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

<ol>
	<li>Rao, M. S., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+mechanisms+of+neuronal+circuit+formation+in+layer+4+of+the+somatosensory+cortex+via+intravital+imaging.">Elucidating mechanisms of neuronal circuit formation in layer 4 of the somatosensory cortex via intravital imaging.</a> Neuroscience Research. 167, 47-53 (2021).</li><li>Iwasato, T., Erzurumlu, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+tactile+sensory+circuits+in+the+CNS.">Development of tactile sensory circuits in the CNS.</a> Current Opinion in Neurobiology. 53, 66-75 (2018).</li><li>Martini, F. J., Guillam&#243;n-Vivancos, T., Moreno-Juan, V., Valdeolmillos, M., L&#243;pez-Bendito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+activity+in+developing+thalamic+and+cortical+sensory+networks.">Spontaneous activity in developing thalamic and cortical sensory networks.</a> Neuron. 109 (16), 2519-2534 (2021).</li><li>Ackman, J. B., Burbridge, T. J., Crair, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+waves+coordinate+patterned+activity+throughout+the+developing+visual+system.">Retinal waves coordinate patterned activity throughout the developing visual system.</a> Nature. 490 (7419), 219-225 (2012).</li><li>Tritsch, N. X., Yi, E., Gale, J. E., Glowatzki, E., Bergles, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+spontaneous+activity+in+the+developing+auditory+system.">The origin of spontaneous activity in the developing auditory system.</a> Nature. 450 (7166), 50-55 (2007).</li><li>Babola, T. 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=Homeostatic+control+of+spontaneous+activity+in+the+developing+auditory+system.">Homeostatic control of spontaneous activity in the developing auditory system.</a> Neuron. 99 (3), 511-524.e5 (2018).</li><li>Mizuno, H., 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=Patchwork-type+spontaneous+activity+in+neonatal+barrel+cortex+layer+4+transmitted+via+thalamocortical+projections.">Patchwork-type spontaneous activity in neonatal barrel cortex layer 4 transmitted via thalamocortical projections.</a> Cell Reports. 22 (1), 123-135 (2018).</li><li>Mizuno, H., 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=NMDAR-regulated+dynamics+of+layer+4+neuronal+dendrites+during+thalamocortical+reorganization+in+neonates.">NMDAR-regulated dynamics of layer 4 neuronal dendrites during thalamocortical reorganization in neonates.</a> Neuron. 82 (2), 365-379 (2014).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294 (5544), 1071-1074 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240 (1), 237-246 (2001).</li><li>Holtmaat, 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=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li><li>Mizuno, H., Nakazawa, S., 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=In+vivo+two-photon+imaging+of+cortical+neurons+in+neonatal+mice.">In vivo two-photon imaging of cortical neurons in neonatal mice.</a> Journal of Visualized Experiments. 140, e58340 (2018).</li><li>Egashira, T., 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=In+vivo+two-photon+calcium+imaging+of+cortical+neurons+in+neonatal+mice.">In vivo two-photon calcium imaging of cortical neurons in neonatal mice.</a> STAR Protocols. 4 (2), 102245 (2023).</li><li>Cantu, D. 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=EZcalcium:+Open-source+toolbox+for+analysis+of+calcium+imaging+data.">EZcalcium: Open-source toolbox for analysis of calcium imaging data.</a> Frontiers in Neural Circuits. 14, 25 (2020).</li><li>Maruoka, H., 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=Lattice+system+of+functionally+distinct+cell+types+in+the+neocortex.">Lattice system of functionally distinct cell types in the neocortex.</a> Science. 358 (6363), 610-615 (2017).</li><li>Ant&#243;n-Bola&#241;os, 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=Prenatal+activity+from+thalamic+neurons+governs+the+emergence+of+functional+cortical+maps+in+mice.">Prenatal activity from thalamic neurons governs the emergence of functional cortical maps in mice.</a> Science. 364 (6444), 987-990 (2019).</li><li>Guillam&#243;n-Vivancos, T., 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=Input-dependent+segregation+of+visual+and+somatosensory+circuits+in+the+mouse+superior+colliculus.">Input-dependent segregation of visual and somatosensory circuits in the mouse superior colliculus.