Csaba Leranth, M.D. - US grants

Our goal is to determine the anatomical and neurochemical bases of neuronal control of gonadotrophin secretion. The "premium movens" of gonadotrophin secretion is the gonadotrophin hormone releasing hormone (GNRH) produced by a discrete group of hypothalamic neurons. The feed- back effects of estrogens of GNRH release are mediated in part by estrogen sensitive neuronal circuits which control the activity of the GNRH producing neurons. The control of GNRH delivery systems is differently organized in rodent and primates, at least in part because of anatomical differences such as the intranuclear location of both the GNRH producing - and their related estrogen sensitive neurons in primates, as opposed to the internuclear GNRH delivery system in rats where the GNRH neurons are located in the medial preoptic area. The proposed experiments will contribute to the following aims: a) to complete the identification of the primary circuits of estrogen sensitive neurons supporting the GNRH neurons in the rat, b) to pursue the origins of these circuits in the hypothalamus and extrahypothalamic areas, c) to determine the degree of similarity between the internuclear system of GNRH control in the rat and the intranuclear control in the monkey. Electron microscopic double immunostaining combined with degeneration, antero- and retrograde tracer techniques will be employed to answer the following specific questions: 1) What is the anatomical basis of the interactions between the mediobasal hypothalamus GABA-,dopamine-, and opiocortin systems in the rat? 2) What is the interrelationship between GABAergic-, catecholaminergic-, and GNRH-neurons in the rat medial preoptic area? 3) How do mediobasal hypothalamus opioid peptide- containing neurons regulate norepinephrine/epinephrine cells projecting to the mediobasal hypothalamus and medial preoptic ara? 4) Is the catecholaminergic innervation of GNRH neurons in the rhesus monkey analogous to that in the rat? Do monkey GNRH neurons receive direct information from GABAergic progesterone receptor-containing hypothalamic neurons?

neuroanatomy; parasympathetic nervous system; synapses; hippocampus; vasopressins; somatostatin; afferent nerve; pyramidal cells; amines; gamma aminobutyrate; innervation; interneurons; electron microscopy; laboratory rat; immunocytochemistry; microscopy;

The overall aim of the proposed studies is to further elucidate the ultrastructural neurochemical anatomy and interconnections of the septo- hippocampal complex. These studies will aid our understanding of mnemonic functions and human diseases, particularly Alzheimer's disease and temporal lobe epilepsy. Additional analyses of the intrinsic and extrinsic connections of the sexually dimorphic and estrogen/androgen sensitive septal complex, particularly with the syndrome and in understanding how the neuroendocrine and cognitive/associational areas of the central nervous system interact. While there are major differences between the human and rodent hippocampal formations, these structures are very similar in the human and monkey brain. Therefore, in addition to rat studies, we propose to continue the analysis of the hippocampal formation of subhuman primates. Two major lines of inquiry are proposed: a) why some hippocampal neurons and areas are more seizure sensitive than others; and b) how the septal complex mediates bidirectional information between the hippocampus and the neuroendocrine hypothalamus. By using interlinked state-of-the-art morphological techniques, including correlated light and electron microscopic double immunostaining methods, antero and retrograde tracer techniques, and Golgi impregnation, as well as by combining electrophysiological and histochemical methods, the following studies are suggested: 1. Further analysis of the synaptology and transmitter content of seizure-sensitive and seizure-resistant neurons in the rat and monkey hippocampal formation. 2. Neurochemistry of interneurons associated with identified mossy cells. 3. Further characterization of extrinsic hippocampal substance P and calretinin systems. 4. Synaptology and neurochemistry of subcortical circuitries controlling septal area neurons and the septo-hippocampal pathway.

