Effect of antisense knock-down of alpha(2a)- and alpha(2c)-adrenoceptors on the antinociceptive action of clonidine on trigeminal nociception in the rat.

Although activation of alpha(2)-adrenoceptors is known to play an important role in mediating antinociception, the contribution of various alpha(2)-adrenoceptor subtypes in modulating trigeminal nociception remains unknown since subtype specific agonists and antagonists are not available. The present study investigated the functional role of alpha(2)-adrenoceptor subtypes in modulating the N-methyl-D-aspartate-induced nociceptive behavior in the medullary dorsal horn by using antisense oligodeoxynucleotides to selectively knock-down the receptor subtypes. Microinjection of N-methyl-D-aspartate (2 nmol in 10 microl) through a cannula implanted dorsal to the medullary dorsal horn produced a total of 164.9+/-8.8 scratches in the facial region (n=14), and the scratching behavior lasted for 77.8+/-5.2s (n=14). Microinjection of clonidine, an alpha(2)-agonist (7 microg in 5 microl), 15 min prior to administration of N-methyl-D-aspartate, produced a reduction of 71.6% (n=12) in the number of scratches and a reduction of 57.5% (n=12) in the duration. The inhibitory effect of clonidine was blocked by idazoxan (n=4) and yohimbine (n=4), alpha(2) antagonists. In rats pretreated with the antisense probe to the alpha(2A) adrenoceptor, clonidine only produced a reduction of 7.3% in the number of scratches (n=12) and a reduction of 9% in the duration (n=12). The antinociceptive effect of clonidine recovered completely 4 days after termination of the alpha(2A) antisense oligodeoxynucleotide treatment. In contrast to the alpha(2A) antisense-treated animals, clonidine reduced the number of scratches and the duration by 85.5% (n=9) and 82.1% (n=9), respectively, in rats pretreated with the sense probe to the alpha(2A) adrenoceptor. The effect of clonidine was not altered in rats pretreated with the antisense or the sense probes to the alpha(2C) adrenoceptor. In the alpha(2C) antisense pretreated rats, clonidine reduced the number of scratches and the duration by 60.8% (n=11) and 44.5 % (n=11), respectively. In the sense-pretreated rats, clonidine produced a reduction of 69.1% in the number of scratches (n=9) and a reduction of 55.1% in the duration (n=9). In order to assess the effectiveness of the antisense treatment, the receptor expression was examined by immunohistochemistry. Antisense treatment reduced alpha(2A) and alpha(2C) receptor immunoreactivity in the medullary dorsal horn compared to the sense and the vehicle-treated animals. Quantitative image analysis revealed a significant decrease in pixel intensity following the antisense treatment. These results indicate that activation of alpha(2A) adrenoceptor plays an important role in mediating the antinociceptive effect of clonidine in the medullary dorsal horn in the rat.