Name: wholemount immunohistochemistry for revealing complex brain topography
Uploaded: 2012-04-05T13:00:00
Description: Watch this Scientific Journal Video about Wholemount Immunohistochemistry for Revealing Complex Brain Topography at JoVE.com

RVS is supported by new investigator start-up funds from Albert Einstein College of Medicine of Yeshiva University.

1. Animal Perfusion and Cerebellum Dissection
<ol>
 <li>Depending on the protein, perfusion may be essential for successful staining1,2. Transcardiac perfusion is an invasive, non-survival procedure that requires the proper use of anesthetics. Correct training, institutional approval, and IACUC approval are all necessary before attempting the procedure. It is always a good idea to consult the institution's veterinarians to get help in identifying experimental requirements and acquiring the correct training. ...

<ol>
	<li>Sillitoe, R. V., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+Immunohistochemistry:+A+high-throughput+screen+for+patterning+defects+in+the+mouse+cerebellum.">Whole-mount Immunohistochemistry: A high-throughput screen for patterning defects in the mouse cerebellum.</a> J. Histochem. Cytochem. 50, 235-244 (2002).</li><li>Kim, S. -. H., Che, P., Chung, S. -. H., Doorn, D., Hoy, M., Larouche, M., Marzban, H., Sarna, J., Zahedi, S., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Mount+Immunohistochemistry+of+the+Brain.">Whole-Mount Immunohistochemistry of the Brain.</a> Current Protocols in Neuroscience. , (2006).</li><li>Dent, J. A., Polson, A. G., Klymkowsky, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-mount+immunocytochemical+analysis+of+the+expression+of+the+intermediate+filament+protein+vimentin+in+Xenopus.">A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus.</a> Development. 105, 61-74 (1989).</li><li>Sillitoe, R. V., Malz, C. R., Rockland, K., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+compartmentation+of+the+primate+and+tree+shrew+cerebellum:+a+common+topography+of+zebrin+II+in+Macaca+mulatta+and+Tupaia+belangeri.">Antigenic compartmentation of the primate and tree shrew cerebellum: a common topography of zebrin II in Macaca mulatta and Tupaia belangeri.</a> J. Anat. 204, 257-269 (2004).</li><li>Ozol, K., Hayden, J. M., Oberdick, J., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+zones+in+the+vermis+of+the+mouse+cerebellum.">Transverse zones in the vermis of the mouse cerebellum.</a> J. Comp. Neurol. 412, 95-111 (1999).</li><li>Apps, R., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+cortical+organization:+a+one-map+hypothesis.">Cerebellar cortical organization: a one-map hypothesis.</a> Nat. Rev. Neurosci. 10, 670-681 (2009).</li><li>Reeber, S. L., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+expression+of+a+cocaine-+and+amphetamine-regulated+transcript+peptide+reveals+complex+circuit+topography+in+the+rodent+cerebellar+cortex.">Patterned expression of a cocaine- and amphetamine-regulated transcript peptide reveals complex circuit topography in the rodent cerebellar cortex.</a> J. Comp. Neurol. 519, 1781-1796 (2011).</li><li>Sarna, J. R., Marzban, H., Watanabe, M., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+stripes+of+phospholipase+Cbeta3+and+Cbeta4+expression+by+Purkinje+cell+subsets+in+the+mouse+cerebellum.">Complementary stripes of phospholipase Cbeta3 and Cbeta4 expression by Purkinje cell subsets in the mouse cerebellum.</a> J. Comp. Neurol. 496, 303-313 (2006).</li><li>Demilly, A., Reeber, S. L., Gebre, S. A., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofilament+heavy+chain+expression+reveals+a+unique+parasagittal+stripe+topography+in+the+mouse+cerebellum.">Neurofilament heavy chain expression reveals a unique parasagittal stripe topography in the mouse cerebellum.</a> Cerebellum. 10, 409-421 (2011).</li><li>Larouche, M., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+clusters+to+stripes:+the+developmental+origins+of+adult+cerebellar+compartmentation.">From clusters to stripes: the developmental origins of adult cerebellar compartmentation.</a> Cerebellum. 5, 77-88 (2006).</li><li>Marzban, H., Chung, S., Watanabe, M., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+Cbeta4+expression+reveals+the+continuity+of+cerebellar+topography+through+development.">Phospholipase Cbeta4 expression reveals the continuity of cerebellar topography through development.</a> J. Comp. Neurol. 502, 857-871 (2007).