John J. Hablitz - US grants

The primary aim is to examine, comprehensively and quantitatively, the effects of prenatal exposure to alcohol on the anatomy and physiology of the hippocampal mossy-fiber system. Whether previously described structural alterations in the mossy-fiber system are associated with electrophysiological changes will be determined as well as the functional dependence of these changes on the amount and duration of the alcohol exposure. Thus, prenatal exposure to alcohol will be used as a probe for a better understanding of the functioning of the hippocampus as well as for a further delineation of the factors responsible for the fetal alcohol syndrome. The extent and distribution of abnormal mossy fibers will be studied as a function of the time and dose of alcohol exposure in utero. This will be accomplished by quantitative computer analysis of histological sections stained by the Timm's sulfide silver histochemical technique. Intra- and extracellular recording techniques will be used in the in vitro hippocampal-slice preparation to determine whether ethanol-induced anatomical alterations of the mossy-fiber system are associated with changes in the synaptic and membrane properties of CA3 pyramidal neurons.

The primary objective is the investigation of excitatory neurotransmission in the hippocampus. Quantitative neurophysiological methods will be employed to study the response of pyramidal neurons in the CA3 and CA1 subregions to exogenously and endogenously (i.e., synaptically released) applied excitatory amino acids. In vitro hippocampal slices, organotypic hippocampal explant cultures, and acutely isolated pyramidal cells will be used as model systems. The former preparation will be employed in continuing studies using the single-electrode voltage-clamp technique, of (1) the mechanism of action of iontophoretically applied excitatory amino acids, (2) the effects of bath application of selected antagonists, and (3) the role of magnesium in controlling synaptic excitability. Organotypic explant cultures and isolated cells will be used to examine the properties of single glutamate channels in hippocampal neurons using patch-clamp techniques. Quantitative studies of antagonist effects will determine the nature of the amino acid receptor mediating synaptic excitation in the CA1 and CA3 subfields of the hippocampus. The patch-clamp studies will provide insights into the gating mechanisms underlying excitatory synaptic transmission that cannot be obtained by other methods. The proposed studies will advance our understanding of the role played by excitatory amino acid receptors in synaptic transmission in the mammalian cortex. Alterations in such receptors have been implicated in the induction and regulation of long-term potentiation (a type of synaptic plasticity) in the hippocampus. Furthermore, certain glutamate antagonists have been shown to have anticonvulsant properties, a finding of particular significance because of the high seizure susceptibility of the hippocampus. The quantitative information derived from the proposed studies will provide a firm basis for subsequent investigations of the role of excitatory amino acid receptors in synaptic plasticity, learning, and disease processes such as epilepsy.

Neuronal migration disorders resulting in cortical dysplasia, microgyria, and hectrotopias are associated with intractable seizure disorders in humans. We have used the rat freeze lesion model to examine neural mechanisms underlying hyperexcitability in dysplastic cortex. Proposed studies will combine optical imaging and whole-cell voltage-clamp techniques to test specific hypotheses about mechanisms that control excitability in the dysplastic cortex. These mechanisms are alterations in N-methyl-D-aspartate receptors (NMDARs) in local horizontal pathways and remodeling of GABAergic networks. Experiments will investigate if the enhanced spread of voltage sensitive dye signals we observed in slices from dysplastic cortex is due to an increased contribution from NMDARs in intracortical horizontal pathways. It will be determined if blockage of NMDAR activation changes the spatial and temporal extent of cortical circuit activation. We will examine if NR2B subunit containing NMDARs in lesioned cortex confer hyperexcitability through the prolongation of excitatory postsynaptic currents. It is hypothesized that NMDAR EPSCs are prolonged in dysplastic cortex and resemble those seen at earlier stages of development in normal neocortex. Anatomical studies will determine if there are changes in the number or extent of axon collaterals forming horizontal connections. We will also determine the site of origin and pattern of spread of depolarizing GABA waves in dysplastic neocortex. It is hypothesized that reorganization of GABA-ergic networks in dysplastic cortex will result in different patterns and rates of propagation. We will delineate the site of origin and pattern of spread of depolarizing GABA waves in dysplastic neocortex. It is hypothesized that a propagating wave of potassium results in spreading changes in intracellular chloride due to activity of potassium-coupled chloride cotransporters. Changes in extracellular potassium and application of transport blockers are predicted to change or block propagation. Glial cells are coupled by gap junctions and participate in spatial buffering of potassium. Disruption of gap junctions is predicted to change or block propagation. These studies will provide important new information about NMDA and GABA receptors in neocortex. They will also contribute to our understanding of the functional changes at both the cellular and circuit level, responsible for the intrinsic hyperexcitability of dysplastic cortex in the freeze lesion model.

