Understanding behaviour in a complex environment. The sixth annual meeting of the International Behavioural and Neural Genetics Society, New Orleans, LA, USA, November 5–7, 2003
M.-A. Enoch, Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD 20892, USA. E-mail:email@example.com
The 2003 IBANGS meeting turned out to be as lively as its setting, the splendidly colourful city of New Orleans. Ninety two registrants participated in talks and discussions. Attendees originated from many countries and presenters ranged across the spectrum of experience from graduate students to a retired professor. Two days were barely enough to encompass the full program that included four symposia, a plenary lecture, invited talks from the Outstanding Young Investigator Awardees, a selected paper session and a poster session. Although the bulk of the work presented predictably focused on mouse models, nevertheless studies on several other species, ranging in complexity from nematodes to non-human and human primates, were also presented. The sobering take-home message from the meeting was that behavioural neurogenetics has moved on to the stage where researchers are now being confronted with the enormous complexity of the genotype–behavioural phenotype relationship that includes for example gene–environment, gene–gene, and sex effects. In this meeting report we have attempted to summarize the interesting findings and important conclusions from the large variety of presentations.
Anxious mice, monkeys and (wo)men: genetic influences
This first symposium, organized by Christina Barr and Tim Newman (National Institute on Alcohol Abuse and Alcoholism (NIAAA), NIH, USA), reached across species to look at genetic influences on a behaviour that we all think we know well in humans, i.e. anxiety, and which we think we can measure in animals. Specific genetic polymorphisms, particularly in brain monoamines, that may confer increased risk for anxiety and related complex behavioural traits such as alcohol consumption, were discussed. Gene–environment interactions and sexually dimorphic effects of gene variation were also addressed.
Evidence of an association between anxious behaviour in humans and genetic variation in the serotonin transporter (5-HTT or SERT) promoter (Lesch et al. 1996), prompted Andrew Holmes (NIAAA, NIH, USA) to work with 5-HTT knock-out (KO) mice. These mice show elevated levels of synaptic serotonin 5-HT and altered 5-HT receptor function, concomitant with abnormalities in aggression and anxiety-like behaviour. Repeated backcrossing of 5-HTT KO mice onto 129S6 or C57BL/6J strains indicated epistatic, mutation–background gene interactions (Holmes et al. 2003). Data presented at this meeting showed heightened depression-related behaviour in 5-HTT KO mice as assayed on tests for behavioural despair. In a clear parallel with recent human findings (Caspi et al. 2003), genetic deficiency of 5-HTT function increased depression-like behaviour only after repeated exposure to stressors. These data support 5-HTT variation as a risk factor for stress-related disorders, and emphasize the importance of studying mutant mouse models of complex traits in multiple genetic backgrounds and test conditions.
Barr and Newman have used an established model of early life stress in rhesus macaque monkeys, maternal separation with peer group rearing, to look at the impact of gene–environment interactions on alcohol consumption and response to stressors. Tim Newman (NIAAA, NIH, USA) described his work on the interactions between polymorphisms in the dopamine D1 receptor (DRD1) and MAOA and early life stress on voluntary ethanol consumption in adult monkeys. In general, peer-reared animals tend to consume more ethanol in adulthood and the interaction of early life stress and decreased serotonin turnover is associated with lower alcohol sensitivity and higher alcohol consumption (Barr et al. 2003). Haplotype analyses of 5 DRD1 SNPs, driven by a single promoter SNP (-111 T>G), showed significant interactions among sex, rearing, genotype and ethanol consumption, such that peer-reared males with the minor allele drank the most. Similarly, peer-reared males with the high activity MAOA promoter variant had the highest ethanol consumption. Barr showed that female monkeys exposed to early life stress have augmented HPA axis responses to alcohol. The interaction of early life stress and genetic variation in the female macaque's 5-HTT promoter (the short ‘s’ allele) was associated with an exaggerated HPA axis response to stress (Barr et al. 2004), and these animals showed marked increases in their levels of consumption with successive exposures to alcohol (Barr et al. in press). Corticotropin-releasing hormone (CRH) is also a good candidate for variation in response to both stress and ethanol. Newly identified variants in the CRH were described, one of which (-2242 C>G) is located within a glucocorticoid response element and is associated with increased baseline ACTH, increased ACTH release in response to alcohol and increased alcohol consumption. These studies in non-human primates demonstrate the influences of early life stress and sex on the modulation of genetic vulnerability to complex behavioural disorders such as alcohol consumption.
