Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A rapid and precise measure of somite volume
A method of sample preparation, data acquisition, and volumetric analysis that allows the rapid measurement of muscle growth in zebrafish larvae is described. Muscle size can be measured in live animals using fish labeled on their plasma membranes with a membrane-targeted GFP (β-actin:HRAS-EGFP) or mCherry (α-actin:mCherry-CAAX). Larvae were transiently anesthetized using tricaine, mounted in low-melting-point agarose and imaged using confocal fluorescence microscopy. Somite 17 was chosen for analysis of muscle size given its accessibility at the trunk-tail interface³¹. In practice, as in theory, myotome volume can be calculated as the product of myotome length (L) and the average of three cross-sectional area measures (CSA) (Figure 2A, which is referred to as the 4-slice method).

To validate this method, whole myotome confocal Z-stacks of live zebrafish larvae (Figure 2B) were obtained, and somite volume was calculated by multiplying the sum of somite 17 profile areas in each slice (80-100 slices) by the inter-slice distance (1-1.2 µm). A strong correlation was observed between the 4-slice and full stack methods of calculation (Figure 2C). However, the 4-slice method generally gave a slightly larger volume estimate (~2% larger on average) (Figure 2D). This difference could be due either to a) obliquity of the samples giving erroneously large volume measurement or b) the observation that zebrafish myotomes taper along the anteroposterior axis, being smaller toward the tail of the animal. As the YZa, YZm, and YZp CSA measurements are toward the anterior portion of the somite 17 myotome chevron (Figure 2A), the latter interpretation was tested by volumetric analysis of the myotomes of somites 16 and 18. Each somite was about 7% larger than the one behind (Figure 2E). Further analysis revealed that a 2-slice method requiring only a single YZ measurement, located in the middle of the somite where the epaxial and hypaxial halves meet at the horizontal myoseptum (YZm), and the XY slice, gives a reasonably accurate estimate of myotome volume (Figure 2F,G). The 2-slice approach enables more rapid data acquisition when time constraints limit the number of fish that can be scanned. In summary, these data show that myotome volume can be measured rapidly and accurately in live zebrafish larvae. Single larvae were successfully, repeatedly, measured over a 6-day period with this method.

For comparative study of growth of an identified tissue unit, the described method provides reliable and precise volume estimates. To obtain accurate absolute volumes, however, a number of modifications can be applied. First, correction can be made for errors caused by oblique mounting either by tilting the dish or microscope stage to obtain better transverse YZ and parasagittal XY slices during imaging or through using the notochord profile in YZ slices; the cross-sectional minor axis reveals the true diameter of the cylindrical notochord, whereas its major axis reveals the angle and magnitude of obliquity (see Note in point 4.6 above). Second, the location of XY and YZ sectioning during scanning must be selected to reflect, accurately, the desired myotome(s). (See Note in point 4.6 above). Note, however, that the form of the myotome chevrons changes depending on developmental stage and this must be taken into account when selecting YZ scans. Lastly, to determine whether changes in myotome 17 reflect muscle growth throughout the axis, myotomes further into the trunk or tail regions may be measured by the described method.

Repeated measurements reveal somite growth
An advantage of the described method is the ease of repeated analysis on single fish. Individual embryos and larvae can be repeatedly embedded, measured, and released without suffering obvious long-term effects (Figure 2H). The myotome grows detectably both in L and CSA between 2 and 5 dpf, leading to steady increase in volume (Figure 2H). Growth was imaged in this way between 1 and 8 dpf and larvae were also released after imaging and grown to adulthood. Analyses further into the late larval period are expected to be possible, although effects of repeated imaging on feeding behavior would need to be monitored carefully on comparison with siblings that are not imaged. Importantly, development of pigmentation can obscure imaging. Whereas pigmentation is not a problem in properly orientated fish, as the melanophore stripes do not prevent the required measurements, pigment can be more problematic in obliquely mounted samples. The use of pigmentation mutant lines, such as roy;mitfa³⁹, is anticipated, which would extend the time window of growth measurements until the limits of practicable confocal scan depth are reached.

A further advantage of the described procedure is the ease of detailed analysis of growth of single fibers in comparison with their whole myotome over short periods. By mosaic labeling of fibers through DNA injection, a method was developed to detect nuclear acquisition and growth in individual identified fibers over 4 h, and then permit re-analysing at later times (Figure 2I). Moreover, growth of the whole myotome can be measured over 12 h or less²⁶ (and data not shown). By repeatedly measuring the same fish, inter-individual variability is eliminated and a small number of animals can yield statistically robust results²⁶.

