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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Watch this Scientific Journal Video about In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli at JoVE.com

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 ...

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