C. Anthony Altar

Electroconvulsive Seizures Regulate Gene Expression of Distinct Neurotrophic Signaling Pathways

Electroconvulsive therapy (ECT) remains the treatment of choice for drug-resistant patients with depressive disorders, yet the mechanism for its efficacy remains unknown. Gene transcription changes were measured in the frontal cortex and hippocampus of rats subjected to sham seizures or to 1 or 10 electroconvulsive seizures (ECS), a model of ECT. Among the 3500–4400 RNA sequences detected in each sample, ECS increased by 1.5- to 11-fold or decreased by at least 34% the expression of 120 unique genes. The hippocampus produced more than three times the number of gene changes seen in the cortex, and many hippocampal gene changes persisted with chronic ECS, unlike in the cortex. Among the 120 genes, 77 have not been reported in previous studies of ECS or seizure responses, and 39 were confirmed among 59 studied by quantitative real time PCR. Another 19 genes, 10 previously unreported, changed by <1.5-fold but with very high significance. Multiple genes were identified within distinct pathways, including the BDNF–MAP kinase–cAMP–cAMP response element-binding protein pathway (15 genes), the arachidonic acid pathway (5 genes), and more than 10 genes in each of the immediate-early gene, neurogenesis, and exercise response gene groups. Neurogenesis, neurite outgrowth, and neuronal plasticity associated with BDNF, glutamate, and cAMP–protein kinase A signaling pathways may mediate the antidepressant effects of ECT in humans. These genes, and others that increase only with chronic ECS such as neuropeptide Y and thyrotropin-releasing hormone, may provide novel ways to select drugs for the treatment of depression and mimic the rapid effectiveness of ECT.

Deficient Hippocampal Neuron Expression of Proteasome, Ubiquitin, and Mitochondrial Genes in Multiple Schizophrenia Cohorts

BACKGROUND Hippocampal dentate granule neurons are altered in schizophrenia, but it is unknown if their gene expressions change in schizophrenia or other psychiatric diseases. METHODS Laser-captured dentate granule neurons from two groups of schizophrenia and control cases and from major depression and bipolar disease cases were examined for alterations in gene expression using complementary DNA (cDNA) microarrays and reverse transcription polymerase chain reaction (RT-PCR). RESULTS Compared with 24 control cases, the 22 schizophrenia patients in both groups revealed decreases in clusters of genes that encode for protein turnover (proteasome subunits and ubiquitin), mitochondrial oxidative energy metabolism (isocitrate, lactate, malate, nicotinamide adenine dinucleotide [NADH], and succinate dehydrogenases; cytochrome C oxidase; adenosine triphosphate [ATP] synthase), and genes associated with neurite outgrowth, cytoskeletal proteins, and synapse plasticity. These changes were not obtained in 9 bipolar cases or 10 major depression cases and were not associated with age, sex, brain weight, body weight, postmortem interval, or drug history. Brain pH contributed to the variance of some genes but was mostly independent of the disease effect. CONCLUSIONS Decreases in hippocampal neuron gene expression are consistent with brain imaging and microarray studies of the frontal cortex in schizophrenia. A mitochondrial and ubiquitin-proteasome hypofunctioning of dentate granule neurons may contribute to the deficits of schizophrenia.

Quantitative analysis of [3H]spiroperidol binding to rat forebrain sections: Plasticity of neostriatal dopamine receptors after nigrostriatal injury

