Effects On Expressions Of μ, δ And κ Opioid Receptor By Exposure Of Primary Cultured Neurons To Morphine And/or Neurotensin

Objective To establish an in vitro culture model of caudate putamen neurons from neonatal rats, and investigate the effects of morphine and/or neurotensin (NT) on expressions ofμ,δandκopioid receptor (mu-opioid receptor, MOR; delta-opioid receptor, DOR; kappa-opioid receptor, KOR) in primary cultured neurons.Methods This study was performed using cell culture, reverse transcription-polymera-se chain reaction (RT-PCR) and immunocytochemistry method. Cell culture:Neurons derived from caudate putamen of neonatal rats, digested in the presence of trypsin, and mechanically dissociated in DMEM with 10% NCS, were separately cultured in vitro at 37℃and under 5% CO2. The axon length and the area of the cell body were measured at 1, 3,5,7,9 days after culture. The caudate putamen neurons were fixed at the 6 th day and anti-NSE antibody immunocytochemistry was conducted, and with NSE-positive cells resulting in more than 85% of the total cell number.Drug treatment:3 h before experiments, the DMEM with 10% NCS was changed into phenolsulfonphthalein-free DMEM without NCS:1. Morphine or NT DMEM (without NCS) with final concentrations of 10-5mol/L or 10-8mol/L was used, and the cells at 0,4,12,24 and 48 hours after administration were collected.2. Morphine DMEM (without NCS) with final concentrations of 0,10-9,10-8,10-7, 10-6 and 10-5mol/L, and NT DMEM (without NCS) with final concentrations of 0,10-12, 10-10 and 10-8mol/L were used respectively. Cells at 24 h after administration were collected.3. After DMEM without NCS was preteated with NT (final concentrations:0,10-12, 10-10 and 10-8mol/L) for 15 min, morphine (10-6mol/L) was added, and cells at 24 or 48 hours after administration were collected. The total RNA of the harvested cells of each group was isolated with RNA extraction kit, and the effects of morphine and/or NT on gene expression of MOR, DOR and KOR in primary cultured neurons were estimated by RT-PCR. Immunocytochemical staining was employed to assess expression of MOR or DOR proteins.1. In vitro cultured caudate putamen neurons could express NSE, which took on fusiforms, or ellipsoidal, pyramidal or polygonal shapes. There were even enlargements and thick growth cone on some of the neurites. Caudate putamen neurons had their axons and cell bodies growing fastest within the first 3 days, steadily during the 5-7 th days, and aging from the 9 th day on.2. The levels of MOR and DOR mRNA decreased but the level of KOR mRNA significantly increased after administration of morphine (p<0.01), which persisted up to 48 hours. Morphine produced a significant fall and rise of MOR or KOR mRNA at 10-8mol/L, which had a dose-dependent relation within the range of 10-8mol/L-10-5mol/L. The decreased DOR mRNA only occurred at 10-6mol/L (p<0.01).3. NT significantly increased MOR, DOR and KOR mRNA at 10-10 mol/L or 10"8mol/L (p<0.05 or p<0.01), and the effet persisted up to 48 hours.4. The levels of MOR or DOR mRNA were sifnificantly higher in the morphine (10-6mol/L) and NT (10-10mol/L or 10-8mol/L) group than in the morphine (10-6mol/L) group (p<0.01), but there was no significance compared with the normal group (p>0.05).5. The optical density (OD) value of MOR was significantly decreased in morphine group compared with the normal group (p<0.05). Although there was no significance compared with the normal group (p>0.05), the OD value of MOR was sifnificantly higher in the morphine (10-6mol/L) and NT (10-10mol/L or 10-8mol/L) group than in the morphine (10-6mol/L) group (p<0.05 or p<0.01); but the OD value of DOR showed no significant change in all the groups compared with the normal group (p>0.05).1. In vitro cultured caudate putamen neurons got their best condition at 6 and 7 days, which are the optimal timing for doing experiments.2. Long term administration of morphine significantly inhibited the gene expression of MOR and DOR, and increased the gene expression of KOR at the transcription level in primary cultured neurons of rat caudate putamen.3. Long term administration of NT significantly increased gene expression of MOR, DOR and KOR at the transcription level in primary cultured neurons of rat caudate putamen.4. NT blocked the effect of morphine on gene expression of MOR and DOR to some extent.5. Morphine inhibited the expression of MOR protein, and the inhibition could be partly blocked by NT, but DOR protein was almost unaffected by morphine and/or NT.