Biomedical subjects

Pat Levitt

Publications and source records attributed to Pat Levitt.

At least 19 recordsLinked to original sources

PubMed2007

Prenatal exposure to cocaine produces unique developmental and long-term adaptive changes in dopamine D1 receptor activity and subcellular distribution.

Low-dose intravenous cocaine administration to pregnant rabbits causes permanent structural alterations in dopamine-rich cerebral cortical areas, substantially reduced dopamine D1 receptor coupling to G(s)-protein, and deficits in cognitive function. The developmental influences of reduced D1-G(s) coupling and the underlying cellular basis are unknown. Using primary neuronal cultures derived from the medial frontal cortex and striatum of in utero saline- and cocaine-exposed embryos, spontaneous neurite outgrowth of in utero-exposed cortical neurons was greater than in control neurons. In contrast, striatal neurons exposed to cocaine in utero exhibited an entirely opposite adaptive response, with diminished spontaneous neurite outgrowth compared with saline-exposed controls. Control neurons isolated from the two structures also exhibited opposite regulatory responses to the D1 receptor agonist SKF38393 (1-phenyl-2,3,4-5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride), inhibiting outgrowth in cortical cultures and stimulating outgrowth in striatal cultures. The agonist was ineffective in modulating neurite outgrowth of neurons from either structure isolated from cocaine-exposed fetuses, reflecting the reduced D1-Gs coupling. Total D1 receptor number was indistinguishable in neurons from the cocaine- and saline-exposed animals, but cell imaging and receptor binding of differentially isolated membranes showed that the lack of responsiveness was because of greatly reduced cell-surface localization of D1 receptors. These data suggest that prenatal exposure to cocaine causes a novel, long-lasting adaptive response in the subcellular distribution of D1 receptors, resulting in alterations in signaling capacity that have developmental and behavioral consequences.

Adaptation, Physiological↗

PubMed2007

Waved-1 mutant mice are hypersensitive to the locomotor actions of cocaine.

Transforming growth factor-alpha (TGFalpha) is a well-known regulator of many developmental processes, and is expressed heavily in basal forebrain and striatal regions. When TGFalpha is reduced in Waved-1 (Wa-1) mutant mice, brain anatomy, biogenic amines, stress response, and behavior are normal prior to, but altered following puberty. As an initial screen for possible alterations in nigrostriatal and mesolimbic dopamine (DA) systems, we tested adult Wa-1 mutant mice in an open field, following acute injection with cocaine (15 mg/kg). Wa-1 mice exhibited significantly greater ambulatory distance, number of ambulatory episodes, and cocaine-induced motor stereotypies than do controls. These data indicate that adult Wa-1 mice are hypersensitive to the locomotor effects of cocaine and provide a new potential link between neurodevelopmental processes and adult psychostimulant responsiveness.

Analysis of Variance↗

PubMed2006

Genetic analysis of anterior posterior expression gradients in the developing mammalian forebrain.

Intrinsic regulatory factors play critical roles in early cortical patterning, including the development of the anteroposterior (A-P) axis. To identify genes that are differentially expressed along the A-P axis of the developing cerebral cortex, we analyzed gene expression in presumptive frontal, parietal, and occipital cerebral walls of E12.5 mouse using complementary DNA microarrays. We identified 106 genes, including expressed sequence tags (ESTs), expressed in an A-P gradient in the embryonic brain and screened 88 by in situ hybridization for confirmation. Central nervous system (CNS) expression patterns of many of these genes were previously unknown. Others, such as Sfrp1, CoupTF1, and FABP7, were expressed in a manner consistent with previous studies, providing independent confirmation. Two related transcription factors, previously not implicated in CNS development, Fhl1 and Fhl2, were observed to be enriched in posterior and anterior telencephalon, respectively. We studied patterning gradients in Fhl1 knockout mice but observed no changes in gene expression related to A-P regionalization in the Fhl1 knockout mice. These data provide an important set of new candidates for studies of cortical patterning and maturation.

Animals↗

PubMed2006

A genetic variant that disrupts MET transcription is associated with autism.

