Imaging Calcium Dynamics in Lateral-Line Hair Cells of Larval Zebrafish

Take a paralyzed transgenic zebrafish larva mounted in a perfusion chamber.

The larva expresses membrane-localized calcium indicators in the hair cells of the neuromast, a component of the lateral-line system that detects water movement.

The hair cells comprise apical hair bundles and basal presynaptic regions, which form synapses with afferent neurons to transmit signals.

Under a confocal microscope, use low magnification to position a fluid-jet pipette near the larva..

Increase magnification to locate a neuromast, reposition the pipette near it, and switch to

fluorescence imaging.

Deliver fluid pulses to deflect hair bundles, opening mechanotransduction channels for apical cation influx, including calcium ions.

Weakly fluorescent indicators bind to calcium, causing a localized increase in fluorescence.

The cation influx depolarizes the cell, triggering calcium entry through voltage-gated channels at the basal presynapse, where it binds to the indicators and increases fluorescence locally.

Visualize the apical and basal calcium dynamics in real time.

In this procedure, backfill 10 microliter of neuronal buffer into a properly broken fluid jet needle using a gel loading tip. Load the solution evenly to the tip without bubbles. Then insert the needle into the pipette holder attached to the motorized micromanipulator.

Place the perfusion chamber into a circular chamber adapter on the microscope stage. Move the motorized stage so that the larva is in the center of the field of view. Subsequently, turn the circular chamber adapter so that the A-P axis of the larva is roughly aligned with the trajectory of the fluid jet needle.

Using transmitted light and differential interference contrast, bring the larva into focus and center it under the 10x objective. Then raise the 10x objective. Using the motorized micromanipulator, bring the fluid jet needle down into the center of the field of view, so it is illuminated by the transmitted light and barely touching the neuronal buffer.

Lower the 10x objective to focus on the larva in order to confirm its location. Focus the objective on the fluid jet needle. Move the fluid jet needle with the micromanipulator in the x and y-axes until it is in a positioned parallel to the dorsal side of the larva.

Afterward, focus back on the larva. Bring the needle down in the z-axis, and position the needle along the dorsal side of the fish, 1 millimeter away from the body. Carefully move the circular chamber adapter to ensure that the fluid jet needle is aligned along the A-P midline of the larva.

Next, switch to the 60x water objective. Ensure that the objective is immersed in the neuronal buffer. Use the fine focus to locate a neuromast. Subsequently, move the motorized stage to place the neuromast of interest in the center of the field of view.

Keep the fluid jet needle tip along the dorsal side of the fish. Do not touch the fluid jet needle tip to the larva or the chamber surface. Position the fluid jet needle with the micromanipulator so that it is 100 micrometers from the outer edge of the neuromast.

Then focus up to the tips of the apical hair bundles. The bottom of the fluid jet needle should be in focus in this plane. Set the high speed pressure clamp from the manual to external mode to receive input from the imaging software. Zero the high speed pressure clamp by pressing the zero button, and use the set-point knob to set the resting pressure slightly positive. Confirm the resting output of the high speed pressure clamp using a PSI manometer attached to the head stage output.

To determine the pressure needed to stimulate the hair bundles, apply a test stimulus with a 0.125 and 0.25 volt output for 200 to 500 milliseconds. For each test stimulus, measure the distance of deflection of the tips of the hair bundles (the kinocilia). Choose a pressure that moves the bundles a distance of approximately 5 micrometers. Ensure that the tips of the kinocilia remain in focus the entire time.

To acquire single plane images, select a stimulus to deliver during the 80 frame acquisition after frame 30 at three seconds. To measure mechanosensitive calcium responses, focus on the base of the apical hair bundles and start image acquisition. To measure presynaptic calcium responses, focus at the base of the hair cells and start image acquisition.