Pharmacogenetic analysis of the mGlu2/3 agonist LY2140023 monohydrate in the treatment of schizophrenia.

The goal of this study was to identify genetic markers associated with LY2140023 monohydrate response in patients with schizophrenia. Variants in eight candidate genes related to the mechanism of action of LY2140023 or olanzapine were investigated in a genetic cohort collected from two clinical trials. Results from this genetic analysis indicate that 23 single nucleotide polymorphisms (SNPs) were associated with a change in Positive and Negative Syndrome Scale total score in response to LY2140023 at 28 days (P<0.01; false discovery rate <0.2). Sixteen of these SNPs were located in the serotonin 2A receptor (HTR2A). Bioinformatic analyses identified a putative antisense nested gene in intron 2 of HTR2A in the region of the genetic markers associated with LY2140023 response. These data suggest a genetic association exists between SNPs in several genes, such as HTR2A, and response to LY2140023 treatment. Additional clinical trials are needed to establish replication of these results.

The ultimate chip shot: can microarray technology deliver for neuroscience?

The use of cDNA and oligonucleotide microarrays, or 'chips', is emerging as a powerful, new technology in the field of neuroscience for examining gene expression in a high-throughput fashion. The application of microarray technology to the study of brain and behavior has lagged behind other areas of biology such as cancer and yeast genetics due to the challenges presented by the heterogeneous and complex organization of the nervous system. This review provides a brief overview of available microarray technology as well as a description of experimental considerations in planning and implementing a neuroscience-based array study. Successful implementation of microarray technology within the field of neuroscience will provide a molecular approach to studying systems neurobiology, leading to insights into areas ranging from fundamental questions of developmental neurobiology to neurological and psychiatric disorders.

The muscarinic agonist xanomeline increases monoamine release and immediate early gene expression in the rat prefrontal cortex.

BACKGROUND: The muscarinic agonist xanomeline has been shown to reduce antipsychotic-like behaviors in patients with Alzheimer's disease. Because atypical antipsychotic agents increase dopamine release in prefrontal cortex and induce immediate early gene expression in prefrontal cortex and nucleus accumbens, the effect of xanomeline was determined on these indices. METHODS: The effect of xanomeline on extracellular levels of monoamines in brain regions was determined using a microdialysis technique, and changes in expression of the immediate early genes c-fos and zif/268 in brain regions were evaluated using in situ hybridization histochemistry. RESULTS: Xanomeline increased extracellular levels of dopamine in prefrontal cortex and nucleus accumbens but not in striatum. Xanomeline increased expression of c-fos and zif/268 in prefrontal cortex and nucleus accumbens. There was no change in immediate early gene expression in striatum. CONCLUSIONS: Xanomeline increased extracellular levels of dopamine, which is similar to the effects of the atypical antipsychotics clozapine and olanzapine. The regional pattern of immediate early gene expression induced by xanomeline resembled that of atypical antipsychotic agents. Based on the antipsychotic-like activity of xanomeline in Alzheimer's patients and the similarity to atypical antipsychotic agents, we suggest that xanomeline may be a novel antipsychotic agent.

Dopaminergic and muscarinic regulation of striatal enkephalin and substance P messenger RNAs following striatal dopamine denervation: effects of systemic and central administration of quinpirole and scopolamine.

Striatal dopamine depletion produces an increase in enkephalin and a decrease in substance P messenger RNAs. Subsequent systemic administration of either the D2 dopamine agonist, quinpirole, or the muscarinic antagonist, scopolamine, results in the reduction of the lesion-induced elevation in striatal enkephalin messenger RNA. These changes in enkephalin messenger RNA levels may be mediated solely within the striatum or through trans-synaptic circuits involving the striatum. To dissociate these possibilities, we have compared the effects of systemic and central administration of quinpirole and scopolamine on striatal enkephalin and substance P messenger RNAs using in situ hybridization histochemistry. Systemic administration of both quinpirole and scopolamine blocked the elevation of striatal enkephalin messenger RNA normally observed in 6-hydroxydopamine-lesioned rats. In addition, high doses of systemic scopolamine (25 and 50 mg/kg per day) prevented the lesion-induced decrease in striatal substance P messenger RNA levels. In order to determine whether the effects of these drugs are mediated directly within the striatum, central administration of quinpirole and scopolamine were compared. In contrast to systemic administration, intraventricular and intrastriatal infusion of quinpirole but not scopolamine prevented the lesion-induced change in striatal enkephalin messenger RNA. However, neither quinpirole nor scopolamine administered centrally affected the level of substance P messenger RNA in the striatum of 6-hydroxydopamine-induced lesioned animals. Together, these data suggest that changes in D2 receptor activation directly in the striatum are responsible for the effects of quinpirole on enkephalin messenger RNA. In contrast, the effect of systemic scopolamine on striatal enkephalin and substance P messenger RNAs may not be mediated within the striatum.

