Flow cytometry of synaptoneurosomes (FCS) reveals increased ribosomal S6 and calcineurin proteins in activated medial prefrontal cortex to nucleus accumbens synapses

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting YFP-expressing AAV into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg of cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label pre- and post-synaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity (Arc, CaMKII and phospho-CaMKII, ribosomal protein S6, and phospho-S6, calcineurin and phospho-calcineurin) in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, indicating that these increases were due to neural activity per se . S6 levels were also increased by novel context exposure alone in the absence of cocaine. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc. SIGNIFICANCE STATEMENT Memories are formed during learning and stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a flow cytometry procedure for high throughput in-line analysis of synaptoneurosomes (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC to NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.