Strong Static Magnetic Fields Elicit Swimming Behaviors Consistent with Direct Vestibular Stimulation in Adult Zebrafish

<div><p>Zebrafish (<i>Danio rerio</i>) offer advantages as model animals for studies of inner ear development, genetics and ototoxicity. However, traditional assessment of vestibular function in this species using the vestibulo-ocular reflex requires agar-immobilization of individual fish and specialized video, which are difficult and labor-intensive. We report that using a static magnetic field to directly stimulate the zebrafish labyrinth results in an efficient, quantitative behavioral assay in free-swimming fish. We recently observed that humans have sustained nystagmus in high strength magnetic fields, and we attributed this observation to magnetohydrodynamic forces acting on the labyrinths. Here, fish were individually introduced into the center of a vertical 11.7T magnetic field bore for 2-minute intervals, and their movements were tracked. To assess for heading preference relative to a magnetic field, fish were also placed in a horizontally oriented 4.7T magnet in infrared (IR) light. A sub-population was tested again in the magnet after gentamicin bath to ablate lateral line hair cell function. Free-swimming adult zebrafish exhibited markedly altered swimming behavior while in strong static magnetic fields, independent of vision or lateral line function. Two-thirds of fish showed increased swimming velocity or consistent looping/rolling behavior throughout exposure to a strong, vertically oriented magnetic field. Fish also demonstrated altered swimming behavior in a strong horizontally oriented field, demonstrating in most cases preferred swimming direction with respect to the field. These findings could be adapted for ‘high-throughput’ investigations of the effects of environmental manipulations as well as for changes that occur during development on vestibular function in zebrafish.</p></div

Average VCR and VOR responses to increasing pulse rates.

<p><i>A</i>-<i>D</i>, Average head (blue) and eye (red) movement traces, in monkey J and B, evoked using increasing pulse rates of 50 (A), 100 (B), 200 (C) and 300pps (D) at maximum current amplitude. <i>E</i>-<i>G</i>, Plots of peak head and eye velocities as a function of pulse rate for current amplitudes of 50 (E), 75 (F), 100% (G) of the maximum.</p

VCR and VOR response latency and relative contribution to gaze.

<p><i>A</i>-<i>B</i>, Latency of evoked eye or head movements using a 2 standard deviation (A) or slope intercept measurement (B). <i>C</i>, Plots of eye versus head movement amplitude during pulse trains delivered at 50, 100, 200 and 300pps. <i>D</i>, Top panels show the contribution of head and eye to instantaneous gaze velocity during pulse trains delivered at the maximum current amplitude and 300pps. Bottom traces show the contribution of head and eye to cumulative gaze position. The average gaze velocity (top panels) and position (bottoms panels) traces are also plotted for comparison.</p

VCR and VOR responses to increasing current amplitudes.

<p><i>A</i>, Pathways connecting the vestibular nerve to neck or extraocular motoneurons. <i>B</i>-<i>D</i>, Average head (blue) and eye (red) movement traces, in monkey J and B, evoked using current amplitudes of 50 (B), 75 (C), and 100% (D) of the maximum for pulse trains delivered at 300pps lasting 100ms. Gray bars indicate stimulus duration and shading represents standard error. Note that for some velocity traces the standard error is smaller than the line thickness. Movements away from implanted side are upwards. Arrows show rebound effect due to the release of inhibition. Insets show peak head and eye velocities for the corresponding traces. <i>EOM</i>, extraocular motoneuron; <i>INC</i>, interstitial nucleus of Cajal; <i>MRST</i>, medial reticulospinal tract; <i>MN</i>, motoneuron; <i>VST</i>, vestibulospinal tract; <i>VN</i>, vestibular nuclei.</p

Adult zebrafish swimming behavior in a horizontal 4.7 T magnet in darkness with infrared illumination.

<p>a) Traces of zebrafish movement during a 1-minute time interval inside the magnetic field are shown in red and yellow. b) A region of interest (ROI, dotted blue line) in the center of the container was defined, and lines were fit to zebrafish swimming paths crossing the ROI (yellow lines in (a)). Red lines in (a) represent swimming paths during the 1-minute time interval inside the magnetic field that occurred outside the ROI or where no line could be fit across the ROI. The magnet north (N) and south (S) poles and magnetic field vectors are demonstrated in b. Most fish developed a heading preference inside the magnet, increasing velocity along its direction of preference. Preference varied by fish; however, the majority avoided the N/S magnetic field vectors. Some fish in the 11.7 T vertical magnet frequently dove in the direction of the magnetic field vector (a, <i>top row</i>); these fish preferred the N/S magnetic field vectors in the 4.7 T horizontal magnet. Six fish (<i>bottom</i> row) showed no consistent heading preference. c) Box plots showing the number of times a fish crossed the container's central zone over a 30-s interval are shown. Even while kept in darkness (with IR) throughout transit into and out of the horizontal magnet, fish more frequently crossed the central zone while in the magnet than while outside the magnet, suggesting that they normally can detect directional cues in a strong magnetic field without visual input. * indicates statistical significance at p<0.01</p

Interaction of vestibular-driven head and eye movements.

<p><i>A</i>, Average gaze, eye and head position (top panels) and velocity (bottom panels) traces during stimulations when the head was restrained and free. Gray bars indicate stimulus duration and shading represents standard error. <i>B</i>, Plots of average gaze movement amplitude during pulse trains delivered at 50, 100, 200 and 300pps when head-restrained versus free. <i>D</i>, Plots of average eye movement amplitude during pulse trains delivered at 50, 100, 200 and 300pps when head-restrained versus free.</p

Adult zebrafish behavior outside and inside of an 11.7 T vertical magnetic field.

<p>Tracing of adult zebrafish path in visible green light during 1 minute prior to magnetic field entry (a) and during 1 minute inside the magnet (b). X- and y-position coordinates are displayed as a function of time. Upon entry into the magnet, fish swimming becomes erratic, with frequent rolling, tight circling and increased swimming velocity.</p

Box plots of adult zebrafish average swimming velocity demonstrating effect of vision on behavior outside and inside of an 11.7 T vertical magnetic field.

<p>X-axis demonstrates the order of light exposure from left to right. Zebrafish were observed for one minute in each lighting condition. The red, dashed vertical lines represent a transition into or out of the 11.7 T vertical magnet. a) Upon entering the magnetic field, swimming velocity increases. There was, however, no change in swimming velocity when lighting in the magnetic field changed from green (visible) to IR (invisible). b) Box plots are shown comparing mean swimming velocities during two minutes inside the magnet to the first minute after exiting the magnet. For those fish that transitioned out of the magnet in IR light (n = 15, top panel), there was less of a decrease in swimming velocity compared to those fish that transitioned out of the magnet in green light (n = 15, bottom panel). IR, infrared.</p