This method can help answer fundamental questions in the field of hearing and balance. It describes how to detect two important aspects of sensory hair cells function, mechanotransduction and presynaptic activity. The main advantages of this technique are that it allows us to visualize the function of hair cells in a live animal, and to study how collections of hair cells detect and transmit sensory information.

To begin this procedure, use fine forceps and tungsten wire to fashion the pins to be used to immobilize the larva through the head and tail, on the hardened encapsulant. To make head pins, hold a piece of 0.035 millimeter tungsten wire in one hand. Using fine forceps in the other hand, bend the wire one millimeter up from the end, at 90 degrees.

Exchange the forceps for fine scissors, and cut one millimeter after the bend, to create the pin. Then use forceps to insert the pins into the hardened encapsulate on the chamber. Position the larva at the center of the perfusion chamber so that it lies flat on its side against the silicone encapsulant.

Using fine forceps, bring the 0.035 millimeter head pin down perpendicular to the larva. Insert the head pin between the eye and otic vesicle, and down into the encapsulant. Use a second set of forceps to stabilize the larva along its dorsal and ventral side, while pinning.

Ensure that the horizontal part of the pin contacts the larva and does not press all the way into the encapsulant. Angle the pin ventrally, or pointing slightly toward the anterior of the larva, to avoid interfering with subsequent heart injection and hair cell imaging. Using the forceps, insert a 0.025 millimeter tail pin into the notochord, as close as possible to the end of the tail.

To inject alpha bungarotoxin into the larva, backfill three microliters of the solution into the injection needle, using a gel-loading pipette tip. Load the solution evenly to the tip, with no bubbles. Then insert the heart injection needle into a pipette holder attached to a manual micromanipulator.

Under a stereo microscope, position the needle so it is aligned perpendicularly to the AP axis of the anesthetized larva, pointing down at an angle of 30 degrees. Next, connect the pipette holder to the pressure injector. Inject a bolus of alpha bungarotoxin into the solution, to test whether the needle tip is clear.

If no red color is seen, very gently scrape the needle tip against the edge of a pin, and try again, until the needle is clear. Subsequently, advance the needle toward the heart, until it touches the skin outside of the heart. Press the needle into the larva, and look for indentation of the pigment cell on the skin in front of the heart to ensure that the needle is positioned in the correct plane, relative to the larva.

Then, advance the needle farther, until it pierces the skin and enters the heart cavity. Pull the needle back slightly, and inject a bolus of alpha bungarotoxin into the heart cavity. Look for inflation of the heart cavity, or for red dye entering the cavity.

Gently rinse the larva three times, with one milliliter of neuronal buffer to remove the residual MS222. Never remove all of the fluid. Maintain larva in approximately 1 millileter of neuronal buffer in the perfusion chamber.

In this procedure backfill 10 microliter of neronal 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 adaptor so that the AP access to 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. Fuous the objective on the fluid-jet needle. Move the fluid-jet needle with the micromanipulator in the x and y-axis until it is in a position 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 adaptor to ensure that the fluid jet needle is aligned along the AP midline of the larva.

Next, switch to the 60x water objective. Ensure that the objective is emersed in the neuronal buffer. Use the fine focus to locate a neuromast.

Subsequently move the motarized 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 200-500 milliseconds. For each test stimulus, measure the distance of deflection of the tips of the hairbundles, 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 3 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. The steps to visualize the spatiotemporal changes in GCaMP6S intensity, within a neuromast during simulation, are outlined here. Time is represented from left to right according to the time stamp.

The top row shows five of the fourteen temporal bins from a 70-frame GCaMP6S F image sequence. In the second row, the baseline has been removed from each GCaMP6S F binned image to create delta F images. In the third row, delta F images have been converted from gray scale, to red hot lookup table.

In the bottom row, the third row has been overlayed onto the F images in the top row. While attempting this procedure, it is important to remember to make sure the larva is pinned correctly and to use appropriate controls, as described in the manuscript to exclude motion artifacts from your data. Working with alpha bungarotoxin can be hazardous.

It is important to take precautions, such as wearing gloves while handling it.

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