Complex Behavioral Phenotypes Research Articles

Changes in the Expression of G Protein-Coupled Receptor Kinases and β-Arrestins in Mouse Brain During Cannabinoid Tolerance: A Role for Ras-ERK Cascade

The focus of our study was to determine the role of G protein-coupled receptor kinases (GRKs) and beta-arrestins in agonist-induced CB1 receptor modulation during cannabinoid tolerance and their dependence from the extracellular signal-regulated kinase (ERK) cascade. In wild-type mice, chronic Delta9-tetrahydrocannabinol (THC) exposure significantly activated specific GRK and beta- arrestin subunits in all the considered brain areas (striatum, cerebellum, hippocampus, and prefrontal cortex), suggesting their involvement in the adaptive processes underlying CB1 receptor downregulation and desensitization. These events were ERK-dependent in the striatum and cerebellum, because they were prevented in the genetic (Ras-GRF1 knockout mice) and pharmacological (SL327-pretreated mice) models of ERK activation inhibition, whereas in the hippocampus and prefrontal cortex, they appeared to be mostly ERK-independent. In the latter areas, ERK activation after chronic THC increased the transcription factors cyclic adenosine monophosphate response element-binding protein and Fos B as well as a downstream protein known as brainderived neurotrophic factor. As a whole, our data suggest that in the striatum and cerebellum, THC-induced ERK activation could represent a key signaling event to initiate homologous desensitization of CB1 receptor, accounting for the development of tolerance to THC-induced hypolocomotion. In the prefrontal cortex and hippocampus, THC-induced alteration in GRKs and beta-arrestins primarily depends on other kinases, whereas ERK activation could be part of the molecular adaptations that underlie the complex behavioral phenotype that defines the addicted state.

Texture of locomotor path: a replicable characterization of a complex behavioral phenotype

A database of mouse locomotor path in spatial tests can be used to search in silico for behavioral measures that better discriminate between genotypes and are more replicable across laboratories. In this study, software for the exploration of exploration (SEE) was used to search a large database for a novel behavioral measure that would characterize complex movement paths. The database included mouse open-field behavior assessed in 3 laboratories, 7 inbred strains, several pharmacological treatments and hundreds of animals. The new behavioral measure, "path texture", was characterized using the local curvature of the path (the change of direction per unit distance, in degrees/cm) across several spatial scales, starting from scales smaller than the animal's body length and up to the scale of the arena size. Path texture analysis differs from fractal dimension analysis in that it does not assume self-similarity across scales. Path texture was found to discriminate inbred strains with relatively high broad-sense heritability (43%-71%) and high replicability across laboratories. Even genotypes that had similar path curvatures in some scales usually differed in other scales, and self-similarity across scales was not displayed by all genotypes. Amphetamine decreased the path curvature of C57BL/6 mice in small and medium scales, while having no effect on DBA/2J mice. Diazepam dose-dependently decreased the curvature of C57BL/6 mice across all scales, while 2 anxiogenic drugs, FG-7142 and pentylenetetrazole, increased it. Path texture thus has high potential for behavioral phenotyping and the study of drug effects in the mouse.

Genetic segregation of brain gene expression identifies retinaldehyde binding protein 1 and syntaxin 12 as potential contributors to ethanol preference in mice.

Genetic strains of mice represent an important resource for research on the biological determinants of complex diseases and behavioral phenotypes. To date, the approaches used have had little success in identifying causal genes. We have evaluated brain gene expression in C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains using differential display to identify a number of sequences showing significant expression differences between the two strains. These differences were confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Knowing that B6 and D2 mouse strains differ for a number of behavioral phenotypes, we asked whether this difference in brain gene expression could explain any of these traits. Here, we show that the expression of two of these genes, retinaldehyde binding protein 1 (Rlbp1) and syntaxin 12 (Stx12), co-segregate with the ethanol preference phenotype in a B6D2 F2 population. Our results suggest a potential role for Rlbp1 and Stx12 in ethanol preference in mice, a conclusion supported by the location of these genes in quantitative trait loci (QTL) regions for this phenotype. This experimental approach has the potential for a broad application in the assessment of the roles of differentially expressed genes in a variety of complex phenotypes, with the advantage of identifying novel and potentially causal candidate genes directly.

Identification of genetic variants in the neuronal form of tryptophan hydroxylase (TPH2).

