Receptor Mediated Calcium Signaling In Glia And Neurons

Child Health And Human Development

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Abstract

Glial cells monitor and respond to neural activity by conditioning the extacellular milieu, signaling within glial cell networks as well as by sending signals back to neurons. Unlike neurons, which use electrical signals to communicate, glial cells possess a form of Ca2+ based excitability, where they generate and propagate intracellular Ca2+ signals as waves over long distances in response to monitored synaptic activity. We aim to understand the principles and mechanisms that govern intracellular calcium signals in glial cells and neurons. One objective is to understand processes that support temporal and spatial characteristics of Ca2+ signals within cells and between cells. A second objective is to understand the precise nature of glial cell signals in response to neuronal activity and the consequence of such signals to CNS function. In previous work, we developed a detailed description of the mechanism of Ca2+ wave propagation within glial cell processes. We found that wave propagation was saltatory due to the underlying regenerative Ca2+ release process. Regenerative Ca2+ release occurs at wave amplification sites, which are specialized Ca2+ release sites found 5 to 7 micrometers apart along cellular processes. These sites are characterized by high-density patches of endoplasmic reticulum (ER) proteins such as the inositol 1,4,5-trisphosphate receptors (IP3Rs), sarco-endoplasmic reticulum calcium pumps, calreticulin and at least one mitochondrion in close association. This specialization of signaling rafts allows for the enhanced Ca2+ release at these wave amplification sites. In addition to supporting long distance wave propagation, these specialized sites also provide for locally discrete Ca2+ signals. Current research is focused on characterization of the local Ca2+ release sites with very high spatial and temporal resolution using confocal microscopy. The aim is to measure the kinetics of elementary Ca2+ release events. These are the smallest units of local Ca2+ release, presumed to be from clusters of IP3R ion channels on the ER, and are known as Ca2+ sparks and Ca2+ puffs. Such characterization of the kinetics will allow us to test the relative contribution of several different independent cellular processes that regulate the gating properties of the ion channel. We are particularly focused on the role of mitochondria in the regulation of IP3Rs, since mitochondria are always found in close proximity of these ion channels. Changes in mitochondrial membrane potential and intra mitochondrial Ca2+ concentration associated with cytosolic Ca2+ signals are also being investigated. A second goal of this investigation is to identify the ion channels that underlie the local Ca2+ release process. Preliminary experiments show that ER in OP cells contains both IP3Rs and RyRs. We are using pharmacological tools to delineate if the ion channels influence each other's gating. Such interactions are likely since the receptor clusters are found in close proximity in wave amplification sites and since both ion channels require Ca2+ for activation but are also inactivated at high concentrations of Ca2+. Our experiments show that indeed RyR activation modulates kinetics of Ca2+ release through IP3R ion channels. In addition to these functional parameters, we are also studying the morphological correlates of the Ca2+ release function. The distribution of different IP3R isoforms in CNS cells (Glia and neurons) is being studied in both adult and developing rat brains. Fluorescence based immunocytochemistry and high-resolution confocal microscopy are being used in conjunction for this purpose. In collaboration with Dr. Carmen Mannella at the high voltage electron microscopy facility in Wadsworth Medical Center, Albany, NY, we are investigating the relationship between ER membranes and mitochondria in oligodendroglial processes. In addition, other molecular components of the signaling rafts including the scaffolds that hold important members together and anchor them to different subcellular structures are being characterized. Our long-term goal is to describe in detail the nature of communication between neuronal networks and glial cell networks. We are developing experimental models to investigate the physiological consequences of glial cell signals in response to neuronal activity. For this purpose, methods are being currently developed to image glial cell processes in brain slice preparations. The aim is to monitor cell shape and volume changes associated with neuronal activity. Impaired glial cell signaling has been implicated in a number of pathological states in the CNS such as excitotoxicity, brain edema and certain degenerative diseases. It is hoped that a detailed understanding of the glial cell signaling modes will be useful in understanding the pathophysiology of such conditions.