Living synaptic vesicle marker: Synaptotagmin‐GFP

Abstract

Synapses are the site of chemical communication between neurons and between neurons and muscles. The synaptic vesicle (SV) is a prominent presynaptic organelle which contains chemical neurotransmitters and fuses with the plasma membrane to mediate neurotransmission. There are about 50 or so synaptic proteins which are either integral vesicle membrane proteins (e.g., synaptotagmin, syt; and synaptobrevin, syb) or vesicle-associated proteins (e.g., cysteine string protein, CSP; Fernandez-Chacon and Sudhof, 1999). We have transformed Drosophila with a novel syt-eGFP (enhanced GFP) fusion protein, the fluorescence pattern of which colocalizes with native SV proteins at synapses, suggesting that the syt-eGFP fusion protein is correctly localized as an integral SV protein and therefore a good SV marker in living synapses. We demonstrate that the syt-eGFP line can be used to study SV dynamics in vivo by fluorescence recovery after photobleach (FRAP).The syt-eGFP fusion was constructed as shown in Figure 1. The eGFP carries double substitution of Phe 64 to Leu and Ser 65 to Thr and fluoresces 35-fold more intensely than wild-type GFP when excited at 488 nm, based on spectral analysis of equal amounts of soluble protein (Cormack et al., 1996). Four syt-eGFP transgenic lines were generated; one with insertion on the X chromosome, two on the second chromosome, and one on the third chromosome. All of these lines produced clear fluorescence when crossed to a pan-neuronal GAL4 driver (elav-GAL4, see Fig. 2) or a subset neuronal GAL4 driver 4G-GAL4 (data not shown). 4G-GAL4 is identified from an enhancer trap screen for neuronal-specific genes; it starts expression at late embryogenesis panneuronally, but in a subset of motor neurons in the third instar larvae, and enriched in mushroom body in adult brain. The eGFP-positive animals, from embryos to adults, can be readily recognizable under a fluorescence dissecting scope. Stocks with expression of syt-eGFP in all neurons (recombinant chromosome carrying both elav-GAL4 and syt-eGFP on the X chromosome) or subset of neurons (recombinant chromosome carrying both 4G-GAL4 and syt-eGFP on the second chromosome) were established.Multiple lines of evidence indicate that syt-eGFP is present in SVs, with expression similar to the native syt (Fig. 2). First, syt-eGFP is highly enriched in the neuropil region of the ventral nerve cord (VNC) of the embryo (data not shown) and larva ( Fig. 2A, left), as well as in the axonal lobes of the larval mushroom body ( Fig. 2A, right). These neuropil regions are densely packed with neuronal synapses. Second, at neuromuscular junction (NMJ) synapses, where we have higher resolution of single synaptic boutons, the syt-eGFP pattern perfectly matches the staining pattern seen with antibodies against SV-associated proteins (Fig. 2B) FIG. 1.Map of syt-eGFP and syb-eGFP fusion constructs. Syt (accession number M55048) or syb (neuronal synaptobrevin, accession number S66686) coding region was fused to the N-terminal of eGFP (enhanced GFP, catalog number 6...

Mushroom body efferent neurons responsible for aversive olfactory memory retrieval in Drosophila

“…1a-c and Supplementary Fig. 2), the signals of the presynaptic marker Syt::GFP (a fusion of eGFP and the synaptic vesicle protein Synaptotagmin 20 ) were highly enriched in the msmpr and LH but not detectable in the MB vertical lobes ( Fig. 1d-f).…”

“…We blocked the neurotransmission of GAL4-expressing cells during the retrieval of different forms of short-and long-lasting memories using Shi ts [19] , a dominant negative temperature sensitive variant of Dynamin that can reversibly block synaptic vesicle endocytosis at elevated temperatures. Because neurons responsible for memory retrieval should receive synaptic inputs from the MB and project to other parts of the brain, we characterized the polarity of MB extrinsic neurons by expressing a presynaptic marker 20 . We further characterized the identified MB output neurons with regard to neurotransmitter and physiological response properties using immunohistochemistry and functional calcium imaging.…”

Dissection of theDrosophilaneuropeptide F circuit using a high-throughput two-choice assay

“…NPF is required for the NPF activation-induced preference. Distribution of NPF neurons in the central brain (A) and ventral nerve cord (B) of the fly, as visualized by the expression of the cell polarity markers DenMark (dendrites) and Syt::GFP (synaptic terminals) (42,43) driven by the NPF-GAL4 driver. Positions of the large P1 and L1-l neurons, and the FSB are indicated.…”

Genetically encoded dendritic marker sheds light on neuronal connectivity inDrosophila

“…Drosophila neurons exhibit the major kinds of compartmentalization present in mammalian neurons and the fly has emerged as a powerful system to study the establishment and maintenance of neuronal connections (3,4). Almost all studies of neuronal circuits in the fly have relied on genetic markers such as CD8::GFP that outline the morphology of entire cells rather than particular subcellular compartments (5), as well as presynaptic markers such as Synaptotagmin, Synaptobrevin, and Bruchpilot GFP fusion proteins (6)(7)(8)(9)(10)(11). However, more accurate identification and mapping of novel neuronal circuits has been hampered by the lack of a genetically encoded and phenotypically neutral dendritic marker.…”