The authors thank T. Sato, M. Kanbayashi, and S. Kouyama for their technical assistance. This work was supported by JSPS KAKENHI Grant Numbers JP15K14322 and JP16H06143, the Takeda Science Foundation, the Uehara Memorial Foundation, and the Collaborative Research Project of Niigata University Brain Research Institute 2017-2923 (H.M.) and by KAKENHI JP16K14559, JP15H01454, and JP15H04263 and Grant-in Scientific Research on Innovation Areas &#34;Dynamic regulation of Brain Function by Scrap &#38; Build System&#34; (JP16H06459) from MEXT (T.I.).

Experiments should be performed in accordance with the animal welfare guidelines prescribed by the experimenter&#39;s institution.
1. Preparation of Pups for Imaging
NOTE: Pups with sparsely labeled cortical neurons can be obtained by in utero electroporation (IUE) of Supernova vectors5,6. The Supernova system consists of the following two vectors: TRE-Cre and CAG-loxP-STOP-loxP-Gene X-ires-tTA-WPRE. In this system, sparse labeling relies on TRE leakage. In a sparse population of transfected neurons, TRE drives the weak expression of Cre and tTA. Subsequently, only in these cells, the expression of gene X is facilitated by a positive feedback of the tTA-TRE cycles. The achieved sparse and bright labeling allows the visualization of morphological details of individual neurons in vivo. Details of the IUE procedure are not described in this protocol since they have been described elsewhere7,8,9,10,11.
<ol>
	<li>Prepare timed-pregnant mice for IUE.</li>
	<li>Prepare a DNA solution for IUE. For sparse labeling with RFP, use a solution containing pK031:TRE-Cre (5 ng/&#181;L) and pK029:CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE (1 &#181;g/&#181;L) or a solution containing pK031:TRE-Cre (5 ng/&#181;L) and pK273:CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE (1 &#181;g/&#181;L). 
	NOTE: Various proteins can be expressed in the labeled neurons using different combinations of vectors. Also, various genes can be knocked-down or knocked-out specifically in labeled cells5,6 (e.g., a series of vectors for the Supernova system are available from RIKEN BioResource Research Center and from Addgene).</li>
	<li>Perform regular IUE7,8,9,10,11 to label cortical neurons. For the labeling of layer 4 neurons, use embryonic day-14 embryos.</li>
	<li>Wait for pup delivery and growth.</li>
</ol>
2. Surgery
<ol>
	<li>Anesthetize the postnatal day-5 (P5) pup using isoflurane gas (1.0%). Perform a tail-pinch test to check the level of anesthesia. If the pup responds to the pinch, increase the isoflurane concentration (up to 2.0%) or wait until the response disappears. Maintain the pup&#39;s body temperature during surgery using a heating pad.</li>
	<li>Subcutaneously inject an analgesic (carprofen, 5 mg/kg).</li>
	<li>Sterilize the pup&#39;s skin covering the skull by wiping it with 70% ethanol three times.</li>
	<li>Remove approximately 20 mm2 of the skin covering the skull using scissors sterilized with 70% ethanol (Figure 1A).</li>
	<li>Remove the fascia of the skull using sterilized forceps and a clean, sterile cotton swab (Figure 1A).</li>
	<li>Apply tissue adhesive using loading tips to the incised skin surface to stop the bleeding. Do not apply tissue adhesive to the imaging area (Figure 1B) because this makes the opening of the skull more difficult.</li>
	<li>Place the pup on a heating pad (37 &#176;C) and allow it to recover from anesthesia. Wait until the tissue adhesive has dried and solidified (approximately 30 min).</li>
	<li>If necessary, apply more tissue adhesive and wait for it to dry and solidify.</li>
</ol>
3. Cranial Window Preparation
<ol>
	<li>Anesthetize the pup using isoflurane (1.0% - 2.0%) and check the anesthesia level by a tail-pinch test.</li>
	<li>Carefully open the skull with a sterilized razor blade leaving the dura intact (1 mm in diameter) (Figure 1C). Use a gelatin sponge (cut into small pieces, approximately &#60; 2 mm3, using sterilized scissors, and apply them using tweezers) soaked in cortex buffer12 (125 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L glucose, 10 mmol/L HEPES, 2 mmol/L CaCl2, and 2 mmol/L MgSO4; pH 7.4; 300 mOsm/L; room temperature) to stop the bleeding. When opening the skull, apply a cortex buffer to keep the brain surface moist.</li>
