Upregulation of preprodynorphin and preproenkephalin mRNA expression by selective activation of group I metabotropic glutamate receptors in characterized primary cultures of rat striatal neurons

Group I metabotropic glutamate receptors (mGluRs) are positively coupled to phosphoinositide hydrolysis, and are expressed in medium spiny neurons of rat striatum in vivo. By modifying intracellular activities: this group of mGluRs is involved in the regulation of gene expression important for neuro plasticity. To characterize the regulatory role of group I receptors in opi oid peptide mRNA expression in vitro, primary cultures of striatal cells we re prepared from neonatal day-1 rat pups. Cells were cultured in the presen ce of a mitotic inhibitor, cytosine arabinoside, which generated predominan t neuronal cell cultures after 12-14 days in culture as demonstrated by den se immunostaining of more than 90% of cultured cells to a specific marker f or neurons (microtubule-associated protein) but not for astroglial cells (g lial fibrillary acidic protein). The vast majority of neurons (>90%) were a lso verified as GABAergic neurons according to their positive immunoreactiv ity to GABA and glutamic acid decarboxylase-65/67 antibodies. A few large n eurons (<5%) showed high levels of choline acetyltransferase immunoreactivi ty, presumably cholinergic neurons. To confirm group I mGluR expression in cultured neurons, both in situ hybridization and immunocytochemistry were p erformed, which detected moderate levels of mGluR1 and mGluR5 mRNAs and pro tein products in most neurons (>70%), respectively. On this culture system, quantitative in situ hybridization was then performed to quantify changes in preprodynorphin (PPD) and preproenkephalin (PPE) mRNA levels in response to mGluR stimulation. Acute incubation of a non-subgroup selective agonist , 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), increased PPD and PPE mRNA levels in a concentration-dependent manner (176 and 189% over con trol for PPD and PPE after 100 muM ACPD incubation, respectively). Applicat ion of a selective group I agonist, 3,5-dihydroxyphenylglycine (DHPG), prod uced much greater induction of either mRNA (285 and 289% over control for P PD and PPE after 100 muM DHPG incubation, respectively). Go-incubation of a selective group I antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chrome n-1a-carboxamide (PHCCC), blocked both ACPD- and DHPG-induced PPD/PPE expre ssion. These data demonstrate the validity of a neuronal cell culture model for studying the molecular regulation of opioid gene expression in vitro. Selective activation of identified group I mGluRs facilitates constitutive expression of PPD and PPE mRNAs in cultured striatal neurons. (C) 2001 Else vier Science B.V. All rights reserved.