Induction of long-term hyperexcitability by memory-related cAMP signaling in isolated nociceptor cell bodies

Abstract

Persistent hyperactivity of nociceptors is known to contribute significantly to long-lasting sensitization and ongoing pain in many clinical conditions. It is often assumed that nociceptor hyperactivity is mainly driven by continuing stimulation from inflammatory mediators. We have tested an additional possibility: that persistent increases in excitability promoting hyperactivity can be induced by a prototypical cellular signaling pathway long known to induce late-phase long-term potentiation (LTP) of synapses in brain regions involved in memory formation. This cAMP-PKA-CREB-gene transcription-protein synthesis pathway was tested using whole-cell current clamp methods on small dissociated sensory neurons (primarily nociceptors) from dorsal root ganglia (DRGs) excised from previously uninjured ("naïve") male rats. Six-hour treatment with the specific Gαs-coupled 5-HT4 receptor agonist, prucalopride, or with the adenylyl cyclase activator forskolin induced long-term hyperexcitability (LTH) in DRG neurons that manifested 12-24 h later as action potential (AP) discharge (ongoing activity, OA) during artificial depolarization to -45 mV, a membrane potential that is normally subthreshold for AP generation. Prucalopride treatment also induced significant long-lasting depolarization of resting membrane potential (from -69 to -66 mV), enhanced depolarizing spontaneous fluctuations (DSFs) of membrane potential, and produced trends for reduced AP threshold and rheobase. LTH was prevented by co-treatment of prucalopride with inhibitors of PKA, CREB, gene transcription, or protein synthesis. As in the induction of synaptic memory, many other cellular signals are likely to be involved. However, the discovery that this prototypical memory induction pathway can induce nociceptor LTH, along with reports that cAMP signaling and CREB activity in DRGs can induce hyperalgesic priming, suggest that early, temporary, cAMP-induced transcriptional and translational mechanisms can induce nociceptor LTH that might last for long periods. The present results also raise the question of whether reactivation of primed signaling mechanisms by re-exposure to inflammatory mediators linked to cAMP synthesis during subsequent challenges to bodily integrity can "reconsolidate" nociceptor memory, extending the duration of persistent hyperexcitability.

Keywords: Cellular memory; Cyclic AMP signaling; Dorsal root ganglia; Excitability; Hyperexcitability; Inflammatory mediator.

Fig. 1

5-HT4 receptor agonist prucalopride (Ppr) induces long-term hyperexcitability (LTH) in small (≤30 µm soma diameter) sensory neurons. A, Experimental design (created with BioRender.com). All inhibitor studies co-applied Ppr with an inhibitor, except for one study in which the inhibitor was applied after washout of Ppr. B-E, Long-term effects on excitability of treatment with Ppr (1  µM) versus vehicle (0.01 % DMSO). Representative 10-s recordings at RMP B), or held under current clamp at −45 mV (D). Corresponding proportions of neurons exhibiting any AP discharge at RMP (SA), C) or at −45 mV (OA), E). Above each bar is the number of neurons exhibiting discharge over the total number sampled. Comparisons using Fisher’s exact test with p values reported above each bar. Red arrows indicate the largest subthreshold depolarizing spontaneous fluctuation (DSF) in each trace. F-H, Impact of 6-h Ppr treatment on other measures of excitability, RMP (F), AP voltage threshold (G), and rheobase (H). Datasets are graphed as mean ± SEM. Each circle represents the mean value for that condition per day of recording when both vehicle and prucalopride were tested. Comparisons used paired t-tests with p values reported in each panel. p < 0.05 was considered significant. AP, action potential; DRG, dorsal root ganglion; MP, membrane potential; N.S., non-significant; OA, ongoing activity; RMP, resting membrane potential; SA, spontaneous activity; Veh, vehicle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

LTH is not attributable to continuing stimulation by Ppr and can be induced by direct stimulation of adenylyl cyclase. A, 10-s representative traces of neurons recorded when clamped at −45 mV after prior 6-h treatment with either vehicle (“Veh”, 0.01 % DMSO), Ppr (1 µM) alone, Ppr followed by overnight treatment with either 5-HT4 receptor blocker GR113808 (1 µM) or GR113808 alone (“GR”). B, proportions of neurons with OA at −45 mV in each experimental condition. C, 10-s representative traces of neurons recorded at −45 mV after prior 6-h treatment with vehicle (0.01 % DMSO) or adenylyl cyclase activator forskolin (1 µM). D, proportions of neurons with OA at −45 mV. For B and D, the number of neurons exhibiting OA over the total number sampled is reported above each bar. Comparisons performed against the vehicle condition with Fisher’s exact test. In B, p < 0.017 was considered significant after Bonferroni correction for 3 comparisons while in D, p < 0.05 was considered significant for a single comparison. Fsk, forskolin; MP, membrane potential; NS, non-significant; OA, ongoing activity; Ppr, prucalopride; Veh, vehicle.

Fig. 3

Induction of LTH depends upon PKA, CREB, gene transcription, and protein synthesis. A, diagram of the experimental hypothesis with the inhibitors and activators employed for testing. B-E, proportions of neurons with OA at −45 mV treated with either vehicle (0.02 or 0.11 % DMSO, depending upon the inhibitor used), Ppr (1  µM) alone, or (“Ppr + X”) plus an inhibitor of PKA (H-89, 10  µM, B), CREB (666–15, 0.5  µM, C), gene transcription (actinomycin D, ActD, 1  µg/ml, D) or protein synthesis (cycloheximide, Chx, 20  µM, E), and each inhibitor alone. The number of neurons exhibiting OA over the total number sampled is reported above each bar. Comparisons with Fisher’s exact tests followed by Bonferroni correction for multiple comparisons. For comparisons of OA proportions between Ppr against the vehicle or Ppr + inhibitor, the significance level was * p < 0.025, ** p < 0.005, and *** p < 0.0005 for two comparisons. For vehicle against inhibitor alone, p < 0.05 was considered significant. LTH, long-term hyperexcitability; NS, non-significant; OA, ongoing activity.

Fig. 4

Ppr-induced LTH involves potentiation of DSFs. A, magnification of the representative 10-second trace of a Ppr-treated sensory neuron recorded at −45 mV shown in Fig. 1D (right side). APs are clipped to show details of the fluctuations of membrane potential. Red arrow indicates the largest subthreshold DSF that was used as a conservative estimate of AP voltage threshold (represented by red dashed line). B-C, effect of Ppr and of each inhibitor tested on DSF amplitude (B) or the frequencies of large (≥5 mV) DSFs (C). Data are graphed as medians. Each open circle represents the mean DSF value from a single neuron. Comparisons of amplitudes or large DSF frequencies between Ppr and vehicle or each Ppr with inhibitor used Kruskal-Wallis followed by Dunn’s multiple comparisons tests, as were differences between vehicle and each inhibitor alone. p values are reported in each panel and p < 0.05 was considered significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)