There is strong evidence for a genetic predisposition to autism and an intense interest in discovering heritable risk factors that disrupt gene function. Based on neurobiological findings and location within a chromosome 7q31 autism candidate gene region, we analyzed the gene encoding the pleiotropic MET receptor tyrosine kinase in a family based study of autism including 1,231 cases. MET signaling participates in neocortical and cerebellar growth and maturation, immune function, and gastrointestinal repair, consistent with reported medical complications in some children with autism. Here, we show genetic association (P = 0.0005) of a common C allele in the promoter region of the MET gene in 204 autism families. The allelic association at this MET variant was confirmed in a replication sample of 539 autism families (P = 0.001) and in the combined sample (P = 0.000005). Multiplex families, in which more than one child has autism, exhibited the strongest allelic association (P = 0.000007). In case-control analyses, the autism diagnosis relative risk was 2.27 (95% confidence interval: 1.41-3.65; P = 0.0006) for the CC genotype and 1.67 (95% confidence interval: 1.11-2.49; P = 0.012) for the CG genotype compared with the GG genotype. Functional assays showed that the C allele results in a 2-fold decrease in MET promoter activity and altered binding of specific transcription factor complexes. These data implicate reduced MET gene expression in autism susceptibility, providing evidence of a previously undescribed pathophysiological basis for this behaviorally and medically complex disorder.

Alleles↗

PubMed2006

Making the case for a candidate vulnerability gene in schizophrenia: Convergent evidence for regulator of G-protein signaling 4 (RGS4).

Both genetic and environmental factors have been associated with an increased risk for schizophrenia. These factors are not mutually exclusive; a single gene can be a genetic factor (due to a mutation in the gene sequence) and a target of a physiological response to an environmental stimulus, both with the common endpoint of altered expression of the gene. Regulator of G-protein signaling 4 (RGS4) has been implicated as such a gene from three lines of evidence. First, a subset of genetic studies revealed an association between schizophrenia and non-functional polymorphisms in the RGS4 gene. Second, across the cortical mantle the expression of RGS4 mRNA is decreased in a diagnosis-specific manner in subjects with schizophrenia. Third, neurobiological studies demonstrate that RGS4 is highly responsive to environmental stimuli and capable of modulating the function of G-protein coupled neurotransmitter receptors implicated in schizophrenia. RGS4 is an example of a molecule that may underlie increased vulnerability through either genetic or non-genetic mechanisms, which we suggest may be typical of other genes in a complex, polygenic disorder such as schizophrenia.

Animals↗

PubMed2006

Multiple receptors mediate the trophic effects of serotonin on ventroposterior thalamic neurons in vitro.

Serotonin (5-HT) exerts prominent morphogenetic roles during development. For example, somatosensory cortical barrel formation is altered in mouse models characterized by excessive extracellular 5-HT, suggesting that 5-HT affects development of thalamic afferents and/or neocortical target regions. The present study assessed 5-HT effects in primary cultures of fetal ventroposterior thalamic (VPT) neurons. 5-HT produces concentration-dependent trophic effects, with impressive 59% and 106% peak increases in total neurite length and number of branching points, respectively, at a dose of 30 microM 5-HT. The exposure of VPT neurons to specific 5-HT receptor agonists 8-OH-DPAT (5-HT(1A)), CGS-12066A (5-HT(1B)), DOI (5-HT(2A/2C)), and m-CPBG (5-HT(3)), enhances primary neurite length and number of branching points with rank-order potency 5-HT(1B) > 5-HT(2A/2C) = 5-HT(3) > 5-HT(1A) = vehicle. Trophic 5-HT effects on embryonic VPT neurons are thus much more prominent than previously reported, and can be mediated by multiple 5-HT receptor subtypes.

Analysis of Variance↗

PubMed2006

RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion.

Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D(2) dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D(2) dopamine receptor modulation of calcium (Ca(2+)) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M(4) muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4-an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.

Acetylcholine↗

PubMed2006

Evaluation of a susceptibility gene for schizophrenia: genotype based meta-analysis of RGS4 polymorphisms from thirteen independent samples.

BACKGROUND: Associations between schizophrenia (SCZ) and polymorphisms at the regulator of G-protein signaling 4 (RGS4) gene have been reported (single nucleotide polymorphisms [SNPs] 1, 4, 7, and 18). Yet, similar to other SCZ candidate genes, studies have been inconsistent with respect to the associated alleles. METHODS: In an effort to resolve the role for RGS4 in SCZ susceptibility, we undertook a genotype-based meta-analysis using both published and unpublished family-based and case-control samples (total n = 13,807). RESULTS: The family-based dataset consisted of 10 samples (2160 families). Significant associations with individual SNPs/haplotypes were not observed. In contrast, global analysis revealed significant transmission distortion (p = .0009). Specifically, analyses suggested overtransmission of two common haplotypes that account for the vast majority of all haplotypes. Separate analyses of 3486 cases and 3755 control samples (eight samples) detected a significant association with SNP 4 (p = .01). Individual haplotype analyses were not significant, but evaluation of test statistics from individual samples suggested significant associations. CONCLUSIONS: Our collaborative meta-analysis represents one of the largest SCZ association studies to date. No individual risk factor arose from our analyses, but interpretation of these results is not straightforward. Our analyses suggest risk due to at least two common haplotypes in the presence of heterogeneity. Similar analysis for other putative susceptibility genes is warranted.

