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A questionnaire survey undertaken among 142 schoolteachers in North Staffordshire revealed most of the respondents did not feel confident when teaching children who had epilepsy and a minority considered their knowledge of the subject to be adequate. Only four teachers had received recent specific instruction on childhood epilepsy and the majority requested training on epilepsy and other medical conditions. Despite this lack of confidence and specific training, the respondents demonstrated good general knowledge of epilepsy and adequate awareness of the difficulties encountered by epileptic schoolchildren. If optimal care is to be achieved for children with epilepsy, then teachers must feel confident with this subject. School health services have a clear role in ensuring that teachers have sufficient knowledge of childhood epilepsy, that they have adequate support, and that communication between teachers, parents, and paediatricians is encouraged. [ABSTRACT FROM AUTHOR]

JOURNAL/nrgr/04.03/01300535-202409000-00039/figure1/v/2024-01-16T170235Z/r/image-tiff Parkinsonism by unilateral, intranigral β-sitosterol β-D-glucoside administration in rats is distinguished in that the α-synuclein insult begins unilaterally but spreads bilaterally and increases in severity over time, thus replicating several clinical features of Parkinson's disease, a typical α-synucleinopathy. As Nurr1 represses α-synuclein, we evaluated whether unilateral transfected of rNurr1-V5 transgene via neurotensin-polyplex to the substantia nigra on day 30 after unilateral β-sitosterol β-D-glucoside lesion could affect bilateral neuropathology and sensorimotor deficits on day 30 post-transfection. This study found that rNurr1-V5 expression but not that of the green fluorescent protein (the negative control) reduced β-sitosterol β-D-glucoside-induced neuropathology. Accordingly, a bilateral increase in tyrosine hydroxylase-positive cells and arborization occurred in the substantia nigra and increased tyrosine hydroxylase-positive ramifications in the striatum. In addition, tyrosine hydroxylase-positive cells displayed less senescence marker β-galactosidase and more neuron-cytoskeleton marker βIII-tubulin and brain-derived neurotrophic factor. A significant decrease in activated microglia (positive to ionized calcium-binding adaptor molecule 1) and neurotoxic astrocytes (positive to glial fibrillary acidic protein and complement component 3) and increased neurotrophic astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10) also occurred in the substantia nigra. These effects followed the bilateral reduction in α-synuclein aggregates in the nigrostriatal system, improving sensorimotor behavior. Our results show that unilateral rNurr1-V5 transgene expression in nigral dopaminergic neurons mitigates bilateral neurodegeneration (senescence and loss of neuron-cytoskeleton and tyrosine hydroxylase-positive cells), neuroinflammation (activated microglia, neurotoxic astrocytes), α-synuclein aggregation, and sensorimotor deficits. Increased neurotrophic astrocytes and brain-derived neurotrophic factor can mediate the rNurr1-V5 effect, supporting its potential clinical use in the treatment of Parkinson's disease., (Copyright © 2024 Copyright: © 2024 Neural Regeneration Research.)

Dynamic interactions of neurons and glia in the ventral midbrain mediate reward and addiction behavior. We studied gene expression in 212,713 ventral midbrain single nuclei from 95 individuals with history of opioid misuse, and individuals without drug exposure. Chronic exposure to opioids was not associated with change in proportions of glial and neuronal subtypes, however glial transcriptomes were broadly altered, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes. Genes associated with activation of the immune response including interferon, NFkB signaling, and cell motility pathways were upregulated, contrasting with down-regulated expression of synaptic signaling and plasticity genes in ventral midbrain non-dopaminergic neurons. Ventral midbrain transcriptomic reprogramming in the context of chronic opioid exposure included 325 genes that previous genome-wide studies had linked to risk of substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry., (© 2023. Springer Nature Limited.)

Overexpression of neurotrophic factors in nigral dopamine neurons is a promising approach to reverse neurodegeneration of the nigrostriatal dopamine system, a hallmark in Parkinson's disease. The human cerebral dopamine neurotrophic factor (hCDNF) has recently emerged as a strong candidate for Parkinson's disease therapy. This study shows that hCDNF expression in dopamine neurons using the neurotensin-polyplex nanoparticle system reverses 6-hydroxydopamine-induced morphological, biochemical, and behavioral alterations. Three independent electron microscopy techniques showed that the neurotensin-polyplex nanoparticles containing the hCDNF gene, ranging in size from 20 to 150 nm, enabled the expression of a secretable hCDNF in vitro. Their injection in the substantia nigra compacta on day 21 after the 6-hydroxydopamine lesion resulted in detectable hCDNF in dopamine neurons, whose levels remained constant throughout the study in the substantia nigra compacta and striatum. Compared with the lesioned group, tyrosine hydroxylase-positive (TH + ) nigral cell population and TH + fiber density rose in the substantia nigra compacta and striatum after hCDNF transfection. An increase in βIII-tubulin and growth-associated protein 43 phospho-S41 (GAP43p) followed TH + cell recovery, as well as dopamine and its catabolite levels. Partial reversal (80%) of drug-activated circling behavior and full recovery of spontaneous motor and non-motor behavior were achieved. Brain-derived neurotrophic factor recovery in dopamine neurons that also occurred suggests its participation in the neurotrophic effects. These findings support the potential of nanoparticle-mediated hCDNF gene delivery to develop a disease-modifying treatment against Parkinson's disease. The Institutional Animal Care and Use Committee of Centro de Investigación y de Estudios Avanzados approved our experimental procedures for animal use (authorization No. 162-15) on June 9, 2019., Competing Interests: None

Cocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn 2+ ) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn 2+ concentrations in postmortem brain (caudate) tissue from humans who died of cocaine overdose were significantly lower than in control subjects. Moreover, the level of striatal Zn 2+ content in these subjects negatively correlated with plasma levels of benzoylecgonine, a cocaine metabolite indicative of recent use. In mice, repeated cocaine exposure increased synaptic Zn 2+ concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn 2+ were dependent on the Zn 2+ transporter 3 (ZnT3), a neuronal Zn 2+ transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn 2+ . ZnT3 KO mice showed significantly lower electrically evoked DA release and greater DA clearance when exposed to cocaine compared to controls. ZnT3 KO mice also displayed significant reductions in cocaine locomotor sensitization, conditioned place preference (CPP), self-administration, and reinstatement compared to control mice and were insensitive to cocaine-induced increases in striatal DAT binding. Finally, dietary Zn 2+ deficiency in mice resulted in decreased striatal Zn 2+ content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn 2+ release and turnover/metabolism in the striatum, and that synaptically released Zn 2+ potentiates the effects of cocaine on striatal DA neurotransmission and behavior and is required for cocaine-primed reinstatement. In sum, these findings reveal new insights into cocaine's pharmacological mechanism of action and suggest that Zn 2+ may serve as an environmentally derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: A high proportion of opioid drug deaths involve concurrent benzodiazepine use. To reduce the risk of drug overdose, various prescription drug monitoring programs have been implemented. This study examined the impact of concurrent benzodiazepine use on opioid-related deaths, and the utility of the Michigan Automated Prescription System (MAPS) in predicting risk of opioid death., Methods: Wayne County Medical Examiner's Office cases from 2018 were examined in terms of MAPS data and MAPS-derived drug risk scores, as well as postmortem toxicology. Opioid death cases with concurrent benzodiazepine use were compared to non-drug deaths., Results: For cases with a MAPS history for 6 months preceding death, the incidence of opioid prescriptions filled did not differ between groups. In contrast, significantly more opioid death cases had filled a benzodiazepine prescription; alprazolam prescription was the single best predictor of opioid drug death. Groups differed in MAPS-calculated drug risk scores, though these were less predictive of opioid death than some individual measures of prescription drug use. In terms of postmortem toxicology, fentanyl was the best discriminator between cohorts, with significant associations seen for morphine, benzodiazepine, or cocaine use. Similar results were obtained in the subset of subjects filling a prescription within a month of death, except that MAPS risk scores no longer predicted drug deaths., Conclusion: MAPS scores did not adequately predict risk of opioid-related death. Contrary to expectations, prescription opioid use was not correlated with opioid-related death, whereas concurrent use of opioids and benzodiazepines represented a highly significant risk factor., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Midbrain dopaminergic neurons (MDN) represent 0.0005% of the brain's neuronal population and mediate cognition, food intake, and metabolism. MDN are also posited to underlay the neurobiological dysfunction of schizophrenia (SCZ), a severe neuropsychiatric disorder that is characterized by psychosis as well as multifactorial medical co-morbidities, including metabolic disease, contributing to markedly increased morbidity and mortality. Paradoxically, however, the genetic risk sequences of psychosis and traits associated with metabolic disease, such as body mass, show very limited overlap., Methods: We investigated the genomic interaction of SCZ with medical conditions and traits, including body mass index (BMI), by exploring the MDN's "spatial genome," including chromosomal contact landscapes as a critical layer of cell type-specific epigenomic regulation. Low-input Hi-C protocols were applied to 5-10 × 10 3 dopaminergic and other cell-specific nuclei collected by fluorescence-activated nuclei sorting from the adult human midbrain., Results: The Hi-C-reconstructed MDN spatial genome revealed 11 "Euclidean hot spots" of clustered chromatin domains harboring risk sequences for SCZ and elevated BMI. Inter- and intra-chromosomal contacts interconnecting SCZ and BMI risk sequences showed massive enrichment for brain-specific expression quantitative trait loci (eQTL), with gene ontologies, regulatory motifs and proteomic interactions related to adipogenesis and lipid regulation, dopaminergic neurogenesis and neuronal connectivity, and reward- and addiction-related pathways., Conclusions: We uncovered shared nuclear topographies of cognitive and metabolic risk variants. More broadly, our PsychENCODE sponsored Hi-C study offers a novel genomic approach for the study of psychiatric and medical co-morbidities constrained by limited overlap of their respective genetic risk architectures on the linear genome.

Opioid abuse is now the most common cause of accidental death in the US. Although opioids and most other drugs of abuse acutely increase signaling mediated by midbrain dopamine (DA)-synthesizing neurons, little is known about long-lasting changes in DA cells that may contribute to continued opioid abuse, craving, and relapse. A better understanding of the molecular and cellular bases of opioid abuse could lead to advancements in therapeutics. This study comprises, to our knowledge, the first unbiased examination of genome-wide changes in midbrain gene expression associated with human opioid abuse. Our analyses identified differentially expressed genes and distinct gene networks associated with opioid abuse, specific genes with predictive capability for subject assignment to the opioid abuse cohort, and genes most similarly affected in chronic opioid and cocaine abusers. We also identified differentially expressed long noncoding RNAs capable of regulating known drug-responsive protein-coding genes. Opioid-regulated genes identified in this study warrant further investigation as potential biomarkers and/or therapeutic targets for human substance abuse.

