Andersson, D. A. | bioRxiv Science

bioRxiv2026

Pathways regulating the excitability of sympathetic neurons derived from human induced pluripotent stem cells

Postganglionic sympathetic neurons not only regulate target organs but also their own intrinsic activity and can be further modulated by parasympathetic neurons. This crosstalk and automodulatory signalling have been implicated in cardiovascular disorders, with the majority of earlier work employing rodent models. Here, we have used human sympathetic neurons derived from induced pluripotent stem cells (hiPSCs), as a scalable human in vitro system with the aim to investigate these pathways. Efficient differentiation of hiPSCs into sympathetic neurons was confirmed using molecular characterisation for the expression of PHOX2B, DBH, TH, PRPH. We employed Ca2+ imaging and whole-cell patch-clamp electrophysiology, to examine the neuronal functional properties and found that hiPSC-derived sympathetic neurons recapitulate key physiological features of the rodent native counterparts. Most cells responded to nicotine and expressed functional 2 adrenergic and muscarinic receptors, involved in sympathetic autoregulation and parasympathetic crosstalk. We further demonstrated that 2 adrenergic and muscarinic receptors inhibit membrane excitability (increased rheobase, hyperpolarisation, reduced input resistance) and that both types of receptors converge on inwardly rectifying K+ channels (GIRK) as effectors. The GIRK blocker Tertiapin-Q significantly reduced the 2 adrenergic and muscarinic responses, while the activator ML297 mimicked their action. Analysis of mouse stellate scRNA-seq confirmed that the receptors and GIRK subtypes studied here are prominently expressed in native sympathetic neurons. Overall, our data show that GIRK channels play an important role in the regulation of sympathetic neurons excitability and that hiPSC-derived neurons provide an attractive in vitro tool for drug discovery to study sympathetic autoregulation and parasympathetic-sympathetic crosstalk. KEY POINTSO_LIhiPSC-sympathetic neurons recapitulate key cellular pathways of native counterparts C_LIO_LIThey express relevant receptors and ion channels involved in the inhibitory autoregulatory feedback and parasympathetic-sympathetic crosstalk C_LIO_LIUsing a combination of RT-qPCR and functional recordings we identified inwardly rectifying K+ channels as downstream effectors of both 2 adrenergic and M2 muscarinic receptors. We cross-validated our findings with a mouse transcriptomic dataset from thoracic sympathetic ganglia. C_LIO_LIOverall, our data suggest that hiPSC-sympathetic neurons can be employed as a human in vitro platform to study cellular pathways and for drug discovery purposes. C_LI O_FIG O_LINKSMALLFIG WIDTH=119 HEIGHT=200 SRC="FIGDIR/small/727423v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@14e37ddorg.highwire.dtl.DTLVardef@35c534org.highwire.dtl.DTLVardef@260837org.highwire.dtl.DTLVardef@e57af6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract.C_FLOATNO Summary of proposed modulation of sympathetic neuron activity via 2ARs and muscarinic receptors via GIRK channels Functional recordings showed that muscarinic receptors and 2 adrenergic receptors reduce neuronal excitability, this effect was mimicked by activation of inwardly rectifying K+ channels (GIRK). Blockade of GIRK abolished the effects of both types of receptors, demonstrating that they converge on GIRK as a common effector. The muscarinic response was blocked by pertussis toxin, indicating an involvement of Gi/o downstream of the receptor, consistent with the high expression of CHRM2 by RT-qPCR. C_FIG

neuroscience↗

bioRxiv2022

TRPA1 analgesia is mediated by kappa opioid receptors

TRPA1 expressed in peripheral sensory neurons is important for nociception. Pharmacological inhibition or genetic ablation of TRPA1 profoundly reduces normal behavioural sensitivity to noxious cold and mechanical stimulation, as well as sensory neuron responses to mechanical stimulation. TRPA1 inhibition also reverses cold and mechanical hypersensitivities in chronic pain models in vivo. Here we demonstrate that these striking effects of TRPA1 inactivation result from an increased constitutive activity of kappa opioid receptors (KOR) co-expressed with TRPA1 in sensory neurons. Inhibition of KOR in Trpa1-/- mice restores nociception and neuronal activity to the levels observed in wild-type mice and reverses the analgesic effects of TRPA1 antagonism in naive mice and in neuropathic and inflammatory pain conditions. TRPA1 regulation of KOR activity in sensory neurons provides a novel mechanism to produce peripherally mediated analgesia. Our findings suggest that TRP channel regulation of constitutive GPCR activity, may be a process of general physiological importance.

neuroscience↗

bioRxiv2019

Passive transfer of fibromyalgia pain from patients to mice.

Fibromyalgia syndrome (FMS) is a chronic pain condition characterized by widespread pain and tenderness1,2. The etiology and pathophysiology of fibromyalgia are unknown and there are no effective treatments. Here we show that sensory hypersensitivity in FMS is caused by autoantibodies that act by sensitizing nociceptive sensory neurons. Administration of IgG from FMS patients increased mouse pain sensitivities to stimulation with mechanical pressure and cold. In contrast, transfer of IgG depleted samples from FMS patients or IgG from healthy control subjects had no effect on pain sensitivity. Sensory nerve fibres in ex vivo skin-nerve preparations from mice treated with FMS IgG were hypersensitive to mechanical stimulation. Immunohistochemical analysis revealed that IgG from FMS patients specifically labeled satellite glial cells and myelinated fibre tracts, as well as a small number of macrophages and endothelial cells in mouse dorsal root ganglia but not skin, muscle, spinal cord and brain. Our results demonstrate that fibromyalgia pain is caused by IgG autoantibodies that sensitize peripheral nociceptive afferents neurons and suggest that therapies that reduce patient IgG titres may be effective treatments of fibromyalgia pain.

neuroscience↗