</li><li>Blank, M. C., Grinberg, I., Aryee, E., Laliberte, C., Chizhikov, V. V., Henkelman, R. M., Millen, 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=Multiple+developmental+programs+are+altered+by+loss+of+Zic1+and+Zic4+to+cause+Dandy-Walker+malformation+cerebellar+pathogenesis.">Multiple developmental programs are altered by loss of Zic1 and Zic4 to cause Dandy-Walker malformation cerebellar pathogenesis.</a> Development. 138, 1207-1216 (2011).</li><li>Sawada, K., Sakata-Haga, H., Fukui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternating+array+of+tyrosine+hydroxylase+and+heat+shock+protein+25+immunopositive+Purkinje+cell+stripes+in+zebrin+II-defined+transverse+zone+of+the+cerebellum+of+rolling+mouse.">Alternating array of tyrosine hydroxylase and heat shock protein 25 immunopositive Purkinje cell stripes in zebrin II-defined transverse zone of the cerebellum of rolling mouse.</a> Nagoya. Brain Res. 1343, 46-53 (2010).</li><li>Sawada, K., Fukui, Y., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+distribution+of+corticotropin-releasing+factor+immunopositive+climbing+fibers+in+the+mouse+cerebellum:+Analysis+by+whole+mount+immunohistochemistry.">Spatial distribution of corticotropin-releasing factor immunopositive climbing fibers in the mouse cerebellum: Analysis by whole mount immunohistochemistry.</a> Brain Res. 1222, 106-117 (2008).</li><li>Marzban, H., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+architecture+of+the+posterior+zone+of+the+cerebellum.">On the architecture of the posterior zone of the cerebellum.</a> Cerebellum. 10, 422-434 (2011).</li><li>Pakan, J. M., Graham, D. J., Wylie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+visual+mossy+fiber+projections+and+zebrin+expression+in+the+pigeon+vestibulocerebellum.">Organization of visual mossy fiber projections and zebrin expression in the pigeon vestibulocerebellum.</a> J. Comp. Neurol. 518, 175-198 (2010).</li><li>Iwaniuk, A. N., Marzban, H., Pakan, J. M., Watanabe, M., Hawkes, R., Wylie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentation+of+the+cerebellar+cortex+of+hummingbirds+(Aves:+Trochilidae)+revealed+by+the+expression+of+zebrin+II+and+phospholipase+C+beta+4.">Compartmentation of the cerebellar cortex of hummingbirds (Aves: Trochilidae) revealed by the expression of zebrin II and phospholipase C beta 4.</a> J. Chem. Neuroanat. 37, 55-63 (2009).</li><li>Sarna, J. R., Larouche, M., Marzban, H., Sillitoe, R. V., Rancourt, D. E., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+Purkinje+cell+degeneration+in+mouse+models+of+Niemann-Pick+type+C+disease.">Patterned Purkinje cell degeneration in mouse models of Niemann-Pick type C disease.</a> J. Comp. Neurol. 456, 279-291 (2003).</li><li>Sarna, J. R., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+Purkinje+cell+loss+in+the+ataxic+sticky+mouse.">Patterned Purkinje cell loss in the ataxic sticky mouse.</a> Eur. J. Neurosci. 34, 79-86 (2011).</li><li>El-Bizri, N., Guignabert, C., Wang, L., Cheng, A., Stankunas, K., Chang, C. P., Mishina, Y., Rabinovitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SM22alpha-targeted+deletion+of+bone+morphogenetic+protein+receptor+1A+in+mice+impairs+cardiac+and+vascular+development,+and+influences+organogenesis.">SM22alpha-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis.</a> Development. 135, 2981-2991 (2008).</li><li>Mondrinos, M. J., Koutzaki, S., Lelkes, P. I., Finck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-engineered+model+of+fetal+distal+lung+tissue.">A tissue-engineered model of fetal distal lung tissue.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 293, 639-650 (2007).</li><li>Coppola, E., Rallu, M., Richard, J., Dufour, S., Riethmacher, D., Guillemot, F., Goridis, C., Brunet, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epibranchial+ganglia+orchestrate+the+development+of+the+cranial+neurogenic+crest.">Epibranchial ganglia orchestrate the development of the cranial neurogenic crest.</a> Proc. Nat. Acad. Sci. 107, 2066-2071 (2010).</li><li>Kubilus, J. K., Linsenmayer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+guidance+of+embryonic+corneal+innervation:+roles+of+Semaphorin3A+and+Slit2.">Developmental guidance of embryonic corneal innervation: roles of Semaphorin3A and Slit2.</a> Dev. Biol. 344, 172-184 (2010).</li><li>Reeber, S. L., Gebre, S. A., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+mapping+of+afferent+topography+in+three+dimensions.">Fluorescence mapping of afferent topography in three dimensions.</a> Brain Struct. Funct. 216, 159-169 (2011).</li><li>Davis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemistry.">Whole-mount immunohistochemistry.</a> Methods Enzymol. 225, 502-516 (1993).</li></ol>

We have described the technical details required for successful wholemount staining using a versatile immunohistochemical approach for revealing protein expression in the developing and adult brain. By using this approach, complex molecular expression patterns can be analyzed and brain topography appreciated without the need for laborious and time consuming tissue sectioning procedures.
This protocol has been used to reveal the patterned expression of several Purkinje cell proteins in both the...

<table border="1">
<tbody>
 <tr>
 <td>Materials </td>
 <td>Function in protocol</td>
 </tr>
 <tr>
 <td>Perfusion pump (Fisher Scientific/13-876-2)</td>
 <td>Allows for consistent and slow perfusion. </td>
 </tr>
 <tr>
 <td>Sharp-tip Scissors (FST/14081-08)</td>
 <td>General use in perfusion and dissection.</td>
 </tr>
 <tr>
 <td>Blunt-tip Forceps (FST/91100-12)</td>
 <td>To stabilize the heart for insertion of the perfusion needle.</td>
 </tr>
 <tr>
 <td>Forceps (FST by Dumont AA/11210-10)</td>
 <td>For use during dissection of the brain from the skull and to separate the cerebellum from the rest of the brain. These are essential because they have a slightly rounded tip that helps minimize damage to the cerebellum during dissection. </td>
 </tr>
 <tr>
 <td>Nutator (Fisher Scientific)</td>
 <td>Used to keep tissue in motion during incubation periods.&nbsp; </td>
 </tr>
 <tr>
 <td>1.5 mL tube (Sarstedt/Screw Cap Micro Tube)</td>
 <td>All steps of the histochemistry protocol take place in these microtubes. The rounded bottom ensures that the cerebellum stays in motion.&nbsp; </td>
 </tr>
 <tr>
 <td>Perforated spoon (FST/10370-17)</td>
 <td>Used to keep wholemounts in the microtubes while gently decanting out the spent solution. </td>
 </tr>
 <tr>
 <td>Leica MZ16 FA microscope</td>
 <td>Used to examine wholemount staining.</td>
 </tr>
 <tr>
 <td>Leica DFC3000 FX camera</td>
 <td>Used to capture wholemount images. </td>
 </tr>
 </tbody>
</table>
Table 1.
<table border="1">
<tbody>
 <tr>
 <td colspan="9">Example calendar for a typical wholemount experiment</td>
 </tr>
 <tr>
 <td>Day 1</td>
 <td colspan="4">Dent's fix, room temperature, 8 hrs</td>
 <td colspan="4">Dent's bleach, 4&deg;C, overnight</td>
 </tr>
 <tr>
 <td>Day 2</td>
 <td colspan="3">100% MeOH, room temperature, 2x, 30 min each</td>
 <td colspan="3">100% MeOH, Freeze/thaw, 
 4x, 30 min/15 min</td>
 <td colspan="2">100% MeOH, -80&deg;C, overnight</td>
 </tr>
 <tr>
 <td>Day 3</td>
 <td>50% MeOH/50% PBS, room temperature, 60-90 min</td>
 <td colspan="2">15% MeOH/ 85% PBS, room temperature, 60-90 min</td>
 <td colspan="2">100% PBS, room temperature, 60-90 min</td>
 <td>10&mu;g/mL Proteinase K in PBS, room temperature, 2-3 min</td>
 <td>100% PBS, room temperature, 3x, 10 min each</td>
 <td>PMT, 4&deg;C, overnight</td>
 </tr>
 <tr>
 <td>Day 4-5</td>
 <td colspan="8">PMT + 1&deg; antibody + 5% DMSO, 4&deg;C, 48 hrs </td>
 </tr>
 <tr>
 <td>Day 6</td>
 <td colspan="3">PMT, 4&deg;C, 2-3x, 2-3 hrs each</td>
 <td colspan="5">PMT + 2&deg; antibody + 5% DMSO, 4&deg;C, 24 hours (Or begin amplification steps with ABC complex)</td>
 </tr>
 <tr>
 <td>Day 7</td>
 <td colspan="2">PMT, 4&deg;C, 2-3x, 2-3 hrs each</td>
 <td colspan="3">PBT, room temperature, 2 hrs</td>
 <td colspan="3">Incubate in fresh DAB in PBS until optimal staining is visualized</td>
 </tr>
</tbody>
</table>
Table 2.
<table border="1">
<tbody>
 <tr>
 <td colspan="2">Recipes (*=prepare fresh every time)</td>
 </tr>
 <tr>
 <td>PBS (phosphate buffered saline)</td>
 <td>0.1M phosphate buffered saline in deionized water. pH 7.2 (Sigma tablets; P4417)</td>
 </tr>
 <tr>
 <td>PFA (Paraformaldehyde)</td>
 <td>Made and stored frozen as a 20% solution and then diluted to 4% in PBS for the working solution (Fisher Scientific; T353)</td>
 </tr>
 <tr>
 <td>Dent's Fixative3*</td>
 <td>4 parts methanol 
 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4)</td>
 </tr>
 <tr>
 <td>Dent's Bleach3*</td>
 <td>4 parts methanol 
 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4) 
 1 part 30% hydrogen peroxide </td>
 </tr>
 <tr>
 <td>Enzymatic Digestion </td>
 <td>10 &mu;g/ml of Proteinase K (Roche Diagnostics; 03115828001) in PBS. </td>
 </tr>
 <tr>
 <td>PBST</td>
 <td>PBS containing: 
 0.1% Tween-20 (Fisher Scientific, BP337; Triton can also be used in place of Tween-20 in all instances.)</td>
 </tr>
 <tr>
 <td>PMT25*</td>
 <td>PBS containing: 
 2% nonfat skim milk powder (Carnation preferred) 
 0.1% Tween-20 (Fisher Scientific; BP337)</td>
 </tr>
 <tr>
 <td>PBT25*</td>
 <td>PBS containing: 
 0.2% bovine serum albumin (Sigma; B9001S) 
 0.1% Tween-20 (Fisher Scientific; BP337)</td>
 </tr>
 <tr>
 <td>DAB*</td>
 <td>Dissolve one 10-mg tablet of 3,3-diaminobenzidine (Sigma-Aldrich; D5905) in 40 ml of PBS. Add 10 &mu;l of 30% hydrogen peroxide to initiate reaction). </td>
 </tr>
 <tr>
 <td>ABC Complex Solution</td>
 <td>Vectastain kit (Vector laboratories, Inc; PK-4000)</td>
 </tr>
 </tbody>
</table>
Table 3.

wholemount immunohistochemistry for revealing complex brain topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Watch this Scientific Journal Video about Wholemount Immunohistochemistry for Revealing Complex Brain Topography at JoVE.com

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

wholemount-immunohistochemistry-for-revealing-complex-brain-topography

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Neuroscience

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...