Csaba Vadasz

The nature and identification of quantitative trait loci: a community’s view

This white paper by eighty members of the Complex Trait Consortium presents a communitys view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

Cannabinoid CB1 receptor knockout mice exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus accumbens

The mechanisms underlying predisposition to alcohol abuse and alcoholism are poorly understood. In this study, we evaluated the role of cannabinoid (CB1) receptors in (i) voluntary alcohol consumption, and (ii) acute alcohol‐induced dopamine (DA) release in the nucleus accumbens, using mice that lack the CB1 receptor gene (CB1–/–). CB1–/– mice exhibited dramatically reduced voluntary alcohol consumption, and completely lacked alcohol‐induced DA release in the nucleus accumbens, as compared to wild‐type mice. The gender difference, with female mice consuming significantly more alcohol than wild‐type male mice, was observed in wild‐type mice, whereas this gender difference was nonexistent in CB1 mutant male and female mice. There was also a significant gender difference, with the wild‐type, heterozygous, and mutant females consuming significantly more liquid and food than wild‐type, heterozygous and mutant males. However, the total volume of fluid consumption and food intake did not differ between wild‐type, heterozygous, and mutant mice. These results strongly suggest that the CB1 receptor system plays an important role in regulating the positive reinforcing properties of alcohol.

Neurons in the Basal Forebrain Project to the Cortex in a Complex Topographic Organization that Reflects Corticocortical Connectivity Patterns: An Experimental Study Based on Retrograde Tracing and 3D Reconstruction

The most prominent feature of the Basal Forebrain (BF) is the collection of large cortically projecting neurons (basal nucleus of Meynert) that serve as the primary source of cholinergic input to the entire cortical mantle. Despite its broad involvement in cortical activation, attention, and memory, the functional details of the BF are not well understood due to the anatomical complexity of the region. This study tested the hypothesis that basalocortical connections reflect cortical connectivity patterns. Distinct retrograde tracers were deposited into various frontal and posterior cortical areas, and retrogradely labeled cholinergic and noncholinergic neurons were mapped in the BF. Concurrently, we mapped retrogradely labeled cells in posterior cortical areas that project to various frontal areas, and all cell populations were combined in the same coordinate system. Our studies suggest that the cholinergic and noncholinergic projections to the neocortex are not diffuse, but instead, are organized into segregated or overlapping pools of projection neurons. The extent of overlap between BF populations projecting to the cortex depends on the degree of connectivity between the cortical targets of these projection populations. We suggest that the organization of projections from the BF may enable parallel modulation of multiple groupings of interconnected yet nonadjacent cortical areas.

Decreased Oral Self-Administration of Alcohol In κ-Opioid Receptor Knock-Out Mice

BACKGROUND Although a large body of evidence suggests a role for the opioid system in alcoholism, the precise role of mu-, delta-, kappa-, and ORL1-opioid receptors and the physiological significance of their natural genetic variation have not been identified. The method of targeted gene disruption by homologous recombination has been used to knock out (KO) genes coding for opioid receptors, and study their effects on alcohol self-administration. Here we examined the effects of targeted disruption of kappa-opioid receptor (KOR) on oral alcohol self-administration and other behaviors. METHODS Oral alcohol, saccharin and quinine self-administration was assessed in a two-bottle choice paradigm using escalating concentrations of alcohol, or tastant solutions. In preference tests 12% alcohol, 0.033% and 0.066% saccharin, and 0.03 mM and 0.1 mM quinine solutions were used. Open-field activity was determined in an arena equipped with a computer-controlled activity-detection system. Subjects were tested for three consecutive days. Locomotor activity was assessed on days 1 and 2 (after saline injection, i.p.) and on day 3 (after alcohol injection, i.p.). Alcohol-induced locomotor activity was determined as the difference in activity between day 3 and day 2. RESULTS Male KOR KO mice in preference tests with 12% alcohol consumed about half as much alcohol as wild-type (WT) or heterozygous (HET) mice, showed lower preference for saccharin (0.033% and 0.066%) and higher preference to quinine (0.1 mM) than WT mice. Female KOR KO mice showed similar reduction in alcohol consumption in comparison to WT and HET mice. Partial deletion of KOR in HET mice did not change alcohol consumption in comparison to WT mice. In all genotype-groups females drank significantly more alcohol than males. MANOVA of locomotor activity among KO, WT, and HET mice indicated that strain and sex effects were not significant for alcohol-induced activation (p > 0.05), while strain x sex interaction effects on alcohol-induced activation could be detected (F(1,55) = 6.07, p < 0.05). CONCLUSION Our results indicating decreased alcohol consumption, lower saccharin preference, and higher quinine preference in KOR KO mice are in line with previous observations of opioid involvement in maintenance of food intake and raise the possibility that the deficient dynorphin/KOR system affects orosensory reward through central mechanisms which reduce alcohol intake and disrupt tastant responses, either as direct effects of absence of kappa-opioid receptors, or as effects of indirect developmental compensatory changes.

