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Protein phosphorylation is an important mechanism for the posttranslational modulation of ionotropic glutamate receptors and is subject to regulation by changing synaptic inputs. In this study, we investigated the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit phosphorylation by cocaine exposure in the rat dorsal striatum in vivo. We found that acute cocaine challenge followed by 6 days of repeated systemic injections of cocaine (20 mg/kg once daily) enhanced the sensitivity of the GluR1 subunit in its phosphorylation at serine 831 (Ser831) in the dorsal striatum. This enhancement of the sensitivity of Ser831 phosphorylation was reduced, at the receptor and ion channel level, by blocking (1) group I metabotropic glutamate receptors (mGluRs), (2) N-methyl-D-aspartate receptors, and (3) L-type voltage-operated Ca(2+) channels. Similar reduction of the enhancement was also induced, at the protein kinase level, by inhibiting (1) protein kinase C, (2) calcium/calmodulin-dependent protein kinases, and (3) c-Jun N-terminal kinases. In addition, inhibition of protein phosphatase 1/2A or calcineurin increased GluR1-Ser831 phosphorylation in the dorsal striatum of normal rats, whereas inhibition of these phosphatases did not further enhance the Ser831 phosphorylation in rats pretreated with 7 daily injections of cocaine. These data suggest that the phosphorylation of AMPA receptor GluR1 subunits at Ser831 is subject to upregulation by acute and repeated cocaine administration. Complex signaling integrations among glutamate receptors, Ca(2+) channels, protein kinases, and protein phosphatases participate in this upregulation.

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. They are highly expressed in the striatum, where medium spiny neurons (MSNs) are a major population. Given that the properties of ASICs in MSNs are unknown, in this study, we characterized ASICs in MSNs of the mouse striatum. A rapid drop in extracellular pH induced transient inward currents in all MSNs. The pH value for half-maximal activation was 6.25, close to that obtained in homomeric ASIC1a channels. Based on psalmotoxin 1 and zinc sensitivity, ASIC1a (70.5% of neurons) and heteromeric ASIC1a-2 channels (29.5% of neurons) appeared responsible for the acid-induced currents in MSNs. ASIC currents were diminished in MSNs from ASIC1, but not ASIC2, null mice. Furthermore, a drop in pH induced calcium influx by activating homomeric ASIC1a channels. Activation of ASICs increased the membrane excitability of MSNs and lowering extracellular Ca2+ potentiated ASIC currents. Our data suggest that the homomeric ASIC1a channel represents a majority of the ASIC isoform in MSNs. The potential function of ASICs in the striatum requires further investigation.

The interplay between dopamine and glutamate in the basal ganglia regulates critical aspects of motor learning and behavior. Metabotropic glutamate receptors (mGluR) are increasingly regarded as key modulators of neuroadaptation in these circuits, in normal and disease conditions. Using PET, we demonstrate a significant upregulation of mGluR type 5 in the striatum of MPTP-lesioned, parkinsonian primates, providing the basis for therapeutic exploration of mGluR5 antagonists in Parkinson disease.

Cocaine administration upregulates the levels of extracellular glutamate and dopamine in the striatum. Activation of the receptors alters calcium homeostasis in striatal neurons leading to the expression of the endoplasmic reticulum (ER) stress proteins. It was therefore hypothesized that cocaine upregulates the expression of the ER stress proteins, immunoglobulin heavy chain binding protein (BiP), Ire1alpha and perk via glutamate and dopamine receptor activation. A novel glutamate microbiosensor and Western immunoblot analyses were mainly performed to test the hypothesis in the rat dorsal striatum. The results showed that i.p. injection of repeated cocaine (20 mg/kg) for nine consecutive days significantly increased extracellular glutamate levels while acute cocaine injection did not. However, the immunoreactivities (IR) of the ER stress proteins in the dorsal striatum were significantly increased by either acute or repeated cocaine injections as compared with saline controls. Intrastriatal injection (i.s.) of the selective group I metabotropic glutamate receptor (mGluR) antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) or the mGluR5 subtype antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 2 and 25 nmol) significantly decreased repeated cocaine-induced increases in the IR of the ER stress proteins in the injected dorsal striatum. Similarly, the selective D1 antagonist (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390; 0.1 mg/kg, i.p.) or the N-methyl-d-aspartate antagonist dizocilpine/(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-ibenzo[a,d]cyclohepten-5,10-imine maleate (MK801; 2 nmol, i.s.) decreased acute or repeated cocaine-induced the IR of the ER stress proteins in the dorsal striatum. These data suggest that cocaine upregulates expression of the ER stress proteins in striatal neurons via a mechanism involving activation of glutamate and dopamine receptors.

