Michael J. Kuhar - US grants

High affinity choline transport, a process which occurs uniquely in cholinergic nerve terminals, was found to be surprisingly resistant to postmortem changes, provided that the tissue was stored at refrigeration temperatures immediately after death. Uptake appeared to be normal at three days but was somewhat depleted at five days. If tissues were not refrigerated, postmortem changes occurred much more rapidly. Also, activity related changes in choline uptake were reversed at room temperatures in very short times, that is 10-30 minutes. Depolarization of cholinergic synaptosomes results in an activation of high affinity choline uptake. We tested whether there was such an activation of uptake for other compounds in depolarized tissues. It was found that a preliminary depolarization increased the transport of glutamic acid, aspartic acid, glycine and choline. Depolarization had no effect on the transport of a number of other compounds. These results suggest that various transport systems in nerve terminals are affected by increased neuronal activity. Since neuroleptic drugs are critical for the treatment of schizophrenia, a precise anatomical localization for their receptor sites in brain is important. We utilized light microscopic autoradiographic techniques to localize neuroleptic receptors in rat brain. Our results are in agreement with the notion that a critical function of these drugs is to block dopamine receptors.

Recently, and in the past several years, techniques have been developed for the direct measurement of receptor molecules in brain. Many psychotropic drugs exert their action directly or indirectly through these receptors and postmortem studies of human tissues have revealed that these receptors are altered in a variety of neuropsychiatric diseases. Thus, it seems possible that receptor alterations may be the causes of at least some of these diseases. This proposal deals with studying receptors in animal and human brain. A receptor of main interest is the D2-dopamine receptor. Antipsychotic drugs are thought to exert their action, at least in part, by blocking D2-dopamine receptors. The question as to whether or not increases in dopamine receptor levels are at least partly the cause of schizophrenia or are due to drug treatmet has been debated heavily. A goal of this proposal is to localize, map, annd measure dopamine receptors in primate brain. This will be carried out by routine receptor autoradiographic techniques which utilize tissue obtained at autopsy as well as by PET scanning techniques which are noninvasive and allow a measurement of receptors in vivo. The evolution of PET scanning techniques for measuring receptors is a significant advance because, for the first time, it will be possible to measure dopamine receptors and other receptors in clinical populations at various stages of their disease. Another goal of this proposal is to map CRF receptors which are an important component of the hypothalamic-pituitary-adrenal system which is a key physiological system in stress. In general, receptor maps point out sites of drug action in brain and will help explain how various drugs exert their effects. Another goal of this proposal is to explore the possibility of making several technical improvements in our light microscopic autoradiographic techniques. These techniques are being used by ever increasing numbers of people and several difficult technical problems remain. A major question in the field of receptor mapping is which specific cells have the receptors. We will attempt to develop light microscopic methods for identifying specific neurons which produce specific receptor types. Considering all of our goals together, preliminary experiments indicate that significant advances can be made with every goal.

substance abuse related disorder; reproductive system; mental disorders; nervous system; endocrine gland /system; Mammalia; Primates; human tissue; psychology; behavioral /social science research tag;

animal tissue; substance abuse related disorder; psychology; nervous system; Primates; Mammalia; biological products; proteins; behavioral /social science research tag;

animal tissue; substance abuse related disorder; mental disorders; human tissue; Primates; Mammalia; drug screening /evaluation; nervous system; behavioral /social science research tag;

