Constitutive and ligand-dependent GHSR1a activity attenuates CaV2 current and hypothalamic GABA release through distinct mechanisms and signaling pathways.

Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.

Growth hormone secretagogue receptor (GHSR) displays high constitutive activity, independent of its endogenous ligand, ghrelin. Unlike ghrelin-induced GHSR activity, the physiological role of GHSR constitutive activity and the mechanisms that underlie GHSR neuronal modulation remain elusive. We previously demonstrated that GHSR constitutive activity modulates presynaptic Ca 2 voltage-gated calcium channels. Here we postulate that GHSR constitutive activity-mediated modulation of Ca 2 channels could be relevant in the hippocampus since this brain area has high GHSR expression but restricted access to ghrelin. We performed whole-cell patch-clamp in hippocampal primary cultures from E16- to E18-day-old C57BL6 wild-type and GHSR-deficient mice after manipulating GHSR expression with lentiviral transduction. We found that GHSR constitutive activity impairs Ca 2.1 and Ca 2.2 native calcium currents and that Ca 2.2 basal impairment leads to a decrease in GABA but not glutamate release. We postulated that this selective effect is related to a higher Ca 2.2 over Ca 2.1 contribution to GABA release (∼40% for Ca 2.2 in wild-type vs. ∼20% in wild-type GHSR-overexpressing cultures). This effect of GHSR constitutive activity is conserved in hippocampal brain slices, where GHSR constitutive activity reduces local GABAergic transmission of the granule cell layer (intra-granule cell inhibitory postsynaptic current (IPSC) size ∼-67 pA in wild-type vs. ∼-100 pA in GHSR-deficient mice), whereas the glutamatergic output from the dentate gyrus to CA3 remains unchanged. In summary, we found that GHSR constitutive activity impairs IPSCs both in hippocampal primary cultures and in brain slices through a Ca 2-dependent mechanism without affecting glutamatergic transmission.

Voltage-gated Ca 2+ (Ca V ) channels couple membrane depolarization to Ca 2+ influx, triggering a range of Ca 2+-dependent cellular processes. Ca V channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of Ca V channels. GPCRdependent mechanisms that downregulate Ca V channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several Ca V subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of Ca V channels depends on the presence of a Ca V β subunit. Ca V β subunits displace Ca V α 1 subunits from the endoplasmic reticulum. The actions of GHSR on Ca V channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.