Critically Assess The Mechanisms That Mediate Calcium Waves Between Glia And Neurons.

Introduction: Intercellular Ca2+ waves in glial cells were first reported by Cornell Bell et al (1990) [1] closely followed by Charles et al (1991) [2]. These propagating waves of Ca2+ suggest that networks of astrocytes constitute a long-range signaling system within the brain that since has been well investigated. The term glia refers to a diverse set of cell types that carry out distinct functions in neurophysiology. There are four major groups of glial cells: Schwann cells and oligodendrocytes, microglia, NG2-positive glia and astrocytes. Astrocytes on the basis of number, surface area, and volume, are the predominant glial cell type and thus most of the experiments discussed are conducted on this glial subtype and extrapolated to the others. This is an important caveat which should be born in mind throughout this essay. Similar generalizations are made for neurons. In the leech, the astrocyte- neuron ratio is 1:25; in C.elegans 1:6; in rats and mice 1:3 and in humans 3:2. This exponential increase of astrocytes implicates a role for these cells in the evolution of increasingly complex brain functions (Banaclocha, 2007) [3]. Indeed, astrocytes are involved in key aspects of human brain development and function, such as neuronal metabolism, synaptogenesis, homeostasis of the extracellular milieu and cerebral microcirculation. However, in this essay I will focus we focus on the evidence that points towards astrocytes having an important role in synaptic physiology, discussing data that indicates the role that astrocytes play in integrating, processing and regulating synaptic transmission and plasticity through the release of gliotransmitters ( transmitters released by glial cells implicated in rapid glial-neuron and glial-glial communication) between neurons and other astrocytes. I will critically asses the evidence pointing towards the role of astrocytes in cell-to-cell Ca2+ waves, both between other glia and neurons. Cell-to-cell calcium waves between glia: Intercellular Ca2+ waves are believed to propagate through networks of glial cells in culture in one of two ways: by diffusion of IP3 between cells through gap junctions or by release of ATP, which functions as an extracellular messenger. Here I will critically discuss the experimental methods used to determine these findings. Both Cornell Bell et al. (1990)[1] Charles et al (1991) [2] investigated the method of communication between glia using digital fluorescence video imaging. It was found that deformation of the membrane of a single astrocyte with a mechanical stimulus can initiate a cellular response which is communicated to many surrounding cells. It was observed that communication between adjacent cells occurs at specific sites of intercellular contact and the Ca2+ wave often followed a circuitous route throughout a field of the culture further. This evidence suggesting that specific intercellular connections, gap-junctions are required for intercellular communication. However, there is concern about using mechanical stimulation where responses are likely to reflect cellular damage. An alternative approach has been to use caging Ca2+ or IP3. However, there are caveats associated with this approach too which I will state before detail of the experiments is given. Firstly, uncaging either Ca2+ or IP3 within astrocytic cell bodies leads to an intracellular Ca2+ wave that moves rapidly throughout the entire astrocyte from the soma into the processes. This fails to mimic the spatiotemporal dynamics and magnitude of Ca2+ increases normally observed when activating endogenous astrocytic receptors. The second caveat associated with uncaging Ca2+ or IP3 is that this approach does not activate the fabric of endogenous regulators such as PLC, DAG which are normally associated with stimulation of Gq GPCRs. Consequently, alternate signaling cascades participating in astrocytic Gq GPCR responses, which could markedly modify astrocytic responses, are absent when uncaging Ca2+ or IP3. The use of caged IP3 seems to be a better choice over caged Ca2+ because it engages some of the endogenous Ca2+-release mechanisms. Nevertheless, the experiments produced very interestin data. No extracellular rise in Ca2+ was observed suggesting that Ca2+ itself does not appear to mediate intercellular communication and thus another second messenger must travel through gap junctions to propagate the communicated Ca2+ response. Charles et al suggest that this messenger is IP3. In these experiments, mixed glial cell cultures containing different cell types including astrocytes, oligodendrocytes, precursors, and microglia-a cellular heterogeneity similar to that of the central nervous system were used. However, the cell types could not be individually identified in each experiment. This could be an area of criticism as the authors conclude communication occurs between different cell types. However, comparison of the relative percentages of cell types based on immunofluorescence staining and morphologic appearance were used to support their conclusions. Additional evidence for the role of IP3 in glial communication was provided by Sanderson et al (1990) [4]. It was found that iontophoretic injection of IP3 into cultured respiratory tract ciliated cells, evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. This supports the hypothesis that IP3 acts as a cellular messenger mediating communication through gap junctions . Consequently, Charles et al (1992) [5] conducted experiments to observe specifically the role that the most widely expressed gap junction, Cx43 play in Ca2+ signaling using digital fluorescence video microscopy. The C6 glioma cell line used expressed low levels of Cx43 and thus cells were transfected with the cDNA encoding Cx43 (increased mRNA and protein expression of Cx43 were confirmed by the authors). Mechanical stimulation of a single cell in transfected C6 culture was shown to induce a Ca2+ wave that was communicated to multiple surrounding cells. The extent of communication was proportional to the level of Cx43 cDNA expression assessed from degree of bioluminescence produced from luciferase activity which acted as a reporter. In non-transfected cells no such communication was shown. These was thought to provide evidence that intercellular Ca2+ signaling occurs via gap junctions. A question that immediately springs to mind is the extent to which this Ca2+ signaling occurs in the intact nervous system. Technical limitations restricted an immediate answer to this question but recent observations of in intact hippocampal slices demonstrate correlation of the temporal and spatial characteristics of glial Ca2+ signaling in vivo. In order to circumvent the caveats associated with artificially inducing Ca2+ waves, genetically modified mice enable selective activation and inactivation of specific genes such and their proteins. For example, Gq GPCR-mediated Ca2+ signaling in astrocytes was examined in hippocampal tissue-specific KOs of the IP3R2. This model also helped to demonstrated the role of IP3 in the propagation of glial Ca2+ waves within the hippocampus (Petravicz et al., 2008). This explanation of Ca2+ waves was initially challenged by observations that Ca2+ waves could cross cell-free lanes and thus were mediated by an extracellular signal (Figure 1a).