Calcium Dynamics In Spine By FRET Based-on GFP

Calcium ion, as a second messenger, plays a key role in cellular growth and differentiation. In nervous system, calcium involves in synthesis and release of neurotransmitter, and signal transmission, dendritic growth, and spine formation, as well as in synaptic plasticity. Previous studies indicate that excitatory synaptic activity evokes calcium ion triggers diverse signaling pathways through influx into spine. Some results from other groups show that free calcium ion accumulation in spine is required to induce most forms of long-term potentiation and depression, which are molecular mechanism of memory and learning. Quantitative measurement of calcium dynamics in single spines is crucial to understanding the cellular and molecular mechanism underlying the synaptic plasticity. Confocal microscopy and calcium indicators are used to monitor the physiological calcium signal in spine. Confocal microscopy has predominances in aberration-free image and ideal resolution and contrast. GECI is an endogenetic biomacromolecule, so it affects little calcium dynamics. Ratio imaging can measure calcium concentration quantitatively, at the same time, it can avoid feint of fluorescence intensity change due to baseline shift. Cameleon, which is based on fluorescence resonance energy transfer (FRET) and GFP, was used to study calcium dynamics in hippocampal neuron. Firstly, we optimize the protocols of hippocampal primary culture and calcium phosphate transfection; and try to replace 430 nm laser with 458 nm laser for monitoring the FRET ratio of Cameleon. Secondly, we compare dynamical ranges of three Cameleons. Exited with 458 nm laser, dynamical ranges of YC2.1, YC6.1, and YC3.60 are 33%, 70%, 220%, respectively. Finally, we quantify calcium concentration from FRET ratio, and apply in the following: (1) Free calcium ion distribution of resting state. Calcium concentrations in cytoplasm, primacy dendrite, secondary dendrite, third dendrite and apical spine of hippocampal neuron were 104±22 nM, 71±10 nM, 55±6 nM, 40±7 nM, 50±14 nM, respectively. Calcium concentration of apical spine higher than that of parent dendrite shows that spine neck acts as diffusion barrier, isolating spine head from its parent dendrite. Diffusional compartmentalization of calcium would underlie synaptic plasticity. Diversity of calcium concentration in spines, which differed from 13 nM to 80 nM, would be related with maturity and calcium handling mode of spines. (2) Change of calcium concentration in cytoplasm and spine with physiological stimulation. Under stimulation of glutamic acid, [Ca2+]i in soma increased from 100 nM to 1 μM, and [Ca2+]i in spines increased from 50 nM to 300 nM. These results prove that monitoring calcium concentration in spine with Cameleon is feasible. It can be used in studying the calcium dynamics, in addition it's applicable for real-time, less damage and long-playing observation. It might be important to study calcium of spine with stimulations of neurotransmitter and action potential.