Calcium-Based Excitability in Glial Cells

James Russell, DVM, Head, Section on Cellular and Synaptic Physiology
Lynne A. Holtzclaw, BS, Research Assistant
Sundip Patel, BS, Predoctoral Student

We investigate signaling between neurons and glial cells in the central and peripheral nervous systems. The intimate communication between glial cells and neurons is crucial for normal brain development and seems to play a critical role in the plastic functions of the brain. Glial cells monitor and respond to neural activity by conditioning the extracellular milieu, signaling within glial cell networks, and sending signals back to neurons. In the brain, glial cell responses to neural activity take the form of propagated Ca²⁺ waves that spread over long distances in response to synaptic activity. Similarly, the myelinating glial cells (oligodendrocytes and Schwann cells) receive signals from the axons they myelinate, and such signals are essential for the maintenance of the myelin sheath. One of our objectives is to understand different modalities of cell-cell signaling and the processes that support temporal and spatial characteristics of Ca²⁺ signals within and between cells. A second objective is to probe the nature of glial cell signals in response to neuronal activity and the consequence of such signals to central nervous system (CNS) function.

Ca²⁺ signaling between axons and myelinating glia

Patel

We are investigating aspects of signaling between axons and their myelinating glia (Schwann cells and oligodendrocytes) in the peripheral nervous system (PNS) and CNS. The myelin sheath serves as an electrical insulator; it reduces current flow across the axonal membrane in the internode by lowering capacitance and increasing resistance, thereby facilitating saltatory conduction. Myelin is formed by a highly specialized extension of myelinating glial cell (oligodendrocytes in the CNS and Schwann cells in the PNS) membranes that are intimately associated with axons.

Beyond the concept of saltatory conduction, several studies support the view that the axon/myelin/glial cell ensemble or “nodal complex” operates in an integrated manner during conduction of the nerve impulse. Over the last two decades, numerous anatomical and physiological studies have shown that an intricate set of signals might exist between the components of the nodal complex, although details of signaling between axons and the myelinating glia are not available.

We initiated a study aimed at describing the distribution of proteins involved in Ca²⁺ signaling in the axoglial apparatus. We believed that the study would point us to the spatial discreteness, if any, in the signaling between axons and Schwann cells. Initially, we hypothesized that the signaling is localized to specific contact sites between the two cell types where the insulating layer of compact myelin gives way to non-compact myelin and Schwann cell membrane. We first characterized the distribution of Ca²⁺ signaling proteins in the axoglial apparatus of the nodes of Ranvier.

We performed immunohistochemical analysis with antibodies against major proteins expressed by Schwann cells and axons in a cell-specific manner to determine the anatomical arrangements of the two cell types in the nodes of Ranvier. Using specific antibodies against IP3Rs, RyRs, P2Y₁, M₁, and G_q, we then examined the distribution of proteins involved in Ca²⁺ signaling within the nodes. In support of our original hypothesis, we found a highly concentrated distribution of Ca²⁺-signaling proteins in the paranodal loop regions on either side of the nodes of Ranvier (Toews et al., 2007). We are planning a higher-resolution electron microscopic analysis, to be conducted in collaboration with Mark Ellisman, that uses immunocytochemical techniques to describe the precise co-localization of various proteins in the axoglial apparatus.

Toews JC, Schram V, Weerth SH, Mignery GA, Russell JT. Signaling proteins in the axoglial apparatus of sciatic nerve nodes of Ranvier. Glia 2007;55:202-213.

In situ imaging of Schwann cell calcium signals

Atkin,¹ Patel, Holtzclaw; in collaboration with Miyawaki, Pickel

To measure directly Schwann cell Ca²⁺ signals associated with action potential traffic along the axons they myelinate, we developed transgenic mouse lines expressing a fluorescent Ca²⁺-indicator photoprotein within Schwann cells. We used the S100β promoter to target a mutant chameleon protein, YC3.60, to Schwann cells in peripheral nerves and astrocytes in the brain. We obtained a YC3.60 construct from Atsushi Miyawaki and engineered a plasmid construct containing the S100β promoter in tandem. YC 3.60 is designed to express a protein containing the structures of cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and calmodulin, a Ca²⁺ sensor. When the ambient Ca²⁺ concentration increases, the efficiency of fluorescent energy transfer (FRET) between the CFP and YFP increases significantly and is readily measurable. Transfection of such a plasmid in C6 glioma cell lines and HEK 293 cell lines showed that YC3.60 was expressed not by HEK293 cells but only by C6 glioma cells, which express S100β.

In collaboration with James Pickel, we used the plasmid to generate transgenic mouse founders. We established four founder lines expressing YC3.60 fluorescence in glial cells and bred the mice to homozygosity. We used two-photon confocal microscopy to image brain slice preparations obtained from two of the lines and readily visualized individual astrocytes brightly fluorescent with the YC3.60 chameleon. We observed cells with large spongiform process arbors, as well as smaller cells, containing YC3.60. Many of these cells—large and small—exhibited elongated cell somas with several large branches radiating parallel or perpendicular to those of their neighboring glia. Numerous YC3.60–positive astrocytic processes extended to wrap small blood vessels. We found YC3.60 expression in the cell soma, extending into all processes in astrocytes. Dual staining with anti–S100β and anti–GFP antibodies in the cerebellum showed that 78 percent of S100β-positive Bergmann glial cells expressed YC 3.60.

YC3.60 expressed by glial cells in transgenic mice responded to glial cell Ca²⁺ signals in isolated brain slice preparations and isolated, teased peripheral nerve preparations. The average stimulus-induced YFP/CFP ratio change was 65 percent. In most experiments, one or two cells showed very large YFP/CFP ratio changes (over 100 percent) whereas some cells did not respond at all. The largest response was over 300 percent change, or direct stimulation of neural pathways.