DESCRIPTION (Applicant's Abstract): Cocaine amphetamine, and other psychostimulant drugs, cause abuse and addiction by interacting with and altering certain neurochemical systems in brain. Recently, a neurochemical has been discovered which appears to be involved in cocaine's action. This neurochemical has been named CART (fro Cocaine and Amphetamine Regulated Transcript). The goal of this proposal is to clarify how CART peptides mediate or modulate the effects of cocaine and other psychostimulant drugs. A first step in this work is to show that injection of these peptides into the brain causes the same physiological effects that are caused by cocaine. In other words, CART peptides cause increased locomotor activity in rodents and reduced food intake. These are well known effects of the psychostimulant drugs themselves. In addition, we will look at the effects of these drugs at brain receptors; stimulation of physiologic receptors in brain is known to produce second messengers molecule such as cyclic AMP. Further, we will map the distribution of CART peptides in brain so as to fully understand where they are located and how they might influence the brain. We will also look at the effects of blocking their production and availability so as to obtain additional evidence about what they do, and we will prepare to make transgenic mice which either overproduce or have no CART peptide. These experiments will help us to understand how cocaine changes the brain, will show us how these new neurochemicals, the CART peptides, are involved in drug abuse, and may provide new strategies for developing medications to treat cocaine abuse and dependence.

Evidence supporting a relationship between stress and reveals increased rates of dexamethasone resistance and basal hypersecretion of glucocorticoids in normal elderly humans. This relationship has been revealed in clinical studies which are highly relevant, however this approach does not permit the specificity of variables or degree of control which may be obtained using animal models. Comparing older and prime aged female rhesus monkeys in a series of studies has provided a specific test of hypotheses regarding 1) the ability of old females to adapt to stress, 2) the effect of stress on the pituitary-adrenocortical axis and the immune system (using functional measures), 3) the effect of social companionship and reactivity in modulating stress and 4) the effect of HRT on immune system measures in ovariectomized females. During this past year studies have been directed toward assessment of factors that have been shown to predictive of immune perturbation in aging in dividual s. These experiments demonstrated differences in stress reactivity, as indicated by changes in immune function, between prime and old age rhesus monkeys, confirming the primary hypothesis. This observation is consistent with a literature suggesting that aging is characterized by a decreased ability of individuals to adapt to stress, a decrement that is linked is linked to hypothalamic-pituitary-adrenal axis dysregulation. FUNDING NIH / AG13722 $133,704 12/01/1996 - 11/30/99 PUBLICATIONS None P51RR00165-38 1/1/98 - 12/31/98 Yerkes Regional Primate Research Center

When animals are given cocaine or amphetamine, CART mRNA is increased in brain regions associated with the addicting properties of these drugs. Accordingly, we have begun an intensive study of CART mRNA and its products. We have prepared antibodies against CART peptides and shown that CART peptides are, indeed, formed in the brain and found in the same neurones that contain CART mRNA. We are beginning to inject CART peptides to measure their behavioral effects.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A recommendation to Senior Scientist Award recipients is that they teach and publish in ethics. One area of scientific ethics where there is little study is blacklisting. Blacklisting is a shunning, excluding and sometimes harassing of an individual for some alleged offence. However, there are many ethical problems associated with it. It is akin to vigilantism, does not provide due process, may rely on power and force without redress, and does not insure that the punishment fits the crime. It is harmful to the blacklisted who may never receive due process or a hearing, and it is degrading to the black listers and to the bystanders who let it happen. There is little or nothing to recommend blacklisting and it should be legislated against. Another area of activity was in data management and the how, when, and why of data management has been addressed. While there was some latitude in the management of data, key goals and procedures must be kept in mind and maintained. These included the recording, processing, and the storing of data such that it can be easily retrieved, and so that published findings can be verified and supported by the original data. There are many practical ways to do this with some new comprehensive data management software systems to be offered in the future. The final publications from this grant (which ended during the reporting period) have been completed. These include a paper on the ethics of blacklisting, and a paper on proper data management.

