The Section on Cell Biology and Signal Transduction studies the cellular mechanisms of calcium signaling by glial cells in the nervous system. Glial cells and neurons are in intimate communication with each other during central nervous system development and normal brain function. Glial cells monitor and respond to neural activity by conditioning the extacellular milieu, signaling within glial cell networks, and sending signals back to neurons. Such signaling takes the form of propagated Ca2+ waves that spread over long distances in response to synaptic activity. One of our objectives is to understand the processes that support temporal and spatial characteristics of Ca2+ signals within 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.

Wave Amplification Sites
Yagodin,a Sheppard, Simpson,c Russell
In previous work using astrocytes and oligodendrocyte progenitors (OP cells) in culture, the section found that wave propagation is achieved by regenerative Ca2+ release at wave amplification sites, which are specialized Ca2+ release sites found 5 to 7 micrometers apart along cellular processes. The 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 socalled signaling rafts allows for the enhanced Ca2+ release at these sites. Signaling rafts, in addition to supporting long-distance wave propagation, provide for locally discrete Ca2+ signals, which last for only very brief periods (Figure 13).

Figure 13

Local Ca2+ Release, Ca2+ Sparks
Haak, Russell
Using confocal microscopy, the section has recently begun a functional characterization of the signaling rafts in OP cell processes. The aim is to measure the kinetics of elementary Ca2+ release events, Ca2+ sparks and Ca2+ puffs. These are the smallest units of local Ca2+ release, presumed to be from clusters of IP3Rs on the ER. Our studies will allow us to test the relative contribution of the different components of the signaling rafts, including mitochondria, in the regulation of Ca2+ release at the site. A second goal of our investigation is to identify the ion channels that underlie the local Ca2+ release process. Preliminary experiments show that ryanodine receptors (RyRs) and IP3Rs interact in OP cells during agonist-evoked Ca2+ release.

IP3R Distribution in situ
Holtzclaw, Stevanovic-Popovic, Russell
Our current work is focused on immunohistochemical studies to identify the IP3R isoform expressed in different glial cells in situ in both adult and developing rat brains. The data show that, throughout the adult rat brain, astrocytes express only the type 2 IP3R, suggesting that all astrocytic Ca2+ waves may depend on IP3R2 ion channels (Figure 3). In addition, Jelena Stevanovic-Popovic is studying the distribution, biogenesis, and motility of mitochondria in OP cell processes, with a focus on understanding the interactions between mitochondria and ER membranes in the signaling rafts. This work forms part of her graduate thesis work at the Department of Neurosciences, University of Maryland. Organelle-specific dyes, targeted expression of green fluorescent protein–tagged DNA constructs in mitochondria, and vital microscopy are used for studying mitochondrial dynamics.

Molecular Characterization of Signaling Rafts
Holtzclaw, Russell
To test the hypothesis that macromolecular components of the rafts are tethered together by molecular scaffolds, the section is also investigating the molecular composition of signaling rafts. Recent data suggest that some members of the annexin family of proteins may be involved in the functional organization of IP3R oligomers. Furthermore, the molecular scaffold homer binds to IP3R and enhances channel open probability. We are currently identifying the individual proteins in the scaffolds and the functional consequences of their interactions. High-resolution proteomic technologies will be applied to the analysis.

A long-term goal of the section 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, we are developing methods 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