This work was supported by grants from the NIH (DC007681, A.B.), CT Stem Cell grant (A.B.), Pardee foundation (A.B.), Predoctoral Ruth L. Kirschstein National Research Service Awards (NRSA) (S.Z.Y.), and an NSF Graduate Research Fellowship (B.L.). We thank the Bordey lab members for helpful comments on the manuscript. The present material is based upon work partly supported by the State of Connecticut under the Connecticut Stem Cell Research Grants Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Connecticut, the Department of Public Health of the State of Connecticut or CT Innovations, Incorporated.

1. Preparation of Solutions, Dissection, and Vibratome
<ol>
	<li>Solutions must be prepared at the correct osmolarity and pH (Table 1). Compared to artificial cerebrospinal fluid (ACSF), dissection solution is prepared with lower concentrations of sodium and calcium, and higher concentrations of magnesium. This is to minimize excitotoxicity effects during slicing.</li>
	<li>Both dissection and ACSF solutions must be saturated with 95% O2/5% CO2 by bubbling for at least 10 min t...

<ol>
	<li>Peretto, P., Giachino, C., Aimar, P., Fasolo, A., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+formation+and+glial+tube+assembly+in+the+shift+from+neonatal+to+adult+subventricular+zone+of+the+rodent+forebrain.">Chain formation and glial tube assembly in the shift from neonatal to adult subventricular zone of the rodent forebrain.</a> J. Comp Neurol. 487, 407-427 (2005).</li><li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+recording+subventricular+zone+progenitor+cells+in+live+tissue+of+postnatal+mice.">Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice.</a> Front. Neurosci. 4, 10-3389 (2010).</li><li>Lacar, B., Young, S. Z., Platel, J. 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C., Dupuis, A., Boisseau, S., Villaz, M., Albrieux, M., Brocard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrony+of+spontaneous+calcium+activity+in+mouse+neocortex+before+synaptogenesis.">Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesis.</a> Eur. J. Neurosci. 25, 920-928 (2007).</li><li>Young, S. Z., Platel, J. C., Nielsen, J. V., Jensen, N. A., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAA+increases+calcium+in+subventricular+zone+astrocyte-like+cells+through+L-+and+T-type+voltage-gated+calcium+channels.">GABAA increases calcium in subventricular zone astrocyte-like cells through L- and T-type voltage-gated calcium channels.</a> Front. Cell. Neurosci. 4, 8 (2010).</li><li>Bolteus, A. J., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+Release+and+Uptake+Regulate+Neuronal+Precursor+Migration+in+the+Postnatal+Subventricular+Zone.">GABA Release and Uptake Regulate Neuronal Precursor Migration in the Postnatal Subventricular Zone.</a> J. Neurosci. 24, 7623-7631 (2004).</li><li>Platel, J., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+a+whole+mount+preparation+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in a whole mount preparation of the subventricular zone.</a> J. Physiol. (Lond). 586, 3783-3793 (2008).</li><li>Nam, S. C., Kim, Y., Dryanovski, D., Walker, A., Goings, G., Woolfrey, K., Kang, S. S., Chu, C., Chenn, A., Erdelyi, F., Szabo, G., Hockberger, P., Szele, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+features+of+postnatal+subventricular+zone+cell+motility:+a+two-photon+time-lapse+study.">Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.</a> J. Comp. Neurol. 505, 190-208 (2007).</li><li>Lacar, B. L., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+controls+Ca2++activity-dependent+network+synchrony+in+the+adult+neurogenic+forebrain.">GABA controls Ca2+ activity-dependent network synchrony in the adult neurogenic forebrain.</a> , (2007).</li><li>Liu, X., Bolteus, A. J., Balkin, D. M., Henschel, O., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP-expressing+cells+in+the+postnatal+subventricular+zone+display+a+unique+glial+phenotype+intermediate+between+radial+glia+and+astrocytes.">GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes.</a> Glia. 54, 394-410 (2006).</li><li>Wang, D. D., Krueger, D. D., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+and+ionic+signature+of+neuronal+progenitors+of+the+postnatal+subventricular+zone+in+situ.">Biophysical properties and ionic signature of neuronal progenitors of the postnatal subventricular zone in situ.</a> J. Neurophysiol. 90, 2291-2302 (2003).</li><li>Tian, L., Hires, S. A., Mao, T., Huber, D., Chiappe, M. E., Chalasani, S. H., Petreanu, L., Akerboom, J., McKinney, S. A., Schreiter, E. R., Bargmann, C. I., Jayaraman, V., Svoboda, K., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li><li>Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca(2)+indicators.">An expanded palette of genetically encoded Ca(2) indicators.</a> Science. 333, 1888-1891 (2011).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat. Neurosci. 13, 759-766 (2010).</li></ol>

