Article Figures & Data
Figures
- Figure 1.
Paired motor neuron-muscle recordings indicate N-type calcium channel involvement in zebrafish neuromuscular transmission. A, Postsynaptic recordings of EPCs in response to a 1 s, 100 Hz train of action potentials elicited in the motor neuron soma. Untreated fish are compared with individual fish treated with 1 μm ω-conotoxin GVIA, 1 μm ω-conotoxin MVIIC or 2 μm ω-agatoxin IVA. Vertical calibration corresponds to 1 nA for ω-conotoxin GVIA and ω-conotoxin MVIIC and 2 nA for control and ω-agatoxin IVA. Horizontal calibration corresponds to 200 ms. B, Scatter plot of total release from individual experiments, n value (in parentheses), along with cumulative mean ± SD for each treatment. The total release was quantitated as charge entry during the 1 s, 100 Hz train by integrating the postsynaptic current, as previously described (Wen et al., 2010). ***p < 0.001. n.s. corresponds to p = 0.38.
- Figure 2.
Pharmacology of the zebrafish N-type calcium channel expressed in HEK293T cells. A, Sample current traces in 5 mV depolarizing increments from a −80 mV holding potential. The recordings were performed in 1 mm Ba2+ and traces were leak subtracted using p/10 protocol. B, Current–voltage relations of the leak subtracted Ba2+ current for the recording shown in A. C, Examples of time-dependent block of Ba2+ current in response to the three different indicated toxins. Each point corresponds to a measurement of inward current at +10 mV. The horizontal bar above the graph indicates the time during which the toxin was applied to the cell. The insets show the maximal inward current before toxin application (black) and after maximal block was achieved (gray). Calibration bars are labeled in the top panel.
- Figure 3.
Compromised neuromuscular transmission in tb204a mutant fish. A, Paired CaP motor neuron-skeletal muscle recordings from a wild-type and tb204a mutant fish during 1 Hz stimulation. The presynaptic action potential (AP) and associated EPC are shown. The recordings shown are discontinuous for the purpose of presentation. B, Postsynaptic recordings of the tb204a mutant during 1 s, 100 Hz stimulation showing decreased failures and increased EPC amplitude when external calcium was elevated from 2.1 to 10 mm. All residual postsynaptic activity was eliminated by addition of 1 μm ω-conotoxin GVIA. C, Scatter plots of individual experiments comparing wild-type and tb204a mutants release, measured as total charge, during a 1 s, 100 Hz stimulus train. Each point represents the measurement from a single experiment. Overall mean ± SD and n value (in parentheses) are indicated. D, Scatter plot of individual experiments comparing the total charge obtained for tb204a mutants in 2.1 mm Ca2+, 10 mm Ca2+, and 1 μm ω-conotoxin GVIA. ***p < 0.001. E, Fluo-5F measurements of calcium response in the presynaptic boutons of wild-type and tb204a mutant fish during a 100 ms, 100 Hz stimulus train. Each trace represents the average responses of wild-type (black, 4 fish, 52 boutons) and tb204a (red, 4 fish, 59 boutons) boutons. The change in Fluo-5F signal (ΔG) was normalized to the fluorescent fill (R) and expressed in arbitrary units. F, Comparison of normalized Fluo-5F signal for individual boutons in wild-type and tb204a fish. The overall mean values are shown; ***p < 0.001.
- Figure 4.
Cloning and expression of zebrafish CACNA1Ab P/Q-type calcium channel in HEK293T cells. A, Alignments of calcium channel sequences from various species in the region corresponding to the tb204a point mutation. The sequence comparisons represent the S6 membrane spanning region in Domain IV (in box) and intracellular region for both P/Q- and N-type calcium channels in human (hs), mouse (mm), fish (dr), and Xenopus (xt). Zebrafish has two genes for P/Q-type (1Aa and 1Ab) and N-type channel (1Ba and 1Bb). Identical residues are shaded black and similar residues are shaded in gray. The gray bar denotes a pair of residues that can be used to distinguish N- and P/Q-type channels. The arrow denotes the location of the tb204a point mutation from tyrosine (Y) to asparagine (N). B, A dendrogram showing the clustering of the annotated N- and P/Q-type genes for zebrafish with the respective mouse voltage-dependent calcium channel sub-groups according to their sequence similarity. The dendrogram was generated by CLUSTAL W program. The zebrafish calcium channel isoforms in this study are in bold. C, Sample Ba2+ currents from wild-type (top) and mutant (bottom) P/Q-type channels expressed in HEK293T cells. The cells were held at −80 mV and incrementing steps of 5 mV were applied. D, The current–voltage relations for the wild-type (square) and mutant (circle) Ba2+ currents shown in C.
- Figure 5.
Pharmacological profile of the zebrafish CACNA1Ab P/Q-type calcium channel expressed in HEK293T cells. A, Representative recordings showing comparison of the block by 1 μm ω-conotoxin GVIA in 5 mm (filled symbols) and 10 mm (open symbols) Ba2+. The block is represented by the time-dependent reduction from preblock peak current amplitude. Each time point was taken at 5 s interval. The time of pressure application is indicated in each divalent concentration by the horizontal bars. The inset shows the Ba2+ current recorded preapplication (dark trace) and postapplication (light trace) of ω-conotoxin GVIA. The test voltage steps corresponded to +10 mV from a holding potential of −80 mV. B, The block by 1 μm ω-conotoxin MVIIC shown in the same manner as A. C, The block by 2 μm ω-agatoxin IVA is shown in the same manner as A. D, Comparison of the current block by different toxins. Average percentage of block and n value (in parentheses) for each condition are shown.
- Figure 6.
Roscovitine rescues tb204a mutant phenotype. A, Comparison of the touch responses from wild-type, tb204a mutant, and tb204a mutant following treatment with either 200 μm DAP or 100 μm roscovitine. Six consecutive still images taken at 10 ms intervals following mechanical stimulation are shown. Merged images are shown at the end of each row. B, Motion analysis of mechanically induced escape responses using Flote software. The curvature reflects the degree of body axis bend associated with the swimming. C, Barium currents recorded from zebrafish P/Q-type calcium channels expressed in HEK cells before (gray) and 30 s into (black) application of roscovitine. The voltage was stepped from −80 mV to +40 mV for 20 ms before returning to −80 mV. Tail currents shown in the box are expanded in the lower traces and the fits to a single exponential curve shown in red. D, Paired recording traces from untreated and roscovitine treated tb204a mutant fish are shown for 1 Hz stimulation. E, Paired recording traces from an untreated tb204a mutant and a roscovitine treated mutant fish during 1 s, 100 Hz stimulation. F, Comparison of total release during 1 s, 100 Hz stimulation from untreated and roscovitine treated tb204a mutant fish. Each symbol corresponds to the value obtained for individual experiments. The overall mean, SD and n value (in parentheses) for each condition are shown. The mean ± SD previously shown in Figures 1 and 3 for wild-type fish are indicated for the purpose of comparison. ***p < 0.001.