	<li>Remove any buffer and blood from the dural surface using a gelatin sponge. Apply a thin layer of 1.0% low-melting-point agarose (dissolved in cortex buffer) using yellow tips. Using a heat block machine, maintain the temperature of the agarose solution at 42&#176;C until application. 
	NOTE: The draining of buffer and blood must be performed from the side of the craniotomy while taking care that the dry gelatin sponge does not come in contact with the dura. Failure to do so may damage the dura.</li>
	<li>Apply a round glass coverslip (No. 1, 3 mm in diameter) onto the agarose gel layer. Remove all bubbles between the coverslip and the agarose gel layer by pouring an excess of agarose gel between them. Remove the excess gel protruding from under the coverslip using tweezers (Figure 1D and 1E).</li>
	<li>Secure the coverslip using dental cement (Figure 1F).</li>
	<li>Mix the cement powder and the cement liquid. Apply the mixture using yellow tips before it becomes solidified. Do not apply dental cement onto the dura, because this may damage the brain.</li>
	<li>Attach a sterile titanium bar (custom made, approximately 30 mg, see Figure 1G) on the cranial bone using dental cement. Align the titanium bar and the coverslip (on the surface of the dura) in parallel to easily capture images.</li>
	<li>Cover the exposed skull with dental cement (Figure 1H).</li>
	<li>Recover the pup from anesthesia. Keep it on a heater (37 &#176;C) until the dental cement has solidified (1 h).</li>
</ol>
4. Two-photon Imaging
NOTE: The in vivo images in Figure 2 were acquired using a two-photon microscope with a titanium-sapphire laser (beam diameter [1/e2]2: 1.2 mm).
<ol>
	<li>Set the two-photon laser wavelength. For RFP excitation, use 1,000 nm (450 mW/mm2 at 400 &#181;m of depth). 
	NOTE: The laser power should be reduced as the z-position moves up.</li>
	<li>Wipe the surface of the coverslip with 70% ethanol.</li>
	<li>Anesthetize the pup using isoflurane (1.5% - 2.0%) and check the anesthesia level using a tail-pinch test.</li>
	<li>Attach the pup to the titanium plate on the imaging stage using a titanium bar on the pup&#39;s head (Figure 2A and 2B). Adjust the head such that the coverslip is parallel to the objective lens using the goniometer stage (Figure 2B). Maintain body temperature of the pup using a heating pad (37 &#176;C).</li>
	<li>Set the isoflurane concentration to 0.7% - 1.0%. 
	NOTE: A very high isoflurane concentration may cause accidental death of the pup during imaging.</li>
	<li>Place the imaging stage under the objective lens (20X, NA 1.0) of the two-photon microscope (Figure 2B and 2C).</li>
	<li>Apply one drop of water onto the coverslip. Use epi-fluorescence to locate the fluorescent protein-labeled neurons in the area where the dura has been exposed.</li>
	<li>Acquire z-stack images at 1.4-&#181;m intervals. For layer 4 neuron imaging, set the z-width to 150 - 300 &#181;m to image the entire dendritic morphology (Figure 2D and 2E). Use slow scanning and averaging to get clear images showing the neuronal morphology (it usually takes &#62; 20 min to acquire the entire dendritic morphology). 
	NOTE: The following parameters are recommended for imaging. Excitation wavelength: 1,000 nm, scanner: galvanometer type, dichroic mirror: 690 nm, emission filter: 575 - 620 nm bandpass, detector: GaAsP type, gain setting &#62; 100, image size &#62; 512 x 512 &#181;m, field of view &#62; 600 x 600 &#181;m, pixel resolution &#60; 1.2 &#181;m.</li>
</ol>
5. Recovery and Nursing
<ol>
	<li>Detach the pup from the imaging stage.</li>
	<li>Place the pup on a heater (37 &#176;C) and allow it to recover (15 min).</li>
	<li>Feed the pup warm milk using a micropipette at 2-h intervals and gently stimulate the stomach to allow excretion. Confirm that the pup is drinking milk by measuring its body weight.</li>
</ol>
6. Re-imaging
<ol>
	<li>Anesthetize the pup with isoflurane (1.5% - 2.0%), and check the anesthesia level using a tail-pinch test. Attach it to the imaging stage.</li>
	<li>Locate the previously imaged neurons and acquire a z-stack image. The identification of neurons is easy owing to their sparse labeling with Supernova.</li>
	<li>Repeat steps 5.1 - 6.2 until imaging is completed.&#160;NOTE: The pup can be imaged up to 18 h without losing weight. 
	</li>
</ol>