Mary-Anne Enoch (NIAAA, NIH, USA) moved up the evolutionary scale to discuss the array of, and relationships between, measurable and diagnosable anxiety phenotypes in humans: anxious temperament (harm avoidance (HA)), state anxiety, pathological anxiety (clinically diagnosed) and intermediate phenotypes, including low-voltage alpha (LVA) resting EEG. Enoch discussed these phenotypes in the context of studies on men and women from two large community-ascertained populations: urban, affluent US Caucasians and rural, poor, Plains American Indians with a high prevalence of alcoholism. Results were the same in both samples despite very different environmental stressors; women had higher HA than men; clinical anxiety disorders were associated with higher HA in men but not women; alcoholics, both male and female, had higher HA than nonalcoholics. Furthermore, the Met/Met genotype of the functional catechol-O-methyltransferase Val158Met polymorphism was associated with elevated HA and also the LVA intermediate phenotype in women but not men (Enoch et al. 2003). These results illustrate the importance of careful phenotyping in a heterogeneous behaviour such as anxiety and also the existence of sex differences in behaviour involving emotionality.
BDNF: molecule of the moment
There is increasing awareness of the wide-ranging roles that brain-derived neurotrophic factor (BDNF) plays in the regulation of neuronal development, plasticity, function and survival. BDNF has been implicated in mood disorders and schizophrenia, and is a candidate for addiction vulnerability. This symposium, organized by Robert Lipsky (NIAAA, NIH, USA) explored genotypic and phenotypic variation associated with changes in BDNF expression in humans and mice, and discussed the current and potential roles of BDNF in drug action in the CNS. Lipsky described the complex regulation of BDNF that involves at least five highly conserved promoter regions that control transcription differentially to produce three prepro BDNF isoforms that may have unique functional characteristics (Marini et al. in press). In order to identify sequence variants that may affect BDNF regulation and probably hippocampal function in humans, a panel of 480 unrelated, ethnically diverse individuals was screened and four novel sequence variants were identified; two promoter and two exonic. Haplotype analyses incorporating these and other variants in 754 Finnish subjects and 505 American Indians showed that the gene is in one (approximately 70 kb) haplotype block. Reporter constructs have been made for the new variants and are being expressed in neuronal cell lines in order to determine their role in BDNF expression. Because BDNF is known to play an important role in neuronal plasticity and survival, a detailed examination of the molecular genetics of BDNF is justified.
Alexei Morozov (National Institute of Mental Health [NIMH], NIH, USA) used the Cre-loxP system to show that, although hippocampal BDNF can be released from both pre- and postsynaptic cells, it is the presynaptically secreted BDNF that is required for neuronal plasticity (Zakharenko et al. 2003). Loss of BDNF from forebrain areas increased fear-related behaviours.
There is now strong evidence from the work of Mark P Mattson (National Institute on Aging [NIA], NIH, USA) and others that dietary restriction (DR: reduced calorie intake or intermittent fasting) increases lifespan and disease resistance and is neuroprotective, at least in rodents (Mattson et al. 2003). Mattson's work has shown that DR stimulates BDNF transcription in the hippocampus and pyramidal neurons, and BDNF signaling in the brain alters insulin sensitivity and regulates peripheral glucose metabolism (Duan et al. 2003). In contrast, overeating reduces BDNF production. Current work is focusing on the role of reciprocal interactions between BDNF and 5-HT in neuronal plasticity and aging processes, as well as on the development of novel dietary and pharmacological interventions that enhance BDNF and/or 5-HT signaling for use in neurodegenerative disorders. It is likely that abnormalities in both dopaminergic and glutamatergic neurotransmission are implicated in schizophrenia. BDNF is produced by glutamatergic neurons and controls dopamine D3 receptor (DRD3) expression.
