Allan C. Collins - US grants

The proposed studies will explore biochemical, behavioral, and genetic interactions between ethanol and cholinergic drugs, most notably nicotine and oxotremorine. These studies will attempt to test the hypothesis that some of ethanol's acute and chronic effects are mediated via effects on nicotinic and muscarinic receptors. The experiments will assess the effects of acute and chronic ethanol treatment on nicotinic and muscarinic receptors as well as the effect of altering these receptors on acute response to ethanol, tolerance to ethanol, and the ethanol withdrawal syndrome. A battery of tests has been developed to measure acute responses to ethanol, nicotine, and oxotremorine as well as tolerance to ethanol and the ethanol withdrawal syndrom. Nicotine, scopolamine, or oxotremorine will be infused into LS and SS mice, two selected lines of mice that differ in acute response to ethanol, and the influence of the resultant receptor changes on ethanol response will be measured. In addition, the effects of ethanol, in vitro and in vivo, on cholinergic receptors will be measured. Finally, the genetic control of ethanol/nicotine and ethanol/oxotremorine interactions will be estimated. These studies should provide added insight into the reasons for simultaneous use and abuse of alcohol and nicotine.

The proposed research will test the hypothesis that differences in the number or affinity of receptors which bind nicotine account for differences in initial sensitivity or tolerance to nicotine. Our animal models will be inbred strains of mice, and appropriate hybrids and backcrosses, which differ in initial sensitivity or tolerance development. The research will also assess the interactions of nicotine with miscarinic and Alpha-bungarotoxin receptors. Susceptibility to nicotine's effects (acute sensitivity or tolerance) will be quantified using a number of tests, and receptor number and affinity will be measured in several brain regions and tissues (adrenal gland, skeletal muscle, spinal ganglia) in order to investigate the possibility of correlations between tolerance development and receptor alterations. Because one of the strains we propose to study does not develop tolerance to nicotine, it will provide an ideal control for assessing the role of receptor changes in tolerance development. Since genetic factors apparently influence human smoking behavior, the proposed studies should provide data which will be useful in determining whether nicotine plans a role in this genetic effect. The neurochemical studies should be of value in determining whether receptor differences underlie genetic differences in human smoking behavior.

Genetic influences on the use of tobacco products in humans and on the acute response in animals to a challenge dose of nicotine have been well documented. Similarly, genetic factors seem to influence the development of tolerance to nicotine. The intent of the proposed studies is to explore the hypothesis that differences in the number of brain nicotinic receptors, measured by the binding of L-(3H)nicotine and Alpha-(125I)bungarotoxin, underlie these genetically determined differences in response to nicotine. Several laboratories have been studying the brain (3H)nicotine binding site and some investigators suggest that this site is noncholinergic. Experiments are designed in this proposal that should resolve whether the (3H)nicotine binding site is or is not cholinergic. Previous studies in our laboratory have demonstrated that nicotine-induced seizures may be regulated in the mouse by a single gene that also regulates the number of hippocampal nicotinic receptors. Studies are proposed that should increase our understanding of this relationship. We have also demonstrated that chronic nicotine treatment results in a dose- and time-dependent increase in the number of brain nicotinic receptors. This increase parallels the development of tolerance to nicotine. Experiments to explore more fully the relationship between the increase in receptor numbers and tolerance development are planned. These studies will include autoradiographic analyses that should allow a determination as to whether all, or only some, of the nicotinic receptors increase following chronic nicotine treatment. Only some of the acute sensitivity differences and tolerance can be explained by differences in receptor number. Therefore, studies of nicotine-induced ion flux and densitization of the receptor are included. In order to carry out the desensitization studies, an enrichment of the brain nicotinic receptors must be achieved. Lastly, studies designed to measure the nicotine withdrawal syndrome in the mouse are described. Taken together these experiments may prove to be valuable in understanding the genetic regulation of acute sensitivity to nicotine, tolerance to nicotine, and the underlying neurochemical processes that control these behaviors. Such knowledge can be instrumental in designing rational therapies that would aid smokers in terminating tobacco use.

