Name: in vivo calcium imaging of mouse geniculate ganglion neuron responses to taste stimuli
Uploaded: 2021-02-11T15:00:00
Description: Watch this Scientific Journal Video about In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli at JoVE.com

The authors thank S. Humayun for mouse husbandry. Funding for this work has been provided in part by UTSA&#39;s Brain Health Consortium Graduate and Postdoctoral Seed Grant (B.E.F.) and NIH-SC2-GM130411 to L.J.M.

Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas San Antonio.
1. Pre-operative setup
NOTE: Please note that initial setup of equipment is not addressed here, as it will vary based on pump system, microscope, camera, and imaging software used. For setup instructions please refer to instructional materials provided by equipment vendor. For equipment used by the authors, please see the Tabl...

<ol>
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This work describes a step-by-step protocol to surgically expose the geniculate ganglion and visually record the activity of its neurons with GCaMP6s. This procedure is very similar to that described previously17, with a few notable exceptions. First, the use of a head post allows for easy adjustment of head positioning during surgery. Second, regarding stimulus delivery, the approach by Wu and Dvoryanchikov flows taste stimuli through esophageal tubing17, whereas this prot...

A key component of the mammalian peripheral taste system is the geniculate ganglion. In addition to some somatosensory neurons that innervate the pinna of the ear, the geniculate is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate. Similar to other peripheral sensory neurons, the geniculate ganglion neurons are pseudo-unipolar with a long axon projecting peripherally to the taste buds, and centrally to the brainstem nucleus of the solitary tract1. These neurons are activated primarily by the release of ATP by taste receptor cells responding to taste stimuli in the oral cavity2,3. ATP is an essential neurotransmitter for taste signaling, and P2rx receptors expressed by the gustatory ganglion neurons are necessary for their activation4. Given that taste receptor cells express specific taste receptors for a particular taste modality (sweet, bitter, salty, umami, or sour), it has been hypothesized that gustatory ganglion neuron responses to taste stimuli would also be narrowly tuned5.
Whole nerve recordings have shown both the chorda tympani and the greater superior petrosal nerves conduct gustatory signals representing all five taste modalities to the geniculate ganglion6,7. However, this still left questions about the specificity of neuronal responses to a given tastant: if there are taste modality specific neurons, polymodal neurons, or a mixture of both. Single fiber recordings give more information about the activity of individual fibers and their chemical sensitivities8,9,10, but this methodology is limited to collecting data from small numbers of fibers. Similarly, in vivo electrophysiological recordings of individual rat geniculate ganglion neurons give information about the responses of individual neurons11,12,13, but still loses the activity of the population and yields relatively few neuron recordings per animal. In order to analyze the response patterns of neuronal ensembles without losing sight of the activity of individual neurons, new techniques needed to be employed.
Calcium imaging, especially using genetically encoded calcium indicators like GCaMP, has provided this technical breakthrough14,15,16,17,18. GCaMP uses calcium as a proxy for neuronal activity, increasing green fluorescence as calcium levels within the cell rise. New forms of GCaMP continue to be developed to improve the signal to noise ratio, adjust binding kinetics, and adapt for specialized experiments19. GCaMP provides single neuron resolution, unlike whole nerve recording, and can simultaneously measure responses of ensembles of neurons, unlike single fiber or single cell recording. Calcium imaging of the geniculate ganglia has already provided important information about the tuning profiles of these neurons in wild-type mice16,20, and has identified peripheral taste miswiring phenotypes in genetically manipulated mice18.
One major difficulty to applying in vivo calcium imaging techniques to the geniculate ganglion is that it is encapsulated within the bony tympanic bulla. In order to obtain optical access to the geniculate, delicate surgery is required to remove the layers of bones, while keeping the ganglion intact. For that purpose, we have created this guide to help other researchers access the geniculate ganglion and image the GCaMP mediated fluorescent responses of these neurons to taste stimuli in vivo.