Opioid abuse is now the primary cause of accidental deaths in the United States. Studies over several decades established the cyclical nature of abused drugs of choice, with a current resurgence of heroin abuse and, more recently, fentanyl's emergence as a major precipitant of drug-related deaths. To better understand abuse trends and to explore the potential lethality of specific drug-drug interactions, we conducted statistical analyses of forensic toxicological data from the Wayne County Medical Examiner's Office from 2012-2016. We observed clear changes in opioid abuse over this period, including the rapid emergence of fentanyl and its analogs as highly significant causes of lethality starting in 2014. We then used Chi-square Automatic Interaction Detector (CHAID)-based decision tree analyses to obtain insights regarding specific drugs, drug combinations, and biomarkers in blood most predictive of cause of death or circumstances surrounding death. The presence of the non-opioid drug acetaminophen was highly predictive of drug-related deaths, likely reflecting the abuse of various combined acetaminophen-opioid formulations. The short-lived cocaine adulterant levamisole was highly predictive of a short post-cocaine survival time preceding sudden non-drug-related death. The combination of the opioid methadone and the antidepressant citalopram was uniformly linked to drug death, suggesting a potential drug-drug interaction at the level of a pathophysiological effect on the heart and/or drug metabolism. The presence of fentanyl plus the benzodiazepine midazolam was diagnostic for in-hospital deaths following serious medical illness and interventions that included these drugs. These data highlight the power of decision tree analyses not only in the determination of cause of death, but also as a key surveillance tool to inform drug abuse treatment and public health policies for combating the opioid crisis.

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.

The human glial-cell derived neurotrophic factor (hGDNF) gene transfer by neurotensin (NTS)-polyplex nanoparticles functionally restores the dopamine nigrostriatal system in experimental Parkinson's disease models. However, high levels of sustained expression of GDNF eventually can cause harmful effects. Herein, we report an improved NTS-polyplex nanoparticle system that enables regulation of hGDNF expression within dopaminergic neurons. We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element (TRE). Another bifunctional plasmid contained the enhanced green fluorescent protein (GFP) gene. Transient transfection experiments in N1E-115-Nurr1 cells showed that doxycycline (100 ng/mL) activates hGDNF and GFP expression. Doxycycline (5 mg/kg, i.p.) administration in rats activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific doxycycline-regulated system suitable for nanomedicine-based treatment of Parkinson's disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Maintenance of the drug-addicted state is thought to involve changes in gene expression in different neuronal cell types and neural circuits. Midbrain dopamine (DA) neurons in particular mediate numerous responses to drugs of abuse. Long noncoding RNAs (lncRNAs) regulate CNS gene expression through a variety of mechanisms, but next to nothing is known about their role in drug abuse. The proportion of lncRNAs that are primate-specific provides a strong rationale for their study in human drug abusers. In this study, we determined a profile of dysregulated putative lncRNAs through the analysis of postmortem human midbrain specimens from chronic cocaine abusers and well-matched control subjects (n = 11 in each group) using a custom lncRNA microarray. A dataset comprising 32 well-annotated lncRNAs with independent evidence of brain expression and robust differential expression in cocaine abusers is presented. For a subset of these lncRNAs, differential expression was validated by quantitative real-time PCR and cellular localization determined by in situ hybridization histochemistry. Examples of lncRNAs exhibiting DA cell-specific expression, different subcellular distributions, and covariance of expression with known cocaine-regulated protein-coding genes were identified. These findings implicate lncRNAs in the cellular responses of human DA neurons to chronic cocaine abuse. Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes, but little is known about their potential role in drug abuse. In this study, we identified lncRNAs differentially expressed in human cocaine abusers' midbrains. One up-regulated antisense lncRNA, tumor necrosis factor receptor-associated factor 3-interacting protein 2-antisense 1 (TRAF3IP2-AS1), was found predominantly in the nucleus of human dopamine (DA) neurons, whereas the related TRAF3IP2 protein-coding transcript was distributed throughout these cells. The abundances of these transcripts were significantly correlated (left) suggesting that TRAF3IP2-AS1 may regulate TRAF3IP2 gene expression, perhaps through local chromatin changes at this locus (right)., (© 2015 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Background: The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) influences nigral dopaminergic neurons via autocrine and paracrine mechanisms. The reduction of BDNF expression in Parkinson's disease substantia nigra (SN) might contribute to the death of dopaminergic neurons because inhibiting BDNF expression in the SN causes parkinsonism in the rat. This study aimed to demonstrate that increasing BDNF expression in dopaminergic neurons of rats with one week of 6-hydroxydopamine lesion recovers from parkinsonism. The plasmids phDAT-BDNF-flag and phDAT-EGFP, coding for enhanced green fluorescent protein, were transfected using neurotensin (NTS)-polyplex, which enables delivery of genes into the dopaminergic neurons via neurotensin-receptor type 1 (NTSR1) internalization., Results: Two weeks after transfections, RT-PCR and immunofluorescence techniques showed that the residual dopaminergic neurons retain NTSR1 expression and susceptibility to be transfected by the NTS-polyplex. phDAT-BDNF-flag transfection did not increase dopaminergic neurons, but caused 7-fold increase in dopamine fibers within the SN and 5-fold increase in innervation and dopamine levels in the striatum. These neurotrophic effects were accompanied by a significant improvement in motor behavior., Conclusions: NTS-polyplex-mediated BDNF overexpression in dopaminergic neurons has proven to be effective to remit hemiparkinsonism in the rat. This BDNF gene therapy might be helpful in the early stage of Parkinson's disease.