Early life stressors~have profound and lasting impact on behavior and neuroendocrine function. Indeed, clinical epidemiological studies have shown that childhood abuse and/or neglect is a significant risk factor for the development of adult psychopathology. These effects are due to changes in brain morphology and neurocircuit function that are only partially documented. Clarification of these changes could lead to new treatment strategies to ameliorate the deficiencies caused by the early life stress or by genetic factors. It has been known for many decades now that CNS catecholaminergic systems play a major role in the regulation of behavior. Depression has long been linked to dysfunction of dopaminergic and/or noradrenergic systems in the brain. This is not surprising as dopaminergic and noradrenergic systems have been linked to reward/motivation and to vigilance, respectively. These circuits also participate in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Clinical depression as well as many animal models of depression-like syndrome are characterized by aberrant vigilance expressed as anxiety, anhedonia, and dysregulation of the HPA axis. The current project will focus on a comparison Of the development and function of these important neural systems in an epigenetic (early life stress) model of depression-like syndrome in rodents. Additionally, a limited number of studies will be performed on a early liter stress nonhuman primate model. Neurochemical, molecular, behavioral, and electrophysiological approaches will be used to characterize these neural systems in these animal models throughout neonatal development and maturity under basal, stress (acute and chronic) conditions, and in response to antidepressant treatment. Both males and females will be used. We hypothesize that alterations in the development and/or function of dopaminergic and/'or noradrenergic neural systems underlie the altered mood, reward, reinforcement, and motor function in these animal models. Further, we postulate that catecholaminergic neurocircuits may become "sensitized" by early life stress leading to enhanced vulnerability to the effects of later exposure to adverse life events thus increasing their risk of developing major depression or related mood disorders. Multiple interactions with other preclinical and clinical components of the Emory University Center for the Neuroscience of Mental Disease will benefit these studies both in terms of the uniformity of the animal models and their treatment but also in terms of intellectual cross-fertilization.

When animals are given cocaine or amphetamine, CART mRNA is increased in brain regions associated with the addicting properties of these drugs. Accordingly, we have begun an intensive study of CART mRNA and its products. We have prepared antibodies against CART peptides and shown that CART peptides are, indeed, formed in the brain and found in the same neurones that contain CART mRNA. We are beginning to inject CART peptides to measure their behavioral effects. FUNDING NIH / DA10732 $146,184 12/01/98 - 11/30/00 PUBLICATIONS Couceyro, P., Paquet, M., Koylu, E., Kuhar, M.J. and Smith, Y. Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in myenteric plexus neurons of the rat ileum and co-localization with choline acetyltransferase. Synapse 30:1-8, 1998. Elias, C.F., Lee, C., Kelly, J., Aschkenasi, C., Ahima, R.S., Couceyro, P.R., Kuhar, M.J., Saper, C.B. and Elmquist, J.K. Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron 21:1375-1385, 1998. Koylu, E.O., Couceyro, P.R., Lambert, P.D. and Kuhar, M.J. Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J. Comp. Neurol. 391:115-132, 1998. Lambert, P.D., Couceyro, P.R., McGirr, K.M., Dall Vechia, S.E., Smith, Y. and Kuhar, M.J. CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29:293-298, 1998. P51RR00165-38 1/1/98 - 12/31/98 Yerkes Regional Primate Research Center

In order to treat effectively and repair the disease of drug addiction, the critical mediators and defects in the brain must be known. While some mediators and defects or changes due to drugs are known, all the critical ones may not be fully known or understood. This proposal deals with a relatively new neurochemical that appears to play a role in the action of psychostimulant drugs such as cocaine and methamphetamine. This neurochemical is actually a small group of peptides referred to as CART peptides. They were discovered by giving animals cocaine or amphetamine, and CART peptides are found in many brain regions involved in reward and reinforcement. Moreover, some CART peptides, upon injection into the brain, produce effects that are psychostimulant-like suggesting that they may mediate, at least partly, some of the actions of these drugs. This proposal further characterizes the behavioral effects of CART peptides and explores the neurochemical and neuroanatomical bases for these effects. All known active CART peptides will be injected into brain regions associated with reward and reinforcement and it will be noted if they produce effects similar to those produced by psychostimulants. The interaction of CART peptides with cocaine and dopamine will be carefully analyzed. The precise location of endogenously occurring CART peptides will be delineated by light and electron microscopy in drug dependence-related neurocircuits. Taken together, these findings will substantially increase our knowledge of the role of CART peptides in the action of psychostimulant drugs, may enhance our understanding of the process of drug dependence, and may suggest targets for development of medications for psychostimulant abusers.

