Microscopy was performed at the UNC Neuroscience Microscopy Core (RRID:SCR_019060), supported in part by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124. Figure 1 was created with BioRender.com. The images and data in Figure 4 are reprinted from a previous publication9 with permission from the publisher.

<ol>
	<li>Stogsdill, J. 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=Astrocytic+neuroligins+control+astrocyte+morphogenesis+and+synaptogenesis.">Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis.</a> Nature. 551 (7679), 192-197 (2017).</li><li>Akdemir, E. S., Huang, A. Y., Deneen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytogenesis:+where,+when,+and+how.">Astrocytogenesis: where, when, and how.</a> F1000Research. 9, (2020).</li><li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains.">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 22 (1), 183-192 (2002).</li><li>Houades, V., 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=Shapes+of+astrocyte+networks+in+the+juvenile+brain.">Shapes of astrocyte networks in the juvenile brain.</a> Neuron Glia Biology. 2 (1), 3-14 (2006).</li><li>Zuchero, J. B., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+in+mammalian+development+and+disease.">Glia in mammalian development and disease.</a> Development. 142 (22), 3805-3809 (2015).</li><li>Srinivasan, R., 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=New+transgenic+mouse+lines+for+selectively+targeting+astrocytes+and+studying+calcium+signals+in+astrocyte+processes+in+situ+and+in+vivo.">New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo.</a> Neuron. 92 (6), 1181-1195 (2016).</li><li>Testen, A., Kim, R., Reissner, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+three-dimensional+imaging+of+individual+astrocytes+using+confocal+microscopy.">High-resolution three-dimensional imaging of individual astrocytes using confocal microscopy.</a> Current Protocols in Neuroscience. 91 (1), 92 (2020).</li><li>Takano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemico-genetic+discovery+of+astrocytic+control+of+inhibition+in+vivo.">Chemico-genetic discovery of astrocytic control of inhibition in vivo.</a> Nature. 588 (7837), 296-302 (2020).</li><li>Baldwin, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HepaCAM+controls+astrocyte+self-organization+and+coupling.">HepaCAM controls astrocyte self-organization and coupling.</a> Neuron. 109 (15), 2427-2442 (2021).</li><li>Amberg, N., Hippenmeyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+dissection+of+candidate+genes+in+mice+using+mosaic+analysis+with+double+markers.">Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers.</a> STAR Protocols. 2 (4), 100939 (2021).</li><li>Dumas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+electroporation+of+multiaddressable+genome-integrating+color+(MAGIC)+markers+to+individualize+cortical+mouse+astrocytes.">In utero electroporation of multiaddressable genome-integrating color (MAGIC) markers to individualize cortical mouse astrocytes.</a> Journal of visualized experiments: JoVE. (159), e61110 (2020).</li><li>Garcia-Marques, J., Nunez-Llaves, R., Lopez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-glia+from+pallial+progenitors+produce+the+largest+clonal+clusters+of+the+brain:+time+frame+of+clonal+generation+in+cortex+and+olfactory+bulb.">NG2-glia from pallial progenitors produce the largest clonal clusters of the brain: time frame of clonal generation in cortex and olfactory bulb.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 34 (6), 2305-2313 (2014).</li><li>Clavreul, S., 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=Cortical+astrocytes+develop+in+a+plastic+manner+at+both+clonal+and+cellular+levels.">Cortical astrocytes develop in a plastic manner at both clonal and cellular levels.</a> Nature Communication. 10 (1), 4884 (2019).</li><li>O'Donnell, J., Ding, F., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+functional+states+of+astrocytes+during+sleep+and+wakefulness:+Is+norepinephrine+the+master+regulator.">Distinct functional states of astrocytes during sleep and wakefulness: Is norepinephrine the master regulator.</a> Current Sleep Medicine Reports. 1 (1), 1-8 (2015).</li></ol>

The authors have no conflicts of interest.

This protocol describes an established method for analyzing astrocyte territory volume and astrocyte tiling in the mouse cortex, detailing all of the major steps beginning with perfusion and ending with image analysis. This protocol requires brains from mice that express fluorescent proteins in a sparse or mosaic population of astrocytes. Outside of this requirement, mice of any age may be used for this protocol, with only minor adjustments to perfusion settings and the volume of freezing media added to the embedding mol...

