Name: single-cell profiling of developing and mature retinal neurons
Uploaded: 2012-04-19T12:00:00
Description: Watch this Scientific Journal Video about Single-cell Profiling of Developing and Mature Retinal Neurons at JoVE.com

1. La disociación de la célula <ol><li> Un diagrama de flujo delineando el protocolo que se muestra es la Figura 1. Para los números de catálogo de los reactivos particulares utilizados en este protocolo, por favor refiérase a la Tabla 1. Disección de la retina en un baño de PBS. Durante la disección, lo mejor es eliminar el vítreo y el cristalino ya que mantener con la retina puede impedir la disociación. No siempre es críticamente importante para eliminar todo el epitelio...

<ol>
	<li>Tietjen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptional+analysis+of+neuronal+progenitors.">Single-cell transcriptional analysis of neuronal progenitors.</a> Neuron. 38, 161-175 (2003).</li><li>Trimarchi, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+heterogeneity+of+developing+retinal+ganglion+and+amacrine+cells+revealed+through+single+cell+gene+expression+profiling.">Molecular heterogeneity of developing retinal ganglion and amacrine cells revealed through single cell gene expression profiling.</a> J. Comp. Neurol. 502, 1047-1065 (2007).</li><li>Trimarchi, J. M., Cho, S. H., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+expressed+preferentially+in+the+developing+peripheral+margin+of+the+optic+cup.">Identification of genes expressed preferentially in the developing peripheral margin of the optic cup.</a> Dev. Dyn. 238, 2327-2327 (2009).</li><li>Cherry, T. J., Trimarchi, J. M., Stadler, M. B., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+diversification+of+retinal+amacrine+interneurons+at+single+cell+resolution.">Development and diversification of retinal amacrine interneurons at single cell resolution.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 9495-9500 (2009).</li><li>Roesch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+of+retinal+Muller+glial+cells.">The transcriptome of retinal Muller glial cells.</a> J. Comp. Neurol. 509, 225-238 (2008).</li><li>Ramos, 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=Evidence+for+diversity+in+transcriptional+profiles+of+single+hematopoietic+stem+cells.">Evidence for diversity in transcriptional profiles of single hematopoietic stem cells.</a> PLoS Genet. 2, e159 (2006).</li><li>Chiang, M. K., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcript+analysis+of+pancreas+development.">Single-cell transcript analysis of pancreas development.</a> Dev. Cell. 4, 383-393 (2003).</li><li>Trimarchi, J. M., Stadler, M. B., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+retinal+progenitor+cells+display+extensive+heterogeneity+of+gene+expression.">Individual retinal progenitor cells display extensive heterogeneity of gene expression.</a> PLoS ONE. 3, e1588 (2008).</li><li>Nelson, S. B., Sugino, K., Hempel, 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=The+problem+of+neuronal+cell+types:+a+physiological+genomics+approach.">The problem of neuronal cell types: a physiological genomics approach.</a> Trends Neurosci. 29, 339-345 (2006).</li><li>Stevens, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+diversity:+too+many+cell+types+for+comfort.">Neuronal diversity: too many cell types for comfort.</a> Curr. Biol. 8, R708-R710 (1998).</li><li>Cepko, C. L., Austin, C. P., Yang, X., Alexiades, M., Ezzeddine, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+determination+in+the+vertebrate+retina.">Cell fate determination in the vertebrate retina.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 589-595 (1996).