Harold K. Kimelberg - US grants

The serotonin signalling system is involved in many cortical brain functions. Its effective extracellular concentrations are controlled by release and uptake systems. In this proposal we plan to study further the high affinity, fluoxetine-sensitive serotonin uptake system we have previously described in primary astrocyte cultures. We will now focus on its development as a function of the age of the animals from which the cultures are derived (E16-P6), to see if we can get an indication when this property is developmentally established in situ. We will also further examine the influence of serum and different defined factors, since we have found that fetal bovine serum increases serotonin uptake in astrocyte cultures. We will attempt to identify the factor or class of factors in serum responsible. We will also examine the effects of co-culture with neurons to see if neuronal influences can stimulate the serotonin uptake system. These studies should give us some clue as to the mechanisms of induction of serotonin uptake. To find evidence that fluoxetine-sensitive serotonin uptake is present in mature astrocytes in situ we will evaluate uptake in astrocytes prepared by density gradient centrifugation or tissue-print techniques, and do further studies to localize uptake sites in situ after intraventricular injection of [3H] serotonin. Uptake will be measured using [3H] serotonin for astrocytes isolated by the density gradient method, and autoradiography to study uptake both in these cells and cells obtained by the tissue-print method. Autoradiography enables uptake to be studied at the single cell level and will be coupled with immuno-staining. We will see if fluoxetine- sensitive serotonin uptake occurs in type 1 and type 2 astrocytes in culture and will use other markers for oligodendrocytes, neurons and microglia to specifically identify all the cells that may take up serotonin in mixed cultures. We will extend our studies to evaluate the presence of serotonin receptors in cultured and bulk-isolated astrocytes as a function of the same conditions used to study serotonin uptake. There are only a few very preliminary reports on the presence of serotonin receptors in primary astrocyte cultures and because of the importance of these receptor systems in the brain and the numerous receptor systems already convincingly demonstrated on astrocytes in culture, the existence of any of the multiple subtypes of serotonin receptors needs to be critically evaluated. We propose that some of the variability seen in the other studies could be due to effects of different growth conditions. These studies should enable us to say when uptake systems develop in astrocytes, what influences this development and whether and how serotonin receptors are localized in astrocytes. From a broader viewpoint they should provide information needed to critically evaluate what role astrocytes may play in the all-important CNS serotonin signalling system.

Our objectives are to study the basic ion transport processes involved in th swelling of mammalian brain astrocytes, using characterized primary astrocyte monolayer cultures from neonatal rat brains. We will study the cells' responses to exposure to hypo- and hypertonic medium to characterize the ion transport processes involved in volume control. We will also study the effects of possible endogenous effectors of astrocytic swelling under isosmotic conditions, such as lactic acid, L-glutamate, norepinephrine and raised [K+]o, on ion transport, cell volume and intracellular pH. We will use different radioactive tracers to measure ion transport, cell volume and intracellular pH in the attached monolayer cultures. We will use standard electrophysiology to monitor membrane potential and resistance changes, and chloride ion specific microelectrodes to directly study rapid changes in the intracellular activity of this ion. Using detached cells we will follow light scattering to look at rapid volume changes and different fluorescent probes to study membrane potential, intracellular pH and [Ca2+]i under comparable conditions, using a flow cytofluorometer. Astrocytes comprise 20-30% of the mammalian central nervous system (CNS). Classically they were relegated to the role of nerve glue or "neuroglia" and even after their cellular identity was established around the turn of the century they were still considered to be analogous to connective tissue with unspecified supportive roles for neurons. It has become clear that their properties are extremely complex, and their ion transport properties appear to be particularly important for their function. The studies should advance our knowledge in this area and add to an understanding of the role(s) in the mammalian brain of these little-studied cells.

biomedical equipment purchase;

