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Major depressive disorder is a common form of mental illness. Many brain regions are implicated in the pathophysiology and symptomatology of depression. Among key brain areas is the striatum that controls reward and mood and is involved in the development of core depression-like behavior in animal models of depression. While molecular mechanisms in this region underlying depression-related behavior are poorly understood, the glutamatergic input to the striatum is believed to play a role. In this study, we investigated changes in metabotropic glutamate (mGlu) receptor expression and signaling in the striatum of adult rats in response to prolonged (10-12 weeks) social isolation, a pre-validated animal paradigm modeling depression in adulthood. We found that mGlu5 receptor protein levels in the striatum were increased in rats that showed typical depression- and anxiety-like behavior after chronic social isolation. This increase in mGlu5 receptor expression was seen in both subdivisions of the striatum, the nucleus accumbens and caudate putamen. At subcellular and subsynaptic levels, mGlu5 receptor expression was elevated in surface membranes at synaptic sites. In striatal neurons, the mGlu5-associated phosphoinositide signaling pathway was augmented in its efficacy after prolonged social isolation. These data indicate that the mGlu5 receptor is a sensitive substrate of depression. Adulthood social isolation leads to the up-regulation of mGlu5 receptor expression and function in striatal neurons.

Activation of group I metabotropic glutamate receptors (mGluRs) up-regulates transcription factor cyclic AMP response element-binding protein (CREB) and Elk-1 phosphorylation via extracellular signal-regulated kinase 1/2 (ERK1/2) in the striatum in vivo. Protein phosphatase 1/2A further regulates immediate early gene expression by inactivating (dephosphorylating) CREB. In this study, using semi-quantitative immunohistochemical and western blot analyses and in situ hybridization histochemistry, we found that intrastriatal infusion of the protein phosphatase 1/2A inhibitor okadaic acid (0.005, 0.05 and 0.5 nmol) increased CREB and Elk-1 phosphorylation and c-Fos immunoreactivity in the injected dorsal striatum in a dose-dependent manner. In addition, okadaic acid (0.05 and 0.5 nM) increased c-fos mRNA expression in the dorsal striatum in a dose-dependent manner. Intrastriatal infusion of the group I agonist 3,5-dihydroxyphenylglycine (DHPG) at 100 and 250 nM also increased CREB and Elk-1 phosphorylation. Pre-treatment of okadaic acid (0.05 nm) did not alter DHPG-induced increases in the phosphorylation of the two transcription factors. These data suggest that protein phosphatase 1/2A in striatal neurons is tonically active in dephosphorylating CREB and Elk-1 and thus suppressing constitutive c-fos mRNA and protein expression. Inhibition of the phosphatase 1/2A may contribute to the group I mGluR-regulated phosphorylation of these transcription factors and c-fos expression.

The Galphaq protein-coupled metabotropic glutamate receptor subtype-5 (mGluR5) is densely expressed in medium spiny projection neurons of striatum. Emerging evidence suggests a significant role of mGluR5 in the addictive plasticity of striatal neurons that is likely derived from inducible cellular gene expression related to stimulation of mGluR5 and associative signaling proteins. In this study, we found that activation of mGluR5 with a selective agonist (RS)-2-chloro-5-hydroxy-phenylglycine (CHPG) induced a rapid and transient phosphorylation of a transcription regulator Elk-1 in cultured striatal neurons from rat E19 embryos or neonatal day-1 pups. The Elk-1 phosphorylation was dose-dependent and occurred in neurochemically identified GABAergic neurons, but not glia. A series of experiments further demonstrated that the CHPG-stimulated Elk-1 phosphorylation was mediated through selective activation of mGluR5-regulated phospholipase C and associative second messenger system, i.e. 1,4,5,-triphosphate-sensitive Ca2+ release. Moreover, the Elk-1 phosphorylation was partially dependent on mGluR5-mediated co-activation of NMDA, but not kainate/AMPA receptors and L-type voltage-operated Ca2+ channels. Using an immediate early gene c-fos as a report of inducible gene expression, we found that CHPG induced marked c-fos mRNA expression. The c-fos induction kinetically corresponded to the Elk-1 phosphorylation and was attenuated by antisense oligonucleotides that selectively knocked down Elk-1 proteins. These results indicate that glutamatergic tone on mGluR5 is positively coupled to Elk-1 phosphorylation in striatal neurons via multiple signaling mechanisms involving Ca2+ release and NMDA activation, and the mGluR5-mediated Elk-1 phosphorylation facilitates gene transcription.

