Shanaz M. Tejani-Butt, Ph.D. - US grants

Much research into either biochemical abnormalities associated with depression and related psychiatric disorders or the mechanism(s) of action of psychotropic drugs has focused on brain monoamines, in particular norepinephrine and serotonin (5HT). The tricyclic antidepressants and some of the newer second generation drugs are potent inhibitors of the neuronal uptake of norepinephrine (NE) and/or serotonin. These effects are important since neuronal uptake is the primary inactivating mechanism for released monoamines and alterations in neuronal uptake may be related to the clinical action of these drugs. A major advance in recent years has been the identification of high affinity binding sites for tricyclic antidepressants, in particular, sites in the brain to which labelled imipramine binds. These binding sites have been found to be associated with uptake sites for 5HT. The development of this tool has facilitated greatly our ability to study the regulation of the 5HT uptake site and whether it shows alterations in neuropsychiatric disorders. Post-mortem samples from suicides have shown a decreased density of sites in brain compared with control samples. Also, imipramine binding sites are present on human platelets and several studies have shown a decrease in the density of these sites in depressed patients. The NE uptake site in brain has been studied far less than the uptake site for 5HT. Most probably, this is due to the absence of a suitable radioligand for this site. So far, studies have been done with labelled desipramine and more recently with labelled mazindol. Labelled desipramine is not an ideal ligand for labelling uptake sites associated with NE as it gives a high amount of non-specific binding and binds to low affinity sites in addition to high affinity sites. These low affinity sites are unrelated to NE uptake sites. Labelled mazindol is also not suitable for labelling uptake sites for NE since in most brain areas only a small proportion of its binding is to these sites. Both ligands are unsuitable for studying the NE uptake sites in vivo as the binding in vivo decreases at physiological temperatures. We propose to develop radioligands for the measurement of sites associated with the uptake of NE in the brain. Compounds selected for study have been reported previously to have high affinity and selectivity for NE uptake sites. Potential ligands will be radiolabelled with tritium to high specific activity and their binding properties will be investigated both in vitro and in vivo. In vitro binding will be assessed using both homogenate preparations and quantitative autoradiography of slide-mounted thin brain sections. If successfully developed, such a ligand would allow many types of studies to be performed on this uptake site, analogous to those reported already on the uptake site for 5HT.

Several studies have reported that the noradrenergic (NA) and the serotoninergic (5-HT) systems are affected in Alzheimer's Disease (AD). Since these systems appear to play a role in many diverse functions such as memory, learning and attention, it would be important to study degenerative changes in these systems that may be associated with AD. To date, little work has been done to study the role of the presynaptic uptake system for norepinephrine (NE) and serotonin (5-HT) in neurodegenerative diseases. A measure of the changes in the density of uptake sites for NE and 5-HT could provide specific information regarding the integrity of these neuronal systems in AD. Our hypotheses are that the density of uptake sites for NE and 5-HT will be decreased in the cell body areas, given previous reports of decreased cell counts in the locus coeruleus (LC) and dorsal raphe nucleus (DR). We also expect to find a loss of presynaptic sites for NE and 5-HT in the terminal field areas given reports of synaptic pathology in AD. In this proposal, we will investigate whether uptake sites for NE and 5-HT are altered in the LC, DR, cortex (CTX) and hippocampus (HIP) in AD by selective radiolabelling of uptake sites for NE (with 3H-nisoxetine) and 5-HT (with 3H- cyanoimipramine) in tissue sections from different brain regions using the technique of quantitative autoradiography. This technique offers several advantages over previously used methods in that it provides a quantitative measure of the density of uptake sites as well as a detailed anatomical map of the location of these sites. Neuropathological characterization of AD involves the formation of neurofibrillary tangles (NFT) and amyloid plaques (AP) which are thought to contribute to neuronal degeneration in the CTX and HIP in AD and reports of significant synaptic loss suggest that the presynaptic terminal system may play an important role in the pathological events that occur in AD. In this proposal, we will investigate whether NFT, AP and synaptophysin (SYP) (a presynaptic terminal protein) share the same anatomical localization as the NE and 5-HT presynaptic uptake sites that are undergoing degeneration in the cell body and terminal field areas. For these studies, brain sections adjacent to those used for the quantitative autoradiographic analysis of NE and 5-HT uptake sites will be used. These sections will be labelled immunohistochemically using monoclonal antibodies specific for tau proteins to determine the relative abundance of NFT, antibodies for beta-amyloid or A4 peptide to study AP formation and SY38 to study SYP immunoreactivity. The results of this study should provide a quantitative measure of the regional distribution and integrity of the NE and 5-HT systems in AD, as well as determine whether the pathological formation of tangles and plaques and synaptic decline in AD can be associated with losses in the NA and/or 5-HT presynaptic systems within the same brain regions.