</li><li>Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+molecular+markers+of+bipolar+cells+in+the+murine+retina.">Identification of molecular markers of bipolar cells in the murine retina.</a> J. Comp. Neurol. 507, 1795-1810 (2008).</li><li>Chang, H. H., Hemberg, M., Barahona, M., Ingber, D. E., Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+noise+controls+lineage+choice+in+mammalian+progenitor+cells.">Transcriptome-wide noise controls lineage choice in mammalian progenitor cells.</a> Nature. 453, 544-547 (2008).</li><li>Levsky, J. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+and+the+myth+of+the+average+cell.">Gene expression and the myth of the average cell.</a> Trends Cell Biol. 13, 4-6 (2003).</li><li>Livesey, F. J., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+neural+cell-fate+determination:+lessons+from+the+retina.">Vertebrate neural cell-fate determination: lessons from the retina.</a> Nat. Rev. Neurosci. 2, 109-118 (2001).</li><li>Masland, R. H., Raviola, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confronting+complexity:+strategies+for+understanding+the+microcircuitry+of+the+retina.">Confronting complexity: strategies for understanding the microcircuitry of the retina.</a> Annu. Rev. Neurosci. 23, 249-284 (2000).</li><li>Blackshaw, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+mouse+retinal+development.">Genomic analysis of mouse retinal development.</a> PLoS Biol. 2, e247 (2004).</li><li>Livesey, F. J., Young, T. L., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+gene+expression+program+of+mammalian+neural+progenitor+cells.">An analysis of the gene expression program of mammalian neural progenitor cells.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 1374-1379 (2004).</li><li>Chowers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+genes+preferentially+expressed+in+the+retina+using+a+custom+human+retina+cDNA+microarray.">Identification of novel genes preferentially expressed in the retina using a custom human retina cDNA microarray.</a> Invest. Ophthalmol. Vis. Sci. 44, 3732-3741 (2003).</li><li>Clarke, G. C., Leavitt, R. A., Andrews, B. R., Hayden, D. F., Lumsden, M. R., J, C., McInnes, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one-hit+model+of+cell+death+in+inherited+neuronal+degenerations.">A one-hit model of cell death in inherited neuronal degenerations.</a> Nature. 406, 195-199 (2000).</li><li>McEvoy, J. F. -. O., Zhang, J., Nemeth, J., Brennan, K., Bradley, R., Krafcik, C., Rodriguez-Galindo, F., Wilson, C., Xiong, M., Lozano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexpression+of+normally+incompatible+developmental+pathways+in+retinoblastoma+genesis.">Coexpression of normally incompatible developmental pathways in retinoblastoma genesis.</a> Cancer Cell. 20, 260-275 (2011).</li><li>Gelder, R. N. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. U.S.A. 87, 1663-1667 (1990).</li><li>Ginsberg, S. D., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profile+analysis+within+the+human+hippocampus:+comparison+of+CA1+and+CA3+pyramidal+neurons.">Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons.</a> J. Comp. Neurol. 487, 107-118 (2005).</li><li>Iscove, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+is+faithfully+preserved+in+global+cDNA+amplified+exponentially+from+sub-picogram+quantities+of+mRNA.">Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA.</a> Nat. Biotechnol. 20, 940-943 (2002).</li><li>Subkhankulova, T., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+linear+and+exponential+amplification+techniques+for+expression+profiling+at+the+single-cell+level.">Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.</a> Genome Biol. 7, R18 (2006).</li></ol>

Un número cada vez mayor de los estudios están revelando robusta célula a célula de la variabilidad en las poblaciones que se creían ser más homogéneos en cuanto a su expresión de genes 6,8. En al menos un caso, esta expresión de los genes &quot;ruido&quot; se ha demostrado que desempeña una importante función biológica 13. Las diferencias de expresión génica entre las células individuales se oculta utilizando tejidos tradicionales de todo el método. Estos experimentos generar el pe...

Reaction mixtures:
Cell Lysis buffer
<table border="0">
	<tbody>
		<tr>
			<td>0.45 &mu;</td>
			<td>10X reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2)</td>
		</tr>
		<tr>
			<td>0.23 &mu;l</td>
			<td>10% NP-40</td>
		</tr>
		<tr>
			<td>0.23 &mu;l</td>
			<td>0.1M DTT</td>
		</tr>
		<tr>
			<td>0.05 &mu;l</td>
			<td>RNase Inhibitor (40 U/&mu;l)</td>
		</tr>
		<tr>
			<td>0.05 &mu;l</td>
			<td>SUPERase-In (20U/&mu;l)</td>
		</tr>
		<tr>
			<td>0.13 &mu;l</td>
			<td>Modified Oligo d(T) primer 
			(TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT)</td>
		</tr>
		<tr>
			<td>0.09 &mu;l</td>
			<td>dNTPs (2.5 mM each)</td>
		</tr>
		<tr>
			<td>3.27 &mu;l</td>
			<td>dH20 (use molecular biology grade for all reaction mixtures)</td>
		</tr>
	</tbody>
</table>
Tailing Reaction Mixture
<table border="0">
	<tbody>
		<tr>
			<td>0.18 &mu;l</td>
			<td>100 mM dATP</td>
		</tr>
		<tr>
			<td>0.6 &mu;l</td>
			<td>10X reaction buffer(100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2)</td>
		</tr>
		<tr>
			<td>4.62 &mu;l</td>
			<td>dH2O</td>
		</tr>
		<tr>
			<td>0.3 &mu;l</td>
			<td>TdT (400 U/&mu;l)</td>
		</tr>
		<tr>
			<td>0.3 &mu;l</td>
			<td>RNase H (2 U/&mu;l)</td>
		</tr>
	</tbody>
</table>
PCR Reaction Mixture
<table border="0">
	<tbody>
		<tr>
			<td>10 &mu;l</td>
			<td>10x Ex-Taq HS Buffer with Mg2+</td>
		</tr>
		<tr>
			<td>10 &mu;l</td>
			<td>2.5 mM dNTPs</td>
		</tr>
		<tr>
			<td>0.2 &mu;l</td>
			<td>Modified Oligo d(T) primer (10 &mu;g/&mu;l)</td>
		</tr>
		<tr>
			<td>1 &mu;l</td>
			<td>Ex-Taq HS Polymerase (5U/&mu;l)</td>
		</tr>
		<tr>
			<td>68.8 &mu;l</td>
			<td>dH2O</td>
		</tr>
	</tbody>
</table>
10X One-Phor All Buffer
<table border="0">
	<tbody>
		<tr>
			<td>0.5M</td>
			<td>Potassium acetate</td>
		</tr>
		<tr>
			<td>0.1M</td>
			<td>Tris acetate (pH 7.6)</td>
		</tr>
		<tr>
			<td>0.1M</td>
			<td>Magnesium acetate</td>
		</tr>
	</tbody>
</table>
Solutions:
10X Phosphate buffered saline (1 liter)
80g NaCl 
2g KCl 
14.4 g Na2PO4 
2.4 g KH2PO4 
adjust to pH 7.4 with NaOH and bring the volume to 1 liter with water
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Vertical needle puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 750</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcapillary tubes</td>
			<td>Sigma</td>
			<td>P0674</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Corning filter tips (100-1000 &mu;l</td>
			<td>Fisher Scientific</td>
			<td>07-200-265</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hank's balanced salt solution (1X)</td>
			<td>Lonza BioWhittaker</td>
			<td>10-508F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES buffer (1M)</td>
			<td>MP Biomedicals</td>
			<td>091688449</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>04716728001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>papain</td>
			<td>Worthington</td>
			<td>LS03126</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>AM2682</td>
			<td>These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear.</td>
		</tr>
		<tr>
			<td>SUPERase-In</td>
			<td>Applied Biosystems</td>
			<td>AM2694</td>
			<td>These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear.</td>
		</tr>
		<tr>
			<td>Modified Oligo d(T) primer (gel purified)</td>
			<td>Oligos ETC</td>
			<td>&nbsp;</td>
			<td>We have used primers purchased from many different companies and have seen the most reliable and reproducible results from Oligos ETC.</td>
		</tr>
		<tr>
			<td>T4 gene 32 protein</td>
			<td>New England Biolabs</td>
			<td>M0300L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Exonuclease I</td>
			<td>New England Biolabs</td>
			<td>M0293S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TdT</td>
			<td>Roche</td>
			<td>3 333 574</td>
			<td>As with other reagents, using a TdT enzyme from other companies gave more variable results.</td>
		</tr>
		<tr>
			<td>RNase H</td>
			<td>Invitrogen</td>
			<td>18021-014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ex-Taq HS Polymerase</td>
			<td>Takara</td>
			<td>RR006A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin N6-ddATP</td>
			<td>Enzo Biosciences</td>
			<td>42809</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. Specific reagents and equipment.

single-cell profiling of developing and mature retinal neurons

Poblaciones altamente especializados, pero muy pequeña de células juegan un papel importante en muchos tejidos. La identificación de células de tipo de marcadores específicos y programas de expresión génica para subconjuntos de células muy raras ha sido un reto con estándar de tejidos de todo el enfoque. Perfiles de expresión génica de las células individuales permite un acceso sin precedentes a los tipos de células que comprenden sólo un pequeño porcentaje del total del tejido 1-7. Además, esta técnica puede utilizarse para examinar los programas de expresión génica que se transitoriamente expresados ​​en un pequeño número de células durante las transiciones dinámicas de desarrollo 8. Este tema de la diversidad celular surge repetidamente en el sistema nervioso central (SNC), donde las conexiones neuronales puede ocurrir entre células muy diversos 9. El número exacto de distintos tipos de células que no se conoce con precisión, pero se ha estimado que puede haber hasta 1.000 tipos diferentes de la corteza itself 10. La función (s) de los complejos circuitos neuronales puede confiar en algunos de los tipos raros neuronales y los genes que expresan. Al identificar nuevos marcadores y ayudando a clasificar molecularmente diferentes neuronas, el enfoque de una sola célula es particularmente útil en el análisis de tipos de células en el sistema nervioso. También puede ayudar a dilucidar los mecanismos de desarrollo neuronal mediante la identificación de genes expresados ​​diferencialmente y vías genéticas durante las primeras etapas del desarrollo neuronal progenitora. Como un pañuelo de papel simple, de fácil acceso a la diversidad neuronal considerable, la retina de los vertebrados es un excelente sistema modelo para estudiar los procesos de desarrollo celular, la diferenciación neuronal y la diversificación de las neuronas. Sin embargo, como en otras partes del sistema nervioso central, esta diversidad celular puede presentar un problema para la determinación de las vías genéticas que conducen a la retina progenitores a adoptar un destino específico de célula, sobre todo teniendo en cuenta que fotorreceptores de los bastones conforman el mayoría de la población total de células de retina 11. Aquí se presenta un método para la identificación de las transcripciones expresadas en células de la retina individuales (Figura 1). La técnica del perfil de una sola célula permite la evaluación de la cantidad presente de la heterogeneidad dentro de las diferentes poblaciones celulares de la retina, 2,4,5,12. Además, este método ha revelado una gran cantidad de nuevos genes candidatos que pueden jugar un papel (s) en el destino de la célula de toma de decisiones que se producen en la población de células progenitoras de la retina 8. Con algunos ajustes sencillos para el protocolo, esta técnica puede ser utilizada para diferentes tejidos y tipos de células.

Watch this Scientific Journal Video about Single-cell Profiling of Developing and Mature Retinal Neurons at JoVE.com

Single-cell Profiling of Developing and Mature Retinal Neurons

Un método para el aislamiento de células de la retina individuales y la amplificación posterior de sus ADNc se describe. Una sola célula de la transcriptómica revela el grado de heterogeneidad presente celular en un pañuelo de papel y descubre nuevos genes marcadores de poblaciones celulares raras. El protocolo de acompañamiento se puede ajustar para adaptarse a diferentes tipos de células.

Una sola célula de perfiles de neuronas de la retina en desarrollo y maduro

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

single-cell-profiling-of-developing-and-mature-retinal-neurons

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Grabaciones de patch clamp de neuronas de ratón de retina en una rebanada preparación adaptada a la oscuridad

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Investigación

Neurociencias

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

Perfiles de voltaje de expresión del ARNm de potasio en las neuronas Canal nigral con una sola celda de RT-PCR Técnicas

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

De corte de carga: una herramienta útil para examinar el grado de acoplamiento Marcador brecha unión entre las neuronas retinianas

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Simultáneas registros de células de fotorreceptores y las neuronas de segundo orden en una rebanada preparación retina Anfibios

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

RNA-Seq de una sola célula de subconjuntos definidos de células de ganglio retiniano

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Aislamiento de núcleos de adultos de la médula espinal para secuenciación masivamente paralela solo núcleo ARN

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.

Secuenciación de ARN unicelular de las neuronas de ratón fluorescencia etiquetada con clasificación Manual y doble transcripción In Vitro con recuentos absolutos secuenciación (DIVA-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Imágenes en vivo de lapso de tiempo y cuantificación de la dinámica de ramas dendríticas rápidas en el desarrollo de neuronas drosophila

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporación Unicelular A Través De Diferentes Cultivos Organotípicos Slice De Ratón Excitatorio Hipocampal Y Neuronas Inhibitorias Específicas De Clase

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Cocultura de neuronas ganglionas retinal de rata axotomizada con glia olfativa, como modelo in vitro de regeneración axonal adulta

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...