Temporal dissociation between changes in striatal enkephalin and substance P messenger RNAs following striatal dopamine depletion.

Changes in the levels of enkephalin and substance P messenger RNA expression were examined in the striatum following dopamine depletion resulting from unilateral injection of 6-hydroxydopamine into the substantia nigra. In response to striatal dopamine depletion, the levels of enkephalin messenger RNA were elevated, whereas substance P messenger RNA was decreased within all regions of the striatum. Examination of the striatal peptide messenger RNAs between one and 21 days after the injection of 6-hydroxydopamine revealed a temporal dissociation between changes in enkephalin and substance P messenger RNAs. Within one day of the 6-hydroxydopamine injection, substance P messenger RNA was significantly decreased by 30% at all levels of the striatum. This decrease was maintained for up to 21 days after the lesion. In contrast, striatal enkephalin messenger RNA was not significantly elevated until three days following the injection of 6-hydroxydopamine, after which there was a gradual increase up to 21 days. In order to correlate alterations in peptide messenger RNA expression with 6-hydroxydopamine-induced changes in striatal dopamine innervation, tissue punches from the striatum were examined for dopamine content at one, two, three and seven days after the lesion. One day after the lesion, striatal dopamine levels were significantly increased by 47%. In contrast, within two days tissue dopamine content was reduced by 77% compared to control levels. A further decrease of 90% or more was observed at three and seven days after the lesion. Taken together, these data demonstrate a temporal dissociation between changes in enkephalin and substance P messenger RNA levels following 6-hydroxydopamine-induced striatal dopamine depletions. This temporal dissociation may reflect a differential response of enkephalin and substance P messenger RNAs to alterations in dopamine release and subsequent receptor activation.

Partial striatal dopamine depletion differentially affects striatal substance P and enkephalin messenger RNA expression.

Near total striatal dopamine denervation results in a decrease in substance P and an increase in enkephalin messenger RNA expression in the striatum. It is unknown whether partial depletions of striatal dopamine content produce similar changes in these peptide messenger RNAs. To test whether compensations in dopamine synthesis and release following partial dopamine denervation prevent the lesion-induced alterations in substance P and enkephalin messenger RNAs, varying concentrations of 6-hydroxydopamine were injected unilaterally into the substantia nigra. Seven days after injection of 6-hydroxydopamine (2-16 micrograms) or vehicle, in situ hybridization histochemistry was used to examine tyrosine hydroxylase messenger RNA in the substantia nigra and substance P and enkephalin messenger RNAs in the striatum. The extent of the dopamine depletion was determined by measuring striatal dopamine tissue content. The decrease in tyrosine hydroxylase messenger RNA paralleled the change in striatal tissue dopamine content. Substance P messenger RNA was decreased in all lesioned rats. In contrast, a significant increase in enkephalin messenger RNA was not detected until striatal dopamine was reduced to 10% of control levels. These results suggest that compensations within the residual dopamine system are not sufficient to maintain normal striatal substance P messenger RNA levels in partially denervated animals, but are sufficient to maintain normal striatal enkephalin messenger RNA expression.

In vitro biocytin injection into perinatal mouse brain: a method for tract tracing in developing tissue.

Injection of biocytin provides an effective method for labeling axonal projections. Several difficulties arise when this technique is employed in fetal or early postnatal animals in vivo, including limited access to injection sites and extended post-injection survival periods. To circumvent these problems, we adapted the technique of extracellular biocytin injection for use in explanted brain hemispheres of developing mice. Briefly, entire brain hemispheres from perinatal mice (E16-P9) were removed and placed in oxygenated aCSF in a brain slice recording chamber. Following visually guided injection of biocytin (2%) into the prelimbic cortex, the brains were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) for varying periods of time and then immersion-fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. The next day, the brains were sectioned and processed for biocytin histochemistry using the avidin-biotin-complex method. We examined the method of injection, electrode type, time of injection, and post-injection incubation period. We found that in E16-P9 animals iontophoresis of biocytin using 8- to 12-megaohm patch clamp electrodes for a duration of 10 min provides optimal axonal labeling. Post-injection incubation times of four or more hours are sufficient for labeling fine caliber collaterals as well as axon bundles that reach distances over 3 mm. In vitro injection of biocytin into explanted brain hemispheres provides a quick and easy method for tract tracing in developing brains.