The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson's disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring dopamine neurons in Parkinson's disease.

The development of new therapeutic strategies for the treatment of complex brain disorders such as drug addiction is likely to be advanced by a more complete understanding of the underlying molecular pathophysiology. Although the study of postmortem human brain represents a unique resource in this regard, it can be challenging to disentangle the relative contribution of chronic pathological processes versus perimortem events to the observed changes in gene expression. To begin to unravel this issue, we analyzed by quantitative PCR the midbrain expression of numerous candidate genes previously associated with cocaine abuse. Data obtained from chronic cocaine abusers (and matched control subjects) dying of gunshot wounds were compared with a prior study of subjects with deaths directly attributable to cocaine abuse. Most of the genes studied (i.e., tyrosine hydroxylase, dopamine transporter, forkhead box A2, histone variant H3 family 3B, nuclear factor kappa B inhibitor alpha, growth arrest and DNA damage-inducible beta) were found to be differentially expressed in chronic cocaine abusers irrespective of immediate cause of death or perimortem levels of cocaine, suggesting that these may represent core pathophysiological changes arising with chronic drug abuse. On the other hand, chemokine C-C motif ligand 2 and jun proto-oncogene expression were unaffected in cocaine-abusing subjects dying of gunshot wounds, in contrast to the differential expression previously reported in cocaine-related fatalities. The possible influence of cause of death and other factors on the cocaine-responsiveness of these genes is discussed.

Chronic drug abuse, craving, and relapse are thought to be linked to long-lasting changes in neural gene expression arising through transcriptional and chromatin-related mechanisms. The key contributions of midbrain dopamine (DA)-synthesizing neurons throughout the addiction process provide a compelling rationale for determining the drug-induced molecular changes that occur in these cells. Yet our understanding of these processes remains rudimentary. The postmortem human brain constitutes a unique resource that can be exploited to gain insights into the pathophysiology of complex disorders such as drug addiction. In this study, we analyzed the profiles of midbrain gene expression in chronic cocaine abusers and well-matched drug-free control subjects using microarray and quantitative PCR. A small number of genes exhibited robust differential expression; many of these are involved in the regulation of transcription, chromatin, or DA cell phenotype. Transcript abundances for approximately half of these differentially expressed genes were diagnostic for assigning subjects to the cocaine-abusing vs control cohort. Identification of a molecular signature associated with pathophysiological changes occurring in cocaine abusers' midbrains should contribute to the development of biomarkers and novel therapeutic targets for drug addiction.

Altered activity of the human dopamine transporter gene (hDAT) is associated with several common and severe brain disorders, including cocaine abuse. However, there is little a priori information on whether such alterations are due to nature (genetic variation) or nurture (human behaviors such as cocaine abuse). This study investigated the correlation between seven markers throughout hDAT and its mRNA levels in postmortem ventral midbrain tissues from 18 cocaine abusers and 18 strictly matched drug-free controls in the African-American population. Here, we show that one major haplotype with the same frequency in cocaine abusers versus drug-free controls displays a 37.1% reduction of expression levels in cocaine abusers compared with matched controls (P=0.0057). The most studied genetic marker, variable number tandem repeats (VNTR) located in Exon 15 (3'VNTR), is not correlated with hDAT mRNA levels. A 5' upstream VNTR (rs70957367) has repeat numbers that are positively correlated with expression levels in controls (r(2)=0.9536, P=0.0235), but this positive correlation disappears in cocaine abusers. The findings suggest that varying hDAT activity is attributable to both genetics and cocaine abuse., (© 2011 The Authors, Addiction Biology © 2011 Society for the Study of Addiction.)

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.

Cocaine-related fatalities can pose forensic challenges, particularly when accompanied by excited delirium (ED) syndrome and interventions by law enforcement and medical personnel. A recent report concluded that elevated heat shock protein 70 (HSP70) expression in autopsy brain samples constitutes a reliable forensic biomarker for the identification of ED as a cause of death. The present study quantified the abundance of both HSPA1A and HSPA1B gene (HSP70-encoding) transcripts in midbrain specimens from a series of cocaine-related fatalities and matched drug-free control subjects. HSP70 expression was increased significantly in cocaine abusers as a group compared to control subjects, irrespective of the presence or absence of ED. Furthermore, elevated HSP70 expression was predictive of a period of survival between cocaine use and death that included medical and/or police intervention. The present data do not support the assertion that HSP70 expression is a reliable brain biomarker for identifying ED as a cause of death., (© 2012 American Academy of Forensic Sciences.)