The long-term objective of the proposed studies is to further characterize the properties of glutamate receptors in the mammalian neocortex. These studies will increase our understanding of excitatory synaptic transmission in higher cortical structures and the control of excitability in central neurons. Patch clamp recordings from visually identified cells in slices of neocortex maintained in vitro will be used to assess the role of glutamate inotropic and metabotropic receptors (mGluRs) in regulating synaptic excitation. Biophysical studies will focus on interneurons in layer 1 to determine how glutamate receptors regulate activity in local cortical circuits. The overall goal is to better understand the role of glutamate as a neurotransmitter and novel neuromodulator in the neocortex. Proposed studies will examine the pharmacological properties of a novel EPSP observed in the presence of high concentrations of NMDA and non-NMDA receptor antagonists. It is hypothesized that this response results from activation of G-protein coupled mGluRs. The ability of mGluR antagonists and/or intracellular GDP-beta-S to block this response will be determined. Additional studies will determine if Ca2+ -permeable AMPA-receptors mediate EPSCs in identified interneurons in rat neocortex. EPSCs will be examined for rectification in I-V relations and Ca2+ permeability will be determined from reversal potential shifts measured in solutions with differing extracellular Ca2+ concentrations. Comparison will be made with whole cell responses to bath applied kainic acid, a non-desensitizing activator of AMPA receptors. In further studies, the ability of mGluR agonists to modulate EPSCs and IPSCs in identified interneurons will be examined. Despite their marked expression in the neocortex, the role of mGluRs in normal synaptic transmission in this brain region is unclear. The goal of the proposed studies is to examine the effects of mGluR activation on excitability of identified interneurons in rat neocortex and determine the mechanisms underlying these actions. It is hypothesized that terminals making synapses on layer I interneurons express different classes of mGluRs, resulting in unique responses to mGluR agonists. The direct postsynaptic response of interneurons to activation of mGluRs will be studied in neocortical interneurons under whole-cell voltage clamp conditions. it is hypothesized that certain classes of layer I cells express mGluRs coupled to IP3 formation resulting in intrinsic membrane oscillations in response to mGluR agonists. Higher nervous functions, including learning and memory acquisition, are believe to involve glutamate mediated synaptic transmission. Neurological diseases such as epilepsy, stroke and neurodegenerative disorders may result from altered glutamate receptor functioning or overstimulation. The proposed studies will increase our understanding of the role of glutamate as a neurotransmitter and provide a better understanding of cortical excitability under normal and pathophysiological conditions.

The UAB Predoctoral Neuroscience Training Program will fill the need for a singular coordinated and broad-based predoctoral training program for the entire campus, provide a regime for appropriate monitoring of student progress throughout their graduate career and serve as a basis for a standardized and outstanding graduate curriculum with flexibility for various tracks which span departmental boundaries. Each student will achieve a broad research background through active participation in formal courses, informal seminars and journal clubs, and through hands- on research opportunities. The curriculum emphasizes the multi- disciplinary and quantitative aspects of modern Neuroscience, a diversity of laboratory research training experiences and the development of skills in critical reading, writing, speaking and teaching. The first year, multi-disciplinary core curriculum will provide students with the breadth of knowledge of necessary fos successful scholarship in Neuroscience. Courses taken the first year will provide the student with background in molecular and cellular neuroscience, biochemistry, integrative neuroscience, cell biology, and developmental neuroscience. These established courses have been offered for at least the last five years. Students also carry out research preceptorships in at least three independent laboratories prior to identifying a permanent mentor. In the first year, there will be a hands-on intensive laboratory-based course in the fundamentals of molecular biology techniques and cellular neurophysiology. In the second year, students will begin pre-dissertation lab research with their permanent mentor and take electives meeting the student's specific needs and interests. Each student will be required in their second year to participate in the Medical Neuroscience course in order to expand their understanding of the disease relatedness of basic neuroscience principles. In addition, the career in traditional academic institutions, the program's graduates will be prepared to become research team members and group leaders in some of the expanding number of biotechnology companies and established pharmaceutical companies. Moreover, they will be positioned for entrepreneurial initiatives to foster intellectual property development and cooperative university- biotechnology efforts in brain-related drug design and development.