The aim of this proposal is to continue our work on volume regulation in astrocytes. We will examine the causes, regulation and consequences of astrocytic swelling, a feature common to many pathological states. As a model system we use a well- characterized in vitro model of astrocytes; primary astrocyte cultures prepared from neonatal rat brain, and we swell them using hypotonic medium or addition of putative endogenous effectors. In this manner we can isolate the swelling and volume regulation processes and examine the direct effects of activators or inhibitors without the problems and limitations associated with in situ studies. We will use tracer methods or an extracellular electrical impedance system to study the magnitude, and high voltage electron microscopy to study as [3H] taurine, [3H] glutamate and [3H] D-aspartate of high pressure liquid chromatography to study the authentic processes will be done. Electrophysiology, specifically membrane potential and whole cell voltage clamp will also be performed. Fluorescent probe methods will be used to study intracellular Ca2 and pH during swelling and immunocytochemistry and protein chemistry to study any possible cytoskeletal changes. Finally, effects of hypotonic medium on efflux of glutamate and taurine from brain slices will also be done. The overall hypothesis to be tested is that astrocytic swelling in pathological states is deleterious and by preventing such swelling we can prevent neurological deficit or death in conditions such as trauma, ischemia and hypoxia in which astroglial swelling is known to occur. One mechanism we wish to test is that astrocytic swelling leads to the release of potential neurotoxic compounds such as L-glutamate present in astrocytes in relatively high amounts and that a class of inhibitors, already shown by us and others to be effective, in improving or preventing death from experimental head trauma or a model of Reye's syndrome, acts by preventing massive efflux of glutamate from swollen astrocytes.

This center will study the potential role of astrocytes as mediators of extracellular environment in the early stages following brain injury. The center will also develop clinical indices of cerebrovascular pathophysiology which will be useful in the valuation of therapies. Also, an attempt will be made to demonstrate a cause and effect relationship between specific aspects of astroglial function and cerebral blood flow regulation abnormalities following injury. Projects are: 1) "Release of cytotoxins and vasoactive agents from astrocytes." This project will test the hypothesis that blockage of anion transport inhibits swelling of astrocytes and/or release of excitotoxins from astrocytes. A second hypothesis will examine the effect of ethanol on astrocytes. A third hypothesis will involve adenosine, H+, and K+ release from astrocytes. 2) "Mechanisms of injury and protection by anion channel inhibition." This project will test hypothesis concerning the mechanisms whereby anion channel inhibition ameliorates brain injury by histopathological techniques. A second hypothesis will concern the effects of ethanol levels on respiratory control after head injury. 3) "Assessment of brain oxygenation by continuous jugular venous oximetry". These clinical studies will attempt to verify new techniques for quantifying the extent of metabolism and blood flow malregulation both temporally and regionally in severely injured patients. We will determine if arterial-venous oxygen saturation differences can be used to continuously estimate cerebral oxygenation status. A goal is to establish parameters that can be used as endpoints for therapy assessment. The core will consist of an administrative office (Core Unit A), and a data center and statistical computer facility (Core Unit B). Overall goals are to establish the means for conducting clinical research on head injured patients and to develop and test specific hypotheses both in vitro and in vivo regarding the mechanisms whereby secondary injury occurs.

DESCRIPTION (Adapted from Applicant's Abstract): Astrocytes are well-known to have almost a full complement of receptors for neurotransmitters. Much of this work, however, has derived from studies on astrocytes in primary cultures, and it remains unclear whether, when or where these receptors are present on astrocytes in situ. The aims of this proposal are to use astrocytes within a few hours of isolation (acute isolation) from different brain regions of postnatal 1 day to older rats, to more directly address these questions. We have been able to prepare acutely isolated preparations in our laboratory, using enzymatic or mechanical dissociation and trituration onto coverslips, and have shown that they can be used to determine the presence or absence of receptor systems linked to ion channels or intracellular Ca2+ increases. The large number of receptors exhibited by primary astrocyte cultures and the key role that receptors and transmitters play in a vast number of neurological disorders, makes it necessary to clearly establish which ones exist on astrocytes in situ and their properties and functions. Studies on astrocytes in slices using conventional or confocal microscopy with electrophysiological or fluorescence techniques are alternative approaches with different advantages and disadvantages. Immunocytochemistry or other visualizing techniques, combined with different levels of microscopic analysis provides important but static data. The acutely isolated cells complements these other approaches. The receptors to be studied will be ATP (P2), adenosine (P1), glutamate and serotonin receptors, and will be studied as a function of the age of the animal and several brain regions. Intracellular changes in (Ca2+) will be studied by microspectrofluorometry, and receptor-linked changes in K+ and C1 channel activity by patch-clamp, single-channel techniques. Up-regulation in culture of receptors not found in the acutely isolated cells will be checked by culturing the cells in serum-containing or serum-free chemically supplemented medium. We have evidence that such up-regulation occurs for both 5-HT2A and P2Y receptors, which are not seen on acutely isolated astrocytes. The problem of false negatives, i.e., receptors not found because of possible damage during isolation, will be approached by immunocytochemistry for the membrane receptor protein, checking that the same cell gives a Ca2+ response to other transmitters, checking for whether cells other than astrocytes in the preparation do show that receptor response, and by use of different enzymes or mechanical dissociation.