Nanomedicine has focused on targeted neurotrophic gene delivery to the brain as a strategy to stop and reverse neurodegeneration in Parkinson's disease. Because of improved transfection ability, synthetic nanocarriers have become candidates for neurotrophic therapy. Neurotensin (NTS)-polyplex is a "Trojan horse" synthetic nanocarrier system that enters dopaminergic neurons through NTS receptor internalization to deliver a genetic cargo. The success of preclinical studies with different neurotrophic genes supports the possibility of using NTS-polyplex in nanomedicine. In this review, we describe the mechanism of NTS-polyplex transfection. We discuss the concept that an effective neurotrophic therapy requires a simultaneous effect on the axon terminals and soma of the remaining dopaminergic neurons. We also discuss the future of this strategy for the treatment of Parkinson's disease., From the Clinical Editor: This review paper focuses on nanomedicine-based treatment of Parkinson's disease, a neurodegenerative condition with existing symptomatic but no curative treatment. Neurotensin-polyplex is a synthetic nanocarrier system that enables delivery of genetic cargo to dopaminergic neurons via NTS receptor internalization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The transcription factor NURR1 plays a pivotal role in the development and maintenance of neurotransmitter phenotype in midbrain dopamine neurons. Conversely, decreased NURR1 expression is associated with a number of dopamine-related CNS disorders, including Parkinson's disease and drug addiction. In order to better understand the nature of NURR1-responsive genes and their potential roles in dopamine neuron differentiation and survival, we used a human neural cellular background (SK-N-AS cells) in which to generate a number of stable clonal lines with graded NURR1 gene expression that approximated that seen in DA cell-rich human substantia nigra. Gene expression profiling data from these NURR1-expressing clonal lines were validated by quantitative RT-PCR and subjected to bioinformatic analyses. The present study identified a large number of NURR1-responsive genes and demonstrated the potential importance of concentration-dependent NURR1 effects in the differential regulation of distinct NURR1 target genes and biological pathways. These data support the promise of NURR1-based CNS therapeutics for the neuroprotection and/or functional restoration of DA neurons.

Although recent data suggest that some long non-coding RNAs (lncRNAs) exert widespread effects on gene expression and organelle formation, lncRNAs as a group constitute a sizable but poorly characterized fraction of the human transcriptome. We investigated whether some human lncRNA sequences were fortuitously represented on commonly used microarrays, then used this annotation to assess lncRNA expression in human brain. A computational and annotation pipeline was developed to identify lncRNA transcripts represented on Affymetrix U133 arrays. A previously published dataset derived from human nucleus accumbens was then examined for potential lncRNA expression. Twenty-three lncRNAs were determined to be represented on U133 arrays. Of these, dataset analysis revealed that five lncRNAs were consistently detected in samples of human nucleus accumbens. Strikingly, the abundance of these lncRNAs was up-regulated in human heroin abusers compared to matched drug-free control subjects, a finding confirmed by quantitative PCR. This study presents a paradigm for examining existing Affymetrix datasets for the detection and potential regulation of lncRNA expression, including changes associated with human disease. The finding that all detected lncRNAs were up-regulated in heroin abusers is consonant with the proposed role of lncRNAs as mediators of widespread changes in gene expression as occur in drug abuse., (© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.)

Background: Glutamatergic transmission in the amygdala is hypothesized as an important mediator of stimulus-reward associations contributing to drug-seeking behavior and relapse. Insight is, however, lacking regarding the amygdala glutamatergic system in human drug abusers., Methods: We examined glutamate receptors and scaffolding proteins associated with the postsynaptic density in the human postmortem amygdala. Messenger RNA or protein levels were studied in a population of multidrug (seven heroin, eight cocaine, seven heroin/cocaine, and seven controls) or predominant heroin (29 heroin and 15 controls) subjects., Results: The amygdala of drug abusers was characterized by a striking positive correlation (r > .8) between α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptor subunit 1 (GluA1) and postsynaptic density protein-95 (PSD-95) mRNA levels, which was not evident in control subjects. Structural equation multigroup analysis of protein correlations also identified the relationship between GluA1 and PSD-95 protein levels as the distinguishing feature of abusers. In line with the GluA1-PSD-95 implications of enhanced synaptic plasticity, Homer 1b/c protein expression was increased in both heroin and cocaine users as was its binding partner, dynamin-3. Furthermore, there was a positive relationship between Homer 1b/c and dynamin-3 in drug abusers that reflected an increase in the direct physical coupling between the proteins. A noted age-related decline of Homer 1b/c-dynamin-3 interactions, as well as GluA1 levels, was blunted in abusers., Conclusions: Impairment of key components of the amygdala postsynaptic density and coupling to the endocytic zone, critical for the regulation of glutamate receptor cycling, may underlie heightened synaptic plasticity in human drug abusers., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Genetic markers in the serotonin transporter are associated with panic disorder (PD). The associated polymorphisms do not include the serotonin transporter-linked polymorphic region and display no obvious functional attributes. A common polymorphism (rs3813034) occurs in one of the two reported polyadenylation signals for the serotonin transporter and is in linkage disequilibrium with the PD-associated markers. If functional, rs3813034 might be the risk factor that explains the association of the serotonin transporter and PD., Methods: Quantitative polymerase chain reaction on human brain samples (n = 65) and lymphoblast cultures (n = 71) was used to test rs3813034 for effects on expression of the polyadenylation forms of the serotonin transporter. rs3813034 was also tested for association in a sample of PD cases (n = 307) and a control sample (n = 542) that has similar population structure., Results: The balance of the two polyadenylation forms of the serotonin transporter is associated with rs3813034 in brain (p < .001) and lymphoblasts (p < .001). The balance of the polyadenylation forms is also associated with gender in brain only (p < .05). Association testing of rs3813034 in PD identified a significant association (p = .0068) with a relative risk of 1.56 and 1.81 for the heterozygous and homozygous variant genotypes, respectively., Conclusions: rs3813034 is a functional polymorphism in the serotonin transporter that alters the balance of the two polyadenylation forms of the serotonin transporter. rs3813034 is a putative risk factor for PD and other behavioral disorders that involve dysregulation of serotonergic neurotransmission., (2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Chronic abuse of cocaine is known to cause neuroadaptive changes in the nucleus accumbens (NAc) and ventral tegmental area (VTA). In addition, altered expression of the myelin-related genes MBP, MOBP, PLP1 as well as of MAL2 in NAc was recently reported by gene array analysis in brains from cocaine abusers. In the present study we used in situ hybridization to quantify transcript expression of these four genes, as well as for the myelin-related transcripts encoding quaking, EDG2, claudin-11, transferrin, CNP, and MAG in caudate, putamen, internal capsule, and NAc in postmortem brain from cocaine abusers and matched comparison subjects. Most transcripts were not different between these groups in these striatal regions, and contrary to previous reports, we did not detect any changes in the NAc. However, expression of the transcript encoding PLP1 was significantly decreased in ventral and dorsal regions of the caudate, putamen, and in the internal capsule. Additionally, expression of claudin-11 and transferrin was decreased in the caudate and internal capsule, respectively. PLP1 is expressed at very high levels in oligodendrocytes and is essential in maintaining stability of myelin sheets. Based on these findings, altered expression of PLP1 in most areas of the striatum suggests that widespread changes to the myelin structure could be associated with the adaptive changes following chronic cocaine abuse.

