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 Table of Materials.
<ol>
	<li>Ensure liquid flows through all vehicle (water) and tastant lines. If line is blocked, disconnect and flush with water. If the line is kinked, massage until liquid flows. Ensure that liquid starts and stops on cue.</li>
	<li>Once all lines are confirmed unblocked, run vehicle for 10 s then close all valves.</li>
	<li>Ensure imaging software is ready with all required variables (e.g., trial length, file names, frame rate, etc). Using &#181;Manager, an open-source image acquisition software package, input 200 ms into the field labeled Exposure Time for a frames per second of 5Hz, select x2 under binning, and press the button labeled Live. When the video starts, press the button on the left side labeled ROI. This will result in a 512x512 field of view.</li>
</ol>
2. Anesthetizing and immobilizing the animal
NOTE: The following protocol is a terminal procedure optimized for mice of either sex weighing 18-35 g. It is recommended for use with animals between 10 and 12 weeks of age. It may be used with transgenic animals expressing Genetically Encoded Calcium Indicators (GECIs) such as the Snap25-GCaMP6s, or animals stereotaxically injected with viral GECIs. Gloves, lab coat, and face mask should be worn for entirety of protocol.
<ol>
	<li>Scruff animal and perform an intraperitoneal injection of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). Assess the depth of anesthesia via toe pinch before continuing.</li>
	<li>Shave the top of the head amd the surgical area at the front of the neck.</li>
	<li>Turn on the heating pad and place the animal prone on the pad.</li>
	<li>Apply ointment to the animal&#39;s eyes to avoid drying of the eyes.</li>
	<li>Make an incision (~1 cm) at the midline of the head to expose the animal&#39;s skull. Remove connective tissue using a sterile swab so that the bare bone is accessible. Use a cotton tipped applicator to ensure the skull is dry.</li>
	<li>Apply vet bond to the skull. Be sure to cover the exposed skull. Wait for the glue to dry.</li>
	<li>In a Petri dish lid, mix and apply a layer of dental cement to skull. The back end of the cotton tipped applicator used in step 2.5 will work well for this process. Place headpost on top of the dental cement and apply a second layer of dental cement to sandwich the headpost in place on the skull.</li>
	<li>Let it sit until the dental cement is dry and solid. Break the cotton tipped applicator in half and use the pointed ends to poke the dental cement to test. If the dental cement does not yield to being poked the animal may be turned to a supine position.</li>
</ol>
3. Tracheotomy
<ol>
	<li>Apply pre-surgical scrub to surgical area. Post-scrub, make a midline incision ~ 2 cm in the skin of the throat from the sternum to the chin.</li>
	<li>Retract the skin and sub-maxillary glands, being sure to fully expose the digastric muscles.</li>
	<li>Find the seam in the paratracheal musculature, separate it with blunt dissection, and retract open.</li>
	<li>Carefully cut an opening in the top of the trachea large enough to fit polyethylene tubing (I.D. 0.86 mm, O.D. 1.27 mm). Do not cut more than halfway through the diameter of the trachea. Insert tubing into the trachea towards the lungs.</li>
	<li>Reposition retractors to release paratracheal musculature and retract the submaxillary glands.</li>
	<li>Glue paratracheal musculature together over tubing with a minimal amount of veterinary glue (see Figure 1A).</li>
</ol>
4. Breaking open the tympanic bulla
<ol>
	<li>Gently tease desired digastric muscle (left or right) up and pull apart the connective tissue. Cut at the anterior end of the muscle, avoiding blood vessels, and pull back posteriorly until clear of the tympanic bulla.</li>
	<li>Tilt the head back slightly to lift the tympanic bulla. Locate the branch of the carotid artery anterior to the posterior insertion point of the digastric muscle. Feel just posterior to this blood vessel for the convex structure of the tympanic bulla.</li>
	<li>Look for a seam in the musculature at this location (see Figure 1B). Using two sets of fine forceps, blunt dissect at the seam until the bone of the tympanic bulla is visible. Use retractors to keep a clear view of the tympanic bulla.</li>
	<li>Find the seam running anterior to posterior on the bulla (see Figure 1C). Using a surgical probe, poke a hole in the bone at the center of this seam. Use a set of fine end scissors to cut a circular area in the bone, taking care not to cut blood vessels anterior, posterior, and deep beneath the bulla.</li>
