We would like to thank Drs. Kathy Albers and Brian Davis for the use of their Leica Microscope and Metamorph program, Charles Warwick for helping to build our thermal Peltier device, and Dr. Raymond Sekula for helping with troubleshooting the surgical preparation. This work was supported by grants from the National Institutes of Health: F31NS125993 (JYG), T32NS073548 (JYG), and R01NS122784 (MSG and RS).

All experiments involving the use of animals in research were performed in accordance with standards put forth by the National Institutes of Health and the International Association for the Study of Pain and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (protocol #22051100). At the end of each experiment, rats were euthanized via exanguination with cardiac perfusion of ice-cold phosphate-buffered saline (PBS), an approach approved by the American Veterinary Medical Association and the University of Pittsburgh IACUC.
1. GCaMP induction
<ol>
	<li>Order time-pregnant Sprague Dawley rats so that pups can be injected at the appropriate time after birth.</li>
	<li>Spray the gloves with 70% EtOH before handling each rat pup. This will deter the dam from cannibalizing the young.</li>
	<li>Swab rat pups (P1-2) with 70% EtOH and anesthetize on ice for 3 min.</li>
	<li>Inject 15 &#181;L of AAV9-CAG-WPRE-GCaMP6s-SV40 (Addgene) intraperitoneally using a 25 &#181;L sterile, gas-tight Hamilton syringe.</li>
</ol>
2. Trigeminal ganglion exposure surgery
<ol>
	<li>Administer anesthetic cocktail (55 mg/kg ketamine, 5.5 mg/kg xylazine, 1 mg/kg acepromazine) intraperitoneally based on body weight to 6-8 week-old rats (approximately 150-200 g). 
	NOTE: This is generally sufficient to maintain a surgical plane of anesthesia as assessed by the absence of a withdrawal reflex to noxious pinch of a hindpaw. However, supplement with isoflurane via a nose cone if the animals become light.</li>
	<li>Once fully anesthetized, shave the hair and whiskers of the head and face.</li>
	<li>Mount the rat to a stereotaxic frame with ear bars and place a heating pad (~37 oC) underneath to maintain body temperature.</li>
	<li>Monitor vital signs (heart rate, respiration rate, blood oxygen saturation) with a mouse oximeter or comparable. 
	NOTE: Body temperature is monitored by a rectal probe and maintained by placing rats on a feedback controlled circulating water blanket.</li>
	<li>Place gauze dipped in ice-cold saline on the head to constrict blood vessels to minimize bleeding. Using a size 15 scalpel, make a midline incision of the skin and muscle over the skull.</li>
	<li>Use blunt dissection of the skin and muscle to expose the skull.</li>
	<li>Using a &#188; round drill bit, carefully perforate the skull cap to expose the forebrain. Then, use rongeurs (2.5 mm cup) to carefully cut through the skull.</li>
	<li>Using a size 15 scalpel, make an incision into the brain (Bregma: -3.80) and the olfactory bulb. 
	NOTE: Cutting more caudally past this point will result in rat death</li>
	<li>Use a spatula to carefully disconnect the dura from the skull and gently lift the severed brain to reveal the TG and the base of the skull. 
	NOTE: Additional use of ice-cold saline perfusion throughout the extraction will minimize bleeding and optimize the following dissection field.</li>
	<li>Using a cautery pen, stem any bleeding resulting from the extraction. 
	&#8203;NOTE: Cutting the dura will result in inevitable bleeding. It is best to prepare ice-cold aCSF (119 mM NaCl, 26.2 mM NaHCO3, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgCl2, 10 mM glucose, 2.5 mM CaCl2) to bathe the skull cavity to help constrict blood vessels while maintaining neuronal health.</li>
</ol>
3. GCaMP6s imaging
NOTE: Given the size and density of these neurons, the imaging and data acquisition system used (objective, microscope, light source, camera) will determine the number of GECI+ cells visualized. The light source, objective, and camera will also determine the parameters used for image acquisition, including exposure time and image capture rate. While multiphoton and confocal techniques can be used depending on the experiment&#39;s parameters, epifluorescence microscopy may be sufficient to resolve many cells. Any image acquisition package can be used. Ideally, the stimulus application is time-locked with the image acquisition software package.
<ol>
	<li>Place the skull cavity underneath the objective and bring the TG into focus using visible light.</li>
	<li>The excitation wavelength of GCaMP6s is 496 nm, and its emission wavelength is 513 nm; thus, use the appropriate dichroic and filter cubes to locate GCaMP+ cells. Adjust the focus to locate and resolve the area of the ganglia in which neurons are responding to stimuli applied to the receptive field of interest.</li>
	<li>Use a 10x objective to enable visualizations of most neurons in the TG responsive to mechanical stimuli applied to a 1 cm2 region of the face16. Use a 20x dry objective with a long working distance (10.8 mm) to increase resolution.</li>
	<li>Use acquisition software (e.g., Metamorph) to collect fluorescence data over time and in response to stimulus application.
	<ol>
		<li>To minimize photo-bleaching of the neurons, use as short exposure time as possible to detect baseline and evoked increases in fluorescence. With the 20x, 0.40 NA air objective, a 120 W mercury halide light source, and a complementary metal-oxide-semiconductor (CMOS) camera used in the present study, an exposure time of 300 ms, and an image acquisition rate of 3 Hz, obtain stable baseline recordings for &#62;90 min. 
		NOTE: 1) These image acquisition parameters must be determined empirically. 2) Mechanical (brush, punctate, vibration, pinch), thermal (heat and cold), and chemical (capsaicin, menthol, inflammatory mediators) may be applied to the receptive field. A relatively inexpensive subjective approach is to apply the stimuli by hand. More objective and reproducible approaches are recommended, however, where feedback-controlled actuators can be used for repeated application at a known force. While feedback-controlled Peltier devices are useful for controlled heating and cooling, they are not ideal for the thermal stimulation of a curved face. Feedback-controlled infrared light sources are an alternative for heating, and cold spray is a less than ideal alternative for cooling.</li>
	</ol>
	</li>
</ol>

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

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Dr. Gold was receiving grant support from Grunenthal during the development of this preparation. There was no overlap in the focus of the Grunenthal study and the preparation described in this manuscript. Neither of the other authors has any other potential conflicts of interest to disclose.

Here, we demonstrate a quick, non-invasive way of generating a GECI rat for imaging the TG. We chose a CAG promotor to drive and maintain high levels of gene expression. While previous studies suggest that other AAV serotypes may efficiently drive gene expression in DRG neurons39, our results are consistent with a recent study involving intraperitoneal injection of AAV in neonates32, indicating that the AAV9 serotype is highly efficient in the infection of rat neonatal sens...

Somatosensation, the neural encoding of mechanical, thermal, and chemical stimuli impinging on the skin or other bodily structures, including muscles, bone, and viscera, starts with activity in primary afferent neurons that innervate these structures1. Single unit based electrophysiological approaches have provided a wealth of information about the afferent subtypes involved in this process as well as how their stimulus-responses properties may change over time1,2,3. However, while there remains strong evidence in support of the labeled line theory, which suggests specific sensory modalities are conveyed by specific subpopulation(s) of neurons, the ability of many subpopulations of neurons to respond to the same types of mechanical, thermal, and chemical stimuli suggests the majority of somatosensory stimuli are encoded by multiple subpopulations of neurons4,5. Thus, a better understanding of somatosensation will only come with the ability to study the activity of 10&#39;s, if not hundreds, of neurons simultaneously.
Advances in optical approaches with the relatively recent advent of confocal and, subsequently, multiphoton and digital imaging techniques have facilitated the ability to perform relatively non-invasive population-level analyses of neuronal activity6,7. One of the last hurdles in the application of this technology has been the development of tools to enable the optical assessment of neural activity. Given the speed of an action potential that can start and end in less than a millisecond, a voltage-sensitive dye with the capacity to follow changes in membrane potential at the speed of an action potential would be the ideal tool for this purpose. But while there has been tremendous progress in this area7,8,9,10, the signal-to-noise ratio for many of these dyes is still not quite high enough to enable a population analysis of hundreds of neurons at the single cell level. As an alternative approach, investigators have turned to monitoring changes in intracellular Ca2+ concentration ([Ca2+]i). The limitations with this strategy have been clear from the start and include the fact that an increase in [Ca2+]i is an indirect measure of neural activity11; that an increase in [Ca2+]i may occur independently of Ca2+ influx associated with the activation of voltage-gated Ca2+ channels (VGCCs)12,13; that the magnitude and duration of a Ca2+ transient may be controlled by processes independent of VGCC activity11,12,14; and that the time-course of Ca2+ transients far exceeds that of an action potential15. Nevertheless, there are a number of significant advantages associated with the use of Ca2+ as an indirect measure of neural activity. Not the least of these is the signal-to-noise ratio associated with most Ca2+ indicators, reflecting both the magnitude of the change in intracellular Ca2+ and the fact that the signal is arising from the three-dimensional space of the cytosol rather than the two-dimensional space of the cell membrane. Furthermore, with the development of genetically encoded Ca2+ indicators (GECI&#39;s), it is possible to take advantage of genetic strategies to drive the expression of the Ca2+ indicators in specific subpopulations of cells, facilitating population-level analyses in intact preparations (e.g., see16).
