Bag of Tricks: Extracellular Vesicles as a Modulator for Neurotransmission

    Neuronal communication underlies all behaviors across animals with a nervous system. In Chapter 3 of Principles of Neurobiology, we learned that neurons communicate with each other through electrical and chemical synapses, with the latter being the more prevalent form. One of the most critical steps of chemical synaptic transmission is the release of neurotransmitters from the presynaptic terminal. This process is achieved by fusion of synaptic vesicles which is triggered by Ca²⁺ entry and mediated by SNARE proteins (Section 3.3–3.5). But besides neuronal activities, are there other means of neuronal signaling that modulate synaptic vesicle release?

In recent years, a type of synapse-independent transcellular signaling in the nervous system has gained more attention—the intercellular material transfer via small (30–1000 nm in diameter) membrane bound particles called extracellular vehicles (EVs). EVs carry a cargo of proteins, lipids, and nucleic acids, thus can trigger a variety of physiological responses in the recipient neuron. However, not much is known about the impact of EV-mediated inter-neuronal communications has on neurotransmission. In a recent paper, Vilcaes et al. carefully explored this aspect of EV function.

To study the role EVs play in synaptic vesicle release and neurotransmission, Vilcaes et al. isolated EVs from a glia-free hippocampal neuron culture and added them to other cultured hippocampal neurons after washout of the original media. They recorded miniature inhibitory postsynaptic current (mIPSC) using whole cell patch recording (see Section 14.21 and Box14-3 for details) at varies time points after the addition of EVs. Very interestingly, they observed increased mIPSC frequency at as early as 30 minutes after the addition of EVs (Vilcaes et al., Figure 3B-C). This effect is dependent on calcium but is independent of neuronal activity, as this increase in frequency is abrogated by the addition of the calcium chelator, BAPTA-AM, but not by the sodium channel blocker, TTX (Vilcaes et al., Figure 3E-F).

How is the modulation of neurotransmitter release in EV recipient neurons achieved? One possibility is that EVs are tuning molecules involved in synaptic vesicle release. The v-SNARE, synaptobrevin-2 (syb2), is the most abundant synaptic vesicle protein and is also present in EVs as revealed by the EV proteome (Vilcaes et al., Figure 1). Previous study has illustrated that a few copies of syb2 can trigger synaptic vesicle fusion. And it has been proposed that increasing the number of SNAREs may increase release probability of neurotransmitters. So, could syb2 be the active ingredient in EVs for augmenting neurotransmitter release?

To validate that syb2 in EVs can be incorporated into synaptic vesicles of the recipient neurons, the authors isolated EVs containing syb2 fused to the pH-sensitive GPF pHluorin and added those EVs to the media of neurons that do not express any florescent proteins. Using live imaging followed by immunostaining of GFP and synapsin 1, they found syb2-pHluorin is incorporated to 3.8% of synapsin 1 positive boutons (Vilcaes et al., Figure 4). Then, to test if those syb2 can constitute functional SNARE complexes, the authors used wild-type EVs to rescue neurotransmission in syb2 knockout neurons. They were able to partially rescue spontaneous but not evoked neurotransmission (Vilcaes et al., Figure 5), which the authors attributed to the small percentage of synapses with EV syb2 incorporation. Finally, they confirmed that syb2 in EVs is responsible for the modulation of neurotransmission by showing that syb2 knockout EVs cannot increase mIPSC frequency.

The study by Vilcaes et al. discovered a novel contributor of synaptic vesicle release regulation. Using electrophysiology, proteomic profiling, and live imaging, the authors elegantly dissected the mechanism through which neuronal EVs augment neurotransmitter release via functional integration of syb2. This paper opens up many new questions to study for both EV biology and synaptic vesicle release regulation. For some examples: How are EV proteins trafficked into synaptic vesicles? Do the contents of EV preferentially target a selected pool of synaptic vesicles or do they integrate promiscuously? Why is only spontaneous inhibitory neurotransmission modulated? Does neuronal activity change the composition of EVs? And what physiological effect(s) augmented spontaneous neurotransmitter release EV has in vivo? EV is indeed a “bag” of many “tricks”, and future studies will tell us more about its biology and function!

Schematic summary of mechanism of EV-mediated augmentation of spontaneous neurotransmission. See Vilcaes et al. 2021 for more details.

Reference

A.A. Vilcaes, N.L. Chanaday, E.T. Kavalali. Interneuronal exchange and functional integration of synaptobrevin via extracellular vesicles. Neuron, 109, 971–983 (2021). Link