Paulo Kofuji - US grants

DESCRIPTION: The overall goal of the proposed research is to define the role of the benzodiazepine (BZ) binding site in the GABA/A receptors of mice during neuronal development, synaptic function and certain behaviors. Gene targeting by Cre-mediated recombination on engineered lox/P sites will be used. The BZ binding site will be disrupted in vivo either by conditional gene knockout of the GABA/A recently gamma/2 subunit or engineering a specific point mutation in the GABA/A receptor alpha/1 subunit. In both mouse lines the total number of GABA/A receptors is expected to be comparable to the number found in the wild type animal, but the affinity for BZ ligands is expected to be drastically reduced. The specific aims are as follows: 1. To remove the high affinity BZ binding site of GABA/A receptors in specific brain regions in mice by targeted deletion of an essential exon in the beta/2 subunit. The spatial and temporal control of the knockout of this subunit in mice brain will be performed by intercrossing with another mice that is transgene expressing Cre recombinase in specific brain regions. 2. To disrupt the high affinity BZ binding site type I in GABA/A receptors by targeted mutations of a specific amino acid in the alpha/1 subunit. The targeted amino acid is the resides His 100 which when mutated to Arg yields receptors which are essentially insensitive to BZ agonists, despite the normal gating by GABA. 3. To investigate the impact of lack of BZ modulation in both mouse lines for CNS function, in particular, regarding neuronal development with synaptic transmission. The analyses will involved electrophysiological, radio-ligand and immunocytochemical techniques. 4. To investigate, in both mouse lines, the impact of lack of a BZ modulation on the behavioral effects of several BZ ligands (each with a different intrinsic activity) and of other substances such as ethanol, volatile anesthetics and barbiturates.

DESCRIPTION (provided by applicant): The central focus of this renewal will be on the molecular understanding of the macromolecular organization and functions of glial potassium, bicarbonate and water transport proteins in the mammalian retina. How transport proteins work together to coordinate the transport of solutes and water across glial cell membranes is a fundamental question in neurobiology and retinal physiology. In the preceding period, we have identified the critical role of Kir4.1 channels in the regulation of extracellular potassium concentration in the retina. This study consists of a series of steps aimed to further elucidate the composition, architecture and functions of water channels (AQP4), potassium channels (Kir4.1) and sodium bicarbonate cotransporters in retinal glia. The specific aims are the following: 1) to verify the hypothesis that potassium and water channels associate with the dystrophin glycoprotein complex (DGC) in Muller cell membranes, 2) to test the hypothesis that extracellular ligand signaling to the DGC is crucial for the highly localized expression of potassium and water channels in Muller cells, 3) determine the molecular identity of sodium bicarbonate cotransporter systems in Muller cells and verify whether they associate with the DGC, 4) extend our studies of the function of Kir4.1 channels to the retinal and optic nerve astrocytes. Potassium and water channels and sodium bicarbonate cotransporters play a critical role in potassium and acid-base buffering in retina and defects in their subunit assembly and macromolecular organization may be implicated in human diseases. The molecular understanding of the events that are central to their regulated expression is therefore essential for developing our knowledge in the area of retinal function in health and in diseases.

