Imaging the Functional Organization of Zebrafish Hindbrain Segments during Escape Behaviors  Donald M O'Malley, Yen-Hong Kao, Joseph R Fetcho  Neuron  Volume 17, Issue 6, Pages 1145-1155 (December 1996) DOI: 10.1016/S0896-6273(00)80246-9

Figure 1 Identified Reticulospinal Neurons Observed in the Brain of the Living Zebrafish (A) Confocal projection of the array of reticulospinal neurons labeled by a bilateral injection of calcium green dextran into caudal spinal cord. Labeled neurons, shown in horizontal section, include the bilateral Mauthner cells (arrows) as well as clusters of neurons in successive hindbrain segments. (B) Confocal projection, from another fish, of the Mauthner cell and its two homologs (MiD2cm and MiD3cm, left and right arrows, respectively) labeled with calcium green dextran by a more selective unilateral injection into the ventral portion of caudal spinal cord. The axons and dendrites of the cells are visible and aid in their identification when viewed either in optical sections or in three-dimensional reconstructions. Rostral is to the left in both panels. Scale bars represent 25 μm. Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)

Figure 2 Comparison of Calcium Green and Lucifer Yellow Dextran (A) Response of a calcium green dextran labeled Mauthner cell during an escape elicited by a head tap. Panels show successive images (in pseudocolor) taken at 400 ms intervals. The color scale on the left represents fluorescence intensity (blue, lowest; red, highest) for both (A) and (B). The asterisk marks the frame in which the tap and escape occurred. A movement artifact from the escape is evident at the top of this frame. Since these images are acquired line by line at 2 ms/line and the movement artifact lasts only about 30 lines (60 ms), this response was the result of a brief movement, as occurs in escapes. The fluorescence of the cell increases quickly, but decays slowly over subsequent frames. The size of the increase was about 50%, as illustrated in the inset plot of fluorescence versus time. The starting fluorescence level was normalized to 100% in the plot. (B) Fluorescence images of a Mauthner cell filled with the nonindicator, lucifer yellow dextran. An escape was elicited in the frame marked by the asterisk. The inset shows that the fluorescence did not change. This control experiment demonstrates that movement artifact cannot explain the fluorescence changes observed in cells labeled with calcium green dextran. The data point for the frame containing the escape was omitted from the plot because movement of the cell during the escape precludes measurement of the fluorescence of the cell. Scale bars represent 10 μm in (A) and (B). Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)

Figure 3 Relationship between Action Potentials and Fluorescence Determined by Antidromic Stimulation of the Mauthner Cell (A) The fluorescence change in a Mauthner cell soma is plotted for a series of trials in which the axon of the Mauthner cell was stimulated electrically in spinal cord 1, 2, 5, or 10 times to elicit a comparable number of action potentials. Stimulus parameters were 10 μA current strength, 0.2 ms pulse duration, and 50 ms interstimulus interval. The stimulus strength was determined by gradually increasing the strength to a level just sufficient to elicit a response. Given the relatively large size of the Mauthner axon and the proximity of the electrode to it, this is likely to result in a reasonably selective activation of the Mauthner axon. The increase in fluorescence is roughly 10% for one stimulus and increases with additional stimuli, although the increase is not linearly related to the number of stimuli. (B) shows a second set of stimuli for the same cell in (A) to illustrate the reproducibility of the relationship between stimuli and fluorescence. The baseline fluorescence is normalized to 100% to allow easy assessment of the percentage increase; however, the absolute baseline fluorescence was relatively stable, with little change from trial to trial. Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)

Figure 4 Responses of the Mauthner Cell near Threshold for Escapes Plots of the fluorescence of the soma of a calcium green dextran-labeled Mauthner cell from a series of successive trials at 2 min intervals in which the strength of a head tap was varied around the threshold for escapes. The voltage applied to the piezoelectric head tapper in each trial is indicated, with larger voltages associated with stronger taps. The asterisks mark those trials in which an escape occurred. Fluorescence increases occurred only in conjunction with escapes, even at stimulus strengths near threshold for escapes, as defined by 50% response probability (about 13 V in this experiment). The fluorescence responses (30%–40% increases) were similar in all escapes independent of whether near-threshold or suprathreshold stimuli were used. Successive points are at 400 ms intervals. Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)