The long range goal of this project is to anatomically dissect the intrinsic circuit organization of the prefrontal cortex at light and electron microscopic levels in normal humans, monkeys, and in selected instances, schizophrenic brains. Emphasis continues to be placed on the cytoarchitecture, local circuits, and monoaminergic innervation of associational and selected sensory cortical regions, with reference to findings obtained on schizophrenic brains in this and other laboratories. The project is subdivided along three lines of inquiry. 1) In ongoing investigations of neuropathological changes in schizophrenic cortex, we propose to add a new cohort of cases as well as expand cytometric investigation to include area 8, the human frontal eyefields, and area 45, Broca's area; we hope to confirm and extend evidence of increased cell density and decreased neuropil obtained in Brodmann's area 9 and 46, and determine whether cellular changes are specific to association areas and, within these areas, to specific neuronal populations; 2) Studies of intrinsic circuitry in human prefrontal cortex will address whether increased cell density in schizophrenics is due to loss or rearrangement of intracortical connections and/or atrophy of dendritic arborizations using fluorescent tracers, e.g., diI, and Golgi impregnations, respectively. Inputs to identified inhibitory interneurons will also be examined in rhesus monkeys and human cortex in immunocytochemical analyses at the light and electron microscopic level; 3) We will proceed with the ultrastructural characterization of the synaptic architecture of chemically defined afferents on identified cortical targets, guided by the hypothesis that norepinephrine, serotonin, and acetylcholine each have preferential patterns of termination within the cortical mantle just as we demonstrated for the dopaminergic innervation. Double labeling immunocytochemical paradigms will also be used to reveal anatomical substrates of the interaction among these systems in the prefrontal cortex of rhesus monkeys, and ultimately in the human brain. The availability of neurotransmitter - and receptor-specific antibodies and mRNA probes which recognize receptor-synthesizing neurons, some of which are being developed in Project III of this proposed Center, will allow us to gain a comprehensive view of pre-and postsynaptic elements of specific neurotransmitter systems and thereby to gain a more explicit understanding of their psychoactive efforts.

DESCRIPTION: (Adapted from applicant's abstract): Recent evidence indicates that estrogen treatment in ovariectomized rats enhances performance on memory tasks. Similarly, human observations suggest a beneficial effect of estrogen treatment on cognitive function in Alzheimer's disease. However, those cellular targets of gonadal hormones that are directly involved in memory processes are ill defined. The only solid observations are that systemic hormonal manipulations result in changes in the density of spines and alterations in the intensity of immunostaining for NMDA receptor of hippocampal CAl pyramidal cells. However, hippocampal principal neurons themselves do not, only a small population of interneurons contains nuclear estrogen receptor. On the other hand, neurons in subcortical areas, including the medial septum diagonal band of Broca (MSDB), supramammillary area (SUM), and median raphe (MR) contain nuclear estrogen receptors, and these structures are associated with the generation/regulation of hippocampal theta activity and long term potentiation. Furthermore, hippocampal theta activity, which is greatly influenced by the changing levels of circulating estrogen, in conjunction with long-term potentiation is believed to be involved in memory processes. Therefore, the hypothesis that estrogen, in addition to influencing the hippocampus directly, regulates mnemonic processes by affecting these subcortical areas will be tested by experiments designed to examine the effects of intra-MR, -MSDB, and -SUM administration of estrogen in ovariectomized rats on: 1) changes in dendritic spine density of hippocampal CAl pyramidal cells; 2) mRNA and peptide levels of ionotropic glutamate receptors in the hippocampus; 3) hippocampal theta activity.

[unreadable] DESCRIPTION (Provided by applicant): The purpose of this proposal is to determine which subcortical structures are involved in gonadal hormonal actions on the hippocampus, in female rats. Animal studies, as well as human observations demonstrated that estrogen has a beneficial effect on hippocampus-associated cognitive functions and symptoms of Alzheimer's patients. The cellular targets of gonadal hormones in these processes are ill-defined. Experiments demonstrated that systemic hormonal manipulations result in dramatic changes in the density of CA1 hippocampal area pyramidal cell dendritic spines. However, there are no convincing data that hippocampal CA1 area pyramidal cells are the direct targets of estrogen. On the other hand, subcortical areas that regulate the activity of the hippocampus, including the medial septum diagonal band of Broca (MSDB) and supramammillary area (SUM) contain nuclear estrogen receptors. Therefore, we hypothesize that gonadal hormones, in addition to acting directly in the hippocampus, regulate hippocampal synaptic plasticity by affecting these estrogen receptor-containing subcortical structures that regulate hippocampal electric activity. This view is supported by recent observations that systemic estrogen administration to fimbria/fornix-transected ovariectomized (OVX) rats does not restore, but local estrogen administration into the supramammillary area dramatically increases CA1 area pyramidal cell spine synapse density. To further elaborate these observations, the following questions will be addressed: (a) is there a difference between the effect of unilateral versus bilateral estrogen implants into the SUM? (b) What is the transmitter content of SUM neurons involved in this process? (c) Are both the MSDB cholinergic and GABAergic septo-hippocampal neurons involved in mediating estrogenic effect to the hippocampus? and (d) Do SUM neurons mediate estrogenic effect to the hippocampus directly or indirectly, via the MSDB?

