Marina Brodsky

Dextromethorphan suppresses both formalin-induced nociceptive behavior and the formalin-induced increase in spinal cord c-fos mRNA.

&NA; The injection of dilute formalin results in a stereotyped nociceptive behavioral response. Administration of dextromethorphan (s.c.) but not saline, 30 min prior to intraplantar formalin injection prevents this nociceptive response in a dose‐dependent manner. In addition, intraplantar formalin reliably induces c fos mRNA in the ipsilateral spinal dorsal horn as assessed with quantitative solution hybridization at 30 min postinjection. No change in c‐fos mRNA was detected in the contralateral spinal dorsal horn, nucleus raphe magnus, periaqueductal grey, medial thalamus, or sensorimotor cortex. Pretreatment with dextromethorphan at 60 mg/ kg s.c., 30 min prior to formalin resulted in a suppression of c fos induction, so that c fos mRNA levels in the ipsilateral spinal dorsal horn of animals receiving dextromethorphan prior to formalin did not differ from controls. These data indicate that dextromethorphan suppresses formalin nociceptive behavior and one of the biochemical consequences of formalin nociception, i.e., induction of c‐fos mRNA.

CNS levels of mu opioid receptor (MOR-1) mRNA during chronic treatment with morphine or naltrexone

The CNS levels of mu opioid receptor (MOR-1) mRNA were determined by solution hybridization in rats treated chronically with morphine or naltrexone. Morphine treatment (2 x 75 mg pellets were implanted SC on Day 1 and 2 more on Day 4) resulted in the development of tolerance to morphines antinociceptive (analgesic) effect, as assessed by the hot plate procedure on treatment Day 7. Following the hot plate test, selected CNS regions were obtained by microdissection. The levels of MOR-1 mRNA in pg/microgram RNA ranged from 0.7 in sensorimotor cortex to 15.3 in medial thalamus. MOR-1 mRNA levels were not altered in the dorsal horn of spinal cord, nucleus raphe magnus, periaqueductal grey, hypothalamus, medial thalamus, or sensorimotor cortex. In a separate experiment, a 2 day exposure to naltrexone (2 x 30 mg pellets) had no effect on CNS levels of MOR-1 mRNA; however, after an 8 day exposure a decrease was detected in the nucleus raphe magnus (by 28%), hypothalamus (by 21%), and medial thalamus (by 27%). Chronic exposure to morphine or naltrexone did not result in alterations in the size of full-length MOR-1 mRNA from rat brain, or in the size of the region protected by the MOR-1 riboprobe (i.e., the entire coding region). Thus, the neuroadaptive processes associated with the development of analgesic tolerance to morphine do not involve concurrent changes in the steady-state levels of MOR-1 mRNA. Chronic treatment with naltrexone appears to produce a region-specific downregulation of MOR-1 mRNA levels, which may be secondary to the naltrexone-induced increase in mu receptor binding.

Mu and delta opioid receptor analgesia, binding density, and mRNA levels in mice selectively bred for high and low analgesia

The present study examined mu and delta opioid analgesia, receptor binding, and receptor mRNA levels in lines of mice from two selective breeding projects of relevance to opioid analgesia. Large differences were observed in the analgesic potency of [d-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO), [d-Pen2,5]enkephalin (DPDPE), and [d-Ala2]deltorphin II (DELT), selective mu, delta1, and delta2 opioid receptor agonists, respectively, in mice selectively bred for high (HA) and low (LA) swim stress-induced analgesia (SIA). HAR and LAR mice, selectively bred for high and low levorphanol analgesia, respectively, display equally large differences in their analgesic sensitivity to DAMGO, modest differences in sensitivity to DPDPE, and no differences in sensitivity to DELT. These sizable genotypic differences in analgesic potency were accompanied by HA/LA and HAR/LAR differences in whole-brain homogenate [3H]DPDPE and/or [3H]DELT, but paradoxically not [3H]DAMGO, binding. Solution hybridization of mRNA extracts encoding mu (MOR-1) or delta (DOR-1) opioid receptors indicated some regional differences in gene expression between high and low lines. Surprisingly, differences in these in vitro markers were often in the direction of LAR>HAR. The present data indicate that selection for either SSIA or levorphanol analgesia produces differential effects on mu and delta opioid analgesia that are accompanied by alterations on in vitro assays, the significance of which remains to be determined. The data are discussed with regard to the utility of in vitro biological markers and genetic models of analgesia.

