Emergence of nociceptive functionality and opioid signaling in human induced pluripotent stem cell-derived sensory neurons.

ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

Ca2+ -calmodulin feedback mediates sensory adaptation and inhibits pheromone-sensitive ion channels in the vomeronasal organ.

The mammalian vomeronasal organ (VNO) mediates the regulation of social behaviors by complex chemical signals. These cues trigger transient elevations of intracellular Ca(2+) in vomeronasal sensory neurons (VSNs), but the functional role of such Ca(2+) elevations is unknown. We show that stimulus-induced Ca(2+) entry plays an essential role as a negative feedback regulator of VSN sensitivity. Electrophysiological VSN responses undergo effective sensory adaptation that requires the influx of Ca(2+) and is mediated by calmodulin (CaM). Removal of the Ca(2+)-CaM feedback eliminates this form of adaptation. A key target of this feedback module is the pheromone-sensitive TRPC2-dependent cation channel of VSNs, as its activation is strongly inhibited by Ca(2+)-CaM. Our results reveal a previously unrecognized CaM-signaling pathway that endows the VSNs with a mechanism for adjusting gain and sensitivity of chemosensory signaling in the VNO.

Homeostatic control of sensory output in basal vomeronasal neurons: activity-dependent expression of ether-à-go-go-related gene potassium channels.

Conspecific chemosensory communication controls a broad range of social and sexual behaviors. In most mammals, social chemosignals are predominantly detected by sensory neurons of a specialized olfactory subsystem, the vomeronasal organ (VNO). The behavioral relevance of social chemosignaling puts high demands on the accuracy and dynamic range of the underlying transduction mechanisms. However, the physiological concepts implemented to ensure faithful transmission of social information remain widely unknown. Here, we show that sensory neurons in the basal layer of the mouse VNO dynamically control their input-output relationship by activity-dependent regulation of K(+) channel gene expression. Using large-scale expression profiling, immunochemistry, and electrophysiology, we provide molecular and functional evidence for a role of ether-à-go-go-related gene (ERG) K(+) channels as key determinants of cellular excitability. Our findings indicate that an increase in ERG channel expression extends the dynamic range of the stimulus-response function in basal vomeronasal sensory neurons. This novel mechanism of homeostatic plasticity in the periphery of the accessory olfactory system is ideally suited to adjust VNO neurons to a target output range in a layer-specific and use-dependent manner.