ANIMAL MODELS IN ALCOHOL RESEARCH

1. ANIMAL MODELS IN ALCOHOL RESEARCH Richard A. Deitrich Ph.D. University of Colorado Alcohol Research Center

2. We assume that biochemical, physiological, pharmacological and genetic discoveries in simpler animals have some counterpart in human biology. While this is the underlying assumption that everyone makes, it behooves us to treat this assumption with considerable caution. Not only is the human organism much more complex than any animal model, humans may solve biological problems in different ways than even our closest relatives in the animal kingdom. We assume that biochemical, physiological, pharmacological and genetic discoveries in simpler animals have some counterpart in human biology. While this is the underlying assumption that everyone makes, it behooves us to treat this assumption with considerable caution. Not only is the human organism much more complex than any animal model, humans may solve biological problems in different ways than even our closest relatives in the animal kingdom.

3. WHY HAVE ANIMAL MODELS Develop Better Methods of Diagnosis, Treatment and Prevention of Human Alcohol Abuse and Alcoholism Advancement of Knowledge: Scientific Value-Independent of Human Health What we discover in the use of animal models advances the knowledge base of biology, however if that is the only goal, then we need to be applying to NSF, not NIH for support of our work. Ultimately, studies in animals must be translated into better diagnosis, treatment and prevention of alcohol abuse and alcoholism. What we discover in the use of animal models advances the knowledge base of biology, however if that is the only goal, then we need to be applying to NSF, not NIH for support of our work. Ultimately, studies in animals must be translated into better diagnosis, treatment and prevention of alcohol abuse and alcoholism.

4. ADVANTAGES OF ANIMAL MODELS Simpler systems Isolate individual actions of ethanol Carry out procedures not possible in humans Shorter generation times Shorter life spans This is a good news/bad news situation. For each advantage, there is a disadvantage to animal models. Animals provide a simpler system with which to work, however, if the system is so simple, that the human does not any longer utilize it, then it becomes irrelevant to our major goal. We can isolate individual actions of ethanol on a variety of systems, but lose the ability to discover the intricate interconnections between systems. We deal with animals with shorter generation times, and shorter life spans and thus can apply selective genetic pressue to develop animal models, but we lose the ability to study long term alcohol effects. The shorter life spans present a subtle problem. When we treat animals with ethanol, the rate at which it disappears from the body is not all that different from the rate in humans. However, the time that the ethanol is in the body represents a much larger fraction of the animal's life time than a similar dose in a human. This is a good news/bad news situation. For each advantage, there is a disadvantage to animal models. Animals provide a simpler system with which to work, however, if the system is so simple, that the human does not any longer utilize it, then it becomes irrelevant to our major goal. We can isolate individual actions of ethanol on a variety of systems, but lose the ability to discover the intricate interconnections between systems. We deal with animals with shorter generation times, and shorter life spans and thus can apply selective genetic pressue to develop animal models, but we lose the ability to study long term alcohol effects. The shorter life spans present a subtle problem. When we treat animals with ethanol, the rate at which it disappears from the body is not all that different from the rate in humans. However, the time that the ethanol is in the body represents a much larger fraction of the animal's life time than a similar dose in a human.