We screened the complete protein coding sequence of the newly identified neuronal form of tryptophan hydroxylase (TPH2) for genetic variants. Genomic DNA samples from 24 African-Americans and 24 Caucasian-Americans in the Coriell human variation collection were screened by denaturing high-performance liquid chromatography followed by sequencing. We identified a number of genetic variants in both the coding and exon-flanking intronic sequences. Only one variant was identified that predicts a structural change in the TPH2 protein, and this was seen in only one out of 96 chromosomes. The gene for TPH2 contains a number of polymorphisms that might serve as useful markers for association analyses of complex behavioral phenotypes or as actual risk factors. Structural polymorphisms are extremely rare in TPH2 and cannot therefore act as substantial risk factors for behavioral disorders in African-American and Caucasian populations.

Epileptic encephalopathies and their relationship to developmental disorders: do spikes cause autism?

Epileptic encephalopathies are progressive clinical and electroencephalographic syndromes where deterioration is thought to be caused by frequent seizures and abundant EEG epileptiform activity. Seizures occur in approximately 10-15% of children with pervasive developmental disorders (PDD) and 8-10% have epileptiform EEG abnormalities without seizures. Thirty percent of children with PDD have regression of social behavior and language at 2-3 years of age. Some authors speculate that the regression is caused by epileptiform activity even in the absence of overt clinical seizures ("autism with epileptic regression") and suggest that elimination of the epileptiform activity, either medically or surgically, should lead to improvement in behavior. This review examines the data showing that interictal epileptiform discharges are associated with transient clinical dysfunction and discusses the implications of these observations for autistic behavioral abnormalities. The results of resective surgery, vagal nerve stimulation, and multiple subpial transaction on children with autism and epileptiform EEG abnormalities are also discussed. I conclude that there is no evidence that interictal discharges per se cause (or contribute to) the complex behavioral phenotype of autism. There is no justification to support the use of anticonvulsant medication or surgery in children with PDD without seizures; that is, there is no evidence that treatment to eliminate EEG spikes will have a therapeutic effect on the behavioral abnormalities of PDD and autism.

Building a More Perfect Beast: APP Transgenic Mice with Neuronal Loss

While there have been a number of successful attempts to develop transgenic mice that express mutant amyloid precursor protein (APP) and develop amyloid deposits that have features similar to those in aging and Alzheimer’s disease (AD),1 until now there has been either no neuronal loss or less than convincing evidence in favor of neuronal loss. In this issue of The American Journal of Pathology, Schmitz and colleagues2 provide compelling evidence for age-dependent neuronal loss in the hippocampus of bigenic transgenic mice expressing a double mutation in APP (Swedish APP mutation APPK670N, M671L (APPsw) and APPV717I, under the mouse Thy1 promoter) and mutant presenilin-1 (PS-1 M146L under the pHMG promoter). The neuronal loss was ascertained by unbiased stereologic methods on Nissl-stained sections. The animals showed significant (30 to 35%) neuronal loss in the hippocampal pyramidal layer compared to controls, which was disproportionate to the degree of amyloid deposition. No neuronal loss was detected in the dentate fascia of the hippocampus, the only other region assessed for neuronal loss. While the regions analyzed are of interest, if the model is to have relevance to AD, it will be important in future studies to extend neuronal counts to limbic and association cortical regions, which are especially vulnerable to amyloid deposition in humans. The behavioral consequences, if any, of neuronal loss in this model are not described in this report or other descriptions of this model3 and are left to future work.

Molecular mechanisms of drug addiction

Regulation of gene expression is one mechanism by which drugs of abuse can induce relatively long-lasting changes in the brain to cause a state of addiction. Here, we focus on two transcription factors, CREB (cAMP response element binding protein) and ΔFosB, which contribute to drug-induced changes in gene expression. Both are activated in the nucleus accumbens, a major brain reward region, but mediate different aspects of the addicted state. CREB mediates a form of tolerance and dependence, which dampens an individual’s sensitivity to subsequent drug exposure and contributes to a negative emotional state during early phases of withdrawal. In contrast, ΔFosB mediates a state of relatively prolonged sensitization to drug exposure and may contribute to the increased drive and motivation for drug, which is a core symptom of addictive disorders. A major goal of current research is to identify the many target genes through which CREB and ΔFosB mediate these behavioral states. In addition, future work needs to understand how CREB and ΔFosB, acting in concert with numerous other drug-induced molecular changes in nucleus accumbens and many other brain regions, interact with one another to produce the complex behavioral phenotype that defines addiction.