The striatum is a key structure of basal ganglia controlling extrapyramidal motor activity and processing addictive plasticity of abused substances. Glutamatergic transmission that is enriched in the striatum regulates a variety of striatal neuronal activities via selective activation of ionotropic and metabotropic glutamate receptors (mGluRs). In this study, the interaction between N-methyl-D-aspartate (NMDA) receptors and group I mGluRs (mGluR1 and mGluR5 subtypes) in activating a phosphorylation cascade to a transcription factor cAMP response element-binding protein (CREB) was investigated in primary cultures of E18 or postnatal day 1 striatal neurons. We found that activation of NMDA receptors with NMDA rapidly and concentration-dependently increased the number of neurons expressing phosphorylated CREB (pCREB) as revealed by immunocytochemistry. The increased pCREB expression by NMDA was sensitive to an NMDA antagonist MK801. Co-incubation of a subthreshold dose of a group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) that itself did not alter basal pCREB expression augmented NMDA-induced CREB phosphorylation. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride blocked the DHPG augmentation of NMDA-induced CREB phosphorylation, while the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester did not. Interestingly, the protein kinase C inhibitors chelerythrine and Gö6983 also prevented DHPG from enhancing CREB phosphorylation induced by NMDA. Whereas a low dose of the protein kinase C activator phorbol 12-myristate 13-acetate mimicked the DHPG potentiation. These results indicate a facilitatory regulation of an NMDA cascade to CREB phosphorylation by concurrent glutamatergic tone on mGluR5, which is probably processed via an intracellular signaling pathway involving protein kinase C.

The group I metabotropic glutamate receptors (mGluRs) are positively coupled to phospholipase C. Through phospholipase C, group I mGluR activation increases intracellular concentrations of diacylglycerol which is known as a strong activator of protein kinase C (PKC). This study investigated the putative role of PKC in the regulation of transcription factor phosphorylation induced by group I mGluR activation in the rat striatum in vivo. We found that the group I agonist 3,5-dihydroxyphenylglycine (DHPG) injected into the dorsal striatum (caudate-putamen) increased phosphorylation of the two transcription factors, cAMP response element-binding protein (CREB) and Elk-1, and extracellular signal-regulated kinase 1/2 (ERK1/2) in the injected striatum. Inhibition of PKC with GF109203X significantly attenuated DHPG-stimulated CREB, Elk-1, and ERK1/2 phosphorylation. Activation of PKC with intracaudate injection of 12-O-tetradecanoylphorbol-13-acetate (TPA) mimicked DHPG actions in facilitating the phosphorylation of CREB, Elk-1, and ERK1/2. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors with the non-competitive antagonist MK801 or the competitive antagonist AP5 attenuated TPA-induced CREB, Elk-1, and ERK1/2 phosphorylation. Similarly, inhibition of Ca(2+)/calmodulin-dependent protein kinases (CaMK) with KN62 also resulted in a significant attenuation of TPA induction of the three phosphoproteins. The data obtained from this study indicate that selective activation of PKC is needed for the group I agonist-induced CREB, Elk-1, and ERK1/2 phosphorylation in striatal neurons. Activated PKC may, at least in part, facilitate the phosphorylation of transcription factors via an NMDA/CaMK-sensitive pathway., (Copyright 2002 IBRO)

In vivo activation of group I metabotropic glutamate receptors (mGluRs) upregulates phosphorylation of cyclic AMP response element-binding protein (CREB), Elk-1 and extracellular signal-regulated kinases (ERK) in striatal neurons. To evaluate putative roles of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in CREB, Elk-1 and ERK phosphorylation, the CaMKII inhibitor, KN62, was infused simultaneously with the group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG), into the rat dorsal striatum. The results showed that DHPG (125, 250, and 500 nmol) increased phosphorylated (p) CaMKII immunoreactivity (IR) in a dose-dependent manner. KN62 (50 nmol) significantly attenuated 500 nmol DHPG-induced pERK, pElk-1 and pCREB IR in the ipsilateral dorsal striatum. These data indicate that pCaMKII is a possible upstream effector that is responsible for the regulation of CREB, Elk-1 and ERK phosphoproteins in response to group I mGluR stimulation in striatal neurons.

Cyclic AMP response element-binding protein (CREB) is a major transcriptional activator at the calcium and cAMP response-element (CaCRE). Phosphorylated (p)CREB facilitates gene expression in striatal neurons. Elk-1 is another transcriptional regulator at the serum response element in the upstream promoter region of the CaCRE. Elk-1 is phosphorylated by extracellular signal-regulated kinases (ERK) and may also contribute to the regulation of gene expression. To evaluate putative roles of group I metabotropic glutamate receptors (mGluRs) in CREB, Elk-1, and ERK phosphorylation, the group I selective agonist, 3,5-dihydroxyphenylglycine (DHPG), was infused into the dorsal striatum at doses of 125, 250, or 500 nmol in freely moving rats. Semi-quantitative immunohistochemistry demonstrated that DHPG significantly increased levels of pCREB, pElk-1, and pERK immunoreactivity of ipsilateral dorsal striatum in a dose dependent manner. The increased immunoreactivity by 500 nmol DHPG was significantly blocked by intrastriatal infusion of the group I selective antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC, 25 nmol), but not by the group II/III antagonist, (RS)-alpha-methylserine-o-phosphate monophenyl ester (MSOPPE, 25 nmol). These data suggest that group I mGluR activation is positively linked to signaling cascades resulting in CREB, Elk-1, and ERK phosphorylation in the striatum in vivo.

Neuronal nitric oxide synthase (nNOS) is normally expressed in one population of intrinsic interneurons of the striatum. Production of nitric oxide in the nNOS-containing neurons is sensitive to dopamine stimulation. Using quantitative in situ hybridization, the present study investigated the alteration in basal nNOS mRNA expression in striatal nNOS-containing neurons of mice treated with the psychostimulant amphetamine or a full D1 dopamine receptor agonist, SKF-82958. A single systemic injection of amphetamine induced a dose-related change in striatal nNOS mRNA expression. Whereas amphetamine at 4 mg/kg decreased basal levels of nNOS mRNA in both the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum, the drug at a higher dose (12 mg/kg) increased nNOS expression in the two regions. Similarly, an acute systemic injection of SKF-82958 decreased and increased nNOS mRNA levels in the dorsal and ventral striatum at 2 and 4 mg/kg, respectively. These data indicate that constitutive nNOS expression in nitric oxide-producing neurons of the mouse striatum is regulated by dopaminergic transmission. Altered nNOS expression may result in changes in nitric oxide synthesis and thus contribute to biological actions of dopamine stimulants.

The purpose of this study was to investigate the effects of intrastriatal blockade of GABA(A) receptors on dopamine D(1)/D(2) receptor interactions in the intact rat striatum. Muscarinic receptors mediate the ability of the D(2) receptor antagonist, eticlopride, to block an increase in striatonigral neuropeptide messenger RNA stimulated by the full D(1) agonist, SKF-82958. However, because D(2) receptor antagonists activate striatopallidal neurons, it is possible that increased GABA release from local medium spiny axon collaterals also contributes to the ability of eticlopride to block the effects of SKF-82958. This hypothesis was addressed by infusing the GABA(A) receptor antagonist, bicuculline, into the dorsal striatum in rats treated with eticlopride and SKF-82958. In contrast to the actions of the muscarinic antagonist, scopolamine, bicuculline did not affect the increase in behaviors induced by SKF-82958 or the ability of eticlopride to block them. Quantitative in situ hybridization demonstrated that bicuculline did not significantly affect basal preprodynorphin messenger RNA, nor did it affect the ability of eticlopride to decrease SKF-82958-induced preprodynorphin messenger RNA. However, the level of the preprodynorphin hybridization signal in bicuculline plus SKF-82958-treated rats was significantly lower than in saline plus SKF-82958-treated rats. In contrast, bicuculline, eticlopride or SKF-82958 by themselves increased basal preproenkephalin messenger RNA. However, there was no significant interaction among bicuculline, eticlopride and SKF-82958 on preproenkephalin messenger RNA levels.These data indicate that blockade of striatal GABA(A) receptors has only a subtle effect on acute dopamine agonist-induced changes in gene expression. These results are discussed in the context of local intrastriatal interactions.

Metabotropic glutamate receptor 1 (mGluR1) is a G-protein-coupled receptor and is expressed in the medium spiny projection neurons of mouse striatum. To define the role of mGluR1 in actions of psychostimulant, we compared both motor behavior and striatal neuropeptide mRNA expression between mGluR1 mutant and wild-type control mice after a single injection of amphetamine. We found that acute amphetamine injection increased motor activity in both mutant and control mice in a dose-dependent manner (1, 4, and 12 mg/kg, i.p.). However, the overall motor responses of mGluR1 -/- mice to all three doses of amphetamine were significantly greater than those of wild-type +/+ mice. Amphetamine also induced a dose-dependent elevation of preprodynorphin mRNA in the dorsal and ventral striatum of mutant and wild-type mice as revealed by quantitative in situ hybridization. In contrast to behavioral responses, the induction of dynorphin mRNA in both the dorsal and ventral striatum of mutant mice was significantly less than that of wild-type mice in response to the two higher doses of amphetamine. In addition, amphetamine elevated basal levels of substance P mRNA in the dorsal and ventral striatum of mGluR1 mutant mice to a similar level as that of wild-type mice. There were no differences in basal levels and distribution patterns of the two mRNAs between the two genotypes of mice treated with saline. These results demonstrate a clear augmented behavioral response of mGluR1 knockout mice to acute amphetamine exposure that is closely correlated with reduced dynorphin mRNA induction in the same mice. It appears that an intact mGluR1 is specifically critical for full dynorphin induction, and impaired mobilization of inhibitory dynorphin system as a result of lacking mGluR1 may contribute to an augmentation of motor stimulation in response to acute administration of psychostimulant.