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M., De Paola, V., Caroni, P., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS Biology. , e272 (2005).</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>Luo, W., 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=Supernova:+A+Versatile+Vector+System+for+Single-Cell+Labeling+and+Gene+Function+Studies+in+vivo.">Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.</a> Scientific Reports. 6, 35747 (2016).</li><li>Mizuno, H., Hirano, T., Tagawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+activity-dependent+cortical+wiring:+formation+of+interhemispheric+connections+in+neonatal+mouse+visual+cortex+requires+projection+neuron+activity.">Evidence for activity-dependent cortical wiring: formation of interhemispheric connections in neonatal mouse visual cortex requires projection neuron activity.</a> The Journal of Neuroscience. 27 (25), 6760-6770 (2007).</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>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>Mizuno, H., Hirano, T., Tagawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-synaptic+and+post-synaptic+neuronal+activity+supports+the+axon+development+of+callosal+projection+neurons+during+different+post-natal+periods+in+the+mouse+cerebral+cortex.">Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex.</a> European Journal of Neuroscience. 31 (3), 410-424 (2010).</li><li>Matsui, A., Yoshida, A. 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W., 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=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</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>Zong, H., Espinosa, J. S., Su, H. H., Muzumdar, M. D., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+double+markers+in+mice.">Mosaic analysis with double markers in mice.</a> Cell. 121 (3), 479-492 (2005).</li><li>Young, P., 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=Single-neuron+labeling+with+inducible+Cre-mediated+knockout+in+transgenic+mice.">Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice.</a> Nature Neuroscience. 11 (6), 721-728 (2008).</li><li>Liu, J., 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=Neonatal+Repeated+Exposure+to+Isoflurane+not+Sevoflurane+in+Mice+Reversibly+Impaired+Spatial+Cognition+at+Juvenile-Age.">Neonatal Repeated Exposure to Isoflurane not Sevoflurane in Mice Reversibly Impaired Spatial Cognition at Juvenile-Age.</a> Neurochemical Research. 42 (2), 595-605 (2017).</li><li>Kondo, M., Kobayashi, K., Ohkura, M., Nakai, J., Matsuzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+of+the+medial+prefrontal+cortex+and+hippocampus+without+cortical+invasion.">Two-photon calcium imaging of the medial prefrontal cortex and hippocampus without cortical invasion.</a> eLIFE. 6, e26839 (2017).</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 nothing to disclose.