The metabotropic glutamate receptor subtype 5 (mGluR5) is densely expressed in striatal projection neurons. As a G protein-coupled receptor, mGluR5 may initiate an intracellular cascade that conveys extracellular signals to gene expression. This study investigated the possible role of mGluR5 in the inducible phosphorylation of a nuclear transcription factor, cAMP response element-binding protein (CREB), in primary cultures of striatal neurons from rat E19 embryos or neonatal day-1 pups. We found that selective activation of mGluR5 with a selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) induced a rapid and transient increase in phosphorylated CREB immunoreactivity in striatal neurons as analyzed by both immunocytochemistry and western blot. The increase in CREB phosphorylation was concentration-dependent, and seen in neurochemically identified GABAergic neurons. Pre-treatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) blocked the CHPG phosphorylation of CREB. In contrast, the mGluR1 antagonist, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOet) did not alter CHPG-stimulated CREB phosphorylation. The mGluR5 antisense oligonucleotides, but not their controls, selectively reduced basal mGluR5 levels as well as CREB phosphorylation in response to CHPG addition. Lastly, using an immediate early gene c-fos as a reporter of inducible gene expression downstream to phosphorylated CREB, we found that CHPG induced a rapid and transient increase in c-fos mRNA levels in cultured neurons as revealed by quantitative in situ hybridization. The increase in c-fos was kinetically correlated well with the CREB phosphorylation and blocked by MPEP and the CREB antisense oligonucleotides. These results demonstrate a positive linkage from surface mGluR5 to CREB phosphorylation, which is able to facilitate immediate gene expression in striatal neurons.

Increasing evidence supports the critical role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors in psychostimulant action. These receptors are regulated via a phosphorylation-dependent mechanism in their trafficking, distribution, and function. The hippocampus is a brain structure important for learning and memory and is emerging as a critical site for processing psychostimulant effects. To determine whether the hippocampal pool of AMPA receptors is regulated by stimulants, we investigated and characterized the impact of amphetamine (AMPH) on phosphorylation of AMPA receptors in the adult rat hippocampus in vivo. We found that AMPH markedly increased phosphorylation of AMPA receptor GluA1 subunits at serine 845 (S845) in the hippocampus. The effect of AMPH was dose dependent. A single dose of AMPH induced a rapid and transient increase in S845 phosphorylation. Among different hippocampal subfields, AMPH primarily elevated S845 phosphorylation in the Cornu Ammonis area 1 and dentate gyrus. In contrast to S845, serine 831 phosphorylation of GluA1 and serine 880 phosphorylation of GluA2 were not altered by AMPH. In addition, surface expression of hippocampal GluA1 was up-regulated, while the amount of intracellular GluA1 fraction was concurrently reduced in response to AMPH. GluA2 protein levels in either the surface or intracellular pool were insensitive to AMPH. These data demonstrate that the AMPA receptor in the hippocampus is sensitive to dopamine stimulation. Acute AMPH administration induces dose-, time-, site-, and subunit-dependent phosphorylation of AMPA receptors and facilitates surface trafficking of GluA1 AMPA receptors in hippocampal neurons in vivo. Acute injection of amphetamine increased phosphorylation of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 subunits at a protein kinase A (PKA)-sensitive site (S845) in the rat hippocampus. This increase was dose- and time-dependent and correlated with an increase in surface GluA1 expression. Thus, amphetamine can upregulate GluA1 phosphorylation and surface trafficking of GluA1 in hippocampal neurons in vivo.

