George Battaglia - US grants

The long term objective of this proposal is to understand how cocaine abuse during pregnancy can contribute to the development in offspring, of subsequent clinical disorders involving dysfunction of brain serotonin(5-HT) systems. Dysfunctional 5-HT systems have been implicated in anxiety, depression, suicidal behaviors and preference for alcohol. In adult animals, cocaine perturbs 5-HT neurons by decreasing their firing rate, inhibiting 5-HT synthesis and inhibiting 5-HT uptake. During gestation, 5-HT plays a critical trophic role in the maturation of 5-HT neurons and target cells receiving 5-HT projections. Our hypothesis is that in utero exposure to cocaine produces perturbations of fetal 5-HT systems that result in long-term deficits in the functional status and integrity of brain 5-HT systems in adult progeny. Our preliminary data support our hypothesis of long-term biochemical and functional deficits in progeny brain 5-HT systems following in utero exposure to cocaine. The goal of the present research is to establish the extent of cocaine-induced changes in adult progeny with respect to the following specific aims: (1) To Determine the Functional Status of Brain 5-HT Neurons by measuring (a) 5-HT/5-HIAA content in terminal and cell body regions and the corresponding number and affinity of 5-HT uptake sites in these regions, (b) the viability of 5-HT terminals by the ability of 5-HT releasers to stimulate ACTH and renin secretion, as well as, to reduce (deplete) brain 5-HT content; (2) To Determine the Functional Status of Postsynaptic 5-HT Receptors with respect to cocaine-induced changes in (a) the stimulation of ACTH and renin secretion by directly acting 5-HT1 and 5-HT2/1C serotonin agonists, and (b) the adaptational responsiveness of 5-HT receptors by investigating the dynamics of 5-HT1 and 5-HT2 receptor turnover following receptor inactivation; and (3) To Determine the Neuroanatomic Specificity of In Utero Cocaine-Induced Effects on 5-HT Pathways by measuring regional densities of 5-HT uptake sites, and 5-HT1 & 5-HT2 receptor subtypes using in vitro autoradiographic techniques. These studies should identify discrete brain regions where cocaine- induced receptor alterations may produce functional consequences. Methods: Pregnant rats will be treated b.i.d. from gestational day 13-20 and all progeny fostered at birth. Male and female progeny of pregnant rats from 3 Treatment Groups: (1) Saline-Injected Ad-Lib Fed; (2) Saline-Injected Pair-Fed; and (3) Cocaine-Injected (15 mg/kg) will be compared at a prepubescent time (PD30). Male progeny will also be investigated at postpubescent time (PD70). Significance: Since cocaine ("Crack") abuse among women of childbearing age is becoming increasingly prevalent, these studies will provide timely and important information regarding neurological deficits which may develop in offspring due to in utero cocaine-induced dysfunction of 5-HT systems. In this regard, changes in the stimulation of plasma hormones in response to 5-HT drugs may provide an important clinical marker for in utero cocaine-induced central 5-HT dysfunction in humans.