Toward a molecular neuropsychiatry of neurodegenerative diseases.

Quantitative neuropsychiatry has provided increasingly precise descriptions of behavioral phenotypes associated with neurodegenerative disorders. Degenerative diseases of the brain are disturbances of protein metabolism, with failure of protein degredation by the ubiquitin-proteosome system, production of neurotoxic peptide oligomers, and accumulation of intracellular protein deposits. Abnormalities of amyloid beta peptide, alpha-synuclein protein, and hyperphosphorylated tau protein account for more than 90% of degenerative dementias. Functionally related neuroanatomical systems have shared metabolic characteristics and common vulnerabilities to protein dysmetabolism, providing the basis for phenotypes that reflect the underlying proteotype. Patients with alpha-synuclein disorders are particularly prone to hallucinations, delusions, and rapid eye movement sleep behavior disorder. Patients with tauopathies manifest disproportionate disinhibition and apathy, and may exhibit compulsions. Alzheimer's disease is a triple proteinopathy with abnormalities of A-beta, tau, and alpha-synculein leading to a complex behavioral phenotype. This molecular approach to neuropsychiatry may assist in understanding the mechanisms of degenerative diseases, provide insight into the pathophysiology of neuropsychiatric symptoms, and contribute to monitoring disease-modifying therapies.

A number no greater than the sum of its parts: the use and abuse of heritability.

Though widely used in quantitative genetics, in the study of human variation perhaps no statistic is more easily misinterpreted than heritability. While the contribution of genetic heritage to complex biological and behavioral phenotypes cannot be lightly dismissed, nonetheless we remain profoundly ignorant of how that legacy plays out in any environmental context. To be sure, it is not reducible to a single number. Nor does the preference among anthropologists for analyzing biological rather than behavioral phenotypes improve what heritability can reasonably say about the sources of human variation. This paper discusses the meaning of heritability, the methods for its estimation, the fallacies underlying its misuse, and its utility for inquiries in evolutionary anthropology and epidemiology. Progress in anthropological genetics will be realized through greater sophistication in study designs, including the measurement of environmental (physical and sociocultural) variation and the judicious choice of phenotypes for study. Elucidating the ontogenetic processes underlying adaptive plasticity is particularly critical to understanding the evolution of human biological variation. Such advancements will also shed light on the feasibility of genotype-targeted biomedical treatments. Failure to appreciate the limits of such approaches can divert resources from demonstrably effective environmentally based health interventions that benefit entire populations. Simplistic notions of genetic determinism should be challenged for the sake of our theories and the well-being of larger communities. As exemplified by the work of Frank B. Livingstone, anthropological genetics is at its best when incorporating anthropology into the study of human phenotypic variation.

H.R. Chin and S.O. Moldin (eds): Methods in Genomic Neuroscience

H.R. Chin and S.O. Moldin (eds): Methods in Genomic Neuroscience

Different Requirements for cAMP Response Element Binding Protein in Positive and Negative Reinforcing Properties of Drugs of Abuse

Addiction is a complex process that relies on the ability of an organism to integrate positive and negative properties of drugs of abuse. Therefore, studying the reinforcing as well as aversive components of drugs of abuse in a single model system will enable us to understand the role of final common mediators, such as cAMP response element-binding protein (CREB), in the addiction process. To this end, we analyzed mice with a mutation in the alpha and Delta isoforms of the CREB gene. Previously we have shown that CREB(alphaDelta) mutant mice in a mixed genetic background show attenuated signs of physical dependence, as measured by the classic signs of withdrawal. We have generated a uniform genetically stable F1 hybrid (129SvEv/C57BL/6) mouse line harboring the CREB mutation. We have found the functional activity of CREB in these F1 hybrid mice to be dramatically reduced compared with their wild-type littermates. These mice maintain a reduced withdrawal phenotype after chronic morphine. We are now poised to examine a number of complex behavioral phenotypes related to addiction in a well defined CREB-deficient mouse model. We demonstrate that the aversive properties of morphine are still present in CREB mutant mice despite a reduction of physical withdrawal. On the other hand, these mice do not respond to the reinforcing properties of morphine in a conditioned place preference paradigm. In contrast, CREB mutant mice demonstrate an enhanced response to the reinforcing properties of cocaine compared with their wild-type controls in both conditioned place preference and sensitization behaviors. These data may provide the first paradigm for differential vulnerability to various drugs of abuse.