Group III metabotropic glutamate (mGlu) receptors are negatively coupled to adenylate cyclase and are distributed pre-synaptically in the striatum. A behavioral study previously conducted in this laboratory shows that activation of this group of mGlu receptors attenuates acute amphetamine-stimulated motor activity. By administering a group III selective agonist or antagonist via the dialysis probe, the present study employed in vivo microdialysis to evaluate the capacity of the group III selective agents to alter extracellular levels of dopamine in the dorsal striatum of normal and amphetamine-treated rats. It was found that the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4) dose-dependently (1, 10 and 100 microM) reduced basal levels of extracellular dopamine. In contrast, the group III antagonist alpha-methyl-4-phosphonophenylglycine (MPPG) dose-dependently (10, 50 and 250 microM) elevated the basal release of extracellular dopamine. This elevation was antagonized by co-perfusion of L-AP4. Perfusion of 5-microM amphetamine through the dialysis probe increased extracellular dopamine in the dorsal striatum. Co-perfusion of L-AP4 (100 microM) significantly reduced amphetamine-stimulated dopamine levels, whereas co-perfusion of L-AP4 (100 microM) and MPPG (100 microM) did not alter the capacity of amphetamine to elicit dopamine release. The data obtained from this study demonstrate the presence of a tonically active glutamatergic tone on group III mGlu receptors in the dorsal striatum to pre-synaptically regulate basal dopamine release in an inhibitory fashion. Moreover, activation of L-AP4-sensitive group III mGlu receptors can suppress the phasic release of dopamine induced by a dopamine stimulant amphetamine.

Group III metabotropic glutamate receptors (mGluRs) are negatively coupled to adenylate cyclase through G-proteins. Activation of this group of mGluRs shows an inhibition of dopaminergic transmission in the forebrain. To define the role of striatal group III mGluRs in the regulation of basal and dopamine-stimulated motor behavior, the recently developed agonist and antagonist relatively selective for group III mGluRs were utilized to pharmacologically enhance and reduce group III mGluR glutamatergic tone in the dorsal striatum of chronically cannulated rats. Bilateral injections of a group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), did not alter basal levels of motor activity at three doses surveyed (1, 10, and 100 nmol). Neither did intracaudate injection of a group III antagonist, alpha-methyl-4-phosphonophenylglycine (MPPG), at 10, 30, and 100 nmol. However, pretreatment with L-AP4 (10 and 100 nmol) dose dependently blocked hyperlocomotion induced by acute injection of cocaine (20 mg/kg, i.p.), amphetamine (2.5 mg/kg, i.p.), or apomorphine (1 mg/kg, s.c.). The behavioral activity induced by cocaine was much more sensitive to L-AP4 than that induced by amphetamine and apomorphine. At 100 nmol, L-AP4 completely blocked cocaine effect whereas amphetamine- and apomorphine-stimulated behaviors were blocked only by 28% and 31%, respectively. The blocking effect of L-AP4 on cocaine action was reversed by pretreatment with MPPG. MPPG itself did not modify behavioral responses to cocaine, amphetamine, or apomorphine. These data indicate that the glutamatergic tone on the group III mGluRs is not active in the regulation of basal and acute dopamine-stimulated motor activity. However, enhanced group III mGluR glutamatergic transmission by an exogenous ligand is capable of suppressing behavioral responses to acute exposure of dopamine stimulants.

Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium-sized spiny projection neurons of striatum. Activation of this group of the mGluRs modifies neuronal physiology through stimulation of phosphoinositide hydrolysis and intracellular Ca(2)+ release. Unlike the ionotropic glutamate receptors that mediate rapid synaptic transmission, activation of the mGluRs produces long-lasting actions brought about by modulation of activities of intracellular effectors. In this study, the role of the group I mGluRs in the modulation of extrapyramidal motor function was examined using a group I selective agonist, 3, 5-dihydroxyphenylglycine (DHPG), in chronically cannulated rats. Bilateral injections of DHPG at a series of subtoxic doses (20, 40, 80, and 160 nmol) into the central part of the dorsal striatum produced hyperlocomotion and a unique stereotypical behavior (spontaneous and repetitive twitching movement of the head and forepaws) in a dose-dependent manner. The characteristic twitchy behavior usually commenced 30 min to 1 h, and could last as long as 10 to 12 h, after a single injection of the group I agonist. The behavioral responses to DHPG administration were markedly antagonized by intrastriatal injection of the group I antagonist PHCCC (10 nmol), but not the group II/III antagonist MSOPPE (10 nmol). Intrastriatal administration of 20 nmol dantrolene, an inhibitor of intracellular Ca(2)+ mobilization, also prevented DHPG-stimulated motor activities. However, blockade of dopamine D(1) receptors with systemic administration of SCH-23390 (0.1 mg/kg, SC) did not alter the ability of DHPG to provoke behavioral activities. These data indicate that selective activation of the DHPG-sensitive group I mGluRs in the striatum can produce long-lasting stimulation of motor activity. DHPG-induced motor stimulation involves the mobilization of intracellular Ca(2)+ stores, but appears to be independent of D(1) dopaminergic transmission.