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			<td>1 x #5 Inox Forceps</td>
			<td>Fine Science Tools</td>
			<td>NC9792102</td>
			<td></td>
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		<tr>
			<td>1ml Syringe with luer lock</td>
			<td>Fisher Scientific</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>2 x #3 Inox Forceps</td>
			<td>Fine Science Tools</td>
			<td>M3S 11200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge Blunt Dispensing Needle</td>
			<td>Fisher Scientific</td>
			<td>NC1372532</td>
			<td></td>
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			<td>3M Vetbond</td>
			<td>Fisher Scientific</td>
			<td>NC0398332</td>
			<td></td>
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			<td>4-40 Machine Screw Hex Nuts</td>
			<td>Fastenere</td>
			<td>3SNMS004C</td>
			<td></td>
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			<td>4-40 Socket Head Cap Screw</td>
			<td>Fastenere</td>
			<td>3SSCS04C004</td>
			<td></td>
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			<td>Absorbent Points</td>
			<td>Fisher Scientific</td>
			<td>50-930-668</td>
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			<td>Acesulfame K</td>
			<td>Fisher Scientific</td>
			<td>A149025G</td>
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			<td>Artificial Tears</td>
			<td>Akorn</td>
			<td>59399-162-35</td>
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			<td>BD Allergist Trays with Permanently Attached Needle</td>
			<td>Fisher Scientific</td>
			<td>14-829-6D</td>
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			<td>Blunt Retractors</td>
			<td>FST</td>
			<td>18200-09</td>
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			<td>Breadboard</td>
			<td>Thor Labs</td>
			<td>MB8</td>
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			<td>Citric Acid</td>
			<td>Fisher Scientific</td>
			<td>A95-3</td>
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			<td>Cohan-Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-02</td>
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			<td>Contemporary Ortho-Jet Liquid</td>
			<td>Lang</td>
			<td>1504</td>
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			<td>Contemporary Ortho-Jet Powder</td>
			<td>Lang</td>
			<td>1520</td>
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			<td>Cotton Tipped Applicators</td>
			<td>Fisher</td>
			<td>19-062-616</td>
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			<td>Custom Head Post Holder</td>
			<td>eMachineShop</td>
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			<td>See attached file 202410.ems</td>
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			<td>Custom Metal Head Post</td>
			<td>eMachineShop</td>
			<td></td>
			<td>See attached file 202406.ems</td>
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			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
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			<td>Digital Camera, sCMOS OrcaFlash4 Microscope Mounted</td>
			<td>Hamamatsu</td>
			<td>C13440</td>
			<td></td>
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		<tr>
			<td>Disection Scope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
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		<tr>
			<td>Hair Clippers</td>
			<td>Kent Scientific</td>
			<td>CL7300-Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>IMP</td>
			<td>Fisher Scientific</td>
			<td>AAJ6195906</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Ketaved</td>
			<td>NDC 50989-996-06</td>
			<td></td>
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			<td>LED Cold Light Source</td>
			<td>Leica Mcrosystems</td>
			<td>KL300LED</td>
			<td></td>
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		<tr>
			<td>Luer Lock 1/16&#34; Tubing Adapters</td>
			<td>Fisher</td>
			<td>01-000-116</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series Optical Posts</td>
			<td>Thorlabs</td>
			<td>MS2R</td>
			<td></td>
		</tr>
		<tr>
			<td>MPG</td>
			<td>Fisher Scientific</td>
			<td>AAA1723230</td>
			<td></td>
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		<tr>
			<td>MXC-2.5 Rotatable probe Clamp</td>
			<td>Siskiyou</td>
			<td>14030000E</td>
			<td></td>
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			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>50-947-346</td>
			<td></td>
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			<td>petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
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			<td>Pressurized air</td>
			<td>Airgas</td>
			<td>AI Z300</td>
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			<td>Quinine</td>
			<td>Fisher Scientific</td>
			<td>AC163720050</td>
			<td></td>
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		<tr>
			<td>Self Sticking Labeling Tape</td>
			<td>Fisher Scientific</td>
			<td>159015R</td>
			<td></td>
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			<td>Silicone Pinch Valve Tubing 1/32&#34; x 1/16&#34; o.d. (per foot)</td>
			<td>Automate Scientific</td>
			<td>05-14</td>
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			<td>Sola SM Light Engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
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			<td>Snap25-2A-GCaMP6s-D</td>
			<td>JAX</td>
			<td>025111</td>
			<td></td>
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		<tr>
			<td>Student Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
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		<tr>
			<td>Surgical Probe</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6067</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Probe Holder</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6061</td>
			<td></td>
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		<tr>
			<td>Thread</td>
			<td>G&#252;termann</td>
			<td>02776</td>
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			<td>BD Intramedic Tubing</td>
			<td>Fisher Scientific</td>
			<td>22-046941</td>
			<td></td>
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			<td>Two Stage Gas Regulator</td>
			<td>Airgas</td>
			<td>Y12FM244B580-AG</td>
			<td></td>
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			<td>Tygon vinyl tubing - 1/16&#34;</td>
			<td>Automate Scientific</td>
			<td>05-11</td>
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			<td>Valvelink8.2 digital/manual controller</td>
			<td>Automate Scientific</td>
			<td>01-18</td>
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			<td>Valvelink8.2 Pinch Valve Perfusion System</td>
			<td>Automate Scientific</td>
			<td>17-pp-54</td>
			<td></td>
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			<td>Xylazine</td>
			<td>Anased</td>
			<td>NADA# 139-236</td>
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</table>

in vivo calcium imaging of mouse geniculate ganglion neuron responses to taste stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Following the protocol, a transgenic Snap25-GCaMP6s animal was sedated, geniculate ganglia were exposed, and tastant was applied to the tongue while video was recorded. The aim of the experiment was to define which tastants elicited responses from each cell. Tastants (30 mM AceK, 5 mM Quinine, 60 mM NaCl, 50 mM IMP + 1 mM MPG, 50 mM Citric Acid)18 were dissolved in DI water and were applied to the tongue for 2 s separated by 13 s of DI water.
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In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

