Animal use follows approved breeding and animal use protocols that conform to the Animal Care and Use Guidelines of Colorado State University.
NOTE: The protocol below describes the preparation and culture method for the long-term incubation and intermittent imaging of hippocampal slices. A single hippocampal slice is attached to a specially prepared photoetched coverslip using a plasma clot, and then the coverslips are sealed onto the flat side of a drilled-out roller tube, which is maintaine...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+stimuli+induce+persistent+ADF/cofilin-actin+rods+that+disrupt+distal+neurite+function.">Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function.</a> Nature Cell Biol. 2 (9), 628-636 (2000).</li><li>Rahman, 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=Cofilin+rods+and+aggregates+concur+with+tau+pathology+and+the+development+of+Alzheimer's+disease.">Cofilin rods and aggregates concur with tau pathology and the development of Alzheimer's disease.</a> J Alzheimers Dis. 42 (4), 1443-1460 (2014).</li><li>Bamburg, J. R., Bernstein, B. 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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=Production+and+use+of+replication-deficient+adenovirus+for+transgene+expression+in+neurons.">Production and use of replication-deficient adenovirus for transgene expression in neurons.</a> Methods Cell Biol. 71, 387-416 (2003).</li><li>K&#252;gler, S., Kilic, E., B&#228;hr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+synapsin+1+gene+promoter+confers+highly+neuron-specific+long-term+transgene+expression+from+an+adenoviral+vector+in+the+adult+rat+brain+depending+on+the+transduced+area.">Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area.</a> Gene Ther. 10 (4), 337-347 (2003).</li><li>Mi, 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=A+genetically+encoded+reporter+for+real-time+imaging+of+cofilin-actin+rods+in+living+neurons.">A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.</a> PLOS ONE. 8 (12), 83609 (2013).</li><li>Wang, L., Blouin, V., Brument, N., Bello-Roufal, M., Francois, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+recombinant+adeno-associated+vectors.">Production and purification of recombinant adeno-associated vectors.</a> Methods Mol Biol. 807, 361-404 (2011).</li><li>Matsushita, 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=Adeno-associated+virus+vectors+can+be+efficiently+produced+without+helper+virus.">Adeno-associated virus vectors can be efficiently produced without helper virus.</a> Gene Therapy. 5 (7), 938-945 (1998).</li><li>Ward, P., Walsh, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+integration+of+rAAV+vector+into+the+AAVS1+region.">Targeted integration of rAAV vector into the AAVS1 region.</a> Virology. 433 (2), 356-366 (2012).</li><li>Akerboom, 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=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J Neurosci. 32 (40), 13819-13840 (2012).</li><li>Kim, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production+and+purification.">Lentivirus production and purification.</a> Methods Mol Biol. 1382, 107-114 (2016).</li><li>Huang, L., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoprocessing+of+human+immunodeficiency+virus+type+1+protease+miniprecursor+fusions+in+mammalian+cells.">Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells.</a> AIDS Res Ther. 7, 27 (2010).</li><li>Bordat, A., Houvenaghel, M. C., German-Retana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibson+assembly:+an+easy+way+to+clone+polyviral+full-length+infectious+cDNA+clones+expressing+an+ectopic+VPg.">Gibson assembly: an easy way to clone polyviral full-length infectious cDNA clones expressing an ectopic VPg.</a> Virol J. 12, 89 (2015).</li><li>Er, J. C., 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=NeuO:+a+fluorescent+chemical+probe+for+live+neuron+labeling.">NeuO: a fluorescent chemical probe for live neuron labeling.</a> Angew Chem Int Ed Engl. 54 (8), 2242-2246 (2015).</li><li>Bernstein, B. W., Chen, H., Boyle, J. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+actin-ADF/cofilin+rods+transiently+retards+decline+of+mitochondrial+potential+and+ATP+in+stressed+neurons.">Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons.</a> Am J Physiol Cell Physiol. 291 (5), 828-839 (2006).</li><li>Cichon, 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=Cofilin+aggregation+blocks+intracellular+trafficking+and+induces+synaptic+loss+in+hippocampal+neurons.">Cofilin aggregation blocks intracellular trafficking and induces synaptic loss in hippocampal neurons.</a> J Biol Chem. 287 (6), 3929-3939 (2012).</li><li>Stine, W. B., Dahlgren, K. N., Krafft, G. A., LaDu, M. 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C., 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=Amyloid+beta+dimers/trimers+potently+induce+cofilin-actin+rods+that+are+inhibited+by+maintaining+cofilin+phosphorylation.">Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin phosphorylation.</a> Mol Neurodegener. 6, 10 (2011).</li><li>Walsh, K. 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=Amyloid-&#946;+and+proinflammatory+cytokines+utilize+a+prion+protein-dependent+pathway+to+activate+NADPH+oxidase+and+induce+cofilin-actin+rods+in+hippocampal+neurons.">Amyloid-&#946; and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.</a> PLOS ONE. 9 (4), 95995 (2014).</li><li>Woo, J. 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The roller tube method described here allows for long-term culturing and high-resolution live imaging of sliced brain tissue. One major issue with the slice technique as applied here is in the mounting and maintenance of slices. Coverslip coatings that support slice adhesion, promote slice thinning by enhancing the outgrowth of neurites and migration of cells out of the slice; thus, we avoided the use of these substrates. The insertion of amino groups onto the glass by treatment with 3-aminopropyltriethoxysilane improved...

