This work was supported by grants from the National Eye Institute R01EY017925 and R21EY020985 and funding from the Dana &amp; Whitehall Foundations to P.K. We also thank Matthew Petrella for assistance with surgical procedures; Grace Dion for tracing the dendrites shown in Figure 5A; and Pratik Chhatbar for comments on the manuscript.

1. Surgical Preparation
<ol>
	<li>Induce anesthesia and continuously monitor heart rate, end tidal CO2, EEG, and temperature. All procedures were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina and were based on those we previously published9,15.</li>
	<li>Expose the dorsal surface of the skull by cutting the skin with a scalpel blade. Dissect the connective tissues overlying the bone using a Brudon curette. Clean the bone using cotton tipped...

<ol>
	<li>Blasdel, G. G., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dyes+reveal+a+modular+organization+in+monkey+striate+cortex.">Voltage-sensitive dyes reveal a modular organization in monkey striate cortex.</a> Nature. 321, 579-585 (1986).</li><li>Grinvald, A., Lieke, E., Frostig, R. D., Gilbert, C. D., Wiesel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+cortex+revealed+by+optical+imaging+of+intrinsic+signals.">Functional architecture of cortex revealed by optical imaging of intrinsic signals.</a> Nature. 324, 361-364 (1986).</li><li>Bonhoeffer, T., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iso-orientation+domains+in+cat+visual+cortex+are+arranged+in+pinwheel-like+patterns.">Iso-orientation domains in cat visual cortex are arranged in pinwheel-like patterns.</a> Nature. 353, 429-431 (1991).</li><li>Ohki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+ordered+arrangement+of+single+neurons+in+orientation+pinwheels.">Highly ordered arrangement of single neurons in orientation pinwheels.</a> Nature. 442, 925-928 (2006).</li><li>Shmuel, A., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+for+direction+of+motion+and+its+relationship+to+orientation+maps+in+cat+area+18.">Functional organization for direction of motion and its relationship to orientation maps in cat area 18.</a> J. Neurosci. 16, 6945-6964 (1996).</li><li>Ohki, K., Chung, S., Ch'ng, Y. H., Kara, P., Reid, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+with+cellular+resolution+reveals+precise+micro-architecture+in+visual+cortex.">Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex.</a> Nature. 433, 597-603 (2005).</li><li>Li, Y., Van Hooser, S. D., Mazurek, M., White, L. E., Fitzpatrick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+with+moving+visual+stimuli+drives+the+early+development+of+cortical+direction+selectivity.">Experience with moving visual stimuli drives the early development of cortical direction selectivity.</a> Nature. 456, 952-956 (2008).</li><li>Bonhoeffer, T., Kim, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whose+Cortical+Column+Would+that+Be.">Whose Cortical Column Would that Be.</a> Frontiers in Neuroanatomy. 4, 16 (2010).</li><li>Kaschube, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universality+in+the+evolution+of+orientation+columns+in+the+visual+cortex.">Universality in the evolution of orientation columns in the visual cortex.</a> Science. 330, 1113-1116 (2010).</li><li>Villeneuve, M. Y., Vanni, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+valproic+acid+exposure+alters+functional+organization+in+the+primary+visual+cortex.">Early valproic acid exposure alters functional organization in the primary visual cortex.</a> Exp. Neurol. 228, 138-148 (2011).</li><li>Bock, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+anatomy+and+in+vivo+physiology+of+visual+cortical+neurons.">Network anatomy and in vivo physiology of visual cortical neurons.</a> Nature. 471, 177-182 (2011).</li><li>Rochefort, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+direction+selectivity+in+mouse+cortical+neurons.">Development of direction selectivity in mouse cortical neurons.</a> Neuron. 71, 425-432 (2011).</li><li>Mrsic-Flogel, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+regulation+of+eye-specific+responses+in+visual+cortex+during+ocular+dominance+plasticity.">Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity.</a> Neuron. 54, 961-972 (2007).</li><li>Bonhoeffer, T., Grinvald, A., Toga, A. W., Mazziotta, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Imaging+Based+on+Intrinsic+Signals.">Optical Imaging Based on Intrinsic Signals.</a> Brain mapping: The Methods. , 55-97 (1996).</li><li>Kerr, J. N., Greenberg, D., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+input+and+output+of+neocortical+networks+in+vivo.">Imaging input and output of neocortical networks in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 14063-14068 (2005).</li><li>Hofer, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+connectivity+and+response+dynamics+of+excitatory+and+inhibitory+neurons+in+visual+cortex.">Differential connectivity and response dynamics of excitatory and inhibitory neurons in visual cortex.</a> Nat. Neurosci. 14, 1045-1052 (2011).</li></ol>

No conflicts of interest declared.