Genetic influences on the use of tobacco products in humans and on the acute response in animals to a challenge dose of nicotine have been well documented. Similarly genetic factors seem to influence the development of tolerance to nicotine. The intention of the proposed studies is to explore the hypothesis that differences in the number of brain nicotinic receptors, measured by the binding of L-(3H)nicotine and (125I)-bungarotoxin, underlie these genetically determined differences in response to nicotine several laboratories have been studying the brain (3H)nicotine binding site, and the investigators suggest that this site is non- cholinergic. Experiments are designed in this proposal that should resolve whether the (3H)nicotine binding site is or is not cholinergic. Previous studies in our laboratory have demonstrated that nicotine-induced seizures may be regulated in the mouse by a single gene that also regulates the number of hippocampal nicotinic receptors. Studies are proposed that should increase our understanding of this relationship. We have also demonstrated that chronic nicotine treatment results in a dose- and time-dependent increase in the number of brain nicotine receptors. This increase parallels the development of tolerance to nicotine. Experiments to explore more fully the relationship between the increase in receptor numbers and tolerance development are planned. These studies will include autoradiographic analyses that should allow a determination as to whether all, or only some, of the nicotinic receptors increase following chronic nicotine treatment. Only some of the acute sensitivity differences and tolerance can be explained by differences in receptor number. Therefore, studies of nicotine- induced ion flux and desitization of the receptor are included. In order to carry out the desensitization studies, an enrichment of the brain nicotinic receptors must be achieved. Lastly, studies designed to measure the nicotine withdrawal syndrome in the mouse are described. Taken together these experiments may prove to be valuable in understanding the genetic regulation of acute sensitivity to nicotine, tolerance to nicotine, and the underlying neurochemical processes that control these behaviors. Such knowledge can be instrumental in designing rational therapies that would aid smokers in terminating tobacco use.

Human smoking appears to be influenced in part by genetic factors. Genotype may influence who does or does not smoke and may influence the course and severity of withdrawal responses observed when smoking is terminated. Similarly, genetic factors are important in the regulation of the responses of mice to either acute or chronic nicotine exposure. as well as in the expression of brain receptors which may regulate the responses to nicotine. The studies outlined in this proposal will examine the role of genotype in the regulation of nicotine effects on acute sensitivity, tolerance development and withdrawal. Simultaneously the genetic regulation of putative nicotinic receptor levels, the changes in these levels during treatment and after withdrawal will be investigated. Six inbred strains that differ in response to nicotine and expression of putative nicotinic receptors will be used as parental stocks for both diallel crosses land classical genetic crosses. The parental strains and their crosses will be used to study the influence of genetic factors on: 1) regulation of the acute responses to nicotine and regulation of the expression of nicotinic binding sites, their subtypes and function with the goal of determining the relationship between acute responsiveness and receptors; 2) regulation of tolerance development and changes in the expression of nicotinic receptor number and function with chronic nicotine treatment with the goal of understanding the biochemical basis for the development of itolerance to nicotine; 3) expression of a withdrawal syndrome and the relationship between the extent of tolerance development, the magnitude of receptor changes, and !the severity of the withdrawal; and 4) control of tolerance development after the exposure to nicotinic agents other than nicotine itself. The results obtained in this study should be valuable in establishing an animal model for nicotine tolerance and dependence with an emphasis on the role of genotype in regulating the physiological and biochemical factors that control nicotine responsiveness.

biomedical equipment purchase; protein purification; high performance liquid chromatography;

DESCRIPTION (Applicant's Abstract): This proposal seeks support for the applicant for a K05 level Research Scientist Award. The application, if funded, would serve to reduce the applicant's teaching and administrative assignments to a minimal level and would increase the time available to the applicant to pursue research. Most of the research that will be carried out during the tenure of this grant will use genetic strategies (classical genetic methods, molecular methods and null mutants/transgenics) to help identify nicotinic receptor subtypes that modulate specific behavioral and neurochemical responses to nicotine. A major goal is to identify mechanisms that explain genetically-based variability in initial sensitivity to nicotine and in the development of tolerance to nicotine. These studies will focus on identifying naturally-occurring polymorphisms in neuronal nicotinic receptor genes in the mouse and on assessing how/whether these polymorphisms alter behavioral and neurochemical responses to nicotine. These studies will be augmented by studies utilizing mice that have had specific nicotinic receptor genes deleted (null mutants). Hopefully, these studies will identify potential mechanisms that would explain how genetic factors influence tobacco use by humans.

