Burrows, Sean
(2021)
Calcium signal propagation in astrocytes.
MRes thesis, University of Nottingham.
Abstract
Astrocytes are glial cells that play many roles in maintaining homeostasis of the central nervous system and modulating both vascular and neuronal networks. Astrocytes express a range of neurotransmitter receptors that allow for communication from the neuron to astrocyte and mediate feedback and modulation of signal transmission at the synapse. Astrocyte calcium signalling caused by receptor activation is responsible for the release of gliotransmitters capable of modulating synaptic activity. The calcium signals of the glia have complex spatiotemporal properties that range from transients, isolated to astrocyte microdomains, to wide intracellular calcium waves capable of propagating within cellular networks. The range that the calcium response covers will determine how many synapses (and blood vessels) could be modulated by feedback from glia. In this study we investigated the factors that control signal propagation in astrocytes using two experimental models: cultured primary astrocytes, and Bergmann glial cells in acutely isolated cerebellar slices.
Initially, primary cultures of astrocytes loaded with Fluo-5F indicator were used to investigate whether ATP release from astrocytes contributed to the calcium response generated by the excitatory neurotransmitter, glutamate. A broad-spectrum antagonist of P2 receptors (PPADS) did not affect either the initial response of astrocytes to glutamate exposure, or the ongoing oscillations in calcium observed during a late stage of continuous stimulation. We next screened purinergic receptor antagonists to identify the receptors linked to ATP responses in the cells, and showed that responses in cultured cells were most sensitive to suramin.
Next, we developed a method to bulk load acutely isolated cerebellar slices with Fluo5F in order to examine Bergmann glial responses to stimulation, without the need for patch clamp recordings. The unipolar morphology of cerebellar Bergmann glia, with long fibres that project microdomains to enclose the synapses throughout the molecular layer of the cerebellar cortex, allows the spatial range of calcium signals to be visualised. Here responses to parallel fibre stimulation were investigated by using spatial and temporal components to isolate the Bergmann glia contribution to the overall fluorescence changes.
Short bursts of electrical stimulation of the parallel fibres at 30-100 Hz in 5-10 pulses reliably generates calcium responses within the Bergmann glia that spread along the Bergmann fibres, and are sensitive to pharmacological inhibition by PPADS (P2 receptor antagonist), CPCCOEt (mGluR1 receptor antagonist) and NBQX (AMPA receptor antagonist). We were able to reproduce these results from single cell recordings using the bulk loading technique, suggesting that the glial signal can be effectively detected from the mixed population of cells in the molecular layer. In contrast to cultured cells, however, suramin had little impact on calcium signals in Bergmann glia, indicating that results from primary culture cannot be generally translated into other, more intact, preparations.
Previous work within our laboratory has shown that stimulation of the parallel fibres with a protocol that induces long term potentiation at the synapse (16 Hz for 15 s) dramatically decreases the spatial range of the Bergmann glia calcium responses generated by the 30 Hz tetanus. This result suggests “spatial plasticity” may be a feature of glial calcium signalling. This depression of 30 Hz responses was also reproduced with the bulk loading method, and we finally used a broad-spectrum serotonergic receptor antagonist, asenapine, to test the hypothesis that serotonergic signalling contributes to the glial spatial plasticity. However, there was no statistically significant effect.
We have successfully validated a less technically demanding method for monitoring the Bergmann glia calcium responses within slices and confirmed the principle for screening signalling pathway candidates that could affect the spatial range and plasticity of astroglial calcium signals. This should accelerate progress in understanding how the pattern of synaptic activity in a neuronal network is related to the range of calcium signals generated in adjacent glial networks.
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