Name: in vivo visualization of spontaneous activity in neonatal mouse sensory cortex at a single-neuron resolution
Uploaded: 2023-11-21T14:00:00.000+00:00
Duration: 6 min 18 s
Description: Read this Scientific Article Protocol about Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging at JoVE.com

This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Transformative Research Areas (B) (22H05092, 22H05094) and for Scientific Research Grants 20K06876, AMED under Grant Number 21wm0525015, the Takeda Science Foundation, the Naito Foundation, the Kato Memorial Bioscience Foundation, the Kowa Life Science Foundation, NIG-JOINT (24A2021) (to H.M.); and Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grants 19K06887 and 22K06446, the Kodama Memorial Fund for Medical Research, the Uehara Memorial Foundation, the Kato Memorial Bioscience Foundation, and the Takeda Science Foundation (to N.N-T.). We thank Dr. Takuji Iwasato for the TCA-RFP mice.

All the experiments were conducted in accordance with the guidelines for animal experimentation of Kumamoto University and the National Institute of Genetics and approved by the animal experimentation committees.
1. In utero electroporation (IUE)
<ol>
	<li>Mate male TCA-RFP mice of ICR background with female wild-type ICR mice. Observe the vaginal plug to check for mating early morning of the next day. Observe the abdomen to check for pregnancy 2 weeks later.</li>
	<li>Prepare plasmid solution containing 5 ng/&#181;L TRE-nCre, 1 &#181;g/&#181;L CAG-loxP-stop-loxP-GCaMP6s-ires-tTA-WPRE, and 0.02 % Trypan Blue in ddH2O to sparsely label neurons by GCaMP using the Supernova system8.</li>
	<li>Prepare an analgesic solution containing 0.5 mg/mL carprofen and 0.01 mg/mL buprenorphine. Protect it from light and keep it at room temperature.</li>
	<li>At embryonic day (E)14, perform IUE as follows. The IUE methods are essentially the same as previous reports9,10,11.
	<ol>
		<li>Wipe the lab bench with 70% ethanol or glutaraldehyde solution. Sterilize all surgical instruments by autoclaving. Wear a mask and a lab coat to reduce the risk of infection to the mouse.</li>
		<li>Prepare glass micropipettes using a micropipette puller. Take the plasmid mixture solution into the glass micropipette using an aspirator tube.</li>
		<li>Induce anesthesia to the pregnant mice at E14 using isoflurane (2.0% in air). Keep the mice anesthetized during the procedure with isoflurane (1.2% in air). Pinch the toe to ensure that the anesthesia is deep enough. Use ophthalmic ointment to prevent eye dryness.</li>
		<li>Disinfect the abdominal area at least three times with alternating rounds of iodine-based scrub and 70% ethanol. Place a sterile drape to cover the surgical area. Make a midline incision and expose the uterus onto the drape.</li>
		<li>Repeat dripping warm saline onto the uterus to prevent it from drying or cooling until abdomen closure (Step 1.4.8).</li>
		<li>Inject the plasmid solution into one side of the lateral ventricle of embryos one by one using a micropipette and an aspirator tube.</li>
		<li>Use a forceps-type electrode to pinch the embryo heads and deliver square electric pulses (40 V, 50 ms) five times at 1 s intervals using an electroporator.</li>
		<li>Return the uterus to the abdominal cavity. Apply ~3 mL of warm saline into the cavity. Suture the peritoneal membrane and the abdominal skin. Administer the analgesic solution at 10 &#956;L/g weight (carprofen at 5 &#956;g/g weight and buprenorphine at 0.1 &#956;g/g weight) under the skin at the back of the neck.</li>
	</ol>
	</li>
	<li>Place the mouse in a recovery cage on a heating pad (37 &#176;C) to recover from anesthesia. Lay the mouse on its belly to prevent its tongue and saliva from choking the throat. Keep the mouse apart from other mice and monitor it until it recovers to move normally. Return the mouse to the cage.</li>
	<li>After birth, perform genotyping to check for RFP allele, and euthanize the pups without the allele. This step is recommended, especially if imaging is done during the second postnatal week when the TCA-RFP signal is weak and difficult to check at step 2.2.7.</li>
</ol>
2. Cranial window surgery
<ol>
	<li>Prepare the cortex buffer containing 125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl2, and 2 mM MgSO4 in ddH2O (pH adjusted to 7.4 with 1 M NaOH)12&#160;before the day of surgery. Sterile the buffer using a vacuum filter. 
	NOTE: The buffer can be kept at 4 &#176;C for up to 3 months. The required volume is 5-10 mL per pup.</li>
	<li>Perform the following steps at P3-12. Also see the previous reports that have described this procedure13,14.
	<ol>
		<li>Mix 50 mg of agarose with 5 mL of cortex buffer and completely dissolve the agarose by heating. Take some of the solution into a 1.5 mL tube and keep it at 42 &#176;C.</li>
		<li>Take some cortex buffer into a 50 mL conical tube and keep it at room temperature. Take some saline into a container (e.g., a cap of a 50 mL conical tube) and keep it at room temperature.</li>
		<li>Prepare an analgesic solution containing 0.01 mg/mL buprenorphine. Protect it from light and keep it at room temperature.</li>
		<li>Sterilize all surgical instruments by autoclaving.&#160;Disinfect the fluorescence stereo microscope with 70% ethanol.</li>
		<li>Use an isoflurane vaporizer to induce anesthesia in the pup with isoflurane (2.0% in air). Pay attention to the pup and take it out when its breathing slightly slows down. 
		NOTE: If the anesthesia is prolonged, the pup&#39;s breathing may cease for a few seconds. Even if the pup&#39;s breathing is resumed, prolonged anesthesia may decrease the pup&#39;s cerebral blood flow and cause irreversible brain damage.</li>
		<li>Disinfect the pup head at least three times with alternating rounds of iodine-based scrub and 70% ethanol. Place the pup onto the heating pad (35 &#176;C) under a fluorescence stereo microscope. Always keep the pup anesthetized with isoflurane (1.5%-2.5% in air).</li>
		<li>Select the pups expressing TCA-RFP and GCaMP by observing the fluorescence through the skull. Euthanize the pups that do not express both.</li>
		<li>Remove the scalp above the cerebral hemispheres as widely as possible carefully so as not to cause bleeding. Rub the skull with a sterile saline-soaked cotton swab to remove connective tissue.</li>
		<li>After the skull dries, adhere the incised scalp surface to the skull with a tissue adhesive.</li>
		<li>Transfer the pup onto a 37 &#176;C heating pad to recover from anesthesia. Wait for at least 15 min to let the adhesive solidify. 
		NOTE: Pause for up to 1 h before moving on to the next step. Prepare other pups in a similar manner if needed during this time.</li>
		<li>Anesthetize the pup with isoflurane. Place the pup onto the heating pad (37&#160;&#176;C) under a fluorescence stereo microscope while anesthetized (1.5%-2.5% isoflurane in air). 
		NOTE: If the anesthesia prolongs more than the time for the endpoint (60 min), euthanize the pup by decapitation during anesthetized with isoflurane.</li>
		<li>Mark the GCaMP-expressing location on the skull with a sterile permanent marker. Apply cortex buffer onto the location.</li>
		<li>Insert the corner of a razor blade into the skull. Push the blade slowly to shave off the skull and make a hole. Pinch off the cracked skull with tweezers and remove it.</li>
		<li>Check that the cranial hole is successfully made by observing blood vessels in the hole. If bleeding occurs, quickly rinse the hole with cortex buffer using a micropipette. Repeat rinsing until the bleeding stops completely.</li>
		<li>Apply a drop of cortex buffer to the cranial hole and place in a sterile 3-mm-diameter round coverglass over the hole. Wipe away the excess buffer with non-woven fabric. Wait until the area around the glass dries.</li>
		<li>Apply warm agarose solution around the glass edge using a micropipette. Because too much agarose may reduce the fluorescent signals, remove the excess solution under the glass by gently pushing the glass from above.</li>
		<li>Remove the agarose on the glass or distant from the glass edge using tweezers. Leave agarose only around the outer glass perimeter.</li>
		<li>Wait until the agarose solidifies. If agarose shrinkage makes a space under the glass, add agarose solution from the side to cover the entire glass edge. Remove any liquid from the skull surface with non-woven fabric.</li>
		<li>Mix acrylic resin powder and liquid in a rubber container. Aspirate the mixture with a micropipette and pour it to cover the agarose surrounding the glass edge with resin. 
		NOTE: Because the resin solidifies soon after mixing the powder and liquid, they need to be mixed repeatedly before applying. The amounts needed in each mixing are ~500 &#181;L for liquid and ~0.15 g for powder.</li>
		<li>Fix a titanium bar with resin on the contralateral hemisphere. Keep the bar angle parallel to the cover glass. Fix the entire skull surface with resin.</li>
		<li>Administer the analgesic solution at 10 &#956;L/g weight (buprenorphine at 0.1 &#956;g/g weight) under the skin at the back of the neck. Return the pup to a 37 &#176;C heating pad for anesthesia recovery. Wait for &#62;60 min for resin solidification before imaging. 
		&#8203;NOTE: Pause for 1-5 h before imaging. Perform surgery on other pups in a similar manner during this time.</li>
	</ol>
	</li>
</ol>
3. Two-photon calcium imaging
<ol>
	<li>Attach a two-axis goniometer with a titanium plate to a stage plate with XY positioning beneath the microscope. Set up a heating pad (35 &#176;C) on the stage.</li>
	<li>Turn on the scanning software with the following conditions: Pixels, 512 x 512; Bidirectional, ON; Averaging, none; imaging area, 600 x 600 &#181;m with a 20x objective. Set the settings so that the scan rate is faster than 1 Hz.</li>
	<li>Place the pup on the heating pad, and fix the head-mount titanium bar to the titanium plate with screws. Keep the pup anesthetized by placing a tube port for isoflurane (1.5-2.0% in air).</li>
	<li>Adjust the window angle horizontally by the goniometer. Turn on the backlight and observe the brain surface with a 5x objective lens, and select the imaging area by XY positioning.</li>
	<li>Put eye drops on the cranial window. Switch the objective to a 20x water-immersion lens. Observe the cortical surface to confirm that blood flow is seen on the brain surface.</li>