Presynaptic alterations associated with enhancement of evoked release and synthesis of norepinephrine in hippocampus of chronically cold-stressed rats.

We have previously demonstrated that prior exposure to chronic cold stress does not alter basal levels of norepinephrine (NE) release or synthesis in hippocampus of rat. However, in response to a subsequent novel stressor, an enhancement of both of these noradrenergic parameters is observed in the chronically stressed animals relative to naive controls. In the present experiments, we have examined whether the biochemical sensitization of NE release and synthesis produced by chronic stress can be demonstrated by local depolarization of the noradrenergic nerve terminals with elevated K+. The local application of elevated K+ in dorsal hippocampus resulted in a greater increase in extracellular NE and 3,4-dihydroxyphenylacetic acid (DOPAC) in chronically stressed rats than in naive controls. It is proposed that in dorsal hippocampus, extracellular NE and DOPAC provide measures of NE release and biosynthesis, respectively. Therefore, these data suggest that local depolarization, similar to novel stress, elicits both enhanced NE release and synthesis in chronically stressed rats. One factor that is known to modulate both of these processes is the presynaptic alpha-2 adrenergic receptor. Therefore, we examined whether a change in the sensitivity of these receptors might contribute to the altered noradrenergic responsivity observed in chronically stressed rats. Local administration of clonidine, an alpha-2 receptor agonist, produced a decrease in extracellular NE and DOPAC in both naive and chronically stressed rats. The dose-response curve for the effect of clonidine on NE was shifted to the left in the latter group. In addition, local administration of idazoxan, an alpha-2 receptor antagonist, produced a greater increase in extracellular NE and DOPAC in the chronically stressed rats than in naive controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of streptozotocin-induced diabetes on cell proliferation in the rat dentate gyrus.

Recent evidence has shown an association between diabetes mellitus and deficits in learning and memory. However, the mechanism by which cognitive abilities are impaired in diabetes has not been identified. The dentate gyrus of the hippocampus plays a significant role in spatial learning and memory. Studies in rodents show that learning tasks enhance neurogenesis in the dentate gyrus of the adult hippocampus. To investigate whether cognitive deficits in diabetes may be related to alterations in hippocampal neurogenesis, we measured the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells, an indicator of cell proliferation, in the dentate gyrus in an animal model of diabetes. Streptozotocin-induced diabetes produced a dramatic decrease in cell proliferation in the dentate gyrus as compared to controls. The results from this study suggest a potential role for alterations in neurogenesis in the cognitive decline observed in diabetes mellitus.

Enhanced tyrosine hydroxylation in hippocampus of chronically stressed rats upon exposure to a novel stressor.

We have used microdialysis to measure the in vivo level of tyrosine hydroxylation in hippocampus of the freely moving rat. An inhibitor of aromatic amino acid decarboxylase, NSD-1015, was administered through the dialysis probe and the resulting accumulation of 3,4-dihydroxyphenylalanine (DOPA) in extracellular fluid of hippocampus was quantified. Administration of the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, decreased extracellular DOPA to undetectable level. In addition, both systemic and local application of clonidine, an alpha 2-adrenergic agonist, produced a decrease in extracellular DOPA. In response to acute tail shock, a significant increase in extracellular DOPA was observed. Thus, it appears that in vivo accumulation of DOPA after local administration of NSD-1015 provides a reliable index of hippocampal tyrosine hydroxylation. We have used this technique to investigate whether prior exposure to chronic stress alters the in vivo level of tyrosine hydroxylation in hippocampus under basal conditions as well as in response to a novel stressor. In rats previously exposed to chronic cold stress, the basal accumulation of extracellular DOPA did not differ from naive controls. Acute tail shock, however, produced a significantly greater and more prolonged elevation in extracellular DOPA of chronically stressed rats. These data suggest that enhanced biosynthetic capacity of noradrenergic terminals may be one mechanism underlying adaptation to chronic stress.

Prior exposure to chronic stress results in enhanced synthesis and release of hippocampal norepinephrine in response to a novel stressor.

