Role of the nucleus raphe magnus in antinociception produced by ABT-594: immediate early gene responses possibly linked to neuronal nicotinic acetylcholine receptors on serotonergic neurons

Abstract

Recently, a novel cholinergic channel modulator, (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), was shown to produce potent analgesia in a variety of rodent pain models when administered either systemically or centrally into the nucleus raphe magnus (NRM). The purpose of the present study was to investigate the possible supraspinal contribution of ABT-594 by assessing its ability to induce expression of the immediate early gene c-fos, a biochemical marker of neuronal activation, in the NRM of rats. Putative serotonergic neurons in the NRM, a medullary nucleus proposed to be involved in descending antinociceptive pathways, were identified immunohistochemically using a monoclonal antibody (mAb) against tryptophan hydroxylase. ABT-594 (0.03-0.3 micromol/kg, i.p.) produced a dose-dependent induction of Fos protein that was blocked by the central nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (5 micromol/kg, i.p.) but not by the peripheral nAChR antagonist hexamethonium (15 micromol/kg, i.p.). Immunohistological studies using mAb 299 revealed the expression of alpha4-containing nAChRs in the NRM. The alpha4 immunostaining was dramatically reduced by pretreating (30 d) animals with the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which was previously shown to substantially attenuate the antinociceptive actions of ABT-594. In a double immunohistochemical labeling experiment, coexpression of the serotonin marker tryptophan hxdroxylase and the alpha4 nAChR subunit in NRM neurons was observed. These results suggest that the analgesic mechanism of ABT-594 may in part involve the activation of the NRM, a site where alpha4-containing nAChRs are expressed by serotonergic neurons.

Fig. 1.

Immunohistochemical identification of the nucleus raphe magnus (NRM). The rat NRM is located (open square) in the rostral ventromedial medulla and is centered just above the pyramidal tract at the base of the brainstem (A) (adapted from Paxinos and Watson, 1997). Serotonergic neurons of the NRM (B) were immunohistochemically identified using an mAb against tryptophan hydroxylase in a saline-treated rat. Scale bar, 100 μm.

Fig. 2.

Photomicrographs of Fos protein in the NRM after ABT-594. Induction of c-Fos expression in the NRM of rats is shown 2 hr after systemic administration of ABT-594 at either 0, 0.03, 0.1, or 0.3 μmol/kg intraperitoneally. The nuclear immunostaining associated with Fos expression is highest in the 0.3 μmol/kg-treated rat.White hatched triangle represents area of NRM analyzed for FLI. Scale bar, 100 μm.

Fig. 3.

Graphs of ABT-594 Fos protein induction. Systemic administration of ABT-594 (0.03–0.3 μmol, i.p.) in rats (n = 6 per group) produced a dose-dependent induction of Fos expression in the NRM (F_(3,20) = 7.336; p = 0.0017). Fos-like immunoreactivity (FLI) was quantified and expressed as the percent of saline-treated control FLI mean ± SEM [hatched lines represent ± control SEM; *p < 0.01 vs saline-treated rats; protected least significant difference (PLSD) post hoctest].

Fig. 4.

Graphs of ABT-594 Fos protein induction and the effects of nicotinic antagonism. Administration of the nAChR antagonist mecamylamine (5 μmol/kg, i.p.) 15 min before ABT-594 (0.3 μmol/kg, i.p.) prevents ABT-594-induced Fos expression in the NRM of rats (n = 4–6 per group) 2 hr later (A). Administration of the peripheral nAChR antagonist hexamethonium (15 μmol/kg, i.p.) 15 min before ABT-594 (0.3 μmol/kg, i.p.) did not prevent ABT-594-induced Fos expression in the NRM of rats (n = 4–6 per group) 2 hr later (B). FLI is expressed as the percent of saline control FLI mean ± SEM (*p < 0.05 compared with saline-treated rats; +p < 0.05 as compared with ABT-594-treated rats; PLSD post hoc test).

Fig. 5.

Photomicrographs of the α4 nAChR subunit in the NRM. α4 immunostaining (mAb 299) was observed in the NRM (A) of a saline-treated rat. At higher magnification (B), the α4 subunit is seen primarily in the cytosol of the soma. Expression of α4 is also seen, but to a lesser extent, in the neuropil. Scale bars: A, 100 μm; B, 10 μm.

Fig. 6.

Photomicrographs of tryptophan hydroxylase and the α4 nAChR subunit in the NRM and the effects of a serotonergic neurotoxin. Expression of tryptophan hydroxylase is shown in the NRM of rats 30 d after either an intra-NRM injection of 0.3 μl of PBS (A) or 3.75 nmol of 5,7-DHT (B). Expression of the α4 nAChR subunit is shown in the same animals again, 30 d after either a single intra-NRM injection of 0.3 μl of PBS (C) or 3.75 nmol of 5,7-DHT (D). The serotonin neurotoxin resulted in almost a complete loss of serotonergic neurons and expression of α4-containing nAChRs in the NRM. Scale bar, 100 μm.

Fig. 7.

Photomicrographs of double labeling of tryptophan hydroxylase and the α4 nAChR subunit in the NRM. Using a fluorescein-conjugated secondary Ab, tryptophan hydroxylase immunostaining (A) shows almost complete overlap compared with the DAB chromogenic immunostaining of α4-containing neurons (B) in the NRM of a saline-treated rat. Double-labeled neurons are indicated by arrows. Scale bar, 100 μm.