Spike-and-wave neocortical patterns in rats : genetic and aminergic control

Spontaneously occurring and drug-induced high voltage spike-and-wave electroencephalogram patterns were examined in inbred rats of the Fischer 344 and Buffalo strains and of the random-bred Sprague-Dawley strain at different ages. In addition, tyrosine hydroxylase activity and dopamine D2 receptor density were determined in the substantia nigra, corpus striatum, olfactory tubercle and ponsmedulla areas of Fisher 344 and Buffalo animals. High voltage spike-and-wave episodes were present in 87.5% of the 3-month-old and in 100% of the older Fischer 344 rats. High voltage spike-and-wave episodes were completely absent in 3-month-old Buffalo and Sprague-Dawley animals but could be induced by systemic injection of pentylenetetrazol and at an older age they appeared in 58.3% (12-month) and 71.4% (greater than 26-month) of the subjects of these strains. The incidence and duration of high voltage spike-and-wave episodes were significantly higher/longer in Fischer 344 rats than in the age-matched Buffalo and Sprague-Dawley animals. The dopamine blocker acepromazine induced a several-fold increase of the incidence and duration of high voltage spike-and-wave episodes in 3-month-old Fischer 344 rats, but failed to induce high voltage spike-and-wave episodes in Buffalo animals at this age. However, acepromazine also triggered high voltage spike-and-wave episodes in Buffalo rats when they were pretreated with subthreshold doses of pentylenetetrazol. Tyrosine hydroxylase activity was significantly higher in the substantia nigra, corpus striatum and olfactory tubercle of the Fischer 344 strain than in Buffalo rats. The higher tyrosine hydroxylase activity was paralleled with significantly higher D2 binding values in the corpus striatum and olfactory tubercle of Fischer 344 rats. These findings suggest that the neocortical high voltage spike-and-wave phenotype is genetically mediated and that the inbred Fischer 344 and Buffalo rats with defined bilineal origin will facilitate future works aimed at the identification of genetic elements involved in the generation of neocortical high voltage spike-and-wave episodes. The significant genotype x age interaction supports the suggestion, however, that high voltage spike-and-wave episodes are likely to be influenced by more than one gene; some of them are probably related to the regulation of brain aminergic systems.

Microarray analysis of gene expression in rat hippocampus after chronic ethanol treatment.

It is thought that changes in gene expression in the brain mediate chronic ethanol-induced complex behaviors such as tolerance, dependence, and sensitization, and also relate to ethanol-induced brain toxicity. Using high-density filter-based cDNA microarrays (GeneFilters), we analyzed the expression of over 5000 genes in the dorsal hippocampus of rats treated with 12% ethanol or tap water for 15 months. Ethanol-induced changes in gene expression were particularly prominent in two groups of genes. One group consisted of oxidoreductases, including ceruloplasmin, uricase, branched-chain alpha-keto acid dehydrogenase, NADH ubiquinone oxidoreductase, P450, NAD+-isocitrate dehydrogenase, and cytochrome c oxidase, which may be related to ethanol-induced oxidative stress. The other group of genes included ADP-ribosylation factor, RAS related protein rab10, phosphatidylinositol 4-kinase, dynein-associated polypeptides, and dynamin-1, which seem to be involved in membrane trafficking. The results may reveal some of the pathways involved in ethanol-induced pathophysiological changes.