Stimulation of Schaffer collaterals in hippocampal slices evoked robust Ca²⁺ signals in astrocytes in the stratum radiatum and stratum moleculare-lacunosum, as judged by YC3.60 fluorescence change. In six slice preparations, the stimulus-evoked YFP/CFP ratio increase occurred in about 80 percent of 74 cells; in each case, we recorded extracellular field potentials. When we measured Ca²⁺ signals in spongiform protoplasmic astrocytes, stimulus-evoked fluorescence changes were apparent within discrete local regions of the cell. Many of these regions represent glial microdomains within the amorphous spongiform morphology of the cell. The cellular Ca²⁺ response elicited by neural stimulation spreads as a wave through the cell. The glial microdomains of high activity are reminiscent of previously described small (less than 2µm) astrocytic terminal sheaths that enwrap single synapses or groups of synapses.

One of the transgenic mouse lines showed abundant YC3.60 fluorescence within all Schwann cells in the peripheral nerves while astrocytes in the CNS did not contain appreciable fluorescence. We used mice derived from the same transgenic line to investigate action potential–dependent Ca²⁺ signals in Schwann cells. We imaged sciatic nerves isolated from these mice with 2-photon confocal microscopy, stimulated the nerve bundles with a suction electrode, and recorded compound action potentials during stimulation. However, in numerous trials, we found no stimulus-associated Ca²⁺ signals in Schwann cells that myelinate the axons. Given that we cannot state with any certainty that the recorded compound action potential represents stimulation of all axons in the bundle, we are unable to draw clear conclusions without further experimentation. One previously published study showed that, in isolated frog nerve bundles, action potential generation resulted in Schwann cell Ca²⁺ signals, albeit only with stimulation at 50 Hz for many minutes. Similar experiments have not been replicated in mammalian axons.

While action potential–dependent Ca²⁺ signals were not detectable in isolated sciatic nerve bundles, application of exogenous purinergic agonists readily elicited Ca²⁺ signals as revealed by YC 3.60 fluorescence changes. The rank order of potency of purinergic agonists was ATP>UTP>2MeSATP, suggesting the presence of P2Y-subtype purinergic receptor on Schwann cells. Furthermore, these purinergic agonist–elicited Ca²⁺ signals persisted in the complete absence of Ca²⁺ ions in the extracellular medium, suggesting that P2X-type purinergic receptors did not contribute to the signal. In support of this conclusion, prolonged exposure to UTP to deplete intracellular Ca²⁺ stores while abolishing a response to ATP did not evoke a response to a P2X-selective purinergic agonist. Our experiments showed that Schwann cells in situ express a functional p2Y-subtype of purinergic receptor. It is likely that this receptor system might be involved in Schwann cell responses to acute nerve injury. It is well known that, following injury, Schwann cells retract, leading to initial demyelination followed by regeneration.

The strategy of directing expression of Ca²⁺ indicator photoproteins in a cell-specific manner has proven extremely valuable for investigating glial cells’ physiological responses during nervous system function both in isolated preparations and in situ. One drawback in the design of Ca²⁺ indicator proteins has been the use of calmodulin, a ubiquitous nervous system Ca²⁺ sensor found abundantly in all cells. In addition, all nervous system cells have a number of calmodulin-binding proteins that can bind to the indicator in a Ca²⁺-dependent manner and thus interfere with the indicator’s responses. Moreover, the photoproteins CFP and YFP, which were used in YC 3.60, are relatively dim compared with more modern mutant proteins with brighter fluorescent yields. Two such proteins are cerulean (cyan) and venus (yellow). Oliver Griesbeck of the Max Planck Institute in Germany designed a novel indicator protein (called CerTn-L15) in which he replaced the calcium sensor with the Ca²⁺-binding region of troponin-C of chicken muscle. While calmodulin and troponin-C are similar in their Ca²⁺-binding affinity, troponin C does not bind to the proteins to which calmodulin binds in a Ca²⁺-dependent manner. Hence, the troponin C–based sensor CerTn-L15 is superior to YC3.60 in this regard. We are now generating transgenic mice expressing CerTn-L15 in astrocytes, Schwann cells, and oligodendrocyte progenitors (OP cells). We plan to use the glial fibrillary acidic protein (GFAP) promoter to target to astrocytes and the cyclic nucleotide phosphodiesterase (CNP) promoter to target to Schwann cells and OP cells. Once we have generated such transgenic mice, we should have the tools we need to investigate signaling in all three types of glial cells. We are already using the ROSA-26 locus to target the photoprotein, which should yield robust expression in cells, and are ensuring cell-specific expression by using cre recombinase–containing constructs that target cell-specific promoters.

Odling K, Albertsson C, Russell JT, Mårtensson LG. An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva. J Exp Biol 2006;209:956-964.
Weerth SH, Holtzclaw LA, Russell JT. Signaling proteins in raft-like microdomains are essential for Ca²⁺ wave propagation in glial cells. Cell Calcium 2007;41:155-167.

¹Stan Atkin, BS, former Student Training Fellow

Collaborators

Mark Ellisman, PhD, National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA
Atsushi Miyawaki, MD, PhD, RIKEN, Tokyo, Japan
James Pickel, PhD, Laboratory of Genetics, NIMH, Bethesda, MD

For further information, contact james@helix.nih.gov.

Calcium-Based Excitability in Glial Cells

Ca2+ signaling between axons and myelinating glia

In situ imaging of Schwann cell calcium signals

Collaborators

Ca²⁺ signaling between axons and myelinating glia