Phosphodiesterase 1B differentially modulates the effects of methamphetamine on locomotor activity and spatial learning through DARPP32-dependent pathways: evidence from PDE1B-DARPP32 double-knockout mice.

Mice lacking phosphodiesterase 1B (PDE1B) exhibit an exaggerated locomotor response to D-methamphetamine and increased in vitro phosphorylation of DARPP32 (dopamine- and cAMP-regulated phosphoprotein, M r 32 kDa) at Thr34 in striatal brain slices treated with the D1 receptor agonist, SKF81297. These results indicated a possible regulatory role for PDE1B in pathways involving DARPP32. Here, we generated PDE1B x DARPP32 double-knockout (double-KO) mice to test the role of PDE1B in DARPP32-dependent pathways in vivo. Analysis of the response to d-methamphetamine on locomotor activity showed that the hyperactivity experienced by PDE1B mutant mice was blocked in PDE1B-/- x DARPP32-/- double-KO mice, consistent with participation of PDE1B and DARPP32 in the same pathway. Further behavioral testing in the elevated zero-maze revealed that DARPP32-/- mice showed a less anxious phenotype that was nullified in double-mutant mice. In contrast, in the Morris water maze, double-KO mice showed deficits in spatial reversal learning not observed in either single mutant compared with wild-type mice. The data suggest a role for PDE1B in locomotor responses to psychostimulants through modulation of DARPP32-dependent pathways; however, this modulation does not necessarily impact other behaviors, such as anxiety or learning. Instead, the phenotype of double-KOs observed in these latter tasks may be mediated through independent pathways.

A network of control mediated by regulator of calcium/calmodulin-dependent signaling.

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.

Ovarian function and morphology after deletion of the DARPP-32 gene in mice.

A plethora of systemic and local signaling molecules regulate ovarian function, but how different signaling molecules interact within an ovarian target cell is not known. Here we report that endocrine cells of the ovary express a phosphoprotein, DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein of Mr 32,000), which integrates signaling molecules in neurons. We thus hypothesized that DARPP-32 might act in a similar way in ovarian endocrine cells and therefore studied whether DARPP-32 gene deletion has consequences for ovarian functions in mice. Reproductive performance of adult mutants did not differ from wild-type females, as judged from numbers of litters and pups delivered. Similar steroid levels in mutant and wild-type mice ruled out gross abnormalities in the hypothalamic-pituitary-ovarian axis. However, an analysis of ovarian morphology, using serially sectioned ovaries, revealed several differences. Ovaries of young adult mutant mice at 2 - 3 months contained luteinized follicles, but fewer corpora lutea. At 5 - 6 months, large cysts were found in mutant mice, as well as reduced numbers of preantral follicles and antral follicles. Interstitial cell hypertrophy and degeneration was marked in all mutant ovaries at this age. Thus, while the lack of DARPP-32 does not overtly alter reproductive performance in adult mice, it is associated with progressive alterations and derangements of growth and development of ovarian follicles, suggesting premature ovarian ageing. This implies that ovarian DARPP-32 is involved in follicular development, presumably by integrating effects of signaling molecules, which act together to ensure efficient follicular development.

DARPP-32 and regulation of the ethanol sensitivity of NMDA receptors in the nucleus accumbens.

The medium spiny neurons of the nucleus accumbens receive both an excitatory glutamatergic input from forebrain and a dopaminergic input from the ventral tegmental area. This integration point may constitute a locus whereby the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors promotes drug reinforcement. Here we investigate how dopaminergic inputs alter the ethanol sensitivity of NMDA receptors in rats and mice and report that previous dopamine receptor-1 (D1) activation, culminating in dopamine and cAMP-regulated phosphoprotein-32 kD (DARPP-32) and NMDA receptor subunit-1 (NR1)-NMDA receptor phosphorylation, strongly decreases ethanol inhibition of NMDA responses. The regulation of ethanol sensitivity of NMDA receptors by D1 receptors was absent in DARPP-32 knockout mice. We propose that DARPP-32 mediated blunting of the response to ethanol subsequent to activation of ventral tegmental area dopaminergic neurons initiates molecular alterations that influence synaptic plasticity in this circuit, thereby promoting the development of ethanol reinforcement.