Pierre Sokoloff (INSERM, France) showed that chronic NMDA receptor blockade produced schizophrenia-type behaviour in mice as well as neurochemical abnormalities found in human schizophrenia; up-regulation of BDNF and DRD3 receptors and down-regulation of reelin. These abnormalities could be corrected by DRD2/DRD3 blockers. BDNF was presented as a candidate for regulating DRD3 expression because BDNF down regulates reelin, and is itself up-regulated by NMDA receptor blockade. In addition the NMDA receptor blockade effects are suppressed in BDNF heterozygous mice. Chronic antipsychotic treatment normalized BDNF expression. Thus BDNF may be the link between dopamine and glutamate dysfunction in schizophrenia and may represent a new therapeutic drug target. Indeed, BDNF may already play an important role in the mechanisms of current CNS drug action in mood disorders.
Eero Castren (University of Helsinki, Finland) discussed his findings that antidepressant drugs increase the production and release of BDNF in prefrontal cortex and hippocampus and increase the activation of the BDNF receptor trkB. Furthermore, by using transgenic mice with reduced BDNF signaling, his group has shown that normal BDNF signaling is required for the behavioural responses typically produced by antidepressant drugs. Increased BDNF signaling may induce the formation and stabilization of synaptic connectivity that gradually leads to an improvement in mood (Saarelainen et al. 2003).
Drug reward: knocking out one gene is not always enough
Scott Hall (National Institute on Drug Abuse [NIDA], NIH, USA), the organizer of this symposium, discussed evidence that cocaine-induced conditioned place preference (CPP) was not altered by deletion of any single monoamine transporter in KO mice, although combined knockout of DAT and SERT did eliminate cocaine reward. Indeed, in many single gene KOs cocaine is more rewarding and, interestingly, fluoxetine and nisoxetine have rewarding effects in some KOs that are not observed in wild type (WT) mice, indicating that profound alterations in the pharmacological basis of cocaine reward have occurred in these mice. In an ambitious effort to further characterize the polygenic basis of drug reward, data from several strains of multiple gene KO mice were presented, combining different monoamine transporters and receptors (DAT/5-HT1A, DAT/5-HT1B and SERT/DRD2 double KO mice). Although none of these combinations eliminated the rewarding effects of cocaine, deletion of 5-HT1B, 5-HT1A, SERT and DRD2 alone all increased the rewarding effects of cocaine. The project continues by examining KOs of combinations of dopaminergic and serotonergic genes that may underlie the elimination of cocaine reward observed in DAT/SERT double KO mice, and how manipulations of multiple genes may interact to determine sensitivity to the rewarding effects of cocaine.
Ichiro Sora (Tohoku University, Tokyo, Japan) showed data on microdialysis in different brain areas, nucleus accumbens (NAc), caudate putamen (CPu) and prefrontal cortex (PFc) in mutant mice. Like Hall's work, combinations of SERT and DAT KO mice were used to determine DA and 5-HT activity. In all three brain areas, cocaine-induced extracellular 5-HT increase was found in the DAT KO mice, whereas the mice showed cocaine-induced extracellular DA increase in CPu and PFc, but not NAc. Knocking out both transporters had rather limited additional effects and it is important to realize that in many cases baseline levels of extracellular 5-HT and DA are increased in response to the absence of the transporter, which complicates the interpretation of some of the effects.
Barak Caine (McLean Hospital, Boston, USA) talked about cocaine self-administration in mice lacking dopamine D1, D2 or D3 receptors. Mice are trained to poke their nose into a hole in order to get a reward. After training, mice are prepared through surgery for self-administration of cocaine. DRD1 KO mice do not self-administer cocaine, whereas DRD2 KO mice show increased self administration to the extent that they die of an overdose with unlimited cocaine access. DRD3 KO mice show self-administration that is similar to WT mice. Data on cocaine-stimulant effects on locomotion were presented in a study using 12 different mouse strains. This work shows important variety across background strains used to generate KO mice and it also elucidates the difference between self-administration vs. locomotor activity studies.