Mice deleted for the DiGeorge/velocardiofacial syndrome region show abnormal sensorimotor gating and learning and memory impairments.

Del22q11 syndrome is caused by heterozygous deletion of an approximately 3 Mb segment of chromosome 22q11.2. Children diagnosed with del22q11 syndrome commonly have learning difficulties, deficits of motor development, cognitive defects and attention deficit disorder. They also have a higher than normal risk for developing psychiatric disorders, mainly schizophrenia, schizoaffective disorder and bipolar disorder. Here, we show that mice that are heterozygously deleted for a subset of the genes that are deleted in patients have deficits in sensorimotor gating and learning and memory. The finding of sensorimotor gating deficits is particularly significant because patients with schizophrenia and schizotypal personality disorder show similar deficits. Thus, our deletion mouse models at least two major features of the del22q11-associated behavioral phenotype, and as such, represents an animal model of this complex behavioral phenotype. These findings not only open the way to pharmacological analyses that may lead to improved treatments, but also to the identification of gene/s that modulate these specific behaviors in humans.

(Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features.

Fragile X syndrome is a common cause of mental retardation involving loss of expression of the FMR1 gene. The role of FMR1 remains undetermined but the protein appears to be involved in RNA metabolism. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macroorchidism and behavioral abnormalities. As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. We observed that the YAC transgene supported production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism was absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, we performed a more thorough evaluation of the Fmr1 knockout phenotype using additional behavioral assays that had not previously been reported for this animal model. The mouse displayed reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein did produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype.

Attention Deficit Hyperactivity Disorder: Advances in Cognitive, Neurobiological, and Genetic Research

Conceptual and technological advances in cognitive neuroscience and molecular genetics have the potential to identify the pathogenesis of psychiatric disorders. This article reviews the application of these technologies to the scientific study of attention deficit hyperactivity disorder. It begins with a summary of shifts in conceptualization and scientific study of this common condition. This is followed by a critical review of findings from recent cognitive, neuroimaging, and genetic studies. The available data do not yet permit an integration across these different levels of enquiry, but implicate problems in response inhibition, dysfunction of frontostriatal networks, and genetic factors in the pathogenesis of this complex behavioral phenotype. The review closes with suggestions for future interdisciplinary research.

Genetic analysis of dyslexia and other complex behavioral phenotypes.

In this review, we discuss recent data on the genetics of developmental dyslexia and consider broader issues involved in the search for genes influencing complex behavioral phenotypes. These issues include 1) the need for a sophisticated analysis of the phenotype and the need for interdisciplinary collaboration between geneticists and cognitive neuroscientists, 2) the likelihood of genetic heterogeneity and non-Mendelian inheritance and the necessity for linkage methods to deal with these issues, and 3) how association analyses complement linkage analyses.

TIC DISORDERS

The picture that emerges is one of complex behavioral phenotypes that alter over the course of CNS development. While many cases are mild and may not come to medical attention, others are chronic and disabling. The aim of this article is to provide an overview of recent progress in understanding phenomenology, epidemiology, genetics, neurobiology, and treatment of tic disorders.

Advantages and limitations of nonhuman primates as animal models in genetic research on complex diseases.

The genetic similarity between humans and nonhuman primates makes nonhuman primates uniquely suited as models for genetic research on complex physiological and behavioral phenotypes. By comparison with human subjects, nonhuman primates, like other animal models, have several advantages for these types of studies: 1) constant environmental conditions can be maintained over long periods of time, greatly increasing the power to detect genetic effects; 2) different environmental conditions can be imposed sequentially on individuals to characterize genotype-environment interactions; 3) complex pedigrees that are much more powerful for genetic analysis than typically available human pedigrees can be generated; 4) genetic hypotheses can be tested prospectively by selective matings; and 5) essential invasive and terminal experiments can be conducted. Limitations of genetic research with nonhuman primates include cost and availability. However, the ability to manipulate both genetic and environmental factors in captive primate populations indicates the promise of genetic research with these important animal models for illuminating complex disease processes. The utility of nonhuman primates for biomedical research on human health problems is illustrated by examples concerning the use of baboons in studies of osteoporosis, alcohol metabolism, and lipoproteins.