Fluoxetine (Prozac ) and other serotonin-selective reuptake blockers (SSRIs) are being increasingly used to treat mood disorders in children. In adults, fluoxetine increases postsynaptic 5HT2A receptor signalling. In contrast, our data reveal that when administered prior to maturation, fluoxetine 5HT2A receptor signal transduction, an effect that is opposite to that produced in adults. However, virtually no preclinical data exist regarding the immediate or long-term changes in 5HT systems due to prepubescent SSRI treatment. The long-term objective of this proposal is to understand the mechanisms and persistence of adaptive changes in 5HT receptor systems produced by pubescent exposure to SSRIs. Because the clinical effectiveness of SSRIs is associated with adaptive changes in 5HT signal transduction, our HYPOTHESIS is that (1) prepubescent fluaxetine treatment will produce different neuroadaptations in postsynaptic 5HT1A and 5HT2A signal transduction than produced by adult treatment, and (2) the effects of prepubescent SSRIs will persist into adulthood and consequently, alter the ability of 5HT systems to respond to subsequent SSRI administration during adulthood. Aim 1 will determine the dose-dependence of fluoxetine-induced changes in postsynaptic 5HT1A and 5HT2A receptors and receptor-mediated neuroendocrine responses, and will establish the treatment dose for subsequent studies. Aim 2 will determine the biochemical mechanisms responsible for fluoxetine-induced adaptation(s) in postsynaptic 5HT1A and 5HT2A receptor systems, by investigating changes in specific components of the signal transduction pathway between 5HT receptors and their respective second messenger enzymes. Aim 3 and Aim 4 will investigate the longer-term effects of prepubescent fluoxetine treatment on changes in postsynaptic 5HT signal transduction. Postsynaptic 5HT1A (aim 3) and 5HT2A (aim 4) receptor systems will be studied with respect to: (1) the persistence of prepubescent fluoxetine-induced 5HT adaptations into adulthood and (2) the regulation of 5HT receptor systems in response to subsequent adult fluoxetine administration following prepubescent exposure. These studies will provide important new information about the mechanisms underlying the immediate and long-term adaptive changes in brain 5HT signalling due to prepubescent fluoxetine treatment. These studies will also elucidate the status of serotonergic function in adults treated previously with SSRIs as juveniles in order to predict how these individuals will respond to subsequent antidepressant treatment as adults. This information is critical to the effective use of SSRIs in treating mood disorders in children and in treating adults treated previously with SSRIs as juveniles.

DESCRIPTION (applicant's abstract): Various mood disorders are being increasingly diagnosed in offspring exposed to cocaine in utero. In humans, impairments in serotonin (5-HT) function are associated with disorders such as anxiety, depression and increased impulsivity and aggression. During the initial funding period, we identified long-term neurochemical and functional impairments in brain 5-HT systems in rat offspring exposed prenatally to cocaine. Thus, the long-term objective of this research is to determine the effectiveness of clinically prescribed serotonin-selective reuptake inhibitors (SSRIs) to treat mood disorders in offspring resulting from prenatal exposure to cocaine. The clinical efficacy of SSRIs is related to their ability to produce neuroadaptive changes in 5-HT systems. This renewal application will determine if clinically used SSRIs, such as paroxetine (Paxil), will be effective in reversing the serotonergic deficits in rats produced by prenatal cocaine exposure. Our HYPOTHESIS is that SSRIs will be effective in restoring brain 5-HT function and producing neuroadaptive changes in 5-HT receptor signal transduction in prenatal cocaine-exposed offspring. This proposal will determine the mechanisms responsible for 5-HT impairments and the restoration of 5-HT function by SSRIs in offspring using biochemical, neurochemical and neuroendocrine measures to study pre- and postsynaptic components of 5-HT pathways. Each aim will study the mechanism of neuroadaptive changes in different components of the 5-HT signal transduction pathway due to prenatal cocaine and subsequent postnatal SSRI treatment. Aim 1 will focus on presynaptic 5-HT terminal function; aim 2 will investigate the sensitivity of somatodendritic 5-HT1A autoreceptors on 5-HT cell bodies; and aims 3 & 4 will investigate postsynaptic function of 5-HT1A and 5-HT2A receptor signal transduction systems, respectively. Because neuroendocrine challenge can also be used in humans, the correspondence between neurochemical changes in brain induced changes in 5-HT-mediated neuroendocrine responses will provide the foundation to assess prenatal cocaine-induced changes in 5-HT function in human offspring. In addition, our studies will identify the mechanisms mediating SSRI-induced neuroadaptive changes in 5-HT systems in offspring impaired by prenatal cocaine. Data obtained from the proposed studies will be important in predicting the potential efficacy of SSRIs in treating disorders in individuals previously exposed to cocaine in utero. To our knowledge, these studies are the first to determine the utility of SSRIs to treat impairments in 5-HT function due to prenatal cocaine exposure.

