CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish

Abstract

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function.SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model.

Keywords: circuit function; electrophysiology; myelin; myrf; oligodendrocyte; zebrafish.

Figure 1.

myrf^ue70 mutants display a gross reduction in the level of CNS myelination at the adult and larval stages. A, top, myrf gene structure composed of 27 exons. Red arrowhead marks the location of the mutation in exon 2. Scale bar: 1000 bp. Schematic created using http://wormweb.org. Middle, Wild-type and mutant nucleotide sequences spanning the mutagenesis site. The gRNA target site (red line) and restriction enzyme (RE) recognition site (green line) are labeled. Bottom, Amino acid sequence indicating that the myrf^ue70 mutation results in shift in the open reading frame leading to downstream coding for a premature stop codon (*). B, The relative concentration of mbp mRNA is reduced by 95% in mutants (0.04 ± 0.03 au) compared with wild types (1.003 ± 0.13 au, p = 0.0002, unpaired t test, N = 3 adult brains per genotype). Error bars represent mean ± SD. C, Transverse section of the spinal cord in an adult myrf^ue70 sibling showing extensive myelination of ventral spinal cord (dashed box). 20× objective. Scale bar: 100 µm. D, TEM images of the spinal cord in the region of the ventral spinal tract (outlined in C) in myrf^ue70 adult siblings (top) and mutants (bottom). Panels i–iv display different fields of view within the region of interest. Thick myelin sheaths are clearly visible in siblings, particularly surrounding the Mauthner axon. There is a lack of myelin surrounding the Mauthner axon in the mutant sample, and distinct reduction in the level of myelination in the remainder of surrounding spinal cord. Occasional hypomyelinated and dysmyelinated axons can be observed in the mutant samples. Scale bar: 5 µm (panels i–iii) and 1 µm (panel iv). m, Mauthner axon. E, top, Brightfield images of myrf^ue70 wild-type and mutant larvae at 6 dpf. Black box defines the anatomic region imaged across animals. Scale bar: 0.5 mm. Bottom, Confocal microscopy images of the spinal cord at 6 dpf in myrf^ue70 Tg(mbp:eGFP-CAAX) larvae. Scale bar: 20 µm.

Figure 2.

myrf^ue70 mutants display CNS-specific hypomyelination at 6 dpf. A, TEM images of the myelinated tracts in the dorsal (top row) and ventral spinal cord (bottom rows). Scale bar: 1 µm. B, Schematic of the transverse section of a 6 dpf larval zebrafish at the level of the urogenital opening. Inset, transverse section of the spinal cord at the same level. Myelinated (green) axons are located in the ventral and dorsal spinal tracts of the spinal cord (CNS) as well as the posterior lateral line (PNS). m, Mauthner axons. C, TEM images of the posterior lateral line at 6 dpf. Scale bar: 1µm. D, The average number of myelinated axons in one hemi-spinal cord is reduced by 66% in mutants (wild types: 35.29 $\pm$ 7.83 myelinated axons, mutants: 12.00 $\pm$ 4.34 myelinated axons, p ≤ 0.0001, unpaired t test, N = 7 wild types, N = 8 mutants). E, The number of myelinated axons in the PNS is similar between genotypes (wild types: 7.33 $\pm$ 1.53 myelinated axons, mutants: 9.00 $\pm$3.83 myelinated axons, p = 0.52, unpaired t test, N = 3 wild types, N = 4 mutants). F, G-ratio of Mauthner axons in wild-type and mutant siblings (wild types: 0.48 $\pm$ 0.009, mutants: 0.80 $\pm$ 0.08, p = 0.0009, unpaired t test). For D-F, error bars represent mean ± SD. G, g-ratios for myelinated axons for small caliber (area <0.3 µm²) and large caliber (area >0.3 µm²) myelinated axons. The g-ratio of small caliber axons is similar between groups [wild types: 0.57 (0.52–0.62), mutants: 0.59 (0.52–0.70), p = 0.51, Mann–Whitney test, n = 53 myelinated axons in wild types, n = 17 myelinated axons in mutants error bars represent median and IQR]. The g-ratios for large caliber axons are significantly higher in mutants than wild-type siblings (wild types: 0.60 $\pm$ 0.08, mutants: 0.71 $\pm$0.08, p ≤ 0.0001, unpaired t test, n = 33 myelinated axons in wild types, n = 19 myelinated axons in mutants error bars represent mean ± SD).