Case-Control Studies↗

PubMed2006

Critical appraisal of DNA microarrays in psychiatric genomics.

Transcriptome profiling using DNA microarrays are data-driven approaches with the potential to uncover unanticipated relationships between gene expression alterations and psychiatric disorders. Studies to date have yielded both convergent and divergent findings. Differences may be explained, at least in part, by the use of a variety of microarray platforms and analytical approaches. Consistent findings across studies suggest, however, that important relationships may exist between altered gene expression and genetic susceptibility to psychiatric disorders. For example, GAD67, RGS4, DTNBP1, NRG1, and GABRAB2 show expression alterations in the postmortem brain of subjects with schizophrenia, and these genes have been also implicated as putative, heritable schizophrenia susceptibility genes. Thus, we propose that for some genes, altered expression in the postmortem human brain may have a dual origin: polymorphisms in the candidate genes themselves or upstream genetic-environmental factors that converge to alter their expression level. We hypothesize that certain gene products, which function as "molecular hubs," commonly show altered expression in psychiatric disorders and confer genetic susceptibility for one or more diseases. Microarray gene expression studies are ideally suited to reveal these putative disease-associated molecular hubs and to identify promising candidates for genetic association studies.

DNA↗

PubMed2006

Transforming growth factor-alpha induces sex-specific neurochemical imbalance in the stress- and memory-associated brain structures.

Transforming growth factor-alpha (TGFalpha) is a well-known regulator of many developmental processes. However, its role in adult nervous system is yet unclear. Studies have shown that TGFalpha can regulate stress and memory behavior in adult mice. When TGFalpha is reduced in Waved-1 (Wa-1) mutant mice, the stress response and memory are impaired predominantly in males and only after puberty. To determine the neurochemical changes resulting from the reduced TGFalpha levels that could explain the reported behavioral outcomes, biogenic amine and amino acid levels were determined in the brain regions associated with stress and memory. Interestingly, sex-specific alterations in neurochemical levels were detected, including elevated noradrenaline and reduced glutamate levels in striatum of Wa-1 males, increased noradrenaline and reduced serotonin metabolite levels in hippocampus of Wa-1 females, reduced serotonin metabolite levels in cortex and amygdala of Wa-1 females, and reduced noradrenaline, dopamine, serotonin, glutamate and glycine levels in hypothalamus of Wa-1 females compared to their respective controls. Increased dopamine turnover in cortex and reduced dopamine and serotonin turnover in amygdala were observed in both male and female Wa-1 mice. The data indicate sex-specific alterations of specific neurochemicals as a result of reduced TGFalpha expression, which may underlie sex-dependent stress response and memory impairment in Wa-1 mice.

Animals↗

PubMed2006

Genetic or pharmacological inactivation of the dopamine D1 receptor differentially alters the expression of regulator of G-protein signalling (Rgs) transcripts.

Dysregulation of dopamine (DA) receptor signalling induces specific changes in behaviour, neuronal circuitry and gene expression in the mammalian forebrain. In order to better understand signalling adaptations at the molecular level, we used high-density oligonucleotide microarrays (Codelink Mouse 20K) to define alterations in the expression of transcripts encoding regulator of G-protein coupled receptor signalling in dopamine D1 receptor knockout mice (Drd1a-KO). Regulator of G-protein signalling (Rgs) 2, Rgs4, and Rgs9 were significantly decreased in the striatum (STR) of Drd1a-KO mice. These changes were confirmed by in situ hybridization, and were also observed in the nucleus accumbens (NAc). In contrast, analysis of the medial frontal cortex (MFC) revealed a significant decrease in Rgs17 expression exclusively, and a modest up-regulation of Rgs5 transcript. The expression of these gene products were not significantly altered in the dopamine-poor visual cortex (VC). The Drd1a-KO mouse, and a rabbit model of in utero cocaine exposure, in which D1R signalling is permanently reduced, possess analogous morphological and functional alterations in dopamine-modulated brain circuits; thus we also examined long-lasting changes in RGS transcript expression following prenatal exposure to cocaine. In sharp contrast to the Drd1a-KO, Rgs2 and Rgs4 were unchanged, and Rgs9 and Rgs17 transcripts were increased in prenatal cocaine-exposed progeny. These data suggest that an absolute absence of D1R signalling (Drd1a-KO) and hypomorphic D1R signalling (prenatal cocaine) produce common alterations in neuronal morphology, but distinct outcomes in molecular neuroadaptations.

