Primary Striatal Cultures Research Articles

Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II

In neurodegenerative disorders associated with primary or secondary mitochondrial defects such as Huntington's disease (HD), cells of the striatum are particularly vulnerable to cell death, although the mechanisms by which this cell death is induced are unclear. Dopamine, found in high concentrations in the striatum, may play a role in striatal cell death. We show that in primary striatal cultures, dopamine increases the toxicity of an N-terminal fragment of mutated huntingtin (Htt-171-82Q). Mitochondrial complex II protein (mCII) levels are reduced in HD striatum, indicating that this protein may be important for dopamine-mediated striatal cell death. We found that dopamine enhances the toxicity of the selective mCII inhibitor, 3-nitropropionic acid. We also demonstrated that dopamine doses that are insufficient to produce cell loss regulate mCII expression at the mRNA, protein and catalytic activity level. We also show that dopamine-induced down-regulation of mCII levels can be blocked by several dopamine D2 receptor antagonists. Sustained overexpression of mCII subunits using lentiviral vectors abrogated the effects of dopamine, both by high dopamine concentrations alone and neuronal death induced by low dopamine concentrations together with Htt-171-82Q. This novel pathway links dopamine signaling and regulation of mCII activity and could play a key role in oxidative energy metabolism and explain the vulnerability of the striatum in neurodegenerative diseases.

Neuroprotection by Hsp104 and Hsp27 in Lentiviral-based Rat Models of Huntington's Disease

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of glutamine repeats in the huntingtin (htt) protein. Abnormal protein folding and the accumulation of mutated htt are hallmarks of HD neuropathology. Heat-shock proteins (hsps), which refold denatured proteins, might therefore mitigate HD. We show here that hsp104 and hsp27 rescue striatal dysfunction in primary neuronal cultures and HD rat models based on lentiviral-mediated overexpression of a mutated htt fragment. In primary rat striatal cultures, production of hsp104 or hsp27 with htt171-82Q restored neuronal nuclei (NeuN)-positive cell density to that measured after infection with vector expressing the wild-type htt fragment (htt171-19Q). In vivo, both chaperones significantly reduced mutated-htt-related loss of DARPP-32 expression. Furthermore, hsps affected the distribution and size of htt inclusions, with the density of neuritic aggregates being remarkably increased in striatal neurons overexpressing hsps. We also found that htt171-82Q induced the up-regulation of endogenous hsp70 that was co-localized with htt inclusions, and that the overexpression of hsp104 and hsp27 modified the subcellular localization of hsp70 that became cytoplasmic. Finally, hsp104 induced the production of endogenous hsp27. These data demonstrate the protective effects of chaperones in mammalian models of HD.

Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

Mechanisms involved in the formation of dopamine‐induced intracellular bodies within striatal neurons

Recent studies demonstrated that methamphetamine (METH) produces intracellular bodies which are reminiscent of those occurring during degenerative disorders. In vivo studies demonstrate the occurrence of these morphological alterations both in the dopamine (DA) neurons of the substantia nigra and striatal cells. These consist of neuronal bodies staining for a variety of antigens belonging to the ubiquitin-proteasome pathway. The formation of these intracellular bodies both in the substantia nigra and PC12 cells depends on the presence of endogenous DA. In the present study, we analyze the mechanisms which lead to METH-induced intracellular bodies within non-dopaminergic striatal neurons. We found that METH is no longer able to produce inclusions in vivo, in striatal cells, when striatal DA is lost. Similarly, in vitro, in primary striatal cell cultures which do not possess DA, METH administration does not produce inclusions. On the other hand, administration of DA to striatal cell cultures produces neuronal inclusions and cell death, which are both related to the inhibition of the ubiquitin-proteasome system and activation of specific-DA receptors. In line with this, we produced subcellular alterations by administering dopamine agonists.

Mice heterozygous for neurotrophin-3 display enhanced vulnerability to excitotoxicity in the striatum through increased expression of N-methyl-d-aspartate receptors

The striatum is one of the brain areas most vulnerable to excitotoxicity, a lesion that can be prevented by neurotrophins. In the present study, intrastriatal injection of the N-methyl-d-aspartate receptor (NMDAR) agonist quinolinate (QUIN) was performed in mice heterozygous for neurotrophin-3 (NT3 +/−) or brain-derived neurotrophic factor (BDNF +/−) to analyze the role of endogenous neurotrophins on the regulation of striatal neurons susceptibility to excitotoxic injury. QUIN injection induced a decrease in dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) protein levels that was higher in NT-3 +/− than in BDNF+/− or wild type animals. This enhanced susceptibility was specific for enkephalin- and tachykinin-positive projection neurons, and also for parvalbumin-positive interneurons. However the excitotoxic damage in large interneurons was not modified in NT-3 +/− mice compared with wild type animals. This effect can be related to the regulation of NMDARs by endogenous NT-3. Thus, our results show that there is an age-dependent regulation of NMDAR subunits NR1 and NR2A, but not NR2B, in NT-3 +/− mice. The deficit of endogenous NT-3 induced a decrease in NR1 and NR2A subunits at postnatal day (P) 0 and P3 mice respectively, whereas an upregulation was observed in 12 week old NT-3 +/− mice. This differential effect was also observed after administration of exogenous NT-3. In primary striatal cultures, NT-3 treatment induced an enhancement in NR2A, but not NR2B, protein levels. However, intrastriatal grafting of NT-3 secreting-cells in adult wild type mice produced a down-regulation of NR2A subunit. In conclusion, NT-3 regulates the expression of NMDAR subunits modifying striatal neuronal properties that confers the differential vulnerability to excitotoxicity in projection neurons and interneurons in the striatum.

Sp1 Is Up-regulated in Cellular and Transgenic Models of Huntington Disease, and Its Reduction Is Neuroprotective

Interactions between mutant huntingtin (Htt) and a variety of transcription factors including specificity proteins (Sp) have been suggested as a central mechanism in Huntington disease (HD). However, the transcriptional activity induced by Htt in neurons that triggers neuronal death has yet to be fully elucidated. In the current study, we characterized the relationship of Sp1 to Htt protein aggregation and neuronal cell death. We found increased levels of Sp1 in neuronal-like PC12 cells expressing mutant Htt, primary striatal neurons, and brain tissue of HD transgenic mice. Sp1 levels were also elevated when 3-nitropropionate (3-NP) was used to induce cell death in PC12 cells. To assess the effects of knocking down Sp1 in HD pathology, we used Sp1 siRNA, a heterozygous Sp1 knock-out mouse, and mithramycin A, a DNA-intercalating agent that inhibits Sp1 function. The three approaches consistently yielded reduced levels of Sp1 which ameliorated toxicity caused by either mutant Htt or 3-NP. In addition, when HD mice were crossed with Sp1 heterozygous knock-out mice, the resulting offspring did not experience the loss of dopamine D2 receptor mRNA characteristic of HD mice, and survived longer than their HD counterparts. Our data suggest that enhancement of transcription factor Sp1 contributes to the pathology of HD and demonstrates that its suppression is beneficial.

Galanin attenuates cyclic AMP regulatory element‐binding protein (CREB) phosphorylation induced by chronic morphine and naloxone challenge in Cath.a cells and primary striatal cultures

Repeated morphine administration leads to molecular alterations of the neural circuitry in the locus coeruleus and nucleus accumbens. These changes include increased activity of several components of the cAMP signaling pathway that are thought to be associated with psychological and somatic signs of opiate withdrawal. The neuropeptide galanin has been shown to attenuate cAMP signaling in multiple cell types. The current study demonstrates that acute galanin treatment blocks the consequences of increased cAMP signaling following chronic opiate administration and withdrawal in Cath.a cells and primary cultures of striatal neurons as measured by phosphorylation of the transcription factor cAMP regulatory element-binding protein (CREB). In addition, galanin-mediated attenuation of CREB phosphorylation is independent of galanin-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in Cath.a cells. These data suggest that galanin receptors may serve as an additional potential therapeutic target for the treatment of opiate withdrawal.

Memantine attenuates staurosporine-induced activation of caspase-3 and LDH release in mouse primary neuronal cultures

Developmental aspects of pro- and antiapoptotic action of some NMDA receptor antagonists in the central nervous system have been postulated. In order to further elucidate this problem, we investigated effect of memantine, an uncompetitive NMDA receptor antagonist and staurosporine alone and in combination on caspase-3 activity and lactate dehydrogenase (LDH) release in primary hippocampal, neocortical and striatal cell cultures on 7 and 12 days in vitro. The data showed that the vulnerability of neuronal cells to induction of caspase-3 activity by staurosporine was higher on 7 DIV than on 12 DIV, whereas staurosporine-mediated LDH release increased with days in vitro in striatal culture only. A specific inhibitor of caspase-3, AcDEVDCHO (60 μM), completely abolished the effect of staurosporine on this enzyme's activity, but only partially attenuated staurosporine-induced LDH release in hippocampal cells. Memantine alone (0.05–2.0 μM) did not induce any cytototoxic effect but attenuated the staurosporine-induced caspase-3 activity and LDH release in hippocampal cultured neurons on each investigated day in vitro. In striatal culture, memantine had a moderate inhibitory effect on staurosporine-evoked LDH release only on 7 DIV with no significant influence on caspase-3 activity. As for neocortical cultures, memantine partially inhibited staurosporine-induced neuronal injury only on 7 DIV. These data showed that the induction of caspase-3 activity by staurosporine was more profound in immature cells, however, the staurosporine neurotoxicity, as reflected by LDH release, only partially depended on caspase-3 activation and stage of cell development. Furthermore, memantine attenuated staurosporine-induced apoptosis more efficiently in hippocampal cultures than in neocortical and striatal ones, which points to tissue specificity of effects of this neuroprotectant.

FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington's disease

There is no satisfactory treatment for Huntington's disease (HD), a hereditary neurodegenerative disorder that produces chorea, dementia, and death. One potential treatment strategy involves the replacement of dead neurons by stimulating the proliferation of endogenous neuronal precursors (neurogenesis) and their migration into damaged regions of the brain. Because growth factors are neuroprotective in some settings and can also stimulate neurogenesis, we treated HD transgenic R6/2 mice from 8 weeks of age until death by s.c. administration of FGF-2. FGF-2 increased the number of proliferating cells in the subventricular zone by approximately 30% in wild-type mice, and by approximately 150% in HD transgenic R6/2 mice. FGF-2 also induced the recruitment of new neurons from the subventricular zone into the neostriatum and cerebral cortex of HD transgenic R6/2 mice. In the striatum, these neurons were DARPP-32-expressing medium spiny neurons, consistent with the phenotype of neurons lost in HD. FGF-2 was neuroprotective as well, because it blocked cell death induced by mutant expanded Htt in primary striatal cultures. FGF-2 also reduced polyglutamine aggregates, improved motor performance, and extended lifespan by approximately 20%. We conclude that FGF-2 improves neurological deficits and longevity in a transgenic mouse model of HD, and that its neuroprotective and neuroproliferative effects may contribute to this improvement.

Amyloid β‐peptide(1–42) and hydrogen peroxide‐induced toxicity are mediated by TRPM2 in rat primary striatal cultures

Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.

Progressive and selective striatal degeneration in primary neuronal cultures using lentiviral vector coding for a mutant huntingtin fragment

A lentiviral vector expressing a mutant huntingtin protein (htt171-82Q) was used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed the transgene so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein Hsp70, and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post-infection, a significant increase in TUNEL-positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether the trophic factors CNTF and BDNF that were found neuroprotective in acute HD models could prevent striatal degeneration in a chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that viral vectors coding for mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.

953. Progressive and Selective Striatal Degeneration in Primary Neuronal Cultures Using Lentiviral Vector Coding Mutant Huntingtin Fragment

Top of pageAbstract Lentiviral vectors expressing a mutant huntingtin protein (htt171-82Q) were used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed mutant huntingtin so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein HSP70 and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post infection, a significant increase in TUNEL|[ndash]|positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether, the trophic factors CNTF and BDNF which were found neuroprotective in acute HD models could prevent striatal degeneration in the present chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that using viral vectors coding for mutated Htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.

Adeno‐associated virus‐mediated gene transfer of human aromatic l‐amino acid decarboxylase protects mixed striatal primary cultures from l‐DOPA toxicity

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.

Role of Protein Phosphatase 2A in mGluR5-regulated MEK/ERK Phosphorylation in Neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

Molecular mechanisms involved in the adenosine A1 and A2A receptor‐induced neuronal differentiation in neuroblastoma cells and striatal primary cultures

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.

Serofendic acid, a sulfur-containing diterpenoid derived from fetal calf serum, attenuates reactive oxygen species-induced oxidative stress in cultured striatal neurons.

We previously identified a novel endogenous substance, serofendic acid, from a lipophilic extract of fetal calf serum. Serofendic acid protects cultured cortical neurons against the cytotoxicity of glutamate and nitric oxide. Here, we reported the protective effect of serofendic acid on reactive oxygen species-induced oxidative stress using primary rat striatal cultures. In addition, we compared the neuroprotective effect and the radical-scavenging activity of serofendic acid with those of dimethyl sulfoxide (DMSO), because serofendic acid possesses a DMSO structure. Paraquat caused neuronal death, which was inhibited by a cell-permeable superoxide dismutase (SOD) mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (Mn-TBAP); a cell-permeable SOD/catalase mimetic, EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride]; and a ferrous ion chelator, 2,2'-dipyridyl, in rat striatal cultures. Serofendic acid (10-100 microM) suppressed the neurotoxicity of paraquat, whereas DMSO (10-100 microM) did not. By contrast, higher concentrations (30-300 mM) of DMSO ameliorated the paraquat-induced cell death. Furthermore, H(2)O(2) induced neurotoxicity, which was prevented by EUK-134 and 2,2'-dipyridyl. Serofendic acid (10-100 microM) also protected striatal neurons against the H(2)O(2)-induced toxicity. Higher concentrations (30-300 mM) of DMSO ameliorated H(2)O(2)-induced neuronal death, whereas lower concentrations (10-100 microM) did not. Electron spin resonance spectrometry with a spin-trapping technique revealed that serofendic acid and DMSO had approximately the same ability to inhibit the formation of the hydroxyl radical (.OH). These results suggest that the.OH-scavenging activity of serofendic acid is attributable to its DMSO structure and that the remaining components such as the atisane structure play an important role in eliciting neuroprotection at a concentration range of 10 to 100 microM.

Transcriptional activation of phosphodiesterase 7B1 by dopamine D1 receptor stimulation through the cyclic AMP/cyclic AMP-dependent protein kinase/cyclic AMP-response element binding protein pathway in primary striatal neurons.

Phosphodiesterase (PDE) 7B, a cAMP-specific PDE which is dominantly expressed in striatum, is expected to be involved in dopaminergic signaling in striatal neurons. Here we show, for the first time, the involvement of the dopaminergic signaling pathway in transcriptional activation of rat PDE7B in primary striatal culture. RT-PCR analysis revealed that dopamine, D1 agonist, forskolin and 8-Br-cAMP stimulation potentiated PDE7B transcription in striatal neurons, while D2 agonist failed to activate the PDE7B transcription. Pre-treatment with D1 antagonist abolished the dopamine- or D1 agonist-induced transcriptional activation of PDE7B. The activation of PDE7B transcription by these stimulators was completely ablated by pre-treatment of the cells with a cAMP-dependent protein kinase inhibitor, H-89. RT-PCR using splice variant-specific primers revealed that transcription of PDE7B1, but not of other splice variants, was activated by D1 agonist. We determined the putative transcription start site of PDE7B1, a brain-specific splice variant of PDE7B, by 5'-RACE and identified a promoter region of PDE7B1. Sequence analysis of the PDE7B1 promoter revealed the presence of a canonical cAMP-response element at 166 bp upstream of the putative transcription start site. The cAMP-responsiveness of the PDE7B1 promoter was demonstrated by functional promoter analysis using the luciferase reporter system. Deletion and mutation of the cAMP-response element site in the PDE7B1 promoter abolished the forskolin-induced activation of the PDE7B1 promoter activity. Electrophoretic mobility shift assay showed the binding of cAMP-response element binding protein to the PDE7B1 promoter. These data demonstrate the dopamine D1 receptor-mediated transcriptional activation of PDE7B through the cAMP/cAMP-dependent protein kinase/cAMP-response element binding protein pathway in striatal neurons.

Caffeine regulates neuronal expression of the dopamine 2 receptor gene.

The psychoactive drug caffeine influences neuronal physiology; however, it is unknown whether it can dynamically alter the expression of genes that influence neurotransmission. Here, we report that caffeine stimulates transcription of the dopamine 2 receptor (D2R) gene in PC-12 cells and primary striatal cultures and increases D2R protein expression in the striatum. Physiological doses of caffeine and the specific adenosine 2A receptor antagonist 8-(3-chlorostyryl) caffeine both increased the activity of a D2R/luciferase reporter construct within 24 h, and simultaneous treatment with 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), a specific adenosine 2A receptor agonist, eliminated this effect. Tests of additional constructs revealed that specific regions of the D2R promoter (-117/-75) and 5'-untranslated region (+22/+317) were required for activation of D2R gene expression by caffeine. In primary striatal cultures, caffeine increased spontaneous firing of neurons between 12 and 80 min after treatment, whereas it increased D2R mRNA expression after only 4 h. These results indicate that regulation of D2R gene expression by caffeine occurs after the initial physiological response has subsided. In vivo, female mice treated with a dose of caffeine (50 mg/kg) showed 1.94- and 2.07-fold increases in D2R mRNA and protein expression, respectively. In contrast, male mice exhibited a 31% decrease in D2R mRNA expression and showed no changes in D2R protein expression. Collectively, these results demonstrate for the first time that caffeine alters D2R expression in neurons. They also suggest that caffeine consumption can lead to sexually dimorphic patterns of gene expression in the brain.

D2 dopamine receptors modulate Galpha-subunit coupling of the CB1 cannabinoid receptor.

CB(1) cannabinoid (CB(1)) and D(2) dopamine (D(2)) receptors are known to couple to the G protein Galpha(i/o). It has been reported that concurrent activation of D(2) receptors and CB(1) receptors, in primary striatal neuronal culture, promotes functional CB(1) receptor coupling to Galpha(s) resulting in elevations in intracellular cyclic AMP levels. We now report that in the absence of D(2) receptors, acute activation of CB(1) receptors inhibits cyclic AMP accumulation, whereas the presence of D(2) receptors promotes CB(1)-stimulated cAMP accumulation, presumably through Galpha(s). This Galpha(s) subunit switching was not prevented by pertussis toxin treatment and occurred in the presence and absence of D(2) receptor activation. Thus, coexpression of the D(2) receptor with the CB(1) receptor was sufficient to switch the coupling of the CB(1) receptors from Galpha(i/o) to Galpha(s). Persistent activation of D(2) receptors resulted in heterologous sensitization of adenylate cyclase to subsequent stimulation by forskolin, whereas the persistent activation of CB(1) receptors did not. Additional studies in human embryonic kidney cells cotransfected with D(2) and CB(1) receptors revealed that persistent activation (18 h) of D(2) receptors induced a switch of CB(1) receptor coupling from Galpha(s) to Galpha(i/o). This D(2) receptor-induced effect allowed for CB(1) receptor-mediated inhibition of cyclic AMP accumulation. The present studies suggest D(2) receptors may have a significant modulatory role in determining the G protein coupling specificity of CB(1) receptors.

Primary striatal neuronal culture.

AbstractThe incredible in vivo complexity of nervous system and activity oftentimes prevents the neuroscientist from attempting to understand how individual neural cells or a specific phenotype of neural cells contribute to a given function of the nervous system. To elucidate intercellular and intracellular mechanisms, the complexity therefore needs to be reduced experimentally. Primary neural culture is an in vitro approach attempting to reduce this complexity, and establishes a relatively purified model for studying the nervous system at a single cell level. Because the culture environment can be readily controlled, neural cultures can be set up to possess biological properties similar or distinct to the original tissue. Research data from the culture study can therefore be valuable to predict the behavior of specific neural cells in vivo.KeywordsStriatal TissueMitotic InhibitorNeural CultureSpecific Neural CellLarge Pale NucleusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.