Critical Steps in the Protocol and Troubleshooting:
The most critical step of the protocol is the removal of the skull (Protocol step 3.2). Upon insertion, the razor blade often adheres to the dura, causing dural bleeding and damage to the brain. This can be avoided by adding a drop of cortex buffer on the skull and removing the skull in cortex buffer.
Bleeding from the dura and the skin after cranial window preparation leads to occlusion of the window. To avoid this, the tissue adhesive and dental cement used should be allowed to completely dry before proceeding to the next step. Multiple pups with a cranial window should be prepared since it is difficult to completely avoid bleeding. In general, if the cranial window remains clear and stable for 2 - 3 h post-surgery, the pup can be used for time-lapse imaging.
Significance of the Method with Respect to Existing/Alternative Methods:
Here, we have described a method for in vivo two-photon imaging of the neonatal cortex. This protocol has several merits compared with previously reported methods. These are listed as follows.
1) Cortical neurons can be sparsely and brightly labeled by transfecting Supernova vectors using IUE. IUE has been widely used for the labeling of cortical neurons during developing stages. However, a simple IUE is unsuitable for the imaging of individual neurons since neurons may be labeled too densely7,8,9,10. Moreover, using the Supernova system, various types of fluorescent proteins can be used for labeling sparse populations of neurons. For example, using Supernova-mediated sparse labeling of a genetically encoded calcium indicator GCaMP13,14, we have recently performed a functional analysis of individual neurons in the developing cortex layer 4 (P3 to P13)15.
2) The method described in this study is suitable for elucidating the molecular mechanisms underlying neuronal circuit development. The Supernova system allows sparsely labeled cell-specific knockout of any gene. Thus, the dynamics of a neuron containing a specific gene disruption can be observed. For this purpose, genetics-based systems such as MADM16 and SLICK17 have been previously reported. However, because the breeding of mouse lines is essential for these systems, they require much time and cost than the protocol described here.
3) This study utilizes a razor blade for skull removal. This minimizes bleeding from the dura and allows the opening of a wide area of the skull (&#60; 2 mm in diameter). Using this procedure, it was possible to observe the spontaneous activity of layer 4 neurons in the entire large barrel area within the somatosensory cortex15.
Limitations of the method:
A disadvantage of in vivo imaging of the mouse neonatal brain, compared with the imaging of transparent animals such as zebrafish larvae and Xenopus tadpoles, is the lower spatial and temporal resolution. Slow scanning and averaging should be performed for yielding clear images of the neuronal morphology because more light scattering occurs in the mouse brain. Light scattering may possibly be reduced using a longer wavelength laser for fluorescent protein excitation and proteins with longer fluorescence emission wavelengths.
Another limitation of the present protocol could be that the surgery for cranial window implantation may affect the formation of a normal cortical circuit due to brain inflammation12. However, there is a high likelihood that in vivo imaging is more physiological compared with in vitro imaging such as time-lapse imaging of the brain slice preparation, which should also give rise to severe inflammation by ischemia and slicing. It has also been reported that repetitive exposure to isoflurane may affect several neuronal processes18. Control experiments should be performed for verifying the appropriateness of results obtained by in vivo imaging. In the case of our imaging of layer 4 neurons in the somatosensory cortex, we confirmed a normal increase in total dendritic length and acquisition of orientation bias of dendritic projections of spiny stellate neurons5,20.
Recent studies reported &#62; 1-mm-depth imaging in an adult mouse brain wherein the dura was removed during surgery19. On the other hand, we have been able to report up to 400-&#181;m-depth imaging in neonates5. Since the dura cannot be removed in neonates and this, in turn, leads to a high light scattering, we consider that deep imaging in neonates is more challenging than in adults. Future improvement in fluorescent probes, lasers, and detectors should allow deep imaging in neonatal brains.
Thus far, we have reported 18-hour time-lapse imaging using the present protocol5. Recently, we have succeeded in 72-hour time-lapse imaging by improving the protocol20. We will continue to refine the protocol presented here (e.g., longer-term, higher time, and/or spatial resolution imaging) for revealing dynamic mechanisms of neuronal circuit development.

The precise wiring of neuronal circuits in the cerebral cortex is essential for higher brain functions including perception, cognition, and learning and memory. Cortical circuits are dynamically refined during postnatal development. Studies have investigated the process of cortical circuit formation using histological and in vitro culture analyses. However, the dynamics of circuit formation in living mammals has remained mostly unexplored.
Two-photon microscopy has been widely used for the in vivo analyses of neuronal circuits in the adult mouse brain1,2. However, owing to technical challenges, only a limited number of studies have addressed neuronal circuit formation in newborn mice. For example, Carrillo et al. performed the time-lapse imaging of climbing fibers in the cerebellum in the second postnatal week3. Portera-Cailliau et al. reported the imaging of axons in cortical layer 1 in the first postnatal week4. In the present study, we summarize a protocol for the observation of layer 4 cortical neurons and their dendrites in newborn mice. Results obtained by applying this protocol, which includes two methodologies, are reported in our recent publication5. First, we use the Supernova vector system5,6 for labeling individual neurons in the neonatal brain. In the Supernova system, the fluorescent proteins used for neuronal labeling are exchangeable and labeled cell-specific gene knockdown and editing/knockout analyses are also possible. Second, we describe a surgical procedure for cranial window preparation in fragile neonatal mice. Together, these methodologies allow the in vivo observation of individual neurons in neonatal brains.