Increased blood pressure and loss of anp-induced natriuresis in mice lacking DARPP-32 gene.

Atrial natriuretic peptide (ANP) is an important regulator of sodium metabolism and indirectly of blood pressure. Evidence has accumulated that ANP regulates sodium metabolism through a cascade of steps involving an increase in the level of cGMP, activation of cGMP-dependent protein kinase (PKG), and inhibition of renal tubular Na+, K+-ATPase activity. One of the major substrates for PKG is DARPP-32. In the present study we observed that ANP does not induce natriuresis in mice that lack DARPP-32. In contrast, there was a 4-fold increase in urinary sodium excretion following ANP administration to wild type mice. ANP as well as Zaprinast, a selective inhibitor of cGMP phosophodiesterase, inhibited renal Na+, K+-ATPase activity in wild type mice but had no such effect in mice lacking DARPP-32. Mean arterial blood pressure, measured in conscious animals, was significantly increased in DARPP-32 deficient mice as compared to wild type mice. The results confirm that DARPP-32 acts as a third messenger in the ANP signaling pathway in renal tissue and suggest an important role of DARPP-32 in the maintenance of normal blood pressure.

Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum.

In the striatum, dopamine D(1) and adenosine A(2A) receptors stimulate the production of cAMP, which is involved in neuromodulation and long-lasting changes in gene expression and synaptic function. Positive coupling of receptors to adenylyl cyclase can be mediated through the ubiquitous GTP-binding protein Galpha(S) subunit or through the olfactory isoform, Galpha(olf), which predominates in the striatum. In this study, using double in situ hybridization, we show that virtually all striatal efferent neurons, identified by the expression of preproenkephalin A, substance P, or D(1) receptor mRNA, contained high amounts of Galpha(olf) mRNA and undetectable levels of Galpha(s) mRNA. In contrast, the large cholinergic interneurons contained both Galpha(olf) and Galpha(s) transcripts. To assess the functional relationship between dopamine or adenosine receptors and G-proteins, we examined G-protein levels in the striatum of D(1) and A(2A) receptor knock-out mice. A selective increase in Galpha(olf) protein was observed in these animals, without change in mRNA levels. Conversely, Galpha(olf) levels were decreased in animals lacking a functional dopamine transporter. These results indicate that Galpha(olf) protein levels are regulated through D(1) and A(2A) receptor usage. To determine the functional consequences of changes in Galpha(olf) levels, we used heterozygous Galpha(olf) knock-out mice, which possess half of the normal Galpha(olf) levels. In these animals, the locomotor effects of amphetamine and caffeine, two psychostimulant drugs that affect dopamine and adenosine signaling, respectively, were markedly reduced. Together, these results identify Galpha(olf) as a critical and regulated component of both dopamine and adenosine signaling.

Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia.

Neuropathological and brain imaging studies suggest that schizophrenia may result from neurodevelopmental defects. Cytoarchitectural studies indicate cellular abnormalities suggestive of a disruption in neuronal connectivity in schizophrenia, particularly in the dorsolateral prefrontal cortex. Yet, the molecular mechanisms underlying these findings remain unclear. To identify molecular substrates associated with schizophrenia, DNA microarray analysis was used to assay gene expression levels in postmortem dorsolateral prefrontal cortex of schizophrenic and control patients. Genes determined to have altered expression levels in schizophrenics relative to controls are involved in a number of biological processes, including synaptic plasticity, neuronal development, neurotransmission, and signal transduction. Most notable was the differential expression of myelination-related genes suggesting a disruption in oligodendrocyte function in schizophrenia.

Motivational effects of ethanol in DARPP-32 knock-out mice.

DARPP-32 (dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa) is an important component of dopaminergic function in brain areas thought to be important for drug and alcohol addiction. The present experiments characterized the acquisition of ethanol-induced conditioned taste aversion, ethanol-induced conditioned place preference, and ethanol self-administration in DARPP-32 knock-out (KO) mice compared to wild-type (WT) controls. For taste conditioning, KO and WT mice received access to 0.2 m NaCl solution followed immediately by intraperitoneal injection of 0-4 gm/kg ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. For place conditioning, KO and WT mice received eight pairings of a tactile stimulus with ethanol (2 gm/kg, i.p.), and a different stimulus with saline. Ethanol produced increases in locomotor activity during conditioning, with KO mice showing higher activity levels after ethanol compared to WT mice. WT mice, but not KO mice, acquired conditioned preference for the ethanol-paired stimulus. In the self-administration procedure, KO and WT mice were trained to lever press for access to 10% v/v ethanol. Subsequently, the mice had 23 hr/d access to food, ethanol, and water. Response patterns were determined using 0-30% v/v ethanol concentrations. WT mice displayed concentration-dependent responding for ethanol. Responding on the ethanol lever by KO mice did not change as a function of ethanol concentration. Saccharin (0.2% w/v) was subsequently added to the ethanol mixture, and responding was examined at 0, 5, 10, and 20% ethanol concentrations. Ethanol responding increased in both genotypes, although WT mice showed higher rates at all concentrations.

Dopamine and cAMP-regulated phosphoprotein 32 kDa controls both striatal long-term depression and long-term potentiation, opposing forms of synaptic plasticity.

A complex chain of intracellular signaling events, critically important in motor control, is activated by the stimulation of D1-like dopamine (DA) receptors in striatal neurons. At corticostriatal synapses on medium spiny neurons, we provide evidence that the D1-like receptor-dependent activation of DA and cyclic adenosine 3',5' monophosphate-regulated phosphoprotein 32 kDa is a crucial step for the induction of both long-term depression (LTD) and long-term potentiation (LTP), two opposing forms of synaptic plasticity. In addition, formation of LTD and LTP requires the activation of protein kinase G and protein kinase A, respectively, in striatal projection neurons. These kinases appear to be stimulated by the activation of D1-like receptors in distinct neuronal populations.

Dopamine D(1) receptor-induced gene transcription is modulated by DARPP-32.

The role of the dopamine- and cyclic AMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32) in dopaminergic regulation of gene transcription in striatum and globus pallidus was examined. Mice with targeted disruption of the gene encoding DARPP-32, its homologue, inhibitor-1, or both, were used. Pharmacological characterization showed that mutant mice had normal basal levels of dopamine D(1) and D(2) receptors and adenosine A(2A) receptors. Basal expression levels of the striatonigral-specific neuropeptides substance P and prodynorphin and the immediate early genes c-fos and NGFI-A were also unaltered in mutant mice. A full D(1) receptor agonist, SKF 82958, up-regulated the expression of these neuropeptides and immediate early genes significantly more in wild-type mice than in mice lacking DARPP-32. Moreover, the additive stimulation of SKF 82958 and quinelorane, a D(2) receptor agonist, on c-fos mRNA in globus pallidus was significantly decreased in DARPP-32 and DARPP-32/I-1 knockout mice. No changes in dopamine receptor-induced gene expression were found in I-1 knockout mice. These results demonstrate an important involvement of DARPP-32 in dopamine receptor-mediated regulation of gene expression both in striatal neurons, which are enriched in DARPP-32, and in pallidal neurons, which do not contain DARPP-32.

DARPP-32 knockout mice exhibit impaired reversal learning in a discriminated operant task.

The current study was conducted to examine the performance of mice with a targeted deletion of the gene for DARPP-32 in a discriminated operant task using food reinforcement. DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission. Initially, wild-type and DARPP-32 knockout mice were trained to nose-poke for food on a continuous reinforcement schedule. The minimum response requirement was increased every 5 days until the animals were responding on an FR-15 schedule of reinforcement. At the completion of extensive operant training, reversal learning was assessed. Wild-type and DARPP-32 knockout mice exhibited equivalent performance during acquisition of this task, with both groups increasing operant responding as the schedule of reinforcement was raised. However, significant differences in discrimination learning were observed during the reversal phase, with DARPP-32 knockout mice requiring significantly more trials to reach criterion than wild-type controls. These results provide evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory.

Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo.

The activation of cAMP-dependent protein kinase regulates the physiological activity of AMPA-type glutamate receptors. In this study, phosphorylation of the AMPA receptor subunit GluR1 at Ser(845) was increased in neostriatal slices by activation of D1-type dopamine receptors and by inhibitors of protein phosphatase 1/protein phosphatase 2A. In contrast, Ser(831), a residue which, when phosphorylated by protein kinase C or calcium/calmodulin-dependent kinase II, increases AMPA receptor channel conductance, was unaffected by either D1 or D2 receptor agonists in neostriatal slices. The phosphorylation of Ser(845), but not Ser(831), was strongly increased in neostriatum in vivo in response to the psychostimulants cocaine and methamphetamine. The effects of dopamine and psychostimulants on the phosphorylation of GluR1 were attenuated in dopamine and cAMP-regulated phosphoprotein M(r) 32 kDa (DARPP-32) knock-out mice. These results identify DARPP-32 and AMPA-type glutamate receptors as likely essential cellular effectors for psychostimulant actions.

Drugs of abuse modulate the phosphorylation of ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in the basal ganglia.

ARPP-21 is a cyclic AMP-regulated phosphoprotein of M(r) 21 kDa that is enriched in the cell bodies and terminals of medium-sized spiny neurons in the basal ganglia. Using a new phosphorylation state-specific antibody selective for the detection of ARPP-21 phosphorylated on Ser(55), we have demonstrated that activation of dopamine D1 receptors increased the level of ARPP-21 phosphorylation in mouse striatal slices. Conversely, activation of D2 receptors caused a large decrease in ARPP-21 phosphorylation. Treatment of mice with either methamphetamine or cocaine resulted in increased ARPP-21 phosphorylation in vivo. Studies using specific inhibitors of protein phosphatases and experiments in mice bearing a targeted deletion of the gene for DARPP-32, a dopamine-activated inhibitor of protein phosphatase-1, indicated that protein phosphatase-2A is primarily responsible for dephosphorylation of ARPP-21 in mouse striatum. These results demonstrate that phosphorylation and dephosphorylation of ARPP-21 are tightly regulated in the striatum. We speculate that ARPP-21 might mediate some of the physiologic effects of dopamine and certain drugs of abuse in the basal ganglia.

D(1) dopamine receptor activation reduces GABA(A) receptor currents in neostriatal neurons through a PKA/DARPP-32/PP1 signaling cascade.

Dopamine is a critical determinant of neostriatal function, but its impact on intrastriatal GABAergic signaling is poorly understood. The role of D(1) dopamine receptors in the regulation of postsynaptic GABA(A) receptors was characterized using whole cell voltage-clamp recordings in acutely isolated, rat neostriatal medium spiny neurons. Exogenous application of GABA evoked a rapidly desensitizing current that was blocked by bicuculline. Application of the D(1) dopamine receptor agonist SKF 81297 reduced GABA-evoked currents in most medium spiny neurons. The D(1) dopamine receptor antagonist SCH 23390 blocked the effect of SKF 81297. Membrane-permeant cAMP analogues mimicked the effect of D(1) dopamine receptor stimulation, whereas an inhibitor of protein kinase A (PKA; Rp-8-chloroadenosine 3',5' cyclic monophosphothioate) attenuated the response to D(1) dopamine receptor stimulation or cAMP analogues. Inhibitors of protein phosphatase 1/2A potentiated the modulation by cAMP analogues. Single-cell RT-PCR profiling revealed consistent expression of mRNA for the beta1 subunit of the GABA(A) receptor-a known substrate of PKA-in medium spiny neurons. Immunoprecipitation assays of radiolabeled proteins revealed that D(1) dopamine receptor stimulation increased phosphorylation of GABA(A) receptor beta1/beta3 subunits. The D(1) dopamine receptor-induced phosphorylation of beta1/beta3 subunits was attenuated significantly in neostriata from DARPP-32 mutants. Voltage-clamp recordings corroborated these results, revealing that the efficacy of the D(1) dopamine receptor modulation of GABA(A) currents was reduced in DARPP-32-deficient medium spiny neurons. These results argue that D(1) dopamine receptor stimulation in neostriatal medium spiny neurons reduces postsynaptic GABA(A) receptor currents by activating a PKA/DARPP-32/protein phosphatase 1 signaling cascade targeting GABA(A) receptor beta1 subunits.

Synapsins as mediators of BDNF-enhanced neurotransmitter release.

We examined enhancement of synaptic transmission by neurotrophins at the presynaptic level. In a synaptosomal preparation, brain-derived neurotrophic factor (BDNF) increased mitogen-activated protein (MAP) kinase-dependent synapsin I phosphorylation and acutely facilitated evoked glutamate release. PD98059, used to inhibit MAP kinase activity, markedly decreased synapsin I phosphorylation and concomitantly reduced neurotransmitter release. The stimulation of glutamate release by BDNF was strongly attenuated in mice lacking synapsin I and/or synapsin II. These results indicate a causal link of synapsin phosphorylation via BDNF, TrkB receptors and MAP kinase with downstream facilitation of neurotransmitter release.

Requirement for DARPP-32 in progesterone-facilitated sexual receptivity in female rats and mice.

DARPP-32, a dopamine- and adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (32 kilodaltons in size), is an obligate intermediate in progesterone (P)-facilitated sexual receptivity in female rats and mice. The facilitative effect of P on sexual receptivity in female rats was blocked by antisense oligonucleotides to DARPP-32. Homozygous mice carrying a null mutation for the DARPP-32 gene exhibited minimal levels of P-facilitated sexual receptivity when compared to their wild-type littermates. P significantly increased hypothalamic cAMP levels and cAMP-dependent protein kinase activity. These increases were not inhibited by a D1 subclass dopamine receptor antagonist. P also enhanced phosphorylation of DARPP-32 on threonine 34 in the hypothalamus of mice. DARPP-32 activation is thus an obligatory step in progestin receptor regulation of sexual receptivity in rats and mice.

Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons.

The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

Effects of chronic 'Binge' cocaine administration on plasma ACTH and corticosterone levels in mice deficient in DARPP-32.

The product of the DARPP-32 gene mediates intracellular signals initiated by the binding of dopamine to its receptors. Cocaine administration leads to increased activation of dopamine receptors, and causes activation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) axis. We determined the effects of chronic 'binge' pattern cocaine on HPA activity in mice containing a targeted disruption of the DARPP-32 gene. Mice received three daily injections of cocaine (15 mg/kg/injection) for 14 days, and were sacrificed 30 min after the last injection. We measured the levels of plasma adrenocorticotropin (ACTH) and corticosterone which reflect HPA activity. In wild-type controls, 'binge' cocaine administration significantly increased plasma ACTH and corticosterone levels. In contrast, DARPP-32-deficient mice failed to show a significant elevation of either plasma ACTH or corticosterone levels following 'binge' cocaine. The results indicate that DARPP-32 plays a role in mediating the stimulatory effects of cocaine on the HPA axis.

D(2) dopamine receptors induce mitogen-activated protein kinase and cAMP response element-binding protein phosphorylation in neurons.

Dopamine, by activating D(1)- and D(2)-class receptors, plays a significant role in regulating gene expression. Although much is known about D(1) receptor-regulated gene expression, there has been far less information on gene regulation mediated by D(2) receptors. In this study, we show that D(2) receptors can activate the mitogen-activated protein kinase (MAPK) and the cAMP response element-binding protein (CREB) in neurons. Treatment of brain slices with the D(2) receptor agonist quinpirole induced rapid phosphorylation of MAPK and CREB. The neuroleptic drug eticlopride, a highly selective D(2) receptor antagonist, blocked the quinpirole-induced phosphorylation of MAPK and CREB. D(2) receptor-induced MAPK phosphorylation depended on intracellular Ca(2+) elevation, protein kinase C activation, and MAPK kinase activation, but not on the protein tyrosine kinase Pyk2, even though quinpirole stimulated Pyk2 phosphorylation. D(2) receptor-induced CREB phosphorylation was mediated by activation of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase, but not MAPK. The dopamine and cAMP-regulated phosphoprotein DARPP-32 also was required for the regulation of MAPK and CREB phosphorylation by D(2) receptors. Our results suggest that MAPK and CREB signaling cascades are involved in the regulation of gene expression and other long-term effects of D(2) receptor activation.