Long-term actions of interleukin-1beta on delay and tonic firing neurons in rat superficial dorsal horn and their relevance to central sensitization

Abstract

Background: Cytokines such as interleukin 1beta (IL-1beta) have been implicated in the development of central sensitization that is characteristic of neuropathic pain. To examine its long-term effect on nociceptive processing, defined medium organotypic cultures of rat spinal cord were exposed to 100 pM IL-1beta for 6-8 d. Interleukin effects in the dorsal horn were examined by whole-cell patch-clamp recording and Ca(2+) imaging techniques.

Results: Examination of the cultures with confocal Fluo-4 AM imaging showed that IL-1beta increased the change in intracellular Ca(2+) produced by exposure to 35-50 mM K+. This is consistent with a modest increase in overall dorsal horn excitability. Despite this, IL-1beta did not have a direct effect on rheobase or resting membrane potential nor did it selectively destroy any specific neuronal population. All effects were instead confined to changes in synaptic transmission. A variety of pre- and postsynaptic actions of IL-1beta were seen in five different electrophysiologically-defined neuronal phenotypes. In putative excitatory 'delay' neurons, cytokine treatment increased the amplitude of spontaneous EPSC's (sEPSC) and decreased the frequency of spontaneous IPSC's (sIPSC). These effects would be expected to increase dorsal horn excitability and to facilitate the transfer of nociceptive information. However, other actions of IL-1beta included disinhibition of putative inhibitory 'tonic' neurons and an increase in the amplitude of sIPSC's in 'delay' neurons.

Conclusion: Since spinal microglial activation peaks between 3 and 7 days after the initiation of chronic peripheral nerve injury and these cells release IL-1beta at this time, our findings define some of the neurophysiological mechanisms whereby nerve-injury induced release of IL-1beta may contribute to the central sensitization associated with chronic neuropathic pain.

Figure 1

A. Time course of media exchanges in spinal cord organotypic cultures, establishment of defined medium and time course of IL-1β application. Nerve Growth Factor (NGF) was 20 ng/ml. Serum was progressively removed by successive dilutions during exchanges of medium. Pen/Strep/Amp = 5 units/mL penicillin G, 5 units/mL streptomycin, and 12.5 ng/mL amphotericin B, Ara-C/U/5 FDU = uridine, cytosine-β-D-arabino-furanoside (AraC), and 5-fluorodeoxyuridine (all at 10 μM). B. Comparison of time course of present IL-1β studies with previous studies of sciatic chronic constriction injury (CCI) [21]. Time scale in days refers to age of experimental animals. C. Photomicrograph obtained from an acutely isolated spinal cord slice. The central canal appears as the light area just to the right of the upper centre of the picture. The ventral surface of the cord is to the left D. Photomicrograph obtained from an organotypic culture, Sections in C and D both immuno-reacted for the microglial marker Iba-1. Calibration bar in both plates is 50 μm.

Figure 2

Effects of 6–8 d exposure of the dorsal hornregion of organotypic cultures to 100 pM IL-1β A. Typical time courses of changes in Fluo 4-AM fluorescence, indicating changes in free intracellular [Ca²⁺] in response to raising [K⁺] in superfusate from 2.5 to 20 mM. Traces are recordings from somata of cells marked as regions of interest for the analysis software. B and C. Quantification of Fluo-4 fluorescence changes in response to 20, 35 and 50 mM K⁺ from 26 control and 26 IL-1β treated neurons. Changes in amplitude in response to 50 mM K⁺ are increased in cytokine-treated slices (p = 0.02). For area under curve (AUC) significant increases are seen for both 35 mM (p < 0.01) and 50 mM K⁺ (p < 0.001).

Figure 3

A – E. Illustrations of discharge pattern of tonic, delay, irregular, phasic and transient neurons in organotypic culture in response to current commands as illustrated. Membrane potential was set to -60 mV. F. Comparison of the percentages of these 5 neuronal types in control (BSA) and cytokine-treated cultures Data from 48 control neurons and 35 interleukin-treated neurons. Numbers over each column refer to number of cells in each category.

Figure 4

A – D. Bar graphs illustrating effect of IL-1β on amplitude and interevent interval of sIPSC's and sEPSC's in tonic and delay cells. Error bars = S.E.M., graphs produced from same data as used for cumulative probability plots. E – L. Reanalysis of the data in A-D shown as cumulative probability plots. For EPSC's in tonic cells (E and F), 800 events from 8 neurons analyzed in control (minimum # of events analyzed per cell = 100), 563 events from 6 neurons analyzed in IL-1β (minimum # of events analysed per cell = 63); For IPSC's in tonic cells (G and H), 600 events from 6 neurons analyzed in control (minimum # of events analyzed per cell = 100), 239 events analyzed in 5 neurons in IL-1β (minimum # of events analysed per cell = 37); For EPSC's in delay cells (I and J), 699 events analyzed in 7 control neurons (minimum # of events analysed per cell = 99), 500 events analyzed in five neurons in IL-1β (minimum # of events analyzed per cell = 100); For IPSC's in delay cells (K and L), 751 events analyzed in 4 control neurons (minimum # of events analysed per cell = 151), 1085 events analyzed in 6 neurons in IL-1β (minimum # of events analysed per cell = 85). M and N. Sample recordings of sEPSCs at -70 mV and sIPSC's at 0 mV.