Given the number of genetic tools now available in mice, it should be no surprise that GECI&#39;s have been used most extensively in this species. Mouse lines with constitutive GECI expression in subpopulations of sensory neurons have been developed7,16,17. With the development of mouse lines expressing recombinases in specific cell types, it is possible to use even more sophisticated strategies to control GECI expression15. However, while these tools are ever more powerful, there are a number of reasons why other species, such as rats, might be more appropriate for some experimental questions. These include the larger size, facilitating a number of experimental manipulations that are difficult, if not impossible, in the smaller mouse; the ease of training rats in relatively complex behavioral tasks; and at least some evidence that biophysical properties and expression patterns of several ion channels in rat sensory neurons may be more similar to that observed in human sensory neurons than are&#160;the same channels in mouse relative to the human18.
While the transduction of somatosensory stimuli generally occurs in the peripheral terminals of primary afferents, the action potential initiated in the periphery must pass through the structure that houses primary afferent somata, referred to as dorsal root (DRG) or trigeminal (TG) ganglia before reaching the central nervous system19. While there is evidence that not every action potential propagating along a primary afferent axon will invade the cell body20, a consequence of the fact the primary afferent somata are connected to the main afferent axon via a T-junction19, the majority of action potentials initiated in the periphery appear to invade the soma21. This confers three experimental advantages when using GECIs to assess population coding in primary afferents: the large size of the cell body relative to the axons further increases the signal to noise when using [Ca2+]i as an indirect measure of afferent activity; the DRG are generally easy to access; and assessing activity at a site that is spatially remote from the afferent terminals minimizes the potential impact of the surgery needed to expose the ganglia on the stimulus-response properties of the afferent terminals. However, because TG are located beneath the brain (or above the palette), they are far more difficult to access than DRG. Furthermore, while there are many similarities between DRG and TG neurons, there is a growing list of differences as well. This includes the roughly somatotopic organization of neurons in the TG22, unique structures innervated, different central terminal termination patterns23,24,25,26, and now a growing list of differences in both gene expression27,28 and functional receptor expression29. In addition, because we are interested in the identification of peripheral mechanisms of pain, the relatively large number of pain syndromes that appear to be unique to the trigeminal system (e.g., migraine, trigeminal neuralgia, burning mouth syndrome) that appear to involve aberrant activity in primary afferents30,31,32, suggests that the TG needs to be studied directly.
Thus, while stimulus-response properties of TG neurons have been studied with GECIs in the mouse16, because the reasons listed above suggest that the rat may be a more appropriate species to address a variety of experimental questions, the purpose of the present study was to develop an approach to use GECIs to study TG neurons in the rat. To achieve this, we utilized a viral approach to drive the expression of the GECI GCaMP6s in the peripheral nervous system. We then removed the forebrain to allow access to the TG. Finally, mechanical and thermal stimuli were applied to the face while neuronal responses were assessed under fluorescent microscopy. Together, these data support a role for utilizing the rat to investigate changes in the TG under many states, expanding the toolkit for investigators interested in sensory coding in the trigeminal system.