The effect of the irreversible μ-opioid receptor antagonist clocinnamox on morphine potency, receptor binding and receptor mRNA

In these experiments, the effect of the irreversible mu-opioid receptor antagonist clocinnamox on the potency of morphine, opioid receptor binding and mu-opioid receptor mRNA was examined. Mice were injected with clocinnamox (0.32-12.8 mg/kg) and the analgesic potency of morphine was examined 24 h later. Clocinnamox produced a dose-dependent decrease in the potency of morphine; and at the higher dose of clocinnamox the maximal analgesic effect was not observed following doses of morphine in excess of 500 mg/kg s.c. In saturation binding studies in brain, clocinnamox (0.32-25.6 mg/kg) dose-dependently decreased mu-opioid ([3H][D-Ala2,MePhe4,Gly-ol5]enkephalin; DAMGO) receptor Bmax with relatively minimal effects on Kd. Binding to delta-opioid receptor ([3H][D-Pen2,D-Pen5]enkephalin; DPDPE) and kappa-opioid receptor ([3H](5,7,8)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec -8-yl) benzeneacetamide; U69,593) was not affected by clocinnamox. The effect of clocinnamox was time-dependent in that the greatest changes in morphine potency and mu-opioid receptor density were observed within 24 h of administration and decreased with time (336 h). Although mu-opioid receptor density was decreased to less than 30% of control 24 h following clocinnamox (12.8 mg/kg) and had increased to 80% by 5 days, a solution hybridization assay for mu-opioid receptor mRNA transcript revealed no changes in the steady-state levels of this mRNA. These studies indicate that clocinnamox is an irreversible antagonist at the mu-opioid receptor since it appears to selectively affect receptor density with minimal effects on affinity. Furthermore, clocinnamox produces time- and dose-dependent changes in Bmax and these changes appear to be unrelated to changes in mu-opioid receptor mRNA. It is possible that the repopulation of brain by mu-opioid receptors following clocinnamox is mediated by an existing pool of receptors that are activated following treatment.

Region-specific changes in NMDA receptor mRNA induced by chronic morphine treatment are prevented by the co-administration of the competitive NMDA receptor antagonist LY274614.

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphines analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.

Quantitation of μ-opioid receptor (MOR-1) mRNA in selected regions of the rat CNS

The mu opioid receptor (MOR-1) mRNA was quantified in rat CNS by a sensitive solution hybridization (SH) technique, employing a 32P-labeled riboprobe derived from the coding region of MOR-1 cDNA. In a Northern blot analysis this riboprobe hybridized to a 14 kb form of rat MOR-1 mRNA. The linear range of SH assay extends from 1 to 250 pg of MOR-1 sense transcript (equivalent to 9.3-2325 pg of MOR-1 mRNA). A microdissection technique for reproducible sampling of selected CNS regions, followed by the SH assay, allowed for a quantitative study of MOR-1 mRNA distribution. The highest levels of MOR-1 mRNA were present in medial thalamus (17.8 +/- 0.3 pg/micrograms RNA), and the lowest in the cerebellum (0.4 +/- 0.1 pg/microgram RNA). Hypothalamus, dorsal spinal horn, nucleus raphe, periaqueductal gray, and sensorimotor cortex contained intermediate levels. This distribution closely parallels the pattern of mu receptor binding, suggesting that both the mRNA and the receptor protein are colocalized within most of the regions studied.