Normal development of the neocortex is critically dependent upon precise signals mediated by specific neuromodulatory systems, such as serotonin (5-HT). Abnormal 5-HT signaling produces a number of long-term anatomical and physiological consequences including alterations in synapse structure and density, and alterations of cortical circuit activity. Such 5-HT-induced effects are correlated with mental retardation and a number of behavioral disorders, including learning deficits, depression, schizophrenia, attentional disorders, and sleep disturbances. While the evidence for 5-HT in shaping normal cortical development is abundant, little is known about the specific components of the 5-HT signaling system that regulate normal neocortical is abundant, little is known about the specific components of the 5-HT signaling system that regulate normal neocortical development. The goal of the present proposal is to examine the role of specific 5-HT signaling components (receptors, transporters) on modulation of synaptogenesis and circuitry in the neocortex. Critical to understanding the role of 5-HT in neocortical development is a determination of the specific modulatory role on 5-HT on cortical circuits and the underlying mechanisms. The hypothesis to be tested is that 5-HT facilitates circuit activity later in development and in mature animals. The specific 5-HT receptors that mediate this developmental switch will be determined, with the hypothesis being that the facilitation is due to transient expression of specific 5-HT receptors in developing pyramidal neurons. 5-HT signaling is determined by the availability of 5-HT levels. However, how SERTs regulate synaptogenesis and circuit signaling during development is not known. The hypothesis to be tested is that SERT expression correlates with 5-HT levels in order to maintain appropriate extracellular 5-HT levels in cortex. Furthermore, it is hypothesized that disruption of SERT function will lead to changes in normal synaptogenesis and circuit signaling. These data will not only provide information regarding the role of specific 5-HT receptors and SERTs in cortical development, but will also be important in understanding the mechanisms underlying abnormal development of cortex related to inappropriate 5-HT signaling.

DESCRIPTION (provided by applicant): The prefrontal cortex (PFC) plays an important role in governing a number of behaviors, including motivation, emotion learning and memory. The PFC receives a dopaminergic projection from the ventral tegmental area (VTA) which has been specifically implicated in cognitive and neuropsychiatric processes. Dopamine (DA) is believed to be an endogenous neuromodulator in the cerebral cortex and to be important for normal brain function. Clinical and experimental studies have also implicated DA in the pathogenesis of a number of neurological and psychiatric disorders, including epilepsy and schizophrenia. The overall goal of this research is to understand the role of DA in the modulation of activity in local neocortical circuits. The cerebral cortex, particularly the prefrontal cortex (PFC), is heavily innervated by dopaminergic afferents, suggesting this system plays a prominent role in regulating neuronal excitability. Despite the wealth of evidence supporting a role for DA in cognition, neuropsychiatric processes and neurological disorders, our knowledge of the function of DA receptors at the circuit and single cell level is incomplete. It is hypothesized that the net effort of DA will be determined by the interaction of changes in excitatory and inhibitory synaptic activity and alterations in intrinsic neuronal excitability. Specifically, it is planned: (1) to determine if DA receptors positively modulate excitatory inputs to layer II/III PFC pyramidal neurons via a mechanism involving Dl receptors, (2) to ascertain if evoked inhibitory postsynaptic currents (IPSCs) are negatively modulated by DA. Studies will determine if this is a presynaptic effect of Dl receptors mediated by activation of PKA and (3) to characterize and compare the postsynaptic effects of DA in pyramidal cells and fast spiking interneurons. The proposed experiments will provide important new information regarding the role of specific DA receptors in the regulation of local cortical circuits. These data will be important not only in understanding normal cortical functioning, but also in understanding the mechanisms underlying abnormal processes such as schizophrenia, epilepsy and Parkinson's disease, relatedto inappropriate DA signaling.

DESCRIPTION: The in vivo Behavioral Assessment Core will provide both service-oriented behavioral phenotyping and research staff training to investigators using rodents. One of the major goals of the Core will be the development and operation of a consolidated facility in which transgenic mice can reliably and consistently be tested using the most-accepted battery of behavioral tests, the SHIRPA (Rogers et al., 1997), along with expanded maze testing. The battery will include a primary behavioral screen, sensory and motor tests (including rotorod, spontaneous locomotor activity, walking coordination, etc.), an open field test for emotionality and exploratory activity and cognitive testing (including elevated plus-maze, Morris water maze, holeboard maze and eight-arm radial maze tasks). Most of the tasks will be automated, thereby greatly decreasing any potential investigator bias from the results. In most instances, the Initial phenotyping of a transgenic line will be carried out by the staff of the Core, who will be blinded to the genetic background of the mice. In those instances in which the investigator is interested in further characterizing the detailed behavior of a line of mice or a treatment, the staff and Director will train personnel from the investigator's lab to conduct more extensive studies, using the equipment of the Core. The Core will provide a similar assessment of rat behavior in apparatuses designed for these animals. For both rats and mice, the Core will, when deemed appropriate, test the animals using more sophisticated cognitive tasks for characterizing small differences between groups. For all animals tested, the Core will provide initial statistical analyses of resulting data in the reports to the investigators. The Core will also afford research personnel from the participating laboratories the opportunity to train in the use of these behavioral testing methods. This will particularly benefit investigators who require extended testing beyond the complexity offered in the standardized protocols of the Core. The Core will also assist investigators in the development of tools that are needed by them for a more detailed assessment of specific behaviors.

