Genome‐wide association study of theta band event‐related oscillations identifies serotonin receptor gene HTR7 influencing risk of alcohol dependence

Event‐related brain oscillations (EROs) represent highly heritable neuroelectrical correlates of human perception and cognitive performance that exhibit marked deficits in patients with various psychiatric disorders. We report the results of the first genome‐wide association study (GWAS) of an ERO endophenotype—frontal theta ERO evoked by visual oddball targets during P300 response in 1,064 unrelated individuals drawn from a study of alcohol dependence. Forty‐two SNPs of the Illumina HumanHap 1 M microarray were selected from the theta ERO GWAS for replication in family‐based samples (N = 1,095), with four markers revealing nominally significant association. The most significant marker from the two‐stage study is rs4907240 located within ARID protein 5A gene (ARID5A) on chromosome 2q11 (unadjusted, Fisher's combined P = 3.68 × 10^?6). However, the most intriguing association to emerge is with rs7916403 in serotonin receptor gene HTR7 on chromosome 10q23 (combined P = 1.53 × 10^?4), implicating the serotonergic system in the neurophysiological underpinnings of theta EROs. Moreover, promising SNPs were tested for association with diagnoses of alcohol dependence (DSM‐IV), revealing a significant relationship with the HTR7 polymorphism among GWAS case–controls (P = 0.008). Significant recessive genetic effects were also detected for alcohol dependence in both case–control and family‐based samples (P = 0.031 and 0.042, respectively), with the HTR7 risk allele corresponding to theta ERO reductions among homozygotes. These results suggest a role of the serotonergic system in the biological basis of alcohol dependence and underscore the utility of analyzing brain oscillations as a powerful approach to understanding complex genetic psychiatric disorders. © 2010 Wiley‐Liss, Inc.     

Age-related increases in within-person variability: delta and theta oscillations indicate that the elderly are not always old

Behavioral and electrophysiological data related to performance in an auditory Go/NoGo task were analyzed in young and older adults in the present study. Especially, differences in within-person variability in behavior and neural activity between young and older adults and changes in topography of slow event-related oscillations (EROs) were of interest. Within-person variability in behavior was assessed by reaction time (RT) variability. Event-related delta and theta oscillations were analyzed using time-frequency transformation, which can give information on the time-course of single trial event-related EEG spectral power enhancement and intertrial phase-locking (ITC). In contrast to our previous visual Go/NoGo study, no under-recruitment of task-relevant brain regions was found for the auditory theta and delta EROs. Young did not differ from older adults in RT variability or in single trial delta/theta ITC. Altered recruitment of brain activity at advanced age was indicated, first, by stronger early theta phase-locking in older compared to young adults and, second, by a Go-specific lateralization of delta/theta activity. We conclude that within-person variability may increase with age, but the degree depends on performance level and the modality investigated.     

Brain oscillations are highly influenced by gender differences.

There are few studies describing gender differences in event related oscillations (EROs). In the present report we demonstrate that gender differences are apparent in EROs even during simple visual stimulation, possibly activating very basic sensory networks. The data of 32 (16 males) healthy subjects were recorded from thirteen different scalp locations (F(3), F(4), C(z), C(3), C(4), T(3), T(4), T(5), T(6), P(3), P(4), O(1), O(2)). Analysis was performed in the delta (0.5-3.5 Hz), theta (5-8.5 Hz), alpha (9-13 Hz), beta (15-24 Hz), and gamma (28-48 Hz) frequency ranges. The results showed that the maximum peak-to-peak delta response amplitudes for women were significantly higher than for men over occipital, parietal, central and temporal electrode locations. There were also differentiations in the beta and gamma oscillatory responses. These gender differences were most pronounced over the electrode site O(2), that is, over primary visual areas. It is suggested that this study might serve as a standard to investigate gender differences in electrophysiology.     