Group II metabotropic glutamate receptors (mGluRs) are distributed both pre- and postsynaptically in the striatum. By bilaterally administering a subgroup-selective agonist or antagonist into the dorsal striatum of chronically cannulated rats, this study examined the role of striatal group II mGluRs in the regulation of basal and dopamine-stimulated motor behavior. Intrastriatal injection of a group II agonist, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, 0.01, 0.1 and 1 nmol), dose-dependently reduced basal levels of motor activity. Pretreatment of rats with intrastriatal DCG-IV at a higher dose (1 nmol), but not a lower dose (0.01 nmol), produced complete or partial blockade of hyperlocomotion induced by acute injection of amphetamine (2.5 mg/kg, i.p.) or apomorphine (1 mg/kg, s.c.), respectively. Blockade of group II mGluRs by intrastriatal injection of a group II antagonist, (RS)-alpha-methylserine-O-phosphate monophenyl ester (10 nmol), was found to: (i) induce a moderate locomotion by itself; (ii) augment amphetamine-stimulated behaviors and (iii) attenuate DCG-IV-induced reduction of basal and amphetamine-stimulated motor activity. These data demonstrate that the group II mGluRs in the striatum play a significant role in the inhibitory modulation of tonic and phasic motor activity, which is most likely processed through both pre- and postsynaptic mechanisms.

Metabotropic glutamate receptors (mGluR) are densely expressed by striatal medium spiny neurons. Activation of mGluR in this brain region alters local transmitter release and behaviors of experimental animals. In particular, mGluR regulate transcription factor and neuropeptide gene expression in striatal neurons through their connections with multiple intracellular effectors. This prominent involvement of mGluR in overall cellular activity is pivotal for the development of neuronal plasticity underlying long-term adaptive changes in cellular physiology related to a variety of neurologic disorders. Accumulating evidence demonstrates that the subtypes of mGluR have distinct effects on gene expression: group I subtypes facilitating, and group II/III subtypes inhibiting, gene expression. Thus, the mGluR can be considered as promising targets in the development of novel therapeutic drugs that can relieve neurologic disorders resulting from dysfunction of the striatum.

Metabotropic glutamate receptors are coupled to multiple intracellular second messenger systems through G-proteins and densely expressed by medium spiny projection neurons in the rat striatum. In chronically-cannulated rats, this study demonstrated that pharmacological activation of metabotropic glutamate receptors by intrastriatal injection of a selective agonist, ACPD, elevated immediate early gene c-fos and zif/268 mRNA expression in the injected dorsal striatum as revealed by quantitative in situ hybridization. The elevation of both c-fos and zif/268 was dose-dependent and the responsiveness of c-fos to ACPD at each dose surveyed was greater than that of zif/268. Induction of the two mRNAs was rapid and transient as increases in the 2 mRNAs became evident as early as 30 min, reached a peak at 1 h, and returned to normal levels 3 (c-fos) or 6 (zif/268) h, after ACPD injection. Coadministration of the selective metabotropic glutamate receptor antagonist, MCPG, with ACPD markedly attenuated ACPD-stimulated c-fos, but not zif/268, expression. Pretreatment with the ionotropic NMDA receptor antagonist, CPP, had no effect on ACPD-stimulated c-fos expression, but partially attenuated ACPD-stimulated zif/268 expression. Blockade of D1 dopamine receptors with SCH-23390 did not alter the ability of ACPD to induce the expression of these genes. These data demonstrate a difference between the profound induction of c-fos and zif/268 gene expression in response to specific activation of metabotropic glutamate receptors in striatal neurons. Furthermore, c-fos induction was independent of D1 dopaminergic and NMDA glutamatergic transmission, whereas zif/268 induction was mediated, at least in part, by NMDA receptors., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

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., (Copyright 1998 Elsevier Science B.V.)