Primary culture of dissociated neurons from regions of rodent brain is an important tool used by researchers to observe responses to pathologically implicated stimuli. However, such studies have the disadvantage of looking at neurons in only 2D and without their glial support system. Furthermore, unless grown under conditions of very high density (640 neurons/mm2 or about 16% of surface area) in which it becomes impossible to follow the random outgrowth of a dendrite or axon for more than a short distance from its cell body, hippocampal neuronal viability over 4 weeks declines significantly1, limiting the use of dissociated cultures for extended studies of age-related pathologies. The culturing of slices prepared from rodent brain is an attractive option that overcomes these limitations by maintaining an organized cell architecture and viability for weeks or months. Conditions for maintaining many different regions of rodent brain in slice culture have been described2.
Two major methods are widely used for long-term culture of brain slices: culturing on membranes at the air-liquid interface3 or culturing on coverslips in sealed tubes allowed to rotate in a roller incubator to provide aeration4. Slices cultured on membranes can be directly imaged with high-resolution fluorescence microscopy using an upright microscope and water immersion objectives5. Alternatively, slices cultured on membranes have been transferred to glass bottom dishes to achieve good resolution of dendritic spines using an inverted microscope6. However, both methods of imaging slices grown on membranes are open systems that require medium changes and often use antifungal and/or antibiotics to prevent or reduce contamination5,6. Slices on a membrane at the air-medium interface maintain excellent morphology and survival, but returning to precise locations during repetitive imaging at high magnification is extremely difficult unless the experiment is following only small groups of cells expressing a fluorescent marker. Although slices grown on membranes have been used with viral-mediated expression of transgenes5,6, biosafety protocols may require an enclosed culture system be employed for certain viral vectors that are used for expressing fluorescently tagged proteins and reporters of cell physiology. Furthermore, immersion objectives require decontamination between samples that will be followed in culture5. One major application of membrane interface cultures is combining high-resolution imaging with electrophysiology at single time points7.
The roller tube method with coverslips inside the plastic tube does not permit any electrophysiology or high-resolution imaging without removing the coverslip. Thus, this method has been most often applied to long-term studies in which post-fixation observations have been made8. Described here is a method that utilizes the roller tube culture technique but on drilled-out tubes with slices on coverslips that can be imaged repetitively for as long as the cultures are maintained. The enclosed system requires no medium change for imaging and utilizes photoetched coverslips to provide fiducial marks that allow imaging at high magnification, after days or weeks, the precise fields previously imaged.
We apply this method to examine changes in the rodent hippocampus, a major brain region involved in memory and learning. The rodent hippocampus is often studied as a model for pathological or age-related changes observed during development of cognitive impairment9, such as those that occur in AD. Our method is particularly well suited to study pathological changes that develop within a single slice over time in response to environmental changes, such as increases in A&#946; peptides, which is characteristic of AD8. One pathology associated with human and rodent AD brain is the presence of cofilin-actin aggregates and rods, the latter containing bundles of filaments in which cofilin and actin are in a 1:1 molar ratio10,11,12. Rods have been observed in fixed slices of rat hippocampus following A&#946; treatment, as well as within a live rodent brain slice expressing cofilin-GFP subjected to hypoxia8, and they may contribute to the synaptic dysfunction seen in AD and stroke. Here we use this new culturing method to observe the time course and distribution within slices of expressed exogenous chimeric fluorescent proteins introduced by different viruses. We then utilize the neuronal specific expression of a cofilin reporter construct to follow the development of cofilin rod and aggregate pathology in hippocampal slices in response to treatment with soluble A&#946; oligomers (A&#946;o).