Identification of QTLs Influencing Alcohol Preference in the High Alcohol Preferring (HAP) and Low Alcohol Preferring (LAP) Mouse Lines

The High- and Low-Alcohol Preferring (HAP1/LAP1 and HAP2/LAP2) mouse lines were developed by selective breeding for differences in alcohol preference. They represent the only extant selectively bred mouse lines developed for this alcohol phenotype. Therefore, they provide a unique resource for QTL detection and mapping. Importantly, neither of the replicate lines is inbred and therefore, novel study designs can be employed to detect loci contributing to alcohol preference. Two independent studies, with very different approaches, were conducted in the HAP and LAP replicate lines. In Study 1, microsatellite markers were genotyped in the replicate HAP1/LAP1 and HAP2/LAP2 mice in QTL regions nominated by other mouse RI and F2 studies in order to detect divergence of allele frequencies in the two oppositely selected lines. Significant differences in allele frequencies were observed in the HAP1/LAP1 mice with markers on chromosome 9 (p<0.01). In the HAP2/LAP2 mice, significant differences in allele frequencies were identified on chromosomes 2 and 9 (p<0.01). In Study 2, a genome-wide screen was performed in a sample of 432 HAP1×LAP1 F2 animals and a QTL on chromosome 9 (LOD=5.04) was found which met criteria for genome wide significance (p<0.001). Gender specific analyses supported a greater effect of the QTL among female mice (LOD=5.19; p<0.0008) than male mice (LOD=1.19). This study provides additional evidence and confirmation that specific regions on chromosomes 9 and perhaps 2 are important for alcohol preference.     

Affect-Related Behaviors in Mice Selectively Bred for High and Low Voluntary Alcohol Consumption

There is considerable evidence for the existence of comorbidity between alcohol-use disorders and depression in humans. One strategy to elucidate hereditary factors affecting the comorbidity of these disorders is to use genetic animal models, such as mouse lines selectively bred for voluntary ethanol consumption. We hypothesized that mice from lines that were bred for high-alcohol preference would manifest increased depression-like phenotypes compared to low-alcohol preferring mice. Mice that were bi-directionally selected and bred on the basis of their High- (HAP) or Low-Alcohol Preference (LAP) were tested in the open-field (OFT), dark–light box (DLB), forced swim (FST), and learned helplessness tests (LH). The study was conducted in two independently derived replicates. In the OFT, both HAP2 and HAP3 mice showed higher levels of general locomotion compared to LAP mice. However, only HAP2 mice spent more time in the center compared to LAP2 mice. In the DLB, there was a slightly higher anxiety-like phenotype in HAP mice. In both FST and LH, we observed higher depression-like behaviors in HAP mice compared to LAP mice, but this was limited to the Replicate 2 mice. Overall, we identified affect-related behavioral changes in mouse lines bred for high-alcohol preference. Notably, the Replicate 3 lines that showed fewer depression-like behaviors also manifest smaller differences in alcohol intake. These data suggest that there may be overlap between genes that predispose to excessive alcohol intake and those underlying affect-related behaviors in the mouse.     

Delta and theta oscillations as risk markers in adolescent offspring of alcoholics.

BACKGROUND: Visual P300 is consistently lower in alcohol-dependent individuals, their offspring and subjects at risk. Delta and theta event-related oscillations (ERO) are the major contributors to the P300 signal. The total and evoked power in delta and theta bands in the 300 to 700 ms post-stimulus window (corresponding to the zone of P300 maxima) was compared between adolescent offspring of alcoholics (high-risk) and age-matched normal controls (low-risk), to assess the utility of the risk markers. METHODS: EEG was recorded during the performance of a visual oddball task. The S-transform algorithm decomposed the EEG signals into different frequency bands and the group differences in total and evoked power in the oscillatory responses during the P300 time window (300 to 700 ms) were analyzed using a multivariate design. Similar analysis was performed on P300 peak amplitude for the target. RESULTS: The high-risk group showed significantly lower parietal post-stimulus evoked and total power in the delta band for targets. A decrease in total power was seen centrally and parietally in the theta band. The P300 peak amplitude in the parietal electrodes was also significantly lower in the high-risk group. CONCLUSIONS: The decreased total theta power and total and evoked delta power for visual targets in high risk individuals may serve as an endophenotypic marker in the development of alcoholism and other disinhibitory disorders. The differences seen between the offspring of alcoholics and controls may have a cholinergic basis. The ERO measures appear to be more robust than the P300 amplitude in differentiating the groups.     