In animal models of Parkinson's disease, a supersensitive response to dopamine (DA) is associated with a switch in the coupling of striatal DA D1 receptors from a cyclic AMP/protein kinase A signaling pathway to one involving extracellular signal-regulated kinase/mitogen-associated protein kinase. In this study, we found that generation of organotypic striatal cultures, with concomitant loss of DA innervation, led to a downregulation in preprotachykinin-A gene expression, which was reinstated by D1 receptor activation in an extracellular signal-regulated kinase/mitogen-associated protein kinase-dependent manner. These data demonstrate that acute organotypic slice cultures recapitulate important changes in D1 receptor-mediated signal transduction seen in DA-denervated animals, providing a valuable model system to study denervation effects on DA signaling and striatal gene expression.

Several lines of evidence suggest that valproate, a drug used in the treatment of mania and bipolar disorders, epilepsies, and addictions, may modulate dopamine transporter (DAT) function, yet the effects of valproate on DAT gene expression have not been directly assessed. Utilizing a human dopaminergic cell line and rat midbrain dopamine (DA) neurons in organotypic culture, we found that valproate increased endogenous DAT gene expression in a concentration- and time-dependent manner. Given previous data demonstrating that members of the specificity protein (Sp) family of transcription factors are strong trans-activators of DAT gene transcription, we investigated the Sp-dependence of valproate effects. Valproate-induced transcription of a DAT reporter construct was significantly attenuated by coexpression of a dominant negative form of Sp, mutation of a Sp-responsive cis-element, or expression in a Sp-null cellular background (SL-2 cells). Valproate significantly altered Sp protein abundance in both dopaminergic model systems employed. In summary, valproate treatment significantly increased DAT gene expression in a Sp transcription factor-dependent manner. Some of valproate's therapeutic effects may involve activation of DAT gene expression.

Recently we showed that the neurotensin polyplex is a nanoparticle carrier system that targets reporter genes in nigral dopamine neurons in vivo. Herein, we report its first practical application in experimental parkinsonism, which consisted of transfecting dopamine neurons with the gene coding for human glial cell line-derived neurotrophic factor (hGDNF). Hemiparkinsonism was induced in rats by a single dose of 6-hydroxydopamine (30 microg) into the ventrolateral part of the striatum. We showed that transfection of the hGDNF gene into the substantia nigra of rats 1 week after the neurotoxin injection produced biochemical, anatomical, and functional recovery from hemiparkinsonism. RT-PCR analysis showed mRNA expression of exogenous hGDNF in the transfected substantia nigra. Western blot analysis verified transgene expression by recognizing the flag epitope added at the C-terminus of the hGDNF polypeptide, which was found mainly in dopamine neurons by double immunofluorescence techniques. These data indicate that the neurotensin polyplex holds great promise for the neuroprotective therapy of Parkinson disease.

Drug abuse is thought to induce long-term cellular and behavioral adaptations as a result of alterations in gene expression. Understanding the molecular consequences of addiction may contribute to the development of better treatment strategies. This study utilized high-throughput Affymetrix microarrays to identify gene expression changes in the post-mortem nucleus accumbens of chronic heroin abusers. These data were analyzed independently and in relation to our previously reported data involving human cocaine abusers, in order to determine which expression changes were drug specific and which may be common to the phenomenon of addiction. A significant decrease in the expression of numerous genes encoding proteins involved in presynaptic release of neurotransmitter was seen in heroin abusers, a finding not seen in the cocaine-abusing cohort. Conversely, the striking decrease in myelin-related genes observed in cocaine abusers was not evident in our cohort of heroin subjects. Overall, little overlap in gene expression profiles was seen between the two drug-abusing cohorts: out of the approximately 39,000 transcripts investigated, the abundance of only 25 was significantly changed in both cocaine and heroin abusers, with nearly one-half of these being altered in opposite directions. These data suggest that the profiles of nucleus accumbens gene expression associated with chronic heroin or cocaine abuse are largely unique, despite what are thought to be common effects of these drugs on dopamine neurotransmission in this brain region. A re-examination of our current assumptions about the commonality of molecular mechanisms associated with substance abuse seems warranted.

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.

Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.

Preprotachykinin-I (PPT) gene expression is regulated by a number of stimuli that signal through cyclic AMP (cAMP)-mediated pathways. In the present study, forskolin, an adenylyl cyclase stimulator, significantly increased PPT mRNA levels in PPT-expressing RINm5F cells, an effect paralleled by an increase in PPT promoter-luciferase reporter construct activity. The forskolin-induced stimulation of PPT transcription was protein kinase A dependent (PKA), as shown by blockade with the PKA inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide. We found that the activation protein 1/cAMP response element (AP1/CRE) site centered at -196 relative to the transcription start site was important for basal and forskolin-induced PPT promoter activity. Because of the involvement of PKA and the similarity of the AP1/CRE element to consensus CRE sequences, we investigated the role of CRE-binding protein (CREB) in the regulation of the PPT promoter. Surprisingly, overexpression of a dominant-negative CREB (i.e. CREB-A) did not affect basal or forskolin-induced PPT promoter activity. Furthermore, binding of CREB to the PPT promoter AP1/CRE site was not demonstrable in electrophoretic mobility shift assays. Rather, our experiments suggested that c-Jun is a member of the complex that binds to this site. We conclude that, at least in RINm5F cells, cAMP-mediated up-regulation of PPT gene expression does not involve CREB or CREB-related transcription factor recruitment to the AP1/CRE site.

Regulator of G-protein signaling 9-1 (RGS9-1) and RGS9-2 are highly related RGS proteins with distinctive C termini arising from alternative splicing of RGS9 gene transcripts. RGS9-1 is expressed in photoreceptors where it functions as a regulator of transducin. In contrast, RGS9-2 is abundantly expressed in the brain, especially in basal ganglia, where its specific function remains poorly understood. To gain insight into the function of RGS9-2, we screened a human cDNA library for potential interacting proteins. This screen identified a strong interaction between RGS9-2 and alpha-actinin-2, suggesting a possible functional relationship between these proteins. Consistent with this idea, RGS9-2 and alpha-actinin-2 coimmunoprecipitated after coexpression in human embryonic kidney 293 (HEK-293) cells. Furthermore, endogenous RGS9-2 and alpha-actinin-2 could also be coimmunoprecipitated from extracts of rat striatum, an area highly enriched in both these proteins. These results supported the idea that RGS9-2 and alpha-actinin-2 could act in concert in central neurons. Like alpha-actinin-2, RGS9-2 coimmunoprecipitated NMDA receptors from striatal extracts, suggesting an interaction between RGS9-2, alpha-actinin-2, and NMDA receptors. Previous studies have shown that alpha-actinin mediates calcium-dependent inactivation of NMDA receptors. In HEK-293 cells expressing NMDA receptors, expression of RGS9-2 significantly modulated this form of NMDA receptor inactivation. Furthermore, this modulation showed remarkable preference for NMDA receptor inactivation mediated by alpha-actinin-2. Using a series of deletion constructs, we localized this effect to the RGS domain of the protein. These results identify an unexpected functional interaction between RGS9-2 and alpha-actinin-2 and suggest a potential novel role for RGS9-2 in the regulation of NMDA receptor function.

Nurr1 (NR4A2) is an orphan nuclear receptor required for the development and maintenance of the dopaminergic phenotype in neurons of the ventral midbrain. This study demonstrates that multiple splice variants of nurr1 are produced in rat and human dopamine neurons. Formed by alternative RNA splicing in exon 7, nurr1a has a truncated carboxy-terminus, nurr1b has an internal deletion in the ligand-binding domain and nurr1c, newly identified in this study, has a novel carboxy-terminus produced by a frame shift downstream of the splice junction. Alternative RNA splicing in exon 3 produces the isoform known as the transcriptionally-inducible nuclear receptor (TINUR), lacking the amino-terminus. Nurr2 and the newly identified nurr2c are produced by utilization of both exon 3 and exon 7 alternative splice sites. In rat midbrain, variants other than full-length nurr1 constitute 20-35% of NR4A2 transcripts. Transfection studies in dopaminergic SK-N-AS cells demonstrate that nurr1a, nurr1b, nurr1c and TINUR have significantly reduced transcriptional activities compared with full-length nurr1, while nurr2 and nurr2c are inactive. Furthermore, in these experiments, nurr2 and nurr2c both act as dominant negatives. Production of these nurr1 variants in vivo as demonstrated here could represent a novel regulatory mechanism of nurr1 transcriptional activity and therefore, dopaminergic phenotype.

Most CNS studies using recombinant adeno-associated virus type 2 (rAAV-2) vectors have focused on gene delivery for the purpose of gene therapy. In the present study, we examined the feasibility of using rAAV-2 vectors to study the regulation of preprotachykinin-A (PPT-A) promoter activity in striatal medium spiny projection neurons. An rAAV-2 vector incorporating a PPT promoter fragment (shown previously to confer some cell-specificity of expression in vitro) coupled to a green fluorescent protein (GFP) reporter gene was stereotaxically injected into the rat striatum. Since medium spiny projection neurons represent the predominant neuronal type (90-95%) in the striatum, we predicted that the vast majority of GFP-expressing cells would be of this phenotype. Surprisingly, the transgene was actually expressed in a similar number of medium spiny projection neurons and interneurons, while glial expression of GFP was not observed. A preponderance of GFP-expressing interneurons was immunoreactive for the marker neuronal nitric oxide synthase (nNOS). Our results suggest that viral vector-related events that occur during transduction are the determining factor in the pattern of transgene expression observed, while the influence of the transgene promoter appears to be secondary, at least under the conditions employed.

The dopamine transporter (DAT) is a plasma membrane transport protein expressed exclusively within a small subset of CNS neurons. It plays a crucial role in controlling dopamine-mediated neurotransmission and a number of associated behaviors. This review focuses on recent data elucidating the role of the dopamine transporter in neurotoxicity and a number of CNS disorders, including Parkinson disease, drug abuse, and attention deficit hyperactivity disorder (ADHD).

The dopamine transporter is a plasma membrane protein that controls extracellular concentrations of the neurotransmitter dopamine. The physiological importance of the DAT provides the impetus for studies aimed at understanding the molecular mechanisms underlying regulation of the DAT gene. In this study, we identified a DAT-expressing neuroblastoma cell line (SK-N-AS) and employed it to investigate the transcriptional regulation of the human DAT gene. Two GC boxes (located at -130 and -60, respectively, relative to the transcriptional start site) were identified as important cis-acting elements mediating DAT promoter activity in dopaminergic SK-N-AS cells. Utilizing Sp-deficient Drosophila Schneider line (SL-2) cells, we showed that both Sp1 and Sp3 are strong activators of DAT transcriptional activity. Differential binding of Sp1 and Sp3 to the two GC boxes was demonstrated by electrophoretic mobility shift assays and super-shift assays. Our results indicate that the Sp1 family of proteins plays an important role in controlling the expression of the dopamine transporter gene within dopaminergic neurons.

Brain-specific regulator of G protein signaling 9 (RGS9-2) is a member of a family of proteins that can function as GTPase-activating proteins for heterotrimeric G proteins. In the present study, we examined the intracellular distribution of RGS9-2 in native brain tissue and transfected cells. Immunocytochemical and immunoblot experiments revealed an unexpectedly high proportion of RGS9-2 within the nuclei of forebrain neurons. A similar intracellular distribution was seen in transfected COS-7 cells. The RGS9 binding partner G(beta5) further enhanced the nuclear localization of RGS9-2, but did not affect the strongly cytoplasmic localization of RGS9-1, the retinal form of RGS9. Deletion construct analysis revealed that the unique polyproline-rich C-terminus of brain-specific RGS9-2 contains sequences necessary and sufficient to target RGS9 to the nucleus of COS-7 cells, as well as cultured striatal neurons. Furthermore, RGS9-2 transfection increased the transcriptional activity of a neuronal gene construct normally expressed in RGS9-positive neurons, suggesting that nuclear RGS9 directly or indirectly regulates transcription in vivo. The nuclear localization of RGS9-2 suggests a heretofore-unanticipated role for this brain-specific protein in transducing signals to the nuclei of forebrain neurons.

During development, survival of midbrain dopamine neurons and specification of their phenotype are dependent upon the intracellular expression of a number of transcription factors, including Engrailed 1, Pitx3, and Nurr1. The role of these transcription factors in the maintenance of the dopaminergic phenotype is less clear. In the present study, we show that each of these transcription factors is robustly expressed in adult dopamine neurons in human midbrain, and that cocaine abuse is associated with a significant decrease in the abundance of Nurr1 and Pitx3 in these cells. These data suggest that cocaine abuse leads to a partial loss of dopaminergic phenotype., (Copyright 2004 Lippincott Williams & Wilkins)

Chronic cocaine abuse induces long-term neural adaptations as a consequence of alterations in gene expression. This study was undertaken to identify those transcripts differentially regulated in the nucleus accumbens of human cocaine abusers. Affymetrix microarrays were used to measure transcript abundance in 10 cocaine abusers and 10 control subjects matched for age, race, sex, and brain pH. As expected, gene expression of cocaine- and amphetamine-regulated transcript (CART) was increased in the nucleus accumbens of cocaine abusers. The most robust and consistent finding, however, was a decrease in the expression of a number of myelin-related genes, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin-associated oligodendrocyte basic protein (MOBP). The differential expression seen by microarray for CART as well as MBP, MOBP, and PLP was verified by RT-PCR. In addition, immunohistochemical experiments revealed a decrease in the number of MBP-immunoreactive oligodendrocytes present in the nucleus accumbens and surrounding white matter of cocaine abusers. These findings suggest a dysregulation of myelin in human cocaine abusers.

Chronic exposure to cocaine induces long-term adaptations that are likely to involve changes in transcription factor expression. This possibility has not been examined in the cocaine-exposed human brain. The transcription factor nurr1 is highly expressed in rodent midbrain dopamine neurons and is essential for their proper phenotypic development. Here we show that human NURR1 gene expression is robust within control subjects and reduced markedly within the dopamine neurons of human cocaine abusers. NURR1 is known to regulate transcription of the gene encoding the cocaine-sensitive dopamine transporter (DAT). We show here that DAT gene expression also is reduced markedly in the dopamine neurons of NURR1-deficient cocaine abusers, suggesting that NURR1 plays a critical role in vivo in controlling human DAT gene expression and adaptation to repeated exposure to cocaine.

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 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.

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.

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.