5. HOW DO WE KNOW IF WE HAVE BEEN SUCCESSFUL? Does the animal model resemble: Human behavior? Human physiology? Human biochemistry? Human genetics? Do findings in animals predict any effects in humans? Since our goal is to provide information about how ethanol acts in humans, the models that we chose and the techniques that we use to study them must have some relevance to human biology. Human behavior is so much more complex than that of any animal, we cannot hope to duplicate human reactions completely in any animal model. We do have an advantage however and that is that we can study simpler behaviors in the absence of complications of more complex ones. Animal physiology, especially in mammals, is similar to that of humans as is animal and human biochemistry. It is largely a matter of faith that human genetic influences on human behavior, physiology and biochemistry will be similar to that in animals. The close correspondence (synteny) between the basic structure of the human and mouse genome gives us great confidence that this will prove to be the case. The ultimate test of all of these factors will be the ability to take a finding in animals and use it to predict a behavior,a physiological or biochemical reaction in humans. Given the effort in the mouse and human genome, it is likely that genes, whose functions are discovered in the animal will provide the answer to the function of a similar genes in humans. Since our goal is to provide information about how ethanol acts in humans, the models that we chose and the techniques that we use to study them must have some relevance to human biology. Human behavior is so much more complex than that of any animal, we cannot hope to duplicate human reactions completely in any animal model. We do have an advantage however and that is that we can study simpler behaviors in the absence of complications of more complex ones. Animal physiology, especially in mammals, is similar to that of humans as is animal and human biochemistry. It is largely a matter of faith that human genetic influences on human behavior, physiology and biochemistry will be similar to that in animals. The close correspondence (synteny) between the basic structure of the human and mouse genome gives us great confidence that this will prove to be the case. The ultimate test of all of these factors will be the ability to take a finding in animals and use it to predict a behavior,a physiological or biochemical reaction in humans. Given the effort in the mouse and human genome, it is likely that genes, whose functions are discovered in the animal will provide the answer to the function of a similar genes in humans.

