Inflammatory mediator-induced modulation of GABAA currents in human sensory neurons

Abstract

The purpose of the present study was to characterize the properties of GABAA receptor currents in human sensory neurons. Neurons were obtained from adult organ donors. GABAA currents were recorded in isolated neurons. Both large inactivating low affinity currents and smaller persistent high affinity currents were present in all of the 129 neurons studied from 15 donors. The kinetics of human GABAA currents were slower than those in rat sensory neurons. GABA currents were completely blocked by bicuculline (10 µM), and persistent currents were activated by the δ-subunit preferring agonist, THIP. The GABA current equilibrium potential was ~20 mV more hyperpolarized than in rat neurons. Both low and high affinity currents were increased by inflammatory mediators but via different second messenger pathways. These results highlight potentially important species differences in the properties of ion channels present in their native environment and suggest the use of human sensory neurons may be a valuable tool to test compounds prior to use in humans.

Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures

“…Human MRGPRX1 can be activated by both Mrgpra3 and Mrgprc11 agonists, although key differences in the pharmacological profiles of these receptors demonstrate distinct molecular mechanisms in mice versus humans 13–17 . In support of these fundamental biological differences, GABA A receptor-mediated currents in cultured human sensory neurons were recently found to exhibit distinct biophysical features from those in rodents, demonstrating the importance of this type of preclinical approach 18–21 .…”

“…Several recent studies have identified species differences and validated molecular mechanisms using human sensory neurons, but few have provided a detailed description of extraction or culturing methodologies 18–21 . Here, we provide a detailed surgical extraction protocol for post-mortem access to human dorsal root ganglia (hDRG) immediately after organ procurement from consented donors.…”

“…Our studies using hDRG show that these adaptations maintain neuronal viability in transport and address several issues that arise from dissociation of substantially larger tissues with more abundant connective tissue than rodent DRG. 22 Notable differences between this protocol and those previously described include the specific types of enzymes used (papain versus trypsin and DNase 18 , or proprietary enzyme mixture 19,21 ) and the duration of enzymatic digestion (up to 3.5 hours in some studies). The type of culture medium and use of neurotrophic factors has also varied between studies, with several studies utilizing DMEM/F12-based media and all studies supplementing with varying concentrations of NGF and/or GDNF.…”

“…Following these procedures will produce viable dissociated cultures of hDRG neurons, but recent publications indicate that it may also be possible to culture human sensory neurons in the presence of other types of culture media 18,19,21 . Investigators may decide to incorporate the same enzymatic digestion, plating protocol, or culture media they have previously used in animal model studies if the goal is to make direct comparisons to those prior studies.…”

Metabotropic Glutamate Receptor 2/3 (mGluR2/3) Activation Suppresses TRPV1 Sensitization in Mouse, But Not Human, Sensory Neurons

“…Therefore, using primary human neurons to validate preclinical rodent findings is an appealing strategy to improve the translational success of basic pain research findings. With this goal in mind, we and others have established approaches to obtain and use human sensory neurons to better understand human nociceptor physiology ( Baumann et al, 1996 ; Anand et al, 2006 ; Davidson et al, 2014 ; Han et al, 2015 ; Zhang et al, 2015 ; Sapio et al, 2016 ; Valtcheva et al, 2016 ; Rostock et al, 2017 ).…”