Accepted

Induced pluripotent stem cells (iPSC) have enabled the generation of various difficult to access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdN) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdN, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored.Here 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 time 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) via 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 hiPSCdN-based drug discovery.

To explore alterations in thalamic subfield volume and iron accumulation in individuals with post-traumatic headache (PTH) relative to healthy controls.

Classically, to electrically excite C-nociceptors, rectangular pulses are used with a duration close to the estimated chronaxie of C-fibres (about 2 ms). Recent results using slow depolarizing stimuli suggest longer chronaxies. We therefore set out to optimize C-fiber stimulation based on recordings of single C-nociceptors in-vivo and C-fiber compound-action-potentials (C-CAP) ex-vivo using half-sine shaped stimuli of durations between 1 and 250ms. Single fiber (n=45) recording in pigs revealed high chronaxie values for C-touch fibers (15.8 ms), polymodal- (14.2 ms) and silent-nociceptors (16.8 ms). Activation thresholds decreased 2-3fold in all fiber classes when increasing the duration of half-sine pulses from 1 to 25 ms (p<0.05). C-CAPs strength-duration curves of the pig saphenous nerve (n=7) showed the highest sensitivity for half-sine durations between 10 and 25 ms. Half-maximum currents for C-CAPS were reduced 3fold compared to rectangular pulses (p<0.01) whereas the opposite was found for A-fiber compound action potentials. Psychophysics in humans (n=23) revealed that half-sine stimulus durations >10 ms reduced detection thresholds, pain thresholds and stimulus current amplitudes required to generate a pain rating of 3 on an 11-point Numeric Rating Scale (NRS) as compared to 1 ms rectangular pulses (p<0.05). Increasing the duration from 1 to 25 ms led to a 4fold amplitude reduction for pain-thresholds and stimuli caused an axon-reflex flare. Excitability of single polymodal nociceptors in animals paralleled human psychophysics and we conclude optimized half-sine pulses facilitate C-nociceptor activation. PERSPECTIVE: Electrical stimulation with longer lasting half-sine wave pulses preferentially activates C-nociceptors and changes in the strength duration curve may identify nociceptor hyperexcitability in patients with neuropathic pain.

Osteoarthritis (OA) is a multifactorial degenerative disease marked by the progressive deterioration of articular cartilage with inflammation of the synovium. OA's main symptoms include pain and function loss. Monosodium Iodoacetate (MIA) experimental model is widely-used for OS induction since it produces symptoms comparable to those occurring in humans.