<meta charset="utf8"></head><body>Mappatura dell'attività neuronale nello sviluppo della corteccia sensoriale del topo

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<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 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.

<meta charset="utf8"></head><body>Autore in primo piano: Decifrare la formazione di circuiti neurali dalla microscopia a due fotoni e dall'imaging di singoli neuroni

Visualizzazione in vivo dell'attività spontanea nella corteccia sensoriale del topo neonatale con risoluzione di un singolo neurone

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

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

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Neuroscience

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

578 Views.  Kagoshima University. Lo scopo della nostra ricerca è quello di studiare la formazione di circuiti neurali nella corteccia cerebrale in via di sviluppo. Il nostro obiettivo è comprendere il ruolo dell'attività neuronale in questo processo. Negli ultimi anni, la microscopia a due fotoni ha permesso di osservare il processo di formazione del circuito neuronale negli animali viventi.Abbiamo sviluppato un metodo per l'imaging di singoli neuroni nella corteccia cerebrale neon...

Video: Autore in primo piano: Decifrare la formazione di circuiti neurali dalla microscopia a due fotoni e dall'imaging di singoli neuroni

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.

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.