Spike-and-wave epilepsy in rats: Sex differences and inheritance of physiological traits

Spontaneously occurring spike-and-wave patterns were examined in seven to eight-month-old rats of the inbred Fischer 344 and Brown Norway strains and their F1 and F2 hybrids. Neocortical activity and movement were monitored for 12 night h. Spike-and-wave episodes were identified by a three-layer back-propagation neural network. The incidence, average duration and total duration of spike-and-wave episodes were significantly higher in F1 males and F2 hybrids than in the parental strains. Male rats of the Brown Norway strain had significantly more and longer episodes than females, whereas no sex differences were present in Fischer rats. The average intraepisodic frequency of spike-and-wave patterns was significantly lower in Fischer rats than in the other groups and significantly higher in males than females. Tremor (myoclonic movements) associated with spike-and-wave episodes was absent or of very small amplitude in Fischer rats but frequent and of large amplitude in Brown Norway rats and their F1 and F2 descendants. Most of the interstrain differences were limited to male rats. Spike-and-wave episodes recurred at predictable short-term (10-30 s) and long-term (15-30 min) periods. The long-term oscillation corresponded to a similar fluctuation of motor activity. The maximum probability of spike-and-wave patterns occurred at a relatively narrow range of delta power (0-3.1 Hz) of the background EEG activity. Systemic administration of the adrenergic alpha-2 agonist, clonidine, increased the incidence of spike-and-wave episodes several-fold. The total duration of spike-and-wave episodes in the clonidine sessions (15 min) and night sessions (12 h test) correlated significantly. We suggest that several genes interact with maturational, environmental and endocrine factors, resulting in sex differences, and produce the variety of EEG and behavioral findings encountered. In addition, we submit that the clonidine test may be useful in genetic investigations of human absence epilepsies. The findings of this work demonstrate that genetic manipulation of rodents is a promising method for producing analogous models for the various forms of human absence epilepsies.

Volumetric structural magnetic resonance imaging (MRI) of the rat hippocampus following kainic acid (KA) treatment

An in vivo MRI study employing a high field (7T) magnet and a T1- and T2-weighted imaging sequence with subsequent histopathological evaluations was undertaken to develop and evaluate MRI-based volumetric measurements in the rat. The brain structures considered were the hippocampus, the cingulate cortex, the retrosplenial granular cortex and the ventricles. Control (n=3) and kainic acid (KA; n=4) treated rats were scanned 10 days following the manifestation of stage four seizures. The MRI images exhibited anatomical details (125 microm in-plane resolution) that enabled volumetric analysis with high intra-rater reliability. Volumetric analysis revealed that KA-treated rats had significantly smaller hippocampi, and a significant increase in ventricular size. The cingulate cortex and the retrosplenial granular cortex did not differ in volume between the two groups. The histological observations supported the MRI data showing neuronal loss and neuronal degeneration in CA1 and CA3 of the hippocampus, which was accompanied by strong microglia activation. These data demonstrate a reliable and valid method for the measurement of the rat hippocampus in vivo using MRI with a high field magnet, thereby providing a useful tool for future studies of rodent models of neuro-degenerative diseases.

Genetic and Pharmacological Manipulations of the CB1 Receptor Alter Ethanol Preference and Dependence in Ethanol Preferring and Nonpreferring Mice

Recent studies have indicated a role for the endocannabinoid system in ethanol‐related behaviors. This study examined the effect of pharmacological activation, blockade, and genetic deletion of the CB1 receptors on ethanol‐drinking behavior in ethanol preferring C57BL/6J (B6) and ethanol nonpreferring DBA/2J (D2) mice. The deletion of CB1 receptor significantly reduced the ethanol preference. Although the stimulation of the CB1 receptor by CP‐55,940 markedly increased the ethanol preference, this effect was found to be greater in B6 than in D2 mice. The antagonism of CB1 receptor function by SR141716A led to a significant reduction in voluntary ethanol preference in B6 than D2 mice. A significant lower hypothermic and greater sedative response to acute ethanol administration was observed in both the strains of CB1 −/− mice than wild‐type mice. Interestingly, genetic deletion and pharmacological blockade of the CB1 receptor produced a marked reduction in severity of handling‐induced convulsion in both the strains. The radioligand binding studies revealed significantly higher levels of CB1 receptor‐stimulated G‐protein activation in the striatum of B6 compared to D2 mice. Innate differences in the CB1 receptor function might be one of the contributing factors for higher ethanol drinking behavior. The antagonists of the CB1 receptor may have therapeutic potential in the treatment of ethanol dependence. Synapse 62:574–581, 2008. Published 2008 Wiley‐Liss, Inc.

Tau Phosphorylation and Cleavage in Ethanol-Induced Neurodegeneration in the Developing Mouse Brain

Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3β (GSK-3β) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3β and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3β inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3β and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.