Serotonin1A (5-HT1A) receptors mediate diverse signaling cascades via different G-proteins that are involved in various physiological functions and play a role in the etiology and/or treatment of mood disorders. Mood disorders are associated with increased activation of the HPA axis that is normalized by antidepressants. In neuroendocrine neurons in the adult hypothalamus, fluoxetine (Prozac(R)) and other serotonin-selective reuptake inhibitors (SSRIs) desensitize 5-HT1A activation of plasma hormones such as ACTH and oxytocin. The long-term objective of this proposal is to develop better treatment strategies by understanding the mechanisms invoked by various classes of agonists in mediating 5-HT receptor signaling of multiple intracellular pathways upon acute versus long-term repetitive drug administration. Because the clinical effectiveness of various drugs including the SSRIs is associated with adaptive changes in 5-HT1A receptor signaling, it is critical to understand the mechanisms by which 5-HT1A receptors mediate signaling in neuroendocrine neurons in response to the acute and chronic exposure to different classes of 5-HT agonists. Based on our initial findings that 5-HT1A receptors can couple to different populations of G proteins to activate both MAP kinase and hormone responses via pathways that act independently upon acute receptor activation but exhibit "cross-talk" resulting in desensitization of 5-HT1A mediated neuroendocrine response upon repetitive drug administration, we HYPOTHESIZE: MAP kinase activation plays an integral role in the neuroadaptive changes in 5-HT1A receptor-mediated hormone signaling in oxytocin and CRF containing neuroendocrine neurons in response to SSRIs and SNRI drugs. This will be tested by the four aims of this project;Aim 1 will determine effectiveness of different classes of agonists to traffick 5-HT1A receptors to Gai/o proteins and activate hypothalamic MAP kinase and/or Gaz-proteins to stimulate hormone responses;Aim 2;will determine the specific Ga-protein subtypes that mediate 5-HT1A receptor agonist activation of MAP kinase in the hypothalamic PVN and the localization of 5-HT1A activated MAP kinase (pERK) to oxytocin- and CRF-positive neurons;Aim 3 will determine the mechanisms by which serotonin/norepinephrine reuptake inhibitors (SNRIs) desensitize 5-HT1A receptor signaling of hormone responses in oxytocin and CRF-containing neuroendocrine neurons and the requirement of MAP kinase in mediating SNRI-induced desensitization, and Aim 4 will determine the efficacy of repetitive administration of different classes of serotonergic agonists to desensitize hypothalamic 5-HT1A receptor mediated MAP kinase signaling. These studies will provide important new information regarding the novel mechanisms of 5-HT1A receptor signaling pathways in hypothalamic neurons and the mechanisms by which clinically used antidepressant drugs may produce adaptive changes in signaling pathways mediated by different G proteins. These studies will elucidate role of MAP kinase in regulating the responsiveness of neuroendocrine neurons to various classes of drugs used clinically. This information is critical to identifying the mechanisms that may contribute to delays in the onset of clinical efficacy or the "side effects" of SSRIs, SNRIs or other drugs developed to treat various psychopathologies and mood disorders involving 5-HT1A receptors.

