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 neuroplasticity. To characterize the regulatory role of group I receptors in opioid peptide mRNA expression in vitro, primary cultures of striatal cells were prepared from neonatal day-1 rat pups. Cells were cultured in the presence of a mitotic inhibitor, cytosine arabinoside, which generated predominant neuronal cell cultures after 12-14 days in culture as demonstrated by dense immunostaining of more than 90% of cultured cells to a specific marker for neurons (microtubule-associated protein) but not for astroglial cells (glial fibrillary acidic protein). The vast majority of neurons (>90%) were also verified as GABAergic neurons according to their positive immunoreactivity to GABA and glutamic acid decarboxylase-65/67 antibodies. A few large neurons (<5%) showed high levels of choline acetyltransferase immunoreactivity, presumably cholinergic neurons. To confirm group I mGluR expression in cultured neurons, both in situ hybridization and immunocytochemistry were performed, which detected moderate levels of mGluR1 and mGluR5 mRNAs and protein 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 control for PPD and PPE after 100 microM ACPD incubation, respectively). Application of a selective group I agonist, 3,5-dihydroxyphenylglycine (DHPG), produced much greater induction of either mRNA (285 and 289% over control for PPD and PPE after 100 microM DHPG incubation, respectively). Co-incubation of a selective group I antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), blocked both ACPD- and DHPG-induced PPD/PPE expression. 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.
Mao
L
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Division of Pharmacology, School of Pharmacy, University of Missouri -Kansas City, 2411 Holmes Street, Rm. M3-C15, Kansas City, MO 64108, USA.
Wang
J Q
JQ
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000166:cytology; D003515:Cycloleucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004399:Dynorphins / Q000032:analysis / Q000235:genetics*; D004745:Enkephalins / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives* / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D009714:Nucleus Accumbens / Q000166:cytology; D010641:Phenotype; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D004745:Enkephalins; C578937:Grm5 protein, rat; D018696:Neuroprotective Agents; D011498:Protein Precursors; D012333:RNA, Messenger; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; C036448:pre-prodynorphin; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; D008734:Methoxyhydroxyphenylglycol; D004399:Dynorphins; C033660:preproenkephalin; C010117:3,4-dihydroxyphenylglycol