6. GENETIC METHODS INBRED STRAINS: These are animals in which at least 20 generations of brother-sister matins have resulted in virtually all alleles except for the sex chromosomes being identical. This results in the experimental variability being due entirely to environment and not genetics. It is crucial that in carrying out inbred strain comparisons, as many strains as possible are tested since the "n" is the number of strains tested, not the number of animals tested. OUTBRED LINES: These animals are deliberately kept as genetically heterogeneous as possible. Normally 10 to 40 breeding pairs are kept. Even this results in some inbreeding over time, the fewer the breeding pairs, the more inbreeding will occur. Experimental variability is due to genetics, environment, and genetic/environment interactions. These animals are useful for fine genetic mapping because the linkage groups have been broken up. They are also used as the starting point for selective breeding. Often they are the result of deliberate intercrossing of a number of inbred strains, normally at least 8. Both mouse and rat outbred lines that have been carefully maintained are available. SELECTIIVE BREEDING: This process is usually started by choosing an ethanol- related behavioral trait that is to be genetically selected from a group of outbred animals. All animals are given the same dose of ethanol and the behavior measured. The animals exhibiting the highest scores are bred together (maintaining outbreeding conditions) and those with the lowest scores are bred together. The high and low scoring groups are not bred together. This process is repeated at each generation until there is no further separation between the groups. Inbreeding of the selected lines is often started at this point. The animals are useful in biochemical and physiological studies since the two groups should differ only in those traits that are related to the behavior that was selected. By making crosses between the selected lines or strains, it is possible to genetically may Quantitative Trait Loci responsible for the trait selected for. Congenic strains: These are very useful strains for isolation of specific regions of the genome responsible for a given behavioral action. The process can be accomplished by breeding methods or by marker assisted development of the strains, if there are genetic markers flanking the region of the genome that are to be transferred onto another background. For example it has been possible to place a small segment of the genome of the Short Sleep animals on an otherwise Long Sleep background and vice versa (Johnson and Bennett). Transgenic animals: In this procedure, the gene expressing a protein of interest is inserted randomly into the genome of an animal, thus providing an animal that overexpresses that protein. The disadvantage of this process is that the insertion is random and may have unknown genetic consequences. The background on which the transgenic animal is made is also a complicating factor. Tissue and developmental specific transgenic animals can be developed and are more desirable. Knockout animals: In this procedure a gene is inactivated so that there is a deficiency of the protein for which the gene codes. Again the background is important. Again conditional knockout animals whereby the gene to be knocked out can be eliminated later in development is an important advance. Also tissue specific knockouts are desirable. Knockin animals. In this situation a gene which has previously been knocked out can be rescued by replacing the gene in the proper position in the genome. This constitutes the best evidence that the phenotype which was absent in the knockout animal and present in the knockin animal was dependent on the gene in question and not due to some other aberration of the genome functioning. In addition an extra gene can be knocked in at the proper place in the genome, in contrast to the transgenic animal where the insertion is random. Random mutations: In this procedure mutations are induced by radiation or by chemical means and the offspring tested for various phenotypes. This process has been advocated as an alternative to selective breeding or comparison of inbred strains with subsequent Quantitative Trait Loci determination. Obviously it will work best on organisms with large numbers of offspring and short generation time. However, it has been successfully applied to rodents also. Candidate genes: In this process, data from behavioral, biochemical, neurochemical, or neurophysiological studies (or a combination of all) identifies a possible pathway for investigation. The genes which control the proteins involved in such a pathway can be searched for in animals that exhibit the desired phenotype, transgenic, knockout or knockin animals can be made utilizing this information. Perhaps in this area the background is of utmost importance since it has been found that a behavior attributed to a gene in one background cannot be duplicated in another background. INBRED STRAINS: These are animals in which at least 20 generations of brother-sister matins have resulted in virtually all alleles except for the sex chromosomes being identical. This results in the experimental variability being due entirely to environment and not genetics. It is crucial that in carrying out inbred strain comparisons, as many strains as possible are tested since the "n" is the number of strains tested, not the number of animals tested. OUTBRED LINES: These animals are deliberately kept as genetically heterogeneous as possible. Normally 10 to 40 breeding pairs are kept. Even this results in some inbreeding over time, the fewer the breeding pairs, the more inbreeding will occur. Experimental variability is due to genetics, environment, and genetic/environment interactions. These animals are useful for fine genetic mapping because the linkage groups have been broken up. They are also used as the starting point for selective breeding. Often they are the result of deliberate intercrossing of a number of inbred strains, normally at least 8. Both mouse and rat outbred lines that have been carefully maintained are available. SELECTIIVE BREEDING: This process is usually started by choosing an ethanol- related behavioral trait that is to be genetically selected from a group of outbred animals. All animals are given the same dose of ethanol and the behavior measured. The animals exhibiting the highest scores are bred together (maintaining outbreeding conditions) and those with the lowest scores are bred together. The high and low scoring groups are not bred together. This process is repeated at each generation until there is no further separation between the groups. Inbreeding of the selected lines is often started at this point. The animals are useful in biochemical and physiological studies since the two groups should differ only in those traits that are related to the behavior that was selected. By making crosses between the selected lines or strains, it is possible to genetically may Quantitative Trait Loci responsible for the trait selected for. Congenic strains: These are very useful strains for isolation of specific regions of the genome responsible for a given behavioral action. The process can be accomplished by breeding methods or by marker assisted development of the strains, if there are genetic markers flanking the region of the genome that are to be transferred onto another background. For example it has been possible to place a small segment of the genome of the Short Sleep animals on an otherwise Long Sleep background and vice versa (Johnson and Bennett). Transgenic animals: In this procedure, the gene expressing a protein of interest is inserted randomly into the genome of an animal, thus providing an animal that overexpresses that protein. The disadvantage of this process is that the insertion is random and may have unknown genetic consequences. The background on which the transgenic animal is made is also a complicating factor. Tissue and developmental specific transgenic animals can be developed and are more desirable. Knockout animals: In this procedure a gene is inactivated so that there is a deficiency of the protein for which the gene codes. Again the background is important. Again conditional knockout animals whereby the gene to be knocked out can be eliminated later in development is an important advance. Also tissue specific knockouts are desirable. Knockin animals. In this situation a gene which has previously been knocked out can be rescued by replacing the gene in the proper position in the genome. This constitutes the best evidence that the phenotype which was absent in the knockout animal and present in the knockin animal was dependent on the gene in question and not due to some other aberration of the genome functioning. In addition an extra gene can be knocked in at the proper place in the genome, in contrast to the transgenic animal where the insertion is random. Random mutations: In this procedure mutations are induced by radiation or by chemical means and the offspring tested for various phenotypes. This process has been advocated as an alternative to selective breeding or comparison of inbred strains with subsequent Quantitative Trait Loci determination. Obviously it will work best on organisms with large numbers of offspring and short generation time. However, it has been successfully applied to rodents also. Candidate genes: In this process, data from behavioral, biochemical, neurochemical, or neurophysiological studies (or a combination of all) identifies a possible pathway for investigation. The genes which control the proteins involved in such a pathway can be searched for in animals that exhibit the desired phenotype, transgenic, knockout or knockin animals can be made utilizing this information. Perhaps in this area the background is of utmost importance since it has been found that a behavior attributed to a gene in one background cannot be duplicated in another background.

