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>
	...

Neuronal Activity Mapping in Developing Mouse Sensory Cortex

<ol>
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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. 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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">
	<tbody>
		<tr>
			<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>
		</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>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>
</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.

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

507 vues.  Kagoshima University. Le but de notre recherche est d’étudier la formation des circuits neuronaux dans le cortex cérébral en développement. Notre objectif est de comprendre le rôle de l’activité neuronale dans ce processus. Ces dernières années, la microscopie à deux photons a permis d’observer le processus de formation des circuits neuronaux chez les animaux vivants.Nous avons développé une méthode d’imagerie de neurones uniques dans le cortex cérébra...

Vidéo: Pleins feux sur l’auteur : Déchiffrer la formation des circuits neuronaux à partir de la microscopie à deux photons et de l’imagerie des neurones uniques

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...

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evaluation and manipulation of neural activity using two-photon holographic microscopy

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural...

long-term imaging of identified neural populations using microprisms in freely moving and head-fixed animals

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

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imaging single cell-level activity in neonatal mice using two-photon microscopy

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

in vivo two-photon microscopy of single nerve endings in skin

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

polarization-sensitive two-photon microscopy for a label-free amyloid structural characterization

Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid...

Characterizing Spherulites with Polarization-Sensitive Two-Photon Microscopy

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in vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

in vivo wide-field and two-photon calcium imaging from a mouse using a large cranial window

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

laser cell ablation in intact drosophila larvae reveals synaptic competition

Author Spotlight: Investigating the Mechanisms of Neural Circuit Assembly and Synapse Formation in &lt;i&gt;Drosophila &lt;/i&gt;

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous...

Single-Neuron Ablation in &lt;i&gt;Drosophila &lt;/i&gt; for Studying Nervous System Adaptation

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intravital two-photon microscopy of the transplanted mouse lung

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

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serial two-photon tomography of the whole marmoset brain for neuroanatomical analyses

Author Spotlight: Mapping Primate Brain Connectivity 

Serial two-photon tomography (STPT) imaging is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. Here we describe a protocol for implementing it for marmoset brains to better understand their structural features.

Volumetric Imaging and Axonal Projection Mapping in the Marmoset Brain

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long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon...

visualizing shifts on neuron-glia circuit with the calcium imaging technique

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional...

in vivo imaging of biological tissues with combined two-photon fluorescence and stimulated raman scattering microscopy

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

two-photon calcium imaging in neuronal dendrites in brain slices

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain...

in vivo two-photon imaging of experience-dependent molecular changes in cortical neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges.  An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

two-photon microscopy for the study of tendons

Two-Photon Microscopy for the Study of Tendons

This article outlines the process of preparing, setting up, and imaging tendons using multiphoton microscopy. Additionally, it covers the application of SHG for analyzing collagen fibril alignment and the creation of a 3D representation of tendons. This methodology proves highly valuable in characterizing tendon cells and their ECM during injury and...

two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

in vivo two-photon imaging of cortical neurons in neonatal mice

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...

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

two-photon calcium imaging of forebrain activity in behaving adult zebrafish

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

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.