John Crabbe, Ph.D.Portland Alcohol Research Center

Department of Behavioral Neuroscience

Oregon Health & Science University

VA Medical Center

Genetic Animal ModelsNeurobiology of Disease Toolbox

May 2, 2007

Collaborators

PortlandPam MettenKari BuckJohn BelknapDeb FinnTamara PhillipsJeanna WheelerHelen Kamens

University of Windsor)Doug Wahlsten

Monell Chemical Senses InstituteAlex Bachmanov

University of TexasSue Bergeson

G X E Interaction

Risk Factors for Substance Abuse

GENETIC ENVIRONMENTAL

Specific genes

FamilyPeersWorkComorbidityEarly onset

Alcoholism

Depression Anxiety

Substance Abuse is Comorbid with Many Psychiatric Diagnoses and Personality Traits

Alcoholism

ADHD Impulsivity

Substance Abuse is Comorbid with Many Psychiatric Diagnoses and Personality Traits

Behaviors are Usually Complex Genetic Traits

MULTIGENIC: Several genes contribute

POLYGENIC: Each gene exerts only a small influence

Such traits are quantitative (distributed continuously) rather than qualitative (all-or-none) in populations

Genes A,B,C, and D converge to influence Trait X, and may interact (epistasis)

Genes B,C, and D all influence both Traits X and Y (pleiotropy)

Genetic influences on traits depend on the environment, and differentially so throughout lifespan

Thanks to Kathleen Merikangas

Data Supporting Genetic Influences

• 4-fold increased in risk in 1o relatives

• Adopted away children have 4-fold increased risk

• Identical vs fraternal twins (2:1 risk ratio)

• Work with genetic animal models

What Behaviors do We Currently Model in Animals?

• Self-administration

• Intoxication

• Tolerance and/or sensitization

• Withdrawal severity

• Reinforcement

DSM-IV Diagnostic Criteria for Substance Dependence

• Tolerance

• Withdrawal

• Use greater than intended

• Loss of control

• Much time spent…,

• at expense of important activities

• Persistence despite problems

(4 symptoms within a year)

Courtesy of Russ Fernald

Advantages of the Mouse for Genetic Studies

• A dense map of the mouse genome now exists

• The mouse and human genetic maps are very similar (~ 80%)

• Mice are a reasonably intelligent mammalian species

Inbred Strains

• Within an inbred strain, all same-sex animals are essentially clones

• Within a strain, individual differences are largely due to environmental effects.

• Across inbred strain mean responses, genetic differences are the most likely influence.

Uses of Inbred Strains

• “Quiet” genetic background on which to explore targeted gene effects

• Identify genetically correlated traits (i.e., pleiotropic gene effects)

• Explore environmental dependence of genetic effects (i.e., GXE interaction), i.e., the instability of genetic correlations across tasks and/or environmental conditions

McClearn & Rodgers, Q J Stud Alc 20:691 (1959)

Two-bottle Preference for 10% EtOH vs Water in 3 Inbred Mouse Strains

C57BL/6 DBA/2

C3H/NT

Wahlsten, et al, PNAS 103:16364 (2006)

Two-bottle Preference, 10% EtOH vs Water

How Not to Use Inbred Strains

Low progesterone leads to high sensitivity to sigma1 receptor ligands which leads to increased cocaine reward, and cocaine dependence

C57SW

Brain progesterone content (ng/g)

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Brain progesterone is negatively correlated with cocaine CPP

Brain progesterone content (ng/g)

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Brain progesterone is negatively correlated with cocaine CPP

r = -1.00

df = n - 2

Brain progesterone content (ng/g)

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Brain progesterone is negatively correlated with cocaine CPP

r = -1.00

df = n - 2

Each strain is essentially many clones of one individual

Brain progesterone content (ng/g)

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Brain progesterone is negatively correlated with cocaine CPP

DBA

r = -.95

Brain progesterone content (ng/g)

Co

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Brain progesterone is negatively correlated with cocaine CPP

DBA

C3H

Brain progesterone content (ng/g)

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Brain progesterone isn't correlated with cocaine CPP

DBA

C3H

r = -.07

Logue et al, Alcohol Clin Exp Res 22:1919 (1998)

Animals with high alcohol preference have low scores on an impulsivity task

Letwin et al. J Neurosci. 20:5277 (2006)

Strain differences in expression patterns of 188 genes in specific brain regions

CR = cerebellum

PG = peri-acqueductal gray

TL = temporal lobe

PF = prefrontal cortex

VS = ventral striatum

Letwin et al. J Neurosci. 20:5277 (2006)

41 genes increased expression

28 genes decreased expression

Strain (genetic) correlation of gene expression with ethanol-stimulated activity

The Mouse Phenome Project

• Data are being collected from 40 inbred mouse strains

• 679 biochemical, physiological and behavioral variables are in this database as of April 18, 2007

• Genetic correlations can be explored with archived data, or your own data

http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home

Issues with Null Mutant Mice

• Developmental compensation

• Genetic background effects

• Intrinsic limitations of background strain

• Limited generalizability of phenotypes

The real issue with null mutant mice is that the addictions are not single gene disorders