Calcium imaging of SVZ cells has been used to study patterns in spontaneous activity in neuroblasts10, receptor channel expression in both neuroblasts and astrocytes4,6,8 and astrocytic calcium waves3. Since cells in the SVZ are either immature or have glial properties, they do not fire action potentials11,12, meaning that millisecond changes in voltage potential indicative of activity in mature networks is not applicable in this region. Therefore, capturing the slower calcium ...

<table border="1">
	<tbody>
		<tr>
			<td>Solute</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Dissection (mM)</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>Dissection: 147 mM 
			ACSF: 0 mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>Dissection: 42 mM 
			ACSF: 126 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td>Dissection: 2.5 mM 
			ACSF: 2.5 mM</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td>Dissection: 4.33 mM 
			ACSF: 1 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>Dissection: 1.25 mM 
			ACSF: 1.25 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>Dissection: 10 mM 
			ACSF: 10 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>Dissection: 26 mM 
			ACSF: 26 mM</td>
		</tr>
		<tr>
			<td>CaCl2.2H2O</td>
			<td>Sigma</td>
			<td>C3306</td>
			<td>Dissection: 1.33 mM 
			ACSF: 2 mM</td>
		</tr>
	</tbody>
</table>
Table 1. Chemical list and recipes of dissection solution and ACSF.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT 1000S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Surehold or 3M</td>
			<td>Surehold 3G Super Glue or 3M Vet-Bond</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection tools</td>
			<td>Roboz or Ted Pella</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluo-4 AM calcium-sensitive dye</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green BAPTA-1 AM calcium-sensitive dye</td>
			<td>Invitrogen</td>
			<td>O6807</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pluronic F-127 20% solution in DMSO</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Upright confocal microscope</td>
			<td>Olympus</td>
			<td>FV300 or FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water-immersion objectives</td>
			<td>Olympus</td>
			<td>LUMPlanFl 40 x W/IR (NA 0.80); LUMPlanFl 60 x W/I (NA 0.90)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Sutter</td>
			<td>MPC-200/MPC-325/MPC-385</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pressure controller</td>
			<td>Parker Hannifin</td>
			<td>Picospritzer</td>
			<td>&lt;3 PSI during application</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter or Narshige</td>
			<td>Sutter P-97 or Narshige PP-830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass pipettes</td>
			<td>Sutter</td>
			<td>BF150-110-10</td>
			<td>I.D.:1.10, O.D.: 1.50</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Harvard Apparatus</td>
			<td>Model 720</td>
			<td>flow rate: 1 ml/min</td>
		</tr>
		<tr>
			<td>Chamber bath</td>
			<td>Warner Instruments</td>
			<td>RC-26 GLP</td>
			<td>Low profile allows for objective clearance</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Tygon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-324B/344B</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Materials/equipment list.

preparation of acute subventricular zone slices for calcium imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

preparation-of-acute-subventricular-zone-slices-for-calcium-imaging

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13.0K 次观看.  Yale University School of Medicine. 一个方法来加载脑室下区（SVZ）细胞中的钙离子指示剂染料记录活性钙。产后SVZ包括神经祖细胞和神经母细胞的细胞排列紧密。而不是使用清洗负载注入染料内的组织可以更好的染料扩散的压力。