	<li>Turn off the backlight and scan the brain surface using one-photon mode. Increase the laser power to make the green autofluorescence of the glass and the brain surface visible.</li>
	<li>Lower the isoflurane concentration to 1.0%-1.5%. Cover the microscope to avoid light leaks. Switch the scanning software to two-photon mode.</li>
	<li>Adjust the laser power and detector gain appropriate for scanning GCaMP and RFP signals. Find the depth where TCA-RFP signals are seen. Make sure that the depth is layer 4, ~300 &#181;m lower than the brain surface at P6. Select the imaging area where many GCaMP-expressing neurons are seen.</li>
	<li>Start timelapse imaging of GCaMP and RFP signals. If two-channel simultaneous scanning is inapplicable, capture GCaMP and TCA-RFP images before the imaging.</li>
	<li>Stop isoflurane to weaken the anesthesia and observe spontaneous activities for ~20 min. Monitor the pup&#39;s movements during imaging using an infrared camera. Resume isoflurane anesthesia (2% in air) immediately if any response indicating distress is observed.</li>
	<li>After the pup stops moving, repeat the imaging from Step 3.9. Change the imaging area if needed.</li>
	<li>Euthanize the pup with an overdose of isoflurane. Fix the brain by transcardial perfusion of saline and 4% PFA, followed by post-fixation in 4% PFA overnight to prepare tangential slices and perform immunohistochemistry. Otherwise, euthanize the pup with an overdose of anesthetic followed by decapitation.</li>
	<li>If the dam has no other pups, euthanize the mother mouse with an overdose of anesthetic followed by cervical dislocation.</li>
</ol>
4. Analysis
<ol>
	<li>Start MATLAB and run EZcalcium toolbox15&#160;to open a graphical user interface (GUI) &#39;Initial GUI&#39;.</li>
	<li>Compensate for the image frame drifts due to mouse movements or other causes.
	<ol>
		<li>Click Motion Correction in the Initial GUI to open the Motion Correction GUI. Click &#39;Add Files...&#39; to load a TIF file of the imaging data.</li>
		<li>Set the settings as follows: Non-rigid Motion Correction, blank; Upsampling Factor, 50; Max Shift, 15; Initial Batch Size, 200; Bin Width, 200. Click &#39;Run Motion Correction&#39; to execute the correction. Motion-corrected imaging data will be saved automatically. 
		NOTE: The settings should be adjusted to the imaging data properties. If the drifts of some frames are not compensated because of nonlinear frame distortions or movement of the cortex in the depth direction, open the original imaging data without correction in ImageJ Fiji and remove the frames, and restart step 4.2.</li>
	</ol>
	</li>
	<li>Detect neurons and draw regions of interest (ROIs).
	<ol>
		<li>Click Automated ROI Detection in the Initial GUI to open the ROI Detection GUI. Click Add Files... to load the motion-corrected imaging data.</li>
		<li>Set the settings as follows: Initialization, Greedy; Search Method, Ellipse; Deconvolution, Constrained FOOPSI-SPGL1; Autoregression, Decay; Estimated # of ROIs, 60 (more than double the number visually detected is recommended); Estimated ROI Width, 17 (~20 &#181;m); Merge Threshold, 0.95; Fudge Factor, 0.95; Spatial Downsampling, 1; Temp. Downsampling, 1; Temp. Iterations, 5. 
		NOTE: The settings should be adjusted to the imaging data properties.</li>
		<li>Click Run ROI Detection to execute the detection. ROI data will be saved automatically.</li>
		<li>Click ROI Refinement in the Initial GUI to open the ROI Refinement GUI. Click Load Data to load the ROI data. Select the ROIs that had low activity frequency (&#60;1 Hz), located under the skull, or contained other neurons&#39; neurites. Click Exclude ROI to exclude the ROIs from the analysis.</li>
		<li>Select the Data Export Format to XLSX and click Export Data to obtain an Excel file with raw dF/F values. The dF/F uses equation (1), where F is the average intensity of pixels in every frame, F0 is the baseline signal intensity, and Fb is the background fluorescence. 
		<img alt="Equation 1" src="/files/ftp_upload/65899/65899eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1)</li>
	</ol>
	</li>
	<li>Calculate the Pearson&#39;s correlation coefficient of dF/F between ROIs and make a correlation coefficient matrix. Only use dF/F after anesthesia has weakened and spontaneous activity has begun to occur (~10 minutes after stopping isoflurane).</li>
	<li>Define the barrel edges from the TCA-RFP image using Fiji. Categorize the ROIs to their respective barrels or septa. Compare the pairwise correlation within the same barrel and that between different barrels.</li>
	<li>Generate 1,000 to 10,000 surrogate data by randomly shuffling the correspondence between ROI and Ca2+ traces. Calculate the average correlation coefficient within individual barrels in each surrogate data, and evaluate the statistical significance of the correlation in the real data. 
	NOTE: If one of 10,000 surrogates has a value higher than the real value, the statistical significance is 0.0001. The analyses described in steps 4.5 and 4.6 can be conducted for pooled data from multiple animals, as performed elsewhere7,16.</li>
</ol>

