Michael Davis - US grants

The research proposes to investigate physiological and pharmacological mechanisms of classical conditioning using the potentiated startle effect, where acoustic startle amplitude in rats is increased in the presence of a light previously paired with a shock. In this paradigm conditioning is measured by the conditioned stimulus's (CS) effect on a reflex (acoustic startle). In the rat, the latency of acoustic startle is 8 msec. in the hindleg. We have delineated the neural pathway that mediates startle. To test where CS presentation modulates transmission along this pathway, startle will be elicited electrically at various points along the startle circuit before and after CS presentation in trained rats or control rats in which lights and shocks were randomly paired. To determine how complex the 'conditioned circuit' might be rats will be conditioned and then in testing the interval between light onset and tone onset will be varied to determine the minimum time the CS has to be on before startle is potentiated. Preliminary evidence indicates that only about 20 msec. may be required. Given retinal delays, this suggests an extraordinarily short transit time between activation of retinal ganglion cells and modification of acoustic startle. Finally, to determine the neurotransmitters involved in the expression of potentiated startle, selected drugs will be administered systemically or locally shortly before testing to see if they block or change potentiated startle.

Cellular substrates of acoustic and tactile startle responses and their modification by habituation, sensitization, and associative learning will be investigated. Startle is a short latency brainstem and spinal reflex. We have attempted to delineate the neural circuit that mediates acoustic startle and are using this information to determine points within this neural pathway where plasticity occurs so as to affect the final behavioral output. Earlier work showed that short-term habituation of electrically elicited startle occurred when the reflex was elicited through early parts of the startle pathway, whereas sensitization occurred when elicited from caudal points of the pathway. We will now more precisely determine where short-term habituation occurs within the startle pathway and then extend these techniques to analyze long-term habituation and sensitization. A consistent component of the startle reflex in rats is a rapid backward movement of the pinna which displays habituation, pre- pulse inhibition and modification by prior fear conditioning. We will determine the neural pathway that mediates this component of startle using chemical lesions and anterograde and retrograde tracing techniques. The neural pathway that mediates startle elicited by an air-puff will also be investigated. To begin a cellular analysis of startle plasticity, single unit activity will be recorded within the division of the facial motor nucleus that projects to the pinna muscles. We have devised a system which allows us to record single units using conventional glass micropipettes and microdrives in an awake, but restrained rat in a modified stereotaxic apparatus. Rats recorded in this way show acoustically-elicited pinna reflexes that can be quantified and measured simultaneously with single unit activity in the facial motor nucleus. This information should allow us to assess the feasibility of carrying out a cellular analysis of startle plasticity that eventually could be applied to each of the nuclei along the startle pathway.

startle reaction; reflex; neurochemistry; auditory reflex; NMDA receptors; inhibitor /antagonist; cerebellar cortex; enzyme inhibitors; association learning; conditioning; corticotropin releasing factor; intraarterial administration; laboratory rat;

DESCRIPTION (provided by applicant): The overall goals of this project are to understand neural systems involved in conditioned fear using the fear potentiated startle test as a measure. Progress over the last 10 years has focused on the amygdala and its direct projection to a critical part of the acoustic startle pathway. In this application we will evaluate the role of the mesencephalic reticular formation, near the border of the deep white layers of the superior colliculus (deep SC/Me), in fear-potentiated startle. We will also test whether fear-potentiated startle results from activation of GABA containing neurons in the lateral division of the CeA (lateral CeA), which then inhibit GABA containing projection neurons in the medial CeA, thereby disinhibiting the deep SC/Me, leading to an increase in startle. During the prior grant period we found that over-expression of CREB (cAMP response element binding protein) dramatically increased fear conditioning. Here we propose to localize more exactly within the basolateral amygdala (Bla) where over-expression of CREB will facilitate fear conditioning using viral vector gene transfer. We also found that experimental extinction (presentation of lights in the absence of shock following fear conditioning) led to an up regulation of gephyrin mRNA, critical for the inhibitory neurotransmitters glycine and GABA. Here we will determine whether treatments that block (local AP5 or Map kinase inhibitors in Bla) or facilitate (D-cycloserine given systemically or directly into Bla) will alter gephyrin mRNA upregulation. We also found fear conditioning induced several genes within and afferent to the amygdala. Here we will determine whether treatments that block (local AP5 or Map kinase inhibitors in Bla) or facilitate acquisition (HSV-CREB) of fear potentiated startle will block or facilitate gene expression in amygdala and other areas. Finally, we will employ a discrimination procedure we developed to determine or GABA antagonists or inactivation of the hippocampus, septal nucleus, bed nucleus stria terminalis or frontal cortex will affect discrimination, inhibition and generalization. The work is relevant to human anxiety disorders and promises to elucidate critical events in the formation and elimination of fear and anxiety.

Aim 1. The role of enhanced release of excitatory amino acids and an up- regulation of cAMP pathways in the locus coeruleus (LC) in the expression of opiate withdrawal measured behaviorally will be evaluated. The effects of local infusion into the LC of excitatory amino acid antagonists, dextromethorphan, or cAMP-dependent protein kinase inhibitors will be measured in morphine-dependent rats to see if these local treatments will reduce opiate withdrawal. Possible additive effects of local infusion into the LC of excitatory amino acid inhibitors and cAMP-dependent protein kinase inhibitors or of clonidine and excitatory amino acid antagonists will also be tested. The ability of local infusion into the LC of carbachol or the phosphodiesterase inhibitor IBMX to produce withdrawal- like behavior in non-dependent rats, including Lewis vs. Fisher strains, will also be tested. Aim 2. Animal models to measure the anhedonic aspects of drug withdrawal that can be classically conditioned to the experimental context in which withdrawal occurs will be developed. These will include the conditioned place aversion test and potentiation of the acoustic startle reflex when animals are reintroduced to a context in which they previously experienced opiate withdrawal. Parameters to produce withdrawal induced context potentiated startle and its context specificity will be explored. Effects of lesions of the amygdala or nucleus accumbens (NAc) on the expression vs. acquisition of opiate-withdrawal induced place aversion or withdrawal induced context potentiated startle will be tested. The effects of local infusion of excitatory amino acid antagonists into the LC, NAc or the amygdala on the expression vs. acquisition of these measures will be tested. The effectiveness of local infusion of carbachol into the LC in producing place aversion or context potentiated startle to the context in which carbachol was infused will be measured. Aim 3. Animal models to measure the hedonic aspects of drug intake using classical conditioning of stimuli associated with drug intake will be developed. These will include the conditioned place preference and conditioned reinforcement paradigms. Effects of lesions of the NAc or amygdala on the expression vs. acquisition of conditioned place preference or conditioned reinforcement will be studied. Effects of local infusion of excitatory amino acid antagonists into the NAc or the amygdala on the expression vs. acquisition of these behaviors will be tested. Effects of chronic morphine exposure and precipitated withdrawal on morphine conditioned place preference and conditioned reinforcement, as well as the effects of drug 'priming' on reinstatement of conditioned reinforcement will be evaluated.

Neuroanatomical and neurochemical mechanisms involved in conditioned fear and anxiety will be investigated in rats and in humans using the acoustic startle reflex. In both rats and humans, startle amplitude can be increased by eliciting the reflex in the presence of a cue previously paired with a shock (fear-potentiated startle equal explicit cue conditioning) as well as exposure to the experimental situation where fear conditioning occurred (context conditioning). In rats, lesions of the amygdala block both explicit cue conditioning and context conditioning, whereas lesions of the bed nucleus of the stria terminalis (BNST), which also projects to the startle pathway, block context conditioning but not explicit cue conditioning. Hence, the Bnst may be especially important for context conditioning because it receives a heavy inputs from the hippocampus. The test this, effects of chemical lesions of either the lateral and basolateral amygdala, the dorsal vs. Ventral hippocampus of the bed nucleus of the stria terminalis will be given either before or after training to asses the possible differential effects of these lesions on both the acquisition and expression of explicit cue and contextual fear conditioning. Patients with post-traumatic stress syndrome (PTSD) seem to display abnormal context conditioning buy normal explicit cue conditioning. Because PTSD has been associated with a dysregulation of brain norepinephrine (NE), and central beta- adrenergic receptors are important for modulation of aversive memories, we hypothesize that context conditioning will be more sensitive than explicit cue conditioning to manipulations of central NE. In rats and healthy humans the effects of the drugs propranolol, yohimbine, or buspirone given systemically will be tested on both context and explicit cue conditioning. We hypothesize that propranolol will be more effective in blocking the acquisition of context conditioning than explicit cue conditioning, that yohimbine will be more effective in facilitating the acquisition of context conditioning than explicit cue conditioning, and that buspirone will block the expression of explicit cue conditioning but not the expression of contextual fear conditioning. In PTSD patients and age-matched healthy controls the effects of propranolol will be evaluated on context conditioning vs. Explicit cue conditioning. Similar predictions are made for these patients excepts that the magnitude of context conditioning is expected to be greater in PTSD vs age-matched controls and this difference in startle magnitude may be normalized by propranolol.

DESCRIPTION (adapted from applicant's abstract): Neural systems involved in fear and anxiety will be studied using changes in the amplitude of the acoustic startle response, a short-latency reflex that can be elicited in all mammals. Acoustic startle can be increased by presentation of a brief light previously paired with shock (fear-potentiated startle), by sustained exposure to the same light even without prior light-shock pairings (light-enhanced startle), or by intraventricular infusion of corticotropin releasing hormone (CRH-enhanced startle). The PI and colleagues found that lesions or chemical inactivation of the bed nucleus of the stria terminalis (BNST) blocked light-enhanced or CRH-enhanced startle but not fear-potentiated startle. Conversely, lesions or chemical inactivation of the central nucleus of the amygdala blocked fear-potentiated startle but had no effect on light-enhanced or CRH-enhanced startle. The proposed experiments will further characterize similarities and differences between these structures in stress-induced enhancement of the startle reflex. Studies will test whether there is additivity of fear-potentiated startle, light-enhanced startle, and CRH-enhanced startle and whether intraventricular or local infusion into the BNST of the CRH antagonist alpha-helical CRH9-41 will block light-enhanced startle. Induction of c-fos in the amygdala vs. the BNST during presentation of a conditioned fear context vs. an unconditioned anxiogenic context will be measured. Other studies will measure how activation of CRH receptors in the BNST may ultimately increase startle at the level of the brainstem. This will be done by first delineating the neural pathway linking the BNST to the nucleus reticularis pontis caudalis. Previously, these investigators found that if rats were first given extensive overtraining and then given amygdala lesions, relearning could occur with further training. Other studies will evaluate whether the BNST can 'take over' for the amygdala in fear-potentiated startle using lesion, chemical inactivation and IC-fosI methodologies.

DESCRIPTION (adapted from applicant's abstract): Neural systems involved in fear and anxiety will be studied using changes in the amplitude of the acoustic startle response, a short-latency reflex that can be elicited in all mammals. Acoustic startle can be increased by presentation of a brief light previously paired with shock (fear-potentiated startle), by sustained exposure to the same light even without prior light-shock pairings (light-enhanced startle), or by intraventricular infusion of corticotropin releasing hormone (CRH-enhanced startle). The PI and colleagues found that lesions or chemical inactivation of the bed nucleus of the stria terminalis (BNST) blocked light-enhanced or CRH-enhanced startle but not fear-potentiated startle. Conversely, lesions or chemical inactivation of the central nucleus of the amygdala blocked fear-potentiated startle but had no effect on light-enhanced or CRH-enhanced startle. The proposed experiments will further characterize similarities and differences between these structures in stress-induced enhancement of the startle reflex. Studies will test whether there is additivity of fear-potentiated startle, light-enhanced startle, and CRH-enhanced startle and whether intraventricular or local infusion into the BNST of the CRH antagonist alpha-helical CRH9-41 will block light-enhanced startle. Induction of c-fos in the amygdala vs. the BNST during presentation of a conditioned fear context vs. an unconditioned anxiogenic context will be measured. Other studies will measure how activation of CRH receptors in the BNST may ultimately increase startle at the level of the brainstem. This will be done by first delineating the neural pathway linking the BNST to the nucleus reticularis pontis caudalis. Previously, these investigators found that if rats were first given extensive overtraining and then given amygdala lesions, relearning could occur with further training. Other studies will evaluate whether the BNST can 'take over' for the amygdala in fear-potentiated startle using lesion, chemical inactivation and IC-fosI methodologies.

Project 6 will use the acoustic startle reflex to assess the behavioral consequences of early life stressors at various stages of development. The acoustic startle reflex has a number of advantages that makes it a useful measure to assess the long-term behavioral consequences of early maternal separation. Its non-zero baseline allows both excitatory and inhibitory effects to be measured. The startle reflex can be elicited by different types of sensory stimuli and shows several forms of plasticity, such as habituation, sensitization, prepulse inhibition, and modulation by fear or anxiety. In addition, acoustic startle has a well-defined neural pathway that allows one to determine where these various forms of plasticity alter neural transmission so as to affect the behavior. Because of this, it should be possible to more fully determine the neural mechanisms that allow early life stressors to produce permanent changes in behavior. The neural pathway that mediates the acoustic startle reflex receives a direct input from the amygdala and the bed nucleus of the stria terminalis (BNST). Certain phenomena, such as the elevation of startle in the presence of a conditioned fear stimulus (the fear-potentiated startle effect), depend on projections from the central nucleus of the amygdala to the startle pathway. Other phenomena, such as the elevation of startle produced by unconditioned anxiogenic events such as exposure to a very bright light or intraventricular infusion of corticotropin releasing factor (CRF), depend on projections from the BNST to the startle pathway. Because repeated maternal separation leads to increased expression of corticotropin releasing hormone messenger RNA (CRF mRNA) in the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST), brain structures critically involved in fear, anxiety, and regulation of the HPA axis, Project 6 will evaluate whether postnatal environmental events such as handling and maternal separation will alter either the amplitude or plasticity of the startle reflex, and, if so, how these changes might relate to changes in the amygdala or BNST. In Aims 1-3 baseline startle amplitude, startle habituation, prepulse inhibition, fear- potentiated startle, and light-enhanced startle will be measured at various times in development and in adulthood in HMS 180, HMS 15 and AFR rats. These studies will assess whether early life stressors produce permanent changes in attention, anxiety or fear. In Aims 4 and 5 we will measure how CRF given intraventricularly or yohimbine given systemically, or directly into the bed nucleus of the stria terminalis will affect startle amplitude at various points in development and in adulthood in HMS180, HMS15 and AFR rats. These studies should provide a direct test of the behavioral consequences of a change in regulation of CRF or catecholamines flowing early life stressors.

The goals of the research proposed in this application are to study the neural circuitry and neurochemicals involved in positive and negative valence as measured by modulation of the acoustic startle reflex in rats. Our basis work in rodents on the circuitry of startle increased by negative valence, termed the fear-potentiated startle effect , has served as part of the basic science foundation in the CSEA for the study of startle modulation in humans. Thus far, however, our laboratory has not worked with positive valence in rats and how this might modulate startle amplitude. However, Dr. Michael Koch, one of the CSEA affiliate investigators, has now devised a method for quantifying positive valence measured as a decrease in startle amplitude when the reflex is elicited in the presence of a cue previously paired with food. He also has shown that dopamine in the nucleus accumbens is important for this pleasure-attenuated startle effect . This work now allows us to begin to use changes in rodent startle to delineate the circuitry involved in positive emotional valence similar to our work on the circuitry of negative emotional valence. Studies proposed in the current application will try to define in neural terms how negative valence leads to an elevation in startle amplitude and how positive valence leads to a decrease in startle amplitude. Double labeling studies will be used to determine whether different groups of cells in the basolateral amygdala project to the central nucleus of the amygdala vs. the nucleus accumbens. Retrograde tracing in combination with FOS-like immunocytochemistry will be used to determine whether cells in the basolateral amygdala that project to the central nucleus of the amygdala or cells in the central nucleus of the amygdala that project to the startle pathway are activated by lights paired with footshock, but not by lights paired with food. Similar studies will test whether cells in the basolateral amygdala that project to the nucleus accumbens are activated by lights paired with food, but not by lights paired with footshock. Based on recent data, we hypothesize that the intensity of both negative and positive valence, (i.e., the level of arousal) depends on the level of dopamine in either the amygdala or the nucleus accumbens during testing. Hence, we will also test the effects of local infusion of dopamine agonists into these brain regions on both fear-potentiated and pleasure-attenuated startle. Finally, we will test the effects of glutamate in the nucleus accumbens on both the acquisition and expression of pleasure- attenuated startle using local infusion of NMDA and AMPA agonists and antagonists in the nucleus accumbens and amygdala.

DESCRIPTION (adapted from applicant's abstract): Neural systems involved in fear and anxiety will be studied using changes in the amplitude of the acoustic startle response, a short-latency reflex that can be elicited in all mammals. Acoustic startle can be increased by presentation of a brief light previously paired with shock (fear-potentiated startle), by sustained exposure to the same light even without prior light-shock pairings (light-enhanced startle), or by intraventricular infusion of corticotropin releasing hormone (CRH-enhanced startle). The PI and colleagues found that lesions or chemical inactivation of the bed nucleus of the stria terminalis (BNST) blocked light-enhanced or CRH-enhanced startle but not fear-potentiated startle. Conversely, lesions or chemical inactivation of the central nucleus of the amygdala blocked fear-potentiated startle but had no effect on light-enhanced or CRH-enhanced startle. The proposed experiments will further characterize similarities and differences between these structures in stress-induced enhancement of the startle reflex. Studies will test whether there is additivity of fear-potentiated startle, light-enhanced startle, and CRH-enhanced startle and whether intraventricular or local infusion into the BNST of the CRH antagonist alpha-helical CRH9-41 will block light-enhanced startle. Induction of c-fos in the amygdala vs. the BNST during presentation of a conditioned fear context vs. an unconditioned anxiogenic context will be measured. Other studies will measure how activation of CRH receptors in the BNST may ultimately increase startle at the level of the brainstem. This will be done by first delineating the neural pathway linking the BNST to the nucleus reticularis pontis caudalis. Previously, these investigators found that if rats were first given extensive overtraining and then given amygdala lesions, relearning could occur with further training. Other studies will evaluate whether the BNST can 'take over' for the amygdala in fear-potentiated startle using lesion, chemical inactivation and IC-fosI methodologies.

DESCRIPTION (adapted from applicant's abstract): Neural systems involved in fear and anxiety will be studied using changes in the amplitude of the acoustic startle response, a short-latency reflex that can be elicited in all mammals. Acoustic startle can be increased by presentation of a brief light previously paired with shock (fear-potentiated startle), by sustained exposure to the same light even without prior light-shock pairings (light-enhanced startle), or by intraventricular infusion of corticotropin releasing hormone (CRH-enhanced startle). The PI and colleagues found that lesions or chemical inactivation of the bed nucleus of the stria terminalis (BNST) blocked light-enhanced or CRH-enhanced startle but not fear-potentiated startle. Conversely, lesions or chemical inactivation of the central nucleus of the amygdala blocked fear-potentiated startle but had no effect on light-enhanced or CRH-enhanced startle. The proposed experiments will further characterize similarities and differences between these structures in stress-induced enhancement of the startle reflex. Studies will test whether there is additivity of fear-potentiated startle, light-enhanced startle, and CRH-enhanced startle and whether intraventricular or local infusion into the BNST of the CRH antagonist alpha-helical CRH9-41 will block light-enhanced startle. Induction of c-fos in the amygdala vs. the BNST during presentation of a conditioned fear context vs. an unconditioned anxiogenic context will be measured. Other studies will measure how activation of CRH receptors in the BNST may ultimately increase startle at the level of the brainstem. This will be done by first delineating the neural pathway linking the BNST to the nucleus reticularis pontis caudalis. Previously, these investigators found that if rats were first given extensive overtraining and then given amygdala lesions, relearning could occur with further training. Other studies will evaluate whether the BNST can 'take over' for the amygdala in fear-potentiated startle using lesion, chemical inactivation and IC-fosI methodologies.

