Imaging Calcium in Hippocampal Presynaptic Terminals With a Ratiometric Calcium Sensor in a Novel Transgenic Mouse

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.

Changes in presynaptic calcium signalling accompany age‐related deficits in hippocampal LTP and cognitive impairment

“…SyGCaMP2 is expressed selectively in excitatory and inhibitory presynaptic terminals (Al‐Osta et al, ). The recorded signals within CA1 can originate not only from Schaffer collateral fibre terminals but also from any presynaptic terminals of neurons that are activated synaptically including intrinsic interneurons or CA1 pyramidal cell axon collaterals.…”

“…Images of mCherry fluorescence revealed the basic distribution of the sensor and show that expression was present throughout the brain but particularly high in the deeper layers of the cortex, the hippocampus, thalamus and fibre tracts, especially those surrounding the hippocampus including the fornix, corpus callosum and fimbria. We have previously shown that within the hippocampus, expression is absent in the cell bodies within the pyramidal cell layer from CA3 to CA1 and subiculum and targeted to both excitatory and inhibitory presynaptic terminals (Al‐Osta et al, ). Relative age‐dependent increases in the SyG:mCh ratio were evident in the hippocampus but also notably in the retrosplenial cortex.…”

“…The generation, expression patterns and characteristics of SyG37 transgenic mice incorporating a SyGCaMP2‐mCherry transgene have already been described in detail (Al‐Osta et al, ). Briefly, SyGCaMP2‐mCherry was created by fusing mCherry to the C‐terminus of SyGCaMP2, which is, in turn, a fusion of GCaMP2 to the C‐terminus of synaptophysin‐1 (Dreosti, Odermatt, Dorostkar, & Lagnado, ).…”

“…Briefly, SyGCaMP2‐mCherry was created by fusing mCherry to the C‐terminus of SyGCaMP2, which is, in turn, a fusion of GCaMP2 to the C‐terminus of synaptophysin‐1 (Dreosti, Odermatt, Dorostkar, & Lagnado, ). A Thy1‐2 promotor was used which produces founder‐dependent neuronal expression that appears from P7‐P10 onward and which is stable throughout adulthood (Al‐Osta et al, ). mCherry was used both to facilitate identification of synaptic boutons and as a reference to allow ratiometric quantification of expression levels and calibration of absolute calcium levels.…”

“…Using a transgenic mouse that expresses a genetically encoded, ratiometric calcium sensor exclusively in presynaptic terminals (Al‐Osta et al, ), we have examined the role of presynaptic calcium in synaptic transmission in the CA1 region of the hippocampus in young adult mice and compared how transmission properties and presynaptic calcium signalling are affected in mice with measured cognitive and synaptic deficits associated with age‐related hippocampal dysfunction. We show that aging is accompanied by elevated calcium influx into individual presynaptic terminals and a chronic increase in absolute residual presynaptic calcium that affects transmission properties in the CA1 region of the hippocampus.…”