The selective M4 muscarinic receptor toxin, MT3, was used in vivo to evaluate the role of M4 receptors in cholinergic inhibition of neuropeptide mRNA expression in striatonigral neurons. Unilateral injection of the muscarinic toxin 3 (0.04-4 nmol) into the dorsal striatum of chronically-cannulated rats elevated basal levels of preprodynorphin, substance P and preproenkephalin mRNAs in the ipsilateral dorsal striatum as revealed by quantitative in situ hybridization. Pretreatment with muscarinic toxin 3 also augmented amphetamine (2.5 mg/kg, i.p.)-stimulated preprodynorphin and substance P expression in the dorsal striatum in a manner similar to that observed after the muscarinic antagonist, scopolamine. Since muscarinic toxin 3 has a much greater affinity for muscarinic M4 receptors than for other subtypes, it is possible that muscarinic toxin 3, by interacting with the muscarinic M4 subtype, regulates basal and/or dopamine-stimulated striatal neuropeptide gene expression.

Systemic administration of the muscarinic receptor antagonist, scopolamine, augments, whereas the muscarinic receptor agonist, oxotremorine, attenuates behaviors (locomotion and stereotypies) and preprodynorphin (PPD) and substance P (SP) gene expression in striatonigral neurons induced by the indirect dopamine receptor agonist, amphetamine (AMPH). In contrast, systemic scopolamine blocks, whereas oxotremorine augments, AMPH-stimulated preproenkephalin (PPE) gene expression in striatopallidal neurons. This study investigated the site of action of these effects by administering scopolamine and oxotremorine directly into the striatum and assessing the expression of neuropeptide mRNAs with quantitative in situ hybridization. Unilateral injection of scopolamine into the dorsal striatum augmented, and oxotremorine attenuated, AMPH (2.5 mg/kg, i.p.)-stimulated behaviors. Intrastriatal scopolamine at a concentration of 62 mM, but not 6.2 mM, increased basal levels of PPD and SP mRNAs in the dorsal striatum. In addition, both 6.2 and 62 mM scopolamine significantly augmented AMPH-stimulated PPD and SP mRNA levels. Intrastriatal infusion of 1.6 or 8.1 mM oxotremorine did not alter basal levels of striatal PPD and SP mRNAs. However, both concentrations of oxotremorine completely blocked AMPH-stimulated SP mRNA and oxotremorine at 8.1 mM blocked AMPH-stimulated PPD mRNA. In contrast, PPE induction by AMPH was blocked by 62, but not 6.2, mM scopolamine. Both concentrations of oxotremorine tended to augment basal and AMPH-stimulated PPE mRNA in the dorsal striatum but the trend was not significant. These data demonstrate an inhibition of striatonigral, and facilitation of striatopallidal, gene expression through activation of local striatal muscarinic receptors, which is consistent with the changes seen after systemic administration of muscarinic agents. Therefore, muscarinic cholinergic regulation of basal and stimulated expression of neuropeptide mRNA is processed within the striatum.

This study investigated the effects of pharmacological blockade or stimulation of muscarinic receptors on constitutive and amphetamine-stimulated preprodynorphin, substance P and pre-proenkephalin gene expression in rat striatum. Acute administration of the non-selective muscarinic antagonist, scopolamine (2.5, 5 and 10 mg/kg, s.c.), caused a dose-dependent increase in preprodynorphin and substance P, but not preproenkephalin, messenger RNA expression in the dorsal and ventral striatum as revealed by quantitative in situ hybridization. In contrast, acute injection of the non-selective muscarinic receptor agonist, oxotremorine (0.125, 0.25 and 0.5 mg/kg, s.c.), caused a dose-dependent increase in basal levels of preproenkephalin messenger RNA in the dorsal striatum, without causing a significant effect on constitutive striatal preprodynorphin and substance P expression. Pretreatment with scopolamine (2.5 mg/kg, s.c.) significantly augmented striatal induction of preprodynorphin and substance P messenger RNA induced by acute injection of amphetamine (1.25 and 2.5 mg/kg, i.p.), whereas scopolamine blocked amphetamine-stimulated striatal preproenkephalin expression. Pretreatment with oxotremorine (0.25 mg/kg, s.c.) significantly attenuated amphetamine (1.25 and 2.5 mg/kg, i.p.)-stimulated striatal preprodynorphin and, to a lesser degree, substance P messenger RNA expression. Oxotremorine tended to increase amphetamine-stimulated preproenkephalin messenger RNA expression, but the effect did not reach statistical significance. In addition, scopolamine increased spontaneous, and enhanced amphetamine-stimulated, behavioral activity, whereas oxotremorine attenuated amphetamine-stimulated behaviors. These data support the concept that cholinergic transmission, via interaction with muscarinic receptors, inhibits basal and D1 receptor-stimulated striatonigral dynorphin/substance P gene expression and facilitates striatopallidal enkephalin gene expression.

In chronically cannulated rats, microinjection of a competitive metabotropic glutamate receptor (mGluR) antagonist, (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), into the dorsal striatum at doses of 0.4, 2 and 10 micrograms/1 microliter did not affect basal levels of preprodynorphin, substance P and preproenkephalin mRNAs in the dorsal striatum as revealed by quantitative in situ hybridization. However, intrastriatal MCPG (0.08, 0.4 and 2 micrograms/1 microliter) dose-dependently attenuated increases in the three mRNA expression induced by acute amphetamine injection (2 mg/kg, i.p.). MCPG had no significant effect on spontaneous, and amphetamine-stimulated, behavioral activities. These data indicate that activation of MCPG-sensitive mGluRs is necessary for upregulation of striatal neuropeptide mRNA expression in response to amphetamine exposure. However, the mGluR activity is not implicated in maintaining basal levels of the peptide gene expression in the striatum.

It is generally accepted that the widely used, partial dopamine D(1) receptor agonist, SKF-38393, does not induce immediate early gene expression in striatal projection neurons unless D(1) receptors are sensitized and uncoupled from D(2) receptors by 6-hydroxydopamine lesions or reserpine treatment. In contrast, this study demonstrates, using quantitative in situ hybridization, that the full D(1) receptor agonist, SKF-82958, induced robust expression of c-fos and zif/268 messenger RNAs in the intact rat striatum, especially in the entire shell and medial and ventral core areas of the nucleus accumbens and olfactory tubercle, and in the cerebral cortex, 45 min after one injection. The induction of the striatal immediate early genes is characterized by (i) induction in only medium-sized spiny neurons, (ii) dose-dependent induction, which correlates well with dose-dependent increases in motor activity, and (iii) blockade by the D(1) receptor antagonist, SCH-23390. The muscarinic cholinergic receptor antagonist, scopolamine, which itself did not alter striatal gene expression, profoundly augmented the behaviors and expression of the two immediate early genes in the ventral and dorsal striatum induced by 0.1, 0.5 and 2.0 mg/kg SKF-82958. However, scopolamine attenuated basal, and SKF-82958-stimulated, expression of c-fos and zif/268 messenger RNAs in the cortex. Scopolamine also enabled SKF-38393 to induce locomotor stimulation and c-fos and zif/268 messenger RNA expression in the normosensitive striatum of the rat when SKF-38393 alone caused no such changes. These data demonstrate an ability of SKF-82958 to induce immediate early gene messenger RNA expression in normosensitive dorsal and ventral striatum. Furthermore, intrinsic muscarinic receptor-mediated cholinergic transmission in the striatum may provide an activity-dependent inhibitory control on striatal D(1) receptor stimulation.

Our previous work has demonstrated a dose-dependent induction of striatal preprodynorphin (PPD) in response to a single injection of the psychostimulants amphetamine (AMPH) or methamphetamine (METH). In the present study, dose-response effects of acute administration of these stimulants on preproenkephalin (PPE) mRNA expression in the rat striatum were investigated with quantitative in situ hybridization histochemistry 3 h after injection. Acute AMPH or METH at equimolar doses (3.75, 7.5, 15, and 30 mumol/kg) significantly increased PPE mRNA expression in dorsal (caudoputamen), but not ventral (nucleus accumbens), striatum in a dose-dependent fashion. In addition, the role of D1 and D2 dopamine receptors in mediating AMPH- and METH-stimulated PPE and PPD expression was also evaluated by using subtype-specific antagonists. Pretreatment of rats with SCH 23390 (0.1 mg/kg, i.p.), a selective D1 receptor antagonist, completely blocked acute AMPH (21 mumol/kg, i.p.)- or METH (21 mumol/kg, i.p.)-induced PPE as well as PPD mRNA expression in the caudoputamen. Pretreatment with eticlopride (0.5 mg/kg, i.p.), a selective D2 receptor antagonist, also blocked PPD induction by the two stimulants, but PPE induction was unaffected. Furthermore, SCH 23390 decreased, and eticlopride elevated, constitutive PPE mRNA levels in the caudoputamen. Neither antagonist had a significant effect on the basal level of PPE or PPD mRNA in the nucleus accumbens. These results demonstrate a clear dose-related responsiveness of PPE gene expression in striatal neurons in response to acute administration of amphetamines, although the intensity of the response is far less than that for striatal PPD. Furthermore, both D1 and D2 subtypes of dopamine receptors mediate AMPH- and METH-stimulated striatal PPD mRNA expression, whereas D1 receptor activation alone mediates amphetamine-stimulated PPE mRNA expression in the rat striatum.

This study tested the role of N-methyl-D-aspartate and kainate/AMPA receptors in mediating mRNA expression of the immediate early gene zif/268 and the opioid peptide genes preprodynorphin and preproenkephalin in rat forebrain following a single injection of methamphetamine. At 3 h after acute methamphetamine [4 mg/kg, intraperitoneally (IP)], quantitative in situ hybridization histochemistry revealed that zif/268 mRNA expression was increased in the dorsal striatum (caudoputamen) and in the sensory cortex. Preprodynorphin was increased in both dorsal and ventral striatum (nucleus accumbens) and preproenkephalin was increased in the dorsal striatum. Pretreatment with (+ or -)-3-(2-carboxypiperazin-4-yl)-propyl-1 -phosphonic acid (CPP) (10 mg/kg, IP), an N-methyl-D-aspartate receptor antagonist, blocked the methamphetamine-induced zif/268 mRNA expression in the striatum and in the region of sensory cortex representing the upper limb and nose. 6,7-Dinitro-quinoxaline-2,3-dione (DNQX) (100 mg/kg, IP), a kainate/AMPA receptor antagonist, did not reduce the ability of methamphetamine to induce zif/268 mRNA in striatal and cortical neurons. Furthermore, both antagonists caused a parallel blockade of methamphetamine-stimulated preprodynorphin mRNA expression in the dorsal and ventral striatum but did not significantly affect methamphetamine-stimulated preproenkephalin mRNA expression. CPP and DNQX reduced basal levels of zif/268 mRNA in cortical and striatal neurons but did not affect the constitutive expression of the two opioid mRNAs in the striatum. Neither antagonist had a significant effect on methamphetamine-induced hyperlocomotion and stereotypies. These results demonstrate that both N-methyl-D-aspartate and kainate/AMPA receptor-mediated glutamatergic transmission is linked to modulation of the methamphetamine-stimulated oploid peptide gene expression in rat forebrain. Furthermore, N-methyl-D-aspartate receptors participate in methamphetamine-stimulated zif/268 expression.

Semi-quantitative immunocytochemistry was used to investigate the levels of cyclase response element-binding protein, phosphorylated cyclase response element-binding protein, Fos and Fos-related antigen immunoreactivity in the striatum of rats after acute or repeated amphetamine administration. Rats were perfused 20 min (phosphorylated cyclase response element-binding protein) or 2 h (cyclase response element-binding protein, phosphorylated cyclase response element-binding protein, Fos, Fos-related antigen) after a single injection (5 mg/kg, i.p.) or five daily injections of amphetamine. The latency to onset of stereotypical behaviors was significantly reduced in rats exposed to repeated amphetamine as compared to acute amphetamine, indicating development of behavioral sensitization. Cyclase response element-binding protein immunoreactivity was not altered in the dorsal or ventral striatum following acute or repeated amphetamine. Phosphorylated cyclase response element-binding protein immunoreactivity was significantly induced 20 min, but not 2 h, following acute amphetamine, whereas a significant induction of phosphorylated cyclase response element-binding protein immunoreactivity was found 20 min and 2 h after repeated amphetamine in the dorsal striatum only. Fos immunoreactivity was significantly induced in the dorsal striatum following acute and repeated amphetamine. Fos immunoreactivity in the core of the nucleus accumbens was significantly increased following repeated amphetamine only. Acute amphetamine induced, and repeated amphetamine further augmented, Fos-related antigen immunoreactivity in the dorsal striatum, while not affecting Fos-related antigen immunoreactivity in the nucleus accumbens. These data demonstrate that repeated amphetamine administration results in a prolonged induction of phosphorylated cyclase response element-binding protein and Fos-related antigen immunoreactivity in the dorsal striatum, indicating that alterations in striatal gene expression associated with the development of behavioral sensitization may be mediated, in part, by these transcription factors.

The role of kainate/AMPA excitatory amino acid receptors in D-amphetamine (AMPH)-induced behavioral changes and the induction of immediate early gene and preprodynorphin (PPD) mRNA in various regions of rat forebrain was investigated with quantitative in situ hybridization histochemistry. Three hours after a single injection of AMPH (5 mg/kg, i.p.), PPD mRNA and mRNA of the transcription factor zif/268, but not c-fos, was increased in dorsal striatum (caudate). Zif/268 mRNA was also increased in the sensorimotor cortex. Pretreatment of rats with DNQX, a kainate/AMPA receptor antagonist, did not affect the behaviors elicited by AMPH. However, the AMPH-stimulated increase in PPD and zif/268 mRNA levels in striatum, but not zif/268 mRNA in cortex, was blocked by DNQX pretreatment. In contrast, DNQX alone attenuated basal (constitutive) levels of zif/268 mRNA expression in sensorimotor cortical, but not in striatal, neurons. These studies indicate that kainate/AMPA receptors mediate the induction of zif/268 and PPD mRNA expression in the caudate nucleus induced by a single injection of AMPH. The fact that DNQX blocked genomic, but not behavioral, responses to acute AMPH suggests that kainate/AMPA receptor mechanisms may be involved in the long-term (possibly sensitizing) effects, rather than the acute effects, of the drug. In addition, tonic kainate/AMPA receptor stimulation may play a key role in maintaining constitutive expression of the zif/268 gene in cortical neurons.