Julie G. Hensler - US grants

The regulation of receptors in brain is important in explaining certain effects of psychotherapeutic drugs. Historically, studies of receptor regulation have focused on the effect of chronic under- or over-exposure of a receptor to its neurotransmitter. The regulation of receptors and responses may also occur through interactions between receptor subtypes for a particular neurotransmitter. The overall objective of this proposal is to investigate whether interactions between the 5-HT1A and 5-HT2 receptors are involved in their regulation. Treatments that result in a desensitization of 5-HT1A receptor-mediated responses also regulate 5-HT2 receptor number and/or the sensitivity of 5-HT2 receptor-mediated responses. The studies proposed in the first specific aim are designed to explore, using cells maintained in culture, whether interactions between the second messenger systems linked to each subtype occur and contribute to regulatory phenomena. Activation of protein kinase C, an integral part of the second messenger system to which the 5-HT2 receptor is coupled, leads to the desensitization of the 5-HT1A receptor. Thus, 5-HT2 receptor activation may play a role in the desensitization of the 5-HT1A receptor. P11 cells, which express 5-HT2 receptors coupled to phosphoinositide (PI) hydrolysis, will be transfected with the 5-HT1A receptor in order to study the interactions between these serotonin receptor subtypes and their respective second messenger systems on the same cell. Whether activation of 5-HT2 receptors causes desensitization of 5-HT1A receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase and/or changes in 5-HT1A receptor binding will be determined. The effect of activation of the adenylyl cyclase cascade on 5-HT2 receptor stimulated PI hydrolysis and 5-HT2 receptor binding will also be investigated. The regulation of 5-HT1A receptors in P11 cells expressing both serotonin receptor subtypes will be compared to its regulation in Chinese Hamster Ovary (CHO) cells, transfected to express only the 5-HT1A receptor. In the second specific aim, studies are proposed to test in vivo whether the regulation of 5-HT1A receptors occurs as a result of activation of 5-HT2 receptors, or as a result of the desensitization or down regulation of 5-HT2 receptors and whether intact serotonergic neurons are required for this to occur. Rats will receive a single injection of the 5-HT2 receptor agonist DOI, or single injection of the antagonist mianserin, or chronic treatment with the 5- HT2 receptor antagonist ketanserin. Treatment with DOI causes activation of 5-HT2 receptors but no down regulation of 5-HT2 receptors at the time when measurements will be done. By contrast, these treatments with antagonists have been shown to down regulate 5-HT2 receptors. The sensitivity and function of both serotonin receptor subtypes will be assessed in the same animal and in the same brain region, specifically 5-HT1A receptor-mediated inhibition of adenylyl cyclase in cortical homogenates and 5-HT2 receptor stimulated PI hydrolysis in cortical slices. This combined approach should provide important information that will contribute to our understanding not only of the interactions between serotonin receptor subtypes and their respective second messenger systems but also of the regulation of the 5-HT1A receptor in vivo.

serotonin receptor; receptor sensitivity; gene induction /repression; receptor expression; enzyme activity; receptor coupling; receptor binding; protein kinase C; adenylate cyclase; ketanserin; serotonin; serotonin inhibitor; hydrolysis; drug resistance; lipid metabolism; phosphatidylinositols; antidepressants; phorbols; forskolin; CHO cells; laboratory rat; transfection; autoradiography;

[unreadable] DESCRIPTION (provided by applicant): Brain-derived neurotrophic factor (BDNF) promotes serotonergic neurotransmission and the structural plasticity of serotonergic (5rHT) neurons in the adult brain. Impaired 5-HT function and decreases in BDNF have been implicated in the pathophysiology of major depression and anxiety disorders. However, 5-HT and BDNF neurotransmission may become compromised in aging and therefore play important roles in the pathogenesis of age-related neurodegenerative disorders, as well as depression. This application describes exploratory research characterizing in a BDNF deficient mouse the function of the serotonin transporter (SERT) and somatodendritic 5-HT1A autoreceptor, proteins that play a key role in regulating 5-HT neuro- transmission. BDNF () mice exhibit age-dependent structural and neurochemical deficits in central 5-HT neurons. Prior to the appearance of gross structural deficits, BDNF () mice at 3-6 months of age display region-specific abnormalities in 5-HT neurotransmission, and an attenuation of somatodendritic 5-HT1A autoreceptor function in the median but not dorsal raphe nucleus. Preliminary data indicate that SERT function in the CAS region of hippocampus is markedly attenuated in BDNF () mice at 5, but not 2 months of age when compared to wild-type mice. The techniques employed in the proposed studies, quantitative autoradiography and in vivo high-speed chronoamperometry, offer the anatomical resolution necessary for us to address the hypothesis that in BDNF deficient mice, age-dependent abnormalities in SERT or somatodendritic 5-HT1A autoreceptor function will be observed for the 5-HT neurons arising from the median raphe nucleus. As