</ol>
5. Exposing the geniculate
<ol>
	<li>Within this hole is a convex bit of bone, this is the cochlea. Anterior to the cochlea is a muscle, the tensor tympani (see Figure 1D). Using the spring scissors, cut the tensor tympani and remove it.</li>
	<li>Perform a toe pinch. If animal responds, give a ketamine/xylazine mixture ata 1/3 dose for redosing.</li>
	<li>Prepare irrigation fluid and a suction line. Using the surgical probe, poke a hole in the cochlear promontory. Immediately irrigate the liquid that flows out and remove it with suction. This liquid will flow more or less continuously from this point and will need to be addressed periodically.</li>
	<li>Enlarge the hole in the cochlea. Take care with the blood vessel encircling the cochlea to the posterior and lateral edge.</li>
	<li>Tilt the mouse&#39;s head forward. Locate the hole in the temporal bone beneath what was the cochlea (see Figure 1E). Take note of the ridge anterior to this hole, this ridge sits directly over the seventh nerve.</li>
	<li>Insert a surgical probe into the hole and carefully lift the temporal bone to expose the seventh nerve (see Figure 1F). Take stock of how much of the seventh nerve is visible and if the geniculate is not fully exposed, tilt the animal&#39;s head back and attempt to pull up bone from anterior to the nerve.</li>
	<li>If the ganglion is still not fully visible, pull up more bone from beneath. Be very careful not to place the probe deep beneath the bone as doing this may damage the geniculate.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62172/62172fig1v2.jpg" /> 
Figure 1: Surgical exposure of the geniculate ganglion. (A) Image of the mouse neck cavity post tracheotomy. Arrow is pointing to the digastric muscle lying over the surgical area explored in the rest of the figure. (B) Image of region under the previously indicated digastric muscle. Arrow indicates the seam in musculature for blunt dissection. (C) Image of the Tympanic Bulla. Arrow indicates seam in the bone to break with a surgical probe. (D) Image of surgical area after opening the bulla. Lower left arrow indicates the cochlea, upper arrow points to the tensor tympani. Boxed line indicates area in (E) and (F). (E) Image of surgical area after cochlea has been broken and the contents removed. White arrow indicates where to place surgical probe referenced in protocol step 5.6. (F) An image of the exposed geniculate ganglion. Arrow indicates body of the seventh nerve, dashed triangle surrounds the geniculate ganglion. Panels A-B, Scale = 5 mm. Panels C-F, Scale = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62172/62172fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Run tastant panel
<ol>
	<li>Use suction to remove liquid from over the geniculate. Optionally place an absorbent point to help mitigate seepage and aid in microscope navigation.</li>
	<li>Place the animal on absorbent pad under the microscope. Locate the geniculate ganglion: useful landmarks include the hole left in the bulla, the hole in the temporal bone, and the seventh nerve. Using the FITC/GFP filter on the epifluorescence scope, check for individual GCaMP-expressing geniculate ganglion neurons. A 10x objective (workind distance 10mm) will provide sufficient resolution to track the activity of individual cells, but a 20x objective (working distance 12 mm) can also be used.</li>
	<li>Place dispensing needle for tastant line firmly in animal&#39;s mouth. Place a Petri dish below the animal&#39;s mouth to catch fluid.</li>
	<li>Ensure that the camera is viewing the microscope&#39;s field of view. Synchronize start of the video recording with the start of tastant presentation.</li>
	<li>During recording, watch live feed for responses, drift, and seepage.</li>
	<li>If seepage occurs, suction the liquid until the view of the geniculate is clear and repeat. If drift occurs, check that all parts of the head post are firmly tightened. If no responses occur check that liquid is flowing and that the microscope and camera are focused on the proper location without anything obscuring the field of view.</li>
	<li>Repeat until desired number of videos have been obtained. Gently ease retractors, then repeat steps 3-6 on the opposite side.</li>
	<li>After the desired videos have been obtained for all desired ganglia, euthanize the animal via cervical dislocation.</li>
</ol>

