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.
We aim to understand the nature of these signals in glial cells. In order to achieve 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 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 YC3.60positive 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 antiS100βand antiGFP antibodies in the cerebellum showed that 78 percent 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 64.845 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. In six slice preparations, the stimulus-evoked YFP/CFP ratio increase occurred in 77.216.9 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 spread as a wave through the cell. The glial microdomains of high activity are reminiscent of previously described, small (less than 2m) 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. This work has since been published. 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 potentialdependent 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 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. 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.
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