We identify a selective essential function for Syt7 in asynchronous release. Our data suggest that multiple synaptotagmins cooperate at a given synapse to mediate the vast majority of all Ca2+-triggered neurotransmitter release. Using quantitative RT-PCR, we measured the expression levels of synaptotagmins and Doc2 proteins in cultured hippocampal neurons.
Among the Ca2+-binding protein mRNAs tested, Syt7 (a Ca2+-dependent synaptotagmin EGFR cancer [Li et al., 1995, Sugita et al., 2001 and Sugita et al., 2002]) was expressed at highest levels, with an ∼3-fold greater abundance than Syt1 (Figure 1A). In addition, Doc2A, Doc2B, and Doc2C were coexpressed at ∼10-fold lower, but still rather high mRNA levels. Since the analyses in Figure 1A were performed on mixtures of neurons, we asked whether individual neurons may express subsets of Ca2+-binding proteins. Cytoplasm from individual neurons was collected by aspiration through a patch pipette, and quantitative
RT-PCR with Fluidigm technology (Pang et al., 2011b) was used to measure the mRNA levels of 20 genes in 20 single neurons (Figure 1B). We found that all neurons coexpressed the Ca2+-binding synaptotagmins Syt1 and Syt7 at high levels, and most neurons selleck additionally coexpressed two other synaptotagmins, Syt4 and Syt11, that do not bind Ca2+ (von Poser et al., 1997 and Dai et al., 2004). Furthermore, most neurons coexpressed Doc2A, Doc2B, and Doc2C at similar, substantial levels (Figure 1B).
No significant expression differences were observed between isothipendyl various Doc2 isoforms in single neurons. Thus, individual hippocampal neurons coexpress multiple synaptotagmin and Doc2 isoforms at high levels. The high and universal expression of Syt7 in all neurons is intriguing given the lack of a known neuronal function for Syt7. However, the finding that Doc2A and Doc2B are coexpressed in hippocampal neurons differs from a previous finding suggesting that hippocampal neurons express only Doc2A (Yao et al., 2011), although it is consistent with other previous studies (Verhage et al., 1997). This issue is important because only the Doc2A knockdown (KD) but not the Doc2B KD was found to decrease asynchronous neurotransmitter release in hippocampal neurons (Yao et al., 2011). The selective effect of the Doc2A KD led to the proposal that Doc2 proteins are general Ca2+ sensors for release and that the Doc2A KD had a selective effect on release in hippocampal neurons because only Doc2A is expressed in these neurons. Other studies, however, did not observe a change in evoked neurotransmitter release in Doc2A- and Doc2B-deficient neurons (Groffen et al., 2010 and Pang et al., 2011a), and our finding of coexpression of Doc2A and Doc2B in hippocampal neurons is also at odds with the hypothesis that Doc2A is a selectively expressed Ca2+ sensor for asynchronous release in hippocampal neurons.