DESCRIPTION (Adapted from applicant's abstract): Our major hypothesis for the last funded period was that reversal of EAA transporters and activation of Volume-Regulated Anion Channels (YRACs) are major sources of EAAs in rat cerebral ischemia and that inhibition of these routes of release would be neuroprotective. In support of the first part of this hypothesis, we found that elevated extracellular [K+] induced EAA release due to both reversal of the EAA transporter and activation of VRACs in primary asfrocyte cultures. In vivo, microdialysis studies in a rat temporary global ischemia model established that application via a microdialysis probe of dihydrokainaxe, an inhibitor of the astrocyte-specific EAA transporter GLT-1, or DNDS an anion channel inhibitor, led to potent suppression of EAA levels during the ischemic episode. If applied together these compounds reduced EAA levels in ischemia by over 80 percent. To check whether inhibition of VRACs is neuroprotective, we chose, on the basis of its high blood-brain barrier permeability, the estrogen receptor antagonist/agonist tamoxifen (TAM) that is also an efficient inhibitor of VRACs in vitro. In the rat middle cerebral artery occlusion model (rMCAO), 5 mg/kg TAM reduced infarction volume by up to 80 percent if applied just before the 2 hour ischemic episode or 3 h after initiation of ischemia. We propose to continue these studies along two lines. One will be devoted to molecular identification of VRACs and intracellular signalling events involved in the volume-dependent release of EAAs in primary astrocyte cultures. Our hypotheses for this part of the project are that more than one VRAC is involved in volume-dependent amino acid release, one or more of these channels are incorporated in calveolae signaling complexes and calmodulin and tyrosine kinases are involved in their volume-dependent activation. The second line of the study will be to explore the molecular mechanisms of TAM neuroprotection and evaluation of its therapeutic window with different dosages and with different durations of reversible middle cerebral artery occlusion. Our hypothesis here is that TAM is highly neuroprotective in rMCAo because it has multiple protective effects. These include inhibition of VRACs, suppression of Ca24 about/calmodulin-dependent nitric oxide production, and/or antioxidant action. We also cannot exclude that some portion of the protection may also be mediated by brain estrogen receptors. This second part of the project will test all these possibilities in animal studies. Both aspects of the project will add new basic knowledge on VRACs, with the potential for understanding their functions in the brain. The second half of the project that deals with neuroprotection has direct potential clinical implications, as TAM is known to be well tolerated in humans being widely used for breast cancer treatment.

LAY SUMMARY; ION CHANNELS in HIPPOCAMPAL ASTROCYTES. Harold K. Kimelberg and Min Zhou

Voltage gated ion channels for the small cations sodium (Na+ ) and potassium (K+ ) are responsible for the wave of depolarization that travels down the neuronal axon and excites other neurons to which it is linked by synapses, where the depolarization releases neurotransmitters which rapidly diffuse across the synaptic cleft to stimulate the next neuron, and so on,. The formation of circuits by such connected neurons are thought to underlie how the brain works. The properties of the Na+ and K+ ion channels in neurons have been extensively studied. The other major type of brain cells are called glia and they are not excitable. Current studies have now shown that these cells can contain the same type of voltage sensitive sodium and potassium channels as are found in neurons, and also voltage dependant chloride channels. This is surprising as the function of these channels is thought to be to modulate neuronal excitability and the glia are unexcitable.
This project will use a freshly isolated preparation of one of the main kinds of glial cells the astroglia to identify what ion channels are present in these cells and to help understand their functions. Our work to date shows that this preparation can be divided into two different classes of astroglia based on a different repertoire of channels. Knowledge of the detailed properties of these channels will give us important clues as to their functions. We will test one hypothesis that one type of astrocyte but not the other has K+ channels and Cl- channels designed to allow it take up K+ plus Cl- when K+ is released via the outward rectifying K+ channel on neurons during impulse conduction. (293 words).