A Conditional Deletion of the NR1 Subunit of the NMDA Receptor in Adult Spinal Cord Dorsal Horn Reduces NMDA Currents and Injury-Induced Pain

To determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, the NMDAR1 (NR1) subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein are reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, are reduced 86–88% in lamina II neurons. The spatial NR1 knock-out does not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. Our results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury

Quantitation of NMDA receptor (NMDAR1) mRNA levels in the adult and developing rat CNS

A rapid and sensitive solution hybridization assay was used to quantitate N- methyl- d- aspartate (NMDA) receptor mRNA levels in the central nervous system (CNS) of rat, mouse and human. A riboprobe labelled with 32P was prepared from a plasmid containing a 1413 base sequence from the cDNA for the functional rat NMDA receptor subunit, NMDAR1. Using a full length sense transcript as the calibration standard, the assay reliably measures 8 pg of NMDAR1 mRNA. When expressed as pg of NMDAR1 mRNA/μg total cellular RNA, the highest levels in the adult rat CNS are in the olfactory bulb (20.9 pg/υg RNA) and the lowest levels are in the spinal cord (5.2 pg/υg RNA). Intermediate levels were found in frontal cortex, hippocampus, cerebellum and whole brain. In the mouse CNS the highest levels of NMDAR1 mRNA were found in the olfactory bulb (12.9 pg equivalents/υg RNA), followed closely by hippocampus, frontal cortex and cerebellum. Mouse spinal cord (4.4 pg equivalents/υg RNA) had the lowest levels of NMDAR1 mRNA. The NMDAR1 riboprobe hybridizes with the same size transcripts in Poly(A) + RNA from rat, mouse and human brain. In the developing rat, NMDAR1 mRNA levels in frontal cortex and hippocampus increased nearly 3 fold from postnatal day 3 to day 15 and approximately doubled from day 15 to day 67 (adult). Therefore, from postnatal day 3 to adult (day 67) frontal cortex and hippocampus levels of NMDAR1 mRNA increased nearly 6 fold. In contrast, cerebellum and brain stem showed no change in NMDAR1 mRNA levels when postnatal days 3 and 15 were compared while the levels were increased 2 fold when day 15 and day 67 were compared. These changes were not a function of alterations in total cellular RNA. These results demonstrate significant differences in frontal cortex and hippocampus when compared to cerebellum and brain stem in the postnatal development of NMDAR1 mRNA

Isolation and stereochemical identification of a metabolite of naltrexone from human urine / Nithiananda Chatterjie, James M. Fujimoto, Charles E. Inturrisi, Sandra Reorig, Richard I.H. Wang, David V. Bowen, Frank H. Field, and Donald D. Clarke Department of Pharmacology, Cornell University Medical College (N.C., C.E.I.), Department of Pharmacology, Medical College of Wisconsin (J.M.F., S.R.), Veterans Administrative Center, Wood, Wisconsin (R.I.H.W.), Mass Spectrometry Service Laboratory, The Rockefeller University (D.V.B., F.H.F.), and Department of Chemistry, Fordham University (D.D.C.)

Pooled urine samples from patients receiving 100-200 mg of naltrexone per day orally were extracted; the basic (alkaloid) compounds derived were isolated by preparative thin-layer chromatography. The major metabolite of naltrexone was found to be an epimer of N-cyclopropylmethyl- 14-hydroxy-7,8-dihydronormorphine wherein the 6-keto group of naltrexone had been reduced to yield the 6β-hydroxy epimer (an isomorphine). This conclusion was based on infrared, mass, and nuclear magnetic resonance spectra studies. Furthermore, the reduction product formed in vitro in a soluble chicken liver enzyme system from naltrexone and an in vivo metabolite of naloxone derived from the chicken were found to have the more commonly expected 6α-hydroxy orientatio

Estradiol replacement in ovariectomized rats is antihyperalgesic in the formalin test

A subcutaneous implant of 17 beta-estradiol or progesterone provides steady-state serum hormone levels from 7 to 24 days after implantation and allows the evaluation of the effects of the replacement with these hormones on phase 1 and phase 2 formal in-induced behaviors in ovariectomized (OVX) rats. Graded doses of 17 beta-estradiol (5% to 40%) reduce formalin-induced behavior by 35% to 49% during phase 2 but not during phase 1, as; measured with an automated formalin apparatus. The maximal response is seen with 20% 17 beta-estradiol. The antihyperalgesic effect of 20% 17 beta-estradiol is significant at 8 days after implantation and persists at 21 days. In contrast, graded doses of progesterone have no effect on either phase of formalin. The estrogen receptor antagonist tamoxifen completely prevents the antihyperalgesic effect of the 20% 17 beta-estradiol implant. Formalin-induced behaviors during phase 2 are significantly less in proestrus females and OVX rats given 20% 17 beta-estradiol compared with OVX control rats. Also, the formalin-induced increase in serum corticosterone is attenuated in OVX control rats compared with proestrus females and OVX rats given 20% 17 beta-estradiol. These results indicate that estrogen replacement in OVX rats restores the maximal corticosterone response to tonic pain and, by an estrogen receptor-mediated process, inhibits tonic pain. Pe rspective: Hormone replacement (HR) therapy remains a widely used modality. We used a pharmacokinetically based rat HR model that results in continuous physiological levels of 17 beta-estradiol to demonstrate the analgesic (antihyperalgesic) effects of estrogen replacement in an inflammatory pain model (formalin). These results suggest a potentially important consequence of HR therapy. (C) 2007 by the American Pain Society