Target‐related parietal P3 and medial frontal theta index the genetic risk for problematic substance use

Theoretical and empirical work suggests that problematic substance use (PSU) is associated with individual differences in prefrontal cortex activity. While research has strongly linked parietal P3 amplitude reduction (P3AR) to genetic risk for problematic substance use, few studies have tested whether prefrontal EEG measures are sensitive to this genetic liability. In addition to P3, oddball target detection tasks elicit medial frontal theta power, reflecting attentional allocation, and parietal delta, indexing decision making or stimulus‐response link updating. Midfrontal theta and parietal delta may index neurocognitive processes relevant to PSU beyond P3AR. The present investigation examined the etiological relationship between PSU and P3, frontal theta, and parietal delta in a large twin sample (N = 754). EEG was recorded during a visual oddball task. Greater PSU was associated with reduced target P3 amplitude and midfrontal theta/parietal delta power, and increased mean reaction time and reaction time variability (RTV; indexing attentional fluctuations). P3, theta, and RTV, but not delta or mean RT, explained unique variance in PSU (R² = 0.04). Twin biometric modeling indicated a genetic relationship between PSU and P3, theta, and RTV. Theta accounted for distinct genetic variance in PSU beyond P3 and RTV. Together, 23% of the total additive genetic variance in PSU was explained by the three endophenotypes. Results replicate P3AR as an endophenotype and provide support for additional behavioral (RTV) and neurophysiological (midfrontal theta) endophenotypes of PSU. Reduced theta and greater RTV may reflect variations in a prefrontal attentional network that confers genetic risk for substance use problems.     

Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice

Because of intrinsic differences between humans and mice, no single mouse model can represent all features of a complex human trait such as alcoholism. It is therefore necessary to develop partial models. One important feature is drinking to the point where blood ethanol concentration (BEC) reaches levels that have measurable affects on physiology and/or behavior (>1.0 mg ethanol/ml blood). Most models currently in use examine relative oral self-administration from a bottle containing alcohol versus one containing water (two-bottle preference drinking), or oral operant self-administration. In these procedures, it is not clear when or if the animals drink to pharmacologically significant levels because the drinking is episodic and often occurs over a 24-h period. The aim of this study was to identify the optimal parameters and evaluate the reliability of a very simple procedure, taking advantage of a mouse genotype (C57BL/6J) that is known to drink large quantities of ethanol. We exchanged for the water bottle a solution containing ethanol in tap water for a limited period, early in the dark cycle, in the home cage. Mice regularly drank sufficient ethanol to achieve BEC>1.0 mg ethanol/ml blood. The concentration of ethanol offered (10%, 20% or 30%) did not affect consumption in g ethanol/kg body weight. The highest average BEC ( approximately 1.6 mg/ml) occurred when the water-to-ethanol switch occurred 3 h into the dark cycle, and when the ethanol was offered for 4 rather than 2 h. Ethanol consumption was consistent within individual mice, and reliably predicted BEC after the period of ethanol access. C57BL/6J mice from three sources provided equivalent data, while DBA/2J mice drank much less than C57BL/6J in this test. We discuss advantages of the model for high-throughput screening assays where the goal is to find other genotypes of mice that drink excessively, or to screen drugs for their efficacy in blocking excessive drinking.     