</a> Science. 377 (6608), 845-850 (2022).</li><li>Cardin, J. A., Crair, M. C., Higley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscopic+imaging:+Shining+a+wide+light+on+large-scale+neural+dynamics.">Mesoscopic imaging: Shining a wide light on large-scale neural dynamics.</a> Neuron. 108 (1), 33-43 (2020).</li><li>Chen, J. L., Voigt, F. F., Javadzadeh, M., Krueppel, R., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+population+dynamics+of+anatomically+defined+neocortical+networks.">Long-range population dynamics of anatomically defined neocortical networks.</a> eLife. 5, e14679 (2016).</li><li>Ota, K., 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=cell-resolution,+contiguous-wide+two-photon+imaging+to+reveal+functional+network+architectures+across+multi-modal+cortical+areas.">cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas.</a> Neuron. 109 (11), 1810-1824 (2021).</li><li>Zariwala, H. 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+Cre-dependent+GCaMP3+reporter+mouse+for+neuronal+imaging+in+vivo.">A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.</a> The Journal of Neuroscience. 32 (9), 3131-3141 (2012).</li><li>Murakami, T., Matsui, T., Uemura, M., Ohki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+strategy+for+development+of+the+hierarchical+visual+network+in+mice.">Modular strategy for development of the hierarchical visual network in mice.</a> Nature. 608 (7923), 578-585 (2022).</li><li>Pnevmatikakis, E. 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=Simultaneous+denoising,+deconvolution,+and+demixing+of+Calcium+imaging+data.">Simultaneous denoising, deconvolution, and demixing of Calcium imaging data.</a> Neuron. 89 (2), 285-299 (2016).</li><li>Shemesh, O. 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. A., Peters, J., Lohmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+and+central+inputs+shape+network+dynamics+in+the+developing+visual+cortex+in+vivo.">Peripheral and central inputs shape network dynamics in the developing visual cortex in vivo.</a> Current Biology. 22 (3), 253-258 (2012).</li><li>Nakagawa, N., Hosoya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+dynamics+in+microcolumnar+gap+junction+network+of+developing+neocortical+pyramidal+neurons.">Slow dynamics in microcolumnar gap junction network of developing neocortical pyramidal neurons.</a> Neuroscience. 406, 554-554 (2019).</li><li>Valiullina, F., 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=Developmental+changes+in+electrophysiological+properties+and+a+transition+from+electrical+to+chemical+coupling+between+excitatory+layer+4+neurons+in+the+rat+barrel+cortex.">Developmental changes in electrophysiological properties and a transition from electrical to chemical coupling between excitatory layer 4 neurons in the rat barrel cortex.</a> Frontiers in Neural Circuits. 10, 1 (2016).</li><li>Avitan, L., 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=Spontaneous+and+evoked+activity+patterns+diverge+over+development.">Spontaneous and evoked activity patterns diverge over development.</a> Elife. 10, e61942 (2021).</li><li>M&#246;lter, J., Avitan, L., Goodhill, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+neural+assemblies+in+calcium+imaging+data.">Detecting neural assemblies in calcium imaging data.</a> BMC Biology. 16 (1), 143 (2018).</li><li>Nakazawa, S., 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=Differential+dynamics+of+cortical+neuron+dendritic+trees+revealed+by+long-term+in+vivo+imaging+in+neonates.">Differential dynamics of cortical neuron dendritic trees revealed by long-term in vivo imaging in neonates.</a> Nature Communications. 9 (1), 3106 (2018).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>20&#215; 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>CaCl2&#8226;2H2O</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>
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		<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>MgSO4&#8226;7H2O</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&#160;</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>
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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.

