Robert M. Sapolsky - US grants

In the rat, the hippocampus loses neurons with age, and cumulative exposure to glucocorticoids (GCs), the adrenocortical stress hormones, plays a major role in such exposure. My prior work suggests that GCs disrupt energy metabolism and thus compromise the ability of neurons to survive metabolic challenges. As evidence, varied insults that damage the hippocampus are more toxic in rates exposed to elevated GC concentrations and are less so in adrenalectomized rates. Such insults include the excittoxin kainic acid, the antimetabolite 3- acetylpyridine and hypoxia-ischemia. My proposed studies will 1) determine whether this model of GC- induced hippocampal damage applies to senescent neuron loss, 2) study the cellular mechanisms of this GC toxicity, and 3) examine the neuroendocrine consequences of the neuron loss. Part I: I will examine whether additional hippocampal insults also have their toxicities modulated by GC milieu. These will include epileptogenic excitotoxins and heavy metals. Since these acute insults may be of limited relevance to the gradual neuron loss of aging, I will then determine whether milder chronic insults also have their toxicities modulated by GCs. Part II: I will determine whether GCs are the sole damaging agents. I will expand my prior work with hippocampal neuron cultures to examine whether GCs endanger cells directly. I will study the steroidal and tissue specificity of the phenomenon, as well as identify the mediating receptors. Part III: I will study whether GCs exacerbate abnormal cellular parameters that accompany and may mediate neuron death. These will include excitatory neurotransmitter release, calcium influxes and lactic acidosis. Part IV: I will determine whether the aged hippocampus is preferentially sensitive to the synergy between GCs and varied insults, and whether GCs produce more extreme changes in the previously mentioned cellular parameters in the aged hippocampus. Part V: The hippocampus is a mediator the inhibitory negative feedback actions of GCs upon the adrenocortical axis. Using hypophysial portal cannulation methods, I will examine whether the hippocampal neuron loss of aging is associated with hypersecretion of corticotropic releasing factor or related peptides. If so, this would explain the GC hypersecretion of the aged rat which, in turn, further damages the hippocampus and impairs its function.

This action is to award funds for a Presidential Young Investigator. Dr. Robert Sapolsky has already embarked on a brilliant career in neuroendocrinology research. His work focuses on three basic questions: 1) What are the neuroendocrine abnormalities that underly syndromes of glucocorticoid hypersecretion; 2) What are the consequences of excessive exposure to glucocorticoids, in particular, what role do glucocorticoids play in mediating neuron death during aging and following neuropathologic insults; and 3) What are the cellular mechanisms by which glucocorticoids are neurotoxic? Dr. Sapolsky has already studied negative-feedback regulation of glucocorticoid secretion, focusing on the role of the hippocampus as an inhibitory negative-feedback brake upon adrenocorticoid activity, its role in terminating glucocorticoid secretion at the end of stress and the failure of this type of regulation in aged rats. He has refined and originated methods for quantitative measures for steroid receptors in the brain in order to examine the relationship between occupancy of hippocampal glucocorticoid receptors and the initiation of inhibitory hippocampal signals to the hypothalamus.

In the rat, the hippocampus loses neurons with age, and cumulative exposure to glucocorticoids (GCs), the adrenocortical stress hormones, plays a major role in such exposure. My prior work suggests that GCs disrupt energy metabolism and thus compromise the ability of neurons to survive metabolic challenges. As evidence, varied insults that damage the hippocampus are more toxic in rates exposed to elevated GC concentrations and are less so in adrenalectomized rates. Such insults include the excittoxin kainic acid, the antimetabolite 3- acetylpyridine and hypoxia-ischemia. My proposed studies will 1) determine whether this model of GC- induced hippocampal damage applies to senescent neuron loss, 2) study the cellular mechanisms of this GC toxicity, and 3) examine the neuroendocrine consequences of the neuron loss. Part I: I will examine whether additional hippocampal insults also have their toxicities modulated by GC milieu. These will include epileptogenic excitotoxins and heavy metals. Since these acute insults may be of limited relevance to the gradual neuron loss of aging, I will then determine whether milder chronic insults also have their toxicities modulated by GCs. Part II: I will determine whether GCs are the sole damaging agents. I will expand my prior work with hippocampal neuron cultures to examine whether GCs endanger cells directly. I will study the steroidal and tissue specificity of the phenomenon, as well as identify the mediating receptors. Part III: I will study whether GCs exacerbate abnormal cellular parameters that accompany and may mediate neuron death. These will include excitatory neurotransmitter release, calcium influxes and lactic acidosis. Part IV: I will determine whether the aged hippocampus is preferentially sensitive to the synergy between GCs and varied insults, and whether GCs produce more extreme changes in the previously mentioned cellular parameters in the aged hippocampus. Part V: The hippocampus is a mediator the inhibitory negative feedback actions of GCs upon the adrenocortical axis. Using hypophysial portal cannulation methods, I will examine whether the hippocampal neuron loss of aging is associated with hypersecretion of corticotropic releasing factor or related peptides. If so, this would explain the GC hypersecretion of the aged rat which, in turn, further damages the hippocampus and impairs its function.