the striatum receives 5-HT projections arising predominantly from the dorsal raphe nucleus, and the hippocampus receives 5-HT projections arising predominantly from the median raphe nucleus, we will focus on these areas of brain. We will characterize the function of the somatodendritic 5- HT1A autoreceptor (Aim #1) and SERT (Aim #2) in BDNF () versus wild-type mice at three discrete ages: (i) 2 months of age, presumably before the interactive effects of BDNF gene deletion and aging would affect 5-HT function, (ii) 5 months of age, a time-point to coincide with previous studies, and (iii) 10 months of age, before a loss of 5-HT innervation to the forebrain occurs. Region-specific and age-dependent changes in BDNF protein levels will be determined in selected brain regions of wild-type and BDNF () mice (Aim #3). [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Brain-derived neurotrophic factor (BDNF) promotes serotonergic neurotransmission and the structural plasticity of serotonergic (5rHT) neurons in the adult brain. Impaired 5-HT function and decreases in BDNF have been implicated in the pathophysiology of major depression and anxiety disorders. However, 5-HT and BDNF neurotransmission may become compromised in aging and therefore play important roles in the pathogenesis of age-related neurodegenerative disorders, as well as depression. This application describes exploratory research characterizing in a BDNF deficient mouse the function of the serotonin transporter (SERT) and somatodendritic 5-HT1A autoreceptor, proteins that play a key role in regulating 5-HT neuro- transmission. BDNF () mice exhibit age-dependent structural and neurochemical deficits in central 5-HT neurons. Prior to the appearance of gross structural deficits, BDNF () mice at 3-6 months of age display region-specific abnormalities in 5-HT neurotransmission, and an attenuation of somatodendritic 5-HT1A autoreceptor function in the median but not dorsal raphe nucleus. Preliminary data indicate that SERT function in the CAS region of hippocampus is markedly attenuated in BDNF () mice at 5, but not 2 months of age when compared to wild-type mice. The techniques employed in the proposed studies, quantitative autoradiography and in vivo high-speed chronoamperometry, offer the anatomical resolution necessary for us to address the hypothesis that in BDNF deficient mice, age-dependent abnormalities in SERT or somatodendritic 5-HT1A autoreceptor function will be observed for the 5-HT neurons arising from the median raphe nucleus. As the striatum receives 5-HT projections arising predominantly from the dorsal raphe nucleus, and the hippocampus receives 5-HT projections arising predominantly from the median raphe nucleus, we will focus on these areas of brain. We will characterize the function of the somatodendritic 5- HT1A autoreceptor (Aim #1) and SERT (Aim #2) in BDNF () versus wild-type mice at three discrete ages: (i) 2 months of age, presumably before the interactive effects of BDNF gene deletion and aging would affect 5-HT function, (ii) 5 months of age, a time-point to coincide with previous studies, and (iii) 10 months of age, before a loss of 5-HT innervation to the forebrain occurs. Region-specific and age-dependent changes in BDNF protein levels will be determined in selected brain regions of wild-type and BDNF () mice (Aim #3). [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Brain-derived neurotrophic factor (BDNF) plays a fundamental role in determining the plasticity and functional architecture of neurons in the adult brain. A frequent, non-conservative polymorphism in the human BDNF gene has been recently identified, which results in impaired secretion of BDNF and may be one factor that increases the susceptibility of an individual to psychiatric disorders brought about by stress, such as major depression. Mice offer a unique opportunity to examine interactions between environmental factors, such as stress, and genetic factors, such as a deficiency in BDNF, in determining behavior. We have interesting preliminary data that indicate that the mild stress of handing and injection of saline, which does not alter behavior in wild-type mice, produces depression-like behavior in male BDNF() mice. Our overall hypothesis is that BDNF deficient mice are more vulnerable to the effects of mild stress than the wild-type mice. We will use intraperitoneal injection of saline as a mild handling stress. The injection of saline will be used as a temporally discrete and simple stressor to which mice easily habituate. Because BDNF promotes activity- dependent synaptic plasticity, and may have a role in the modulation of responses to repeated stress, we hypothesize that mice deficient in BDNF will not habituate to repeated injections of saline. We will measure plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone as a physiological indication of the reactivity of BDNF() mice to stress immediately following the acute (3 injections of saline over 24 hours) and chronic (once daily injection of saline for 7 days) handling stress procedures (Specific Aim 1). In this exploratory application, rather than attempt to model the complex syndrome of major depression, we will model symptoms of depression that are hallmarks of this disorder such as (a) despair, and (b) loss of interest in normally pleasurable activities or anhedonia. To this end, we will measure in BDNF() mice immobility in the forced swim test (Specific Aim 2) and