7. GENETIC METHODS

8. SELF ADMINISTRATION Preference: Discussion of methods. �Forced� Choice Preference in �addicted� animals Acceptance Reinforcement �Working for a drink� There are many self administration paradigms in use. Unfortunately, many investigators do not compare their methods with others resulting in contradictory or inconsistent results abound in the literature. Most comonally animals are given a choice between a drinking bottle containing ethanol at some low concentration and water. The fluid level in the bottles is measured, usually each day, and the positions switched. Variations in concentration of the ethanol, presence of other chemicals either in the ethanol or as a third choice, environmental changes etc all make for the possibility of a very complex testing situation. Often the animal is given no choice for several days, thus forcing the intake of at least some ethanol solution. After a prolonged period of forced intake, the animals become tolerant and dependent (�addicted�). Self administration in such animals is used as a model of �craving� in humans. Acceptance is the process where by animals are water deprived and then given a choice of water or ethanol, or sometimes just of ethanol alone. The amount taken in is referred to as �acceptance� but does not always correlate very well with preference in the same animal strain. Many operant techniques are available in which the animal performs some task for a reward. Some animals will work for ethanol, either available in the drinking fluid or injected directly into the stomach or into the brain. There are many self administration paradigms in use. Unfortunately, many investigators do not compare their methods with others resulting in contradictory or inconsistent results abound in the literature. Most comonally animals are given a choice between a drinking bottle containing ethanol at some low concentration and water. The fluid level in the bottles is measured, usually each day, and the positions switched. Variations in concentration of the ethanol, presence of other chemicals either in the ethanol or as a third choice, environmental changes etc all make for the possibility of a very complex testing situation. Often the animal is given no choice for several days, thus forcing the intake of at least some ethanol solution. After a prolonged period of forced intake, the animals become tolerant and dependent (�addicted�). Self administration in such animals is used as a model of �craving� in humans. Acceptance is the process where by animals are water deprived and then given a choice of water or ethanol, or sometimes just of ethanol alone. The amount taken in is referred to as �acceptance� but does not always correlate very well with preference in the same animal strain. Many operant techniques are available in which the animal performs some task for a reward. Some animals will work for ethanol, either available in the drinking fluid or injected directly into the stomach or into the brain.