Syntax facit saltum: Computation and the genotype and phenotype of language

Human language has long captured the imagination of biological researchers. However, the gulf separating ‘computation’, ‘biology’, and ‘language’ has been equally long-standing-in large measure resulting from the gap between linguistic and biological description: we do not expect to literally find a ‘passive grammar rule’ inside a person’s head. Similarly, we evidently do not find a corresponding passive rule-specific ‘dysphasia’ that destroys one’s ability to say, “This problem was solved by the student” while sparing, along with the rest of the grammar, the ability to say “The student solved this problem”. The puzzle is a classic biological one: how to bridge between a ‘genotype’ and a ‘phenotype’, in this case perhaps the most complex behavioral phenotype we know. Linguistics has cast natural languages’s intricate and ultimately behavioral ‘outer form’ or ‘phenotype’ at an abstract level far removed form language’s computational and biological ‘inner form’ or ‘genotype’. Thus, even though linguistic science’s successful program the past 40 years has resulted in perhaps the richest description of a human ‘genotype to phenotype’ mapping that we know of-the initial substrate for human language and how language develops in an individual-until recently, progress at a more ‘reductionist’ level has been far more halting. The aim of this article is to show how recent developments in linguistic theory dubbed the “minimalist program” (MP) [l, 21 might help bridge the biology-language ‘abstraction gap’, because the minimalist program shows that despite its apparent surface complexity, language’s core might in fact be much simpler than has previously been supposed. For neuroscientists pursuing clues left by the linguistic phenotypes’s ‘fault lines’ down to the level of the real genotype, as exemplified in the work of Gopnik and colleagues [3, 4, 51, or even the so-called ‘candidate gene approach’, this is a promising development. The minimalist program is eliminative in exactly the right sort of way, since it boils down all syntactic relations and rules, including transformations, to feature matching-thus tying linguistic theory more tightly than ever to accounts such as Gopnik’s model of Specific Language Impairment. More generally, the minimalist program serves as a case study for how a complex behavioral phenotype can emerge from the interactions of a much simpler ‘genotype’. In particular, the minimalist program posits that the human ‘syntactic engine’consists of just three components: (1) words, that is, word semantics and word morpho-phonology; (2) word features; and (3) a simple operation that glues together or merges words and word complexes. This article demonstrates how just these three components interact to yield many, perhaps all, of the ‘special design features’ of human syntax. By ‘design features’ we simply mean

Mapping quantitative trait loci for open-field behavior in mice.

By performing a whole genome screen in an F2 intercross of two strains of mice (A/J and C57BL/6J), which differ markedly in their behavioral response to a brightly lit open field (O-F), we have mapped several quantitative trait loci (QTL) for this complex behavioral phenotype. QTL on chromosomes 1 and 10 were identified that affect both initial ambulation in the O-F (initial "response to novelty" ambulation) (lod of 7.1 and 8.8, respectively) and vertical rearings (lod of 4.5 and 8.5, respectively). For habituated O-F behavior, QTL were identified on chromosomes 3 and 10 for ambulation (lod of 4.1 and 14.7, respectively) and on chromosomes 1, 10, and 19 for vertical rearings (lod of 5.8, 6.0, and 4.7, respectively). The QTL on chromosome 1 (near D1Mit116; 101 cM) was specific for initial O-F ambulation behavior, whereas the QTL on chromosome 10 (near D10Mit237; 74 cM) affected both initial and habituated rearing behavior. Additional suggestive QTL (lod, > 2.8) were mapped to chromosomes 1, 8, 11, 15, and 19. The QTL on chromosomes 1, 10, and 19 individually explain from 3.2 to 12.7%. Collectively, the multiple independent QTL explain from 16.3 to 24.1% of the F2 population's phenotypic variance, depending on the trait. These identified QTL should prove useful for dissecting the genetic and behavioral dimensions of O-F behavior, fostering an understanding of individual differences.

Pathways to sociopathy: Twin analyses offer direction

AbstractUnderstanding the bases of complex behavioral phenotypes, such as sociopathy, is assisted by an evolutionary approach, in addition to other theoretical perspectives. Unraveling genetic and environmental factors underlying variant forms of sociopathy remains a key challenge for behavioral science investigators. Twin research methods (e.g., longitudinal analyses; twins reared apart) offer informative means of assessing novel hypotheses relevant to sociopathic behaviors.