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			<td height="40" style="height:40px;width:280px;">Bottoms from 15 cm culture dishes</td>
			<td style="width:220px;">VWR Scientific</td>
			<td style="width:191px;">25384-326</td>
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			<td height="40" style="height:40px;">Phillips Head Machine Screws (#10-32)</td>
			<td>Ace Hardware</td>
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			<td>2.5&#34; long and 3/16&#34; in diameter</td>
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			<td>ACE Hardware</td>
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			<td>ACE Hardware</td>
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			<td>ACE Hardware</td>
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			<td>5/16&#34;, thick; 5/8&#34;, hole diameter; 1.125&#34;, OD</td>
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			<td height="40" style="height:40px;">Polyethylene tubing (5/16&#34;; OD, 3/16&#34;; ID)</td>
			<td>ACE Hardware</td>
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			<td>Cut to 1.8&#34; length</td>
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			<td>ACE Hardware</td>
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			<td>Ace Hardware</td>
			<td>ProTech Model 1201</td>
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			<td>VWR</td>
			<td>62407-076</td>
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			<td>Ace Hardware</td>
			<td>Diablo freud brand</td>
			<td>Drill bits for cutting plastic.</td>
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			<td>Ace Hardware</td>
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			<td>Ace Harware</td>
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			<td>Ace Hardware</td>
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			<td height="40" style="height:40px;">Swivel Head Deburring Tool, 5&#34;</td>
			<td>Ace Hardware</td>
			<td>26307</td>
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			<td style="width:220px;">Grace Bio-Labs</td>
			<td style="width:191px;">666581</td>
			<td>0.5 mm Thickness</td>
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			<td>Office Max</td>
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			<td>thepunchbunch.com</td>
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			<td height="40" style="height:40px;">Phototeched Coverslips, 12 mm diameter</td>
			<td>Bellco Glass, Inc.</td>
			<td>1916-91012</td>
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			<td height="40" style="height:40px;">3-aminopropyltriethoxylane</td>
			<td>Sigma-Aldrich</td>
			<td>A3648</td>
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			<td>Sigma-Aldrich</td>
			<td>179124</td>
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			<td height="40" style="height:40px;">#5 Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">McIlwain Tissue Chopper</td>
			<td>Ted Pella, Inc.</td>
			<td>10180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Double Edge Razor Blades</td>
			<td>Ted Pella, Inc.</td>
			<td>121-6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Whatman Filter Paper</td>
			<td>VWR</td>
			<td>28450-182</td>
			<td>Cut into 5.8 cm diameter circles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-chloro-trifluoro-ethylene (Aclar)</td>
			<td>Ted Pella, Inc.</td>
			<td>10501-10</td>
			<td>Cut into 5.8 cm diameter circles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#21 Surgical Blade</td>
			<td>VWR Scientific</td>
			<td>25860-144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#5 Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spatula, stainless with tapered end</td>
			<td>VWR</td>
			<td>82027-518</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gey&#39;s Balanced Salt Solution</td>
			<td>Sigma-Aldrich</td>
			<td>G9779&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>15023-021</td>
			<td>25% (w/v) Solution, 0.2 mm filter sterilized</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chicken Plasma</td>
			<td>Cocalico Biologicals</td>
			<td>30-0300-5L</td>
			<td>Rehydrate in sterile water, centrifuge at 2500 x g 30 min at 4 &#176;C, quick freeze aliquots in liquid nitrogen and store at&#160; -80 &#176;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thrombin, Bovine</td>
			<td>Fisher</td>
			<td>60-516-01KU</td>
			<td>150 units /ml in GBSS/Glucose, quick freeze aliquots in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell Roller System</td>
			<td>Bellco Biotech</td>
			<td>SciERA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Roller Incubator</td>
			<td>Forma</td>
			<td>Model 3956</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">N21-MAX</td>
			<td>ThermoFisher Scientific</td>
			<td>AR008</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pen/Strep (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">200 mM Glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>15023-021</td>
			<td>25% (w/v) Solution, 0.2 mm filter sterilized</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal A</td>
			<td>ThermoFisher Scientific</td>
			<td>10888-022&#160;</td>
			<td>Complete Medium: 48 mL Neurobasal A, 1 mL N21-MAX, 0.625 mL 200 mM Glutamine, 0.180 mL 25% Glucose, 0.250 mL 100x pen/strep.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Third generation lentivirus packaging</td>
			<td>Life Technologies</td>
			<td>K4975-00</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">159 K cutoff centrifugal filters (Centricon)</td>
			<td>EMD Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lentiviral cloning system (InFusion)</td>
			<td>Clonetech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plasmids 30323, 50856, 51279</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neuronal cell viability dye (NeuO)</td>
			<td>Stemcell technologies</td>
			<td>1801</td>
			<td>Thaw once and quick freeze in 4 &#181;L aliquots. Store at -20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope objectives</td>
			<td>Olympus</td>
			<td>air: 4X, 20; oil: 40X, 60X,</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spinning disc confocal system</td>
			<td>Yokagawa</td>
			<td>CSU22</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope EMCCD camera</td>
			<td>Photometrics</td>
			<td>Cascade II</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Linear encoded (x,y), piezo z flat top stage</td>
			<td>ASI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope lasers and integration</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEK293T cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human Plasmin</td>
			<td>Sigma Aldrich</td>
			<td>P1867</td>
			<td>0.002 U/mL in 0.1% bovine serum albumin (0.2 mm filter sterilized), quick freeze in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
	</tbody>
</table>

