This work was supported by National Institutes of Health Grants R01DE026677 and R01DE031477 (to Y.S.K.), UTHSCSA startup fund (Y.S.K.), a Rising STAR Award from University of Texas system (Y.S.K.), and Craniofacial Oral-biology Student Training in Academic Research (COSTAR) National Institute of Health Grant 5T32DE014318 (J.S.).

All procedures described here were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio.
NOTE: Once started, animal surgery (step 1) and imaging (step 2) must be completed in a continuous manner. Data analysis (step 3) may be performed later.
1. Surgery and securing the animal for right side L5 DRG imaging
NOTE: Bo...

<ol>
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The authors declare no competing financial interests.

Persistent pain is present in a wide range of disorders, debilitating and/or reducing the quality of life for about 8% of people29. Primary sensory neurons detect noxious stimuli on the skin, and their plasticity contributes to persistent pain8. While neurons can be studied in cell culture and explants, doing so removes them from their normal physiological context. Surgical exposure of the DRG, followed by Pirt-GCaMP3 Ca2+ imaging, permits the study of primary se...

Primary sensory neurons directly innervate the skin and carry somatosensory information back to the central nervous system1,2. Dorsal root ganglia (DRGs) are cell body clusters of 10,000-15,000 primary sensory neurons3,4. DRG neurons present diverse size, myelination levels, and gene and receptor expression patterns. Smaller diameter neurons include pain-sensing neurons and larger diameter neurons typically respond to non-painful mechanical stimuli5,6. Disorders in the primary sensory neurons such as injury, chronic inflammation, and peripheral neuropathies can sensitize these neurons to various stimuli and contribute to chronic pain, allodynia, and pain hypersensitivity7,8. Therefore, the study of DRG neurons is important in understanding both somatosensation generally and many pain and itch disorders.
Neurons firing in vivo are essential to somatosensation, but until recently, tools to study intact ganglia in vivo have been limited to relatively small numbers of cells9. Here, we describe a powerful method for studying the action potentials or activities of neurons on a population level in vivo as an ensemble. The method employs imaging based on cytoplasmic Ca2+ dynamics. The Ca2+ sensitive fluorescent indicators are good proxies for measuring cellular activity due to the normally low concentration of cytoplasmic Ca2+. These indicators have allowed simultaneous monitoring of hundreds to several thousands of primary sensory neurons in mice9,10,11,12,13,14,15,16 and rats17. The method of in vivo Ca2+ imaging described in this study can be used to directly observe populational level responses to mechanical, cold, thermal, and chemical stimuli.
The phosphoinositide-binding membrane protein, Pirt is expressed at high levels in almost all (&#62;95%) primary sensory neurons18,19 and can be used to drive the expression of the Ca2+ sensor, GCaMP3, to monitor neuron activity in vivo20. In this protocol, techniques are described for performing in vivo DRG surgery, Ca2+ imaging, and analysis in the right side lumbar 5 (L5) DRG of Pirt-GCaMP3 mice14 using confocal laser scanning microscopy (LSM).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anased Injection (Xylazine)</td>
			<td>Covetrus, Akorn</td>
			<td>33197</td>
			<td></td>
		</tr>
		<tr>
			<td>C Epiplan-Apochromat 10x/0.4 DIC</td>
			<td>Cal Zeiss</td>
			<td>422642-9900-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>McKesson</td>
			<td>24-106-1S</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13007-12</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Temperature Controller Heating Pad</td>
			<td>FHC</td>
			<td>40-90-2-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Ceramic Coated Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-50</td>
			<td></td>
		</tr>
		<tr>
			<td>FHC DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluriso (Isoflurane)</td>
			<td>MWI Animal Health, Piramal Group</td>
			<td>501017</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16221-14</td>
			<td></td>
		</tr>
		<tr>
			<td>GelFoam</td>
			<td>Pfizer</td>
			<td>09-0353-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketaset (Ketamine)</td>
			<td>Zoetis</td>
			<td>KET-00002R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminescent Green Stage Tape</td>
			<td>JSITON/ Amazon</td>
			<td>B803YW8ZWL</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrx VIP 3000 Isoflurane Vaporizer</td>
			<td>Midmark</td>
			<td>91305430</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting spring scissors</td>
			<td>Roboz</td>
			<td>RS-5677</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Rectal Thermistor Probe</td>
			<td>FHC</td>
			<td>40-90-5D-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Roboz</td>
			<td>RS-6812</td>
			<td></td>
		</tr>
		<tr>
			<td>Pirt-GCaMP3 C57BL/6J mice</td>
			<td>Johns Hopkins University</td>
			<td>N/A</td>
			<td>Either sex can be imaged equally well. Mice should be at least 8 weeks old due to weak or intermittent Pirt promoter expression in younger mice.</td>
		</tr>
		<tr>
			<td>SMALGO small animal algometer</td>
			<td>Bioseb In vivo Research Instruments</td>
			<td>BIO-SMALGO</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Kopf Model 923-B</td>
			<td>923-B</td>
			<td></td>
		</tr>
		<tr>
			<td>td-Tomato C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>7909</td>
			<td></td>
		</tr>
		<tr>
			<td>Top Plate, 6 in x 10 in</td>
			<td>Newport</td>
			<td>290-TP</td>
			<td></td>
		</tr>
		<tr>
			<td>TrpV1-Cre C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>17769</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 800 confocal microscope</td>
			<td>Cal Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen 2.6 Blue Edition Software</td>
			<td>Cal Zeiss</td>
			<td>Zen (Blue Edition) 2.6</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64826/64826fig04.jpg" /> 
Figure 4: Representative images of L5 dorsal root ganglia of Pirt-GCaMP3 mice. (A,D) Single frame high resolution scans of L5 dorsal root ganglia of Pirt-GCaMP3 mice are shown. (B,E). Average intensity projections of 15 frames of Pirt-GCaMP3 L5 DRG ganglia from panel A and panel D, respectively...

Watch this Scientific Journal Video about In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia at JoVE.com

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.

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 ...

The number of neurons monitored using this method allows collection of neuron network data that would be impossible using older or other methods. The main advantage of this technique is the ability to monitor almost 2, 000 primary sensory neurons at once, responding directly to stimuli applied to the hind paw. This procedure is especially well suited to studying therapeutic interventions for peripheral allodynia and pain hypersensitivity.This method can be adapted to studying trigeminal or geniculate ganglia or used with genetically encoded sensors for voltage or cell signaling molecules, such as cyclic AMP. It takes practice to perform this surgery. You can practice on up to six dorsal root ganglia on a single animal.Demonstrating the procedure will be myself, with assisting from John Shannonhouse and Mr.Ruben Gomez. To begin, place the mouse on a heated pad to maintain the body temperature at 37 degrees Celsius. Locate the lumbar enlargement by feeling the pelvic bone of the mouse.Then, shave the back of the mouse above the area of lumbar enlargement. Using scissors, make a three-sided rectangular incision above the lumbar enlargement, and fold the skin away with forceps. Use the 13-millimeter spring dissection scissors to make three to four millimeters incisions on the right side of the spine.Use scissors to cut back the skin and muscles to the sides to expose the spine. Then, using eight-millimeter scissors, cut away the muscle and connective tissue to clean the transverse process of the right side L5 DRG. Try to minimize bleeding by using cotton or Gelfoam to absorb the blood.Cut open the right side L5 transverse process using Friedman-Pearson rongeurs or strong fine forceps, being careful to not touch the DRG. Next, move the mouse and heating pad onto the custom stage. Use stage tape to secure the animal and the heating pad in place.Place the nose of the animal in the nose cone for continuous isoflurane anesthesia. Secure the right hind paw sticking out to a position off the stage for easy application of the stimuli. Secure the spine in place with the stage clamps over the skin on the vertebrae or the pelvic bone just rostral and caudal to the L5 DRG.Adjust the clamps and the stage to make the surface of the DRG as level as possible. Then, place the stage below the microscope so that the objective is directly eight millimeters above the DRG when lowered. Insert the rectal thermometer.Connect the power lines to the heating pad and the rectal thermometer. Connect the nose cone to the isoflurane gas lines. Use an upright confocal microscope with a 10x 0.4 DIC objective and the associated software for imaging.Use the green FITC filter settings of excitation at 495 nanometers, emission at 519 nanometers, and detection wavelength between 500 to 580 nanometers. Under the microscope, find the surface of the DRG. Adjust the clamps on the stage so the DRG surface is as level as possible and the maximum surface area is visualized in the focal plane.Monitor the animal throughout the procedure to maintain isoflurane anesthesia without overdosing. To load the microscope rapid scanning protocol, use the typical settings of voxel size 2.496 by 2.496 by 16 microns, 512 by 512 pixels, 10 optical slice Z-stack, one Airy unit for 32 micrometers, 1%laser power of 488 nanometers and five milliwatts, pixel time 1.52 microseconds, line time 0.91 milliseconds, frame time 465 milliseconds, LSM scan speed of eight, bidirectional scanning, PMT detector gain 650 volts, and digital gain of one. Take a short, eight-cycle scan of the DRG by clicking on Start Experiment under the Acquisition tab.Create a movie by making an orthogonal projection of scans at one scan per frame over time. Manually check for image clarity and imaging artifacts, such as waves of brightness crossing the DRG. Adjust clamp position and optical section thickness, and repeat this step until a clear, high-quality movie is achieved.Then, load the microscope high-resolution scanning protocol using the typical settings of voxel size 1.248 by 1.248 by 14 microns, 1024 by 1024 pixels, six optical slice Z-stack, 1.2 Airy unit or 39 micrometers, 5%laser power of 488 nanometers and 25 milliwatts, pixel time 2.06 microseconds, line time 4.95 milliseconds, frame time 5.06 seconds, LSM scan speed of six, bidirectional scanning, PMT detector gain at 650 volts, and digital gain of one. Click on the Start Experiment button under the Acquisition tab to make a high-resolution image of the DRG. Load the microscope rapid scanning protocol, and record spontaneous activity in the DRG for 80 cycles.Generate an orthogonal projection movie, and verify that the image is of sufficient quality for analysis. For applying stimuli, set the microscope to perform 15 to 20 scans. Wait for scans one to five to complete to produce the baseline.Apply the stimulus during scans six to 10. Wait at least five minutes following each stimulus before applying the next one to prevent desensitization. For a mechanical press, hold the algometer's pincher with the paw between the paddles without touching the paw.Pinch the paw starting immediately after the end of scan five and stopping immediately after scan 10. Monitor the press force with an algometer, and ensure that it does not exceed 10 g over the desired force. For thermal stimuli, heat a beaker of water to just above the desired temperature.When the water is at the correct temperature, apply the stimulus immediately after scan five by immersing the paw in the water. Pull the beaker away immediately after scan 10. Surgical L5 DRG exposure followed by confocal microscopy allowed up to 1, 800 neurons to be imaged at once using Pirt-GCaMP3 mice.Primary sensory neurons were observed in an ensemble at a populational level for spontaneous calcium transients in the absence of stimuli and in response to stimuli in their normal physiological context. Strong stimuli or noxious heat increased calcium responses. Compared to pressing with 100 g, pressing with 300 g increased the number of neurons producing calcium transients and the delta F by F0 area under the curve.Warm heat in a non-noxious temperature range of 21 and 45 degrees Celsius increased the number of neurons producing calcium transients and the delta F by F0 area under the curve. Non-noxious temperatures activated a smaller number of neurons producing transients compared to a noxious heat stimulus at 57 degrees Celsius. Further, smaller-and medium-diameter neurons produced calcium transient spontaneously and under all stimuli, but larger-diameter neurons only produced calcium transients in response to the 300 g press stimulus.The DRG should be level and optimum optical slice thickness. It should be such that you can detect the maximum number of cells and there is no waviness in the movie. This technique has been used for analyzing sensory modalities on a populational level for somatosensation and taste and to study adjacent neurons activating in pairs or in synchronized clusters contributing to chronic and neuropathic pain.

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2.5K Views.  University of Texas Health Science Center at San Antonio. The number of neurons monitored using this method allows collection of neuron network data that would be impossible using older or other methods. The main advantage of this technique is the ability to monitor almost 2, 000 primary sensory neurons at once, responding directly to stimuli applied to the hind paw. This procedure is especially well suited to studying therapeutic interventions for peripheral allodynia and pain hypersensitivity.This method can be adapted to studying trigemina...