• 1996 saw the first paper reporting an effect of a null mutation on an alcohol response

• By 2006, 141 papers had reported effects of manipulating nearly 100 genes on various alcohol responses

• 76 genes have been studied for effects on alcohol preference drinking

¼ increased, 1/3 decreased, 40% had no effect

Crabbe, Phillips, Harris, Arends & Koob, Addiction Biol 11:195 (2006)

This polygenicity is not just a problem with alcohol due to its peculiar pharmacology

• 1991 saw the first paper reporting an effect of a null mutation on response to an amphetamine-like drug

• By 2007, ~150 papers had reported effects of manipulating 80 genes on various responses

• 115 papers have studies effects on acute locomotor stimulation

Phillips, Kamens & Wheeler, In: Crusio, Sluyter & Gerlai (Eds), Handbook of Behavioral Genetics of the Mouse. In press.

Parental Population

Selection Generation

Maze-BrightMaze-Dull

Plomin, Nat Rev Neurosci (2001); adapted from Tryon, J Comp Psychol (1940)

Advantages of Selection

• Gene frequencies for these affecting the trait increase to produce large cumulative changes in the trait

• Genes acting additively and interactively (epistasis) are captured

• Divergently selected lines can be used to explore biological underpinnings

Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice differ in handling induced convulsion

severity after 72 hr EtOH vapor inhalation

Withdrawal convulsions after 16 selected generations

Responses Correlated with Ethanol Withdrawal in WSP vs WSR Mice

• WSP have more severe withdrawal from barbiturates, benzodiazepines, other sedative drugs

• During withdrawal, WSP has greater enhancement in sensitivity to NMDA, kainic acid, and corticosterone

• WSP shows large increases in L-type Ca++ channels

• WSP chows pronounced changes in GABA-A receptor subunit composition

• WSP shows greater sensitivity to modulators of Cl-

flux at GABA-A channels, including neurosteroids

Quantitative Trait Loci (QTLs)

• Each QTL comprises a region of a chromosome containing a gene affecting the target trait

• Each such QTL indicates one or more genes

• Each QTL usually exerts a modest effect

• Collectively, the multiple QTLs affecting a trait can control a substantial proportion of individual differences in the trait

Mapping QTLs in WSP x WSR F2 Mice

• 440 mice were made dependent and scored for withdrawal severity

• The highest and lowest scoring 20% were genotyped for 82 microsatellite markers spanning the genome

• Five significant associations were found, on chromosomes 1, 4, 8, 11 & 14

Bergeson et al, Mammalian Genome 14:454 (2003)

Bergeson et al, Mammalian Genome 14:454 (2003)

Mapping QTLs in WSP x WSR F2 Mice

DBA/2J (D2) inbred mice have more severe acute ethanol withdrawal than C57BL/6J (B6) inbreds

A single large dose of alcohol is given by injection (4.0 g/kg, i.p.). Handling induced convulsions are assessed 4-12 hours later. Peak withdrawal intensity is at 6-8 hrs post-injection.

0 2 4 6 8 10 12Hours post ethanol injection

0

1

2

3

4

D2

B6

Combined data from RI strains, an F2 segregating population, and short-term selected lines [Buck et al., J. Neurosci 17:3946 (1997) ]

Location of significant QTLs for acute alcohol withdrawal

Boxes denote approximate 95% confidence intervals.

Short term selective breeding for acute alcohol withdrawal severity from B6D2F2 mice

As withdrawal severity increases, frequency of the D2 allele at the b locus on Chr 4 increases.

Metten et al, Behavioural Brain Res. 95:113 (1998)

A congenic strain is identical to its inbred, background strain, except for a small segment of a chromosome that has been introduced from another (donor) inbred strain by repeated backcrossing

x x

x x

x

1

2

3

4

5-10

Two inbred strains F1 hybrid (backcross)

Congenic line

Additional backcross generations

Three chromosomes are shown

Congenic Strains can be Used for QTL Mapping

Trait??

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Introgressed QTL region

QTL is narrowed to the yellow interval

Congenic strains for the Chromosome 4 alcohol withdrawal QTL

Fehr, et al, J. Neuroscience 22:3730 (2002)

Fehr et al, J. Neuroscience 22:3730 (2002)

Capture of B6 alleles confers lower acute ethanol and pentobarbital withdrawal

Additional backcrossing reduced the QTL interval until only two known genes remained, Mpdz and Nfib

Shirley et al., Nat. Neurosci. 7:699 (2004)

Mpdz haplotypes and protein variants in 15 inbred strains, and their acute ethanol withdrawal scores

Fehr et al, J. Neurosci 22:3730 (2002)Metten & Crabbe, Behav. Pharmacol 5:533 (1994)

Shirley et al., Nat. Neurosci. 7:699 (2004)

Summary

• Inbred strains, selected lines, and mice with single genes manipulated are all powerful genetic animal models for exploring substance abuse

• Gene mapping efforts started with QTL mapping based on polymorphisms in gene sequence, and are increasingly using gene expression array methods

• Invertebrate models (Drosophila, C. elegans) and zebrafish have been little used thus far, with the exception of ethanol and a few studies with cocaine.