Genetics of rewarding and aversive effects of ethanol
Chris Cunningham (Oregon Health and Science University [OHSU], Portland, USA) presented the plenary lecture of the meeting. His talk distinguished between self-administration procedures and procedures in which drugs are given by injection to induce various types of learning, such as CPP, place aversion and taste aversion. Moreover, he explained the advantages and disadvantages of self-administration and conditioning models. Most of his presentation was devoted to examining genetic correlations among these phenotypes in mice exposed to ethanol. Several different genetic models were discussed, with a strong emphasis on data from studies involving inbred strains or lines selectively bred for ethanol sensitivity in self-administration or conditioning procedures. In general, there was weak evidence of genetic correlation between CPP and self-administration or either of the other two conditioning phenotypes. However, the data showed a negative correlation between ethanol self-administration and conditioned taste aversion (e.g. Broadbent et al. 2002) and between self-administration and conditioned place aversion. Ethanol's ability to produce both conditioned-rewarding and conditioned-aversive effects was explained by a model in which ethanol is hypothesized to produce an initial short-lived aversive effect followed by a slower, but longer lasting rewarding effect. For further reading, a recent paper focuses on many issues discussed in this plenary lecture (Broadbent et al. 2002).
Genotype–environment interactions: what are they and what shall we do with them?
The first speaker in this symposium, Doug Wahlsten (University of Alberta, Canada) presented data that followed up on an important study by Crabbe et al. (1999). Wahlsten attempted to determine the influence of genotype and environmental factors, such as laboratory and experimenter, on the outcome of behavioural experiments. The original 1999 study was performed in eight ‘strains’ and three institutions, whereas in the follow up study 21 strains were tested in two out of those three institutions (Edmonton and Portland). The new data set shows that in contrast to 1999 there is no difference in open-field activity and elevated plus maze between the two sites, whereas the accelerating rotarod data are site dependent. Overall, labs produced rather comparable data sets, whereas strains showed important differences, and the interaction of strain × lab was in most cases not significant. One point worth noting is that prior to this second study both labs had moved to different locations at their respective universities compared to the 1999 paper. Thus, the greater similarity of results for the elevated plus maze in 2002 must have arisen from changes in the lab environments within a university, because they used the identical apparatus and some of the same strains as in 1999. The second part of the talk focused on stability of behaviour over time within the same strains. Data sets from a number of labs from studies as old as the 1950s and 1960s were compared with more recent findings. Correlations of recent open-field activity data with similar inbred strain studies from 1968 and even 1953 were quite high. For the elevated plus maze, on the other hand, differences with other labs over the past 10 years were large.
Jeffrey Mogil (McGill University, Canada) followed up on this issue of consistency of data sets within the same lab environment by comparing the tail-withdrawal response as a measure for pain responsiveness. Mogil has studied this paradigm for eight years and he collected over 8000 samples in three locations. Importantly, these samples include records about basic parameters such as room temperature, humidity, time of the day and experimenter. Heritability of nociception appears to be only 24%. CART (classification and regression tree analyses) were used to determine those factors that affect nociception. Overall, female mice were more sensitive than males; the first mouse tested is less sensitive than the following ones, and testing in the spring shows lower latencies. However, the most important factor of all in this assay (more important than strain, sex, etc.) is the experimenter! Dr Mogil nicely presented the overall influence of environment, genotype and the interaction as published in 2002 (Chesler et al. 2002).
The final two talks were given by the symposium organizer, Neri Kafkafi (University of Maryland, USA), and Yoav Benjamini (Tel Aviv University, Israel) who described a new computational analysis model designed to identify laboratory environmental factors that influence behavioural traits by using a large data archive. Kafkafi demonstrated the model by comparing open field behaviour of eight mouse genotypes across three laboratories. Benjamini presented the Linear Mixed Model of two-way anova, which recognizes that genotype–laboratory interaction is an inevitable fact of life and uses it as the yardstick for estimating the genotype effect. This method sets a higher benchmark for showing a genotype effect and can be used in single-lab studies for behavioural tests for which the interaction has previously been estimated in multilab studies.
The recipients of the 2003 Young Investigator Awards; two graduate students, a postdoctoral fellow and a junior investigator, were invited to present their research at the meeting. Four lively and stimulating presentations ensued. Liz Hammock (Emory University, GA, USA) described her work with monogamous prairie voles and polygamous montane voles. Brain vasopressin 1a receptors (V1aR) are known to regulate vole social behaviour in a species-specific fashion, probably due to species-specific V1aR distribution patterns. A polymorphic microsatellite expansion has been identified in the 5′ regulatory region of the prairie vole V1aR, which could alter the brain distribution pattern of the receptor between and within species. Preliminary analyses indicate relationships between genotype of this locus and behaviour. This may turn out to be a mechanism for the rapid evolution of complex behaviours.