9. PREFERENCE Concentration Palatability Rate and timing Lick counters Limited access High intake Fading Chemicals- Cyanamide, TIQ�s Concentration: One technique is to increase the concentration of alcohol in the preference paradigm until the animal does not increase the absolute intake of ethanol or actually takes less. This becomes that animal's base line of intake against which all pharmacological manipulations are measured. The taste of 10% lab alcohol in warm water is not very paltatable to most humans. The rate which an animal takes in alcohol and the time of day when it occurs was ignored in much of the early literature. The availability of automatic recording devises such as lick counters has corrected this problem. Often it is the pattern of drinking, not the absolute amount which is of most interest. Related to this is the problem that in order to ascribe the intake to a given animal it was necessary to singly house the animals. This is undoubtedly stressing, but in group housed animals, determining which animal is drinking requires either video taping or implantation of chips in the animal's neck and a devise to detect which animal is drinking at any given time, along with the lick counter. Limited access paradigms are another technique by which one can measure the desirability of alcohol for an animal. In many of these paradigms, depending on the species or strain of animal, they do not take in enough ethanol to become intoxicated, develop tolerance and dependence. To overcome this various methods have been devised to induce the animals to take in more ethanol. One of the more successful methods is the sucrose fading technique whereby the animal is given a sucrose solution at first as a choice and this is gradually replaced by ethanol. Administration of various compounds has also been found to induce intake of large amounts of ethanol. Cyanamide, an aldehyde dehydrogenase and catalase inhibitor, has been used as has ICV injection of tetrahydroisoquinolines. These compounds are condensation products of biogenic amines and aldehdyes, including acetaldehyde. A number of compounds will also decrease preference drinking in animals. The opiate antagonist, naltrexone, will do this and has found limited use in treating human alcoholics. The serotonin uptake inhibitors are effective in animals to decrease drinking but have a checkered success in humans. Acamprosate is being utilized in Europe. Of course, the aversive agents, disulfiram and cyanamide will decrease drinking by causing the accumulation of acetaldehdye in individuals who consume ethanol. Only disulfiram is an approved drug in the US. Concentration: One technique is to increase the concentration of alcohol in the preference paradigm until the animal does not increase the absolute intake of ethanol or actually takes less. This becomes that animal's base line of intake against which all pharmacological manipulations are measured. The taste of 10% lab alcohol in warm water is not very paltatable to most humans. The rate which an animal takes in alcohol and the time of day when it occurs was ignored in much of the early literature. The availability of automatic recording devises such as lick counters has corrected this problem. Often it is the pattern of drinking, not the absolute amount which is of most interest. Related to this is the problem that in order to ascribe the intake to a given animal it was necessary to singly house the animals. This is undoubtedly stressing, but in group housed animals, determining which animal is drinking requires either video taping or implantation of chips in the animal's neck and a devise to detect which animal is drinking at any given time, along with the lick counter. Limited access paradigms are another technique by which one can measure the desirability of alcohol for an animal. In many of these paradigms, depending on the species or strain of animal, they do not take in enough ethanol to become intoxicated, develop tolerance and dependence. To overcome this various methods have been devised to induce the animals to take in more ethanol. One of the more successful methods is the sucrose fading technique whereby the animal is given a sucrose solution at first as a choice and this is gradually replaced by ethanol. Administration of various compounds has also been found to induce intake of large amounts of ethanol. Cyanamide, an aldehyde dehydrogenase and catalase inhibitor, has been used as has ICV injection of tetrahydroisoquinolines. These compounds are condensation products of biogenic amines and aldehdyes, including acetaldehyde. A number of compounds will also decrease preference drinking in animals. The opiate antagonist, naltrexone, will do this and has found limited use in treating human alcoholics. The serotonin uptake inhibitors are effective in animals to decrease drinking but have a checkered success in humans. Acamprosate is being utilized in Europe. Of course, the aversive agents, disulfiram and cyanamide will decrease drinking by causing the accumulation of acetaldehdye in individuals who consume ethanol. Only disulfiram is an approved drug in the US.

10. PREFERENCE Species and Strain Differences Selective breeding :Rats UCha, UChb AA/ANA P/NP HAD/LAD SP/SNP Mice There are many inbred strains of mice and rats that are usually used in alcohol research. There are also many lines of rats that have been selectively bred for ethanol preference. However, only one project (Indiana University) has selected mice for ethanol preference. The details of selective breeding and the use of genetics in animal models is covered in another lecture. There are many inbred strains of mice and rats that are usually used in alcohol research. There are also many lines of rats that have been selectively bred for ethanol preference. However, only one project (Indiana University) has selected mice for ethanol preference. The details of selective breeding and the use of genetics in animal models is covered in another lecture.

11. PREFERENCE This is the question that every investigator must ask of their particular model. In general, drugs that influence ethanol preference in animals does alter ethanol drinking in humans, but usually to a much lesser extent and often not significantly. Thus, the use of alcohol preference in animals as a drug screening tool for use in humans has been disappointing. The second question must await more information from the human studies. The search for QTLs in animal models will be advanced by results from the human studies. In a similar way the search for QTLs in humans will be aided by the findings in animal models. This is the question that every investigator must ask of their particular model. In general, drugs that influence ethanol preference in animals does alter ethanol drinking in humans, but usually to a much lesser extent and often not significantly. Thus, the use of alcohol preference in animals as a drug screening tool for use in humans has been disappointing. The second question must await more information from the human studies. The search for QTLs in animal models will be advanced by results from the human studies. In a similar way the search for QTLs in humans will be aided by the findings in animal models.