Animals↗

PubMed2005

Dissociation of corticothalamic and thalamocortical axon targeting by an EphA7-mediated mechanism.

Molecular mechanisms generating the topographic organization of corticothalamic (CT) circuits, which comprise more than three-quarters of the synaptic inputs onto sensory relay neurons, and their interdependence with thalamocortical (TC) axon development are unknown. Using in utero electroporation-mediated gene transfer, we show that EphA7-mediated signaling on neocortical axons controls the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that mis-express EphA7 do not shift the relative positioning of their pathway within the subcortical telencephalon (ST), indicating that they do not depend upon EphA7/ephrin-A signaling in the ST for establishing this topography. Moreover, mis-expression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. Our results support a model in which EphA/ephrin-A signaling controls independently the precision with which CT and TC projections develop, yet is essential for establishing their topographic reciprocity.

Animals↗

PubMed2005

Prenatal cocaine exposure specifically alters spontaneous alternation behavior.

Our laboratory has previously characterized a rabbit model of gestational cocaine exposure in which permanent alterations in neuronal morphology, cell signaling and psychostimulant-induced behavior are observed. The cellular and molecular neuroadaptations produced by prenatal cocaine occur in brain regions involved in executive function and attention, such as the anterior cingulate and medial prefrontal cortices. Therefore, in the present study, we have measured the effects of prenatal cocaine exposure on specific behavioral tasks in adult offspring whose mothers were treated with cocaine (3mg/kg, twice a day, E16-E25). We assessed non-spatial, short-term memory in a two-object recognition task and found no deficits in memory or exploratory behaviors in cocaine-exposed offspring in this paradigm. We also evaluated a different memory task with a more robust attentional component, using spontaneous alternation in a Y maze. In this task, young adult rabbits exposed to cocaine prenatally exhibited a significant deficit in performance. Deficits in spontaneous alternation can be induced by a wide variety of behavioral and cognitive dysfunctions, but taken together with previous findings in this and other animal models, we hypothesize that prenatal exposure to cocaine alters highly specific aspects of cognitive and emotional development.

Animals↗

PubMed2005

Early experience modifies the postnatal assembly of autonomic emotional motor circuits in rats.

Rat pups that are repeatedly handled and separated from their dam exhibit altered adult behavioral, endocrine, and autonomic responses to stress, but the extent to which early handling and/or maternal separation (H/S) alters the development of circuits that underlie these responses is unknown. The present study tested the hypothesis that early H/S alters the postnatal assembly of synapses within preautonomic emotional motor circuits. Circuit development was traced by synapse-dependent retrograde transneuronal transport of pseudorabies virus (PRV) from the stomach wall. Control and H/S rats were analyzed between postnatal day 6 (P6) and P10, a period of rapid synaptic assembly among preautonomic circuit components. Pups in H/S groups were removed from their dam daily for either 15 min or 3 h beginning on P1, and were injected with virus on P8 and perfused on P10. Quantitative analyses of primary and transsynaptic PRV immunolabeling confirmed an age-dependent assembly of hypothalamic, limbic, and cortical inputs to autonomic nuclei. Circuit assembly was significantly altered in H/S pups, in which fewer neurons in the central amygdala, the bed nucleus of the stria terminalis, and visceral cortices were infected compared with age-matched controls. In contrast, H/S did not alter the assembly of paraventricular hypothalamic inputs to gastric autonomic neurons. H/S-related reductions in limbic and cortical transneuronal infection were similar in pups exposed daily to 15 min or 3 h maternal separation. These findings support the view that environmental events during early postnatal life can influence the formation of neural circuits that provide limbic and cortical control over autonomic emotional motor output.

Age Factors↗

PubMed2005

DNA self-polymers as microarray probes improve assay sensitivity.

DNA microarrays provide a method for determining the expression levels of thousands of genes simultaneously. However, the phenotypic complexity of brain tissue and cross-dilution of transcripts from different sources make it difficult to detect many of the low abundance RNA species. Furthermore, these experiments require significant amounts of starting material, which must often be amplified by one or two rounds of T7 amplification. We have developed a novel microarray probe with increased sensitivity. In this approach, PCR-generated microarray probes are end-ligated into redundant polymers and printed on standard arraying surfaces. These DNA polymer probes result in greatly improved sensitivity over classical monomer probes. Furthermore, polymer microarray sensitivity can be even further improved by incorporation of a biotin adapter into the first strand cDNA during reverse transcription and attachment of a gold particle (Genicon RLS, Invitrogen, CA) in a secondary reaction. This approach allowed us to reliably assess: expression of genes from < 5 microg of total RNA starting material without sample amplification. Finally, the resonance light scattering-labeled microarrays can be archived without fading, allowing re-scanning at a later time.