This technique can answer important functional questions about neuronal responses to taste in the geniculate ganglia, a crucial part of the interior chorda tympani taste pathway. This technique can be used to monitor the real-time reactions of multiple individual neurons in a single experimental trial, as cells recorded per animal are significantly higher than typically observed via the electrophysiological method. With the headpost-mounted mouse in the supine position on a heating pad, make a two centimeter midline incision in the skin over the throat, from the sternum to the chin.And retract the skin and sub-maxillary glands to fully expose the digastric muscles. After locating the seam in the paratracheal musculature, separate the seam with blunt dissection and retract the tissue to open it. Carefully cut an opening in the top of the trachea large enough to fit a piece of polyethylene tubing without cutting more than halfway through the diameter of the trachea and insert the tubing into the trachea toward the lungs.Reposition the retractors to release the paratracheal musculature and retract the sub maxillary glands. Then, use a small amount of veterinary glue to seal the paratracheal musculature together over the tube. To break open the tympanic bulla, gently tease the desired digastric muscle upward and pull the connective tissue apart.Make an incision at the anterior end of the muscle, avoiding the blood vessels. And pull back posteriorly until clear of the tympanic bulla. Tilt the head back slightly to lift the tympanic bulla and locate the branch of the carotid artery anterior to the posterior insertion point of the digastric muscle.Palpate just posterior to this blood vessel for the convex structure of the tympanic bulla and locate a seam in the musculature. Using two sets of fine forceps, blunt dissect at the seam until the bone of the tympanic bulla is visible and use retractors to keep a clear view of the bone. Locate the seam running anterior to posterior on the bulla and use a surgical probe to poke a hole in the bone at the center of the seam, Then, use a set of fine end scissors to cut a circular area in the bone, taking care not to cut blood vessels anterior and posterior to or below the bulla.To expose the geniculate, locate the cochlea. Anterior to the cochlea is the tensor tympani muscle. Use spring scissors to cut and remove this muscle.Use the surgical probe to poke a hole in the cochlear promontory and immediately use suction to aspirate any liquid that flows out of the hole. Enlarge the hole in the cochlea, taking care not to damage the blood vessel encircling the cochlea, to the posterior and lateral edge. And tilt the mouse's head forward to locate the hole in the temporal bone beneath the former cochlear structure.Take note of the ridge anterior to the hole that sits directly over the seventh nerve. And insert a surgical probe into the hole to allow the temporal bone to be carefully lifted to expose the seventh nerve. If the geniculate is not fully visible, gently tilt the animal's head back and attempt to pull up the bone anterior to the nerve.If the ganglia is still obscured, pull up more bone from beneath, taking care not to place the probe deep beneath the bone, as this may damage the geniculate. To run the tastant panel, use suctioned to remove the liquid from over the geniculate and place the mouse on an absorbent pad under a dissecting microscope. Use the hole left in the bulla, the hole in the temporal bone, and the seventh nerve to locate the geniculate ganglion.And use the FITCGFP filter on the epifluorescent scope to check for individual GCaMP expressing geniculate ganglion neurons. Place the dispensing needle for one tastant line firmly into the animal's mouth and place a Petri dish below the mouth to catch any fluid. Synchronize the start of video recording with the start of tastant presentation, watching the live feed for responses, drift, and seepage during the recording.If seepage occurs, suction the liquid until the view of the geniculate is clear and redeliver the tastant. If drift occurs, check that all of the parts of the headpost are firmly tightened. If no response occurs, check that the liquid is flowing and that the microscope and camera are focused on the proper location without anything obscuring the field of view.When all of the desired experiments are completed, gently ease the retractors and repeat the exposure and tastant analysis on the opposite side of the animal. As observed, taste stimuli applied to the tongue should result in a rapid transient increase in GCaMP fluorescence, causing a noticeable change in brightness among the responding neurons. Analysis of the fluorescence video footage allows the generation of traces corresponding to the changes in fluorescence over the baseline response within individual regions of interest over time.Changes in the fluorescence intensity above the threshold level is considered a positive response. In addition to damaging the ganglia, it is important to avoid bleeding. If bleeding occurs, wait for the blood to clot before applying saline and suction to remove the blood from the visual field.Visualization of the geniculate ganglia has allowed researchers to directly measure the responses of neurons to taste-like stimuli and to identify these neurons with tools such as Cre-dependent GCaMP.

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