Elizabeth Gould - US grants

Unlike most mammalian brain regions which undergo neurogenesis only during a discrete developmental period, the rat dentate gyrus shows cell birth, migration and death well into adulthood. The long term objectives of this proposal are to gain a better understanding of these processes and to determine the factors which regulate their occurrence. Developmental studies have shown that postnatal cell birth, migration and death in the rat dentate gyrus are mediated by adrenal steroids and excitatory amino acid input. Since the adult dentate gyrus retains many developmental characteristics, it is possible that adrenal steroids and/or afferent input continue to regulate these cellular processes throughout the life of the animal. This proposal seeks to determine whether dentate gyrus cell birth, migration and survival are affected in adulthood by those factors which control their occurrence developmentally. In an effort to gain a better understanding of the adult dentate gyrus, the following specific aims will be addressed: 1) to examine the differentiation of newly born cells, 2) to investigate the role that adrenal steroids play in regulating cell birth and survival, 3) to determine whether adrenal steroids regulate the migration of newly born cells, 4) to determine whether dentate gyrus neuroblasts and glioblasts express adrenal steroid receptors, and 5) to determine the role that afferent input plays in the regulation of cell birth and survival. These goals will be achieved by combining in vivo 3H-thymidine autoradiography with immunohistochemistry or retrograde tracing in brains of intact adult rats or adult rats which will be subjected to glucocorticoid manipulations or pharmacological receptor blockade. Cell death is a major contributor to the functional decline seen with many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Since most brain regions in the adult do not maintain the ability to create new neurons, cells lost during disease can not be replaced. A better comprehension of a brain region with the unique capacity to generate new cells in adulthood may bring us closer to understanding, as well as influencing, the factors that cause most neuronal populations to lose this ability with time.

In the majority of mammalian brain regions, neurogenesis, migration and programmed cell death occur during a discrete time period that begins during gestation. In contrast, the dentate gyrus of the rat undergoes neuronal birth, migration and death throughout the postnatal period and well into adulthood. This neural region is an ideal system in which to study these processes in the absence of similar changes occurring in afferent or efferent neuronal populations. Since the dentate gyrus develops predominantly during the postnatal period, it is especially vulnerable to early environmental perturbations that may yield permanent effects on brain function. Identification of the factors that regulate the development of the dentate gyrus may elucidate the predisposing mechanisms underlying psychiatric conditions, such as depression and posttraumatic stress disorder, the etiologies of which are thought to involve early life experiences. Moreover, studying the cues that influence the birth, migration and survival of cells in the dentate gyrus may ultimately lead to an understanding of the factors that produce abnormal hippocampal development, as seen in schizophrenia, and potentially to the ability to reverse such developmental pathology by replacing aberrant neurons. The broad, long-term objectives of this proposal are to characterize neuronal birth, migration and survival in the developing dentate gyrus and to identify and study the factors that control these processes during the postnatal period and enable their occurrence in adulthood. Specifically, these experiments will examine the effects of adrenal steroids, excitatory input and cell death on neurogenesis, migration and cell survival in this system. A series of experiments assessing the effects of adrenalectomy or hormone administration, pharmacologic receptor blockade or stereotaxic lesions of afferent populations on the birth and migration of cells will be performed using in vivo 3H-thymidine autoradiography combined with Nissl staining and immunohistochemistry for neuronal and glial specific markers. These markers will also be used to assess the possibility that neuroblasts can be stimulated to divide by dying cells. In addition, the effects of adrenal hormones on the development of excitatory amino acid receptors and dendritic spines will be examined using autoradiography and single-section Golgi impregnation respectively. Collectively, these studies are designed to provide a thorough characterization of neuronal birth, migration and survival in this unusual brain region in order to understand the cues that permit these developmental processes to occur throughout the life of the animal.

The dentate gyrus of the hippocampus is unusual because it continues to produce new neurons in adulthood in a variety of mammals, ranging from rodents to primates. Exposure to learning tasks that require the hippocampus for acquisition enhances the survival of adult-generated neurons in the dentate gyrus. In addition, adult female rats produce more new neurons than males, and these sex differences are positively correlated with sex differences in hippocampal-dependent learning. These findings indicate that learning influences the survival of new hippocampal neurons and raise the possibility that adult-generated granule neurons are involved in hippocampal-dependent learning. The broad, long-term objectives of this proposal are to characterize the influence of ovarian steroids and learning on the production, survival and gene expression of hippocampal granule neurons generated in adulthood in both sexes. Moreover, the potential role that new granule neurons play in hippocampal-dependent learning and memory will be explored. Using associative learning paradigms of trace classical eyeblink conditioning and the Morris spatial water maze, the role of hormones and experience in the production and survival of new granule neurons in males and females will be investigated with markers of proliferating cells, such as 3H-thymidine and bromodeoxyuridine, combined with immunocytochemistry for neuronal and glial markers. Combined in situ hybridization and immunocytochemistry will be used to explore changes in expression of genes important for cell survival, synaptic plasticity and/or learning, such as NMDA receptor subunits, BDNF and bcl-2, specifically in adult-generated granule neurons following associative learning. Furthermore, the function of hippocampal granule neurons generated in adulthood will be explored in animals depleted of new neurons by endocrine manipulations or treatment with selective toxins for proliferating neuronal precursors and then tested on tasks that require the hippocampal region for acquisition. This proposal is relevant to disorders or age- and disease-related cognitive decline which can be ameliorated by ovarian steroids. In addition, these studies may provide insight into mechanisms of neuronal regeneration.