The Complexity of Alcohol Drinking: Studies in Rodent Genetic Models

Risk for alcohol dependence in humans has substantial genetic contributions. Successful rodent models generally attempt to address only selected features of the human diagnosis. Most such models target the phenotype of oral administration of alcohol solutions, usually consumption of or preference for an alcohol solution versus water. Data from rats and mice for more than 50 years have shown genetic influences on preference drinking and related phenotypes. This paper summarizes some key findings from that extensive literature. Much has been learned, including the genomic location and possible identity of several genes influencing preference drinking. We report new information from congenic lines confirming QTLs for drinking on mouse chromosomes 2 and 9. There are many strengths of the various phenotypic assays used to study drinking, but there are also some weaknesses. One major weakness, the lack of drinking excessively enough to become intoxicated, has recently been addressed with a new genetic animal model, mouse lines selectively bred for their high and intoxicating blood alcohol levels after a limited period of drinking in the circadian dark. We report here results from a second replicate of that selection and compare them with the first replicate.     

Seven generations of genetic selection for ethanol dependence in mice

An animal model of alcohol dependence is being produced by selecting mice for differences in severity of ethanol withdrawal seizures. Replicate lines of high-dependence (HA), low-dependence (LA), and control (CA) mice are being developed by within-family selection. After seven generations both (replicate) HA and LA lines have separated significantly. Some of the difference between the replicate pairs of HA and LA in the early generations was due to differences in ethanol consumption. This difference in consumption may be attributable to a difference in metabolic rate or activity level rather than to a difference in ethanol preference. Females are more susceptible to seizures than males; this appears to be due partly to their higher consumption of ethanol during treatment.This research was supported in part by Grant AA-03527 from the National Institute of Alcohol Abuse and Alcoholism to the University of Colorado Alcohol Research Center.     

Selective Breeding for High and Low Alcohol Preference in Mice

High and low alcohol preference (HAP and LAP, respectively) mice were created by 10 generations of bidirectional selection for differences in two-bottle choice alcohol consumption. The progenitors used for selection were HS/lbg mice, which are a genetically defined, out-bred stock. During selection, mice had 24-h, daily access to 10% alcohol (v/v) and water ad libitum for 30 days and were selected based on the alcohol (g/kg) consumed per day over the entire period. Food was available ad libitum. At S10, line means for alcohol consumption differed greatly, with consumption of over 12 g/kg per day in the HAP mice and less than 2 g/kg per day in the LAP mice. Realized heritability for bidirectional selection was approximately 0.2. Female mice consumed more alcohol than male mice. There were no differences between lines in alcohol elimination rate, nor were there line differences in intake of salt or quinine solutions. However, consumption of saccharin solutions was greater in HAP mice than LAP mice, consistent with previous findings of a genetic correlation between sweet preference and alcohol drinking. Because the mouse genome is relatively well characterized, these selected lines should prove a useful tool for assessment of the genetic basis of, and phenotypes that correlate with, alcohol drinking.     

Comparison of focally induced epileptiform activities in C57BL/6 and BALB/c mice by using in vivo EEG recording

Epilepsy characterized by repeated seizures is influenced by genetic factors. Seizure response of inbred mouse strains changes depending on the variety of stimuli including chemical (e.g., pentylenetetrazole, nicotine, cocaine, NMDA, kainate), physical (e.g., auditory) or electrical. In this study, we compared the susceptibilities of C57BL/6 and BALB/c mice strains to penicillin induced epileptiform activity (a focally induced, experimental epilepsy model), by analyzing the spike onset latency, spike amplitude and spike frequency. The power spectrums of baseline EEGs were also investigated. We found no alterations of spike onset latencies between the C57 and BALB mice. However, spike amplitudes and spike frequencies were found to be higher in BALB mice than C57 mice. With regard to EEG power spectrum, absolute power of investigated bands was not different between the two strains. Interestingly, the relative power of all investigated bands differed significantly between two strains. The relative power of delta and theta was lower whereas relative power of alpha, beta and gamma was higher in C57 mice compared to BALB mice. In conclusion, our findings showed that BALB mice are more sensitive to penicillin induced epileptiform activity when compared to C57 strain.     

Different motivational effects induced by the endogenous mu-opioid receptor ligands endomorphin-1 and -2 in the mouse

The present study was designed to investigate the motivational effects of the newly discovered endogenous mu-opioid receptor ligands, endomorphin-1 and endomorphin-2, using the conditioned place preference paradigm in mice. The binding properties of these peptides were first examined using an opioid binding assay. In membranes obtained from the mouse whole brain, the binding of [3H][D-Ala2, NMePhe4, Gly(ol)5]enkephalin (DAMGO; mu), but not of [3H][D-Phe2, D-Phe5]enkephalin (DPDPE; delta) or [3H]U69593 (kappa) selectively and concentration-dependently competed with that of endomorphin-1 and endomorphin-2, indicating that both endomorphin-1 and endomorphin-2 are specific ligands for mu-opioid receptors in the brain. Endomorphin-1 (1-30 nmol/mouse) given i.c.v. produced a dose-related place preference. This effect was abolished by pre-treatment with the mu-opioid receptor antagonist beta-funaltrexamine but not the delta-opioid receptor antagonist naltrindole or the kappa-opioid receptor antagonist nor-binaltorphimine. In contrast, endomorphin-2 (5.6 nmol/mouse) produced place aversion. This aversive effect was inhibited by nor-binaltorphimine as well as beta-funaltrexamine, but not by naltrindole. The place aversion produced by endomorphin-2 was also attenuated by pre-treatment with antiserum against the endogenous kappa-opioid receptor ligand dynorphin A (1-17). These findings indicate that endomorphin-1 may produce its rewarding effect via mu-opioid receptors. On the other hand, the aversive effect induced by endomorphin-2 may be associated with the stimulation of endomorphin-1-insensitive mu-opioid receptors and the activation of dynorphinergic systems in the mouse brain.     

Toward an Animal Model for Antisocial Behavior: Parallels Between Mice and Humans

The goal of this article is to examine whether mouse lines genetically selected for short and long attack latencies are good animal models for antisocial behavior in humans. To this end, we compared male Short and Long Attack Latency mice (SAL and LAL, respectively) with the extremes of the Dunedin Multidisciplinary Health and Development Study (men who persistently displayed antisocial behavior [Persisters] and men who never manifested antisocial behavior [Abstainers]). Groups were compared on the basis of five distinct domains: aggression/violence, reproduction, cognition, behavioral disorders, and endophenotypes. Our observations point to considerable parallels between, on one side, SAL and Persisters, and, on the other side, between LAL and Abstainers (but to a lesser extent). We believe that SAL and LAL are good mouse models to study the development of antisocial behavior and will yield valuable and testable hypotheses with regard to the neurobiological and genetical architecture of antisocial behavior.     

Morphine preference of alcohol-avoiding and alcohol-preferring C57BL mice

C57BL/Uae inbred mice are similar to C57BL/6J and C57BL/10J mice in displaying a high preference for morphine presented in a saccharin vehicle. C57BL/Uae mice are unique among investigated C57BL lines in not showing a high preference for alcohol. This pattern of differences among closely related inbred strains could be useful in investigating commonalities of alcohol and morphine effects.This work was supported by NIDA Grant No. 00828 and NIMH Grant No. 11218.     

Genetic and Neurophysiological Correlates of the Age of Onset of Alcohol Use Disorders in Adolescents and Young Adults

Discrete time survival analysis was used to assess the age-specific association of event-related oscillations (EROs) and CHRM2 gene variants on the onset of regular alcohol use and alcohol dependence. The subjects were 2,938 adolescents and young adults ages 12–25. Results showed that the CHRM2 gene variants and ERO risk factors had hazards which varied considerably with age. The bulk of the significant age-specific associations occurred in those whose age of onset was under 16. These associations were concentrated in those subjects who at some time took an illicit drug. These results are consistent with studies which associate greater rates of alcohol dependence among those who begin drinking at an early age. The age specificity of the genetic and neurophysiological factors is consistent with recent studies of adolescent brain development, which locate an interval of heightened vulnerability to substance use disorders in the early to mid teens.     

