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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 appears 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.

Imaging astrocytic Ca²⁺ signals evoked by neurotransmitters and neuronal activity

In collaboration with James Pickel, we generated transgenic mouse lines expressing YC3.60, a fluorescent Ca²⁺ indicator protein directed to be expressed discretely in astrocytes in the brain and Schwann cells in peripheral nerves by using the human S100β promoter sequence. Three founder lines of mice were bred to homozygosity of which two lines of mice showed astrocytic expression of transgene product in the CNS, while the fourth showed YC 3.60 only in Schwann cells in peripheral nerves. 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 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 and extending into all processes in astrocytes. Dual staining with anti–S100β and anti–GFP antibodies in the cerebellum showed that 78% of S100β-positive Bergmann glial cells expressed YC 3.60.

Glial cell Ca²⁺ signals evoked by exogenous application of neurotransmitter substances (glutamate) and electrical stimulation were detected by the YC3.60 expressed by glial cells in transgenic mice. The average stimulus-induced YFP/CFP ratio change was 64.8±45% in most experiments, although some cells did not respond at all. One or two cells showed very large YFP/CFP ratio changes (over 100%), and the largest response recorded was a 307.11% change.

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 77.2±16.9% 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 spread 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. In addition to astrocytic Ca²⁺ signals following stimulation, spontaneous signals were also readily recorded in brain slice preparations. Both stimulated activity and spontaneous signals were blocked by TTX application (1).

In situ imaging of Schwann cell calcium signals

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. A previous study from our laboratory had showed that proteins involved in Ca²⁺ signaling are concentrated in regions of the Schwann cell around nodes of Ranvier. The paranodal region and the juxtaparanode were rich in ion channels involved in metabotropic Ca²⁺ signaling. This finding suggested that these cellular regions may initiate Schwann cell signals during action potential propagation.

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 this 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. One previously published study had shown 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.

Application of exogenous purinergic agonists to isolated sciatic nerve axons readily elicited Ca²⁺ signals in Schwann cells, 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 receptors 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 is 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.

Future experimental plan

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 similar to calmodulin, the Ca²⁺-binding motif in chicken troponin-C does not bind to the calmodulin-binding proteins in the nervous system of mammals, thus making CerTn-L15 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.

We have initiated a collaborative study with the Department of Orthopedic Surgery, University of California at Irvine (Ranjan Gupta) to investigate Schwann cell signals associated with chronic nerve compression injury. Gupta’s laboratory has developed methods to produce such chronic injuries in mouse sciatic nerves. The collaborative study will aim to expose transgenic mice expressing YC 3.60 in Schwann cells to such injury, in order to record Schwann cell Ca²⁺ signals at various times following injury. We hope to detect acute and chronic Schwann cell signals and will attempt to block these signals using specific receptor antagonists to ameliorate cellular pathology.

Publications

Atkin SD, Patel SS, Kocharyan A, Holtzclaw LA, Weerth SH, Schram V, Pickel J, Russell JT. Transgenic mice expressing a cameleon Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo. J Neurosci Meth 2009 181:212-226.
Besser L, Chorin E, Sekler I, Silverman WF, Atkin S, Russell JT, Hershfinkel M. Synaptically released zinc triggers metabotropic signaling via a zinc-sensing receptor in the hippocampus. J Neurosci 2009 29:2890-2901.

Collaborators

Ranjan Gupta, MD, University of California at Irvine, Irvine, CA
James Pickel, PhD, Laboratory of Genetics, NIMH, Bethesda, MD
Mark Ellisman, PhD, National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA

Contact

For more information, email james@helix.nih.gov or visit http://neuroscience.nih.gov/Lab.asp?Org_ID=265.

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