DESCRIPTION (provided by applicant): This is a competitive renewal application from a successful University of Alabama at Birmingham (UAB) Neuroscience Core Center. The Neuroscience Core Center at UAB builds upon the University's 30 years of contributions to interdisciplinary neuroscience research. The mission of the Core Center is to provide state-of-the-art resources that advance collaborative team approaches to neuroscience. The Behavioral Assessment Core (Core A) provides both service-oriented behavioral phenotyping and research staff training to investigators using rodents. In the renewal, facilities for emotional and fear conditioning, an important emerging area of research within the UAB Neuroscience community, will be added. The Molecular Detection Core (Core B) will offer sensitive, standardized immunohistochemistry and in situ hybridization technologies. The utility of this core will be enhanced by the addition of facilities for tissue sectioning and embedding. An increased focus on protocol development and investigator training will insure that these methods are successfully transferred to individual groups of investigators. The Protein Interaction Core (Core C) services will provide expression cloning of cDNAs and genomic sequences in vectors appropriate for identification of protein interactions, perform two-hybrid screening to identify protein interactions and provide viral vectors for expression of GFP and/or shRNA. New services will include constructs for tandem affinity purification (TAP) and adenoviruses (AAVs) for in vivo applications. Core C will emphasize generation of vectors and viruses that can be used in vivo. The Administrative Core (Core D) will provide administrative and scientific leadership. In the renewal, Core D leadership will also provide active mentorship of junior faculty in Co- Director positions. Collectively, the cores promote multidisciplinary collaboration and exchange of information and technologic advances, attract new investigators to neuroscience research, develop collaborative research activities with other institutions and increase the quality and productivity of funded research projects in a cost-effective manner.

[unreadable] DESCRIPTION (provided by applicant): Intractable seizure disorders in humans are often associated with cortical dysplasia, microgyria, and heterotopias resulting from neuronal migration disorders. We have used the rat freeze lesion model to examine neural mechanisms underlying hyperexcitability in focal cortical dysplasia. N-methyl-D-aspartate (NMDA) receptors are involved in generation of epileptiform discharges in this model and in human cortical dysplasia. The location of the NMDA receptors has not been determined and functional studies in human tissue are limited. Proposed studies will use electrophysiological and two-photon microscopy techniques to test specific hypotheses about the role of NMDA receptors in the freeze lesion model of cortical dysplasia. The applicability of these finding to human epilepsy will be directly tested in tissue slices from patients with focal cortical dysplasia using imaging methods. It is hypothesized that NMDA receptors are located on presynaptic nerve terminals of GABAergic interneurons in the early postnatal period and these receptors are normally developmentally downregulated but persist in cortical dysplasia. Experiments will investigate (1) if presynaptic NMDA receptors are present on inhibitory nerve terminals in neocortex. Whole-cell voltage-clamp recordings of pharmacologically isolated IPSCs will be obtained under conditions where postsynaptic NMDA receptors are blocked. It is hypothesized that NMDA receptor antagonists will decrease the frequency of miniature IPSC in young (PN 12-16) but not older (PN 26-30) sham operated animals. We will also use styryl dye (FM1-43) staining and multiphoton excitation microscopy to visualize vesicular release from inhibitory GABAergic terminals and study NMDA receptor modulation of GABA release. It will be determined if NMDA receptor activaton selectively modulates distinct vesicular pools, the readily releasable pool and the reluctant pool. We will also examine (2) if functional presynaptic NMDA receptors are present in human cortical dysplasia. Previous studies of human cortical dysplasia tissue has shown an increase in NMDA receptor expression, particularly NR2B receptors. It is hypothesized that NMDA receptors are located presynaptically on inhibitory nerve terminals and modulate vesicular release of GABA. These studies will provide important new information about presynaptic NMDA receptors and regulation of GABA release in cortical dysplasia. New insights into the role of presynaptic NMDA receptors in development and regulation of epileptiform activity will be forthcoming. These studies will also increase our understanding of basic mechanisms of transmitter release and its' developmental regulation in human and animal models of focal cortical dysplasia. PUBLIC HEALTH RELEVANCE: Cortical dysplasia is associated with intractable seizure disorders in humans. Up to 43% of patients receiving surgical treatment for intractable seizures have some sort of cortical malformation. Anticonvulsant drug therapy is often ineffective in these patients. Studies in vitro of brain slices prepared from human dysplastic neocortex have demonstrated that this tissue displays intrinsic hyperexcitability. The mechanisms responsible for the inherent epileptogenicity of dysplastic cortex have been incompletely defined. It is proposed to directly examine a previously unexplored mechanism, presynaptic NMDA receptors, using an animal model of cortical dysplasia and tissue samples from human cortical dysplasia. New insights into mechanisms regulating presynaptic release could lead to novel strategies for therapy. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION: The Administrative Core will provide continuous, high quality administrative and communication support for the individual Core Pis and Core Directors and the UAB Neuroscience Core Center investigators.