<ol>
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The authors have no conflict of interest to report.

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 protocol delivers liquid directly into the mouth with a dispensing needle. Either method can be used to successfully evoke geniculate ganglion neuron responses by stimulating the fungiform and palatal taste buds.
A note on maintaining a clear imaging field: after breaking the cochlea, there will be continuous seepage of fluid within the cavity, including directly over the geniculate ganglion. It is also possible that bleeding will obscure the geniculate ganglion. While a small amount of seepage may not be sufficient to prevent imaging, even small amounts of blood can entirely occlude the ganglia. These issues can be addressed in a couple of ways. First, if seepage is relatively minor, it can be removed using a suction line fitted with a blunt dispensing needle between trials. Alternatively, liquid can also be wicked away from the field by carefully placing absorbent points posterior or lateral to the seventh nerve. If the flow is particularly bad, it may be necessary to apply suction to the cavity during imaging. A carefully placed suction line can keep the ganglia clear while being applied to a lateral location, so as to avoid obscuring the ganglia during imaging.
The imaging itself can be accomplished using different styles of microscope setups, each with its attendant advantages and limitations. When using an epifluorescence scope18,21, it is only possible to image the more superficial neurons. Another issue with epifluorescence imaging is that signals from deeper cells will come out as changes in background fluorescence (out of focus light) so be careful with the analysis not to pick up fluorescence changes from other cells in the ROI. For particularly thin structures, such as the geniculate ganglia, these issues may not be problematic. Use of a 2-photon16 or confocal20&#160;microscope can potentially allow for imaging cells in deeper layers.
It is important to highlight a few critical steps and ways to troubleshoot common issues. First, it must be noted that analysis will vary considerably depending on the software used. The open-source software, ImageJ provides tools sufficient for preliminary analysis. First, remove small motion artifacts using the Image Stabilizer plugin22, Next use ImageJ to calculate the change in fluorescence divided by baseline fluorescence (dF/F). This can be accomplished using one of many open source macros for ImageJ23, the referenced macro provides detailed installation notes. For other macros please refer to their documentation. After correcting for dF/F, utilize the forward and reverse buttons at the bottom of the image stack to observe cell responses to stimuli. Using the lasso tool from the tool bar, select fluorescing cells individually. After selecting a cell use Image | Stacks | Plot-Z-Axis. This will provide sufficient information to determine response profiles and analyze the time related events of each region of interest (ROI). More advanced analysis was long the domain of custom scripts in Matlab, R, etc. However, the popularity of calcium imaging has gradually led to the development of multiple open-source resources for analysis including CaImAn, EZCalcium, and more24,25. 
Calcium imaging is a powerful tool for examining the activity of neural ensembles with single neuron resolution. Because of the geniculate ganglion&#39;s small size, this protocol is especially powerful because the entire ganglion can be visualized within a single field. However, there are some limitations to this technique. In addition to the limitations common to all calcium imaging experiments, the surgical approach described here is invasive and must be carried out under anesthesia. This is a terminal procedure - the animal must be euthanized immediately after imaging. Therefore this surgical approach is not appropriate for awake/behaving recordings.
Over the past few years, researchers have used this technique to study response profiles of the neurons of the geniculate ganglion16,20,18. Recent work has focused on potential genetic markers that could be used to manipulate subpopulations within the ganglia and has shown how transgenic Cre driver lines and Cre-dependent GCaMP can be used to identify the response profiles of these populations26. Other work may use GCaMP with photo-activated proteins such as pa-mCherry to first identify and then mark cells activated by tastants to then be used in immunohistochemistry or in situ hybridization27. It may also be possible to utilize calcium dependent photo-convertible proteins such as CaMPARI to the same effect while using experimental methods very similar to those described here28. Regardless of the specific questions and experiments, calcium imaging offers a powerful tool for exploring the response profiles of neurons engaged in any number of activities, and its usefulness in exploring the taste system is only beginning.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 x #5 Inox Forceps</td>