12. Preference Study This measure does not correlate well with performance on the stationary dowel. This is also a function of the speed of the rotating rod. This measure does not correlate well with performance on the stationary dowel. This is also a function of the speed of the rotating rod.

13. ACUTE BEHAVIORAL EFFECTS OF ETHANOL High dose effects: Anesthesia �Sleep Time� Aerial Righting Reflex Development of Tolerance Sensitization SELECTIVE BREEDING Historically high dose ethanol effects were studied first. The test is simple, one measures the time that an animal has lost the righting response. This is done by placing them in a plexiglass trough, or by dropping them from a height onto a soft surface. Normally mice will not lie on their backs and turn over in the trough or will right themselves in the air so that they land on their feet. It is important to obtain a blood ethanol level at the regain of the response (and sometimes at the loss as well) in order to assess effects on the metabolism of ethanol by the procedure being studied. Why give such large doses? One reason is to engage all the neuronal systems that ethanol affects. Various systems in the brain react at low levels of ethanol and others only at high levels. Another complicating factor is the development of tolerance during the time of the loss of function. We now know that tolerance to many of the electrophysiological effects of ethanol begin to develop within minutes if not seconds of the arrival of ethanol at a neuron. This is reflected in behavioral tolerance on a somewhat longer time scale. The problem is that the measure of loss of function is complicated by the development of tolerance during this period. The longer that the ethanol is present, the more tolerance develops, up to some maximum level. Development of sensitization to the effects of ethanol is also possible, but not usually observed. Mice and rats have been selectively bred for sensitivity to high dose ethanol ("sleep time"), and mice have been bred for development of acute functional tolerance. There is accumulating evidence from Schuckit and his colleagues, that insensitivity to ethanol and/or the rapid development of tolerance are the best predictors of the risk of development of alcoholism in humans. Historically high dose ethanol effects were studied first. The test is simple, one measures the time that an animal has lost the righting response. This is done by placing them in a plexiglass trough, or by dropping them from a height onto a soft surface. Normally mice will not lie on their backs and turn over in the trough or will right themselves in the air so that they land on their feet. It is important to obtain a blood ethanol level at the regain of the response (and sometimes at the loss as well) in order to assess effects on the metabolism of ethanol by the procedure being studied. Why give such large doses? One reason is to engage all the neuronal systems that ethanol affects. Various systems in the brain react at low levels of ethanol and others only at high levels. Another complicating factor is the development of tolerance during the time of the loss of function. We now know that tolerance to many of the electrophysiological effects of ethanol begin to develop within minutes if not seconds of the arrival of ethanol at a neuron. This is reflected in behavioral tolerance on a somewhat longer time scale. The problem is that the measure of loss of function is complicated by the development of tolerance during this period. The longer that the ethanol is present, the more tolerance develops, up to some maximum level. Development of sensitization to the effects of ethanol is also possible, but not usually observed. Mice and rats have been selectively bred for sensitivity to high dose ethanol ("sleep time"), and mice have been bred for development of acute functional tolerance. There is accumulating evidence from Schuckit and his colleagues, that insensitivity to ethanol and/or the rapid development of tolerance are the best predictors of the risk of development of alcoholism in humans.

14. DETERMINATION OF HIGH DOSE ETHANOL SENSITIVITY Anesthetic doses vary widely from species to species and from strain to strain. For example the anesthetic dose for long sleep mice is 2.8 gm/kg while the anesthetic dose for short sleep mice is 5.2 gm/kg. The anesthetic dose for the base (HS) stock is 4.2 gm/kg. It is necessary to obtain a blood sample at the regain (and preferably at the loss also) of the righting reflex to detect any development of tolerance or sensitization as well as anesthetic effects. A separate group of animals should be used for determination of the blood ethanol at the loss of the righting reflex. In mice the loss of body temperature is a confounding effect on ethanol�s effects in the brain and metabolism in the liver. Anesthetic doses vary widely from species to species and from strain to strain. For example the anesthetic dose for long sleep mice is 2.8 gm/kg while the anesthetic dose for short sleep mice is 5.2 gm/kg. The anesthetic dose for the base (HS) stock is 4.2 gm/kg. It is necessary to obtain a blood sample at the regain (and preferably at the loss also) of the righting reflex to detect any development of tolerance or sensitization as well as anesthetic effects. A separate group of animals should be used for determination of the blood ethanol at the loss of the righting reflex. In mice the loss of body temperature is a confounding effect on ethanol�s effects in the brain and metabolism in the liver.

15. Determination of High Dose Ethanol Sensitivity This is the behavioral paradigm used extensively by the Addiction Research Foundation Laboratory in Toronto. This is the behavioral paradigm used extensively by the Addiction Research Foundation Laboratory in Toronto.

16. ACUTE BEHAVIORAL EFFECTS OF ETHANOL Low dose effects: Measures of Ataxia Dowel Inclined Plane Moving Belt Rope Roto-rod Grip strength Screen Activity chamber Etc SELECTIVE BREEDING Measures of low dose effects of ethanol require subjecting the animals to some physical task. These tasks are numerous and performance on one does not necessarily correlate with performance on another. When devising any new task it is important to include some measures on the previously established methods. Animals often show tolerance to the task and this tolerance may develop before the first behavioral measure is taken. Frequent testing allows for detection of this tolerance but also introduces the problem of practice effects. In any case practice effects need to be investigated. Selective breeding of rats for performance on the inclined plane has been carried out (Alcohol Tolerant and Alcohol Non-Tolerant rats) in Finland. Selective breeding of mice for tolerance on the dowel task has been carried out at Colorado (High and Low Acute Functional Tolerance, HAFT and LAFT). Recent studies show that the genetic control of sensitivity and tolerance effects depends on the strain used, on the apparatus employed and the time of testing. For example, no overlap in QTLs was found for performance on a roto-rod, stationary dowel, activity chamber or sleep time in mice selectively bred for ethanol effects. Measures of low dose effects of ethanol require subjecting the animals to some physical task. These tasks are numerous and performance on one does not necessarily correlate with performance on another. When devising any new task it is important to include some measures on the previously established methods. Animals often show tolerance to the task and this tolerance may develop before the first behavioral measure is taken. Frequent testing allows for detection of this tolerance but also introduces the problem of practice effects. In any case practice effects need to be investigated. Selective breeding of rats for performance on the inclined plane has been carried out (Alcohol Tolerant and Alcohol Non-Tolerant rats) in Finland. Selective breeding of mice for tolerance on the dowel task has been carried out at Colorado (High and Low Acute Functional Tolerance, HAFT and LAFT). Recent studies show that the genetic control of sensitivity and tolerance effects depends on the strain used, on the apparatus employed and the time of testing. For example, no overlap in QTLs was found for performance on a roto-rod, stationary dowel, activity chamber or sleep time in mice selectively bred for ethanol effects.

17. Mouse on Stationary Dowel

18. Picture of Animal on RotoRod It is this measure that was used to develop the High and Low Alcohol Tolerant mice lines. It is this measure that was used to develop the High and Low Alcohol Tolerant mice lines.

19. Picture of Animal on Inclined Plane This is the behavioral paradigm used to selectively breed the Alcohol Tolerant and Alcohol Non-Tolerant rats in Helsinki, Finland.This is the behavioral paradigm used to selectively breed the Alcohol Tolerant and Alcohol Non-Tolerant rats in Helsinki, Finland.

20. Rat on a Moving Belt

21. TOLERANCE MEASURES Acute Single Dose Tolerance (ASDT): Minutes Acute Functional Tolerance (AFT): Minutes to hours Rapid Tolerance:Hours to days Chronic Tolerance: Days In this slide we present the definitions that are in common use for the various types of tolerance based on the time allowed for its development. In this slide we present the definitions that are in common use for the various types of tolerance based on the time allowed for its development.