Noa Heiman (University of Colorado, USA) also looked at genotype-phenotype relationships, but in humans. Some, but not all, studies have found a relationship between novelty-seeking and a functional VNTR (variable number of tandem repeats) in the DRD4 receptor. However, Heiman did not find this association in a highly comprehensive study of 2185 adolescents.
Daesoo Kim (Korea Institute of Science and Technology, Korea) presented his work on an animal model for novelty and alcohol seeking behaviours. Mice lacking alpha1G T-type Ca2+ channels initially showed markedly increased exploratory behaviour whenever exposed to novel environment. However, they acclimated quickly. These mutants also consumed significantly more alcohol in two bottle choice tests. The present results support the idea that novelty and alcohol-seeking behaviours are mediated by common genetic mechanisms that are modulated by T-type Ca2+ channels.
Karen Szumlinski (Medical University of South Carolina, USA) rounded off the session with a presentation about Homer proteins; regulators of multiple aspects of glutamate receptor signaling. Ventral striatal levels of extracellular glutamate (but not dopamine) are reduced by 50% in Homer2 KO mice that exhibit increased ethanol aversion and sedation and reduced tolerance. Detailed results were presented which suggest that Homer2 regulation of mesocorticolimbic mGluR1a and NMDA function is implicated in ethanol sensitivity and ethanol-induced neural plasticity, both of which contribute to addiction vulnerability.
Poster session: oral presentations
This year the program committee selected several of the submitted posters for short oral presentations. The presenters were: Mara Dierssen (Genomic Regulation Center, Barcelona, Spain), Christopher Janus (University of Toronto, Canada), Diana Jerusalinsky (University of Buenos Aires, Argentina), Philip Newton (University of California, San Francisco, USA), Igor Ponomarev (University of Texas, USA) and Catharine Rankin (University of British Columbia, Canada). Unfortunately, space dictates that only a couple can be discussed here.
Mara Dierssen described the role of the neurotrophin type 3 receptor gene (NTRK3) in anxiety disorders. NTRK3 is the only neurotrophin receptor expressed in the locus coeruleus (LC) and NTRK3 is located in a chromosomal region duplicated in panic disorder patients. A mouse model with overexpression of NTRK3 shows increased volume and number of tyrosine hydroxylase positive neurons in the LC. These mice showed increased anxiety and panic responses and increased FOS expression in LC target areas after administration of panicogenic agents.
Christopher Janus presented a transgenic mouse model for Alzheimer's disease. The TgCRND8 mice carry a double mutation of familial Alzheimer's disease in amyloid precursor protein (APP) gene and are maintained on a hybrid C57BL/6 × C3H background. These mutants showed impaired performance in both the cued and hidden version of the Morris Water Maze. The associative learning of taste aversion was also impaired. However, only spatial learning and memory of TgCRND8 mice was inversely correlated with detergent (SDS)-soluble β-amyloid levels in a brain.
The quality and variety of the presentations and the large number of attendees at this meeting indicated that IBANGS is a healthy and flourishing society. Doug Wahlstein showed that despite using purportedly the same mouse strains and the same protocols, subtle environmental variations between labs and within the same lab over time had strain-specific consequences, and Jeffery Mogil surprised the meeting with the revelation that the most significant variable was the experimenter. Christina Barr and Tim Newman showed how it was possible to dissect the results of environment manipulations in non-human primates to elucidate sex-specific gene–environment interactions. Several speakers raised issues with single KO; Scott Hall and Ichiro Soro showed that at least in drug reward it is necessary to study multiple gene KOs, and Andrew Holmes demonstrated epistatic interactions between KO and background genes. It was suggested that heterozygous KOs might be more informative for understanding certain types of human pathology. Several participants raised the issue of sex differences in the manifestations and origins of behaviour and concluded that it is now time to include, admittedly more complex, studies on female organisms.