In the rat, the aging hippocampus loses neurons, and cumulative exposure to glucocorticoids (GCs), the adrenal stress hormone, plays a major role in such loss; it is prevented by decreasing lifetime GC exposure, while GCs and/or stress accelerate senescent neuron loss. My work suggests that GCs disrupt energy metabolism and comprise the ability of neurons to survive metabolic challenges; thus, excitotoxins, antimetabolites and hypoxia-ischemia are all more damaging to the hippocampus in rats exposed to elevated GC concentrations and are less so in adrenalectomized rats. A similar exacerbation of toxicity of these insults by GCs occurs in primary hippocampal cultures and is GC receptor-mediated. These observations have lead to the development of pharmacological and behavioral interventions that protect the hippocampus from GCs after such insults and during aging, and decrease cognitive impairments arising from hippocampal damage. Of human relevance, we find that chronic stress also damages the primate hippocampus. Despite these advances, it is still not clear how GCs endanger hippocampal neurons. Part I: We have found that GCs inhibit 30% of glucose transport into cultured hippocampal neurons and glia. We will 1) test whether hippocampal glucose utilization in vivo is sensitive to physiological changes in GC concentrations; 2) determine the molecular mechanisms underlying the effect; 3) assess the energetic consequences of a 30% inhibition of glucose transport for hippocampal neurons by measuring phosphocreatine in GC- treated cultures under basal and metabolically-challenged conditions; 4) test whether the elevated GC concentrations typical of aged rats inhibits hippocampal glucose utilization. Part II: We have found that GCs increase the activity of glutamine synthetase in the adult hippocampus. This astrocytic enzyme is the rate-limiting step in a shuttle that provides glutamine to neurons for conversion to the excitotoxin glutamate. 1) Is this GC effect physiological? 2) Do the elevated GC concentrations of the aged rat cause an increase in glutamine synthetase activity? Part III: Glutamate (and other excitatory amino acids -- EAAs) appears to mediate various neurological insults to the hippocampus. Both energy depletion and increased glutamine synthetase activity enhance extracellular EAA concentrations. Thus GCs, via their effects on glucose transport and glutamine synthetase, might do the same; our data suggest that is the case. 1) Do GCs increase extracellular EAA concentrations basally or during metabolic challenges? 2) If so, do GCs enhance EAA release or impair their uptake, and if the latter, is this a neuronal or glial effect? 3) Are these effects attributable to GC effects on glucose uptake and/or on glutamine synthetase? 4) Is this GC effect relevant to the aging hippocampus? Part IV: EAAs ultimately damage neurons by increasing free cytosolic calcium concentrations. Our data show that GCs do the same. 1) Does this occur both basally and during metabolic challenges? 2) Do GCs enhance calcium influx or release from intracellular stores, or diminish efflux? 3) Do these GC effects arise from their inhibition of glucose transport? 4) Do GCs effect calcium trafficking in the aged hippocampus?

In the rat, the aging hippocampus loses neurons, and cumulative exposure to glucocorticoids (GCs), the adrenal stress hormone, plays a major role in such loss; it is prevented by decreasing lifetime GC exposure, while GCs and/or stress accelerate senescent neuron loss. My work suggests that GCs disrupt energy metabolism and comprise the ability of neurons to survive metabolic challenges; thus, excitotoxins, antimetabolites and hypoxia-ischemia are all more damaging to the hippocampus in rats exposed to elevated GC concentrations and are less so in adrenalectomized rats. A similar exacerbation of toxicity of these insults by GCs occurs in primary hippocampal cultures and is GC receptor-mediated. These observations have lead to the development of pharmacological and behavioral interventions that protect the hippocampus from GCs after such insults and during aging, and decrease cognitive impairments arising from hippocampal damage. Of human relevance, we find that chronic stress also damages the primate hippocampus. Despite these advances, it is still not clear how GCs endanger hippocampal neurons. Part I: We have found that GCs inhibit 30% of glucose transport into cultured hippocampal neurons and glia. We will 1) test whether hippocampal glucose utilization in vivo is sensitive to physiological changes in GC concentrations; 2) determine the molecular mechanisms underlying the effect; 3) assess the energetic consequences of a 30% inhibition of glucose transport for hippocampal neurons by measuring phosphocreatine in GC- treated cultures under basal and metabolically-challenged conditions; 4) test whether the elevated GC concentrations typical of aged rats inhibits hippocampal glucose utilization. Part II: We have found that GCs increase the activity of glutamine synthetase in the adult hippocampus. This astrocytic enzyme is the rate-limiting step in a shuttle that provides glutamine to neurons for conversion to the excitotoxin glutamate. 1) Is this GC effect physiological? 2) Do the elevated GC concentrations of the aged rat cause an increase in glutamine synthetase activity? Part III: Glutamate (and other excitatory amino acids -- EAAs) appears to mediate various neurological insults to the hippocampus. Both energy depletion and increased glutamine synthetase activity enhance extracellular EAA concentrations. Thus GCs, via their effects on glucose transport and glutamine synthetase, might do the same; our data suggest that is the case. 1) Do GCs increase extracellular EAA concentrations basally or during metabolic challenges? 2) If so, do GCs enhance EAA release or impair their uptake, and if the latter, is this a neuronal or glial effect? 3) Are these effects attributable to GC effects on glucose uptake and/or on glutamine synthetase? 4) Is this GC effect relevant to the aging hippocampus? Part IV: EAAs ultimately damage neurons by increasing free cytosolic calcium concentrations. Our data show that GCs do the same. 1) Does this occur both basally and during metabolic challenges? 2) Do GCs enhance calcium influx or release from intracellular stores, or diminish efflux? 3) Do these GC effects arise from their inhibition of glucose transport? 4) Do GCs effect calcium trafficking in the aged hippocampus?

In the rat, the aging hippocampus loses neurons, and cumulative exposure to glucocorticoids (GCs), the adrenal stress hormone, plays a major role in such loss; it is prevented by decreasing lifetime GC exposure, while GCs and/or stress accelerate senescent neuron loss. My work suggests that GCs disrupt energy metabolism and comprise the ability of neurons to survive metabolic challenges; thus, excitotoxins, antimetabolites and hypoxia-ischemia are all more damaging to the hippocampus in rats exposed to elevated GC concentrations and are less so in adrenalectomized rats. A similar exacerbation of toxicity of these insults by GCs occurs in primary hippocampal cultures and is GC receptor-mediated. These observations have lead to the development of pharmacological and behavioral interventions that protect the hippocampus from GCs after such insults and during aging, and decrease cognitive impairments arising from hippocampal damage. Of human relevance, we find that chronic stress also damages the primate hippocampus. Despite these advances, it is still not clear how GCs endanger hippocampal neurons. Part I: We have found that GCs inhibit 30% of glucose transport into cultured hippocampal neurons and glia. We will 1) test whether hippocampal glucose utilization in vivo is sensitive to physiological changes in GC concentrations; 2) determine the molecular mechanisms underlying the effect; 3) assess the energetic consequences of a 30% inhibition of glucose transport for hippocampal neurons by measuring phosphocreatine in GC- treated cultures under basal and metabolically-challenged conditions; 4) test whether the elevated GC concentrations typical of aged rats inhibits hippocampal glucose utilization. Part II: We have found that GCs increase the activity of glutamine synthetase in the adult hippocampus. This astrocytic enzyme is the rate-limiting step in a shuttle that provides glutamine to neurons for conversion to the excitotoxin glutamate. 1) Is this GC effect physiological? 2) Do the elevated GC concentrations of the aged rat cause an increase in glutamine synthetase activity? Part III: Glutamate (and other excitatory amino acids -- EAAs) appears to mediate various neurological insults to the hippocampus. Both energy depletion and increased glutamine synthetase activity enhance extracellular EAA concentrations. Thus GCs, via their effects on glucose transport and glutamine synthetase, might do the same; our data suggest that is the case. 1) Do GCs increase extracellular EAA concentrations basally or during metabolic challenges? 2) If so, do GCs enhance EAA release or impair their uptake, and if the latter, is this a neuronal or glial effect? 3) Are these effects attributable to GC effects on glucose uptake and/or on glutamine synthetase? 4) Is this GC effect relevant to the aging hippocampus? Part IV: EAAs ultimately damage neurons by increasing free cytosolic calcium concentrations. Our data show that GCs do the same. 1) Does this occur both basally and during metabolic challenges? 2) Do GCs enhance calcium influx or release from intracellular stores, or diminish efflux? 3) Do these GC effects arise from their inhibition of glucose transport? 4) Do GCs effect calcium trafficking in the aged hippocampus?

It is now believed that the neurobehavioral and neuropathologic indices of AIDS Dementia Complex can arise directly from HIV infection of the brain. Specifically, HIV infection of macrophages and microglia is thought to lead to the release of a neuroendangering factor(s). Attention has focused on the role of the HIV glycoprotein gp120 in this picture; gp120 can damage cultured neurons via indirect activation of a cascade involving glutamatergic synapses, the NMDA receptor and cytosolic calcium mobilization. Furthermore, our work indicate that gp120 can suppress metabolism in neuronal cultures, a notable finding given that the neuropathology of HIV infection appears to be far more about neuronal dysfunction than about neuron death. The proposed studies test the hypothesis that glucocorticoids (GCs), the adrenal steroids released during stress, can worsen the deleterious effects of gp120 in the rat brain. GCs can damage neurons in the hippocampus and impair the capacity of hippocampal, cortical and striatal neurons to survive necrotic insults. This GC-induced endangerment is energetic in nature, probably arising from GCs inhibition of glucose uptake in the brain. As a result, neurons are less capable of the costly tasks of controlling the waves of potentially lethal glutamate and calcium loosened during necrotic insults, thereby exacerbating damage. In testing whether GCs augment the damaging effects of gp120, we will determine a) whether GCs increase gp120-induced calcium mobilization; b) if gp120 causes calcium-dependent cytoskeletal proteolysis and oxidative damaged and, if so, if this is worsened by GCs; c) if GCs exacerbate the suppressive effects of gp120 on metabolism or gp120-induced declines in ATP and phosphocreatine content; e) if GCs increase the toxicity of gp120. Studies will be carried out in tissue slices from cortex, striatum and hippocampus, and from cortical, striatal and hippocampal primary cultures. The effects of both endogenous and synthetic GCs will be tested, as well as of stress itself (in rats from whom slices are generated). We will also test whether any GC effects are reversible with energy supplementation, as with other instances of GC neuroendangerment. GCs are administered in megadose quantities to control the Pneumocystis carinii pneumonia of HIV infection; it seems essential to determine the neurobehavioral and neuropathologic consequences of this. That, coupled with the possible implications of the stressfulness of HIV infection prompt the proposed studies.

The vector core will produce herpes simplex virus (HSV)-based amplicon and retroviral vectors to be used in Projects by Giffard and Steinberg. Vectors will be produced expressing the genes for CuZn-superoxide dismutase (SOD1), manganese SOD (SOD2), glutathione peroxidase (GPX), combinations of the antioxidant genes, p35 and crmA and bcl-2. All the vectors will be "bicistronic," expressing both the gene under investigation and a reporter gene. For each experimental vector, the cognate control vector will contain the gene in question with a stop codon inserted in its center, insuring non-expression. Retroviral vectors to express SOD1, SOD2, catalase, GPx and combinations of the antioxidant genes will be produced. Control vectors will express either the reporter gene beta-galactosidase or a stop codon version of the gene of interest. The continuing purposes of the Vector Core are to ensure a constant supply of vectors for the research groups, insure quality control in the production of the vectors, and to troubleshoot problems encountered in the use of the vectors.

The protective potential of three groups of genes will be studied in ischemia-like injury of brain injury of brain cells from cortex, hippocampus, and striatum, in primary culture. First an antioxidant strategy will be tested by over-expressing CuZn superoxide dismutase (SOD1) using herpes virus and adenoviral vectors to achieve rapid expression in neurons and astrocytes, and retroviral vectors for prolonged, stable expression in astrocytes. Whether acute expression can provide protection will be tested. If this is not protective, the effect of prolonged stable expression and the use of bicistronic vectors, to rapidly express both SOD and a downstream antioxidant vectors, to rapidly express both SOD and a downstream antioxidant enzyme, will be tested. We will test for protective effects, as well as for induction of other antioxidant enzymes. Whether protection correlates with induction of other antioxidant enzymes will be determined. Under conditions where protections seen the extent of oxygen radical production, lipid peroxidation and changes in level of glutathione will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined, as well as the time window in which expression can still protect, since it is important to develop therapeutic strategies that are effective after insults. Second, we will study the ability of Bcl-2 expression to protect in the same injury paradigms. Oxidative status and the time window in which Bcl-2 can protect will be determined. Third we will study the ability of the inducible heat shock protein 70 (HSP70) to protect from these injury again analyzing the time window during which this gene can protect and whether it blocks apoptotic or necrotic cell death. Primary cultures are particularly useful for analyzing mechanisms of ischemic brain injury and mechanisms of protection at the cellular level. Primary cultures of neurons and glial cells and pure astrocyte cultures will be mad3e from hippocampus and striatum. Results will be compared with parallel studies carried out on primary cultures from neocortex. In addition, astrocyte cultures will be produced from transgenic mice made with different gene dosages of SOD1 or MnSOD (SOD2), including knockouts lacking these genes, to determine the importance of these enzymes for astrocyte survival of ischemia-like insults. These studies will provide fresh insight into possible mechanisms of protection by three candidate genes for anti- ischemic gene therapy tested in three brain regions.

DESCRIPTION (provided by applicant): The hippocampus has been studied for its synaptic plasticity, role in cognition, and the neurogenesis that occurs in the adult hippocampus. With this has come an appreciation of endocrine modulators of hippocampal function. Specifically, glucocorticoids (GCs), adrenal steroids secreted during stress, disrupt [or] impair facets of synaptic plasticity and cognition, and inhibit neurogenesis. Estrogen, in contrast, enhances plasticity, cognition and neurogenesis. There has also been progress in the use of viral vectors to deliver transgenes into the CNS. We will use herpes simplex virus-1 vectors in a gene therapy strategy to protect the hippocampus from the disruptive effects of GCs and of stress, and to divert some of those GC effects into salutary estrogenic ones. We have constructed and wish to explore the protective potential of vectors expressing a) an enzyme which degrades GCs; b) a dominant negative GC receptor; c) a chimeric steroid receptor which binds GCs but has the genomic actions of an estrogen receptor. In Aim 1, we will alter these vectors to make them inducible by stress and GCs, as a means to have their expression triggered by insults. We will then characterize their patterns of expression. We will then examine the protective potential of these vectors, examining if the first two spare neurons from adverse GC effects, and if the chimeric vector also generates the salutary estrogenic effects when exposed to GCs. In Aim 2, the endpoint will be long-term potentiation in hippocampal slices generated from rats with differing pre-mortem regimes of GC exposure or stress. In Aim 3, we will study the effects of GCs, stress, and these vectors upon a hippocampal-dependent cognitive task. In Aim 4, we will study neurogenesis in vitro and in the adult hippocampus. We will characterize the effects of GCs, stress and estrogen upon it. We will then determine whether these vectors can protect neurogenesis from the inhibitory effects of GCs and, in the case of the chimeric vector, harness this to produce stimulatory estrogenic effects.