Stressful experiences during early life have been associated with abnormalities in the hypothalamicpituitary-adrenal (HPA) axis, changes in behavior and the development of psychopathology. Studying the cellular changes that occur in the brain in response to developmental stressors may bring us closer to understanding the mechanisms that underlie these functional consequences. Other projects in this Center grant proposal are designed to explore stress-induced changes in the brain at various levels of analysis, from molecular to behavioral. This project will examine the impact of aversive experience during early life on the structure of the brain. The studies proposed here focus on the hippocampal region, an area that is sensitive to stress and has been implicated in psychopathologies, including depression and posttraumatic stress disorder. The hippocampal region undergoes extensive development during the postnatal period that may render this structure potentially vulnerable to environmental perturbations. In particular, the granule neuron population of the dentate gyms is formed predominantly during the postnatal period. In fact, the production of these cells extends well into adulthood in most mammals, from rodents to primates. Previous studies have shown that the production of these hippocampal neurons is suppressed by stress in adulthood. This proposal is designed to explore the consequences of prolonged early life stress on the development of the granule neuron population, the CA3 pyramidal neuron population and the mossy fiber connections between these two cell groups using the rodent and primate models provided by the cores (PI, Plotsky), (PI, Insel). Using a variety of histological methods, including BrdU labeling, 3H-thymidine autoradiography, immunocytochemistry, Golgi impregnation and neuroanatomical tract tracing, the impact of early life stress on the formation and maintenance of hippocampal neurons will be explored. These studies will contribute to the Center's overall purpose to gain a better understanding of the effects of early life stress on brain structure and function and, ultimately, to the development psychopathology.

[unreadable] DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to gain a better understanding of the function of adult neurogenesis in the hippocampus. The following approaches will be used to achieve this general goal: 1) examination of the impact of socially and spatially complex experiences on new neurons, hormones levels and learning; 2) Exploration of the interaction between sleep, new neurons and learning; 3) determination of the impact of new neuron depletion on hippocampal function; and 4) characterization of differences between mature and immature neurons. Living under standard laboratory conditions has a profound negative impact on the survival of new neurons in the hippocampus. Therefore, a major goal of the experiments proposed is to identify the elements of a naturalistic, relatively complex environment that are important for hippocampal neurogenesis. The production, survival and phenotype of new cells in the hippocampus will be assessed with bromodeoxyuridine (BrdU) labeling combined with immunocytochemistry for cell-type specific markers, hormone levels will be assessed via radioimmunoassay and hippocampal function will be assessed by a variety of associative learning tasks, including trace eyeblink conditioning, trace fear conditioning, context fear conditioning and spatial learning in a Morris water maze. These techniques will be applied to animals that have been exposed to different social experiences, deprived of different stages of sleep or pharmacologically depleted of new neurons. A final series of studies will examine the structural and biochemical characteristics of neurons generated in adulthood compared to those generated during development. These experiments will use neuroanatomical tract tracing combined with BrdU labeling, electron microscopy combined with 3H-thymidine labeling and confocal microscopy. Because stress, sleep and the hippocampus have been linked to several psychiatric conditions, many of which have a cognitive component, these results may elucidate the brain mechanisms that underlie the development of psychopathology.