DESCRIPTION (provided by applicant): Focal cortical dysplasia is associated with the development of seizures in children and is present in up to 40% of intractable childhood epilepsies. Transcortical freeze-lesions in the newborn rat reproduce many of the anatomical and electrophysiological characteristics of human focal cortical dysplasias. Although abnormalities in several voltage-dependent currents have been implicated in epilepsy, changes in intrinsic excitability have not been extensively investigated in cortical dysplasia. The broad goal of this proposal is to identify mechanisms whereby alterations in HCN channel expression in specific cell types contribute to hyperexcitability in cortical dysplasia. The role of HCN channels in generation and modulation of synchronized epileptic network activity is poorly understood. Ictal discharges in human dysplastic cortex are partially dependent on synchronous GABA mediated events. The role of HCN channels in modulation of GABAergic interneuron excitability has received little attention. We will test the hypothesis that specific subclasses o layer 5 (L5) pyramidal neurons in dysplastic cortex have changes in intrinsic excitability and hyper-excitable dendrites due to decreases in HCN channels. The mechanism underlying increased excitability will be determined by quantifying changes in currents co-expressed in these cells (Kir and M-currents) and known to be modified in other models of epilepsy. We will also test the hypothesis that (1) excitability of GABAergic interneurons is modulated by HCN channels and that (2) decreases in HCN expression alters intrinsic excitability and temporal summation in identified subtypes of inhibitory interneurons in cortical dysplasia. Finally, we will test if Ih modulates synchronization in GABAergic interneuron networks. It is hypothesized that Ih inhibition will enhance interneuron network activity and this will be altered in cortical dysplasia. Overall, we will provide new information on HCN modulation of cellular and network properties in cortical dysplasia and elucidate how HCN channels influence signaling in GABAergic neurons and networks.

Core D, the Administrative Core, will be responsible for continuous, high quality administrative and communication support for the individual research Core. This support will be directed towards the PIs, Core Directors and the UAB Neuroscience Core Center investigators. The Core will determine the overall research direction based on input from the Core Directors, the Center's Steering Committee, and on responses from the investigator base. In executing these functions, the Administrative Core will conduct the day-to-day operations of the Center and provide a multi-faceted infrastructure that accelerates effective, productive neuroscience research.

Abstract This is the second competitive renewal application for the Training Program in Neurobiology of Cognition and Cognitive Disorders at the University of Alabama at Birmingham (C&CD Program). The C&CD Program is housed in the Department of Neurobiology, and includes 43 faculty from 15 basic science and clinical departments in the School of Medicine (SOM), School of Optometry, and College of Arts and Sciences. The success of the C&CD Program in its recent funding period is evidenced by its robust participation (34 total students), graduation of a talented cadre of doctoral students (25 PhDs graduated-5 URMs), with an average of more than four publications each, and all transitioning to competitive postdoctoral positions or completing clinical training. The overall goal of the C&CD Program is to create the next generation of talented, diverse and independent scholars and leaders in cognitive neurobiology. These responsible individuals will have (1) a solid foundation in molecular, cellular and cognitive neuroscience theories and findings, (2) training in the mechanisms for translating basic research into advancements in the treatment of cognitive disorders, and (3) be well-prepared to employ a multidisciplinary research approach using skill sets encompassing the next generation of tools and technologies to address important questions in cognitive neurobiology. The specific ?value added? components of the C&CD Program include the special clinical/translational training experience in patient- oriented research, interactions with both basic science and clinician scientist mentors, and distinct extracurricular activities. The UAB SOM has planned expansions for the Department of Neurobiology and Clinical Neuroscience with investments in areas relevant to cognition and cognitive disorders, thus providing a supportive environment for the future of the C&CD Program which will benefit substantially from this investment with the addition of dynamic new faculty mentors and research facilities.