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.).

<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 sensory neurons in their normal physiological context using stimuli applied to the hindpaw. A major advantage of this method of Ca2+ imaging is that a large number of neurons can be monitored simultaneously. This method allows analysis at a populational level of modality, neuron diameter, and rare phenomena such as coupled activation10,11,12,13,14,15,16,17,30, providing insights into normal and pathological somatosensation. In addition to neuron diameter as an identifier, specific types of neurons can be genetically labeled with a red fluorophore such as td-Tomato for identification during imaging. This technique allows for observations of specific sensory modalities or of neurons expressing specific receptors14,31. Other applications include imaging other sensory ganglia such as the trigeminal ganglion24,32 or geniculate ganglion23,26 and imaging innervation of the spinal dorsal horn by primary sensory neurons in explants19,33.
Critical steps in the protocol include preventing excessive bleeding or bleeding onto the DRG, control of anesthesia during imaging, leveling of the DRG surface, selecting optimal optical slice thickness, and preventing movement of the DRG during application of stimuli. Excessive bleeding could cause death of the animal or artifacts due to physiological effects of blood loss. Blood loss may cause increases in brightness (cytoplasmic calcium) across vast areas of the DRG. Blood loss could also cause numbness, reducing the number of neurons activating in response to stimuli. Bleeding onto the DRG directly blocks imaging, limiting the number of neurons that can be successfully imaged. Since anesthesia affects neuronal activities and responses, a minimal level of isoflurane anesthesia should be used. A level DRG and optimal optical slice thickness are the most critical parts of the procedure. Incorrectly performing these steps can cause out of focus neurons and waviness during imaging (see Movie1 and Movie 2) that will confound the analysis or make the analysis impossible. Even small movements can make tracking individual neurons across frames impossible and/or create artifacts where a neuron appears to become darker or brighter, causing significant errors.
The system described here also has some significant limitations. Anesthesia interferes with neuron and glial Ca2+ responses25. Because this method is terminal, the same animal cannot be monitored over multiple experiments. Ca2+ transients are an indirect proxy for neuron firing and activity. This method uses Ca2+ transients as a proxy for action potentials. However, other signals such as Gq protein or ryanodine receptor signaling can change cytoplasmic Ca2+ concentrations34,35,36. Other mechanisms have not been reported to cause Ca2+ transients in DRG neurons. However, application of chemical stimuli to the paw is known to increase Ca2+ in spinal dorsal horn astrocytes36.
In conclusion, intact DRG Pirt-GCaMP3 Ca2+ imaging is a powerful tool for studying somatosensation under normal and pathological conditions. The results shown here are representative for normal control mice. Control mice can be compared to pain and disease models or genetic mutants10,15,17,30,31,37. Furthermore, the method presented here can be adapted to other genetically encoded Ca2+ indicators10,11,16 and used to image other sensors such as voltage sensing38,39,40 and cyclic adenosine monophosphate sensing41. While confocal microscopy was used in this study, two-photon microscopy can be used as well11.

