Mutations in deadly seven/notch1a reveal developmental plasticity in the escape response circuit

Abstract

The relatively simple neural circuit driving the escape response in zebrafish offers an excellent opportunity to study properties of neural circuit formation. The hindbrain Mauthner cell is an essential component of this circuit. Mutations in the zebrafish deadly seven/notch1a (des) gene result in supernumerary Mauthner cells. We addressed whether and how these extra cells are incorporated into the escape-response circuit. Calcium imaging revealed that all Mauthner cells in desb420 mutants were active during an elicited escape response. However, the kinematic performance of the escape response in mutant larvae was very similar to wild-type fish. Analysis of the relationship between Mauthner axon collaterals and spinal neurons revealed that there was a decrease in the number of axon collaterals per Mauthner axon in mutant larvae compared with wild-type larvae, indicative of a decrease in the number of synapses formed with target spinal neurons. Moreover, we show that Mauthner axons projecting on the same side of the nervous system have primarily nonoverlapping collaterals. These data support the hypothesis that excess Mauthner cells are incorporated into the escape-response circuit, but they divide their target territory to maintain a normal response, thus demonstrating plasticity in the formation of the escape-response circuit. Such plasticity may be key to the evolution of the startle responses in mammals, which use larger populations of neurons in circuits similar to those in the fish escape response.

Figure 1.

des mutants have supernumerary Mauthner cells. A, B, Mauthner cells in a 30 hr wild-type (A) and des^b420 (B) mutant embryo as revealed by 3A10 antibody labeling and confocal microscopy. Dorsal views with anterior to the top. Scale bar, 40 μm.

Figure 2.

Mauthner cells in des mutants are active during an escape response. A calcium-imaging trial of a des^b420 mutant is shown. A, Mauthner cells in mutant larvae were backfilled with the fluorescent dye, calcium green dextran, at 4 dpf and imaged on confocal microscopy with a 63× objective. B, For quantification purposes, Mauthner cells on the right side of the hindbrain (A, arrow) were numbered 1 through 4. Fluorescence intensities were presented in pseudocolor with red being the brightest. In this trial, the plane of focus was optimized for cell 3. However, as is typically the case, all four cells were noticeably brighter after the escape when compared with the baseline of frames collected before the stimulus. The distortion in frame 6 indicates movement of the fish in response to a tap on the tail. Frames are ordered from left to right with 578 msec between frames. C, Quantification of the increase in fluorescence intensities. The fluorescence intensities for each cell (1-4) were plotted for each frame. The y-axis shows the normalized intensities (ΔF/F). The x-axis shows the time in seconds. Scale bar, 30 μm.

Figure 3.

Mauthner cell lateral dendrites in des mutants are in close apposition to sensory nerves. A-D, Seventy-two hour wild-type (A, C) and mutant (B, D) embryos were labeled with 3A10 antibody and analyzed by confocal microscopy. The trigeminal (Tg) and lateral line (Ll) nerves extend perpendicular to the Mauthner cell (Mth) and are closely juxtaposed to Mauthner cell lateral dendrites (arrow). A and B are the left sides of merged sections of wild-type and mutant embryos; C and D are single optical sections through the wild-type and mutant embryo shown in A and B. All images are dorsal views with anterior to the top. Scale bar, 35 μm

Figure 4.

des^b420 mutant larvae exhibited primarily normal escape responses. A tap with a small polished glass probe elicited escapes. In this trial, the tap was delivered to the left side of the head (bottom left-hand corner). Images were captured at 1000 frames per second. Every third frame (msec) is shown, starting at the initiation of the response (frame 0). After the start of movement, peak turn angle is reached at 15 msec (15). For an example of a wild-type escape response, see Liu and Fetcho (1999). Scale bar, 550 μm.

Figure 5.

The number of collaterals per Mauthner axon is dramatically decreased in des mutants. A, B, Four dpf wild-type (A), des^b420 (B), and des^tp37 larvae were back-labeled from the caudal spinal cord with lysinated rhodamine dextran. The number of axon collaterals was quantitated over a three spinal cord hemisegment region in the midtrunk, and the number of Mauthner cells was counted under transmitted light (C). Arrowheads denote axon collaterals. Scale bar, 5 μm.

Figure 6.

Collaterals from distinct Mauthner axons in des^b420 mutants do not form in the same location. Two Mauthner cell bodies in des^b420 mutants were electroporated with different fluorophores, and their axon collaterals were imaged at 6 dpf. One axon is shown in red (A), and the other is shown in green (B). The merged image (C) shows that the collaterals from the two axons do not form in the same location. Arrowheads denote collaterals. Scale bar, 10 μm