DESCRIPTION (provided by applicant): It is clear that repeated exposure to stress increases the risk of developing and expressing symptoms of human disorders of fear and anxiety, however little is known about the mechanisms by which this occurs. Numerous studies have shown that the hippocampus is profoundly affected by chronic stress. Accordingly, patients with disorders of fear and anxiety often exhibit reduced hippocampal activity, and impairments in hippocampus-dependent learning and memory. In contrast, such patients are reported to exhibit increases in amygdala activity and enhancement of amygdala-dependent learning and memory. Because the amygdala plays an essential role in both innate and learned fear, this region may be a locus of stress-related changes that underlie fear and anxiety disorders. However, almost no work has examined the impact of chronic stress on the amygdala. In the proposed research, we will characterize stress-related changes in the amygdala in rats, using functional measures (behavior and synaptic plasticity) for which stress-related impairments in hippocampal neurons are well documented. We hypothesize that a regimen of chronic stress that negatively impacts hippocampal function across multiple measures will facilitate amygdala function along those same measures, thereby modeling the relationship between stress and disorders of fear and anxiety in humans. We will then examine the efficacy of two gene therapeutic interventions in reversing stress-related enhancement of fear. We will generate stress-inducible herpes simplex-1 viral amplicons designed to express an activity-dependent potassium channel (Kv1. 1), a calcium-dependent potassium channel (SK), the enzyme 11-beta-hydroxysteroid dehydrogenase-2 (11B), or a transdominant negative glucocorticoid receptor (Td). We will examine the impact of overexpressing these proteins in two amygdaloid regions (the basolateral complex or central nucleus) during stress to determine whether reducing either neuronal excitability (Kv1.1 and SK) or the actions of glucocorticoids (11B or Td) in the amygdala during stress restores normative function across behavioral and electrophysiological measures. These studies will be among the first to probe the feasibility of gene therapy in treating models of psychiatric disorders, and will provide important and novel insights into the mechanisms by which chronic stress impacts amygdala function.

DESCRIPTION (provided by applicant): Parasites have evolved various mechanisms for manipulating host behavior for the benefit of the parasite. This proposal concerns an example relevant to understanding fear and anxiety. The protozoan Toxoplasma gondii reproduces sexually in a two-species life cycle. The parasite reproduces in cats, and is then excreted. Infected feces are eaten by rodents, in which Toxoplasma forms cysts in muscle and the CNS. The life cycle is completed when the rodent is predated by a cat. Recent reports indicate that Toxoplasma alters the behavior of infected rodents so as to increase the likelihood of their being predated by cats. Specifically, the parasite blunts the innate aversion of rats for the urine of cats, producing instead an attraction towards cat pheromones. This appears to be a rather specific effect (rather than merely being secondary to damage to the olfactory system or to the CNS in general). Moreover, additional observations suggest that Toxoplasma decreases ethologically-relevant fear in other mammals as well. These findings suggest that Toxoplasma is selectively able to interfere with circuits of fear and anxiety in the rodent, a possibility unstudied to date, to my knowledge. The purpose of this exploratory grant is to examine how broadly Toxoplasma is able to suppress fear-related behaviors in rodents, and to begin to understand the neurobiology of this effect; potentially, this could pave the way for novel approaches to the treatment of anxiety disorders. In Specific Aim 1, we will test the generality of these Toxoplasma effects, examining the behavior of infected rats with a battery of tests of differing facets of fear and anxiety. Controls will be mocked infected or infected with Sarcocystis neurona, a related protozoa that does not reproduce in a carnivore. In Specific Aim 2, we will use a Toxoplasma strain that expresses a reporter gene during its phase of forming CNS cysts in order to do a detailed examination of the neuroanatomy of infection. We hypothesize that Toxoplasma infects brain regions involved in the circuitry of fear and anxiety (in particular, the amygdala). In Specific Aim 3, we will examine the effects of Toxoplasma infection on cell number and volume, and on dendritic morphology in brain regions implicated in the prior two Specific Aims in the effects of Toxo.

[unreadable] DESCRIPTION (provided by applicant): The behavioral manipulation hypothesis posits that parasites can change the behavior of hosts to increase the reproductive fitness of the parasite. The protozoan parasite Toxoplasma gondii fits this description well. Toxoplasma shuttles between an asexual life-cycle in rodents and a sexual life-cycle in cat intestine. In rodents, parasites preferentially localize to the brain. The parasite has evolved the capacity to, once having formed cysts in the brain, abolish the innate fear that rodents have of the odors of cats, and to convert that fear into an attraction; this increases the likelihood of the rodent being predated, thereby completing the parasite's life cycle. Our prior work has shown the robustness of this behavioral phenomenon, the preferential formation of Toxoplasma cysts in the amygdala, and the capacity of the parasite to reduce cell number and dendrite length in the basolateral amygdala. The present grant further explores the neurobiology of Toxoplasma's behavioral effects, in order to better understand both normal and pathological fear. Specific Aim 1 identifies brain regions that are candidates for mediating the behavioral effects of Toxoplasma. A time course study will indicate brain regions where Toxoplasma cysts form prior to the first emergence of the behavioral effects of the parasite. In addition, we will construct a map of brain regions activated in response to cat odor in control rats (using cFos expression as a marker), and then determine whether Toxoplasma blocks such activation in any of those regions. The candidate brain regions identified in this Specific Aim will be studied in the subsequent Aims. Specific Aim 2 studies whether Toxoplasma causes inflammation in local brain regions and whether that contributes to the behavioral syndrome. It will also investigate whether the parasite changes the number of cells (both neurons and glia) in different brain regions, and/or changes the complexity of dendritic processes in neurons. Finally, it will examine whether Toxoplasma blocks the effects of cat odor on extracellular concentrations of glutamate, GABA and dopamine in those candidate brain regions. Specific Aim 3 will explore the reductive bases of Toxoplasma's actions. The first part will be an analysis of protein expression profiles in brain regions implicated in the parasite's behavioral effects, and in the parasite as well. The second will be a conditioned medium experiment, determining whether the medium in which cultured Toxoplasma grows can have the same effects in the brain as the parasite itself. This will be a first step in determining whether a factor secreted by the parasite mediates the behavioral effects. PUBLIC HEALTH RELEVANCE Experiments proposed in this grant explore an unlikely intersection of parasitology and neurobiology, that is, loss of innate fear in rodents due to Toxoplasma infection. Pathological fear is central to several psychiatric disorders. Understanding how Toxoplasma abolishes fear will shed light on how fear is generated in the first place and how we can manage pathological fear. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glucocorticoids (GCs), the adrenal steroids secreted during stress, can compromise the ability of neurons to survive necrotic neurological insults. These deleterious effects have often been viewed as counterbalanced by the benefits of the anti-inflammatory actions of GCs in the injured brain. However, GCs are actually less consistently anti-inflammatory in the injured brain than generally assumed. Moreover, as a marked challenge to dogma, recent work from my lab and others has shown that GCs can even potentiate aspects of inflammation in the injured hippocampus and cortex (while being classically anti-inflammatory elsewhere in the brain). Specifically, the hormone augments the migration of inflammatory cells to the injury site, the expression of and protein levels of pro-inflammatory cytokines, and the activation of the pro- inflammatory transcription factor NFkB. These findings are challenging at the basic science level (i.e., uncovering how GCs can have opposite effects on inflammation in different contexts);moreover, they are of considerable potential clinical relevance, given the enormous use of synthetic GCs to control post-insult inflammation in the human brain. This proposal will study the mechanisms underlying these novel GC actions at a more reductive level than in previous studies. In all experiments, intact rats will be exposed to either LPS, a bacteria-derived molecule which stimulates a robust inflammatory response, or a seizure-inducing excitotoxin, which also causes inflammation in the brain. Specific Aim 1 will analyze how the magnitude and duration of GC exposure, its temporal relationship to one of these inflammatory challenges, and the brain region examined determine whether GCs worsen or blunt the inflammation caused by these challenges. From these data, we will identify the most striking contrasts between conditions where GCs augment versus blunt facets of inflammation. In Specific Aim 2, we will identify gene expression profiles that differentiate between those two states;specifically, we will identify genes whose expression is influenced in a contrasting manner by GCs, depending on whether the hormone is enhancing or inhibiting. Specific Aim 3 will then identify the cell types in which these expression differences are occurring (i.e., whether in neurons, astrocytes, microglia, endothelial cells, neutrophils or macrophages). PUBLIC HEALTH RELEVANCE: Glucocorticoids (GCs, including synthetic corticosteroids such as hydrocortisone or prednisone) are anti-inflammatory, and are widely used to decrease the damaging inflammation that occurs after brain injury. However, a growing literature shows that GCs can actually worsen inflammation following some acute neurological insults. The proposal begins to dissect the molecular mechanisms underlying these unexpected and damaging pro-inflammatory GC effects.