Astrocytes are elaborately branched cells that perform many important functions in the brain1. In the mouse cortex, radial glial stem cells give rise to astrocytes during the late embryonic and early postnatal stages2. During the first three postnatal weeks, astrocytes grow in size and complexity, developing thousands of fine branches that directly interact with synapses1. Concurrently, astrocytes interact with neighboring astrocytes to establish discrete, non-overlapping territories to tile the brain3, while maintaining communication via gap junction channels4. Astrocyte morphology and organization are disrupted in many disease states following insult or injury5, indicating the importance of these processes for proper brain function. Analysis of astrocyte morphological properties during normal development, aging, and disease can provide valuable insights into astrocyte biology and physiology. Furthermore, analysis of astrocyte morphology following genetic manipulation is a valuable tool for discerning the cellular and molecular mechanisms that govern the establishment and maintenance of astrocyte morphological complexity.
Analysis of astrocyte morphology in the mouse brain is complicated by both astrocyte branching complexity and astrocyte tiling. Antibody staining using the intermediate filament glial fibrillary acidic protein (GFAP) as an astrocyte-specific marker captures only the major branches, and vastly underestimates astrocyte morphological complexity1. Other cell-specific markers such as glutamate transporter 1 (GLT-1; slc1a2), glutamine synthetase, or S100&#946; do a better job of labeling astrocyte branches6, but introduce a new problem. Astrocyte territories are largely non-overlapping, but a small degree of overlap exists at the peripheral edges. Because of the complexity of branching, when neighboring astrocytes are labeled the same color, it is impossible to distinguish where one astrocyte ends and the other begins. Sparse or mosaic labeling of astrocytes with endogenous fluorescent proteins solves both problems: the fluorescent marker fills the cell to capture all of the branches and allows for imaging of individual astrocytes that can be distinguished from their neighbors. Several different strategies have been used to achieve sparse fluorescent labeling of astrocytes, with or without genetic manipulation, including viral injection, plasmid electroporation, or transgenic mouse lines. Details on the execution of these strategies are described in previously published studies and protocols1,7,8,9,10,11,12,13.
This article describes a method for measuring astrocyte territory volume from mouse brains with fluorescent labeling in a sparse population of astrocytes (Figure 1). Because the average diameter of an astrocyte in the mouse cortex is approximately 60 &#181;m, 100 &#181;m thick sections are used to improve the efficiency in capturing individual astrocytes in their entirety. Immunostaining is not required but is recommended to enhance the endogenous fluorescent signal for confocal imaging and analysis. Immunostaining may also enable better detection of fine astrocyte branches and reduce photobleaching of endogenous proteins during image acquisition. To improve antibody penetration into the thick sections, and to preserve tissue volume from sectioning through imaging, free-floating tissue sections are used. Analysis of astrocyte territory volume is performed using commercially available image analysis software. Additionally, this protocol describes a method for analysis of astrocyte tiling in tissue sections with mosaic labeling, where neighboring astrocytes express different fluorescent labels. This protocol has been used successfully in several recent studies1,8,9 to characterize astrocyte growth during normal brain development, as well as the impact of genetic manipulation on astrocyte development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 forceps</td>
			<td>Roboz</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL TB Syringe</td>
			<td>Becton Dickinson (BD)</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBS (tris-buffered saline)</td>
			<td></td>
			<td></td>
			<td>30 g Tris, 80 g NaCl, 2 g KCl, HCl to pH 7.4, dH2O to 1 L; store at room temperature (RT)</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Genesee Scientific</td>
			<td>25-106MP</td>
			<td></td>
		</tr>
		<tr>
			<td>1x TBS</td>
			<td></td>
			<td></td>
			<td>100 mL 10x TBS + 900 mL dH2O; store at RT</td>
		</tr>
		<tr>
			<td>1x TBS + Heparin</td>
			<td></td>
			<td></td>
			<td>28.2 mg Heparin + 250 mL 1x TBS; store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Genesee Scientific</td>
			<td>25-107MP</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Sucrose in TBS</td>
			<td></td>
			<td></td>
			<td>15 g sucrose, 1x TBS to 50 mL; store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>4% PFA (paraformaldehyde) in TBS</td>
			<td></td>
			<td></td>
			<td>40 g PFA, 4-6 NaOH pellets, 100 mL 10x TBS, dH2O to 1 L; store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Avertin</td>
			<td></td>
			<td></td>
			<td>0.3125 g tri-bromoethanol, 0.625 mL methylbutanol, dH2O to 25 mL; store at 4 &#176;C; discard 2 weeks after making</td>
		</tr>
		<tr>
			<td>Blocking and antibody buffer</td>
			<td></td>
			<td></td>
			<td>10% goat serum in TBST; store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>CD1 mice</td>
			<td>Charles River</td>
			<td>022</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection vial for brains</td>