DESCRIPTION (provided by applicant): Aversive stress impairs plasticity and neuronal architecture in both the prefrontal cortex and hippocampus. Moreover, stress and elevated glucocorticoids negatively impact behaviors associated with these brain regions, by impairing cognitive function and enhancing anxiety. Recent studies have shown that positive or rewarding stress protects the prefrontal cortex and hippocampus from the damaging effects of elevated glucocorticoids by promoting neuronal growth, enhancing cognitive function and relieving anxiety. Although the prefrontal cortex and hippocampus are interconnected, very little is known about how these brain regions influence one another under conditions of positive or negative stress. The neuropeptide oxytocin may play a protective role under rewarding social situations, but its influence over hippocampal-prefrontal cortex circuitry remains unexplored. This proposal is designed to explore interactions between the hippocampus and prefrontal cortex under environmental conditions that are detrimental compared to those that are beneficial. The role of oxytocin in protecting these brain regions from a potentially damaging hormonal milieu will also be explored. To investigate these issues, we will employ positive and negative social stress paradigms, temporary inactivation of specific brain regions, manipulations of oxytocin receptors, cognitive and anxiety behavioral assays and measures of structural plasticity. The results of these studies will inform us of the interplay between the hippocampus and prefrontal cortex in mediating positive and negative outcomes in response to stress. Mood disorders are associated with cognitive dysfunction and volumetric decreases in the hippocampus and prefrontal cortex. Stress predisposes individuals to develop psychopathology, including depression and anxiety. Understanding the interplay between the hippocampus and prefrontal cortex as these areas respond to experience may enable the development of effective therapies that emphasize growth-promoting mechanisms conducive to improved cognitive function and reduced anxiety. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it is focused on identifying mechanisms that protect the hippocampus and prefrontal cortex from the damaging effects of stress. Since the hippocampus and prefrontal cortex are dysfunctional in depression and anxiety disorders, this work may suggest targets for therapeutic interventions in mood disorders.

DESCRIPTION (provided by applicant): Motor neuron death is a major feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease that leads to paralysis and death. Although most motor neurons die in ALS, some populations are resistant to degeneration. This exploratory proposal is designed to compare gene expression in motor neurons that are resistant to ALS with those that are vulnerable to ALS. The long term objectives of these experiments are to identify genes that confer protection to certain motor neurons and, ultimately, to induce expression of these genes in motor neurons that typically succumb to ALS. To accomplish these goals, we will compare gene expression in the bulbocavernosus and oculomotor nuclei, motor neuron populations that are resistant to ALS, with gene expression in the retrodorsolateral and trigeminal nuclei, motor neuron populations that are vulnerable to ALS, in both wildtype and ALS transgenic rats. The following aims will be investigated: Specific Aim 1: Compare gene expression in populations of motor neurons with differential susceptibility to ALS. These experiments will use gene microarray analysis of motor neurons after laser capture microdissection and RNA amplification. Specific Aim 2: Examine expression of specific genes identified in motor neurons with differential susceptibility to ALS. These experiments will use RT-PCR, in situ hybridization and immunohistochemistry. Specific Aim 3: Determine whether molecules associated with ALS- resistant motor neurons are differentially affected in a transgenic rat model of ALS. These experiments will use the techniques of Specific Aim 2 in transgenic SOD-1 mutant rats. Death of motor neurons is a hallmark of ALS, a paralyzing neurological disease that is fatal. Understanding how some motor neuron populations resist degeneration, even in late stage disease, may provide clues about targets for gene therapy in motor neurons at risk for degeneration in ALS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a degenerative neurological disease that results in paralysis and death. This proposal is designed to explore gene expression in motor neurons with differential resistance to ALS with the goal of identifying mechanisms that protect some motor neurons from death.

DESCRIPTION (provided by applicant): Obesity is a major public health problem affecting over 30% of the US population. In addition to its negative consequences on the rest of the body, obesity is associated with profound neural and cognitive deficits. The long-term objectives of this proposal are to determine the cellular underpinnings of obesity-induced cognitive dysfunction with the ultimate goal of developing novel interventions. Neuroimaging studies have shown that obese humans exhibit decreases in the volume of brain regions supporting higher level cognition, including the prefrontal cortex. In a rodent model of diet-induced obesity, simila findings have been observed, as well as the loss of dendritic spines, primary sites of excitatory synapses, and synaptic proteins. In addition, obesity increases the numbers of microglia in brain regions involved in cognition. Because microglia are known to phagocytose neuronal debris under conditions of disease and damage, a traditional interpretation of these findings is that the increased numbers of microglia in the obese brain facilitate clearance of degenerating synapses. However, developmental studies showing that microglia play an active role in sculpting neural circuitry suggest an alternative possibility: that microglial phagocytosis of synapses is a cause rather than an effect of synaptic loss in obesity, and a contributor to cognitive decline. The focus of this exploratory proposal is to test this hypothesis. First, diet-induced obesity in wildtype and transgenic mice along with immunolabeling, western blots, DiI labeling, electron microscopy and behavioral tasks will be used to determine whether microglial engulfment of synapses is associated with cognitive decline. Second, blockers of microglial activation and microglial phagocytosis combined with the above methods will be used to examine whether microglia are responsible for synapse loss and cognitive decline. Taken together, these experiments will explore the relationship between microglia and synapse loss and help elucidate mechanisms underlying obesity-induced cognitive dysfunction.