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, in the absence of stimuli. Some neurons that produced spontaneous Ca2+ transients are called out with yellow arrows. (C) Average intensity projection of two frames before stimulus and all five frames of stimulus of Pirt-GCaMP3 ganglion from panel A during 100 g hindpaw press. Some neurons that produced Ca2+ transients during the stimulus are called out with yellow arrows. (F-I) Average intensity projections of frames 4 and 5 (before stimulus) (panels A and D) and frames 6-9 (during 100 g press stimulus) of ganglion from panel D with the frames 4-6, frame 4 + 5 + 7, frames 4 + 5 + 8, and frames 4 + 5 + 9 of a 100 g hindpaw press, respectively. (J) Fluoresence intensity&#160;traces of three spontaneously active neurons from panel B (black) corresponding to the panel A ganglion and panel E (red) corresponding to the panel D ganglion. Each neuron&#39;s trace is normalized to its median intensity (shown by the line at Y = 0). (K) &#916;F / F0 traces of three neurons activating in response to 100 g press from panel C (black) corresponding to the panel A ganglion and panels F-I (red) corresponding to the panel D ganglion. Note the X and Y axes are different than in panel J. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Imaging of a large number of neurons using confocal scanning of Pirt-GCaMP L5 DRG 
Surgical L5 DRG exposure followed by confocal microscopy allowed up to 1,800 neurons to be imaged at once using Pirt-GCaMP3 mice (Figure 4). This creates a powerful advantage of being able to simultaneously observe thousands of primary sensory neurons in an ensemble at a populational level in their normal physiological context, both in control and in abnormal conditions. Spontaneous Ca2+ transients in the absence of stimuli (Figure 4B,E and Movie 2) and Ca2+ transients in response to stimuli (Figure 4C,F-I, Movie 3, and Movie 4) can be monitored. The diameters of activating neurons are highly correlated with different nerve fiber groups.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64826/64826fig05.jpg" /> 
Figure 5: Increasing intensity of stimuli enhances Ca2+ responses in ganglia of Pirt-GCaMP3 mice. (A)&#160;Graphs of the numbers of neurons in L5 DRG ganglia showing Ca2+ transients in response to stimuli from two different press intensities are shown: 100 g, n = 8 ganglia; 300 g, n = 5. (B) Graphs of &#916;F / F0 areas under the curve of samples of Ca2+ transients from two different press intensities are shown: 100 g, n = 88 neurons; 300 g, n = 104. (C) Graphs of traces of &#916;F / F0 of 100 g and 300 g press stimuli. Red trace is the mean &#916;F / F0; 100 g, n = 60 neurons; 300 g, n = 57 neurons. (D)&#160;Graphs of the numbers of neurons in L5 DRG ganglia showing Ca2+ transients in response to three different non-noxious and one noxious temperature are shown: 21 &#176;C, n = 4 ganglia; 10 &#176;C, n = 6; 45 &#176;C, n = 7; 57 &#176;C, n = 3. (E) Graphs of &#916;F / F0 areas under the curve of samples of Ca2+ transients from three different non-noxious and one noxious temperature stimulus intensities are shown: 21 &#176;C, n = 40 neurons; 10 &#176;C, n = 60; 45 &#176;C, n = 61; 57 &#176;C, n = 118. (F) Graphs of traces of &#916;F / F0 of 10 &#176;C and 57 &#176;C stimuli. Red trace is the mean &#916;F / F0; 10 &#176;C, n = 60 neurons; 57 &#176;C, n = 118. (G) Graph of percentages of neurons of different diameters (&#60;20 &#181;m, 20-25 &#181;m, &#62;25 &#181;m) producing Ca2+ transients spontaneously and with thermal and mechanical stimuli. Spontaneous activity (Spon), n = 5 ganglia; 45 &#176;C, n = 3; 300 g, n = 3. * p &#60; 0.05, ** p &#60; 0.01, *** p &#60; 0.001. Bar graphs show the means and the SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Number of cells producing Ca2+ transients and Ca2+ transient area under the curve (AUC) in the presence of stimuli 
Strong stimuli or noxious heat increased Ca2+ responses. Compared to pressing with 100 g, pressing with 300 g increased the number of neurons producing Ca2+ transients (Figure 5A, Student&#39;s t-test: t = 2.398, df = 11, p = 0.0354) and the &#916;F / F0 area under the curve of Ca2+ transients (Figure 5B, Welch&#39;s t-test: t = 3.243, df = 187.4, p = 0.0014). Similarly, warm heat in a non-noxious temperature range (21 &#176;C and 45 &#176;C) increased the number of neurons producing Ca2+ transients (Figure 5C, one-way ANOVA F2,14 = 4.853, p = 0.0251, followed by Tukey&#39;s q = 4.380, df = 14, p = 0.0202) and the &#916;F / F0 area under the curve of Ca2+ transients (Figure 5D, Brown-Forsythe ANOVA F2,151.6 = 60.76, p = 0.0029 followed by Welch&#39;s t-test: t = 3.333, df = 96.72, p = 0.0036). Compared to a noxious heat stimulus (57 &#176;C), non-noxious temperatures (21 &#176;C, 10 &#176;C, and 45 &#176;C) activated a smaller number of neurons producing Ca2+ transients (Figure 5E, one-way ANOVA F2,16 = 2.513, p &#60; 0.001, Dunnett&#39;s test, 21&#176;C: q = 9.594, df = 16, p &#60; 0.001; 10 &#176;C: q = 9.583, df = 16, p &#60; 0.001; 45 &#176;C: q = 9.160, df = 16, p &#60; 0.001) and &#916;F / F0 area under the curve of Ca2+ transients at 21 &#176;C and 10 &#176;C (Figure 5D, Brown-Forsythe ANOVA F3,268.4 = 12.98, p &#60; 0.001, Dunnett&#39;s T3 test, 21 &#176;C: t = 5.415, df = 128, p &#60; 0.001; 10 &#176;C: t = 4.398, df = 174.5, p &#60; 0.001; 45 &#176;C: t = 2.079, df = 161.9, p = 0.1127). In this experiment, smaller and medium diameter neurons produced Ca2+ transients spontaneously and under all stimuli, but larger diameter neurons only produced Ca2+ transients in response to the 300 g press stimulus (Figure 5G).
Labeling of neurons expressing specific receptors with td-Tomato in Pirt-GCaMP3 mice 
The cre-inducible reporter td-Tomato can be observed during Pirt-GCaMP3 imaging. Pirt-GCaMP3, td-Tomato mice that expressed Cre under the control of the TrpV1 promoter showed wide scale but not universal labeling of primary sensory neurons (Figure 6A,B). Spontaneous Ca2+ transients were observed in both td-Tomato-labeled neurons and neurons that are not labeled (Figure 6C). Noxious heat was not tested on this experiment, but will be tested in the future experiments.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64826/64826fig06.jpg" /> 
Figure 6: Labeling of TrpV1-positive neurons with td-Tomato in Pirt-GCaMP3 mice. (A)&#160;A maximum intensity projection of spontaneous activity of primary sensory neurons of a TrpV1-Cre, td-Tomato, Pirt-GCaMP3 mouse imaged on the 495 nm excitation/519 nm emission (FITC, green) channel only. Some neurons producing Ca2+ transients are called out with yellow arrows (not labeled with td-Tomato) or red arrows (labeled with td-Tomato). (B) An average intensity projection of the same primary sensory neurons as panel A on the 592 nm excitation/614 nm emission red channel only. (C) A maximum intensity projection of simultaneous&#160;red and green channels of the same primary sensory neurons as in panel A is shown. Some neurons expressing GCaMP3 but not td-Tomato are called out with yellow arrows. White arrows indicate GCaMP positive with no Ca2+ transients and not td-Tomato postive. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1: Spontaneous activity of a Pirt-GCaMP3 ganglion that has not been properly leveled or optimized for optical slice thickness causing waviness. 70 frames per second AVI. Optical slice thickness = 16 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826_Movie1.zip" target="_blank">Please click here to download this Movie.</a>
Movie 2: Spontaneous activity of the same ganglion from movie 1 that has been properly leveled and optimized for optical slice thickness with less waviness. 70 frames per second AVI. Optical slice thickness = 13 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826_Movie2.zip" target="_blank">Please click here to download this Movie.</a>
Movie 3: 100 g hindpaw press of Pirt-GCaMP3 ganglion. 3 frames per second AVI. Stimulus was applied during frames 6-10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826_Movie3.zip" target="_blank">Please click here to download this Movie.</a>
Movie 4: 45 &#176;C thermal stimulus to hindpaw of Pirt-GCaMP3 ganglion. 3 frames per second AVI. Stimulus was applied during frames 6-10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/64826_Movie4.zip" target="_blank">Please click here to download this Movie.</a>
Supplementary File 1: Example analysis spreadsheet <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64826/Example_Analysis_Spreadsheet.xlsx" target="_blank">Please click here to download this File.</a>

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 ...

in-vivo-calcium-imaging-neuronal-ensembles-networks-primary-sensory

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.6K Views.  University of Texas Health Science Center at San Antonio. 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.

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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

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, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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

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Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish