Behavioral Genetics
           Topic #12

      Animal Models of Alcoholism




 Advantages of Animal Models
• Precise con...
Preference/Avidity
• % of times in 14-day period animal selects
  10% ethanol solution vs. tap water (both a
  sweetened w...
Alcohol Preference Comparisons




                                 3
Classical Analysis


                  P1                                         P2




                                 ...
Realized Heritability
S = Selection differential (mean of parents –
  mean of original population)

R = response to select...
Genetic Correlation
Selection concentrates gene variants
underlying, e.g., high sensitivity in the
selected line.

Therefo...
Correlated Responses




   Inbred Strain Comparisons

                     C57BL            BALB/C

  Sleep Times       3...
Withdrawal Severity
• 72 hour exposure to ethanol vapor and
  treatment with pyrazole

• 25 hour withdrawal period where r...
Buck et al. (1997)




                         Replications




Linkage to 1, 4 & 11 have been most consistently reported...
Significance of QTL
• Identifies a region in which to look for
  genes
    – 1 cM (~ 10-12 genes) is probably limit of
   ...
QTL for Withdrawal Severity
    Mouse     % Genetic      Candidate        Human
    QTL       Variance        Genes       ...
Random Mutagenesis
• Strengths:
   – Both monomorphic and polymorphic genes
   – Focusing on large-effect mutations makes ...
Mayfield et al. (2002). Patterns of gene expression are altered in the
   frontal and mortor cortices of human alcoholics....
Overexpression of Dopamine D2
            Receptors (DRD2)
   (Thanos et al. (2001), J of Neurochem, 78: 1094-1103)

 • Lo...
Behavioral Examples
• 5-HT1B Receptor Knock-Outs:
  – Saudou et al (Science, 1994) – intruder aggression
  – Crabbe et al....
Barr et al. (2004). Archives of General Psychiatry, 61: 1146-1152




                           Summary
 • Classical meth...
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Behavioral Genetics Topic #12

  1. 1. Behavioral Genetics Topic #12 Animal Models of Alcoholism Advantages of Animal Models • Precise control over environmental exposure • Manipulation of genetic risk • Explore component phenotypes and their interrelationships OUTLINE • Component Phenotypes: – Alcohol Preference -- Inbred strain comparisons – Alcohol Sensitivity -- Selection studies • Forward Genetics – QTL Analysis - Withdrawal Severity – Random mutation screens • Backward Genetics – Targeted mutations (transgenic, null-mutants) 1
  2. 2. Preference/Avidity • % of times in 14-day period animal selects 10% ethanol solution vs. tap water (both a sweetened with saccharin) • Marked differences, 0-80% • Is it heritable? – Inbred strain comparisons Inbred Strain Comparisons • Inbreeding – mating between genetically related individuals. Increases homozygosity at every loci • Inbred Strains – At least 20 generations of brother-sister mating (at least 98% of segregating loci are fixed) 2
  3. 3. Alcohol Preference Comparisons 3
  4. 4. Classical Analysis P1 P2 F1 F2 Sensitivity Physiological, biochemical, and behavioral response to ethanol in a drug-naïve animal. Is it heritable? – Selection studies 72” Parents Original Mean -4 -3 -2 -1 0 1 2 3 4 70” Children -4 -3 -2 -1 0 1 2 3 4 Kids’ Mean Response to Selection = R = Kids’ Mean – Original Mean 4
  5. 5. Realized Heritability S = Selection differential (mean of parents – mean of original population) R = response to selection (gain=mean of children – mean of original population) = h2S Realized heritability = h2 = R/S Response to Selection Genetic Correlation/Pleiotropy G1 Mediating Behavioral G2 System Phenotype G3 5
  6. 6. Genetic Correlation Selection concentrates gene variants underlying, e.g., high sensitivity in the selected line. Therefore, correlated responses to selection identify genetically correlated traits that may explicate the mechanism of genetic influence. Alcohol Metabolism ADH ALDH ETOH Acetaldehyde Acetate Neurobiological Basis for ETOH Sensitivity? • LS mice are hyper-sensitive to a wide range of hypnotic drugs including: – Benzodiazepines – Barbiturates γ-amniobutyric acid Type A Receptors (GABA receptors) 6
  7. 7. Correlated Responses Inbred Strain Comparisons C57BL BALB/C Sleep Times 38 minutes 138 minutes BrAC at 430 mg/kg 287 mg/kg righting Forward Genetic Approaches (Phenotype-Driven) • Top down approach (i.e., from phenotype to genotype) to identify relevant genes – Quantitative Trait Loci (QTL) Analysis – Mutation Screens – Microarray analysis 7
  8. 8. Withdrawal Severity • 72 hour exposure to ethanol vapor and treatment with pyrazole • 25 hour withdrawal period where relevant withdrawal symptoms are measured • Genetic analysis: – Response to selection (realized h2 = .28) – Not correlated to sensitivity – Moderate correlation with preference QTL Analysis • Linkage analysis in non-human animal species • Based on Recombinant Inbred Strains – Inbred strains that are created from the crossing of two progenitor inbred strains. Each RI strain represents a unique recombination of the alleles that differed in the two parental strains. • BxD RI derived from C57BL x DBA BXD Recombinant Inbred Lines C57BL/6J DBA/2J 00 11 F1 01 F2 25% - 00 50% - 01 25% - 11 20 generations of B-S mating 26 RI Strains 8
  9. 9. Buck et al. (1997) Replications Linkage to 1, 4 & 11 have been most consistently reported and have also been implicated in QTL analysis of pentobarbital withdrawal. Buck & Finn (2000). Addiction, 96: 139-149. 9
  10. 10. Significance of QTL • Identifies a region in which to look for genes – 1 cM (~ 10-12 genes) is probably limit of resolution – Expression studies may reduce to 5-6 genes – Sequence analysis would reduce further • Implicates gene regions in the human Importance of the Mouse Genome • Mouse genome (Nature, December 5, 2002): – 2.5Gb – ~27,000 – 30,500 genes • Relationship to human genome: – ~99% of mouse genes have counterparts (orthologs) in human – ~96% of human genes have orthologs in mouse – Conservation of some non-coding regions – Synteny Mouse Genome Sequencing Consortuim (2002). Initial Sequencing and comparative cnalysis of the mouse genome. Nature, 420: 520-562. 10
  11. 11. QTL for Withdrawal Severity Mouse % Genetic Candidate Human QTL Variance Genes Synteny 1 26% 1q21-q32 4 26% 9p21-p23/ 1p32-22.1 11 12% GABAA 5q32-q35 2 Glutamic acid 2p24-q37/ decarboxylase 11p13 QTL Methodology • Strengths: – Systematic and relatively easily implementable approach to identify linkage for natural variants – Can help to implicate specific candidate genes and syntenic regions in human genome • Limitations: – Linkage identifies regions; limited resolution – Not all genetic variants exist in parent stocks – Will not inform us about invariant genes Random Mutagensis • Expose a large number (thousands) of male mice to a chemical mutagen (ethylnitrosourea, ENU, produces point mutation rates at about 1/1000) • Bred with unexposed females to produce many thousands of F1 progeny. Screen F1 for dominant effects (further breeding is needed to screen for recessive effects) • Confirm outlier is transmitted by further breeding 11
  12. 12. Random Mutagenesis • Strengths: – Both monomorphic and polymorphic genes – Focusing on large-effect mutations makes gene identification easier – In theory, greater likelihood of gene identification in complex systems • Limitations: – Less powerful when there is large background phenotypic variability due to genes or environ – Need efficient, sensitive and reliable behavioral screens Forward Genetic Approaches (Phenotype-Driven) • Top down approach (i.e., from phenotype to genotype) to identify relevant genes – Quantitative Trait Loci (QTL) Analysis – Mutation Screens – Microarray analysis Microarray Analysis • Identify gene expression differences (mRNA) from two samples simultaneously on 1000s of genes • Human Example: Mayfield et al. (J Neurochem, 2002) compared expression of 10,000 genes in post-mortem brains of alcoholics versus controls found 191 that had at least 1.4-fold difference in expression • Animal Example: McBride at al. (2002, Alc: Clin & Exp Research) compared expression of more than 3906 genes in hippocampus of P and NP rats; found 25 that differed in expression at p < .0001. 12
  13. 13. Mayfield et al. (2002). Patterns of gene expression are altered in the frontal and mortor cortices of human alcoholics. J Neurochem, 81: 802-813. Reverse Genetics (Genotype-Driven) • Bottom up approach (i.e., from genotype to phenotype) to determine gene effects – Transgenic: – Knock-out (null mutant): Transgenics • Animal where foreign DNA has been stably incorporated into their germ line 13
  14. 14. Overexpression of Dopamine D2 Receptors (DRD2) (Thanos et al. (2001), J of Neurochem, 78: 1094-1103) • Low levels of DRD2 appear to be involved in reinforcing effects of alcohol – Reduced reinforcement in KO mouse – Alcoholics have reduced levels of DRD2 – Rats selected for ETOH preference have low DRD2 • Thanos et al. experimentally increased expression of DRD2 in (NAc brain region of) rats – Results in sharp reduction in ETOH preference Thanos et al (2001) Null-Mutants (Knock out) • Targeted inactivation of a specific gene • Conditional knock- outs 14
  15. 15. Behavioral Examples • 5-HT1B Receptor Knock-Outs: – Saudou et al (Science, 1994) – intruder aggression – Crabbe et al. (Nature Genetics, 1996) – ethanol consumption and sensitivity • 5-HTT Knock-Outs: – Kelai et al (Science, 1994) – reduced alcohol consumption • D4R Knock-outs: – Dulawa et al. (J. Neuroscience, 1999) less responsive to novelty – Rubinstein et al. (Cell, 1999) – more responsive to activating effects of ETOH, cocaine, methamphetamine Limitations of the Standard Knock-out Procedure • Effects of deleting gene expression may depend on genetic background • Compensatory mechanisms • Cannot necessarily extrapolate from absence of gene to consequences of varying level of gene expression Gene-Environment Interaction Bennett et al. (2002). Molecular Psychiatry, 7: 118-122. 15
  16. 16. Barr et al. (2004). Archives of General Psychiatry, 61: 1146-1152 Summary • Classical methods for establishing heritability – Inbred strains – Selective breeding • Genetic Correlation – Correlated response to selection – Inbred strain correlations • Preference and Sensitivity are heritable and inversely associated (at least for the depressing effects) • Metabolism is not assoc with sleep time but is with preference • Withdrawal susceptibility is largely a genetically independent phenotype Summary • Forward Genetic Approaches (phenotype-driven) – QTL Analysis Candidate genes & candidate regions – Mutation Screens – Microarray analyses • Reverse Genetic Approaches (genotype-driven) – Transgenics – Knockouts • Genotype-environment interaction 16

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