PROJECT SUMMARY Early life adversity is associated with an increased incidence of anxiety disorders in adulthood. Understanding the cellular processes that connect early life stress with excessive anxiety may provide clues for the development of new therapies to treat anxiety disorders. Studies in humans and rodents have identified subregions of the hippocampus as important for anxiety regulation. Neuronal oscillations in both theta and gamma frequency ranges in the hippocampus have been linked to anxious behavior and these oscillations are driven by coordinated activity among a specific class of inhibitory interneurons, the parvalbumin positive (PV+) basket cells. Perineuronal nets (PNNs), extracellular matrix structures that modulate plasticity and have been linked to psychopathology, surround many PV+ basket cells. In mice, early life adversity increases PNN formation around PV+ cells in the ventral hippocampus, raising the possibility that this change leads to early life adversity-induced enhancement of neuronal oscillations and anxiety. Adult neurogenesis in the ventral hippocampus is also impacted by early adverse experience and adult-generated granule cells are known to synapse with PV+ basket cells. The possibility that connections between adult-generated neurons and inhibitory interneurons are altered by postnatal stress, potentially through alterations in PNNs, and thus produce enhanced neuronal oscillations and anxiety remains unknown. This proposal is designed to address the gaps in our understanding about how early life stress alters hippocampal PNNs, adult-generated granule cell connections with inhibitory interneurons, neuronal oscillations and anxiety and to explore the connections among these effects. The proposed experiments will use a mouse model of early life stress, manipulations of PNNs in the hippocampus, retroviral labeling of new granule cells in the hippocampus, immunolabeling combined with confocal microscopy and electron microscopy, transgenic and retroviral-mediated manipulation of adult-generated neuron number, in vivo electrophysiology during behavior and anxiety test analyses in adult male and female mice. The studies in this proposal are designed to achieve three significant and related, but not interdependent, aims regarding the influence of early life stress on hippocampal plasticity in adulthood with the long-term goal of understanding the cellular changes that lead to increased neuronal oscillations and anxiety.

Project Abstract Autism spectrum disorder (ASD) is a heterogeneous condition affecting approximately 1 in 59 children in the US. ASD is characterized by deficits in social interactions, repetitive behaviors and/or restricted interests, and is often associated with intellectual disability. Although ASD is clearly developmental, with diagnoses typically occurring by 2-3 years of age, most people do not outgrow the diagnosis and continue to suffer with dysfunction in adulthood. Adults with ASD experience greater unemployment and social isolation than their peers with other developmental disorders, strongly supporting the need for therapies targeted to adults. A few studies have reported improvements in symptoms of ASD patients with interventions in adulthood, raising the possibility that plastic processes in the adult ASD brain may be enhanced to optimize function. Many brain regions have been implicated in ASD but among them the hippocampus is notable in that it is involved in both social and cognitive behavior and displays ongoing plasticity throughout life. Perineuronal nets (PNNs) are extracellular matrix structures that dampen plasticity and have been linked to neuropsychiatric disease. Studies have found evidence for mutations in genes associated with the extracellular matrix in ASD but previous work has not investigated whether PNNs contribute to social and cognitive dysfunction. Research indicates that PNNs and orthodenticle homeobox 2 (OTX2), a transcription factor important for PNN maintenance, are excessive in ASD mice in the hippocampal CA2 and CA3 regions, areas important for social and contextual/spatial processing. No studies have investigated whether interventions that normalize PNNs and OTX2 in the hippocampus mitigate problematic behaviors associated with ASD. Previous work suggests that ASD mice have reduced postnatal neurogenesis in the hippocampus and since adult-generated neurons contribute to social behavior as well as learning and memory, diminished adult neurogenesis may exacerbate ASD symptoms. Many target sites of new neurons in the hippocampus are surrounded by PNNs and since PNNs are known to inhibit plasticity, their over production in ASD may prevent optimal connections from forming. This proposal will address gaps in our understanding about how aberrant PNNs and their connections with adult-generated neurons contribute to behavioral dysfunction in ASD mice. The experiments will use transgenic and inbred ASD mouse models, manipulations of PNNs and OTX2, retroviral labeling of new neurons, immunolabeling with confocal and electron microscopy, drug and experiential stimulation of neurogenesis and behavioral analyses to explore the efficacy of interventions to mitigate ASD symptoms by normalizing PNNs, reducing OTX2 and optimizing connections between new neurons and PNN+ targets. The proposed work will advance our understanding of how structural plasticity in the hippocampus may be enhanced in the service of improving social and cognitive dysfunction in adults with ASD.