preproenkephalin and 1-amino-1-3-dicarboxycyclopentane

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

Topics: Animals; Animals, Newborn; Cells, Cultured; Corpus Striatum; Cycloleucine; Dynorphins; Enkephalins; Gene Expression; Immunohistochemistry; In Situ Hybridization; Methoxyhydroxyphenylglycol; Neurons; Neuroprotective Agents; Nucleus Accumbens; Phenotype; Protein Precursors; Rats; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; RNA, Messenger

Metabotropic glutamate receptors (mGluR) are coupled to multiple intracellular second messenger systems through G-proteins and densely expressed by medium spiny projection neurons in the rat striatum. Unlike ionotropic glutamate receptors which mediate rapid synaptic transmission, mGluRs are important for relatively long-lasting modulation of neuronal metabotropic activity, possibly including gene expression, in response to cellular stimulation. In this study, the effects of acute injection of the selective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on behavior and striatal neuropeptide mRNA expression were evaluated in chronically-cannulated rats. Unilateral injection of ACPD into the dorsal striatum at doses of 0.8, 4, 20, 100, 500 and 1000 nmol had no significant effect on spontaneous behavioral activity. However, intrastriatal ACPD (0.8, 4, 20 and 100 nmol) dose-dependently elevated preprodynorphin (PPD), substance P (SP) and preproenkephalin (PPE) mRNA expression in the dorsal striatum as revealed by quantitative in situ hybridization. PPD/SP mRNAs showed a biphasic response to a single injection of ACPD as the expression of these two mRNAs was increased at 3 and 6 h, decreased at 11 h, and returned to normal 24 h after ACPD administration. PPE induction in the dorsal striatum was significantly elevated as early as 2 h and remained even 24 h after ACPD was injected. In addition, the PPD and PPE mRNA induction by ACPD was blocked by intrastriatal pretreatment with the selective mGluR antagonist, (+)-alpha-methyl-4-carboxyphenyl-glycine. These data demonstrate a facilitatory regulation of constitutive expression of striatonigral PPD/SP, and striatopallidal PPE, mRNAs by local mGluR-mediated glutamatergic transmission.

Topics: Animals; Corpus Striatum; Cycloleucine; Dynorphins; Enkephalins; Globus Pallidus; In Situ Hybridization; Male; Microinjections; Neurons; Neuropeptides; Protein Precursors; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; RNA, Messenger; Substance P; Substantia Nigra