			<td>Fine Science Tools</td>
			<td>NC9792102</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>3M Vetbond</td>
			<td>Fisher Scientific</td>
			<td>NC0398332</td>
			<td></td>
		</tr>
		<tr>
			<td>4-40 Machine Screw Hex Nuts</td>
			<td>Fastenere</td>
			<td>3SNMS004C</td>
			<td></td>
		</tr>
		<tr>
			<td>4-40 Socket Head Cap Screw</td>
			<td>Fastenere</td>
			<td>3SSCS04C004</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent Points</td>
			<td>Fisher Scientific</td>
			<td>50-930-668</td>
			<td></td>
		</tr>
		<tr>
			<td>Acesulfame K</td>
			<td>Fisher Scientific</td>
			<td>A149025G</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Tears</td>
			<td>Akorn</td>
			<td>59399-162-35</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Allergist Trays with Permanently Attached Needle</td>
			<td>Fisher Scientific</td>
			<td>14-829-6D</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Retractors</td>
			<td>FST</td>
			<td>18200-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Breadboard</td>
			<td>Thor Labs</td>
			<td>MB8</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Fisher Scientific</td>
			<td>A95-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cohan-Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Contemporary Ortho-Jet Liquid</td>
			<td>Lang</td>
			<td>1504</td>
			<td></td>
		</tr>
		<tr>
			<td>Contemporary Ortho-Jet Powder</td>
			<td>Lang</td>
			<td>1520</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Fisher</td>
			<td>19-062-616</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Head Post Holder</td>
			<td>eMachineShop</td>
			<td></td>
			<td>See attached file 202410.ems</td>
		</tr>
		<tr>
			<td>Custom Metal Head Post</td>
			<td>eMachineShop</td>
			<td></td>
			<td>See attached file 202406.ems</td>
		</tr>
		<tr>
			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera, sCMOS OrcaFlash4 Microscope Mounted</td>
			<td>Hamamatsu</td>
			<td>C13440</td>
			<td></td>
		</tr>
		<tr>
			<td>Disection Scope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>LED Cold Light Source</td>
			<td>Leica Mcrosystems</td>
			<td>KL300LED</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>MXC-2.5 Rotatable probe Clamp</td>
			<td>Siskiyou</td>
			<td>14030000E</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>50-947-346</td>
			<td></td>
		</tr>
		<tr>
			<td>petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressurized air</td>
			<td>Airgas</td>
			<td>AI Z300</td>
			<td></td>
		</tr>
		<tr>
			<td>Quinine</td>
			<td>Fisher Scientific</td>
			<td>AC163720050</td>
			<td></td>
		</tr>
		<tr>
			<td>Self Sticking Labeling Tape</td>
			<td>Fisher Scientific</td>
			<td>159015R</td>
			<td></td>
		</tr>
		<tr>
			<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>
			<td></td>
		</tr>
		<tr>
			<td>Sola SM Light Engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap25-2A-GCaMP6s-D</td>
			<td>JAX</td>
			<td>025111</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Thread</td>
			<td>G&#252;termann</td>
			<td>02776</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic Tubing</td>
			<td>Fisher Scientific</td>
			<td>22-046941</td>
			<td></td>
		</tr>
		<tr>
			<td>Two Stage Gas Regulator</td>
			<td>Airgas</td>
			<td>Y12FM244B580-AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon vinyl tubing - 1/16&#34;</td>
			<td>Automate Scientific</td>
			<td>05-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Valvelink8.2 digital/manual controller</td>
			<td>Automate Scientific</td>
			<td>01-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Valvelink8.2 Pinch Valve Perfusion System</td>
			<td>Automate Scientific</td>
			<td>17-pp-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Anased</td>
			<td>NADA# 139-236</td>
			<td></td>
		</tr>
	</tbody>
</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.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62172/62172fig2v2.jpg" /> 