DESCRIPTION (provided by applicant): The Wistar-Kyoto (WKY) rat has been proposed as a useful animal model in which to study the connection between stress responsiveness and vulnerability to depressive behavior. Physiologically, the WKY rat shows greater susceptibility to stress-induced gastric ulcers than other strains. Endocrine studies report that WKY rats have high levels of stress-induced adrenocorticotropin and low levels of corticotropin releasing hormone, suggesting a defective feedback in the hypothalamic-pituitary-adrenal (HPA) axis. Our research has revealed significant differences in central norepinephrine (NE) and serotonin (5-HT) sites in WKY rats when compared to Sprague-Dawley rats. Treatment with desipramine (a NE uptake blocker), but not paroxetine (a 5-HT uptake blocker), decreased immobility time in the Porsolt forced swim test, and reduced ulcer incidence, implicating the NE system in the mediation of depressive behavior in WKY rats. This grant is aimed at further substantiating the value of the WKY model, and is designed to determine the mechanisms that mediate the disease condition. Differential sensitivity to antidepressants with distinct pharmacological actions may provide useful information regarding the underlying substrates that contribute to a selective response in the WKY rat. Thus the central objective is to ascertain whether antidepressants that target specific neurotransmitter sites in the brain, will alleviate depressive behavior and attenuate the animal's responsiveness to stress. This behavioral response is expected to decrease ulcer susceptibility, modify feedback in the HPA axis, and reverse the characteristic NE and 5-HT receptor alterations in the WKY rat. A positive answer to this objective will provide significant information regarding the mechanisms that underlie depressive behavior and ulcer susceptibility in this vulnerable rat strain. The information gained from this study could provide a better understanding of why a clinical response is observed in some populations of depressed patients, while a resistance in treatment response is seen in others.

[unreadable] DESCRIPTION (provided by applicant): Clinical reports suggest a direct connection between alcohol and mood and it is believed that depression can serve as a predisposing factor for alcohol abuse. Stress has been identified as an antecedent to depressive behavior and a relationship can be drawn between exposure to stress and an increase in alcohol consumption. While the mesolimbic dopamine (DA) pathway represents an important pathophysiological target for alcohol abuse, the norepinephrine (NE) system is implicated in the pathogenesis and treatment of depressive illness. Given that the relationship between stress, depression and alcohol abuse remains poorly understood, a useful strategy would be to study these relationships in an animal model that exhibits the desired phenotypes. Previous studies have revealed that the Wistar-Kyoto (WKY) rat strain demonstrates the predisposed characteristics of behavioral depression. WKY rats show greater susceptibility to stress induced gastric ulcers. Chronic stress leads to dysregulation of the hypothalamic-pituitary-adrenal axis in WKY rats. WKY rats exhibit significant differences in their NE and DA profiles at baseline compared to other rat strains. Stress exacerbates depressive behavior and modulates the NE transporter (NET) site in limbic areas in WKY rats. WKY rats consume greater amounts of alcohol, with increases in DA transporter (DAT) sites in specific mesolimbic areas. Interestingly, antidepressant drugs that block NET or DAT not only alleviated depressive behavior but also increased DAT sites in similar mesolimbic areas as was seen following alcohol consumption in WKY rats. These results implicate a role for DA and NE pathways in both of these disorders and suggest that alcohol may be serving to alleviate depressive symptoms in the WKY rats. Given that the pathological consequences of an impaired DA or NE pathway in depressive behavior and alcohol abuse are presently unknown, this proposal will test the hypothesis that a deficit in these pathways exists in WKY rats, which may predispose them to depressive behavior leading to increased alcohol consumption. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The overall goal of this grant is to understand the neurobiological mechanisms that underlie posttraumatic stress disorder (PTSD)-like conditions as well as the treatment of these conditions in an established rodent model. The specific aims are: 1) to delineate the immediate and long term effects of a traumatic experience on sleep architecture, freezing behavior and heart rate changes in Wistar-Kyoto (WKY) and Wistar (WIS) rats and correlate these changes with noradrenergic activity in the prefrontal cortex, amygdala and hippocampus;2) to evaluate whether treatments with prazosin or propranolol will differentially ameliorate the behavioral and neuroanatomical outcomes measured in Aim 1 in the vulnerable WKY vs. resistant WIS rats. The basic approach will be to use fear conditioning, accomplished by pairing tones (conditioned stimulus [CS]) with foot shocks (unconditioned stimulus [US]), to assess conditioned fear. Freezing, ultrasonic vocalization, heart rate and sleep behavior will be compared between the two strains. Treatment with prazosin (11 adrenoceptor antagonist) or propranolol (21 adrenoceptor antagonist) will take place at appropriate timings relative to the initial triggering and subsequent exacerbating events. Following sleep recording, animals will be decapitated, and trunk blood and brains removed for analysis of stress hormones and noradrenergic pre and post-synaptic receptors. It is expected that PTSD-like symptoms will be more pronounced in the stress-reactive WKY rats compared to the more stress-resistant WIS rats. Furthermore, these strain differences will be related to altered expression and function of 11 and 21-adrenoceptors in the prefrontal cortex, amygdala and hippocampus, the three brain regions strongly implicated in the pathophysiology of PTSD. By defining the noradrenergic pathways by which these drugs alleviate PTSD-like conditions, our studies will provide relevant information which would otherwise be difficult to obtain from clinical studies, and will enhance our ability to develop effective treatment strategies to improve the quality of life for PTSD patients. PUBLIC HEALTH RELEVANCE: Our proposed studies are designed to provide information regarding the noradrenergic mechanisms that underlie posttraumatic stress disorder (PTSD)-like conditions in the Wistar-Kyoto rat, an established animal model for studying stress-related psychiatric disorders. The basic approach will be to use a fear conditioning procedure, followed by measurements of freezing, ultrasonic vocalization, heart rate and sleep behaviors. Following sleep recordings, noradrenergic pre and post-synaptic receptors will be measured in the brains of these animals. Treatment with prazosin (11 adrenoceptor antagonist) or propranolol (21 adrenoceptor antagonist) will take place at appropriate timings relative to the initial triggering and subsequent exacerbating events. By defining the noradrenergic pathways by which these drugs alleviate PTSD-like conditions, our studies will provide relevant information which would otherwise be difficult to obtain from clinical studies, and will enhance our ability to develop effective treatment strategies to improve the quality of life for PTSD patients.