DESCRIPTION (provided by applicant) The long-term goal of the proposed studies is to provide novel therapeutic approaches for the treatment of mood disorders that will work more rapidly and more effectively than the currently available medications. The introduction of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac) has revolutionized psychiatry. In addition to their effectiveness in the treatment of depression, SSRIs also have proven effective for the treatment of several mood disorders for which the older medications were ineffective. These disorders include anxiety, obsessive compulsive disorder, aggression, eating disorders and premenstrual syndrome. However, a lag time of about 2-3 weeks exists from the onset of medication until the first signs of improvement appear. This delayed onset of therapeutic effects is a severe problem. Some of the suicides of depressed patients occur during this lag time. Therefore, there is a great need to find therapeutic approaches that will work more rapidly. Studies supporting the parent grant indicate that treatment with SSRIs produces a delayed-onset, homologous desensitization of post-synaptic serotoninlA( 5-HT1A) receptors in the hypothalamus. This desensitization is most likely mediated by increased levels of 5-HT in the synapse and the resulting over-activation of post-synaptic receptors. Studies in depressed patients indicate that the therapeutic effectiveness of SSRIs is dependent on maintaining high levels of 5-HT in the synaptic cleft. The 5-HT reuptake mechanism is the primary mechanism terminating the activation of post-synaptic receptors by 5-HT in the synaptic cleft. However, the release of 5-HT from the nerve terminals is highly regulated by inhibitory 5-HT1A autoreceptors on the soma and dendrites of the serotonergic cells in the raphe nuclei and by inhibitory 5-HT1B/1D autoreceptors on the serotonergic nerve terminals in forebrain regions. Thus, the overall hypothesis is that during SSRI therapy, the inhibitory influences of 5-HT autoreceptors must be overcome to allow SSRIs to increase the levels of 5-HT in the synaptic cleft. The proposed studies will use in vivo microdialysis approaches to investigate the time courses of changes in both 5-HT1A and 5-HT1B/1D autoreceptors, and thus determine whether these receptors contribute to the delay in onset of fluoxetine-induced increase in extracellular levels of 5-HT in several forebrain regions. In addition, the studies will investigate whether combining fluoxetine with selective 5-HT1A and/or 5-HT1B/1D antagonists will accelerate the fluoxetine-induced increase in extracellular levels of 5-HT. The results of the present studies will provide the scientific foundation for the use of specific autoreceptor antagonists as adjunctive therapy with SSRIs to produce a more rapid and effective therapy of mood disorders.

The long term objective of this program is to find novel treatments for the mood disorders associated with cocaine withdrawal. A major problem with cocaine abuse is the return to cocaine use after a period of abstinence (relapse). contributing factor to cocaine relapse is withdrawal-induced anxiety and depression which stimulate re-administration as form of self-medication. Withdrawal from cocaine results in supersensitivity of serotonin-2A (5-HT2A ) receptors. 5-HT2A receptor supersensitivity is associated with depression and anxiety. Therefore, treating 5-HT2A receptor supersensitivity may alleviate the anxiety and depression that contribute to cocaine relapse. However, to date, no studies have investigated the mechanisms responsible for cocaine-induced 5-HT2A receptor supersensitivity. The proposed studies will investigate the mechanisms through which withdrawal from cocaine induces supersensitivity of 5-HT2A receptor-mediated secretion of hormones. In addition, two potential therapeutic approaches will be tested to reverse the supersensitivity of post-synaptic 5-HT2A receptors during cocaine withdrawal. Our hypothesis is that the cocaine-induced changes in sensitivity of 5-HT2A receptors are due to changes in specific components of the intracellular signaling cascade. The proposed studies will investigate signaling mechanisms underlying the supersensitivity of 5-HT2A receptor systems in the hypothalamic paraventricular nucleus after withdrawal from cocaine and their response to treatment. Aim 1 will determine the minimum number of cocaine injection days that will produce supersensitivity of 5-HT2A receptor signaling. One of the characteristics of drug dependence is that a drug must be administered repeatedly before withdrawal effects appear. Aim 2 will determine the onset of supersensitivity of 5-HT2A receptors after exposure to cocaine and to determine whether these effects are irreversible. This study also will establish the treatment parameters to be used in aims 3-4. The cocaine withdrawal effect may be a compensatory supersensitivity of 5-HT2A receptors due to reduced 5-HT release. Thus, Aim 3 will determine how the cocaine withdrawal effects on 5-HT2A receptors can be reversed by increasing the levels of 5-HT in the synapse with selective monoamine oxidase-A (MAO-A) inhibitors. Aim 4 will determine how the cocaine withdrawal effects on 5-HT2A receptors can be reversed by 5-HT2A antagonists. Our results from these studies on the treatment with selective 5-HT2A antagonists and MAO-A inhibitors may lead to novel therapeutic approaches to reverse the supersensitivity of 5-HT2A receptors and hence treat mood disorders associated with cocaine withdrawal and relapse.