Figure 3.

myrf^ue70 mutants have fewer oligodendrocytes which produce less myelin and fail to maintain myelin sheaths over time. A, Confocal images of the spinal cord at 6 dpf in sibling control and myrf^ue70 Tg(mbp:nls-eGFP) larvae. Scale bar: 100 µm. B, Oligodendrocyte numbers in the spinal cord at 6 dpf (wild-type: 304.8 $\pm$ 39.07, mutants: 239.3 $\pm$ 50.48, p = 0.0002, unpaired t test, N = 15 wild types, N = 22 mutants). Error bars represent mean $\pm$ SD. C, Representative confocal images of single oligodendrocytes mosaically labeled with mbp:mCherry-CAAX reporter construct in a wild type (top) and mutant (bottom) at 6 dpf. Scale bar: 15 µm. D, Average myelin sheath number was reduced in myrf^ue70 mutants relative to wild-type siblings at 6 dpf [wild types: 10.50 (7.00–14.00) sheaths per cell, mutants: 7.00 (5.00–10.50) sheaths per cell, p = 0.02, Mann–Whitney test]. Values and error bars represent median and IQR. E, Average myelin sheath length was reduced from 41.83 $\pm$9.68 µm in wild types to 31.35 $\pm$11.49 µm in mutants at 6 dpf (p = 0.002, unpaired t test). Error bars represent mean $\pm$ SD. F, Total myelin produced per oligodendrocyte was reduced from 458.2 $\pm$ 156.4 µm in wild types to 241.1 $\pm$138.6 µm in mutants at 6 dpf (p ≤ 0.0001, unpaired t test). Error bars represent mean $\pm$ SD. D–F, N = 20 wild types, N = 27 mutants. G, Confocal images of a single mutant oligodendrocyte labeled with mbp:mCherry-CAAX at 4 and 6 dpf. A myelin sheath (*) and myelinated neuronal cell body (#) are observed at 4 dpf and subsequently retracted by 6 dpf. Arrowheads label myelin sheaths which are observed to shrink between 4 and 6 dpf. Scale bar: 15 µm. H, Myelin sheaths belonging to wild-type oligodendrocytes demonstrated a net growth of 6.24 $\pm$ 3.43 µm between 4 and 6 dpf, while mutants display net shrinkage of myelin sheaths by −0.31 $\pm$ 4.79 µm (p = 0.003, unpaired t test). Error bars represent mean $\pm$ SD. I, Between 4 and 6 dpf, wild-type oligodendrocytes retracted 0 (0–0) myelin sheaths, while mutants retracted 2 (1–3) myelin sheaths (p = 0.009, Mann–Whitney test). Error bars represent median and IQR. J, Number of abnormal myelin sheaths at 6 dpf [wild types: 0.00 (0.00–0.00); mutants: 2 (0.00–3.00), p ≤ 0.0001, Mann–Whitney test]. Error bars represent median and IQR. H, I, N = 11 wild types, N = 7 mutants. J, N = 20 wild types, N = 27 mutants.

Figure 4.

myrf^ue70 mutants exhibit increased latency to perform startle responses, and a tendency to perform avoidance behavior, in response to defined acoustic stimuli. A, Overview of the neuronal circuitry involved in motor response to auditory stimuli. Startle response (SLC): sensory input from the ear, via the auditory nerve (red), is received at the lateral dendrite of the Mauthner cell body (black). The axon of the Mauthner cell crosses into the contralateral aspect of the spinal cord where it extends along the ventral tract to recruit motor neurons directly along the length of the larvae. Recruitment of motor neurons allows muscle contraction on the side of the body contralateral to the stimulus, allowing a rapid, high-velocity c-bend (motor response) away from the stimulus (inset). Avoidance behavior (LLC): sensory input is detected by prepontine neurons (purple) in the hindbrain, which recruit ipsilateral motor neurons indirectly, resulting in a low-velocity, longer latency, c-bend away from the stimulus. B, Schematic of the behavioral rig. C, Relative frequency histogram displaying the distribution of latencies for behavioral responses in response to acoustic stimuli in wild-type and mutant larvae (N = 24 wild-type larvae, n = 220 events; N = 35 mutant larvae, n = 299 events; Kolmogorov–Smirnov test, p ≤ 0.0001). D, Number and proportion of events (SLC vs LLC) per genotype. E, React rate per fish (median react rate = 100% in both wild types and mutants, p = 0.24, Mann–Whitney test, N = 25 wild-type larvae, N = 38 mutant larvae). Larvae are excluded from subsequent analysis if they exhibit a react rate <70%. F, Average latency values per fish [wild type: 10.55 ms (9.6–16.15 ms), mutants: 17.6 ms (12.9–21.88 ms), p = 0.003, Mann–Whitney test]. G, Average latency of SLC (<16 ms; wild types: 10.03 ± 0.85 ms, mutants: 10.67 ± 0.83 ms, p = 0.006, unpaired t test). H, Average latency of LLC (>16 ms; wild types: 43.20 ± 8.95 ms; mutants: 38.91 ± 10.15 ms, p = 0.28, unpaired t test). I, Mean and SDs values for SLC and LLC responses per genotype. J–M, Analysis of c-bend kinematics. J, Example trace of orientation over time during a behavioral response to an acoustic stimulus. C-bend kinematics are calculated from individual traces for each response per fish. Latency is the time from stimulus onset to behavioral onset (red star). C-bend duration (A) is time from behavior onset to initial turn angle (blue star). Maximum angular velocity is defined as the change in orientation over time (B/A). Turning angle equates to the initial turn angle. K, Initial turn duration (SLC: wild types: 10.06 ± 0.70 ms, mutants: 9.81 ± 0.72 ms, p = 0.20, unpaired t test; LLC: wild types: 14.30 ± 3.65 ms, mutants: 13.25 ± 3.10, p = 0.42, unpaired t test). L, Maximum angular velocity [SLC: wild types: 24°/ms (22.78–28.68°/ms), mutants: 25°/ms (23.10–26.60°/ms), p = 0.73, Mann–Whitney test; LLC: wild types: 16.16 ± 6.61°/ms, mutants: 13.67 ± 4.95°/ms, p = 0.24, unpaired t test]. M, Initial turn angle (SLC: wild types: 121.9 ± 10.80°, mutants: 127.4 ± 9.86°, p = 0.051, unpaired t test; LLC: wild types: 85.11 ± 35.78°, mutants: 83.78 ± 29.07°, p = 0.91, unpaired t test). N, Descriptive statistics (mean ± SD) for c-bend kinematics. For G–H, K–M, SLCs, N = 24 wild types, N = 35 mutant larvae, LLCs, N = 8 wild-types, N = 34 mutant larvae. For E, F, L, values represent median and interquartile range; for G–I, K and M values represent mean ± SD.

