ASTROCYTIC REGULATION OF NEURONAL EXCITABILITY IN VIVO

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It is our conviction that if we are to understand the role of astrocytes in brain, these cells must be studied in the complex geometry, cellular and chemical milieu of brain. While cell culture experiments indicate that astroglia have properties that would enable them to influence neuronal excitability and participate in brain function, there is virtually no evidence that perturbing astrocytes in vivo affects brain function. It is very likely that the void of information supporting a functional role for astrocytes in brain stems from a lack of methods for studying astrocytes in vivo. This proposal has two major goals. First, to develop a model system whereby the properties of astrocytes can be systematically perturbed in vivo such that the role of these properties in brain physiology can be assessed. Second, to test the hypothesis that disruption of gap junction communication between astrocytes leads to increases in extracellular K+ and attendant increases in neuronal excitability. One of the more important hypotheses concerning neuronal-astrocyte interactions is that astrocytes regulate neuronal activity through their ability to maintain extracellular K+ within the narrow limits required for normal neuronal activity. This process is referred to as spatial buffering and is thought to be accomplished by the uptake of K+ through inward rectifying K+ channels and its dissipation into an astrocytic syncytium formed by gap junctions. All indications are that small increases in extracellular [K+] in brain markedly increase neuronal excitability and that this increase in excitability can lead to seizure activity and/or excitotoxicity. To perturb astrocytic properties in vivo, DNA constructs designed to knock-down the expression of specific astrocytic gene products will be delivered into the CA1 and CA3 regions of the hippocampus using adenoviral and adeno-associated viral vectors. These vectors have been reported to transduce brain cells with high efficiency and stability. We will focus our studies in the substratum radiatum of the hippocampus where CA1 and CA3 pyramidal cell dendrites receive excitatory input and are known to be embedded in an astrocyte syncytium. These regions have been used extensively to study neuron excitability, LTP, seizure activity and excitotoxicity. Our long-term goal is to fully understand the role that astrocytes associated with synapses in CA1 and CA3 s. radiatum play under normal and pathological conditions. Four testable hypotheses will be examined. First, that adenoviral and/or adeno-associated viral vectors can be used to transduce astrocytes in vivo. Second, that gene constructs can be used to knockdown gap junctional communication in vitro. Third, that knock-down constructs placed into either adenoviral or adeno-associated viral vectors and injected into the hippocampus reduce gap junction communication in vivo. And fourth, that constructs which knock-down gap junction communication between astrocytes will increase extracellular [K+]and neuronal excitability in situ and in vivo.