Manipulations which change somite volume can readily be detected
The current protocol allows one to interrogate changes in muscle growth under various biological and physiological conditions, such as altered physical inactivity. Anesthetic tricaine, which blocks nerve action potentials by inhibiting voltage-gated Na⁺ channels⁴⁰, was used to induce muscle inactivity in β-actin:HRAS-EGFP larvae. As shown previously²⁶, inactivity for 24 h between the third and fourth dpf greatly reduced myotome volume, indicating that larval muscle growth is activity-dependent (Figure 3A). The effect of inactivity can also be investigated using other detection methods which reveal the structure of the somite, such as mCherry-CAAX (either in a transgenic line or by mRNA injection) or by overnight immersion of larvae in BODIPY dye (Figure 3A). The latter approach, while removing the need to cross fish onto transgenic backgrounds or inject embryos cannot be used for repeated measurements due to toxicity of BODIPY. Thus, the current method allows one reproducibly to measure changes in the volume of muscle tissue.

As described above, genetic marking methods can be used to make repeated measurements of myotome volume, permitting tracking of change in muscle size over successive days in individual fish. As individual fish and entire lays at the same development stage differ in absolute myotome volume (Figure 3A; perhaps due the size or health of eggs), the ability to measure growth of each individual reduces the effects of individual variation by permitting paired sample statistical analyses. Repeatedly measuring the same fish reduces the number of fish needed to detect effects robustly. To illustrate this effect, results from analyzing the growth of each individual from 3 to 4 dpf in populations of active and inactive larvae were compared with analysis of the same two populations using only the single measure of myotome size made at 4 dpf. From lay to lay, larger variation in apparent reduction of myotome size was observed when measuring myotome volume at 4 dpf only compared to measuring 4/3 dpf volume for each individual (Figure 3B). Note the greater range of reduction (68%-91%) in the 4 dpf only measurements, compared to the 4/3 dpf method (78%-89%) and the weak correlation between the two measures. Although, as expected, the mean reduction across all 13 biological replicates was similar in each assessment (Figure 3C) being 82.62 ± 1.01% (mean ± SEM, n = 13) for the 4/3 dpf method and 82.31 ± 1.92% for the 4 dpf only method, the estimated error with the 4 dpf only method was almost double that with the 4/3 dpf method. Thus, quantifying individual growth through repeated measurement is the more accurate method, by eliminating size variability between fish within the same lay, as demonstrated previously²⁶. Nonetheless, as no significant difference in the reduction in myotome volume caused by inactivity was observed when measuring myotome volume at 4 dpf only (Figure 3C), the data suggest that ~6-8 fish are sufficient to average out inter-individual size difference within a lay. Clearly, when size changes are small the 4/3 dpf method is preferred.

Analysis of the cellular basis of growth
Myotome CSA is determined by muscle fiber number and fiber size. Fiber number can be estimated by counting the number of fast and slow fibers on three YZ sections (YZa, YZm, YZp). Although regions of two adjacent myotomes are contained in such YZ sections, as shown by the presence of vertical myosepta (VM) in most sections (Figure 2A), these counts accurately reflect fiber number. Over-counting occurs at VMs due to the tapering of pairs of fibers from each adjacent segment at the VM (Figure 4A). Such overcounting can be accounted for using the following equation: Fiber number = Total fiber count – (fibers in contact with VM) / 2 reference³³. Furthermore, average fiber volume can be determined by dividing myotome volume by the number of fibers. We have used these analyses to reveal that activity controls both cellular aspects of growth (Figure 4B,C).

