Abstract
The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca2+-sensor, the Ca2+ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca2+. It is suggested that the photo-activation of ChRFR facilitated the release of Ca2+ from intracellular Ca2+ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.
Highlights
It is a generally accepted idea that transmitter release from the presynaptic terminal consists of several elementary processes [1,2,3,4,5,6]
As one of the methods to introduce genes of interest into the chick ciliary ganglion (CG) presynaptic terminal, we adopted an in ovo electroporation technique [26,27]
At E14, EGFP fluorescence was detectable in the neurons in the EW nucleus, which innervate the CG through the oculomotor nerve, the axons in the oculomotor nerve and the large calyx-type presynaptic terminals (Fig. 1C–1E and Video S1)
Summary
It is a generally accepted idea that transmitter release from the presynaptic terminal consists of several elementary processes [1,2,3,4,5,6]. Upon invasion of an action potential into a presynaptic terminal, the voltage-dependent Ca2+ channels (VDCCs) are activated by brief depolarization. This is followed by Ca2+ inflow into the nerve terminal according to the electrochemical gradient. The consequent focal elevation of the intracellular Ca2+ concentration triggers a molecular cascade that causes the exocytosis of neurotransmitter in the synaptic vesicles. These vesicles are retrieved by endocytosis, refilled and recycled for the subsequent transmitter release. Each of these elementary processes is the principal target of short- and long-term modulation of the transmitter release
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