neuroinflammation and trigeminovascular activation. In the context of multiple SDs, the cortical inflammatory processes could be facilitated by SDs-evoked microglia activation. These findings may implicate the potential role of innate immunity in migraine pathogenesis.