Visualising the role of presynaptic calcium in hippocampal circuits using a novel, genetically encoded calcium sensor
thesisposted on 21.05.2018, 10:34 by Ibrahim Mahmoud Shaban Al-Osta
In this project we used a combination of electrophysiology and fluorescent imaging to monitor synaptic transmission and calcium signalling in synaptic terminals. The study as a whole intended to examine how presynaptic calcium contributes to normal synaptic transmission within different hippocampal neuronal pathways. To this end, we used a transgenic mouse strain known as SyG37 that stably expresses a calcium sensor, SyGCaMP2-mCherry that is expressed in subsets of CNS neurones under the control of the Thy1 promoter. Our findings indicate that this new ratiometric sensor, in the SyG37 mouse strain, provides an excellent tool for detecting neural activity in acute brain slices. First, we showed that evoked calcium transients can be detected in acute brain slices prepared from SyG37 mice where electrical activation of Schaffer collaterals or mossy fibres elicited large calcium transients in area CA1 and CA3, respectively. Using immunohistochemical techniques, SyGCaMP2-mCherry co-localised with presynaptic proteins such as Bassoon, VGLUT1 and VGAT, confirming that it is expressed presynaptically in both excitatory and inhibitory terminals. Blocking fast glutamatergic and GABA/Glycinerergic transmission reduced the size of calcium transients in CA1 and CA3 by only 25 and 20% respectively indicating that the majority of the signals originated from first order presynaptic terminals. Pharmacologically, manipulating the adenosine receptor signalling pathway showed that the actions of adenosine, via the A1 receptor subtype, were different in the CA3 region compared to those in CA1. Forskolin also caused a small, concentration dependent effect on SyGCaMP2 fluorescence in response to electrical stimulation within both CA1 and CA3 regions with pronounced effects on field potential recordings. Together, with this SyG37 strain of transgenic mouse, it is possible to detect neuronal activity with fast temporal and high spatial resolution without the need for pre-incubation with organic calcium dyes or invasive viral transduction procedures.