sucrose consumption in a two bottle choice test (Specific Aim 3). We also hypothesize that signs of behavioral depression will be reversed by the tricyclic antidepressant desipramine. Because estrogen positively modulates BDNF mRNA and protein within the hippocampus and cortex, which may make female mice less vulnerability to the deleterious effects of stress, we will conduct these proposed exploratory experiments in both male and female mice. BDNF() mice appear to exhibit characteristics consistent with theoretical or proposed etiology of major depressive disorder. In this exploratory application we further examine the validity of this model by determining the effect of mild handling stress on often used indices of behavioral depression. The validity of this model will make it of great interest to further examine in BDNF() mice the neurobiological and neurochemical mechanisms by which BDNF deficiency confers vulnerability to stress. PUBLIC HEALTH RELEVANCE: Brain-derived neurotrophic factor (BDNF) expression in the brain is decreased by stress and increased by chronic antidepressant treatments. A frequent, non-conservative polymorphism in the human BDNF gene has been recently identified, which results in impaired secretion of BDNF and may be one factor that increases the susceptibility of an individual to psychiatric disorders brought about by stress. Thus, stress-related psychiatric disorders, such as major depression, may result from a subtle atrophy or diminished function of BDNF- responsive neurons in the brain.

DESCRIPTION (provided by applicant): The Serotonin Club, founded in 1987, is an international association for biomedical scientists who are interested in research on any aspect of the neurotransmitter serotonin. Serotonin continues to be the focus for research important to substance abuse, pain, cardiovascular function, digestive processes, and especially mental health. Drugs that influence serotonergic neurotransmission are used to treat major depression, schizophrenia, mania, anxiety, eating disorders, panic, and post-traumatic stress disorder. The Serotonin Club sponsors an international meeting on serotonin every other year, and hosts annual lectures and dinners held in association with the American Society for Neuroscience meeting. The Serotonin Club Meeting for 2010 will be held July 9-11 in Montreal. The Club's overall goal is to conduct an international meeting on serotonin research that will be of the highest possible scientific quality in order to attract and educate both young and senior investigators. An innovative feature of the 2010 Meeting program is the addition of one junior scientist, selected from submitted abstracts, to speak per symposium. The primary aim of this R13 application is to provide financial support to students, post-doctoral fellows and junior researchers (i.e. young investigators not more than three years past their postdoctoral period of training) to enable them to attend and participate in the 2010 Serotonin Club Meeting. Bursaries will be provided at two levels: 1. Travel Awards will be given to 12 young investigators (ie. students, post-doctoral fellows, junior researchers), chosen to be featured as the fourth speaker in one of each of the 12 symposia. The award will cover the registration fee, airfare, housing and subsistence costs for each of the young investigators participating in the symposia;2. Outstanding Abstract Awards will be given to 10 young investigators (ie. students, post-doctoral fellows, junior researchers). These awards will cover the registration fee and a portion of expenses to attend the meeting for these young researchers. The purpose of the Travel Awards and the Outstanding Abstract Awards is to support the attendance of young investigators at the Serotonin Club Meeting. The objective of these Awards is to encourage the involvement and career development of young researchers through their exposure to the members of the Serotonin Club and science presented at the Meeting. The opportunity to attend the Serotonin Club Meeting will foster the careers of young scientists by affording them the opportunity to (i) participate in an outstanding scientific program, (ii) learn the most recent, and often unpublished, advances in serotonin research, and (iii) meet and interact with internationally distinguished researchers and scientists with whom they had previously known only through literature. The Serotonin Club recognizes the importance of mentoring to the professional development of junior scientists. All awardees will participate in the Mentorship Program. The Mentoring Program provides formal opportunities for young investigators to benefit from access to more experienced researchers. PUBLIC HEALTH RELEVANCE: Serotonin research is important to substance abuse, pain, cardiovascular systems, digestive processes, and especially mental health. Serotonergic drugs are used to treat depression, schizophrenia, mania, anxiety, eating disorders, panic, and post-traumatic stress disorder. By supporting the attendance of young scientists to the Serotonin Club Meeting, Travel and Outstanding Abstract awards will foster the careers of young scientists by affording them the opportunity to (i) participate in an outstanding scientific program, (ii) learn about the most recent advances in serotonin research, and (iii) meet and interact with internationally distinguished researchers with whom they had previously known only through literature.