Neuronal Activity Mapping in Developing Mouse Sensory Cortex

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+calcium+imaging+of+dense+neural+populations+via+a+cell-body-targeted+calcium+indicator.">Precision calcium imaging of dense neural populations via a cell-body-targeted calcium indicator.</a> Neuron. 107 (3), 470-486 (2020).</li><li>Giovannucci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaImAn+an+open+source+tool+for+scalable+calcium+imaging+data+analysis.">CaImAn an open source tool for scalable calcium imaging data analysis.</a> Elife. 8, e38173 (2019).</li><li>Pachitariu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suite2p:+beyond+10,000+neurons+with+standard+two-photon+microscopy.">Suite2p: beyond 10,000 neurons with standard two-photon microscopy.</a> BioRxiv. , (2017).</li><li>Sitdikova, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+suppresses+early+cortical+activity.">Isoflurane suppresses early cortical activity.</a> Annals of Clinical and Translational Neurology. 1 (1), 15-26 (2014).</li><li>Marques-Smith, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transient+translaminar+GABAergic+interneuron+circuit+connects+thalamocortical+recipient+layers+in+neonatal+somatosensory+cortex.">A Transient translaminar GABAergic interneuron circuit connects thalamocortical recipient layers in neonatal somatosensory cortex.</a> Neuron. 89 (3), 536-549 (2016).</li><li>Tuncdemir, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+somatostatin+interneuron+connectivity+mediates+the+maturation+of+deep+layer+cortical+circuits.">Early somatostatin interneuron connectivity mediates the maturation of deep layer cortical circuits.</a> Neuron. 89 (3), 521-535 (2016).</li><li>Nakazawa, S., Yoshimura, Y., Takagi, M., Mizuno, H., Iwasato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+phase+transitions+in+spatial+organization+of+spontaneous+activity+in+postnatal+barrel+cortex+layer+4.">Developmental phase transitions in spatial organization of spontaneous activity in postnatal barrel cortex layer 4.</a> The Journal of Neuroscience. 40 (40), 7637-7650 (2020).</li><li>Yu, Y. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+electrical+coupling+regulates+neocortical+lineage-dependent+microcircuit+assembly.">Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly.</a> Nature. 486 (7401), 113-117 (2012).</li><li>Siegel, F., Heimel, J. 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The authors have no competing financial interests to declare.

Given that the spontaneous activities emerge from the sensory organ or lower nervous system and travel to the primary sensory area through a pathway equivalent to that of a mature nervous system3, it is crucial to define the primary sensory area and the location of imaged neurons within the area. In this protocol, we addressed this requirement by employing transgenic mice that visualize thalamocortical axons and the Supernova system that expresses GCaMP sparsely8. These tec...

The cerebral cortex contains several sensory areas with distinct functions. The areas receive inputs originating from their corresponding sensory organs, mostly conveyed through the spinal cord or brainstem and relayed via the thalamus1,2. Notably, neurons in each primary sensory area exhibit uniquely synchronized activity during early developmental stages, which also originate from sensory organs or the lower nervous centers, but essentially differ from the activities observed in the mature cortex3.
In neonatal rodents, for example, the primary visual area (V1) displays wave-like activity, which originates in the retina (retinal wave) and propagates through the entire visual pathway while conserving retinotopy4. The primary auditory area (A1) exhibits synchronous activity organized in band-shaped subregions that correspond to the isofrequency bands in the mature brain. The activity emanates from the cochlea&#39;s inner hair cells5,6. The barrel cortex in the primary somatosensory area (S1) shows a patchwork-like activity pattern in which layer 4 neurons within individual barrels, namely, neurons responsive to individual whiskers, are synchronously activated7. Although proposed to originate from the trigeminal ganglion, the source of the activity remains unknown7. Consequently, neonatal activity patterns are specialized both within each primary sensory area and within intra-areal subfields. The simultaneous visualization of neuronal activity and structure of primary sensory areas may facilitate an inquiry into the contribution of these activity patterns to the development of sensory systems.
In this article, we summarized a series of protocols: (1) to visualize individual neuronal activities using sparse labeling of GCaMP and primary sensory areas using TCA-RFP mice that express red fluorescent protein in thalamocortical axons7, (2) to image single cell-level activity in neonatal mice using two-photon microscopy in vivo, and (3) to analyze the activity correlations within S1 barrel cortex. The representative results show patchwork-like synchronized activity within individual barrels of a postnatal day (P)6 mouse. Despite some limitations, this technique can be used for chronic imaging, wide-field imaging across multiple sensory areas, and various manipulation experiments. The multifaceted analysis of neuronal activity during development will enrich our comprehension of brain circuit formation mechanisms.

<table><tbody><tr><td>20&amp;times; objective lens (water immersion)</td><td></td><td></td><td></td></tr><tr><td>250 mL Vacuum Filter/Storage Bottle System</td><td>Corning</td><td>431096</td><td></td></tr><tr><td>4%-paraformaldehyde phosphate buffer solution (4% PFA)</td><td>Nacalai</td><td>09154-85</td><td></td></tr><tr><td>Acrylic resin (UNIFAST II)</td><td>GC</td><td>N/A</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9793</td><td></td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Nacalai</td><td>06731-05</td><td></td></tr><tr><td>Electroporator</td><td>BEX</td><td>GEB14</td><td></td></tr><tr><td>Eye drop (Scopisol)</td><td>Senju Pharmaceutical</td><td>N/A</td><td></td></tr><tr><td>Fluorescence stereo microscope</td><td>Leica</td><td>M165FC</td><td></td></tr><tr><td>Glucose</td><td>Nacalai</td><td>16806-25</td><td></td></tr><tr><td>Heating pad</td><td>Muromachi Kikai</td><td>FHC-HPS</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Isoflurane</td><td>Pfizer</td><td>N/A</td><td></td></tr><tr><td>KCl</td><td>Nacalai</td><td>28514-75</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>131-00405</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-100</td><td></td></tr><tr><td>Multiphoton laser</td><td>Spectra-Physics</td><td>Mai Tai eHP DeepSee</td><td></td></tr><tr><td>Multiphoton microscope</td><td>Zeiss</td><td>LSM 7MP</td><td></td></tr><tr><td>NaCl</td><td>Nacalai</td><td>31320-05</td><td></td></tr><tr><td>Non-woven fabric (Kimwipe)</td><td>Kimberly Clark</td><td>S-200</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Nacalai</td><td>27575-31</td><td></td></tr><tr><td>Plasmid: CAG-loxP-STOP-loxP-GCaMP6s-ires-tTA-WPRE</td><td>Addgene</td><td>pK175</td><td></td></tr><tr><td>Plasmid: TRE-nCre</td><td>Addgene</td><td>pK031</td><td></td></tr><tr><td>Precision calibrated micropipets</td><td>Drummond</td><td>2-000-050</td><td></td></tr><tr><td>Razor blade</td><td>Feather</td><td>FA-10</td><td></td></tr><tr><td>Rimadyl (50 mg/mL Carprofen)</td><td>Zoetis JP</td><td>N/A</td><td></td></tr><tr><td>Round cover glass, 3-mm-diameter&amp;nbsp;</td><td>Matsunami</td><td>CS01078</td><td></td></tr><tr><td>Saline</td><td>Otsuka</td><td>035175315</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Nacalai</td><td>26427-72</td><td></td></tr><tr><td>Stage for imaging living pup (two single-axis translation stage for XY positioning, two-axis goniometer, base plate, adjustable pillar for z positioning)</td><td>ThorLabs</td><td>LT1/M, GN2/M, BM2060/M, MLP01/M</td><td></td></tr><tr><td>TCA-RFP mouse</td><td>N/A</td><td>N/A</td><td>Mizuno et al., 2018a</td></tr><tr><td>Tissue adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Titanium bar</td><td>Endo Scientific Instrument</td><td>N/A</td><td>Custom made (Mizuno et al., 2018b)</td></tr><tr><td>Titanium bar fixing plate</td><td></td><td>N/A</td><td>Custom made (Mizuno et al., 2018b)</td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Tweezers with platinum plate electrode, 5 mm diameter</td><td>BEX</td><td>CUY650P5</td><td></td></tr><tr><td>Wild-type ICR mouse</td><td>Nihon SLC</td><td>Slc:ICR</td><td></td></tr></tbody></table>

in vivo visualization of spontaneous activity in neonatal mouse sensory cortex at a single-neuron resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

Figure 1 shows the representative results of layer 4 neuron activities in the barrel cortex of a P6 pup visualized using the present protocol. Two-photon images of the green channel (GCaMP) and red channel (TCA-RFP) were temporally averaged and shown in Figure 1A. Because TCA-RFP fluorescence was much weaker than GCaMP fluorescence, the GCaMP signal leaked into the red channel (Figure 1A1,A2). Fourteen ROIs were dra...

Read this Scientific Article Protocol about Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging at JoVE.com

Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

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Research

JoVE Journal

Neuroscience

686 Views.  Kagoshima University. Het doel van ons onderzoek is om de vorming van neurale circuits in de zich ontwikkelende hersenschors te onderzoeken. We willen de rol van neuronale activiteit in dit proces begrijpen. In de afgelopen jaren heeft twee-fotonmicroscopie het mogelijk gemaakt om het proces van neuronale circuitvorming bij levende dieren te observeren.We hebben een methode ontwikkeld voor het in beeld brengen van individuele neuronen in de neonatale hersenschors. Door tege...

Video: Auteur in de schijnwerpers: ontcijfering van neurale circuitvorming uit twee-fotonmicroscopie en beeldvorming van één neuron

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Behavior

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

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