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<li>Kügler, S., Kilic, E., Bähr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">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>
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<li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines%2C+zebrafish+and+mice%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PLOS ONE. 6 (4), 18556(2011).</li>
<li>Benskey, M. J., Manfredsson, F. P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/26611582">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/pubmed?term=((Autoprocessing+of+human+immunodeficiency+virus+type+1+protease+miniprecursor+fusions+in+mammalian+cells%5BTitle%5D)+AND+%22AIDS+Res+Ther%22%5BJournal%5D)">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/pubmed?term=((Gibson+assembly%3A+an+easy+way+to+clone+polyviral+full-length+infectious+cDNA+clones+expressing+an+ectopic+VPg%5BTitle%5D)+AND+%22Virol+J%22%5BJournal%5D)">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/pubmed?term=((NeuO%3A+a+fluorescent+chemical+probe+for+live+neuron+labeling%5BTitle%5D)+AND+%22Angew+Chem+Int+Ed+Engl%22%5BJournal%5D)">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/pubmed?term=((Formation+of+actin-ADF%2Fcofilin+rods+transiently+retards+decline+of+mitochondrial+potential+and+ATP+in+stressed+neurons%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">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/pubmed?term=((Cofilin+aggregation+blocks+intracellular+trafficking+and+induces+synaptic+loss+in+hippocampal+neurons%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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. Jr, Dahlgren, K. N., Krafft, G. A., LaDu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+characterization+of+conditions+for+amyloid-beta+peptide+oligomerization+and+fibrillogenesis%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.</a> J Biol Chem. 278 (13), 11612-11622 (2003).</li>
<li>Maloney, M. T., Minamide, L. S., Kinley, A. W., Boyle, J. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta-secretase-cleaved+amyloid+precursor+protein+accumulates+at+actin+inclusions+induced+in+neurons+by+stress+or+amyloid+beta%3A+a+feedforward+mechanisms+for+Alzheimer%27s+disease%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Beta-secretase-cleaved amyloid precursor protein accumulates at actin inclusions induced in neurons by stress or amyloid beta: a feedforward mechanisms for Alzheimer's disease.</a> J Neurosci. 25 (49), 11313-11321 (2005).</li>
<li>Davis, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+beta+dimers%2Ftrimers+potently+induce+cofilin-actin+rods+that+are+inhibited+by+maintaining+cofilin+phosphorylation%5BTitle%5D)+AND+%22Mol+Neurodegener%22%5BJournal%5D)">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/pubmed?term=((Amyloid-%CE%B2+and+proinflammatory+cytokines+utilize+a+prion+protein-dependent+pathway+to+activate+NADPH+oxidase+and+induce+cofilin-actin+rods+in+hippocampal+neurons%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Amyloid-β 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>
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</ol>

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).

<table><tbody><tr><td>Bottoms from 15 cm culture dishes</td><td>VWR Scientific</td><td>25384-326</td><td></td></tr><tr><td>Phillips Head Machine Screws (#10-32)</td><td>Ace Hardware</td><td></td><td>2.5&quot; long and 3/16&quot; in diameter</td></tr><tr><td>Flat Washers #10</td><td>ACE Hardware</td><td></td><td></td></tr><tr><td>Machine Screw Nuts (#10-32)</td><td>ACE Hardware</td><td></td><td></td></tr><tr><td>Rubber Grommets&amp;nbsp;</td><td>ACE Hardware</td><td></td><td>5/16&quot;, thick; 5/8&quot;, hole diameter; 1.125&quot;, OD</td></tr><tr><td>Polyethylene tubing (5/16&quot;; OD, 3/16&quot;; ID)</td><td>ACE Hardware</td><td></td><td>Cut to 1.8&quot; length</td></tr><tr><td>Lock Washer #10</td><td>ACE Hardware</td><td></td><td></td></tr><tr><td>Drill Press, 5 speed&amp;nbsp;</td><td>Ace Hardware</td><td>ProTech Model 1201</td><td></td></tr><tr><td>Nunclon Delta Flat-Sided Tubes</td><td>VWR</td><td>62407-076</td><td></td></tr><tr><td>Drill bits, 3 mm, 6 mm and 15 mm&amp;nbsp;</td><td>Ace Hardware</td><td>Diablo freud brand</td><td>Drill bits for cutting plastic.</td></tr><tr><td>Drill bits for wood, 1.5 cm and 1 mm</td><td>Ace Hardware</td><td></td><td></td></tr><tr><td>Wood file, 1/4&quot; round</td><td>Ace Harware</td><td></td><td></td></tr><tr><td>Spring clips, 16 mm snap holder</td><td>Ace Hardware</td><td></td><td></td></tr><tr><td>Swivel Head Deburring Tool, 5&quot;</td><td>Ace Hardware</td><td>26307</td><td></td></tr><tr><td>Adhesive Silicone Sheet (Secure Seal)</td><td>Grace Bio-Labs</td><td>666581</td><td>0.5 mm Thickness</td></tr><tr><td>6 mm hole punch</td><td>Office Max</td><td></td><td></td></tr><tr><td>12 mm hole punch</td><td>thepunchbunch.com</td><td></td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Phototeched Coverslips, 12 mm diameter</td><td>Bellco Glass, Inc.</td><td>1916-91012</td><td></td></tr><tr><td>Bunsen Burner</td><td></td><td></td><td></td></tr><tr><td>Absolute Ethanol</td><td></td><td></td><td></td></tr><tr><td>Nanopure Water</td><td></td><td></td><td></td></tr><tr><td>3-aminopropyltriethoxylane</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>#5 Dumont Forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>Ted Pella, Inc.</td><td>10180</td><td></td></tr><tr><td>Double Edge Razor Blades</td><td>Ted Pella, Inc.</td><td>121-6</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>VWR</td><td>28450-182</td><td>Cut into 5.8 cm diameter circles</td></tr><tr><td>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><td>#21 Surgical Blade</td><td>VWR Scientific</td><td>25860-144</td><td></td></tr><tr><td>#5 Dumont Forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Spatula, stainless with tapered end</td><td>VWR</td><td>82027-518</td><td></td></tr><tr><td>Gey&#039;s Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>G9779&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;Glucose</td><td>ThermoFisher Scientific</td><td>15023-021</td><td>25% (w/v) Solution, 0.2 mm filter sterilized</td></tr><tr><td>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 &amp;deg;C, quick freeze aliquots in liquid nitrogen and store at&amp;nbsp; -80 &amp;deg;C.</td></tr><tr><td>Thrombin, Topical (Bovine)</td><td>Pfizer</td><td>Thrombin-JMI</td><td>Quick freeze aliquots in liquid nitrogen at 1,000 international units/mL in diluent provided and store at -80&amp;deg;C. Use at 250 units/mL.</td></tr><tr><td>Cell Roller System</td><td>Bellco Biotech</td><td>SciERA</td><td></td></tr><tr><td>Roller Incubator</td><td>Forma</td><td>Model 3956</td><td></td></tr><tr><td>N21-MAX</td><td>ThermoFisher Scientific</td><td>AR008</td><td></td></tr><tr><td>Pen/Strep (100X)</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>200 mM Glutamine</td><td>ThermoFisher Scientific</td><td>25030081</td><td></td></tr><tr><td>Glucose</td><td>ThermoFisher Scientific</td><td>15023-021</td><td>25% (w/v) Solution, 0.2 mm filter sterilized</td></tr><tr><td>Neurobasal A</td><td>ThermoFisher Scientific</td><td>10888-022&amp;nbsp;</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><td>Third generation lentivirus packaging</td><td>Life Technologies</td><td>K4975-00</td><td></td></tr><tr><td>159 K cutoff centrifugal filters (Centricon)</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Lentiviral cloning system (InFusion)</td><td>Clonetech</td><td></td><td></td></tr><tr><td>Plasmids 30323, 50856, 51279</td><td>Addgene</td><td></td><td></td></tr><tr><td>Neuronal cell viability dye (NeuO)</td><td>Stemcell technologies</td><td>1801</td><td>Thaw once and quick freeze in 4 &amp;micro;L aliquots. Store at -20 &amp;deg;C</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>Microscope objectives</td><td>Olympus</td><td>air: 4X, 20; oil: 40X, 60X,</td><td></td></tr><tr><td>Spinning disc confocal system</td><td>Yokagawa</td><td>CSU22</td><td></td></tr><tr><td>Microscope EMCCD camera</td><td>Photometrics</td><td>Cascade II</td><td></td></tr><tr><td>Linear encoded (x,y), piezo z flat top stage</td><td>ASI</td><td></td><td></td></tr><tr><td>Microscope lasers and integration</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>HEK293T cells</td><td>American Type Culture Collection</td><td>CRL-3216</td><td></td></tr><tr><td>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 &amp;deg;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...

Watch this Scientific Journal Video about Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System at JoVE

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 ...

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Research

JoVE Journal

Neuroscience

10.6K Views.  Colorado State University. Hier is een gemodificeerde roller buis methode voor het kweken en intermitterende high-resolution beeldvorming van knaagdier hersenen plakjes vele weken met precieze herpositionering op photoetched coverslips. Neuronale levensvatbaarheid en morfologie van het segment zijn goed onderhouden. Toepassingen van deze volledig gesloten systeem met virussen voor celtype specifieke expressie worden geleverd.

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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

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 ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

A Modified Roller Tube Method for the Long-Term Culture of Rodent Brain Slices

Source: Fixman, B. B., et al. <a href="https://app.jove.com/v/56436">Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System.</a> J. Vis. Exp. (2017).
This video demonstrates a modified roller tube method for the long-term culturing of brain slices from a rodent model. First, the brain slices are attached to coverslips. Then, flat-sided roller tubes with a hole on the flat side are taken, and the coverslips are positioned on the hole using adhesive discs. Finally, a nutrient medium and a carbon dioxide and air mixture are added to the tube, and the tubes are incubated with a rolling motion.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: The protocol below describes ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

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

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