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Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle fusing (Pv) and in the number of vesicles available for immediate release, known as the readily-releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of action potentials. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses.Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release. Significance StatementNeurotransmitter is released through two mechanisms: action potential-evoked and spontaneous vesicle fusion. It is unknown if some synapses specialize in either evoked or spontaneous release with an antagonistic relationship, or if the two forms of release coexist and have a cooperative relationship. We used a robust optical glutamate indicator to measure both forms of release at individual synapses. We found that the frequency of spontaneous release displays significant heterogeneity and is directly related to the size of the readily releasable pool of vesicles. This finding links both mechanisms of neurotransmitter release and suggests an important signaling mechanism to the postsynaptic neuron at individual synapses. 

The fine control of synaptic function requires robust trans-synaptic molecular interactions. However, it remains poorly understood how trans-synaptic bridges change to reflect the functional states of the synapse. Here, we develop optical tools to visualize in firing synapses the molecular behavior of two trans-synaptic proteins, LGI1 and ADAM23, and find that neuronal activity acutely rearranges their abundance at the synaptic cleft. Surprisingly, synaptic LGI1 is primarily not secreted, as described elsewhere, but exo- and endocytosed through its interaction with ADAM23. Activity-driven translocation of LGI1 facilitates the formation of trans-synaptic connections proportionally to the history of activity of the synapse, adjusting excitatory transmission to synaptic firing rates. Accordingly, we find that patient-derived autoantibodies against LGI1 reduce its surface fraction and cause increased glutamate release. Our findings suggest that LGI1 abundance at the synaptic cleft can be acutely remodeled and serves as a critical control point for synaptic function. 

Fluorescent glutamate sensors shed light on the microscopic organization underlining spontaneous neurotransmission. 

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Recently, dynamic Ca 2+ uptake into the ER during electrical activity was shown to play an essential role in synaptic transmission. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca 2+ uptake during electrical activity, as knockdown (KD) of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca 2+ during electrical stimulation. Moreover, our experiments demonstrate that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to voltage. Thus, our data demonstrate that a nonconducting role of Kv2.1 exists through its binding to the ER protein VAMP-associated protein (VAP), which couples ER Ca 2+ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca 2+ handling independent of voltage and reveals a critical pathway for maintaining ER lumen Ca 2+ levels and efficient neurotransmitter release. Taken together, these findings reveal an essential nonclassical role for both Kv2.1 and the ER-PM junctions in synaptic transmission. 

Abstract The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR’s nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines. 

Analysis of the presynaptic action potential’s (AP syn ) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca 2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for K v 1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic K v 1 channel inactivation was mediated by the K v β1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of K v β1 blocked all broadening of the AP syn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic K v channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.