Five compounds (the CRH1 receptor antagonist SB723620, the NK1 antagonist G597599, the SSRI/5HT2A agonist Vilazodone, the antidepressant 8-hydroxy-bupropion, and the type 4 phosphodiesterase inhbitor SB207499), provided by GlaxoSmithKline as part of The Emory-GSK-NIMH Collaborative Mood Disorders Initiative, will be evaluated for anti-fear and anxiolytic activity using fear-potentiated, light-enhanced, and CRH-enhanced startle paradigms (increased startle in the presence of cues that predict shock, during sustained illumination, or following i.c.v. corticotropin-releasing hormone infusions, respectively). Whereas light- and CRH-enhanced startle (which are more akin to anxiety than to fear) are mediated by circuitry that includes the bed nucleus of the stria terminalis (BNST) but not the central nucleus of the amygdala (CeA), fear-potentiated startle is mediated by circuitry that includes the CeA but not the BNST. A central goal of the proposed studies will be to identify differing pharmacological vulnerabilities associated with BNST (anxiety) versus CeA (fear) dependent behaviors. In humans, anxiety disorders are more prevalent in women than in men and, in rats, light-enhanced startle is more robust in females than in males. Interestingly, the BNST is sexually dimorphic in both species. Thus, a second goal will be to compare the influence of gender on BNST- versus CeA-dependent responses, and to evaluate gender influences on drug responses in each model. These same compounds will be evaluated in other laboratories (separate applications) using different behavioral models and non-behavioral assays. It is hoped that this integrated effort will foster new approaches for the rapid evaluation of novel compounds with potential clinical utility.

The major hypothesis that guides studies in this project is that early life stress leads to a reduction in the ability to respond to safety signals. Despite substantial anecdotal evidence in support of this hypothesis, direct tests in humans or animals, using well-controlled behavioral paradigms, have been limited. In both rodents and humans, we have developed a discrimination procedure, referred to as AX+, BX-, that allows for the independent evaluation of excitation and inhibition of fear conditioning measured with the acoustic startle reflex. For example, AX+ could mean that two cues, such as a light and air flow created by a quiet fan presented together, are followed by shock. If so, then BX- would mean that a second cue, such as a tone and the fan, when presented together, would not be followed by shock. In this procedure, A elicits fear, whereas B is a safety signal, because it predicts that no shock will occur. Following such training, one can test whether B, the safety cue, will reduce fear to A by presenting A and B together. We have found in both rats and humans that B comes to inhibit fear following such training and this effect is not due to external inhibition. In humans, we will evaluate effects of early life stress in this paradigm using patients with or without major depressive disorders (MDD), with or without early life stress, as well as patients with social phobia with or without early life stress. In male and female rhesus monkeys we will set up this same procedure and then evaluate effects of early life stress using normal rearing conditions or rearing with repeated maternal separation, our model of early life stress. In addition, we will use another test we believe is a more a measure of anxiety than stimulus specific fear. Both humans and monkeys startle more in the dark than in the light. In rats, a nocturnal animal, we find they startle more in the light than in the dark, and this effect depends on the bed nucleus of the stria terminalis and not the central nucleus of the amygdala. A similar effect is found in rats when startle is enhanced after intraventricular infusion of CRF. Based on the fact that dark or light-enhanced startle, as well as CRF-enhanced startle in rats, involves unpredictable threat, we have suggested these effects may be more akin to anxiety vs. stimulus specific fear. Hence, we will evaluate dark-enhanced startle in these same patient groups, as well as in male and female rhesus monkeys, following normal rearing or rearing with early life stress.

[unreadable] DESCRIPTION (provided by applicant): Disorders of anxiety and fear dysregulation are highly prevalent. It is known that these disorders affect women approximately two times more than they affect men, and this disparity is thought to be related to hormone regulation, yet the underlying mechanism of hormone regulation of the fear circuit in either sex remains unknown. The purpose of this Exploratory/ Developmental Grant proposal, in response to PA-03-169, is to develop a more informative and accurate method to determine the effect that gonadal hormones have on fear in both male and female animals. Here, we will employ a new adapted version of the discrimination training paradigm AX+.BX-, along with the use of highly specific estrogen receptor agonists and an androgen receptor-specific testosterone metabolite, to measure the effect that activation of specific gonadal steroid receptors have on both the activation and inhibition of fear. The specific aims of this project are: 1. to validate the use of the AX+, BX- discrimination learning paradigm as a model for examining hormone modulation of fear acquisition, expression and inhibition. 2. to use this model to examine: the effect of gonadal steroids on fear acquisition, and the effect of gonadal steroids on fear expression, in gonadectomized male and female rats. Data from these studies will begin to elucidate the specific effects that these steroid hormones have on very particular aspects of emotional memory formation and expression in general, and moreover, these studies will help to clarify the contribution that the various gonadal steroids make to fear learning in each individual sex . Results from these investigations would not only speak to the development, and expression of emotional learning within each individual sex, but will also begin to address the gender disparity in the occurrence of PTSD, depression and anxiety illness, and panic disorder. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic 03-MH-101* Biomarkers in mental disorders. The major hypothesis is that post-traumatic stress disorder (PTSD) leads to a reduction in the ability to respond to safety signals. For example, central to the clinical problem of PTSD, which has a ~90% comorbidity with major depressive disorder in patients who had early life stress, is the inability of these patients to inhibit their fear to stimuli reminiscent of their traumatic experience, even in safe conditions. In fact, we now have evidence that the inability to inhibit fear may be a unique biomarker of PTSD. Thus the sound of a car back firing or the smell of sulfur can lead to an intense fear reaction in Viet Nam veterans many years after combat, even in the safety of their own home. Despite substantial anecdotal evidence in support of this hypothesis, direct tests in humans or animals, using well-controlled behavioral paradigms, have been limited. We have now developed in rats, rhesus monkeys and humans an objective measure of safety signal learning and expression using identical paradigms and the acoustic startle reflex in all species. Three cues are used in the form AX+/BX-, where cues A and X in compound (e.g. a light and air blowing from a quiet fan) are paired with an aversive event (+), and a new cue B (e.g. a tone) and the same cue X signal no aversive event (-). Cue A becomes excitatory as the subject learns that A and X presented together predict the US. Cue B becomes inhibitory because B presented with X predicts "safety" from the US. In a critical subsequent transfer test trial, presentation of A and B together (AB) results in a reduced fear response compared with the response to A. In three independent studies PTSD patients sometimes could discriminate AX from BX while others could not. However, in all three studies PTSD patients did not inhibit fear to A on AB test trials. Because PTSD has high comorbidity with early life stress and major depression other studies in our group have now shown that patients with early life stress only or major depression only have normal safety signal learning and only patients with PTSD or PTSD with depression fail in show safety signal learning or expression. Thus, we believe we have an objective way to measure a major biomarker of PTSD. In a prior grant we tested 6 young adult rhesus monkeys, 3 with typical social and mother-infant interactions (controls) and 3 separated from their mothers for variable periods of time each day when they were infants (maternally-separated). 3 control and 1 maternally-separated showed successful AX+, BX- discrimination and all of them had less startle in the presence of AB, vs. A. The other two monkeys, both maternally separated, were never able to discriminate between A+ and B- because they continued to be fearful of both A and B (i.e. no safety signal learning following early life stress). The one maternally separated monkey that learned very well suggests resilience to early life stress in this animal. Currently we are testing the second cohort of 6 monkeys which should be completed in the next 2 months so we will have 12 monkeys in total, 6 control and 6 maternally-separated. As part of a long standing ongoing program at the Yerkes National Primate Center studying the effects of early life stress in rhesus monkeys, a great deal of data on another 36 animals in addition to the 12 we will have tested has been carried out. This includes neuroendrocrine, behavioral, autonomic, neuroimaging and genetic data, as well as banked DNA from all the animals available for further genotyping. We believe this is a tremendous resource that should not be wasted. If we cannot pay the per diems on these animals they will have to be released and we will no longer have access to them. Hence, we propose to finish testing these 36 additional monkeys so that our final sample will be 48 monkeys. This will allow a thorough analysis of how early life stress affects safety signal learning, autonomic, neuroimaging and neuroendrocrine functioning and whether some of these effects can be associated with various genetic variants. It may also detect monkeys that are resilient to early life stress so the measures already collected on them can be correlated with this behavioral measure of resilience. This is a "shovel ready" project. By hiring two new, full time technicians devoted entirely to this project we can get this work done in 2 years and believe it will be money well spent by NIMH. PUBLIC HEALTH RELEVANCE: The major hypothesis is that post-traumatic stress disorder (PTSD) leads to a reduction in the ability to respond to safety signals. We have evidence for this in humans using an objective test of fear inhibition. In this grant we want to evaluate whether early life stress in rhesus monkeys will associated with this deficit using the same objective measure of fear inhibition we also have developed in monkeys. If so would provide the first model of PTSD in rhesus monkeys.

DESCRIPTION (provided by applicant): One of the core symptoms of many anxiety disorders, especially Post-Traumatic Stress Disorder (PTSD), is an inability for fear safe in situations where healthy individuals do feel safe. Thus, animal models of fear conditioning and fear inhibition offer useful tools to determine how these learned fears are diminished or inhibited. We have developed a new paradigm in rodents referred to as AX?, where cues A and X in compound signal an aversive event and cues B and X in compound signal no aversive event (safety). In a critical subsequent transfer test trial, presentation of A and B together (AB) results in a reduced fear response compared with the response to A. Other tests have shown this is bona fide conditioning inhibition and not due to external inhibition. We have now found this to be true in humans and rhesus monkeys where we see transfer of inhibition on AB test trials, in contrast to prior failures to see transfer in humans using the typical conditioned inhibition paradigm. Most importantly, in three independent groups of patients with PTSD we see some discrimination between AX and BX but no transfer on the critical AB test trial, thus detecting a core symptom in PTSD. The R21 phase of this application is to modify our current AX? paradigm into a "working memory" test, which will allow the same monkeys to be tested repeatedly in this new paradigm using sets of pictures as stimuli instead of lights and tones. We will then evaluate in adult monkeys that have sustained neurotoxic lesions of orbital frontal areas 14/25 vs.11/13 vs. 12 in safety signal learning and expression. As a positive control, we will also test these monkeys in reversal learning and reinforce devaluation that is known to be compromised by damage to one or more of these lesions. If successful, the R33 phase will begin to evaluate the development of safety signal learning from year 1 to year 3, a time period when pronounced developmental changes occur in these orbital frontal areas. We believe a "working memory" version of this measure of safety signal learning in which the same animal can be tested repeatedly will provide a major new paradigm to study safety signal learning in psychiatric disorders and to eventually lead to new and better treatments for people with anxiety disorders. This project is clinically relevant because: (1) many emotional disorders in humans, such as anxiety, phobias and post-traumatic stress disorders, are characterized by a resistance to extinguish learned emotional reactions to anxiogenic stimuli or contextual information associated with these anxiogenic stimuli, (2) learned fear in early infancy has strong resistance to extinction that yield anxiety disorders later in life and (3) anxiety disorders have also been reported in several developmental neuropsychiatric disorders, such as autism and schizophrenia, as well as following pediatric traumatic brain injury and early life stress.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. During the reporting period, in other work we found that patients with PTSD do not show normal conditioned inhibition which means we have an objective measure of one of the core symptoms of PTSD, namely the inability to feel safe. We have set up the AX+, BX- in rhesus monkeys at Yerkes and have found good safety signal learning in 3 control and 1 maternally separated monkeys. In the other two separated monkeys they failed show inhibitory learning. This work will now continue in 34 more monkeys funded by a Challenge grant for which I hired a new non-human primate technician, to do this work. She has now completed testing on 12 more monkeys and is almost done with another 6 monkeys.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. During the reporting period, subjects with PTSD failed to inhibit fear and this was correlated with baseline and post-dexamethsone ACTH in PTSD subjects. These results suggest that impaired fear inhibition and associated alterations in HPA feedback may reflect amygdala hyperactivity in subjects with PTSD. FUNDING SOURCE: This grant has now terminated.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. During the reporting period, we have begun to evaluate how serotonin reuptake inhibitors and GSK compounds will affect our measures of fear vs. anxiety and whether over expression of CRF in CRF neurons in the amygdala will provide a novel model of anxiety.

DESCRIPTION (provided by applicant): While many engineering schools and specifically biomedical engineering departments offer summer research experiences whereby students can gain exposure to engineering approaches to medicine, there is no program focused on pediatric diseases. As our ability to detect and treat pediatric congenital and acquired diseases improves, so do opportunities to better manage, treat, and cure these diseases. While exciting, this is an area in need of multidisciplinary research across several disciplines. Emory, Georgia Tech and Children's Healthcare of Atlanta have formed a partnership with this as the sole purpose. The Centers for Pediatric Nanomedicine and Pediatric Innovation are focused on applying engineering principles to addressing pediatric diseases. This program is well supported by all 3 institutes, has maturing projects that are nearing independent funding, and truly extends from Bench-to-Bassinet. While there is much excitement, there is also great opportunity. The next step is to help train the next generation of scientists that can carry this innovative discipline forward. We propose to create a 10-week summer program for 10 undergraduate students to perform interdisciplinary research focused on pediatric nanomedicine. We will integrate with the existing Emory SURE program, reach out to local (and national) universities serving underrepresented minorities, and use the well-developed infrastructure to not only train the students scientifically, but to also help them understand clinical needs, ethics in research and medicine, and career development opportunities.

DESCRIPTION (provided by applicant): Alzheimer's disease currently affects an estimated 5.4 million Americans, and this number is expected to grow significantly in the coming decade. A critical and ultimate goal of Alzheimer's disease research is to improve methods of early diagnosis, so that patients can be identified sooner and, therefore, obtain greater advantage from available therapies. A second critical and more immediate goal is to develop better ways to detect individuals who are at risk or who are already on the trajectory for Alzheimer's but have not yet developed clinical symptoms, so that pharmaceutical companies and researchers can populate their clinical trials with appropriate individuals. A lack of early markers or predictors f impending disease represents a significant roadblock in the ability to develop new drugs that might treat or even cure Alzheimer's disease. Neurotrack Technologies, Inc. is a Palo Alto CA-based startup formed around a suite of behavioral and software technologies designed to facilitate the detection and prediction of Alzheimer's both at home and in the clinical setting. Th centerpiece of Neurotrack is a behavioral assay that uses eye-tracking technology along with data analysis algorithms to assess recognition memory. It is based on decades of research in nonhuman primates and in human amnesic patients by scientists and physicians at Emory University, who have developed a behavioral task that is highly sensitive to memory impairment and which has shown promise both in the early diagnosis of Alzheimer's disease and in the ability to predict the onset of Alzheimer's disease several years before clinical symptoms occur. Our behavioral test assesses recognition memory, a form of memory that critically depends on the integrity of the hippocampal region of the brain, one of the first areas to undergo morphological change in the course of Alzheimer's. Published results from our 5-year NIH-funded longitudinal study revealed that performance on this test was highly accurate in predicting cognitive decline. Neurotrack's technology has the potential to give pharmaceutical companies and research organizations the tools they need to recruit truly appropriate candidates for clinical trials and more effectively measure drug efficacy, speeding up drug discovery and development. This proposal consists of three aims: 1) to develop and optimize a commercial prototype for administering our test, 2) to beta test and refine this prototype with a small group of patients, and 3) to conduct an initial feasibility study comparing our new commercial version of the test to the established predictive assays currently available to researchers and clinicians. These improvements will advance the technology into a viable first generation commercial prototype in preparation for a large, multi-site clinical trial.

? DESCRIPTION (provided by applicant): Adverse remodeling of the myocardium after myocardial infarction speeds progression to heart failure. Some of the major shortcomings in cell therapy are the growth and differentiation of implanted cells. In patients, cells are injectedto an area comprised mostly of noncontractile collagen, far different from their native microenvironment. This study aims to determine whether a biodegradable, modular hydrogel can be used to implant cells and direct their behavior. Dr. Andres Garcia has developed a biocompatible, injectable, smart delivery system based on polyethylene glycol. This material forms a stable hydrogel and degrades as matrix metalloproteases are released. Our preliminary data suggest that adult c-kit positive cardiac progenitor cells (CPCs) currently in human clinical trials, migrate into hydrogels containing growth factors, but the numbers of these cells in vivo is not known. Preliminary data from that Davis laboratory suggests that these cells express the Notch receptor and respond to Jagged- induced activation by increasing differentiation. In a separate system, a Notch-activating peptide termed JAG-1 has been immobilized and CPCs cultured within demonstrate increased expression of cardiac, endothelial, and smooth muscle genes. Completion of the proposed studies will demonstrate a potential role for PEG hydrogel- mediated delivery of CPCs to the post-infarct myocardium for the purpose of enhancing implanted progenitor cell therapy.

Abstract Adverse remodeling of the myocardium after myocardial infarction speeds progression to heart failure. While cell therapy has been met with great enthusiasm, there are numerous shortcomings that prevent long-term functional improvements. Moreover, these cells come from diseased individuals and immunogenicity limits most studies to autologous therapy. Finally, it is widely believed that the main effect of cell therapy is mediated by paracrine effectors and not the cells themselves. Our published studies demonstrate that rat cardiac progenitor cells (CPCs) make exosomes, and when cells are exposed to hypoxia, exosomes are reparative following infarction. Moreover, we were able to demonstrate potential pathways using computational and systems biology. Recently, we have been able to isolate CPCs from pediatric biopsies and show age-related changes in cell therapy in a model of heart failure. Preliminary studies demonstrate that these exosomes also vary in function by age and hypoxia. Therefore, the objective of this proposal is to examine the protective/regenerative capacity of human pediatric CPC exosomes in rat models of ischemia-reperfusion. Additionally, with a large number of patient samples from a wide variety of children, we can perform multivariate analysis to examine the factors that affect various in vivo mechanisms. Factors include patient age, gender, and exposure to hypoxic conditions. Finally, we will expand our model by looking at reparative exosomes from other cell types, CD34+ cells, and examine whether mechanisms of hypoxic exosome function are conserved among different cells. Completion of the proposed studies will determine whether hypoxic exosomes are a beneficial therapy for ischemia-reperfusion injury, as well as determine potential patient factors that contribute to these responses.

Much progress has been made in the last 30 years to improve survival of children with CHD, but what was once a fatal condition is now a manageable long-term disease. Of these, children with Hypoplastic Left Heart Syndrome face the worst outcomes as they are left with a single ventricle (the right ventricle) that ultimately fails. These children are either left with long-term morbidity such as poor exercise tolerance, decreased executive function, and poor social skills. At the worst, they are placed on the transplant list where short-term survival in the highest need cohort is 50%, and grafts have a maximal longevity of roughly 12 years before needing a new heart. The goal of this study is to provide FDA-enabling data for a combination therapy of two Phase 1 FDA approved treatments; cardiac progenitor cells and decellularized porcine cardiac extracellular matrix. Cell therapy is promising in children, but similar to adult studies the cells do not persist for long and are transplanted to a diseased microenvironment. In 3 separate aims, we will examine the efficacy of this combined therapy in both immunocompromised and immune-competent rats, followed by large animal pig studies. These data will not only provide critical data for an FDA IND, but will also shed light on potential physiological mechanisms by which these therapies act. With congenital heart disease occurring in nearly 1% of all births and representing the most common birth defect, the potential impact of this study cannot be understated. Information gleaned from this study will not only improve the lives of nearly 40,000 children per year and their families, but it would also generate greater societal impact in terms of economic and educational outcomes. With both individual therapies under early clinical trials in humans, this proposal is the next logical step toward enhancing pediatric cell therapy.