ABSTRACT
A grant has been awarded to Dr. Feddersen at the University of Minnesota-Twin Cities to acquire
scientific instrumentation that will enhance training and research opportunities concerning the influence
of ions (potassium, sodium, calcium, chloride, etc.) on cell function. Changes in ionic gradients
mediate diverse processes including cellular communication, chemical transportation, information
storage/retrieval and energy production/use. Regulation of ion concentration is the job of selective
channels traversing the membranes of all cells. While ion channel diversity and conservation among
species have been revealed through the recent findings of genome projects, much remains to be learned
about the basic function of ion channel proteins. Seminal advances in ion channel research have been
the focus of both the Nobel prize and Lasker Award in the past year serving to draw the attention of
young investigators. The goal of this proposal is to improve integration of research and research
training in the field of ion channel biology which is well-represented at the University of Minnesota.
Cutting-edge instrumentation will be utilized in multiple laboratory courses and time-shared with
research laboratories where it will enhance 'on-the-job' research training. The award will facilitate three
objectives: 1) improving the teaching capabilities of several laboratory courses, 2) expanding ion
channel research opportunities for undergraduate and graduate students, and 3) providing critical
infrastructure for the research training of non-university students. To meet these objectives seven state-of-
the-art experimental workstations will be assembled and distributed to four courses and at least six
different research labs during the year.
A common method to study ion gradients and the channels that affect them requires the use of
sensitive physiological approaches whereby miniature sensors(electrodes) are delicately placed in a
living specimen. Individual ion channels are most conveniently studied in a simple system utilizing
frog eggs. This approach involves genetic engineering and synthesis of information molecules
(mRNAs) coding for ion channels followed by injection of the mRNA into large, viable egg cells. The
cell's translation machinery converts the mRNA into ion channel proteins that are inserted into the cell
membrane. Electrodes placed in the cell collect minute signals that report ion channel function. Using
this approach researchers will measure, and instructors will teach students how to measure, the
response of channels to various stimuli or blockers in the presence or absence of accessory molecules.
The power of genetic engineering allows the introduction of precise mutations to pinpoint the functional
importance of each part of a channel. Protein expression and functional analysis in frog eggs has
become a standard approach of ion channel and cell surface receptor researchers. The instrumentation
allowed by this award is well matched to that application. The equipment includes microscopes, micro-manipulators, mRNA injectors, computers, analog/digital converters, amplifiers and signal conditioning software that make ion channel recording efficient and informative. The equipment will also be used in basic electrophysiology training and research in more complex specimens such as cells in a variety of
tissues, including neurons in brain slices isolated from the mature nervous system.
Teaching laboratories expose students to the critical observations and techniques that provide the
foundation of advanced life sciences research. The instrumentation awarded will directly support
objective #1 because it will be used to present ion channel training exercises in several undergraduate
and graduate level laboratory courses. Fundamentals taught in laboratory courses require an appropriate
setting to advance research to the frontier of discovery. Therefore, when not needed for course work,
the equipment will become a 'core utility' supplied to Principal Investigator labs for undergraduate and
graduate student research projects. The contemporary, fully compatible equipment will best serve the
diversity of research efforts ongoing at the University of Minnesota and satisfy objective #2. As
University courses become more available and convenient for a greater diversity of students the
equipment will be accessed by non-university researchers and small college educators meeting objective
#3. The research instrumentation will become a distributed and unifying feature across courses and
contemporary research endeavors, as well as, among individual labs within departments and research
sectors. Because overall research training and research will be integrated through this award, cost-sharing
funds were pledged from three separate colleges at the University of Minnesota. The funded proposal will enhance the presentation of laboratory-based education, promote student participation in the achievement of independent research goals and enrich the interaction among non-traditional students and university personnel.

DESCRIPTION (provided by applicant): Melanopsin (Opsin 4), a photopigment found in a small subset of retinal ganglion cells projecting to the suprachiasmatic nucleus and other brain areas, is implicated in nonvisual responses to environmental light such as the pupillary light reflex, seasonal adaptations in physiology, photic inhibition of nocturnal melatonin release, and modulation of sleep, alertness and activity. Because melanopsin containing ganglion cells are few in number and scattered throughout the retina, they are difficult to study. To address these limitations, we engineered a mouse line in which the Enhanced Green Fluorescent Protein (EGFP) is expressed under the control of the mouse melanopsin promoter employing BAC (bacterial artificial chromosome) transgenesis. We will perform two types of studies in these mice: (1) single cell electrophysiological recordings of EGFP positive neurons in whole mount retinas to characterize their functional properties during development, (2) electrophysiological recordings of acutely isolated EGFP positive neurons to characterize their intrinsic properties. Taking advantage of our ability to selectively target the melanopsin expressing neurons we will also engineer an additional mouse line: Opn4tTA line, in which expression of a transcriptional activator will be regulated both reversibly and quantitatively by exposing the transgenic animals to varying concentrations of doxycycline (Dox). The Opn4 tTA line will be used to allow the conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release or conditional overexpression of the inward rectifying potassium channel, Kir2.1, to diminish the excitability of melanopsin expressing neurons. These mouse lines will provide important tools to the neuroscience and vision research community to address the mechanisms of signal transduction in melanopsin expressing cells, their physiological properties, and their roles in vivo. Within the retina of the vertebrate eye there are photoreceptors that capture light to regulate non visual processes such as day night rhythms and the narrowing and widening of the pupil. To capture light, these special cells called intrinsically photoreceptive retinal ganglion cells, require pigments called melanopsins, similar to the opsins that the rod and cone cells use for turning light into vision. While cones and rods have been extensively studied, much less is known about the melanopsin expressing ganglion cells, although they appear to function more like the photoreceptors found in invertebrate eyes. This study will generate novel mouse models that will allow the study of these cells in vivo and in vitro. Determining how the intrinsically photosensitive ganglion cells work may allow the treatment of disorders such as sleep disorders and seasonal depression.

