Kevin L. Behar, M.Phil, Ph.D. - US grants

The concentration of intracellular free calcium ((Ca2+)i) is a key element in the modulation of a host of cellular processes. Increases in (Ca2+)i during ischemia have been implicated in the mechanisms leading to cell death. 19F and 31P nuclear magnetic resonance (NMR) spectroscopy will be used, in conjunction with fluorinated calcium (5-FBAPTA) and intracellular pH (pHi) indicators (di- and tri-fluoromethyl alanine), to measure (Ca2+)i, high energy phosphates, inorganic phosphate, and pHi in the cat brain in vivo. A ventricular method of perfusion will be developed to administer the membrane-permanent acetoxymethyl esters of each indicator, to determine the optimum loading conditions, and to map the distribution of uptake within the NMR volume by (3H)5-FBAPTA autoradiography. Potential effects of the Ca2+ indicator or cerebral energy metabolism and function will be assessed by the 31P NMR spectrum and electroencephalogram in vivo and by regional ATP measurements in coronal sections. Reversible global ischemia will be produced by intrathoracic clamping of the vessels supplying the brain following the intracerebral loading of the Ca2+ and pHi indicators. The effects of elevated inorganic phosphate and low pHi on (Ca2+)i will be assessed during hypercarbia, a condition where energy failure does not occur. The effects of ATP depletion and high inorganic phosphate, without an accompanying change in pHi, will be assessed during insulin-induced hypoglycemia following electrocerebral silence and in the recovery period after glucose administration. The results of this work will provide new information on the concentration of intracellular Ca2+ in vivo, and how its control is related to the energetic status of the brain. This knowledge should lead to a better understanding of the role of calcium in the etiology of brain damage.

DESCRIPTION: (Verbatim from the Applicant's Abstract) For the last 10-15 years our laboratory has had a major role in the development and use of new spectroscopy and imaging techniques in vivo to study the regulation of pathways of brain glucose metabolism. Over the last 4 years of this grant we reported a number of important findings which indicate that release of glutamate and GABA from neurons and their cycling between neurons and glia has a major influence on brain energy metabolism and that the isoforms of a key enzyme of GABA synthesis, glutamic acid decarboxylase (GAD) mediate different proportions of total GABA synthesis. Our central hypothesis is that specific GAD isoforms and supply routes of glutamate carbon play key roles in regulating GABA synthesis and GABA neurotransmitter cycling. We propose the following specific aims: 1a) Determine the relationship between the rates of GABA synthesis, GABA neurotrasmitter cycling, and glutamate/glutamine cycle over a wide range of cortical metabolic activity. 1b) Assess the role of cofactor interaction and phosphorylation of the GAD isoforms in the regulation of GABA synthesis and GABA/glutamine cycling flux. 2) Investigate the role of GAD isoforms in regulation of GABA cycling flux through the two glutamate precursor pathways using GABA-transaminase inhibition and GABA elevation to selectively alter GAD isoform composition. 3) Quantitate the key metabolic pathway fluxes that supply glutamate precursors for GABA synthesis and GABA/glutamine cycling. 4) Determine the relationship between vesicular and non-vesicular GABA, precursor glutamate pathways, and GAD isoforms using diffusion-sensitized MRS in vivo. Using Magnetic Resonance spectroscopy (MRS) and 13C-labeled isotopes in vivo, and enzymatic assays in vitro with pharmacological and molecular biological interventions, we will examine the role of GAD isoforms and glutamate precursors in GABA synthesis and GABA neurotransmitter cycling The uniqueness of this project derives from i) the combination of the these techniques, ii) the track record of the investigator in addressing such questions using MRS, and iii) it complements ongoing clinical investigations of GABA and glutamate metabolism in epilepsy and neuropsychiatric disorders.

magnetic resonance imaging; nervous system; trauma; growth factor; biomedical resource;

DESCRIPTION (provided by applicant): Glucose is the primary substrate of the brain and most of this is oxidized in neurons providing the energy required for functional activity. Under certain conditions, such as chronic hypoglycemia, substantial oxidation of other blood-borne substrates (monocarboxylic acids) can occur. The extent to which alternate substrates can replace glucose in support of brain function however is not known. This issue is of importance in the treatment of type-1 diabetes where hypoglycemia can compromise cognitive and neurological function. Studies in this laboratory have shown that neuronal glucose oxidation in the cerebral cortex is coupled to glutamate/GABA/glutamine neurotransmitter cycling between neurons and astroglia in a near 1:1 stoichiometry. This finding supports a mechanistic model which leads to specific predictions about the fundamental role of glucose in the support of neurotransmitter cycling and the role that alternate substrates could play as oxidative fuels. The central hypothesis of this proposal is that astroglial glycolysis is required for functional support of glutamate/GABA cycling but that neuronal glucose oxidation can be replaced by other fuels. Prolonged exposure to recurrent-hypoglycemia leads to up-regulation of alternate fuel oxidation but does not replace the obligatory need for glucose. The work will involve the application of state-of-the-art In Vivo Magnetic Resonance Spectroscopy (MRS) in conjunction with intravenous infusions of 13C-labeled substrates. We will assess the effects of increasing oxidation of monocarboxylic acids (3-hydroxybutyrate, lactate, and acetate) on the relationship between the rates of glucose utilization and neurotransmitter cycling of glutamate and GABA in the rat neocortex. The studies will be conducted in physiological normal rats and in a rat model of recurrent-hypoglycemia where alternate substrate oxidation is believed to be up-regulated. Results of these studies can be expected to provide fundamental insights into the role of glucose in brain function, the importance of alternate substrate as fuels in chronic hypoglycemia, and the role that these substrates may play in the phenomenon of 'hypoglycemia unawareness'which can arise during intensive insulin therapy in subjects with type-1 diabetes.

