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/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>

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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 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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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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Neuroscience

4.9K Views.  The University of Texas at San Antonio. Deze techniek kan belangrijke functionele vragen beantwoorden over neuronale reacties op smaak in de geniculate ganglia, een cruciaal onderdeel van de inwendige chorda tympani smaakroute. Deze techniek kan worden gebruikt om de real-time reacties van meerdere individuele neuronen in een enkele experimentele proef te volgen, omdat cellen die per dier worden geregistreerd aanzienlijk hoger zijn dan gewoonlijk wordt waargenomen via de elektrofysiologische methode. Met de op de hoofdpost gemontee...

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 Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 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.

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 ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

In Vivo Calcium Imaging of Neuronal Ensembles in the Intact Dorsal Root Ganglia of a Mouse

Source: <a href='https://app.jove.com/v/64826/in-vivo-calcium-imaging-of-neuronal-ensembles-in-networks-of-primary-sensory-neurons-in-intact-dorsal-root-ganglia'>Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. Vis. Exp. (2023).</a>The video demonstrates an in vivo calcium imaging technique to monitor neuronal activity in the dorsal root ganglia (DRG) of a transgenic mouse. DRG neurons express a calcium indicator that increases fluorescence intensity upon binding to calcium. Mechanical or thermal stimuli applied to a hind paw generate action potentials that propagate to the DRG, inducing calcium influx in the neurons and resulting in increased fluorescence emission.

Source: Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. ...

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

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