Ventral striatal regulation of CREM mediates impulsive action and drug addiction vulnerability.

Impulsivity, a multifaceted behavioral hallmark of attention-deficit/hyperactivity disorder (ADHD), strongly influences addiction vulnerability and other psychiatric disorders that incur enormous medical and societal burdens yet the neurobiological underpinnings linking impulsivity to disease remain poorly understood. Here we report the critical role of ventral striatal cAMP-response element modulator (CREM) in mediating impulsivity relevant to drug abuse vulnerability. Using an ADHD rat model, we demonstrate that impulsive animals are neurochemically and behaviorally more sensitive to heroin and exhibit reduced Crem expression in the nucleus accumbens core. Virally increasing Crem levels decreased impulsive action, thus establishing a causal relationship. Genetic studies in seven independent human populations illustrate that a CREM promoter variant at rs12765063 is associated with impulsivity, hyperactivity and addiction-related phenotypes. We also reveal a role of Crem in regulating striatal structural plasticity. Together, these results highlight that ventral striatal CREM mediates impulsivity related to substance abuse and suggest that CREM and its regulated network may be promising therapeutic targets.

Dopamine receptor D1 and postsynaptic density gene variants associate with opiate abuse and striatal expression levels.

Opioid drugs are highly addictive and their abuse has a strong genetic load. Dopamine-glutamate interactions are hypothesized to be important for regulating neural systems central for addiction vulnerability. Balanced dopamine-glutamate interaction is mediated through several functional associations, including a physical link between discs, large homolog 4 (Drosophila) (DLG4, PSD-95) and dopamine receptor 1 (DRD1) within the postsynaptic density to regulate DRD1 trafficking. To address whether genetic associations with heroin abuse exist in relation to dopamine and glutamate and their potential interactions, we evaluated single-nucleotide polymorphisms of key genes within these systems in three populations of opiate abusers and controls, totaling 489 individuals from Europe and the United States. Despite significant differences in racial makeup of the separate samples, polymorphisms of DRD1 and DLG4 were found to be associated with opiate abuse. In addition, a strong gene-gene interaction between homer 1 homolog (Drosophila) (HOMER1) and DRD1 was predicted to occur in Caucasian subjects. This interaction was further analyzed by evaluating DRD1 genotype in relation to HOMER1b/c protein expression in postmortem tissue from a subset of Caucasian subjects. DRD1 rs265973 genotype correlated with HOMER1b/c levels in the striatum, but not cortex or amygdala; the correlation was inversed in opiate abusers as compared with controls. Cumulatively, these results support the hypothesis that there may be significant, genetically influenced interactions between glutamatergic and dopaminergic pathways in opiate abusers.

Improving child protection: a systematic review of training and procedural interventions.

AIM: To synthesise published evidence regarding the effectiveness of training and procedural interventions aimed at improving the identification and management of child abuse and neglect by health professionals. METHODS: Systematic review for the period 1994 to 2005 of studies that evaluated child protection training and procedural interventions. Main outcome measures were learning achievement, attitudinal change, and clinical behaviour. RESULTS: Seven papers that examined the effectiveness of procedural interventions and 15 papers that evaluated training programmes met the inclusion criteria. Critical appraisal showed that evaluation of interventions was on the whole poor. It was found that certain procedural interventions (such as the use of checklists and structured forms) can result in improved recording of important clinical information and may also alert clinical staff to the possibility of abuse. While a variety of innovative training programmes were identified, there was an absence of rigorous evaluation of their impact. However a small number of one-group pre- and post-studies suggest improvements in a range of attitudes necessary for successful engagement in the child protection process. CONCLUSION: Current evidence supports the use of procedural changes that improve the documentation of suspected child maltreatment and that enhance professional awareness. The lack of an evidence based approach to the implementation of child protection training may restrict the ability of all health professionals to fulfil their role in the child protection process. Formal evaluation of a variety of models for the delivery of this training is urgently needed with subsequent dissemination of results that highlight those found to be most effective.

The dopamine transporter gene (SLC6A3) variable number of tandem repeats domain enhances transcription in dopamine neurons.

The dopamine (DAT) and serotonin (SERT) transporter genes both contain variable number of tandem repeats (VNTR) in non-coding gene regions which have been correlated with a predisposition to a variety of CNS disorders. There is considerable homology between individual DAT and SERT repeat DNA sequences, which is reflected in their ability to compete with each other for specific protein binding as demonstrated by electrophoretic mobility shift assay. The SERT VNTR has recently been shown to act as a transcriptional enhancer. Because of the similarities between SERT and DAT VNTRs, the DAT VNTR may also enhance transcription. This study demonstrates by lipid transfection into an immortalized dopaminergic cell line and biolistic transfection into dopamine neurons in neonatal rat midbrain slices that the human nine-repeat DAT VNTR can enhance transcription. This enhancing activity suggests that the DAT VNTR may play a role in regulation of DAT gene expression.

The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders.

The dopamine transporter is a plasma membrane protein that controls the spatial and temporal domains of dopamine neurotransmission through the accumulation of extracellular dopamine. The dopamine transporter may play a role in numerous dopamine-linked neuropsychiatric disorders. We review the cloning and organization of the human dopamine transporter gene, polymorphisms in its coding and noncoding sequence, and emerging data on its transcriptional regulation.

Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism.

The importance of the nuclear receptor nurr1 for the appropriate development of mesencephalic dopamine-synthesizing neurons has been clearly demonstrated through the targeted disruption of the nurr1 gene. The persistence of nurr1 expression in adult tissue suggests a possible role for this transcription factor in the maintenance, as well as development, of the dopaminergic phenotype. To address this issue, we analyzed the effects of nurr1 on the transcriptional expression of the human dopamine transporter gene (hDAT), one of the most specific phenotypic markers for dopaminergic neurons. Nurr1 enhanced the transcriptional activity of hDAT gene constructs transiently transfected into a newly described cell line (SN4741) that expresses a dopaminergic phenotype, whereas other members of the NGFI-B subfamily of nuclear receptors had lesser or no effects. Nurr1 activation of hDAT was not dependent upon heterodimerization with the retinoid X receptor. Unexpectedly, functional analysis of a series of gene constructs revealed that a region of the hDAT 5'-flanking sequence devoid of NGFI-B response element (NBRE)-like sites mediated nurr1 activation. Additional experiments using a nurr1 mutant construct suggest that nurr1 activates hDAT transcription via a novel NBRE-independent mechanism.

Real-time analysis of preprotachykinin promoter activity in single cortical neurons.

Technological limitations have hindered the study of gene elements regulating transcription within CNS neurons. In the present stuides, rat cortical brain slices endogenously expressing the preprotachykinin (PPT) gene were transfected with gene constructs encompassing green fluorescent protein (GFP) under the control of the PPT promoter. These slices were maintained in organotypic culture so that the fluorescence intensity within individual living cells could be quantified using laser scanning confocal microscopy before and after application of stimulatory agents. Combined treatment with forskolin and elevated potassium significantly increased expression of both endogenous PPT mRNA and the PPT promoter-GFP construct. The ability to follow fluorescence changes within single neurons in real time offers a powerful "within-subject" experimental approach for analysis of neural gene promoters.

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration.

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.

Stress-induced C-fos expression in the rat locus coeruleus is dependent on neurokinin 1 receptor activation.

These experiments examined the role of substance P-selective neurokinin 1 receptors in the restraint-induced activation of the rat locus coeruleus. Immunohistochemistry revealed high levels of neurokinin 1 receptor expression in the plasma membrane of tyrosine hydroxylase-positive locus coeruleus neurons. The selective neurokinin 1 receptor antagonists, RP 67580 (5 nmol) and L-760,735 (3.4 nmol), were administered intracerebroventricularly prior to restraint stress, and c-fos protein was measured as an index of locus coeruleus activation. Both antagonists attenuated the restraint-induced increase in locus coeruleus c-fos expression, whereas their inactive enantiomers were ineffective. These results suggest that neurokinin 1 receptors may mediate activation of locus coeruleus neurons during stress. Neurokinin 1 receptor antagonists may prove to be novel therapeutic compounds in the treatment of anxiety and depression.

Characterization of the 5'-flanking region of the human dopamine transporter gene.

The dopamine transporter (DAT) plays a major role in modulating dopamine (DA) neurotransmission by controlling the levels of this neurotransmitter in the extracellular space. We have isolated 8.3 kb of the 5'-flanking regulatory region of the human DAT (hDAT) gene and identified numerous potential elements involved in transcriptional control of the DAT. A series of hDAT-luciferase reporter constructs encompassing increasing amounts of 5'-flanking sequence was utilized in transient transfection assays assessing basal activity and response to selected stimuli. Our results suggest that the proximal hDAT 5'-flanking region displays a strong, nonselective promoter activity that is silenced through regulatory elements present in the distal portion of the 5'-flanking sequence. Although potential cyclic AMP responsive elements (CRE) were identified on the sequence, hDAT constructs were unresponsive to cyclic AMP induction. The transcription factor nurr1 increases the transcriptional activity of several larger hDAT constructs, consistent with the presence of several putative NGFI-B response elements (NBRE). The cloning and functional analysis of an extensive portion of the 5'-flanking regulatory region of the hDAT gene provides further insights into the factors involved in the regulation of this gene.

Molecular characterization of human and rat RGS 9L, a novel splice variant enriched in dopamine target regions, and chromosomal localization of the RGS 9 gene.

A novel splice variant of RGS 9 was isolated from a rat hypothalamus, human retina, and a human kidney (Wilm's) tumor. This variant, termed RGS 9L, differs from the retinal form (termed RGS 9S) identified previously in that it contains a 211- (rat) or 205- (human) amino acid proline-rich domain on the carboxyl terminus. The pattern of RGS 9 mRNA splicing was tissue specific, with striatum, hypothalamus- and nucleus accumbens expressing RGS 9L, whereas retina and pineal expressed RGS 9S almost exclusively. This pattern of mRNA splicing seemed to be highly conserved between human and rodents, suggesting cell-specific differences in the function of these variants. Transient expression of RGS 9L augmented basal and beta-adrenergic receptor-stimulated adenylyl cyclase activity while suppressing dopamine D2 receptor-mediated inhibition. Furthermore, RGS 9L expression greatly accelerated the decay of dopamine D2 receptor-induced GIRK current. These results indicate RGS 9L inhibits heterotrimeric Gi function in vivo, probably by acting as a GTPase-activating protein. The human RGS 9 gene was localized to chromosome 17 q23-24 by radiation hybrid and fluorescent in situ hybridization analyses. The RGS 9 gene is within a previously defined locus for retinitis pigmentosa (RP 17), a disease that has been linked to genes in the rhodopsin/transducin/cGMP signaling pathway.

Tachykinin NK1 receptor antagonists enhance stress-induced c-fos in rat locus coeruleus.

These experiments tested the hypothesis that substance P neurotransmission at tachykinin NK1 receptors in the locus coeruleus is involved in stress-induced activation of the locus coeruleus, using c-fos as an index of activation. Selective tachykinin NK1 receptor antagonists administered systemically did not result in substantial locus coeruleus c-fos expression. Restraint stress resulted in a large number of locus coeruleus c-fos expressing cells. Administration of two selective tachykinin NK1 receptor antagonists prior to restraint resulted in an increase in the number of locus coeruleus c-fos expressing cells, compared to restraint alone. These results suggest that the enhanced c-fos expression observed in response to tachykinin NK1 receptor antagonists combined with stress, could be due to the blockade of tachykinin NK1 receptor-mediated activity at sites other than the locus coeruleus, resulting in an overall activation of the locus coeruleus.

Regulators of G protein signaling: rapid changes in mRNA abundance in response to amphetamine.

This study examined mRNAs encoding regulators of G protein signaling (RGSs) expressed within the striatum and determined whether their expression in the caudate putamen was altered by amphetamine. RT-PCR techniques were used to clone cDNA probes of RGSs expressed within the rat striatum. Northern blot analysis of caudate putamen and nucleus accumbens RNA determined the relative abundance of RGS mRNA expressed within the caudate putamen and adjacent nucleus accumbens to be RGS 2 > RGS 5 > RGS 16 > RGS 4 = RGS 9 > RGS 8 = RGS 3. A single injection of amphetamine rapidly and transiently induced RGS 2 mRNA. The temporal pattern of induction of RGS 2 strongly resembled that of the immediate early gene c-fos. Levels of mRNAs of RGS 3 and 5 steadily increased over a 4-h interval, as did that of the 6.6-kb transcript of RGS 8. The level of RGS 9 mRNA, which shows strong striatal-specific expression, steadily decreased over a 4-h interval, whereas RGS 4 and 16 and the 3.9-kb transcript of RGS 8 were not significantly affected at any point examined. The ability of amphetamine to alter RGS mRNA expression within the caudate putamen suggests these proteins may play an important role in adaptive processes to psychostimulant exposure.

Differential modification of dopamine transporter and tyrosine hydroxylase mRNAs in midbrain of subjects with Parkinson's, Alzheimer's with parkinsonism, and Alzheimer's disease.

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group in accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43-53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.

Serotonin, dopamine and norepinephrine transporter mRNAs: heterogeneity of distribution and response to 'binge' cocaine administration.

Quantitative in situ hybridization was utilized to map the distribution and abundance of the serotonin, dopamine and norepinephrine transporter (SERT, DAT and NET, respectively) mRNAs. SERT mRNA was quantified within the dorsal raphe (DR) and the median raphe (MR), DAT mRNA within the ventral tegmental area -substantia nigra (VTA-SN) region and NET mRNA within the locus coeruleus (LC). SERT mRNA expression within the raphe complex was organized into distinct subregional domains with the rank order of mRNA abundance: ventromedial (vm) DR > dorsomedial (dm) DR > MR > dorsolateral (dl) DR. The relative abundance of DAT mRNA also varied across subregions: SN pars compacta > the parabrachial pigmentosis (PBP) > the intrafascicular (IF). The effects of a 'binge' paradigm of cocaine administration on SERT, DAT and NET mRNA abundance were compared in the brains of behaviorally sensitized rats. Cocaine significantly decreased the abundance of the SERT mRNA within the dlDR and DAT mRNA abundance within the SNc and the PBP, and increased the abundance of the NET mRNA within the LC. Finally, correlational analysis indicated that post-cocaine levels of DAT, SERT and NET mRNAs were not associated with cocaine-induced sensitization.