It is evident from Figure 2A that fibers vary in size across the myotome, a reality that is not reflected in calculated average fiber volume measurements. By drawing around each fiber of somite 17 from two fish, measured fiber cross-sectional area was shown to range from 28 µm² to 217 µm² (Figure 4D). In reality, however, many fibers are angled obliquely within the myotome, so such CSA measures do not reflect the true CSA of a fiber perpendicular to its long axis. Conversely, due to the varying angles of fibers within the myotome, all fibers running at orientations that are not aligned anteroposteriorly have lengths that differ from the myotome length. Despite these caveats, which can only be circumvented either by complete segmentation of the myotome into single fiber volumes or by calculation after measuring the angle of obliquity of each fiber, the measured CSAs provide an estimate of fiber size diversity in each fish. For example, individual fibers decreased in CSA with inactivity, resulting in a shift to the left in the cumulative frequency curve with respect to active (un-anesthetized) control larvae (Figure 4E). As inactive fish have ~10 fewer fibers than active fish (Figure 4B), either the 10 smallest fibers from active un-anesthetized fish (on the assumption that they are the new ones) or the 10 largest (as the alternative extreme) were omitted from the comparison, which showed that most loss of the myotome volume is due to lack of fiber growth, rather than failure of new fiber formation (Figure 4F). Taken together, these data show that the described method allows detailed investigation of the role of physical activity on the formation and growth of muscle tissue.

Reimposition of activity by electrical stimulation
Physical activity is required for muscle growth (Figure 3A). A method to re-impose muscle contractions by electrical stimulation in otherwise inactive larvae, evoking a strong contractile response is described (Figure 5A, Supplemental File 8). Although precise stimulation parameters are described here (Figure 5B) to maximally activate the musculature, the protocol can be altered (by changing current amplitude, frequency, pulse duration, etc.) to control muscle activation and exercise dosage. Thus, the current method provides a controlled activity stimulus with standardized behavior between activity bouts, overcoming an important limitation of current animal models of exercise¹⁸.

The described methods demonstrate the potential of using zebrafish larvae to study various aspects of muscle growth (e.g., hyperplasia and hypertrophy). In particular, myogenesis in zebrafish larvae is shown to be amenable to analysis through pharmacologically-induced inactivity and electrically-induced contractility. The approach allows the study of the molecular mechanisms by which physical activity leads to muscle growth in vivo.

Figure 1: Design of a stimulation chamber for re-introduction of muscle electrical activity to zebrafish larvae. A 6-well cell culture plate fitted with electrodes permits maintenance of larvae in wells made within 2% agarose gel. Custom 4-well combs are made to create rectangle wells (dimensions as indicated) in agarose to maintain position and orientation of individual fish larvae, and to prevent them from contacting the silver wires during vigorous twitching upon electrical stimulation. Please click here to view a larger version of this figure.

Figure 2: Measuring muscle volume in live zebrafish α-actin:mCherry-CAAX (A,B,E) or β-actin:HRAS-EGFP transgenic larvae (H,I). Larvae imaged from lateral view and shown dorsal to top, anterior to left in upper panels (A,B,E). (A) In the 4-slice method, myotome volume was calculated by multiplying the length of somite 17 (L) measured between vertical myosepta (VM) on an XY plane (blue arrow) by the average of three cross-sectional areas (CSA) measured on YZ planes (YZm, YZa, and YZp; dashed yellow lines). (B) In the Full Stack method, the area of somite 17 myotome was measured (examples outlined in yellow) in each slice of a whole Z-stack of a live zebrafish larva and the sum of myotomal areas was multiplied by the inter-slice distance. (C) High concordance between the 4-slice and Full Stack methods. Colors denote individual β-actin:HRAS-EGFP (squares) or α-actin:mCherry-CAAX (circles) fish. (D) Myotome volume is slightly, but significantly, larger when calculated using the 4-slice method. (E) In the tapering zebrafish larvae, more anterior somites are larger than posterior somites, as measured by the 4-slice method. (F,G) Strong correlation between volume measurements made using the average of three CSA sections (4-slice method) or a single YZm CSA (2-slice method). p-values show results of two tailed t-tests with equal variance (D,G) or one way ANOVA with Bonferroni post hoc tests (E). ns, not significant. (H) Measurement of myotome 16 volume, length (L) and cross-sectional area (CSA) from 2 to 5 dpf in three individual fish (colors) expressing β-actin:HRAS-EGFP and myog:H2B-mRFP. YZm images of the green individual at each timepoint are shown above. (I) Single fiber growth measured by automated constant-threshold segmentation. A β-actin:HRAS-EGFP;myog:H2B-mRFP fish mosaically labeled by injection of a CMV:Cerulean plasmid at the 1-2 cell stage. A single Cerulean marked fiber in somite 10 was scanned with high resolution full XYZ stacks repeatedly on a Zeiss LSM880. Images shown are representative single slices (left) and the projection of the three dimensional segmented volume (right). Each datapoint on the graph represents a single scan of the same fiber, at 3 dpf (0 h), after 4 h, and at 5 dpf (54 h). Triplicate scans were made and segmented per time-point to show reproducibility. White arrowheads point to fiber nuclei; note that two nuclei are added between 3 and 5 dpf. Please click here to view a larger version of this figure.