The binding of [3H]spiroperidol to rat coronal sections in vitro was investigated using two procedures: swabbing studies, in which the tissue sections are wiped from the microscope slides after incubation in the presence of [3H]spiroperidol, and autoradiographic studies, in which the autoradiographic negatives are analyzed using computer-assisted densitometry. In the swabbing studies, the pharmacological and kinetic properties of butaclamol-displaceable binding were investigated, and the following results suggest that [3H]spiroperidol binds specifically to only a single site within the basal forebrain of tissue sections and that the site is the dopamine D-2 receptor. The pseudo-first order and first order plots for the rate of association to and dissociation from tissue sections appeared to be linear. Dopamine antagonists, such as haloperidol and butaclamol, were much more effective than dopamine agonists or the serotonin S-2 ligand, ketanserin, in inhibiting [3H]spiroperidol binding. The ability of dopamine agonists to inhibit [3H]spiroperidol binding was markedly reduced by the guanine nucleotide, Gpp(NH)p. Saturation analysis of specific [3H]spiroperidol binding revealed a Kd and Bmax of 0.93 nM and 447 fmol/mg protein, and a Hill coefficient of 1.05. The findings are also compatible with the possibility that [3H]spiroperidol binds to several sites that have identical affinities for this ligand. Densitometric studies were used to assess the effect of lesions on [3H]spiroperidol binding in the neostriatum. Intrastriatal injection of kainic acid substantially reduced 1 microM (+)--butaclamol-displaceable binding, indicating that the receptors are in large part on intrinsic striatal neurons. Neostriatal [3H]spiroperidol binding was investigated 7 days after destruction of the mesotelencephalic dopamine system by the ventral tegmental injection of 6-hydroxydopamine. As determined by saturation analysis, the average values for Kd and Bmax were 0.66 nM and 1212 fmol/mg protein in the intact striatum, and 0.82 nM and 1504 fmol/mg in the denervated striatum. The finding of a significant 23.8% increase in receptor density by the end of the first postoperative week, a period during which behavioral supersensitivity to apomorphine increases rapidly, supports the hypothesis that a proliferation of D-2 receptors underlies the behavioral manifestations of denervation supersensitivity.

Aripiprazole blocks reinstatement of cocaine seeking in an animal model of relapse.

BACKGROUND Aripiprazole (Abilify) is an atypical antipsychotic drug primarily characterized by partial agonist activity at dopamine (DA) D2 receptors and low side effects. Based on pharmacologic properties that include a stabilization of mesocorticolimbic DA activity, a pathway implicated in addiction, aripiprazole was tested for its ability to prevent relapse to cocaine seeking in rats. METHODS We assessed the dose-dependent effects of aripiprazole on conditioned cue-induced and cocaine-primed reinstatement of drug-seeking behavior following chronic intravenous cocaine self-administration in an animal model of relapse. RESULTS Aripiprazole potently and dose-dependently attenuated responding on the previously cocaine-paired lever during both reinstatement conditions, with slightly greater efficacy at reducing conditioned-cued reinstatement. Aripiprazole was effective at doses that failed to alter cocaine self-administration, food self-administration, reinstatement of food-seeking behavior, or basal locomotor activity, suggesting selective effects of aripiprazole on motivated drug-seeking behavior. CONCLUSIONS These results in a relapse model show that aripiprazole can block cocaine seeking without affecting other behaviors. The D2 partial agonist properties of aripiprazole likely account for the blockade of reinstatement of cocaine-seeking behavior. Given its established efficacy and tolerability as a treatment for psychosis, aripiprazole may be an excellent therapeutic choice for reducing craving and preventing relapse in people with cocaine dependency.

Comparison of microarray-based mRNA profiling technologies for identification of psychiatric disease and drug signatures

The gene expression profiles of human postmortem parietal and prefrontal cortex samples of normal controls and patients with bipolar disease, or human neuroblastoma flat (NBFL) cells treated with the mood-stabilizing drug, valproate, were used to compare the performance of Affymetrix oligonucleotide U133A GeneChips and Agilent Human 1 cDNA microarrays. Among those genes represented on both platforms, the oligo array identified 26-53% more differentially expressed genes compared to the cDNA array in the three experiments, when identical fold change and t-test criteria were applied. The increased sensitivity was primarily the result of more robust fold changes measured by the oligonucleotide system. Essentially all gene changes overlapping between the two platforms were co-directional, and ranged from 4 to 19% depending upon the amount of biological variability within and between the comparison groups. Q-PCR validation rates were virtually identical for the two platforms, with 23-24% validation in the prefrontal cortex experiment, and 56% for both platforms in the cell culture experiment. Validated genes included dopa decarboxylase, dopamine beta-hydroxylase, and dihydropyrimidinase-related protein 3, which were decreased in NBFL cells exposed to valproate, and spinocerebellar ataxia 7, which was increased in bipolar disease. The modest overlap but similar validation rates show that each microarray system identifies a unique set of differentially expressed genes, and thus the greatest information is obtained from the use of both platforms.

