Abstract
Genetically encoded calcium indicators (GECIs) have gained widespread use for measurement of neuronal activity but their low expression levels in transgenic mice tend to limit sensitivity. We have developed a transgenic mouse line (SyG37) that expresses a ratiometric calcium sensor, SyGCaMP2-mCherry, that is expressed throughout the brain but targeted to presynaptic terminals. Within the CA1 and CA3 regions of hippocampus of male and female mice, SyGaMP2 fluorescence responds linearly up to 10 electrical stimuli at frequencies up to 100 Hz and it can detect responses to a single stimulus. Responses in single boutons can be measured using multiphoton microscopy. The ensemble amplitude of SyGCaMP2 responses is a function of the number of stimuli applied and the number of contributing boutons. The peak responses and initial rates of calcium influx in single boutons in CA1 and CA3 were similar but the rate of calcium clearance from CA3 boutons after stimulation was significantly faster. In CA1, DNQX reduced SyGCaMP2 responses to Schaffer collateral stimulation to 86% of baseline indicating that 14% of the total response originated from presynaptic terminals of neurones synaptically driven via AMPA receptors. Theta burst stimulation induced long-term potentiation (LTP) of both SyGCaMP2 and fEPSP responses in both young and 18-month-old mice. The proportion of postsynaptically connected terminals increased significantly to 76% of the total after LTP induction. The SyG37 mouse allows stable optical detection of synaptic activation and connectivity at the single bouton level and can be used to characterize the contributions of presynaptic calcium to synaptic transmission and plasticity.
Highlights
Encoded calcium indicators (GECIs) have gained widespread use for measuring neuronal activity
Both green and red fluorescence were markedly brighter in SyG37 mice compared to WT animals indicating that both the green and red fluorescent protein components of SyGCaMP2-mCherry were expressed. mCherry, whose fluorescence is calcium-independent, and a direct indicator of expression levels, was present throughout the brain but was noticeably high in the subiculum of the hippocampus, deeper layers of the cortex, the hippocampus, thalamus and fiber tracts, especially those surrounding the hippocampus including the fornix, corpus callosum and fimbria
Expression of mCherry was co-localized with GCaMP2 and both fluorophores were present throughout the brain but with high levels of expression in the hippocampus, cortex, thalamus as well as the cerebellum
Summary
Encoded calcium indicators (GECIs) have gained widespread use for measuring neuronal activity. Whereas GCaMP2 lacks the sensitivity to detect single action potentials when expressed non- in drosophila neurones (Hendel et al, 2008), it has been used to detect responses in cerebellar parallel fibers through cell specific expression in granule cells (Díez-García et al, 2005) whose presynaptic terminals have very low volumes. Whereas this approach allowed detection in synchronized bundles of activated fibers, specific targeting of GCaMP2 to presynaptic terminals by fusing it to the extracellular facing terminus of the vesicular protein synaptophysin allowed detection of single action potentials (Dreosti et al, 2009) in single, identified presynaptic terminals. Whilst GCaMP2 is not as bright or sensitive as the more recently developed GCaMP6 family of GECIs, it has a much faster response time to calcium (Chen et al, 2013)
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