? DESCRIPTION (provided by applicant): The objective of this proposal is to examine the role of the intrinsic circadian clock in glial cells on the generation of body circadian rhythms and regulation of brain redox homeostasis. Glial cells have emerged as key players in brain information processing given the extensive neuronal-glial and glial-neuronal communication that takes place at central synapses. Glial cells also express an intrinsic molecular clock and release the neuromodulator ATP in a circadian fashion. Also disruption of glial signaling in fruit flies severely disrupt behavioral circadian rhythms. Collectively, these data suggest that glial cells and their intrinsic circadian rhythms have an important role in modulating circadian rhythmicity of central and peripheral clocks. The focus of this exploratory proposal is to examine mice in which glial cell circadian rhythmicity is ablated to examine such hypothesis. Experiments in Specific Aim 1 will examine the circadian rhythmicity at cellular and at whole animal level from mice in which the canonical clock gene Bmal1 is selectively disrupted in astrocytes. Experiments in Specific Aim 2 will examine the oxidative damage and generation of reactive oxygen species in brains of mice in which expression of Bmal1 is selectively ablated in astrocytes. These experiments will explore the role of the intrinsic circadian clock in glial cellsin behavioral and cellular rhythmicity and its role in neuroprotection in mammals. Results from proposed studies may challenge the dogma that the central pacemaker is exclusively driven by neuronal networks by providing the direct experimental evidence that glial intrinsic clock plays a vital role in circadian rhythmicity.

PROJECT SUMMARY/ABSTRACT The overall goal of this research is to better understand how astrocytes in the medial subnucleus of the central amygdala (CeM) modulate conditioned fear behaviors. We recently found using brain slices that exogenous activation of astrocytes in the CeM powerfully regulates synaptic transmission and decreases CeM neuronal activity. However, critical gaps remain in identifying the role of CeM astrocytes in vivo, their contribution during fear conditioned behaviors, and the neurochemical pathways that couple the concerted activity of neuronal and astrocyte networks in the CeM. The goal of this proposal is therefore to address these gaps in knowledge by performing measurements of astrocytic and neuronal activity in the amygdala in freely, behaving mice using optical and genetic methods. In Aim1 we will record the activity of astrocytes in the CeM during the acquisition, expression and extinction of cued fear conditioned behaviors. They will provide the first physiological evidence that astrocytes are specifically and differentially activated during specific phases of conditioned fear responses. In Aim 2 we will determine whether CeM astrocytes have an endogenous role in modulating amygdala-driven responses. It will also further test the role of astrocytic endocannabinoid signaling in these behaviors. In Aim 3 we will determine the cellular and behavioral consequences upon exogenous activation of CeM astrocytes using chemogenetic and optogenetic methods. In summary, the proposed studies will provide a much more detailed picture on the role of astrocytes in the central amygdala and extend our recent ex vivo amygdala slice studies. This knowledge will pave the way for new pharmacological strategies to treat anxiety/fear disorders, by taking advantage of the uncommon profile of receptor expression of astrocytes to regulate their activity. Therefore, our studies will contribute to generate a new strategy to treat neurological diseases, such as anxiety, post-traumatic stress syndrome by targeting a relatively understudied cell type in the amygdala.