Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors

Abstract

Differentiation of presynaptic nerve terminals is mediated, in part, through contact with the appropriate postsynaptic target cell. In particular, studies using dissociated nerve and muscle derived from Xenopus embryos have indicated that the properties of transmitter release from motor neurons are altered after contact with skeletal muscle. This maturation of presynaptic function has further been linked to retrograde signaling from muscle that involves activation of postsynaptic ACh receptors. Using FM1-43 optical determinants of exocytosis, we now compare calcium-mediated exocytosis at neuromuscular junctions of wild-type zebrafish to mutant fish lacking postsynaptic ACh receptors. In response to either high-potassium depolarization or direct electrical stimulation, we observed no differences in the rate or extent of FM1-43 destaining. These data indicate that the acquisition of stimulus-evoked exocytosis at early developmental stages occurs independent of both postsynaptic receptor and synaptic responses in zebrafish.

Figure 1.

FM1-43 labeling of nerve terminals is activity dependent. A, Low-power photograph of a 5-d-old larva. The region of tail corresponding to the photograph in B is outlined. B, A differential interference contrast photograph corresponding to the region of tail muscle outlined in A showing the individual muscle cells and myocomata. The outlined region in B corresponds approximately to the size of the regions shown in C and D. C, High-power fluorescence photograph of the tail from a fish that was loaded with 10 μm FM1-43 in the presence of a high-potassium HBSS. The circles indicate varicosities that were dye loaded. D, High-power fluorescence photograph of the tail of a fish different from that shown in C that was loaded with 10 μm FM1-43 in low-potassium HBSS. The areas shown in both C and D correspond to approximately one tail segment. Scale bars: A, 500 μm; B, 100 μm; C, D, 20 μm.

Figure 2.

FM1-43 label colocalizes with AChR clusters in wild-type fish. A, A 5-d-old wild-type fish colabeled with 0.1 μm Alexa 594-conjugated α-Btx and 10 μm FM1-43. The green FM1-43 labeling of synaptic varicosities (left) and the red labeling of AChR clusters by Alexa 594 α-Btx (middle) colocalize in the merged image (right). B, The same field is shown after 3 min of high-potassium treatment. The area shown corresponds to approximately one tail segment. Scale bar, 20 μm.

Figure 3.

FM1-43 labels nerve in both wild-type and mutant synapses. Secondary motor neurons were visualized in transgenic wild-type and mutant lines of fish that were expressing cytoplasmic GFP. The GFP was constitutively expressed in the motor neurons of all three lines using a neuron-specific Islet1 promoter (top). Three Islet1 fish, corresponding to wild-type, nic-1, and sofa potato, were labeled with 10 μm FM1-43 (middle). Merged images indicate that the motor neuron GFP label colocalizes with the FM1-43 label (bottom). Scale bar, 50 μm.

Figure 4.

High-potassium destaining kinetics of FM1-43-loaded synaptic varicosities in wild-type fish. A, Time-lapse images of three varicosities (indicated by arrows 1-3) during high-potassium-induced destaining. Representative images are shown at 12 sec intervals after perfusion of high-potassium solution. Scale bar, 10 μm. B, Time-course plot of fluorescence loss after background correction for the three individual varicosities shown in A. Time 0 is determined by the slight twitch of muscles with arrival of high-potassium solution. Fluorescence intensity was measured every 4 sec. The time required for decay from 90 to 10% of peak fluorescence determined for each trace is indicated alongside the corresponding legend bar.

Figure 5.

High-potassium destaining kinetics of FM1-43-loaded varicosities in mutant zebrafish. Representative time-lapse images of labeled varicosities (indicated by arrows) are shown at 12 sec intervals for nic-1 (A) and sofa potato (C) after perfusion of high-potassium solution. Scale bars, 10 μm. The associated time course of fluorescence loss for each varicosity is plotted in B and D for each mutant after background correction. Time 0 is determined by the slight twitch of muscles with arrival of high-potassium solution. Fluorescence intensity was measured every 3 sec. The time required for decay from 90 to 10% of peak fluorescence determined for each trace is indicated alongside the corresponding legend bar.

Figure 6.

Destaining kinetics of FM1-43-loaded varicosities induced by electrical field stimulation. Representative time-lapse images of labeled varicosities (indicated by arrows) at 24 sec intervals after the onset of electrical stimulation are shown for wild-type (A), nic-1 (B), and sofa potato (C) fish. Scale bars, 10 μm. The time course of fluorescence loss associated with the corresponding varicosities is shown in A-C. Time 0 corresponds to the onset of electrical stimulation. Fluorescence intensity was measured every 3 sec. The time required for decay from 90 to 10% of peak fluorescence determined for each trace is indicated alongside the corresponding legend bar. In several traces, the final plateau for destaining occurred beyond the time shown in the traces.