Magnesium Involvement in Sleep: Genetic and Nutritional Models

Alterations of peripheral magnesium (Mg) concentration have been reported in association with several behavioral disorders and sleep organization. Blood Mg regulation is under a strong genetic control, whereas brain Mg regulation does not seem to be affected. We have studied peripheral and central levels of Mg and analyzed sleep in two lines of mice selected for low (MGL) and high (MGH) red blood cell (RBC) Mg levels. The same variables were also studied in C57BL/6J mice before and after 3 weeks of Mg deficiency. Whereas blood Mg was highly affected by the selection, brain Mg exhibited only small differences between the two lines. In contrast, Mg deficiency strongly decreased both central and peripheral Mg levels. Sleep analysis indicated that in both models the amount of paradoxical sleep was lower in mice with higher Mg levels. The amplitude of daily variation in sleep and slow-wave sleep delta power was markedly decreased in MGH line. Quantitative electroencephalogram (EEG) analysis also revealed a faster theta peak frequency in MGH mice, irrespective of behavioral states. Central Mg showed significant correlations with the amount of paradoxical sleep and sleep consolidation. However, because the direction of these correlations was not consistent, it is concluded that optimal, (physiological) rather than high or low, Mg levels are needed for normal sleep regulation.     

The effect of aging on EEG brain oscillations related to sensory and sensorimotor functions

PurposeThe question of the present study is whether the brain as a system with gradually decreasing resources maximizes its performance by reorganizing neural networks for greater efficiency.Material/methodsAuditory event-related low frequency oscillations (delta δ – [2, 4] Hz; theta θ – [4.5, 7] Hz; alpha α – [7.5, 12] Hz) were examined during an auditory discrimination motor task (low-frequency tone – right hand movement, high-frequency tone – left hand movement) between two groups with mean age 26.3 and 55 years.ResultsThe amplitudes of the phase-locked δ, θ and α activity were more pronounced with a progressive increase in age during the sensory processing, independent of tone type. The difference between the groups with respect to scalp distribution was tone-independent for delta/theta oscillations, but not for the alpha activity. Age-related and tone-dependent changes in α band activity were focused at frontal and sensorimotor areas. Neither functional brain specificity was observed for the amplitudes of the low-frequency (δ, θ, α) oscillations during the cognitive processing, which diminished with increasing age.ConclusionThe cognitive brain oscillatory specificity diminished with increasing age.     

Rack1对慢性吗啡成瘾小鼠海马BDNF表达和动物行为偏爱的影响

目的：通过干扰C激酶受体1蛋白（Rack1）的表达,探讨Rack1对慢性吗啡成瘾小鼠海马的作用。方法：建立慢性吗啡成瘾小鼠模型,并分析条件性位置偏爱（CPP）效应。应用RT-PCR检测慢性吗啡成瘾小鼠海马中Rack1和脑源性神经营养因子（BDNF）的mRNA表达水平以及免疫组化观察海马中BDNF蛋白的表达情况。结果：与生理盐水组相比,慢性吗啡成瘾组小鼠海马中Rack1和BDNF mRNA和蛋白表达水平升高（P〈0．05）,且吗啡诱导的CPP效应上升（P〈0．05）,与慢性吗啡成瘾组相比,Rack1干扰质粒组小鼠海马中Rack1和BDNF mRNA和蛋白表达水平均明显下降（P〈0．05）,吗啡诱导的CPP效应下调（P〈0．05）。结论：在慢性吗啡成瘾小鼠海马中,Rack1是一个关键因子。