Ultrastructural analysis of synapses after induction of spike-timing-dependent plasticity

Repeated sequential activation of connected neurons causes lasting changes in synaptic strength, a process known as spike-timing-dependent plasticity (STDP). Recently, sequential spike patterns have been induced without electrodes, using two spectrally separated channelrhodopsins. However, due to the difficulty of labeling and localizing the few connecting synapses between the stimulated pre- and postsynaptic neurons (∼1-5 per neuron pair), ultrastructural analysis after STDP has not been reported. Here, we optogenetically induce STDP at CA3-CA1 hippocampal synapses and identify stimulated boutons and spines in CA1 using transmission electron microscopy (TEM). Presynaptic CA3 neurons express vesicle-targeted horseradish peroxidase, cre recombinase, and cre-dependent ChrimsonR, a red light-activatable channelrhodopsin. Postsynaptic neurons express violet light-activatable CheRiff and dAPEX2, an enhanced ascorbate peroxidase. In TEM, presynaptic boutons and postsynaptic spines are readily identifiable with well-preserved ultrastructural features. Our labeling strategy allows ultrastructural analysis of optogenetically manipulated neurons and their synapses.