Figure 5.

Whole cell current-clamp recordings from Mauthner cells demonstrate slower conduction velocity times and abnormal spiking profiles in myrf^ue70 mutants. A, Electrophysiological preparation for recording from Mauthner neuron in a whole-cell current clamp configuration while stimulating with an extracellular monopolar field electrode midway through the spinal cord. B, Resting membrane potential is unchanged in siblings (n = 18 cells): −70.82 ± 2.76 mV, mutants (n = 6 cells): −70.68 ± 1.25 mV, p = 0.9077 at 6 dpf. C, Sample trace of an action potential recorded at 6 dpf in a wild-type fish illustrating the measurement of half-width. Half-width is described as width of action potential (ms) at its half height. D, Half-width of action potential is unchanged [siblings (n = 18 cells): 0.64 ± 0.09 ms, mutants (n = 9 cells): 0.60 ± 0.06 ms, p = 0.2610 at 6 dpf]. E, An example of current–clamp recording from Mauthner neuron in a 6 dpf wild type and mutant following field stimulation (stimulus artifact is indicated by a gray dashed line). Latency is described as time from the onset of stimulus artifact to the peak of action potential. F, Normalized action potential latency is increased in mutants at 6 dpf [siblings (n = 19 cells): 0.80 ± 0.11 ms/mm, mutants (n = 9 cells): 0.97 ± 0.07 ms/mm, p = 0.0003 at 6 dpf]. G, Conduction velocity of Mauthner action potentials is significantly decreased in mutant larvae [siblings (n = 19 cells): 1.27 ± 0.17 m/s, mutants (n = 9 cells): 1.04 ± 0.08 m/s, p 0.0005 at 6 dpf]. H, Sample traces of three subsequent action potentials recorded from the same wild-type Mauthner cell at 6 dpf superimposed and aligned to the peak of stimulus artifact. The area outlined by the rectangle is magnified in the inset and demonstrates slight imprecision of action potential arrival. I, Precision of action potential arrival is comparable in siblings and mutants [siblings (n = 16 cells): 0.0064 ± 0.0019 ms, mutants (n = 8 cells): 0.0062 ± 0.0009 ms, p = 0.8166 at 6 dpf]. J, Sample trace of a train of action potentials fired following 10 stimuli at 1000 Hz at 6 dpf in a myrf^ue70 mutant and sibling. K, Mauthner neurons in mutant larvae do not sustain prolonged action potential trains of high-frequency stimulation [siblings (n = 19 cells): 55.79 ± 10.17% mutants (n = 9 cells): 38.89 ± 17.64% at 6 dpf, p = 0.0014 at 6 dpf]. For B, D, F, G, I, K, error bars represent mean ± SD. Unpaired t test for B, D, F, G, I and a two-way ANOVA for K. Scale bars: 10 mV and 1 ms (C, E, H, J) and 5 mV and 200 μs (H, inset).