Figure 2: Responses of geniculate ganglion neurons to tastants using in vivo GCaMP6s imaging. (A) Epifluorescent image of the geniculate ganglion of a Snap25-GCaMP6s transgenic mouse during baseline as water is perfused over the tongue. Dashed lines indicate the approximate boundaries of the geniculate ganglion. The seventh cranial nerve is labeled as such. (B) Snapshot of the same ganglion in (A) as a sweet tastant (AceK 30 mM) is applied to the tongue of the mouse. Notice several individual neurons increase in fluorescence intensity. Dashed line box indicates sweet responding cell used in (C) end. (C) Traces from five neurons indicating the amplitude of their GCaMP6s mediated fluorescence in response to a panel of tastants comprising sweet (30 mM acesulfame K), bitter (5 mM quinine); salty (60 mM NaCl); umami (50 mM monopotassium glutamate and 1mM inosine monophosphate); and sour (50 mM citric acid). Colored bars show the placement and duration (2 s) of the stimulus over the time course of the experiment. These representative data do not include a response to umami. Individual neurons commonly respond to both bitter and sour stimuli (bottom trace) 16,18,20. Panels A-B, Scale = 5 mm. Panel C, horizontal scale bar indicates 6.5 seconds, vertical scale bar indicates threshold of 4% dF/F. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62172/62172fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As can be seen in Figure 2, taste stimuli applied to the tongue should result in a rapid, transient increase in GCaMP fluorescence, causing a noticeable change in brightness among responding neurons. The video can be analyzed with a variety of software packages to produce traces displaying the changes in fluorescence over baseline (dF/F) over time of individual regions of interest (such as individual neurons), thereby showing the responses of each cell to the tastant panel. In a successful surgery, in a Snap25-GCaMP6s transgenic line, it is typical to see responses in 20-40 neurons within a single ganglion/field of view. This may change depending on the transgenic line used or if AAV-GCaMP is used instead. Note that baseline fluorescence may be affected by a number of factors, including the expression level of the GCaMP, and possible damage to the cells during the surgery. Changes in fluorescence intensity above a threshold level (typically df/f &#62; 3-fold above the average noise)20,21 is considered a positive response.
To determine the timing of stimulus delivery, the time it takes for liquid to flow through the lines should be measured in order to know when a fluid change actually contacts the tongue. To reduce this delay, use a moderate flow rate (5-10 ml/min) and a short length of tubing leading from the perfusion manifold to the oral cavity. Typically, with the stimuli described here, fluorescence starts almost immediately after tastant is applied to the tongue and will begin to fade almost immediately after the tastant is stopped and the oral cavity is washed with vehicle solution. When working with an unknown stimulus it can be helpful to observe the change in fluorescence of a region without responding neurons to compare overall changes in the image.

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 ...

in-vivo-calcium-imaging-mouse-geniculate-ganglion-neuron-responses-to

Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.8K Views.  The University of Texas at San Antonio. 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.

Video: In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems are already found in clinical applications, and are important tools for basic brain research. A particularly interesting recent development is the integration of closed-loop approaches with optogenetics, such that specific patterns of neuronal activity can trigger optical stimulation of selected neuronal groups. However, setting up an electrophysiological system for closed-loop experiments can be difficult. Here, a ready-to-apply Matlab code is provided for triggering stimuli based on the activity of single or multiple neurons. This sample code can be easily modified based on individual needs. For instance, it shows how to trigger sound stimuli and how to change it to trigger an external device connected to a PC serial port. The presented protocol is designed to work with a popular neuronal recording system for animal studies (Neuralynx). The implementation of closed-loop stimulation is demonstrated in an awake rat.

This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided.

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

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