Synapsin-Cre Lentivirus

In presynaptic nerve terminals, synaptic vesicle exocytosis is restricted to specialized sites called active zones. At these sites, neurotransmitter release is determined by the number of releasable vesicles and their probability of release. Vertebrate ELKS proteins are enriched in presynaptic active zones, but their function is unclear. Here, researchers generated ELKS1 knockout mice and found that its constitutive removal resulted in lethality. To avoid lethality and to avoid redundancy between ELKS1 and ELKS2 in synaptic transmission, a conditional genetic approach was used to remove both genes in cultured hippocampal neurons after synapse establishment. Simultaneous removal of ELKS1 and ELKS2 resulted in a 50% reduction in neurotransmitter release from inhibitory synapses, along with a decrease in release probability. Removal of ELKS did not affect the number of synapses or their electron microscopic appearance. Using Ca²⁺ imaging, researchers found that loss of ELKS resulted in a 30% reduction in single action potential-triggered Ca²⁺ influx in inhibitory nerve terminals, consistent with defects in synaptic transmission and release probability. Unlike the loss of the active zone proteins RIM, RIM-BP, or bruchpilot, ablation of ELKS did not result in a significant decrease in the levels of presynaptic Ca²⁺ channels. These results suggest that ELKS is required for normal Ca²⁺ influx in the nerve terminals of inhibitory hippocampal neurons.

To analyze the synaptic function of ELKS, researchers avoided redundancy by crossing ELKS1floxed mice with ELKS2floxed mice. Researchers then generated cultured hippocampal neurons from these mice that either lacked ELKS1 and ELKS2 when infected with cre lentivirus (ELKS cDKO) or had a complete set of ELKS when infected with a control virus (control). The time course of synaptic ELKS disappearance was determined by analyzing the gradual disappearance of ELKS from synapses after infection of neurons with synapsin-cre lentiviruses at DIV 5. Markers for synaptic vesicles at inhibitory and excitatory synapses (VGAT and VGluT1, respectively) display punctate staining patterns at DIV 7 (Figure 1A), consistent with the presence of evoked synaptic currents at DIV 7 in cultured hippocampal neurons. ELKS immunostaining appeared unaffected at DIV 7 but was visibly reduced starting at DIV 9, and ELKS was entirely absent at DIV 13 (Figure 1A). All functional analyses were done from DIV 14 to DIV 18. Quantitative Western blotting with 125I-labeled antibodies confirmed that cre recombination efficiently removed ELKS protein at DIV 14 without affecting protein levels of other presynaptic proteins (Figure 1B). This suggests that removal of ELKS1 and ELKS2 in cultured hippocampal neurons after DIV 5 does not significantly affect the development of neurons and synapses in vitro.

Figure 1. Synaptic protein composition in ELKS cDKO neurons. (Liu C, et al., 2014)