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

## TL;DR

Researchers used optogenetics and electron microscopy to study how synapses change after a type of brain plasticity called STDP.

## Contribution

A new method combining optogenetics and electron microscopy to visualize STDP-induced synaptic changes at the ultrastructural level.

## Key findings

- Optogenetically induced STDP can be studied at the ultrastructural level using electron microscopy.
- Potentiated spines grow and curve around presynaptic boutons after STDP.
- Only a few synapses among thousands are strengthened by STDP.

## Abstract

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.

•We optogenetically induce spike-timing-dependent plasticity•We combine channelrhodopsin with electron-dense labels for electron microscopy•We perform ultrastructural analysis of optogenetically manipulated neurons•We show that paired stimulation drives expansion of axon-spine interface

We optogenetically induce spike-timing-dependent plasticity

We combine channelrhodopsin with electron-dense labels for electron microscopy

We perform ultrastructural analysis of optogenetically manipulated neurons

We show that paired stimulation drives expansion of axon-spine interface

Spike-timing-dependent plasticity (STDP) induces changes in the strength of synaptic connections between neurons that have been spiking together. Whether this results in long-lasting changes in the ultrastructure of individual synapses or whether other changes are responsible for synaptic memory has not been investigated. This gap in our understanding is largely due to technical difficulties. First, it has only recently become possible to induce STDP without patching the postsynaptic neuron. Second, it is not trivial to individually label the pre- and postsynaptic neurons that have undergone STDP. Here, we present a workflow that allows all-optical induction of STDP and subsequent processing of the tissue to find the relevant pre- and postsynaptic structures using transmission electron microscopy.

Optogenetic induction of spike-timing-dependent plasticity leads to the strengthening of a few hippocampal synapses among thousands of synapses that received no paired stimulation. Wang et al. combine channelrhodopsins with electron-dense labels to find the potentiated synapses with electron microscopy and show that potentiated spines grow and curve around their presynaptic boutons.