DESCRIPTION (provided by applicant): CART peptides are neurochemical messengers in the brain that are involved in drug addiction, and they may be especially relevant to psychostimulants like cocaine and methamphetamine. Indeed, administration of cocaine or amphetamine to animals results in rapid upregulation of CART mRNA. Also, when CART peptides are injected into the brain, the animals behave as though they have been given psychostimulants. Locomotor activity increases and they produce signs of being dependent. Because CART peptides are naturally occurring mediators and modulators of these drugs, and because they may make good targets for new medications, they must be well understood. This proposal focuses on the brain mechanisms that control the levels of these peptides. In particular, the research will produce an understanding of how the CART gene is regulated by cellular events such as stimulation by drug and signal transduction pathways. The CART gene "promoter," which regulates expression of the gene and ultimately the level of its peptide product, will be characterized.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This new Training Program in the Neurobiology of Drug Abuse focuses on brain mechanisms of drug use/abuse/dependence. The Program is interdisciplinary and offers training in gene expression, gene profiling, drug receptors, signal transduction, neurotransmitters and neuroanatomy of reward/reinforcement, animal models, behavioral effects of drugs, medications development, and brain imaging in animals and humans. The goal is to produce scientists who have a depth of expertise in their respective approach and a breadth of expertise in the neurobiology of drug abuse. In addition to an emphasis on technical research skills, this program will develop written and oral communications skills, the ability to critically assess papers, data and applications, familiarity with grant/contract/application writing, ethical awareness and knowledge of the cutting edge research problems and questions in the field of drug abuse. "Hands-on" research, courses, journal clubs and meetings comprise the training. The Training Faculty derives from five departments and has a record of outstanding achievements. The core of the Faculty consists of long-standing NIDA-funded investigators, as well as other strong investigators whose work intersects the Drug Abuse area and who have a strong interest in expanding their work more formally in drug abuse. The Faculty includes experienced Full and Associate Professors as well a,' more junior investigators who add substantial expertise and who are committed to developing a program in Drug Abuse research. The Program is designed to support four predoctoral fellows after they have completed their Doctoral Qualifying exams, usually at the end of their second year. In addition, it is designed to support six postdoctoral fellows. The duration of support for any given trainee will vary from one year (the minimum) to three years (rarely, and the maximum) so that many trainees will have the opportunity for support. A large percentage of our previous trainees have desirable positions and have been very productive. [unreadable] [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is a training grant in the neurobiology of drug abuse that supports six predoctoral students after their second year when they make a commitment to work in a lab on a drug abuse problem. This funding also supports four postdoctoral fellows. The training may be with any of the approved faculty listed above. This grant has been renewed and we are in the seventh year of funding.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The last major part of the CART system to be identified is the CART receptor (it is possible that there are several receptors). Screening for medications related to CART peptides requires an ability to work with and express the receptor. While there have been several publications on the receptor, we have begun an intensive study with the goal of improving assays and a cloning of the receptor. The CART receptor binding assay has been standardized, and levels of specific binding in several cell lines have been measured. Specifically specific binding has been found in PC12 cells;when these cells differentiate, the binding increases. Microarray analysis has identified several possible receptors that are increased after differentiation and are therefore receptor candidates. Another approach to finding the CART peptide receptor has been to study known orphan receptors (an orphan is a receptor that is known, but whose activating substrate is unknown). Some orphan receptors have been expressed in cells, but as of this date, it is not yet clear if these are candidates. We continue to make significant progress identifying the CART peptide receptor(s).