22. Determination of Tolerance

23. Determination of Single Dose Tolerance

24. LOW DOSE EFFECTS OF ETHANOL How does it feel? Place Preference Elevated Plus Maze Mirror Box Fecal Boli Activity Boxes Radial Arm Maze Head Poke Drug Discrimination Etc. There are many tests designed to emulate some of the emotional and physiological responses of humans under the influence of ethanol. There are many tasks designed to measure anxiety (plus maze), fear (mirrored box), impulsiveness (head poke/reward paradigm) There are many tests designed to emulate some of the emotional and physiological responses of humans under the influence of ethanol. There are many tasks designed to measure anxiety (plus maze), fear (mirrored box), impulsiveness (head poke/reward paradigm)

25. EFFECTS ON LEARNING What happened yesterday? Morris water maze Other learning paradigms Tolerance? Debates have gone on for many years around the question of whether or not excessive alcohol ingestion in humans is a learned behavior. Similar debate has occurred concerning the effect of experience of humans and animals under the influence of ethanol, i.e. state dependent learning. The whole area of tolerance has learning (i.e. practice effects) as an important variable. Debates have gone on for many years around the question of whether or not excessive alcohol ingestion in humans is a learned behavior. Similar debate has occurred concerning the effect of experience of humans and animals under the influence of ethanol, i.e. state dependent learning. The whole area of tolerance has learning (i.e. practice effects) as an important variable.

26. EFFECTS OF ENVIRONMENT Environment dependent effects Environment independent effects Age Gender Animal husbandry effects Uncontrolled, unknown effects One need only look at the variability of values obtained with inbred strains in various ethanol related behavioral measures to become convinced that environment is a major contributor to the variability. It is seldom possible to attribute more than about 50% of the variability in any measure to genetic effects and we are left with the other 50% uncontrolled environmental variation. One need only look at the variability of values obtained with inbred strains in various ethanol related behavioral measures to become convinced that environment is a major contributor to the variability. It is seldom possible to attribute more than about 50% of the variability in any measure to genetic effects and we are left with the other 50% uncontrolled environmental variation.

27. CHRONIC METHODS Lieber-DeCarli Tsukomoto-French Forced intake-drinking water Sweetened drinking water For years administration of ethanol orally was difficult. Given that most animals will not voluntarily take very much ethanol orally, investigators were left with the choice of intubation or surgically implanting cannulae in the stomach. The advent of the liquid diet by Lieber and DeCarli was a major advance. The use of the sucrose fading technique, utilized by Sampson is an important addition. The French-Tsukamoto method, while employing surgical implantation of cannulae, has been an important advance as well. For years administration of ethanol orally was difficult. Given that most animals will not voluntarily take very much ethanol orally, investigators were left with the choice of intubation or surgically implanting cannulae in the stomach. The advent of the liquid diet by Lieber and DeCarli was a major advance. The use of the sucrose fading technique, utilized by Sampson is an important addition. The French-Tsukamoto method, while employing surgical implantation of cannulae, has been an important advance as well.

28. DEPENDENCE MEASURES Piloerection Elevated Temperature Increased Running Handling Induced Convulsions (HIC) Spontaneous Convulsions Death In this slide we present some of the more common measures of dependence as defined by withdrawal signs. There is a semantic question of whether dependence exists in the absence of withdrawal. This is akin to a biological Heisenberg uncertainty principle. In this slide we present some of the more common measures of dependence as defined by withdrawal signs. There is a semantic question of whether dependence exists in the absence of withdrawal. This is akin to a biological Heisenberg uncertainty principle.

29. TISSUE DAMAGE Liver Brain Fetus Heart Gastro-Intestinal The models used to produce tissue damage in animals could easily take up another hour of discussion, but for brevity the major tissues where damage is found are listed here. The methods of alcohol administration to observe these effects are those discussed previously. Addition of other chemicals to promote or enhance ethanol�s toxic effect is also possible. For example carbon tetrachloride, iron, and glutathione depleting agents have been used. The models used to produce tissue damage in animals could easily take up another hour of discussion, but for brevity the major tissues where damage is found are listed here. The methods of alcohol administration to observe these effects are those discussed previously. Addition of other chemicals to promote or enhance ethanol�s toxic effect is also possible. For example carbon tetrachloride, iron, and glutathione depleting agents have been used.

30. ACKNOWEDGMENTS