Hypothalamic orexin (hypocretin) neurons project to the key structures of the limbic system and orexin receptors, both orexin receptor type 1 (OXR1) and type 2 (OXR2), are expressed in most limbic regions. Emerging evidence suggests that orexin is among important neurotransmitters that regulate addictive properties of drugs of abuse. In this study, we examined the effect of psychostimulant cocaine on orexin receptor protein abundance in the rat limbic system in vivo. Intermittent administration of cocaine (20 mg/kg, i.p., once daily for 5 days) caused a typical behavioral sensitization response to a challenge cocaine injection at a 14-day withdrawal period. Repeated cocaine administration at the same withdrawal time also increased OXR2 protein levels in the nucleus accumbens while repeated cocaine had no effect on OXR1 and orexin neuropeptide (both orexin-A and orexin-B) levels in this region. In contrast to the nucleus accumbens, OXR2 levels in the frontal cortex, the ventral tegmental area, the hippocampus, and the dorsal striatum (caudate putamen) were not altered by cocaine. Remarkably, the up-regulated OXR2 levels in the nucleus accumbens showed a long-lasting nature as it persisted up to 60 days after the discontinuation of repeated cocaine treatments. In contrast to chronic cocaine administration, an acute cocaine injection was insufficient to modify levels of any orexin receptor and peptide. Our data identify the up-regulation of OXR2 in the nucleus accumbens as an enduring molecular event that is correlated well with behavioral plasticity in response to chronic psychostimulant administration. This OXR2 up-regulation may reflect a key adaptation of limbic orexinergic transmission to chronic drug exposure and may thus be critical for the expression of motor plasticity.

Glutamate receptors regulate gene expression in neurons by activating intracellular signaling cascades that phosphorylate transcription factors within the nucleus. The mitogen-activated protein kinase (MAPK) cascade is one of the best characterized cascades in this regulatory process. The Ca(2+)-permeable ionotropic glutamate receptor, mainly the NMDA receptor subtype, activates MAPKs through a biochemical route involving the Ca(2+)-sensitive Ras-guanine nucleotide releasing factor, Ca(2+)/calmodulin-dependent protein kinase II, and phosphoinositide 3-kinase. The metabotropic glutamate receptor (mGluR), however, activates MAPKs primarily through a Ca(2+)-insensitve pathway involving the transactivation of receptor tyrosine kinases. The adaptor protein Homer also plays a role in this process. As an information superhighway between surface glutamate receptors and transcription factors in the nucleus, active MAPKs phosphorylate specific transcription factors (Elk-1 and CREB), and thereby regulate distinct programs of gene expression. The regulated gene expression contributes to the development of multiple forms of synaptic plasticity related to long-lasting changes in memory function and addictive properties of drugs of abuse. This review, by focusing on new data from recent years, discusses the signaling mechanisms by which different types of glutamate receptors activate MAPKs, features of each MAPK cascade in regulating gene expression, and the importance of glutamate/MAPK-dependent synaptic plasticity in memory and addiction.

The Galphaq protein-coupled metabotropic glutamate receptor subtype-5 (mGluR5) is densely expressed in medium spiny projection neurons of striatum. Emerging evidence suggests a significant role of mGluR5 in the addictive plasticity of striatal neurons that is likely derived from inducible cellular gene expression related to stimulation of mGluR5 and associative signaling proteins. In this study, we found that activation of mGluR5 with a selective agonist (RS)-2-chloro-5-hydroxy-phenylglycine (CHPG) induced a rapid and transient phosphorylation of a transcription regulator Elk-1 in cultured striatal neurons from rat E19 embryos or neonatal day-1 pups. The Elk-1 phosphorylation was dose-dependent and occurred in neurochemically identified GABAergic neurons, but not glia. A series of experiments further demonstrated that the CHPG-stimulated Elk-1 phosphorylation was mediated through selective activation of mGluR5-regulated phospholipase C and associative second messenger system, i.e. 1,4,5,-triphosphate-sensitive Ca2+ release. Moreover, the Elk-1 phosphorylation was partially dependent on mGluR5-mediated co-activation of NMDA, but not kainate/AMPA receptors and L-type voltage-operated Ca2+ channels. Using an immediate early gene c-fos as a report of inducible gene expression, we found that CHPG induced marked c-fos mRNA expression. The c-fos induction kinetically corresponded to the Elk-1 phosphorylation and was attenuated by antisense oligonucleotides that selectively knocked down Elk-1 proteins. These results indicate that glutamatergic tone on mGluR5 is positively coupled to Elk-1 phosphorylation in striatal neurons via multiple signaling mechanisms involving Ca2+ release and NMDA activation, and the mGluR5-mediated Elk-1 phosphorylation facilitates gene transcription.