The overall goal of this procedure is to record calcium changes in olfactory bulb neurons in response to temperature drops induced at the nasal epithelium. This method can help answer key questions in the field of affections, such as how the olfactory bulb integrates and processes temperature information acquired in the nose. The first and second order neurons of the olfactory bulb are differentially stained and the affects of temperature fluctuations at the nasal epithelium are measured by calcium imaging.

The visual demonstration of cell electroporation, bolus-loading and activity correlation imaging will facilitate the implementation of these techniques in brain networks other than the olfactory bulb. To begin this procedure, place a tadpole on a gel cushion. Fix it by placing needles around and beware not to poke the needles through its skin.

Next, place the animal under the stereo microscope and focus on the nostrils. Then, place a dye crystal in one of the nasal cavities and wait until it dissolves completely, which should take less than a minute. If the crystals are smaller, add two or three into each cavity until they produce a non-translucent, high-concentrated solution.

After that, place the cathode on the skin of the tadpole and the anode into one of the nostrils. Apply six pulses of 20 volts and 20 milliseconds with approximately 0.5 seconds of stimulus interval. For dyes with a different polarity, the staining results may be improved by placing the cathode into the nostrils.

If unsure about the polarity of the dye, both electrodes can be placed simultaneously in the nostrils and alternate the polarity between single voltage pulses. After sacrificing the animal, use a scalpel to dissect out a block of tissue containing the nasal cavities, the olfactory nerves and olfactory bulbs. Make the first incision close to the left nostril and move the blade forward, cutting alongside the left olfactory nerve and bulb until the telencephalic-diencephalic border.

Repeat likewise on the right side. Next, isolate the preparation from the rest of the nervous system by making a final cut posterior to the telencephalon. Pin the tissue block again by inserting two needles between the olfactory nerves.

Then, put a drop of Frog Ringer solution on the tissue. Bathing the preparation in Ringer solution will prevent the brain tissues from collapsing upon dissection. After that, remove the meninges covering the axon sorting zone and the olfactory bulbs by making three incisions.

Starting from the posterior edge of the tissue block, cut caudorostrally closely along the left olfactory bulb until the entrance point of the olfactory nerves into the bulbs. Repeat this procedure for the right hemisphere. Subsequently, raise the meninges with forceps and make the third cut perpendicular to the previous ones at the axon sorting zone.

After that, transfer the sample to a recording chamber filled with Frog Ringer for further processing and imaging. Make sure that the ventral side is facing upward and secure the sample with a net of nylon fibers spanned over a small platinum frame. For an easier application of stimuli, place the nasal cavities on top of one of the nylon fibers.

In this procedure, fill the micropipette with 10 microliters of the solution and remove the air bubbles by flicking the micropipette. Next, mount the micropipette in a pipette holder. Make sure that pressure can be applied either manually with a syringe or a pneumatic drug ejection device and monitor the applied pressure with a gauge.

Then, lower the pipette onto the surface of the olfactory bulb with the tip pointing into the rostral direction of the preparation. Apply a small and constant positive pressure to the micropipette to prevent clogging of the pipette. Gently insert the micropipette into the tissue and apply a positive pressure.

Confirm the outflow from the pipette by watching for slight tissue movements visible under bright light illumination when the pressure is applied. After 10 minutes of loading, reduce the applied pressure to zero and check the staining. The size of the stained area varies significantly and depends on several parameters, like the amount of injected dye and location of ejection site.

A good staining covers an area of about 100 micrometers by 100 micrometers. Monitor the progressive dye loading of mitral cell somata over time during bolus loading. If the staining intensity or the number of stained mitral cells do not increase, check the pipette for clogging and replace it if necessary.

In this step, monitor the temperature of the cooled Ringer by inserting the clean thermometer probe into the tube. Wait until the temperature drops below one degree Celsius before starting the experiment. Next, use a funnel applicator allowing stimulus delivery concomitantly to the perfusing Ringer so that the water flow in the chamber remains constant and uninterrupted during the stimulus solution release.

Then, position the funnel in such a way that the distal outlet is less than one millimeter away from the olfactory epithelium. Afterward, place a nichrome temperature sensor connected to a a digital thermometer close to the epithelium and the outlet of the funnel applicator. Wire the thermometer output port to a computer to record and visually display voltage changes reflecting small temperature fluctuations.

After that, start the image acquisition and sequentially apply 200 to 400 microliters of cold Ringer, L-histidine, and room temperature Ringer via an electronic pipette with an interstimulus interval of 20 to 30 seconds. For better control of the stimulus application, release the stimulus with a trigger signal sent by the chosen imaging setup to the pipette if possible. In this procedure, load the raw data acquired as a variable to the MATLAB user's workspace, organized as an x, y, z, t matrix, with x and y referring to the lateral dimensions, z, the axial direction, and t, the time course.

Then, call ACI from the MATLAB command line. In the UI, select Prepare Data, then select the variable containing the data and a directory in which the results will be saved. Scroll through the measured z-layers by moving the corresponding slider in the UI to get an overview of the variance map displayed.

After that, enter the size of the ROI into the UI.For a mitral cell soma, adjust the ROI to span approximately 10 micrometers in the lateral and five micrometers in the axial direction. For a glomerulus, the slightly higher values of 20 micrometers laterally and 10 micrometers axially are appropriate. Then, select an ROI containing the gamma glomerulus and additional regions for each visible soma of the surrounding mitral cells by clicking with the middle mouse button onto the center of the cell or glomerulus.

Afterward, close the main UI, which triggers the calculation of correlation maps for all reference traces. The result is automatically saved and displayed. This image shows that the axons of ORNs terminating in the small cluster were stained by electroporation with a non-calcium sensitive dye, Alexa 647 Dextran.

The dotted line outlines the gamma glomerulus. Shown here is an image of the same region in the second measurement channel after bolus loading with a calcium sensitive dye, Fluo-8 AM.Some mitral cell somata were visible but the contrast was limited. Two response traces were plotted for activity correlation imaging, Blue, red and black bars below the traces depict the onset of the application of cold Ringer, histidine at 10 micromolar, and room temperature Ringer as negative control, respectively.

The ACI result of the trace in the highlighted areas responding predominantly to cold Ringer is shown here. And here is the ACI result of the trace in the highlighted areas responding predominantly to histidine. This image shows the overlay of the two ACI maps.

Mitral cells responding to histidine and the innervated glomerulus were easily distinguishable form thermosensitive mitral cells in the gamma glomerulus. This procedure can be performed to investigate whether thermosensitivity and chemosensitivity are in covert and overlapping neuronal networks of the olfactory bulb. After watching this video, you should have a good understanding of how to record temperature processing in the olfactory bulb using cell electroporation in living animals, bolus loading, and activity correlation imaging, also called ACI.

Bolus loading and ACI are powerful tools to observe and compare the activity and connectivity of dozens of neurons in 3D, which makes them readily applicable to different brain regions and neuronal networks.

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