Figure 3: Activity-dependent muscle growth in somite 17. (A) Inhibiting activity for 24 h by application of tricaine (pink) between 3 and 4 dpf reduces myotome volume both in transgenic lines and in non-transgenic fish stained with BODIPY compared to vehicle controls on siblings (blue). Volume quantified by 4-slice method. Symbol shape indicates replicate experiments from distinct lays (biological replicates). Large symbols denote mean ± SEM values. Small faint symbols show the volume of individual replicate larvae. (B) Comparison of myotome volume reduction in inactive fish compared to control siblings determined by single measurement of myotome volume at 4 dpf (4 dpf only, upper schematic) or change in myotome volume between 3 and 4 dpf (4/3 dpf, lower schematic). Each symbol represents the mean volume of myotome 17 in ~5 inactive fish from a single lay divided by the mean myotome 17 volume of ~5 active control siblings. (C) No difference was observed in the mean reduction in muscle growth in inactive fish, when measuring by the 4 dpf only or 4/3 dpf methods. Numbers within bars represent total number of fish analyzed. p-values show results of two way ANOVA with Bonferroni post hoc tests (A) or two tailed t-test with unequal variance (C). Please click here to view a larger version of this figure.

Figure 4: Cellular level changes in muscle growth caused by inactivity. (A) Schematic showing how overcounting occurs at VMs due to double counting of tapering fibers where the blue and red myotomes meet. Note that the average fiber count is 6 but the true mean value is 5.5. The corrected count gives a better approximation. (B,C) Fiber number (B) and average fiber volume (C) are reduced in inactive (pink) compared to active (bue) larvae. Symbol shape indicates replicate experiments from distinct lays (biological replicates). Large symbols denote mean ± SEM values. Smaller faint symbols show the value of individual replicate larvae. Numbers within bars represent total number of fish analyzed. (D) In single larvae, each fiber profile in myotome 17 was outlined and CSA determined. Boxes show mean ± SEM. p-values show results of two-way ANOVA with Bonferroni post hoc tests (B,C) or two tailed t-test with equal variance (D). (E,F) Cumulative frequency curves showing fiber size distribution in active control (blue) and inactive (pink) larvae. Comparison of all fibers (E), or after omission of presumed-nascent small or, at the alternative extreme, large fibers (F) shows that fiber size increase, not increase in fiber number, primarily accounts for activity-driven growth of the myotome. Please click here to view a larger version of this figure.

Figure 5: Brief electrical stimulation evokes tetanic muscle contractions in anesthetised zebrafish larvae. (A) Sequential images (captured from video in Supplemental File 8) showing how electrical stimulation triggers maximal contractions of an anaesthetized larva at 3 dpf. Time scale in seconds. Red boxes indicate movements at the start of three successive 1 s stimulation trains. Each image is a 40 ms exposure. (B) Schematic showing the electrical stimulation regime, in which a 1 s train of 200 high frequency, 20 V electrical impulses is given every 5 s. Please click here to view a larger version of this figure.

Supplemental File 1: Schematics of captured images of perfectly (top left) and imperfectly mounted larvae with respect to the microscope XYZ reference frame (black axes). Myotome (green), notochord (yellow), neural tube (tan), measured myotomal parameters (red), measured notochord parameters (black arrows), and possible or actual fish rotations (blue). Please click here to download this File.

Supplemental File 2: Screenshot showing somite length measurement from XY images in ZEN. Please click here to download this File.

Supplemental File 3: Screenshot showing CSA measurement from YZ images in ZEN. Please click here to download this File.

Supplemental File 4: Screenshot showing somite length measurement from XY images in Fiji/ImageJ. Please click here to download this File.

Supplemental File: Screenshot showing extraction of calibration parameters for YZ images from ZEN. Please click here to download this File.

Supplemental File 6: Screenshot showing calibration of YZ images in Fiji/ImageJ. Please click here to download this File.

Supplemental File 7: Screenshot showing CSA measurement from YZ images in Fiji/ImageJ. Please click here to download this File.

Supplemental File 8: Representative video showing muscle contraction evoked by direct electrical stimulation (length 1 s) of a tricaine-anaesthetized 3 dpf larva. Please click here to download this File.