<table><tbody><tr><td>AAV9-CAG-WPRE-GCaMP6s-SV40&amp;nbsp;</td><td>Addgene</td><td>100844-AAV9</td><td>AAV9-GCaMP6s virus</td></tr><tr><td>ACEpromazine maleate</td><td>Covetrus</td><td>11695-0095-5</td><td>10 mg/mL</td></tr><tr><td>AnaSed (Xylazine) injection</td><td>AKORN Animal Health</td><td>23076-35-9</td><td>20 mg/mL</td></tr><tr><td>CTR5500 Electronics box</td><td>Leica</td><td>11 888 820</td><td>Power Supply</td></tr><tr><td>Cutwell burr drill bit</td><td>Ransom &amp;amp; Randolph</td><td></td><td>&amp;frac14; round</td></tr><tr><td>DM 6000 FS</td><td>Leica</td><td>11 888 928</td><td>Base Stand</td></tr><tr><td>EL6000</td><td>Leica</td><td>EL6000</td><td>Light source with 120 W mercury bulb</td></tr><tr><td>Forceps</td><td>FST</td><td>11252-00</td><td>Dumont No. 05</td></tr><tr><td>Friedman rongeurs</td><td>FST</td><td>16000-14</td><td>2.5 mm cup size</td></tr><tr><td>Friedman-Pearson rongeurs</td><td>FST</td><td>16021-14</td><td>1 mm cup size</td></tr><tr><td>Heating pad (Temperature therapy pad)</td><td>STRYKER</td><td>8002-062-022</td><td></td></tr><tr><td>Ketamine hydrochloride</td><td>Covetrus</td><td>1695-0703-1</td><td>100 mg/mL</td></tr><tr><td>Plan Fluor 20x/0.40</td><td>Leica</td><td>MRH00105</td><td>20x objective, 0.4 NA10.8 mm WD</td></tr><tr><td>Power handle high-temp cautery pen</td><td>Bovie</td><td>HIT1</td><td>handheld Change-A-Tip cautery pen</td></tr><tr><td>Prime 95B</td><td>Photometrics</td><td>Prime 95B</td><td>CMOS Camera</td></tr><tr><td>Saline</td><td>Fisher Scientific</td><td>NC0291799</td><td>0.9% Sterile Saline</td></tr><tr><td>Scalpel blade</td><td>Fisher Scientific</td><td>22-079-701</td><td>size 15 disposable blade</td></tr><tr><td>Spatula</td><td>BRI</td><td>48-1460</td><td>brain spatula</td></tr><tr><td>Spring scissors</td><td>FST</td><td>91500-09</td><td>Student Vannas, 5 mm cutting edge</td></tr><tr><td>Spring scissors</td><td>FST</td><td>15012-12</td><td>Noyes, 14 mm cutting edge</td></tr><tr><td>STP6000 Smart touch panel</td><td>Leica</td><td>11 501 255</td><td>Control Panel</td></tr><tr><td>Syringe</td><td>Hamilton</td><td>80201</td><td>25 &amp;mu;L Model 1702 Luer Tip syringe</td></tr><tr><td>Water heater</td><td>Adroit</td><td>HTP-1500</td><td></td></tr></tbody></table>

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.

Because we have previously had success with the AAV9 serotype for the infection of rat sensory neurons15, we used this serotype for the expression of GCaMP6s in rat TG neurons. We therefore first sought to assess the sensory neuron infection efficiency of AAV9-CAG-GCaMP6s-WPRE-SV40 (AAV9-GCaMP) when this virus was administered to neonatal rat pups20. This virus utilizes the CAG promoter, which drives and maintains high levels of gene expression. Furthermore, AAV9 has been s...

Watch this Scientific Journal Video about Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury at JoVE.com

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.

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 ...

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1.9K Views.  Center for Neuroscience at the University of Pittsburgh. 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. 

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

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in 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, ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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.

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 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 ...

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 ...

Calcium Imaging of Thoracic Segment T1 DRG for Cardiopulmonary Sensory Neurons

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.

This study presents a surgical manipulation to expose the T-DRG in anesthetized mice for in vivo calcium imaging, along with synchronous ECG recordings. This method represents a cutting-edge tool for studying peripheral electric nerve stimulation and thoracic visceral organ inputs, as well as their interactions at the primary sensory level.

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. ...

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

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Chapters in this video

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

In vivo Neuronal Calcium Imaging in C. elegans

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

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

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation