This work was supported by new investigator start-up funds from Albert Einstein College of Medicine of Yeshiva University to RVS.

1. Delivering tracers in vivo using sterile surgery (Sections 1.1-1.16)
Throughout the procedure we advise that standard sterile surgical techniques be used. This includes using sterile gloves, gown, and facemask. Tools should either be autoclaved before use or cleaned thoroughly with water and then ethanol. During the surgery, and especially between animals, clean tissue fragments off the tools and dry sterilize instruments with a hot bead sterilizer (Steri 250 Cat. # 18000-45, Fine S...

<ol>
	<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>Grishkat, H. L., Eisenman, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+spinocerebellar+projection+in+the+prenatal+mouse.">Development of the spinocerebellar projection in the prenatal mouse.</a> J. Comp. Neurol. 363, 93-108 (1995).</li><li>Mesulam, 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> Tracing neural connections with horseradish peroxidase. , (1982).</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+(CART)+peptide+reveals+complex+circuit+topography+in+the+rodent+cerebellar+cortex.">Patterned expression of a cocaine- and amphetamine regulated transcript (CART) peptide reveals complex circuit topography in the rodent cerebellar cortex.</a> Journal of Comparative Neurology. , (2011).</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. , (2011).</li><li>Sillitoe, R. V., Stephen, D., Lao, Z., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed+homeobox+genes+determine+the+organization+of+Purkinje+cell+sagittal+stripe+gene+expression+in+the+adult+cerebellum.">Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum.</a> J. Neurosci. 28, 12150-12162 (2008).</li><li>Sillitoe, R. V., Vogel, M. W., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed+homeobox+genes+regulate+establishment+of+the+cerebellar+afferent+circuit+map.">Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map.</a> J. Neurosci. 30, 10015-10024 (2010).</li><li>Vig, J., Goldowitz, D., Steindler, D. A., Eisenman, L. M. <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+reeler+cerebellum:+segregation+and+overlap+of+spinocerebellar+and+secondary+vestibulocerebellar+fibers+and+their+target+cells.">Compartmentation of the reeler cerebellum: segregation and overlap of spinocerebellar and secondary vestibulocerebellar fibers and their target cells.</a> Neuroscience. 130, 735-744 (2005).</li><li>Vogel, M. W., Prittie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+spinocerebellar+mossy+fiber+projections+are+maintained+in+the+lurcher+mutant.">Topographic spinocerebellar mossy fiber projections are maintained in the lurcher mutant.</a> J. Comp. Neurol. 343, 341-351 (1994).</li><li>Voogd, J., Broere, G., van Rossum, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medio-lateral+distribution+of+the+spinocerebellar+projection+in+the+anterior+lobe+and+the+simple+lobule+in+the+cat+and+a+comparison+with+some+other+afferent+fibre+systems.">The medio-lateral distribution of the spinocerebellar projection in the anterior lobe and the simple lobule in the cat and a comparison with some other afferent fibre systems.</a> Psychiatr. Neurol. Neurochir. 72, 137-151 (1969).</li><li>Wu, H. S., Sugihara, I., Shinoda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+patterns+of+single+mossy+fibers+originating+from+the+lateral+reticular+nucleus+in+the+rat+cerebellar+cortex+and+nuclei.">Projection patterns of single mossy fibers originating from the lateral reticular nucleus in the rat cerebellar cortex and nuclei.</a> J. Comp. Neurol. 411, 97-118 (1999).</li></ol>

No conflicts of interest declared.

We have described the surgical and technical details required for successful axonal and dendrite tracing using a novel fluorescent-based approach for rapidly labeling neural projections in developing and adult mice. Using WGA-Alexa we show how tracers and markers can be used for analyzing patterned circuit topography at high resolution and in three dimensions.
Many tracers are available for tracing neuronal circuits including Cholera toxin subunit B, Biocytin, Neurobiotin, Phaseolus vulgaris ...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment/Reagents</td>
			<td>Model/Catalogue number</td>
			<td>Company</td>
		</tr>
		<tr>
			<td>Bead sterilizer</td>
			<td>Model Steri 250 Cat. # 18000-45</td>
			<td>Fine Science Tools</td>
		</tr>
		<tr>
			<td>Cauterizer</td>
			<td>Cat. # 18000-00</td>
			<td>Fine Science Tools</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Cat. # 300056</td>
			<td>Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Dual stage Glass Micropipette Puller</td>
			<td>Model 001-PC-10</td>
			<td>Narishige</td>
		</tr>
		<tr>
			<td>Micrometer syringe</td>
			<td>Cat. # GS-1100</td>
			<td>Gilmont Instruments</td>
		</tr>
		<tr>
			<td>Small Animal Stereotaxic Instrument</td>
			<td>Model 940-A</td>
			<td>Kopf Instrumentation</td>
		</tr>
		<tr>
			<td>Electrode Manipulator</td>
			<td>Model 960</td>
			<td>Kopf Instrumentation</td>
		</tr>
		<tr>
			<td>Vetcare chamber</td>
			<td>Cat. # 340508</td>
			<td>Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Cat. # 341241</td>
			<td>Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Leica DFC360 FX camera</td>
			<td>DFC360 FX</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>Leica DFC490 camera</td>
			<td>DFC490</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>Leica DM5500 microscope</td>
			<td>DM5500</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>Leica DFC3000 FX camera</td>
			<td>DFC3000 FX</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>Leica MZ16 FA microscope</td>
			<td>MZ16 FA</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>CY3 Filter</td>
			<td>Model # 11600231</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>FITC Filter</td>
			<td>Model # 11513880</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>A4 DAPI/UV filter</td>
			<td>Model # 11504162</td>
			<td>Leica</td>
		</tr>
		<tr>
			<td>Wheat germ agglutinin, Alexa Fluor 488 conjugate</td>
			<td>Cat. #W11261</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>Wheat germ agglutinin, Alexa Fluor 555 conjugate</td>
			<td>Cat. #W32464</td>
			<td>Invitrogen</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Revealing Neural Circuit Topography in Multi-Color at JoVE.com

Revealing Neural Circuit Topography in Multi-Color

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 ...

revealing-neural-circuit-topography-in-multi-color

Research

JoVE Journal

Neuroscience

14.9K visualizações.  Yeshiva University. Nós fornecemos um guia prático para entregar traçadores In vivo E usar a via espinocerebelar como um sistema modelo para demonstrar passos essenciais para a análise do circuito neuronal sucesso em camundongos. Descrevemos em detalhe o nosso protocolo versátil rastreamento que explora aglutinina de gérmen de trigo (WGA), conjugada com fluoróforos Alexa.