<table>
	<tbody>
		<tr>
			<td>pK031. TRE-Cre</td>
			<td>Authors</td>
			<td>-</td>
			<td>Available from RIKEN BRC and Addgene</td>
		</tr>
		<tr>
			<td>pK029. CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE</td>
			<td>Authors</td>
			<td>-</td>
			<td>Available from RIKEN BRC and Addgene</td>
		</tr>
		<tr>
			<td>pK273. CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE</td>
			<td>Authors</td>
			<td>-</td>
			<td>Available from authors</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Wako</td>
			<td>099-06571</td>
			<td></td>
		</tr>
		<tr>
			<td>410 Anaesthesia Unit (isoflurane gas machine)</td>
			<td>Univentor</td>
			<td>8323101</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond (tissue adhesive)</td>
			<td>3M</td>
			<td>084-1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>M&#181;ltiFlex Round (loading tip)</td>
			<td>Sorenson</td>
			<td>13810</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam (gelatin sponge)</td>
			<td>Pfizer</td>
			<td>09-0353-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9793</td>
			<td>Low melting point</td>
		</tr>
		<tr>
			<td>Round-shaped coverslip</td>
			<td>Matsunami</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Unifast 2 (dental cement)</td>
			<td>GC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium bar</td>
			<td>Authors</td>
			<td>-</td>
			<td>Custom made (see Figure 1G)</td>
		</tr>
		<tr>
			<td>Rimadyl (carprofen)</td>
			<td>Zoetis</td>
			<td>-</td>
			<td>Injectable</td>
		</tr>
		<tr>
			<td>2-photon microscope</td>
			<td>Zeiss</td>
			<td>LSM7MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium-sapphire laser</td>
			<td>Spertra-Physics</td>
			<td>Mai-Tai eHPDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium plate</td>
			<td>Authors</td>
			<td>-</td>
			<td>Custom made (see Figure 2A)</td>
		</tr>
		<tr>
			<td>IMARIS, FilamentTracer, MeasurementPro</td>
			<td>BITPLANE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goniometer stage</td>
			<td>Thorlabs</td>
			<td>GN2/M</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figures 2D - 2F show representative results of two-photon time-lapse imaging of layer 4 cortical neurons using the present protocol. For the purpose of analysis, select neurons with clear dendritic morphology throughout the imaging periods. We analyzed the dendritic morphology of imaged neurons using morphological analysis software. Representative dendritic morphology reconstruction is shown in Figure 2F. Neurons showing disconnected dendrites (Figure 2G) should be excluded from analyses, because disconnected dendrites indicate cell death induced by damage during surgery or imaging. In addition, neurons with blurred dendritic tips should be excluded (e.g., the neuron with the arrowhead in Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58340/58340fig1.jpg" /> 
Figure 1: Surgery, cranial window preparation, and attachment of the titanium bar. (A) This panel shows the removal of the skin covering the skull. (B) This panel shows the fixation of the gap between the skin and the skull. Be careful not to apply the bond to the imaging area. (C) This is an image of the exposed dura. A razor blade was inserted between the skull and the dura, and the skull was flapped open to the left of the exposed area (arrowhead). The bone can then be easily removed using forceps. (D) This is a schematic design showing a vertical view of the cranial window. The gap between the cover glass and the dura is filled with a thin layer of agarose gel. The coverslip is fixed to the skull using dental cement. (E) A round-shaped coverslip is placed on the agarose gel layer. (F) This is an image of the secured coverslip. (G) This panel shows the design of the titanium bar. The titanium bar contains two screw holes for attachment to the imaging stage (see Figure 2) and one flat rectangular part that is attached to the pup&#39;s head. (H) The rectangular part of the titanium bar is attached to the pup&#39;s skull using dental cement. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58340/58340fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58340/58340fig2.jpg" /> 
Figure 2: In vivo two-photon imaging of cortical neurons in neonatal mice. (A) This panel shows the design of the titanium plate. The titanium plate has two screw holes for attaching the titanium bar and four screw holes for attaching the goniometer stage, which is placed on the imaging stage. 2.5 mm (in diameter) x 2 mm (in length) screws are used for fixation. (B) This is a representative image of the pup attached to the imaging stage. The pup&#39;s body temperature is maintained using a heater. (C) The pup is anesthetized using isoflurane during the in vivo imaging procedure. (D) This panel shows a representative Z-stack time-lapse image of layer 4 cortical neurons of a P5 pup. The arrowhead indicates the neuron with blurred dendrites, which should be removed from the analysis. (E) This panel shows higher magnification time-lapse images of the neuron with arrows in panel D. Blue arrowheads: dendritic tips that are retracted in 4.5 h, yellow arrowheads: dendritic tips that are elongated in 4.5 h, small white arrowheads: axon of a neighboring cell. (F) This is a 3-D model of the dendritic trace of neuron in the left figure of panel E. Blue circles indicate the position of the cell body. (G) This panel shows representative neurons with disconnected dendrites, which are not included in the analysis. The sample data in panels D - G contain NR1 (an essential subunit of NMDA-type glutamate receptor)-knocked-out neurons (because of limited data available from the authors). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58340/58340fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice at JoVE.com

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.0K Views.  Kumamoto University. 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.

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

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

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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 post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

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 reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

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 presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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 ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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 ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute 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 slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...