[unreadable] DESCRIPTION (provided by applicant): Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of policarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, including baby bottles, as well as the BPA-containing dental prostheses and sealants, considerable potential exists for human exposure to this compound. We have demonstrated for the first time that treatment of ovariectomized female rats with BPA dose- dependently inhibits both the hippocampal synaptoplastic and the positive cognitive effects of estrogen administration. These effects of BPA can already be observed at a dose of 40 [unreadable]g/kg that is below the current U.S. Environmental Protection Agency reference daily limit for human exposure. Moreover, several publications have also raised the same worrysome issue that low- dose BPA might compromise normal sexual development and function of the brain. However, the majority of these studies, including our own, investigating BPA effects on the brain have been performed in rats. Hence, the implications of these alarming results to human health are intensively debated. Therefore, we propose to reveal the effect of BPA on estrogen-induced spine synapse formation in the brain of nonhuman primates, which will provide more relevance to human health. There is ample evidence that remodeling of synaptic contacts might mediate the beneficial effects of estrogen on both hippocampus- and prefrontal cortex-dependent executive functioning, particularly working memory. Thus, we hypothesize that BPA exposure may interfere with estrogen-induced synaptogenesis in the hippocampal CA1 and CA3 areas and dentate gyrus, as well as in the prefrontal cortex of nonhuman primates. This hypothesis will be tested using ovariectomized, adult young vervet monkeys that will be treated with vehicle- (controls), estrogen-, BPA- and estrogen+BPA. Furthermore, regarding the receptor target(s) of BPA, we will test whether BPA interferes with the positive effects of estrogen on the hypothalamic expression of oxytocin (O) and the progestin receptor (PR). Neurons producing PR and O contain different estrogen receptors, ERa and ER[unreadable], respectively. Light and electron microscopic unbiased stereological calculations will be used to determine the total number of spine synapses in the hippocampus and prefrontal cortex, and the number of hypothalamic PR- containing and O-producing neurons. Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of policarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, including baby bottles, as well as the BPA-containing dental prostheses and sealants, considerable potential exists for human exposure to this compound. We have demonstrated for the first time that treatment of ovariectomized female rats with 40 [unreadable]g/kg BPA, which is below the the current U.S. Environmental Protection Agency reference daily limit for human exposure, inhibits both the hippocampal synaptoplastic and the positive cognitive effects of estrogen administration. This study, has been performed in rats. Thus, the implications of these alarming results to human health are intensively debated by the chemical industry. Therefore, we propose to reveal the effect of BPA on estrogen-induced spine synapse formation in the brain of non-human primates, which will provide more relevance to human health. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of polycarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers and baby bottles, there is a wide human exposure to this compound. We have demonstrated for the first time that low-dose BPA treatment of gonadectomized female and male rats and monkeys inhibits the synaptogenic effect of sex steroids in the hippocampus and prefrontal cortex. Mounting evidence indicates that remodeling of hippocampal and prefrontal spine synapses is a morphological basis for learning and memory, suggesting that the negative synaptogenic effects of BPA are associated with impaired cognitive performance. According to the National Toxicology Program, available evidence provides a basis for concern, yet the Food and Drug Administration (FDA) concludes that data collected so far is not sufficient to resolve the problem whether BPA exposure represents a threat to human health. The FDA calls for further studies on BPA that are more relevant to human health. In this application, we propose three specific aims that are designed along guidelines published in the most recent FDA report on BPA. Using our established nonhuman primate model, we will test the hypothesis that BPA- induced loss of hippocampal and prefrontal spine synapses and compromised dopaminergic neurotransmission are associated with impaired cognitive performance. In addition, we will analyze whether the effects of BPA are cumulative and whether they are reversible. Finally, infants and children in particular seem to be at the highest level of risk from BPA exposure, because low- dose BPA interferes with neuronal development in rodents, leading to potentially irreversible alterations in behavior. Our last specific aim is designed to address this problem by analyzing whether perinatal BPA exposure causes irreversible harm in our nonhuman primate model. These studies will provide information on the risks of BPA exposure with most relevance to human health, which will be highly valuable for governing bodies to appropriately regulate the use of BPA. PUBLIC HEALTH RELEVANCE: Recent rodent studies suggest that the xenoestrogen, bisphenol A, at levels found in the environment, is capable of diminishing one's ability to learn and interferes with brain development. Because there are considerable differences between the brains of rodents and humans, this suggestion is intensively debated. Therefore, we will investigate how bisphenol A affects the brain and learning capability of adult and juvenile monkeys, to provide results that are more relevant to human health and may guide future environmental policy about bisphenol A.