modified roller tube method for precisely localized and repetitive intermittent imaging during long-term culture of brain slices in an enclosed system

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

To determine how accurately fiducial marks can be utilized to reimage the same cells within the same fields over time, we examined slices grown on photoetched coverslips (Figure 6A). Neurons were visualized by staining with a vital dye (100 nM for 2 h; does not stain non-neuronal cells), which disappears from neurons over time without harming the cells25. We identified a fiducial mark in a single grid square (<strong c...

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Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

The overall goal of this modified roller tube method for brain slice culture is to provide an enclosed culture system for long-term slice survival. with the capability for repetitive high-resolution florescence imaging. This method can help answer key questions in neuronal development and neurodegenerative disorders that require observations of the same neurons over time, such as localizing key proteins involved in synapse formation or loss.The main advantages of this technique are the fully enclosed culture system that allows safe use of viruses for protein expression and the use of photo etched coverslips to localize the same cells for imaging. We first had the idea for this method when we were culturing slices on coverslips inside roller tubes, but needed a better way to visualize changes occurring in living slices over time. The widespread use of viral vectors to express transgenes and specific cell populations for microscope imaging drove us to develop an enclosed system to protect users and eliminate contamination of microscope objectives.The ability to follow the identical population of cells within brain slices over time allows for studies on development of pathological changes and synapse alteration in rodent models of human neurological disorders. To begin prepare the roller tube rack and make a jig to assist with drilling a hole into the roller tubes as described in the accompanying text protocol. Using the jig, hold in position a flat sided, one centimeter, plastic culture tube, so that the flat side is facing up.Then drill a six millimeter diameter hole with the center one centimeter from the bottom and centered between the sides of the tube. With the swiveling deburring tool smooth the edges of the hole and make four grooves on the inside edge of the hole to facilitate draining of the hole during rotation. Next, rinse the tubes with 70%ethanol and then air-dry them in a biological safety cabinet.Sterilize the tubes in some 12 millimeter punched adhesive silicone rubber disks for 40 minutes under the UV lamp. After 20 minutes reposition the tubes and disks so that all of the exposed surfaces are sterilized. Next peel off the white backing from the adhesive disk, align the holes and affix the silicone rubber to the outside of the tube.Once the slices have been obtained place two microliters of chicken plasma on the center of the photo etched side of a prepared coverslip. Spread the plasma slightly to achieve a three to four milliliter diameter spot. Then use a sterile, narrow-tipped spatula to lift a prepared brain slice.Use closed forceps to keep the slice on the spatula tip while lifting the slice. Touch the spatula to the plasma spot on the coverslip and with closed forceps push the slice onto the coverslip. Add a thin layer of chick plasma on the coverslip before placing the slice and a minimal amount of thrombin treated plasma to cover to cover the slice.If the slice floats it will not remain adhered when the cloud is removed. Next mix 2.5 microliters of plasma and 2.5 microliters of thrombin in a separate tube. Quickly place 2.5 microliters of this mixture over and around the slice and pipette up and down gently to mix it.Remove the clear, plastic covering from the exposed side of the silicone rubber adhesive previously affixed to the roller tube. Then place the coverslip with the brain slice onto the adhesive aligning the slice within the hole. To ensure adhesion apply soft even pressure to the coverslip with the thumb, by pressing the coverslip down evenly, and holding it for a about one minute while transferring it to the biological safety cabinet.The pressure on the coverslip to adhere it to the tube without leaking must be just right and maintained for long enough to eliminate air channels in the salient. Too much pressure will crack the coverslip. In a