DESCRIPTION (provided by applicant): Evidence of altered brain glutamatergic and GABAergic function is reported in a wide array of psychiatric and neurological disorders. Most current treatments for neuropsychiatric illness target the monoamine systems and have limited efficacy. The acknowledgement of this fact has led to an increased drive to develop novel drugs acting through alternative mechanisms. There is now intense focus on the amino acid neurotransmitter systems (glutamate/GABA/glutamine) as targets for treatment, creating the need to identify reliable biomarker assays. In vitro cell culture and brain slice preparations often fail to predict in vivo responses to glutamate- modulating drugs in humans or unanesthetized animals. There is a pressing need for quantitative assays of glutamate/GABA neurotransmission that reflect the in vivo physiological state, avoids anesthesia or postmortem effects, and can be translated more directly to humans. Recently, a novel ex vivo 3D in situ magnetic resonance spectroscopic imaging (MRSI) approach was introduced, which generates high spatial resolution quantitative maps of numerous neurochemicals from the brain's of rodents euthanized by microwave irradiation, preserving neurochemical levels and microstructure. Applied with 13C labeled tracers, high-spatial resolution 2D and 3D maps of 13C-labeled amino acids can be generated. Appropriately validated, rate maps reflecting neuronal (glutamatergic and GABAergic) and astroglial metabolism, and neurotransmitter cycling can be extracted from the data sets. Combined with other neuroimaging modalities (e.g., T1, T2 diffusion-tensor), quantitative measurements of high information content of multiple endpoints for metabolism, structure, and connectivity can be obtained. The addition of other '-omics' end-point measurements are also possible. With all neuroimaging efficiently acquired in the same brain and coordinate space, the proposed assay has significant potential to reveal altered glutamatergic/GABAergic neuronal and glial pathways, accelerating preclinical drug evaluation and treatment response. Development of this methodology would afford investigators opportunity to obtain thousands of precisely defined neurochemical and anatomical data points in a single experiment, in contrast to present methods (e.g., cell-free extracts or tissue slices) which access only one or a few regions at a time. Aim 1 will develop and validate the ex vivo metabolic flux mapping assay against 13C fractional enrichment measured in cell-free extracts, and evaluate the accuracy of the ex vivo flux measurements against in vivo MRS time courses. A double-labeling approach to increase reliability and efficiency will also be evaluated. Aim 2 will develop automated metabolite quantification and analysis methods for the ex vivo 3D MRSI/MRI high density data sets for efficient and unbiased extraction of available information, enhanced data quality, and greatly increased throughput. Aim 3 will apply the ex vivo flux mapping assay to characterize and compare the acute effects of glutamate modulating drugs with a broad range of potential therapeutic effects, on regional rates of glutamate/GABA neurotransmission with receptor activated signaling (phosphoproteins).

? DESCRIPTION (provided by applicant): GABA is the major inhibitory neurotransmitter in the mammalian brain, and dysfunction of the GABAergic system underlies a number of neurological and neuropsychiatric disorders such as depression, schizophrenia and epilepsy. Currently 1H MRS is the only noninvasive method to measure brain GABA in humans. Altered GABA levels detected by MRS occur in a variety of clinical conditions such as depression and epilepsy, and recent reports indicate a strong relationship between GABA concentration and cortical excitability (as assessed by fMRI and gamma EEG) and cortical interconnectivity (as assessed by EEG and resting state fMRI). While the GABA MRS measurement has gained increasing importance as a biomarker in both clinical and basic neuroscience research, key questions about its meaning remain: ? Does the GABA MRS measurement provide an accurate assessment of total brain GABA? ? Does cytoplasmic GABA, which is most likely the primary component of the GABA MRS signal, determine the concentration of extracellular fluid (ECF) GABA, and therefore act as an important mechanism for tonic extrasynaptic GABA inhibition? ? Can the cytoplasmic and vesicular subcomponents of the GABA MRS signal be separately quantitated? ? What is the relation between physiological changes in cellular and extracellular GABA concentration and changes in cortical excitability? We propose to address these questions in a well-established rodent model that allows MRS, fMRI, microdialysis, and electrophysiology to be performed in parallel. In Aim 1 the in vivo MRS visibility of GABA will be determined, and whether the 1H MRS GABA signal can act as a biomarker of extracellular fluid GABA and tonic inhibition, through measurement of ECF GABA, cellular GABA, and cortical excitability. In Aim 2 a novel diffusion-weighted MRS method will be employed to characterize the cytoplasmic and vesicular components to the 1H MRS GABA signal to allow the two components of the GABA inhibition system to be studied in vivo. These aims will leverage our extensive experience and advances in 1H MRS GABA measurements with our strong collaborations with clinical neuroscientists who will provide expertise in microdialysis, electrica recordings, and pharmacological and functional manipulations of the GABAergic system. Successful completion will provide critical methodological validation of the MRS GABA measurement, develop a novel method to differentiate cytoplasmic and vesicular GABA pools, and gain new insight into how physiological and pharmacologically-induced shifts in GABA levels relates to GABA inhibitory function, and thus the meaning of [GABA]MRS as a translational biomarker.