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

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

The purpose of our research is to investigate the formation of neural circuits in the developing cerebral cortex. We aim to understand the role of neuronal activity in this process. In recent years, two-photon microscopy has made it possible to observe the process of neuronal circuit formation in living animals.We have developed a method for imaging single neurons in the neonatal cerebral cortex. By simultaneously observing activity patterns and changes in their morphology, we will be able to clarify which activity patterns are involved in neuronal circuit formation.

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

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.

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

in-vivo-visualization-spontaneous-activity-neonatal-mouse-sensory

Research

JoVE Journal

Neuroscience

Imaging Single Cell-Level Activity in Neonatal Mice Using Two-Photon Microscopy

To begin, affix a two-axis goniometer with a titanium plate to the stage plate for XY positioning under the microscope. Activate the scanning software, set pixels to 512 by 512 bidirectional to on using a 20X objective lens. After performing the cranial window surgery, place the pup expressing GCaMP on one side of the hemisphere on the heating pad.Using screws, secure the head mount titanium bar to the titanium plate. Then adjust the window angle horizontally with the goniometer. Illuminate the brain surface with the backlight and select the imaging area using XY positioning with a 5X objective lens.Under a 20X water immersion lens, administer eyedrops to cranial window. After disabling the backlight, scan the brain surface in one photon mode. Increase the laser intensity to visualize the green autofluorescence from the glass and brain surface.Cover the microscope to prevent external light interference. Start the scanning software to two photon mode for imaging. Then calibrate the laser power and detector gain to optimally visualize GCaMP signals.Locate the depth in an imaging area populated with many GCaMP-expressing neurons and start time-lapse imaging to capture GCaMP signals. Two-photon microscopy revealed layer four neuron activities in the barrel cortex of a postnatal day six pup with green fluorescence. The green GCaMP was more prominent, resulting in signal leakage into the red channel and identification of 14 regions of interest.

Analyzing the Activity Correlations Within S1 Barrel Cortex of Neonatal Mice After Two-Photon Imaging

To begin, initiate MATLAB and execute the EZcalcium toolbox to access the initial GUI. Within the initial GUI, select Motion Correction to open the motion correction GUI. Use the Add Files option to upload a TIF file containing the imaging data.Next, set the non-rigid motion correction to blank, upsampling factor to 50, maximum shift to 15, initial batch size and bin width to 200. Click on Run Motion Correction to initiate the correction. Within the initial GUI, activate automated ROI detection to access the ROI detection GUI.Use the Add Files feature to import the motion-corrected imaging data. Set initialization to greedy, search method to ellipse, deconvolution to constrained FOOPSI-SPGL1, and autoregression to decay. Then set the estimated number of ROIs to 60.Assign the estimated ROI width to 17, merge threshold to 0.95, fudge factor to 0.95, spatial and temporal downsampling to one, and temporal iterations to five. Then click Run ROI Detection to initiate the detection process. In the initial GUI, select ROI Refinement to launch the ROI refinement GUI.Use the Low Data button to import ROI data. Select the ROIs with low activity frequency situated beneath the skull or those overlapping with other neurons/neurites. Click Exclude ROI to exclude those ROIs from subsequent analysis.Calculate the delta F by F values using this equation. Choose XLSX as the data export format and execute export data to generate an Excel file populated with the raw delta F by F values. Compute the Pearson's correlation coefficient for the delta F by F values among the ROIs and construct a matrix of the correlation coefficients.Use Fiji software to delineate the barrel boundaries from the TCA-RFP image. Then assign the ROIs to their corresponding barrels or septa. Compare the pairwise correlation coefficients within identical barrels and distinct barrels.Generate 1, 000 to 10, 000 surrogate datasets by randomly permuting the association between ROI positions and calcium ion traces. In each surrogate dataset, compute the mean correlation coefficient within barrels individually and determine the statistical significance of the correlation. Higher pairwise correlation coefficients were observed within the same sensory processing units than across different units.Activities demonstrated stronger synchrony within the same units despite longer distances, surpassing the correlation with physically closer neurons from different units. The average of correlation coefficients within the same barrels was significantly higher than that calculated from 10, 000 sets of surrogate data. The correlation within the same barrels was significantly strong among three different time windows.

540 Views.  Kagoshima University.  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.

Neuronal Activity Mapping in Developing Mouse Sensory Cortex

Summary