RGS9-2 modulates nociceptive behaviour and opioid-mediated synaptic transmission in the spinal dorsal horn

Abstract

The regulator of G protein signaling 9-2 (RGS9-2) is a constituent of G protein-coupled receptor (GPCR) macromolecular complexes with a major role in regulation of GPCR activity in the central nervous system. Previous in situ hybridization and Western blot studies revealed that RGS9-2 is expressed in the superficial dorsal horn of the spinal cord. In the present study, we monitored tail withdrawal latencies to noxious thermal stimuli and performed in vitro whole-cell patch clamp electrophysiological recordings from neurons in lamina II of the spinal dorsal horn to examine the role of RGS9-2 in the dorsal horn of the spinal cord in nociceptive behaviours and opiate mediated modulation of synaptic transmission. Our findings obtained from RGS9 knockout mice indicate that the lack of RGS9-2 protein decreases sensitivity to thermal stimuli and to the analgesic actions of morphine in the tail immersion paradigm. This modulatory role of RGS9-2 on opiate-mediated responses was further supported by electrophysiological studies showing that hyperpolarization of neurons in lamina II of the spinal dorsal horn evoked by application of DAMGO ([d-Ala2, N-MePhe4, Gly-ol]-enkephalin, a mu opioid receptor agonist) was diminished in RGS9 knockout mice. The results indicate that RGS9-2 enhances the effect of morphine and may play a crucial role in opiate-mediated analgesic mechanisms at the level of the spinal cord.

Fig. 1

RGS9-2 modulates nociceptive thresholds in the hot water tail immersion assay. The tail withdrawal latency values in the 54 °C tail immersion test were significantly higher in RGS9KO mice compared to their RGS9WT littermates (a), suggesting that RGS9-2 plays an essential role in the modulation of spinal nociceptive transmission. Data are presented as mean ± SEM, n = 8 per group, Student’s t-test, *P < 0.05. (b) Shows responses of RGS9WT and RGS9KO mice in the formalin test. Licking and biting behaviour of animals injected with formalin in the plantar surface of the left hindpaw were recorded for 60 min at 10 min intervals. Data are presented as mean ± SEM, n = 12–13 per group.

Fig. 2

RGS9-2 is a positive modulator of morphine analgesia in the tail immersion assay. RGS9KO mice showed decreased sensitivity to morphine in the tail immersion test, at low doses (1 mg/kg s.c.) compared to their RGS9WT controls (n = 11 per genotype). The thermal withdrawal latency is increased in wild-type but not in mutant mice. Both genotypes showed similar analgesic responses (n = 6–8) to morphine at higher doses (3 mg/kg). The analgesic effect of morphine in the tail immersion test is completely blocked following naloxone (NLX) administration (0.3 mg/kg s.c., n = 7–9 per group). Data are expressed as mean MPE ± SEM, *P < 0.05, two way ANOVA followed by Bonferroni post hoc test.

Fig. 3

RGS9-2 is a positive modulator of opiate actions in lamina II neurons. Responses of neurons in lamina II of the spinal dorsal horn to DAMGO are shown in (a). In wild-type animals, 11 of the 57 recorded neurons (from 41 animals) responded to DAMGO; all of them with a prominent hyperpolarization. In RGS9KO animals, none of the 33 recorded neurons (from 22 animals) responded to DAMGO. Scale bars: 20 mV, 1 min. Histogram in (b) shows the amplitude of DAMGO evoked hyperpolarization in RGS9WT and RGS9KO animals. Data are presented as mean ± SEM. The numbers of responding neurons and the total numbers of recorded neurons (in brackets) are indicated above the corresponding column of the histogram.