The use and abuse of tobacco remains one of the major causes of a myriad of health problems. It is well established that nicotine is the most important psychoactve substance in tobacco smoke and that smokers modify their use of tobacco to regulate nicotine intake. Smoking also appears to be regulated by genetic factors in humans. The studies outlined in this proposal will continue to use genetic strategies to attempt to explain variability in response to a first dose of nicotine (initial sensitivity). We have shown that inbred mouse strains differ in initial sensitivity to nicotine and that 35-40% of this variability correlates with variability in nicotinic receptor numbers; strains that have higher numbers of L[3H]- nicotine binding sites are more sensitive to several low dose effects of nicotine whereas strains that have high numbers of alpha[125I]- bungarotoxin binding sites are more sensitive to seizures that are evoked by high doses of nicotine. The studies outlined here will attempt to determine whether differences in brain nicotinic receptor function and desensitization also contribute to strain differences in response to nicotine. Initial studies will focus on two recently developed-essays (dopamine release from striatal synaptosomes and (86)Rb+ efflux from midbrain synaptosomes). Since the dopamine release process is very sensitive to inhibition by neuronal bungarotoxin it seems likely that the striatal nicotinic receptor that mediates this process contains an alpha-3 subunit. In contrast, the (86)Rb+ efflux from midbrain seems to be regulated by an alpha-4-containing receptor. Additional assays will also be developed and characterized by assessing the rank order of potency and efficacy of eight agonists and selected antagonists, especially neuronal bungarotoxin and alpha-bungarotoxin. Data obtained from these studies will be compared with results obtained by others using known nicotinic receptor combinations expressed in frog oocytes to provide a tentative assignment of receptor type. Subsequently, the kinetics of receptor desensitization and resensitization will be determined for each of the functional assays in each of the brain regions; these properties will be compared using brain tissue from mouse strains that differ in sensitivity to nicotine. We have also shown that chronic nicotine treatment results in tolerance to nicotine; some mouse strains develop tolerance at lower infusion doses than are required to elicit tolerance in others. Chronic nicotine infusion also evokes what has come to be known as a paradoxical up-regulation of brain nicotinic receptors. This change in receptor numbers parallels tolerance development in some mouse strains, but not in others. We have speculated that the unexpected up-regulation occurs because chronic nicotine treatment serves to desensitize or perhaps even inactivate brain nicotinic receptors, and have recently obtained evidence that suggests that this may be true, but brain regions may respond differently. The proposed studies will utilize the functional assays that we have developed and intend to develop to determine whether inbred mouse strains that differ in proclivity for developing tolerance do so because of differences in effects of chronic nicotine treatment on receptor function. The effects of chronic nicotine treatment will be compared with the effects of chronic mecamylamine treatment because we have recently observed that chronic antagonist treatment also elicits up-regulation of brain nicotinic receptor numbers. The results of these studies should provide much-needed insight into the role of nicotinic receptors in regulating response to nicotine, and may provide explanations for genetically-determined differences in response to nicotine that may be useful in explaining individual differences among humans in smoking behaviors.

Nicotine is the primary constituent that regulates the maintenance of tobacco use. Many of the biological effects of nicotine arise directly or indirectly through the specific interaction with nicotinic cholinergic receptors. The nicotinic receptor family is composed of many diverse subtypes that display different anatomical locations, subunit compositions, and functional manifestations. Furthermore, chronic exposure of people (through smoking) or animals (through chronic nicotine administration) affects the number and function of nicotinic receptors in the brain. In addition, the effects of nicotine are influenced by the genetic makeup of the individual, either human or animal. However, several major questions remain to be addressed. This project will unite three research groups that have had a long-standing interest in nicotine or nicotinic receptors to investigate the role of nicotinic receptor subtypes in the regulation of response to nicotine after either acute or chronic exposure. Molecular biological, immunochemical, anatomical, biochemical, classical genetic, and pharmacological approaches will be used to study the biological bases of the heterogeneity of response to nicotine. Although it is known that chronic nicotine treatment increases the number of receptors measured by high affinity agonist binding, the precise molecular mechanism for this increase is unclear. Even less is known about those receptors that cannot be detected by ligand binding assays. With the interaction between the Collins group and the Lindstrom group, the effects of chronic nicotine treatment in vivo and in vitro on many nicotinic receptor subtypes can now be studied. The results of these experiments should clarify the role of these subtypes in tolerance development and clarify the molecular mechanisms underlying the changes in receptor levels observed with chronic treatment. Even though the numbers of nicotinic binding sites increase with chronic treatment, functional responses decrease. Because of the complexity of the nicotinic receptor system, a definitive assignment of the structure of the receptors mediating any of several biochemical measures has not been made. The interaction between the Collins group and the Heinemann group will begin to address this question. The determination of the effects of deleting a gene encoding a specific nicotinic receptor subunit on the behavioral, physiological and functional responses to nicotine will be of immense importance in assigning roles to each of the defined subunits. In addition, evaluation of the effects of gene deletion on tolerance development should further clarify the role of a given receptor subunit in the adaptive responses of the nervous system to chronic nicotine treatment. Furthermore, the application of immunological techniques to the knockout mice should provide useful information about the interaction among the remaining receptor subunits in the absence of one or more of the genes. The multidisciplinary approach achieved with this collaboration should expand understanding of the -molecular mechanisms underlying nicotine tolerance and dependence.

nicotinic receptors; receptor sensitivity; drug interactions; ethanol; nicotine; neurotoxicology; choice; longitudinal animal study; brain metabolism; genetic strain; drug tolerance; drug withdrawal; behavioral genetics; receptor expression; oral administration; behavior test; laboratory mouse;

technology /technique development; substance abuse related disorder; taste; nervous system; genetics; ear; biomedical equipment development; Mammalia; bioengineering /biomedical engineering; biomedical resource; eye; human tissue; mental disorders; behavioral /social science research tag;

The deleterious health effects that result from long-term tobacco addiction extract an enormous socioeconomic cost each year, yet astonishingly little is known about the causes of tobacco, or more appropriately, nicotine addiction. Nicotine use and/or treatment elicits a broad array of behavioral and physiological effects that presumably arise subsequent to the binding of nicotine to brain nicotinic cholinergic receptors (nAChR). Brain nAChRs are ligand-gated ion channels that are readily activated and desensitized by nicotinic agonists. Molecular approaches have identified 10 genes that encode for nAChR subunits. The mRNAs for some of these subunits are expressed in many brain regions whereas others appear to be expressed in only a few. The studies outlined in this proposal will attempt, using neurochemical and genetic strategies, to identify the subunit composition of nAChRs that make up several different ligand binding sites and regulate presynaptic processes such as ion flux and neurotransmitter release. These studies will make use of several null mutant (knockout) mice that have specific nAChR subunit genes disrupted. Other studies will assess the effects of chronic nicotine treatment on the number and function of mouse brain nAChRs. Experiments done with cell lines expressing neuronal-type nAChRs have demonstrated that chronic nicotine treatment elicits a permanent inactivation of receptor function. The chronic studies outlined in this proposal will evaluate whether a similar phenomenon occurs in mammalian brain, and whether this effect varies across brain regions and receptor subtype. The information provided will also be useful in testing the hypothesis that changes in nAChR number and function underlie tolerance and dependence on nicotine.

