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 synaptic activity. This 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.
We aim to understand the nature of these signals in glial cells. Towards this end, 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 S100βpromoter 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 use 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. Glial cell 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 64.845 percent in most experiments, although some cells did not respond at all. One or two cells showed very large YFP/CFP ratio changes (over 100 percent), and the largest response recorded was a 307.11 percent 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. Both stimulated activity and spontaneous signals were blocked by TTX application. This work has since been published. We initiated another 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. 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 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. We imaged sciatic nerves isolated from transgenic mice expressing YC 3.60 in Schwann cells with 2-photon confocal microscopy. We stimulated the nerve bundles with a suction electrode, and recorded compound action potentials during stimulation. 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. 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 agonistelicited 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. Furthermore in recent experiments we showed that action potential propagation elicited by electrical stimulation of isolated sciatic nerve bundles induces a robust Ca2+ rise in Schwann cells particularly in the paranodal region. This action potential dependent Schwann cell Ca2+ response was abolished in the presence of purinergic anatagonists which are specific to P2Y2 subtype of receptors. We are attempting to delineate the cellular mechanism that supports action potential evoked ATP release by axons or the Schwann cells. 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. This work is currently being prepared for publication. 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. Recently, Oliver Griesbeck of the Max Planck Institute in Germany designed a novel indicator protein (called CerTn) 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 series of indicators superior to YC3.60. We are now generating transgenic mice expressing one of this class of indicators, TN-XXL in astrocytes, Schwann cells, and oligodendrocyte progenitors (OP cells). We plan to use the astrocyte specific Glt-1 genepromoter 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 will also use cell-specific expression by with cre recombinasecontaining constructs that target cell-specific promoters. We have initiated a collaborative study with the Department of Orthopedic Surgery, University of California at Irvine (Dr. Ranjan Gupta) to investigate Schwann cell signals associated with chronic nerve compression injury. Dr. Guptas laboratory has developed methods to produce such chronic injuries in mouse sciatic nerves. The collaborative study will aim to impose such injury to transgenic mice expressing YC 3.60 in Schwann cells in order to record Schwann cell Ca2+ signals at various times following injury. We hope to learn acute and chronic Schwann cell signals. We will attempt to block these signals using specific receptor antagonists thereby attempt to ameliorate cellular pathology. Atkin, S. D., Patel, S. S., Kocharyan, A., Holtzclaw, L. A., Weerth SH, Schram, V., Pickel, J. and 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. Neurosi. Met. 2009;181:212-226.

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Russell, James T (2010) Imaging calcium signals In vivo: A powerful tool in pharmacology. Br J Pharmacol :
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Atkin, Stan D; Patel, Sundip; Kocharyan, Ara et al. (2009) Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo. J Neurosci Methods 181:212-26
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