biological safety cabinet, add 0.8 milliliters of complete neurobasal a culture medium to each tube.Then flow a a 5%carbon dioxide 95%air mixture through a sterile cotton plugged Pasteur pipette held securely by a clamp. Use this to flush the roller tube with the gas mixture and rapidly cap the tube as it is withdrawn from around the pipette. Next label the tubes with the slice number and rack number.Insert the tubes into a roller rack;ensuring that they are geometrically-balanced. If there is an odd number of tubes add blank tubes to balance. Place the racks in a 35 degree Celsius roller incubator, with rollers turning the roller rack at 10 to 13 revolutions per hour.Tilt the incubator back approximately 5 degrees by lifting its front on to a board. Transfer a tube with a slice culture to a custom-made tube holder like the one shown here. Then the place the tube holder into the stage adapter to keep the coverslip perpendicular to the objective during imaging.Next push the slider on the stage adapter tight against the tube to hold the tube in position. Using a four times objective under bright field trans-illumination, focus on the photo etched grid period under the slice. For the initial imaging session, quickly scan around the slice to locate and record the number for the various regions where higher magnification imaging is desired.Once the regions of interest have been identified, move the stage to the first grid square containing a region of interest. Then switch to the 20 strength error objective and locate a fiducial mark. Next switch to a higher power objective, localize the same fiducial mark, and record the x and y position of the stage.Move the stage to find the other fields of interest nearby and record their x and y offsets from the fiducial mark. The offset positions allow for consistent pinpoint of the same coverslip location when the slice is imaged, even though the original x, y setting of the fiducial mark changes when the tube or stage adapter are removed and replaced. Capture an image Z-stick within each selected field using the either the microscope objective control or a Piezo stage control if one is available.Using a neuronal vital dye, the confocal stack of images show here, highlights the 3D architecture of both neurites and the cell body, but the stain is excluded from the nucleus. To examine time-dependent changes in growth slice morphology and viability within slices during long-term culture, the same slice was labeled with fluorescent neuronal vital dye once per week, 24 hours before imaging. To demonstrate the reproducibility of imaging the identical cells over time, the same field of a slice labeled with neuronal vital dye on day 13, was imaged on days 14, 15, 16, and 17.The position of three cells on each day is shown by a symbol over the nucleus. In addition to the neuronal vital dye, viral-mediated fluorescent proteins can be utilized with this system to label cells. Here both a Calcium sensitive reporter and Cofilin-containing rods are able to be clearly identified 10 days post-infection.Successful implementation of this procedure requires preparation in advance of all tubes, coverslips, and solutions. Being able to obtain slices in a short post-mortem interval is also crucial to success and requires much practice. Once mastered this technique can be done in about 90 minutes to obtain 12-18 hippocampal slices from a mouse pup, including times for dissection and plating.Following this procedure, brain slices can be infected at various times with viruses for achieving cell-type specific expression of fluorescently tagged proteins or silence for gene expression with small interfering RNA or short hairpin RNA to probe gene function. Don't forget that working with recombinant viruses can be hazardous and precautions such as wearing eye protection should always be taken when working with these reagents.

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Research

JoVE Journal

Neuroscience

10.5K visualizações.  Colorado State University. The overall goal of this modified roller tube method for brain slice culture is to provide an enclosed culture system for long-term slice survival. with the capability for repetitive high-resolution florescence imaging. This method can help answer key questions in neuronal development and neurodegenerative disorders that require observations of the same neurons over time, such as localizing key proteins involved in synapse formation or loss.The main advantages of this technique are the...

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