			<td>Fisher Scientific</td>
			<td>03-337-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal acquisition software</td>
			<td>Olympous</td>
			<td>FV31S-SW</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV3000RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12544E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific</td>
			<td>CryoStar NX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat blade</td>
			<td>Thermo Scientific</td>
			<td>3052835</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding mold</td>
			<td>Polysciences</td>
			<td>18646A-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing Medium</td>
			<td></td>
			<td></td>
			<td>2:1 30% sucrose:OCT; store at RT</td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Aves Labs</td>
			<td>GFP1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Scientific</td>
			<td>158920010</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-chicken 488</td>
			<td>Invitrogen</td>
			<td>A-11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit 594</td>
			<td>Invitrogen</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Gibco</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td>N/A</td>
			<td>Version 9.8.0</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal lunch tin</td>
			<td>AQUARIUS</td>
			<td>N/A</td>
			<td>From Amazon, &#34;DIY Large Fun Box&#34;</td>
		</tr>
		<tr>
			<td>Methylbutanol</td>
			<td>Sigma-Aldrich</td>
			<td>152463</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors</td>
			<td>Roboz</td>
			<td>RS-5921</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouting medium</td>
			<td></td>
			<td></td>
			<td>20mM Tris pH8.0, 90% Glycerol, 0.5% N-propyl gallate ; store at 4 &#176;C; good for up to 2 months</td>
		</tr>
		<tr>
			<td>Nailpolish</td>
			<td>VWR</td>
			<td>100491-940</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>02370-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T.</td>
			<td>Fisher Scientific</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil</td>
			<td>Olympus</td>
			<td>IMMOIL-F30CC</td>
			<td>Specific to microscope/objective</td>
		</tr>
		<tr>
			<td>Operating Scissors 6&#34;</td>
			<td>Roboz</td>
			<td>RS-6820</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital platform shaker</td>
			<td>Fisher Scientific</td>
			<td>88861043</td>
			<td>Minimum speed needed: 25 rpm</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Bogrinuo</td>
			<td>N/A</td>
			<td>From Amazon, &#34;Detail Paint Brushes - Miniature Brushes&#34;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet (5.75&#34;)</td>
			<td>VWR</td>
			<td>14672-608</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet (9&#34;)</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>RFP antibody</td>
			<td>Rockland</td>
			<td>600-401-379</td>
			<td></td>
		</tr>
		<tr>
			<td>Sectioning medium</td>
			<td></td>
			<td></td>
			<td>1:1 glycerol:1x TBS; store at RT</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chrloide</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>TBST (TBS + Triton X-100)</td>
			<td></td>
			<td></td>
			<td>0.2% Triton in 1x TBS; store at RT</td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>VWR</td>
			<td>414004-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-bromoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Thermo Scientific</td>
			<td>424570025</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 (high-quality)</td>
			<td>Fisher Scientific</td>
			<td>50-489-120</td>
			<td></td>
		</tr>
		<tr>
			<td>XTSpotsConvexHull</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>custom XTension provide as supplementary material</td>
		</tr>
		<tr>
			<td>Buffers and Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBS</td>
			<td></td>
			<td></td>
			<td>xx mM Tris, xx mM NaCl, xx mM KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>1x TBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x TBS + Heparin</td>
			<td></td>
			<td></td>
			<td>add xx mg Heparin to xx mL of 1x TBS</td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% Sucrose in TBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sectioning medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TBST</td>
			<td></td>
			<td></td>
			<td>0.2% Triton in 1x TBS</td>
		</tr>
		<tr>
			<td>Blocking and antibody buffer</td>
			<td></td>
			<td></td>
			<td>10% goat serum in 1x TBST</td>
		</tr>
		<tr>
			<td>Mouting medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of astrocyte territory volume and tiling in thick free-floating tissue sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

Figure 1&#160;presents a schematic outline of the major steps and workflow for this protocol. Figure 2 shows screenshots of key steps using the image analysis software to generate a surface, generate spots close to the surface, and generate a convex hull. Figure 3 demonstrates the application of this technique to determine astrocyte territory overlap/tiling. In Figure 4, representative results from a pr...

Watch this Scientific Journal Video about Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections at JoVE.com

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

analysis-astrocyte-territory-volume-tiling-thick-free-floating-tissue

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3.2K Views.  University of North Carolina, Chapel Hill. This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Video: Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...