Inhibition of STAT-mediated cytokine responses to chemically-induced colitis prevents inflammation-associated neurobehavioral impairments.

Depression can be associated with chronic systemic inflammation, and production of peripheral proinflammatory cytokines and upregulation of the kynurenine pathway have been implicated in pathogenesis of depression. However, the mechanistic bases for these comorbidities are not yet well understood. As tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which convert tryptophan to kynurenine, are rate-limiting enzymes of the kynurenine pathway, we screened TDO or IDO inhibitors for effects on the production of proinflammatory cytokines in a mouse macrophage cell line. The TDO inhibitor 680C91 attenuated LPS-induced pro-inflammatory cytokines including IL-1ß and IL-6. Surprisingly, this effect was TDO-independent, as it occurred even in peritoneal macrophages from TDO knockout mice. Instead, the anti-inflammatory effects of 680C91 were mediated through the suppression of signal transducer and activator of transcription(STAT) signaling. Furthermore, 680C91 suppressed production of proinflammatory cytokines and STAT signaling in an animal model of inflammatory bowel disease. Specifically, 680C91 effectively attenuated acute phase colon cytokine responses in male mice subjected to dextran sulfate sodium (DSS)-induced colitis. Interestingly, this treatment also prevented the development of anxiodepressive-like neurobehaviors in DSS-treated mice during the recovery phase. The ability of 680C91 to prevent anxiodepressive-like behavior in response to chemically-induced colitis appeared to be due to rescue of attenuated dopamine responses in the nucleus accumbens. Thus, inhibition of STAT-mediated, but TDO-independent proinflammatory cytokines in macrophages can prevent inflammation-associated anxiety and depression. Identification of molecular mechanisms involved may facilitate the development of new treatments for gastrointestinal-neuropsychiatric comorbidity.

DARPP-32/protein phosphatase 1 regulates Rasgrp2 as a novel component of dopamine D1 receptor signaling in striatum.

Dopamine regulates psychomotor function by D1 receptor/PKA-dependent phosphorylation of DARPP-32. DARPP-32, phosphorylated at Thr34 by PKA, inhibits protein phosphatase 1 (PP1), and amplifies the phosphorylation of other PKA/PP1 substrates following D1 receptor activation. In addition to the D1 receptor/PKA/DARPP-32 signaling pathway, D1 receptor stimulation is known to activate Rap1/ERK signaling. Rap1 activation is mediated through the phosphorylation of Rasgrp2 (guanine nucleotide exchange factor; activation) and Rap1gap (GTPase-activating protein; inhibition) by PKA. In this study, we investigated the role of PP1 inhibition by phospho-Thr34 DARPP-32 in the D1 receptor-induced phosphorylation of Rasgrp2 and Rap1gap at PKA sites. The analyses in striatal and NAc slices from wild-type and DARPP-32 knockout mice revealed that the phosphorylation of Rasgrp2 at Ser116/Ser117 and Ser586, but not of Rasgrp2 at Ser554 or Rap1gap at Ser441 or Ser499 induced by a D1 receptor agonist, is under the control of the DARPP-32/PP1. The results were supported by pharmacological analyses using a selective PP1 inhibitor, tautomycetin. In addition, analyses using a PP1 and PP2A inhibitor, okadaic acid, revealed that all sites of Rasgrp2 and Rap1gap were regulated by PP2A. Thus, the interactive machinery of DARPP-32/PP1 may contribute to efficient D1 receptor signaling via Rasgrp2/Rap1 in the striatum.

Forebrain-specific conditional calcineurin deficiency induces dentate gyrus immaturity and hyper-dopaminergic signaling in mice.

Calcineurin (Cn), a phosphatase important for synaptic plasticity and neuronal development, has been implicated in the etiology and pathophysiology of neuropsychiatric disorders, including schizophrenia, intellectual disability, autism spectrum disorders, epilepsy, and Alzheimer's disease. Forebrain-specific conditional Cn knockout mice have been known to exhibit multiple behavioral phenotypes related to these disorders. In this study, we investigated whether Cn mutant mice show pseudo-immaturity of the dentate gyrus (iDG) in the hippocampus, which we have proposed as an endophenotype shared by these disorders. Expression of calbindin and GluA1, typical markers for mature DG granule cells (GCs), was decreased and that of doublecortin, calretinin, phospho-CREB, and dopamine D1 receptor (Drd1), markers for immature GC, was increased in Cn mutants. Phosphorylation of cAMP-dependent protein kinase (PKA) substrates (GluA1, ERK2, DARPP-32, PDE4) was increased and showed higher sensitivity to SKF81297, a Drd1-like agonist, in Cn mutants than in controls. While cAMP/PKA signaling is increased in the iDG of Cn mutants, chronic treatment with rolipram, a selective PDE4 inhibitor that increases intracellular cAMP, ameliorated the iDG phenotype significantly and nesting behavior deficits with nominal significance. Chronic rolipram administration also decreased the phosphorylation of CREB, but not the other four PKA substrates examined, in Cn mutants. These results suggest that Cn deficiency induces pseudo-immaturity of GCs and that cAMP signaling increases to compensate for this maturation abnormality. This study further supports the idea that iDG is an endophenotype shared by certain neuropsychiatric disorders.

Noradrenaline activation of hippocampal dopamine D1 receptors promotes antidepressant effects.

Dopamine D1 receptors (D1Rs) in the hippocampal dentate gyrus (DG) are essential for antidepressant effects. However, the midbrain dopaminergic neurons, the major source of dopamine in the brain, only sparsely project to DG, suggesting possible activation of DG D1Rs by endogenous substances other than dopamine. We have examined this possibility using electrophysiological and biochemical techniques and found robust activation of D1Rs in mouse DG neurons by noradrenaline. Noradrenaline at the micromolar range potentiated synaptic transmission at the DG output and increased the phosphorylation of protein kinase A substrates in DG via activation of D1Rs and ß adrenergic receptors. Neuronal excitation preferentially enhanced noradrenaline-induced synaptic potentiation mediated by D1Rs with minor effects on ß-receptor-dependent potentiation. Increased voluntary exercise by wheel running also enhanced noradrenaline-induced, D1R-mediated synaptic potentiation, suggesting a distinct functional role of the noradrenaline-D1R signaling. We then examined the role of this signaling in antidepressant effects using mice exposed to chronic restraint stress. In the stressed mice, an antidepressant acting on the noradrenergic system induced a mature-to-immature change in the DG neuron phenotype, a previously proposed cellular substrate for antidepressant action. This effect was evident only in mice subjected to wheel running and blocked by a D1R antagonist. These results suggest a critical role of noradrenaline-induced activation of D1Rs in antidepressant effects in DG. Experience-dependent regulation of noradrenaline-D1R signaling may determine responsiveness to antidepressant drugs in depressive disorders.

Subregion-Specific Regulation of Dopamine D1 Receptor Signaling in the Striatum: Implication for L-DOPA-Induced Dyskinesia.

The striatum is the main structure of the basal ganglia. The striatum receives inputs from various cortical areas, and its subregions play distinct roles in motor and emotional functions. Recently, striatal maps based on corticostriatal connectivity and striosome-matrix compartmentalization were developed, and we were able to subdivide the striatum into seven subregions. Dopaminergic modulation of the excitability of medium spiny neurons (MSNs) is critical for striatal function. In this study, we investigated the functional properties of dopamine signaling in seven subregions of the striatum from male mice. By monitoring the phosphorylation of PKA substrates including DARPP-32 in mouse striatal slices, we identified two subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Low D1 receptor signaling in the two subregions was maintained by phosphodiesterase (PDE)10A and muscarinic M4 receptors. In an animal model of 6-hydroxydopamine (6-OHDA)-induced hemi-parkinsonism, D1 receptor signaling was upregulated in almost all subregions including the DL-IR, but not in the IC. When L-DOPA-induced dyskinesia (LID) was developed, D1 receptor signaling in the IC was upregulated and correlated with the severity of LID. Our results suggest that the function of the striatum is maintained through the subregion-specific regulation of dopamine D1 receptor signaling and that the aberrant activation of D1 receptor signaling in the IC is involved in LID. Future studies focusing on D1 receptor signaling in the IC of the striatum will facilitate the development of novel therapeutics for LID.SIGNIFICANCE STATEMENT Recent progress in striatal mapping based on corticostriatal connectivity and striosome-matrix compartmentalization allowed us to subdivide the striatum into seven subregions. Analyses of D1 receptor signaling in the seven subregions identified two unique subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Aberrant activation of D1 receptor signaling in the IC is involved in L-DOPA-induced dyskinesia (LID). Previous studies of LID have mainly focused on the DL-IR, but not on the IC of the striatum. Future studies to clarify aberrant D1 receptor signaling in the IC are required to develop novel therapeutics for LID.

Sex Differences in Dendritic Spine Formation in the Hippocampus and Animal Behaviors in a Mouse Model of Hyperthyroidism.

Thyroid hormones are critical for the regulation of development and differentiation of neurons and glial cells in the central nervous system (CNS). We have previously reported the sex-dependent changes of glial morphology in the brain under the state of hyperthyroidism. Here, we examined sex-dependent changes in spine structure of granule neurons in the dentate gyrus of hippocampus in male and female mice with hyperthyroidism. Using FIB/SEM (focused ion beam/scanning electron microscopy), three-dimensional reconstructed structures of dendritic spines in dentate granule cells were analyzed. Dendritic spine density in granule cells increased significantly in both male and female mice with hyperthyroidism. The decrease in spine volume was observed only in female mice. These findings suggest that hyperthyroidism induces the formation of spines with normal size in male mice but the formation of spines with small size in female mice. To evaluate an outcome of neuronal and previously observed glial changes, behavioral tests were performed. Male mice with hyperthyroidism showed increased locomotor activity in the open field test, while female mice showed elevated immobility time in the tail suspension test, reflecting depression-like behavior. Although direct link between changes in spine and behavioral modifications requires further analysis, our results may help to understand gender-dependent neurological and psychological symptoms observed in patients with hyperthyroidism.

Adolescent psychosocial stress enhances sensitization to cocaine exposure in genetically vulnerable mice.

Development of drug addictive behaviors is modulated by both genetic and environmental risk factors. However, the molecular mechanisms remain unknown. To address the role of adolescent stress in the development of drug addiction, we combined a transgenic mouse model in which a putative dominant-negative form of DISC1 under expressional control of the prion protein promoter is used as a genetic risk factor and adolescent social isolation stress as a gene-environmental interaction (GXE). Repeated cocaine exposure induced greater locomotion in the GXE group than in the other groups. In a conditioned place preference (CPP) test, GXE mice exhibited a significant place preference to the cocaine-conditioned area compared with the other groups. In the nucleus accumbens (NAc) of GXE mice, we found increased enzyme activity of phosphodiesterase-4 (PDE4), predominantly located in NAc D2-receptor-expressing neurons, and enhanced effects of the PDE4 inhibitor rolipram, but not the D1 agonist SKF81297, on the phosphorylation of DARPP-32 and GluA1 at PKA sites. Rolipram injection before cocaine exposure completely inhibited cocaine-induced hyperlocomotion and CPP in the GXE group. These results indicate that GXE enhances sensitivity to repeated cocaine exposure via an increase in PDE4 activity in NAc D2-recptor-expressing neurons, leading to the development of cocaine addictive behaviors.

Obligatory roles of dopamine D1 receptors in the dentate gyrus in antidepressant actions of a selective serotonin reuptake inhibitor, fluoxetine.

Depression is a leading cause of disability. Current pharmacological treatment of depression is insufficient, and development of improved treatments especially for treatment-resistant depression is desired. Understanding the neurobiology of antidepressant actions may lead to development of improved therapeutic approaches. Here, we demonstrate that dopamine D1 receptors in the dentate gyrus act as a pivotal mediator of antidepressant actions in mice. Chronic administration of a selective serotonin reuptake inhibitor (SSRI), fluoxetine, increases D1 receptor expression in mature granule cells in the dentate gyrus. The increased D1 receptor signaling, in turn, contributes to the actions of chronic fluoxetine treatment, such as suppression of acute stress-evoked serotonin release, stimulation of adult neurogenesis and behavioral improvement. Importantly, under severely stressed conditions, chronic administration of a D1 receptor agonist in conjunction with fluoxetine restores the efficacy of fluoxetine actions on D1 receptor expression and behavioral responses. Thus, our results suggest that stimulation of D1 receptors in the dentate gyrus is a potential adjunctive approach to improve therapeutic efficacy of SSRI antidepressants.

Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution.

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels.

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus.

A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission.

Acute effects of resveratrol to enhance cocaine-induced dopamine neurotransmission in the striatum.

Resveratrol is known as an activator of SIRT1, which leads to the deacetylation of histone and non-histone protein substrates, but also has other pharmacological profiles such as the inhibition of monoamine oxidase (MAO)-A and MAO-B. Resveratrol was previously demonstrated to potentiate the rewarding effects of chronic cocaine via activation of SIRT1. However, the role of resveratrol in cocaine responses in the acute phase remains unexplored. Therefore, we investigated the acute effects of resveratrol on cocaine-stimulated dopamine neurotransmission by analyzing protein phosphorylation in neostriatal slices. Treatment with resveratrol (50µM for 30min) enhanced cocaine-induced increases in the phosphorylation of DARPP-32 at Thr34 and GluA1 at Ser845, postsynaptic substrates for dopamine/D1 receptor/PKA signaling, and a cocaine-induced decrease in the phosphorylation of tyrosine hydroxylase at Ser40, a presynaptic substrate for dopamine/D2 receptor signaling. The inhibition of both MAO-A and MAO-B by clorgyline and pargyline, respectively, enhanced the effects of cocaine on DARPP-32 phosphorylation. The acute effect of resveratrol on cocaine-induced DARPP-32 phosphorylation was occluded with inhibition of MAO-A and MAO-B. In behavioral studies, resveratrol (40mg/kg, s.c.) enhanced the increase in locomotor activity induced by acute cocaine administration (10mg/kg, i.p.). Thus, this study provides pharmacological evidence that acute resveratrol enhances cocaine-induced dopamine neurotransmission and behavioral responses, presumably via mechanisms involving the inhibition of dopamine catabolism by MAO-A and MAO-B. Resveratrol may be useful to treat dysregulated dopamine neurotransmission, but it may enhance the risk of developing drug addiction.

Deficiency of schnurri-2, an MHC enhancer binding protein, induces mild chronic inflammation in the brain and confers molecular, neuronal, and behavioral phenotypes related to schizophrenia.

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

Muscarinic receptors acting at pre- and post-synaptic sites differentially regulate dopamine/DARPP-32 signaling in striatonigral and striatopallidal neurons.

Muscarinic receptors, activated by acetylcholine, play critical roles in the functional regulation of medium spiny neurons in the striatum. However, the muscarinic receptor signaling pathways are not fully elucidated due to their complexity. In this study, we investigated the function of muscarinic receptors in the striatum by monitoring DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa) phosphorylation at Thr34 (the PKA-site) using mouse striatal slices. Treatment of slices with a non-selective muscarinic receptor agonist, oxotremorine (10 µM), rapidly and transiently increased DARPP-32 phosphorylation. The increase in DARPP-32 phosphorylation was completely abolished either by a dopamine D(1) receptor antagonist (SCH23390), tetrodotoxin, genetic deletion of M5 receptors, muscarinic toxins for M1 and M4 receptors, or 6-hydroxydopamine lesioning of dopaminergic neurons, whereas it was enhanced by nicotine. Analysis in D(1)-DARPP-32-Flag/D(2)-DARPP-32-Myc transgenic mice revealed that oxotremorine increases DARPP-32 phosphorylation selectively in D(1)-type/striatonigral, but not in D(2)-type/striatopallidal, neurons. When D(1) and D(2) receptors were blocked by selective antagonists to exclude the effects of released dopamine, oxotremorine increased DARPP-32 Thr34 phosphorylation only in D(2)-type/striatopallidal neurons. This increase required activation of M1 receptors and was dependent upon adenosine A(2A) receptor activity. The results demonstrate that muscarinic receptors, especially M5 receptors, act at presynaptic dopaminergic terminals, regulate the release of dopamine in cooperation with nicotinic receptors, and activate D(1) receptor/DARPP-32 signaling in the striatonigral neurons. Muscarinic M1 receptors expressed in striatopallidal neurons interact with adenosine A(2A) receptors and activate DARPP-32 signaling.

Phosphodiesterase 4 inhibition enhances the dopamine D1 receptor/PKA/DARPP-32 signaling cascade in frontal cortex.

RATIONALE: Alteration of dopamine neurotransmission in the prefrontal cortex, especially hypofunction of dopamine D1 receptors, contributes to psychotic symptoms and cognitive deficit in schizophrenia. D1 receptors signal through the cAMP/PKA second messenger cascade, which is modulated by phosphodiesterase (PDE) enzymes that hydrolyze and inactivate cyclic nucleotides. Though several PDEs are expressed in cortical neurons, the PDE4 enzyme family (PDE4A-D) has been implicated in the control of cognitive function. The best studied isoform, PDE4B, interacts with a schizophrenia susceptibility factor, disrupted in schizophrenia 1 (DISC1). OBJECTIVES: We explore the control of mouse frontal cortex dopamine D1 receptor signaling and associated behavior by PDE4. RESULTS: Inhibition of PDE4 by rolipram induced activation of cAMP/PKA signaling in cortical slices and in vivo, leading to the phosphorylation of DARPP-32 and other postsynaptic and presynaptic PKA-substrates. Rolipram also enhanced DARPP-32 phosphorylation invoked by D1 receptor activation. Immunohistochemical studies demonstrated PDE4A, PDE4B, and PDE4D expression in DARPP-32-positive neurons in layer VI of frontal cortex, most likely in D1 receptor-positive, glutamatergic corticothalamic pyramidal neurons. Furthermore, the ability of rolipram treatment to improve the performance of mice in a sensorimotor gating test was DARPP-32-dependent. CONCLUSIONS: PDE4, which is co-expressed with DARPP-32 in D1 receptor-positive cortical pyramidal neurons in layer VI, modulates the level of D1 receptor signaling and DARPP-32 phosphorylation in the frontal cortex, likely influencing cognitive function. These biochemical and behavioral actions of PDE4 inhibitors may contribute to the hypothesized antipsychotic actions of this class of compounds.

Mechanisms for the modulation of dopamine d(1) receptor signaling in striatal neurons.

In the striatum, dopamine D(1) receptors are preferentially expressed in striatonigral neurons, and increase the neuronal excitability, leading to the increase in GABAergic inhibitory output to substantia nigra pars reticulata. Such roles of D(1) receptors are important for the control of motor functions. In addition, the roles of D(1) receptors are implicated in reward, cognition, and drug addiction. Therefore, elucidation of mechanisms for the regulation of dopamine D(1) receptor signaling is required to identify therapeutic targets for Parkinson's disease and drug addiction. D(1) receptors are coupled to G(s/olf)/adenylyl cyclase/PKA signaling, leading to the phosphorylation of PKA substrates including DARPP-32. Phosphorylated form of DARPP-32 at Thr34 has been shown to inhibit protein phosphatase-1, and thereby controls the phosphorylation states and activity of many downstream physiological effectors. Roles of DARPP-32 and its phosphorylation at Thr34 and other sites in D(1) receptor signaling are extensively studied. In addition, functional roles of the non-canonical D(1) receptor signaling cascades that coupled to G(q)/phospholipase C or Src family kinase become evident. We have recently shown that phosphodiesterases (PDEs), especially PDE10A, play a pivotal role in regulating the tone of D(1) receptor signaling relatively to that of D(2) receptor signaling. We review the current understanding of molecular mechanisms for the modulation of D(1) receptor signaling in the striatum.

Role of adrenoceptors in the regulation of dopamine/DARPP-32 signaling in neostriatal neurons.

Studies in animal models of Parkinson's disease have revealed that degeneration of noradrenaline neurons is involved in the motor deficits. Several types of adrenoceptors are highly expressed in neostriatal neurons. However, the selective actions of these receptors on striatal signaling pathways have not been characterized. In this study, we investigated the role of adrenoceptors in the regulation of dopamine/dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) signaling by analyzing DARPP-32 phosphorylation at Thr34 [protein kinase A (PKA)-site] in mouse neostriatal slices. Activation of beta(1)-adrenoceptors induced a rapid and transient increase in DARPP-32 phosphorylation. Activation of alpha(2)-adrenoceptors also induced a rapid and transient increase in DARPP-32 phosphorylation, which subsequently decreased below basal levels. In addition, activation of alpha(2)-adrenoceptors attenuated, and blockade of alpha(2)-adrenoceptors enhanced dopamine D(1) and adenosine A(2A) receptor/DARPP-32 signaling. Chemical lesioning of noradrenergic neurons mimicked the effects of alpha(2)-adrenoceptor blockade. Under conditions of alpha(2)-adrenoceptor blockade, the dopamine D(2) receptor-induced decrease in DARPP-32 phosphorylation was attenuated. Our data demonstrate that beta(1)- and alpha(2)-adrenoceptors regulate DARPP-32 phosphorylation in neostriatal neurons. G(i) activation by alpha(2)-adrenoceptors antagonizes G(s)/PKA signaling mediated by D(1) and A(2A) receptors in striatonigral and striatopallidal neurons, respectively, and thereby enhances D(2) receptor/G(i) signaling in striatopallidal neurons. alpha(2)-Adrenoceptors may therefore be a therapeutic target for the treatment of Parkinson's disease.

Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum.

Phosphodiesterase (PDE) is a critical regulator of cAMP/protein kinase A (PKA) signaling in cells. Multiple PDEs with different substrate specificities and subcellular localization are expressed in neurons. Dopamine plays a central role in the regulation of motor and cognitive functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade, and therefore controlled by PDE activity. We used in vitro and in vivo biochemical techniques to dissect the roles of PDE4 and PDE10A in dopaminergic neurotransmission in mouse striatum by monitoring the ability of selective PDE inhibitors to regulate phosphorylation of presynaptic [e.g., tyrosine hydroxylase (TH)] and postsynaptic [e.g., dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32)] PKA substrates. The PDE4 inhibitor, rolipram, induced a large increase in TH Ser40 phosphorylation at dopaminergic terminals that was associated with a commensurate increase in dopamine synthesis and turnover in striatum in vivo. Rolipram induced a small increase in DARPP-32 Thr34 phosphorylation preferentially in striatopallidal neurons by activating adenosine A(2A) receptor signaling in striatum. In contrast, the PDE10A inhibitor, papaverine, had no effect on TH phosphorylation or dopamine turnover, but instead robustly increased DARPP-32 Thr34 and GluR1 Ser845 phosphorylation in striatal neurons. Inhibition of PDE10A by papaverine activated cAMP/PKA signaling in both striatonigral and striatopallidal neurons, resulting in potentiation of dopamine D(1) receptor signaling and inhibition of dopamine D(2) receptor signaling. These biochemical results are supported by immunohistochemical data demonstrating differential localization of PDE10A and PDE4 in striatum. These data underscore the importance of individual brain-enriched cyclic-nucleotide PDE isoforms as therapeutic targets for neuropsychiatric and neurodegenerative disorders affecting dopamine neurotransmission.

Regulation of DARPP-32 phosphorylation by three distinct dopamine D1-like receptor signaling pathways in the neostriatum.

Dopamine D(1)-like receptors play a key role in dopaminergic signaling. In addition to G(s/olf)/adenylyl cyclase (AC)-coupled D(1) receptors, the presence of D(1)-like receptors coupled to G(q)/phospholipase C (PLC) has been proposed. Benzazepine D(1) receptor agonists are known to differentially activate G(s/olf)/AC and G(q)/PLC signaling. By utilizing SKF83959 and SKF83822, we investigated the D(1)-like receptor signaling cascades, which regulate DARPP-32 phosphorylation at Thr34 (the PKA-site) in mouse neostriatal slices. Treatment with SKF83959 or SKF83822 increased DARPP-32 phosphorylation. The SKF83959- and SKF83822-induced increase in DARPP-32 phosphorylation was largely, but partially, antagonized by a D(1) receptor antagonist, SCH23390, and the residual SCH23390-insensitive increase was abolished by an adenosine A(2A) receptor antagonist. In addition, the SKF83959-induced, SCH23390-sensitive increase in DARPP-32 phosphorylation was enhanced by a PLC inhibitor. Analysis in slices from D(1)R/D(2)R-DARPP-32 mice revealed that both D(1) receptor agonists regulate DARPP-32 phosphorylation in striatonigral, but not in striatopallidal, neurons. Thus, dopamine D(1)-like receptors are coupled to three signaling cascades in striatonigral neurons: (i) SCH23390-sensitive G(s/olf)/AC/PKA, (ii) adenosine A(2A) receptor-dependent G(s/olf)/AC/PKA, and (iii) G(q)/PLC signaling. Interestingly, G(q)/PLC signaling interacts with SCH23390-sensitive G(s/olf)/AC/PKA signaling, resulting in its inhibition. Three signaling cascades activated by D(1)-like receptors likely play a distinct role in dopaminergic regulation of psychomotor functions.

Cell type-specific regulation of DARPP-32 phosphorylation by psychostimulant and antipsychotic drugs.

DARPP-32 is a dual-function protein kinase/phosphatase inhibitor that is involved in striatal signaling. The phosphorylation of DARPP-32 at threonine 34 is essential for mediating the effects of both psychostimulant and antipsychotic drugs; however, these drugs are known to have opposing behavioral and clinical effects. We hypothesized that these drugs exert differential effects on striatonigral and striatopallidal neurons, which comprise distinct output pathways of the basal ganglia. To directly test this idea, we developed bacterial artificial chromosome transgenic mice that allowed the analysis of DARPP-32 phosphorylation selectively in striatonigral and striatopallidal neurons. Using this new methodology, we found that cocaine, a psychostimulant, and haloperidol, a sedation-producing antipsychotic, exert differential effects on DARPP-32 phosphorylation in the two neuronal populations that can explain their opposing behavioral effects. Furthermore, we found that a variety of drugs that target the striatum have cell type-specific effects that previous methods were not able to discern.