Striatal dopamine uptake and swim performance of the aged rat

The striatum and olfactory tubercle of 30-month-old F344 rats contain significantly (21-24%) less dopamine compared with young adult (8-month) animals. However, rats of the two age groups show identical Km and Vmax values for the kinetics of [3H]dopamine uptake into striatal homogenates; uptake into the olfactory tubercle also appeared unaffected in old age. The preservation of dopamine uptake despite reductions in content of the transmitter suggests that the forebrain dopaminergic nerve terminals are intact, but that reduced dopamine synthesis and/or enhanced degradation may occur in the existing terminals. Administration to senescent animals of the dopamine uptake blockers nomifensine or bupropion (but not the norepinephrine uptake blocker desmethylimipramine) improved their swim performance to levels comparable with young adult animals. The findings suggest that amine reuptake may limit the synaptic effectiveness of dopamine released in the aged striatum.

Insulin, IGF-1, and Muscarinic Agonists Modulate Schizophrenia-associated Genes in Human Neuroblastoma Cells

BACKGROUND Genes associated with energy metabolism are decreased in schizophrenia brain and human and rodent diabetic skeletal muscle. These and other similarities between diabetes and schizophrenia suggest that an insulin signaling deficit may underlie schizophrenia. We determined with human SH-SY5Y neuroblastoma and astrocyte cell lines whether insulin or other molecules could modulate genes opposite to their change reported in schizophrenia brain. METHODS Both cell lines were treated with insulin, insulin-like growth factor (IGF)-1, IGF-2, or brain-derived neurotrophic factor (BDNF). Genes whose expression was found with microarrays to be changed by insulin in a reciprocal manner to their change in schizophrenia were used in a 16-gene miniarray to identify small molecules that might mimic insulin. RESULTS Insulin phosphorylated its receptor in the neuroblastoma cells but not in astrocytes and, like IGF-1, increased ERK1/2 and Akt phosphorylation. Insulin and IGF-1 increased the expression of genes decreased in schizophrenia, including those involved in mitochondrial functions, glucose and energy metabolism, hydrogen ion transport, and synaptic function. These gene effects were confirmed and shown to be dose related with the 16-gene miniarrays. Most of 1940 pharmacologically unique compounds failed to alter gene expression, with the exception of muscarinic agonists, which mimicked insulin and IGF-1, and which were blocked by the muscarinic antagonists atropine and telenzepine. CONCLUSIONS Stimulation of muscarinic and insulin/IGF-1 receptors alter genes associated with metabolic and synaptic functions in a manner reciprocal to their changes in schizophrenia. Pharmacologic activation of these receptors may normalize genomic alterations in schizophrenia and better address root causes of this disease.

Altered expression of hippocampal dentate granule neuron genes in a mouse model of human 22q11 deletion syndrome

Hemizygous deletion of a 3 Mb region of 22q11.2 is found in 1/4000 humans and produces 22q11 deletion syndrome (22q11DS). Up to 35% of 22q11DS patients develop schizophrenia, making it the second highest risk factor for schizophrenia. A mouse model for 22q11DS, the Df1/+ mouse, carries a hemizygous deletion in a region syntenic with the human deletion. Df1/+ mice are mostly viable but display deficits in prepulse inhibition and learning and memory, two common traits of schizophrenia thought to result, at least in part, from defects in hippocampal neurons. We used oligonucleotide microarrays and QRT-PCR to evaluate gene expression changes in hippocampal dentate granule neurons of Df1/+ mice versus wild-type littermates (n=12/group). The expression of only 287 genes changed with p value significance below 0.05 by microarray, yet 12 of the 21 Df1 region genes represented on the array showed highly significantly reduced expression compared to wild-type controls (33% on average, p values from 10(-3) to 10(-7)). Variants in two of these genes, COMT and PRODH, have been linked with schizophrenia. Overlap of the 287 genes with the reportedly reduced expression of mitochondrial, ubiquitin/proteasome, and synaptic plasticity genes in schizophrenia dentate granule neurons, was not significant. However, modest increases in expression of mitochondrial electron transport genes were observed in the Df1/+ mice. This perhaps indicates a compensation for mitochondrial dysfunction caused by the strongly reduced expression of the Df1 region-encoded mitochondrial enzymes proline dehydrogenase (Prodh) and thioredoxin reductase 2 (Txnrd2).