The release and synthesis of norepinephrine (NE) in hippocampus were measured in naive and chronically cold-stressed rats in response to acute tail-shock stress. Using in vivo microdialysis, it was determined that the basal extracellular concentrations of NE and 3,4-dihydroxyphenylacetic acid (DOPAC) in hippocampus were the same in the two groups. However, 30 min of intermittent tail shock produced a greater elevation of extracellular NE and 3,4-dihydroxyphenylacetic acid in the chronically cold-stressed rats than in the native controls. In hippocampus, the extracellular concentration of DOPAC may reflect NE biosynthesis, and thus the enhanced DOPAC response in the chronically stressed rats suggests an increase in NE synthesis. In order to investigate this possibility, two further methods of assessing NE biosynthesis were employed. Tyrosine hydroxylase (TH) activity was assayed in vitro in the presence of saturating concentrations of cofactor. No change in maximal TH activity could be detected in hippocampus of chronically cold-stressed rats. In addition, the in vivo rate of tyrosine hydroxylation in cold-stressed rats was measured by the accumulation of 3,4-dihydroxyphenylalanine in tissue following inhibition of aromatic amino acid decarboxylase. It was found that, whereas basal synthesis was the same in both groups of rats, synthesis accompanying a novel stressor was increased to a greater extent in the chronically stressed rats.

Identification of MARPP-58, a morphine- and cyclic AMP-regulated phosphoprotein of 58 kDa, as tyrosine hydroxylase: evidence for regulation of its expression by chronic morphine in the rat locus coeruleus.

Previously, we have identified a number of morphine- and cyclic AMP-regulated phosphoproteins (MARPPs) in the rat locus coeruleus (LC) and other brain regions. We now show that one of these phosphoproteins, a 58 kDa protein designated MARPP-58, is tyrosine hydroxylase. First, MARPP-58 comigrates with immunolabeled, immunoprecipitated, and purified tyrosine hydroxylase on 1- and 2-dimensional electrophoresis. Second, MARPP-58, immunoprecipitated tyrosine hydroxylase, and purified tyrosine hydroxylase yield identical 1-dimensional phosphopeptide maps. Third, MARPP-58 exhibits a regional and subcellular distribution in brain consistent with tyrosine hydroxylase. Identification of MARPP-58 as tyrosine hydroxylase made it possible to determine whether increases in MARPP-58 phosphorylation induced by chronic morphine in the LC reported previously are associated with alterations in enzyme activity and expression in this brain region. We show that chronic treatment of rats with morphine increases levels of tyrosine hydroxylase activity, immunoreactivity, and mRNA in the LC. Induction of the enzyme by chronic morphine was blocked by concomitant treatment of rats with the opiate receptor antagonist naltrexone, indicating that morphine produces this effect through the activation of opiate receptors. Consistent with previous observations that the chronic morphine-induced change in MARPP-58 phosphorylation is specific to the LC, changes observed in enzyme activity, immunoreactivity, and mRNA were not observed in a number of other brain regions studied. The results indicate that chronic morphine regulates the expression of tyrosine hydroxylase specifically in the LC and suggest that such regulation reflects long-term adaptations of LC neurons to chronic morphine at the level of gene expression.

Early patterning of prelimbic cortical axons to the striatal patch compartment in the neonatal mouse.

The striatum receives excitatory input from virtually the entire cerebral cortex. In the adult, this input is segregated into two functionally distinct compartments of the striatum, the patch (striosome) and matrix regions. This study determined whether the patterning of corticostriatal afferents from the prelimbic cortex to the striatal patch compartment develops during the early period of collateral formation or instead at the time of peak synaptogenesis. Initial formation of corticostriatal axon collaterals was observed by embryonic day (E) 19. Quantification of corticostriatal collaterals revealed a significant increase in the number and complexity of collateral branches at postnatal day 6 as compared to E19. Concomitant with the increase in collateral branching, a heterogeneous pattern of collateralization consisting of parallel rows of corticostriatal collaterals was observed in the medial striatum. In addition to the rows, clusters of corticostriatal axons occurred more laterally. These clusters colocalized with patches of dense tyrosine hydroxylase-positive fibers, a marker for the striatal patch compartment in the neonatal mouse. Together, these data indicate that corticostriatal patterning occurs during the period of early axon collateralization resulting in a segregation of corticostriatal axon collaterals from the prelimbic cortex to the striatal patch compartment.

Effects of cold exposure on rat adrenal tyrosine hydroxylase: an analysis of RNA, protein, enzyme activity, and cofactor levels.

Long-term cold exposure (5-7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3-6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48-72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold-induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre- and posttranslational regulation. Our findings also suggest that cold stress-induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.