IBN 98-12301 ZECEVIC The main task of functional neuronal networks is to process information and that task depends critically on how exactly signals are integrated by individual nerve cells that are often anatomically and functionally complex. The principles of information processing in single neurons can only be determined by studying specific neuronal types in different experimental preparations. Conventional studies of information processing in individual neurons are limited to electrical recordings from one or two locations. However, regional electrical properties of neurons and their processes are complex, dynamic and very difficult to analyze in the absence of detailed measurements. To achieve better spatial resolution it is necessary to turn from direct electrical recording to optical measurements. Recent new developments carried out on invertebrate neurons, now permit monitoring membrane potential transients simultaneously at many sites by a special type of optical recording with voltage- sensitive dyes. It is of considerable interest to apply this technique to dendrites of vertebrate CNS neurons in brain slices. The long-term objective of this proposal is to advance our knowledge about the logic of information processing in individual neurons from the vertebrate brain using multiple site optical measurements of membrane potential. The first specific task is to apply voltage-sensitive dye recording methodology, as developed for invertebrate neurons, to vertebrate neurons in a brain-slice preparation. Apart from our preliminary experiments, there is no previous experience in this field. To achieve this goal it is planned to: a) integrate the patch-clamp technique that utilizes infra-red (IR-DIC) video microscopy with the apparatus for fast voltage-sensitive dye recordings based on a 464-element photodiode array and b) optimize the intracellular staining protocol using patch pipettes. The experiments in the next phase will focus on hippocampal CA1 pyramidal nerve cells. The main goal is to determine the functional significance of active propagation of action potentials into the dendrites. A particular aspect of this question will be examined, for which multisite optical recording is uniquely well suited. It is important to test the hypothesis, based on computer simulation and indirect experimental evidence, that the dendritic excitability and dendritic action potential amplitude can be selectively modulated in a limited region of the neuron (particular dendritic branches). Such local changes in excitability would functionally subdivide a neuron. The idea is to monitor optically the main dendritic branches of a pyramidal neuron under control and experimental conditions (local, synaptically-evoked subthreshold depolarizations) that are hypothesized to result in local and selective modifications of dendritic excitability and action potential amplitude. The experimental results will be analyzed and interpreted using a biophysically realistic computer model. Results from these studies will have important implications for synaptic plasticity and some forms of associative learning.