DESCRIPTION (provided by applicant): Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are expressed throughout the brain as well as in the autonomic ganglia. Starting in the mid-1980's multiple neuronal nAChR subunit genes have been identified, cloned and sequenced. Today, we know of a minimum of 11 different subunits that are expressed in mammals, and 9 of these subunits are expressed in the mouse brain. It is believed that neuronal nAChRs are pentameric ligand-gated ion channels that are similar to the well-characterized muscle-type nAChR. Tremendous progress has been made in understanding these receptors using expression systems. For example, studies done in Xenopus oocytes and cell lines indicate that altering the subunit composition of the nAChRs results in tremendous changes in the physiological and pharmacological properties of these receptors. Less is known about the naturally occurring receptors. Questions that are of current interest include: Where are the nAChRs expressed in the brain; What is the subunit composition of the brain nAChRs; What role do these receptors play in synaptic transmission; What role do they play in modulating brain function (behavior), and What is their role in modulating components of the nicotine addiction process? One tool that shows incredible promise for answering these questions is the null mutant and knockin mouse. Over the last few years nAChR subunit null mutants and knockin mice have been made in several laboratories. We have been given many of these mutant animals and more have been promised to us. These animals will be used in several funded research projects. This P30 grant has been submitted to provide support for what is the world's most complete collection of nAChR transgenic mice. Support will allow us to do studies that would otherwise be nearly impossible (given the financial support currently available) such as breeding the mice on multiple genetic backgrounds and breeding double mutants. We hope that this resource will, ultimately, be made available to other researchers in the nAChR field.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This proposal is seeking support, via an institutional training grant, for a training program that is geared towards training pre- and postdoctoral fellows who will pursue research careers that focus on the study of genetic influences on substance abuse. The IBG faculty is distinguished and active in research and IBG scientists have actively pursued studies of genetic influences on substance abuse for over 35 years. Major substance abuse-related research projects are now in progress in both human and animal behavior genetics. The application of biometrical genetic techniques and the development of quantitative trait loci methods allow the mapping of genes that regulate complex polygenic traits. Information from such analyses, along with neurochemical, neuropharmacological, and molecular genetic studies, are being used in attempts to provide an understanding of gene function related to behavior. Facilities are available for gene mapping studies in human, mouse and nematode models, behavioral and biochemical studies in mice and nematodes, and biometrical analyses. [unreadable] [unreadable] Funds are requested to support 4 predoctoral and 2 postdoctoral trainees. Predoctoral trainees receive doctorate degrees from a cooperating academic unit and certification in behavior genetics. Academic requirements in the training program include training in behavior genetics, quantitative and biometrical genetics, theoretical and computer-based statistics, molecular genetics, responsible conduct of research, and courses on behavioral and clinical phenotypes. Additional requirements vary according to the degree granting academic unit. Research experience is an integral part of training. Postdoctoral trainees also pursue a formalized program that emphasizes individual research as well as competence in molecular and quantitative behavior genetics. Other activities in their preparation include: supervision of students and/or technicians, hosting of seminar speakers, guest lecturing, and mandatory attendance in a course on the responsible conduct of research and a weekly journal club. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Many studies, the first done more than 50 years ago, have demonstrated that genes play an important role in regulating the development of alcoholism in humans. Unfortunately, only limited progress has been made towards actually identifying genes that contribute to alcoholism. Animal models may be of some utility in this respect. Many studies done at the University of Colorado, as well as other places, have demonstrated that genes regulate many alcohol-induced and-related behaviors. Much of our early work was devoted to determining whether genes influence alcohol phenotypes. One area of particular success was the development of the long-sleep (LS) and short-sleep (SS) mouse lines. These lines were derived by selective breeding starting from the outbred, heterogeneous stock (HS) mice. In recent years we have been searching for the genes that regulate the sleep time phenotype. Congenic strains and recombinant inbred strains have been developed from the LS and SS mice that will be very useful in identifying genes that regulate the sleep time phenotype. Perhaps of greater importance, these mice have already proven to be of value in studying many other alcohol phenotypes. We have also developed mouse lines (HAFT-LAFT) that differ from acute functional tolerance (first dose tolerance) to alcohol, via selective breeding from the HS stock. These mice are a very valuable and unique resource that must be maintained. This R24 application is being submitted to help guarantee the continued success of alcohol genetics research at the University of Colorado and so that we can continue to supply our valuable animals to other researchers.

The Mouse Genetics Core is responsible for collaborating in the design and execution of experiments that use genetically-defined mouse strains to model the genetic and phenotypic aspects of schizophrenia that the Center has identified in human subjects. Acra7 (acetylcholine receptor alpha7) is the mouse equivalent of the CHRNA7 (CHolinergic Receptor Nicotinic alpha7) gene for the alpha7 nicotinic receptor that the Center will be focusing on in their human work. The experiments rely on acra7 knockout strains and a spontaneously occurring mutation in the inbred DBA/2 mouse strain in the acra7 gene. Dr. Sherry Leonard (Project 0001) proposes to use acra7 knockouts as a model to help interpret results from her human post mortem brain microarray assays. Dr. Diego Restrepo (Project 0002) is currently examining differences in alpha7 nicotinic receptor expression in the olfactory bulb and olfactory behavioral differences between DBA/2 and C3H mice, as a model for the differences he will be examining in schizophrenics. Dr. Robert Freedman (Project 0004) will use congenic mouse strains (the DBA/2 acra7 locus on a C3H background) to investigate the developmental pharmacology of sensory gating abnormalities. He will also use various acra7 null mutant strains to determine the mechanisms of action of perinatal choline treatment. The director of the core, Dr. Allan Collins, is director of the mouse behavioral genetics facility at the Institute for Behavioral Genetics and a NIDA-funded investigator whose principal research interest, the genetic determination of the response to nicotine, formed the scientific basis of the animal model experiments of this application.