Figure 5 Differential Activation of the Mauthner Cell and Its Homologs during Escapes Each row shows a trial consisting of a sequence of images during which an escape was elicited by an ipsilateral touch to the head or tail. The color scale (applies to all figures) represents fluorescence intensity (blue, lowest; red, highest). (A) Response of MiD3cm during an escape elicited by a head tap. MiD3cm is shown at relatively high magnification to illustrate clearly the fluorescence increase. The tap was applied during the third frame. The escape event transiently causes the cell to move out of the frame, which provides a convenient record of the behavioral event. This movement frame is omitted from subsequent rows. (B) Simultaneous imaging of both the Mauthner cell (left) and MiD3cm (far right) during a head stimulus. Both cells respond. (C) Same field as in (B), but with a tail stimulus. The Mauthner cell responds but not MiD3cm. (D) Simultaneous imaging of the Mauthner cell (left) and MiD2cm (right) during a head stimulus. Both cells respond. (E) Same field as in (D), but with a tail stimulus. Only the Mauthner cell responds. Arrows mark the first frame after the escape in (B)–(E). Images were acquired at 400 ms intervals. Scale bars: 10 μm in (A), 15 μm in (B)–(E). The size of the fluorescence increases in each trial were as follows: 41% (A); 62% in Mauthner cell, 21% in MiD3cm (B); 53% in Mauthner cell, 2% in MiD3cm (not significant, see Figure 2) (C); 21% in Mauthner cell, 22% in MiD2cm (D); 67% in Mauthner cell, none in MiD2cm (E). Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)

Figure 6 Quantification of Escape Responses of the Mauthner Cell and Its Homologs over Successive Trials Each row shows a series of successive trials in which we alternately elicited escapes by either head or tail stimuli. Each point represents the mean pixel intensity per frame in a rectangular box placed over the cell (400 ms between frames). The escape was elicited during the sixth frame in each trial. In some trials, the cell moved out of the field during the frame in which the escape occurred. Because the fluorescence of the cell could not be measured when it was not in the frame, there is no data point for those movement frames, leading to a discontinuity in the plot. A 2 min rest was allowed between each trial. Each row is from a different fish. The mean pixel intensity was normalized so that the starting fluorescence baseline is 100 in each trial. This normalization corrects for any slow changes in the baseline brightness of the cells (due to bleaching for example) and allows an easy comparison of successive trials. (Top) The Mauthner cell responds repeatedly during es- capes elicited by either head or tail taps. MiD2cm (middle) and MiD3cm (bottom) cells both respond during escapes elicited by head taps, but not during escapes produced by tail taps. Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)

Figure 7 Responses of the Mauthner Cell and MiD3cm near the Threshold for Escapes (A) The fluorescence of a line (black line in top panel) that passed through both the Mauthner cell (left) and MiD3cm (right) was monitored repeatedly as shown in the bottom panel. Consecutive lines were acquired at 2 ms intervals (vertical scale bar, 200 ms) and are displayed from top to bottom. An escape elicited by a tap to the ipsilateral side of the head is indicated by the arrows. The trace was briefly disrupted by the escape movement, but following the movement, the fluorescence of the vertical bands representing the two cells had increased, indicating that both cells had responded rapidly, within 30 ms of one another. (B) Quantification of responses from the cells in (A) over several trials in which the head was tapped at a strength near threshold for escape (escapes occurred in 67% of the trials). The points show the average intensity of successive sets of 15 lines (i.e. 30 ms per data point) plotted over time. Successive trials, 2 min apart are separated by dashed lines. The starting baseline in each trial was normalized to 100 as in Figure 3. Repeated stimuli at a constant stimulus strength near threshold led to escapes in trials 2, 4, 5 and 6, but not in 1 and 3. When an escape occurred, both the Mauthner cell and MiD3cm responded, but neither responded when there was no escape. Horizontal scale bar, 20 μm. Size of increases in (A): 70% in Mauthner cell; 34% in MiD3cm. Neuron 1996 17, 1145-1155DOI: (10.1016/S0896-6273(00)80246-9)