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. 2012 Sep 6:219:204-13.
doi: 10.1016/j.neuroscience.2012.06.011. Epub 2012 Jun 13.

GDNF induces mechanical hyperalgesia in muscle by reducing I(BK) in isolectin B4-positive nociceptors

J Hendrich  1 , P Alvarez, X Chen, J D Levine
Affiliations

GDNF induces mechanical hyperalgesia in muscle by reducing I(BK) in isolectin B4-positive nociceptors

J Hendrich et al. Neuroscience. .

Abstract

We have assessed the mechanism underlying glial cell-derived neurotrophic factor (GDNF)-induced mechanical hyperalgesia in the gastrocnemius muscle, using patch clamp electrophysiology, in vivo electrophysiology and behavioral studies. Cultured isolectin B4-positive (IB4+) dorsal root ganglion neurons that innervated this muscle were held under current clamp; the majority developed an increase in action potential duration (a factor of increase of 2.29±0.24, compared to 1.13±0.17 in control, P<0.01) in response to GDNF (200 ng/ml) by 15 min after application. They also demonstrated a depolarization of resting membrane potential, but without significant changes in rheobase, action potential peak, or after-hyperpolarization. Large-conductance voltage- and calcium-activated potassium (BK) channels, which have recently been shown to play a role in the repolarization of IB4+ nociceptors, were inhibited under voltage clamp, as indicated by a significant reduction in the iberiotoxin-sensitive current. In vivo single-fiber recording from muscle afferents revealed that injection of iberiotoxin into their peripheral nociceptive field caused an increase in nociceptor firing in response to a 60s suprathreshold stimulus (an increase from 392.2±119.8 spikes to 596.1±170.8 spikes, P<0.05). This was observed in the absence of changes in the mechanical threshold. Finally, injection of iberiotoxin into the gastrocnemius muscle produced dose-dependent mechanical hyperalgesia. These data support the suggestion that GDNF induces nociceptor sensitization and mechanical hyperalgesia, at least in part, by inhibiting BK current in IB4+ nociceptors.

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Conflict of interest statement

The authors declare no conflict of interest

Figures

Figure 1
Figure 1. Effect of GDNF on action potentials recorded from IB4+ sensory neurons innervating the gastrocnemius muscle in the rat
A. Representative action potentials elicited over time in two small-diameter IB4+ neurons that innervated the gastrocnemius muscle, exposed to either 200 ng/ml GDNF or vehicle. The action potential at rheobase is shown before, and 10, 20 and 30 min after GDNF or vehicle application to the bath. B. Mean action potential characteristics (duration, AHP amplitude, action potential peak and RMP) are shown for 7 cells in control (squares) and 8 cells in GDNF (circles) conditions. The factor of increase in action potential duration was significantly greater in GDNF-treated cells (2.29 ± 0.24) than in control (1.13 ± 0.13; P<0.01) at 30 min. Also, the depolarization of RMP was significantly greater in GDNF-treated cells (10.31 ± 2.19 mV) compared to control (4.35 ± 1.64 mV; P<0.05) at 30 min. No significant changes were observed in AHP amplitude or action potential peak.
Figure 2
Figure 2. GDNF inhibits BK current in IB4+ muscle afferents in vitro
A. A control I-V trace (left panel), onset of iberiotoxin block in response to a +60 mV pulse (middle panel), and iberiotoxin-resistant component of the current (right panel) are shown. Subtraction of the iberiotoxin-resistant component from the control I-V trace produced a trace denoted as BK current. B. Mean iberiotoxin-sensitive I-V relationships of control (filled squares; n=10) and GDNF pre-incubated (open circles; n=8) neurons are shown. Mean current density at +60 mV was significantly reduced (P<0.05) in GDNF-treated cells (0.77 ± 0.2 nA/pF) compared to control (2.2 ± 0.5 nA/pF). C. Representative current traces of control (filled square) and GDNF (open circle) pre-incubated neurons. BK currents showed variation both in current size and kinetics within the same conditions. D. Fluorescent micrograph showing IB4 (green) and DiI (red) immunoreactivity in small-to-medium diameter DRG neurons (top panel). The corresponding brightfield micrograph is shown in the bottom panel. Images were taken <4 hours after dissociation. Scale bars represent 50 μm.
Figure 3
Figure 3
Intra-muscular injection of iberiotoxin into the gastrocnemius muscle produces a dose-dependent reduction in mechanical nociceptive threshold. Mechanical threshold was significantly reduced (PB. NS1619 prevents GDNF-induced muscle mechanical hyperalgesia. While muscle mechanical threshold (Pre) is not modified by the treatment (Post) with either NS1619 (n=6, open bars) or its vehicle (n=6, solid bars), NS1619 significantly inhibited the decrease of mechanical threshold induced by GDNF compared to vehicle, at 30 (10.1 ± 1.3% versus 26.5 ± 1.2, respectively) and 60 (9 ± 1.2 versus 30.7 ± 0.9, respectively) min after GDNF injection. ***P<0.001
Figure 4
Figure 4. Iberiotoxin produces increased firing in muscle nociceptors after iberiotoxin injection
A. Bar chart showing the mean response of 12 recorded muscle nociceptors to sustained (60 s) 10 g stimuli, before and after intramuscular injection of iberiotoxin. The mean number of spikes fired increased from 392.2 ± 119.8 before iberiotoxin injection, to 596.1 ± 170.8 after (P<0.05). B. Time course of the mean responses of muscle nociceptors to a suprathreshold 60 s stimulus, before (top panel) and after (bottom panel) iberiotoxin injection (n = 12). Bin width is 1 s. C. The mechanical threshold of muscle nociceptors was not significantly altered by the application of iberiotoxin.
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