DNA↗

PubMed2005

Region- and age-specific deficits in gamma-aminobutyric acidergic neuron development in the telencephalon of the uPAR(-/-) mouse.

We have previously shown that in adult mice with a null mutation in the urokinase-type plasminogen activator receptor (uPAR) gene, maintained on a C57BL/6J/129Sv background, there is a selective loss of GABAergic interneurons in anterior cingulate and parietal cortex, with the parvalbumin-expressing subpopulation preferentially affected. Here, we performed a more detailed anatomical analysis of uPAR(-/-) mutation on the congenic C57BL/6J background. With glutamic acid decarboxylase-67 and gamma-aminobutyric acid (GABA) immunostaining, there is a similar region-selective loss of cortical interneurons in the congenic uPAR(-/-) mice from the earliest age examined (P21). In contrast, the loss of parvalbumin-immunoreactive cells is observed only in adult cortex, and the extent of this loss is less than in the mixed background. Moreover, earlier in development, although there are normal numbers of parvalbumin cells in the uPAR(-/-) cortex, fewer cells coexpress GABA, suggesting that the parvalbumin subpopulation migrates appropriately to the cortex, but does not differentiate normally. Among the other forebrain regions examined, only the adult hippocampus shows a loss of GABAergic interneurons, although the somatostatin, rather than the parvalbumin, subpopulation contributes to this loss. The data suggest that uPAR function is necessary for the normal development of a subpopulation of GABAergic neurons in the telencephalon. It is likely that the late-onset parvalbumin phenotype is due to the effects of an altered local environment on selectively vulnerable neurons and that the extent of this loss is strain dependent. Thus, an interplay between complex genetic factors and the environment may influence the phenotypic impact of the uPAR mutation both pre- and postnatally.

Aging↗

PubMed2005

Direct profiling of the cerebellum by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: A methodological study in postnatal and adult mouse.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) can detect substantial changes in expression of proteins in tissues, such as cancer cells. A more challenging problem is detecting the smaller changes expected in normal development or complex diseases. Here we address methodological issues regarding the acquisition and analysis of MALDI MS data from tissue sections, in a study of mouse cerebellum at different stages of development. Sections of the cerebellar cortex were analyzed at the peak of granule neuron production [postnatal day (P) 7], during synapse formation (P14), and in adults. Data were acquired (Voyager-DEtrade mark STR Biospectrometry Workstation; seven acquisitions of 50 shots per section, 3.5-50 kDa), preprocessed (Data Explorer 4.3), and averaged. Among 846 peaks detected, in at least 50% of at least one group, 122 showed significant group differences (Kruskal-Wallis ANOVA) after Bonferroni correction. Factor analyses revealed two age-related factors, possibly reflecting gradients of expression during development. Predictive analysis of microarrays generated a model from half of the sample that correctly predicted developmental groups for the second half. Intraclass correlation coefficients, measuring within-mouse consistency of peak heights from three tissue sections, were acceptable at lower m/z and for larger peaks at higher m/z. Low mass was the best predictor of significant group differences. The analysis demonstrates that MALDI MS of normal tissue sections at different ages can detect consistent, significant group differences. Further work is needed to increase the sensitivity of the methods and to apply them reliably to brain regions and to subproteomes with relevance to diverse brain functions and diseases.

Age Factors↗

PubMed2005

Regional expression of brain derived neurotrophic factor (BDNF) is correlated with dynamic patterns of promoter methylation in the developing mouse forebrain.

Brain Derived Neurotrophic Factor (BDNF) plays an important role in brain development and plasticity. BDNF gene expression is known to be dynamically regulated during development, but the regulatory controls of normal differential expression are not well understood. Methylation of CpG dinucleotides within gene promoters is emerging as an important epigenetic control mechanism of transcription, and the BDNF complex promoter contains several CpG dinucleotides. We determined BDNF expression in the developing mouse forebrain and examined whether there were correlated patterns of methylation at CpG dinucleotides within the BDNF promoter. The data show that BDNF is dynamically expressed in the mouse forebrain and that expression is correlated with differential methylation specifically at CpG dinucleotides in eIV of the mouse BDNF promoter. These studies demonstrate that DNA methylation of this regulatory region may be an important mechanism controlling differential expression of BDNF during forebrain development.

Animals↗