We present a method to target the labeling of neuronal cell bodies (or dendrites and axons) in pre-determined functional micro-domains of the neocortex. Merging intrinsic signal optical imaging with two-photon microscopy offers the possibility of determining which synapses and cells contribute to the micro-domain of any functional map, whether neuronal selectivity correlates with the location of the neuron in a functional map, and the neuronal circuit components that change with visual experience7 or the appl...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1. Life support/experiment prep</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Webster Vet</td>
			<td>NDC 57319-474-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Midmark</td>
			<td>VIP 3000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Feedback regulated heating blanket</td>
			<td>Harvard Apparatus</td>
			<td>50-7079F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ECG monitor</td>
			<td>Digicare Biomedical</td>
			<td>LifeWindow Lite</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EEG amplifier</td>
			<td>A-M Systems</td>
			<td>1800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EEG display monitor</td>
			<td>Hewlett Packard</td>
			<td>78304A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>End tidal CO2 monitor</td>
			<td>Respironics</td>
			<td>Novametrix Capnoguard 1265</td>
			<td>Optimize ventilation</td>
		</tr>
		<tr>
			<td>Carbide drill burrs for drilling bone</td>
			<td>Henry Schein</td>
			<td>fine (0.5 mm tip) and coarse (1.25 mm tip)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cement for headplate/chamber</td>
			<td>Dentsply</td>
			<td>675571, 675572</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Black Powder Tempera Paint</td>
			<td>Sargent Art Inc.</td>
			<td>22-7185</td>
			<td>Add to cement to improve light shielding and reduce reflections</td>
		</tr>
		<tr>
			<td>Agarose - Type III-A</td>
			<td>Sigma</td>
			<td>A9793</td>
			<td>For minimizing pulsations during intrinsic signal and two-photon imaging</td>
		</tr>
		<tr>
			<td>Coverglass: 5 or 8 mm diameter, 0.17 mm thickness</td>
			<td>World Precision Instruments</td>
			<td>502040, 502041</td>
			<td>For minimizing pulsations during imaging, the coverglass may be cut as needed</td>
		</tr>
		<tr>
			<td>Brudon curettes</td>
			<td>George Tiemann</td>
			<td>105-715-0, 105-715-3</td>
			<td>Cleaning skull surface</td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Ethicon</td>
			<td>W31G</td>
			<td>Quickly stop bleeding</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicator</td>
			<td>Electron Microscopy Sciences</td>
			<td>72308-05</td>
			<td>Clean and dry bone surface</td>
		</tr>
		<tr>
			<td>Dumont #5CO Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-20</td>
			<td>Grab individual layers of dura or pia</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td>Cut dura</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pfizer</td>
			<td>09-0396-05</td>
			<td>To stop bleeding on the dura</td>
		</tr>
		<tr>
			<td>Absorption spears</td>
			<td>Fine Science Tools</td>
			<td>18105-01</td>
			<td>Ultra-fast and lint-free wicking of CSF</td>
		</tr>
		<tr>
			<td>Blackout material</td>
			<td>Thorlabs</td>
			<td>BK5</td>
			<td>Shield craniotomy</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Dye preparation / injection</td>
		</tr>
		<tr>
			<td>Dimethyl Sulphoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pluronic</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 Bapta-1 AM</td>
			<td>Invitrogen</td>
			<td>O6807</td>
			<td>Calcium indicator</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A10438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifugal filter (0.45 &mu;m pore size)</td>
			<td>Millipore</td>
			<td>UFC30HV00</td>
			<td>To remove impurities before injection</td>
		</tr>
		<tr>
			<td>Glass pipette puller</td>
			<td>Sutter Instruments</td>
			<td>P97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Borosilicate glass filamented capillary (1.5 mm outer diameter)</td>
			<td>World Precision Instruments</td>
			<td>1B150F-4</td>
			<td>Dye ejection pipette</td>
		</tr>
		<tr>
			<td>Microloader</td>
			<td>Eppendorf</td>
			<td>5242 956 003</td>
			<td>For loading dye into pipette</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td>To position pipette</td>
		</tr>
		<tr>
			<td>Pressure pulse controller</td>
			<td>Parker Hannifin</td>
			<td>PicoSpritzer III</td>
			<td>For pressure injection of the dye</td>
		</tr>
		<tr>
			<td>Single-cell electroporator</td>
			<td>Molecular Devices</td>
			<td>Axoporator 800A</td>
			<td>For electroporation of the dye</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. Intrinsic imaging</td>
		</tr>
		<tr>
			<td>4x Objective (0.13 NA, 17 mm WD)</td>
			<td>Olympus</td>
			<td>UPLFLN4X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intrinsic hardware / software</td>
			<td>Optical Imaging Inc.</td>
			<td>Imager 3001 / VDAQ</td>
			<td>VDAQ software is used for episodic imaging</td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Adimec</td>
			<td>Adimec-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light source power supply</td>
			<td>KEPCO</td>
			<td>ATE 15-15M</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Optical Imaging Inc.</td>
			<td>HAL 100</td>
			<td>Light intensity at the cortical surface is 3-5 mW</td>
		</tr>
		<tr>
			<td>Green filter (for vascular image)</td>
			<td>Optical Imaging Inc.</td>
			<td>&lambda; = 546 nm (bandpass 30 nm)</td>
			<td>For reference image of surface vasculature</td>
		</tr>
		<tr>
			<td>Red filter (for intrinsic signal)</td>
			<td>Optical Imaging Inc.</td>
			<td>&lambda; = 630 nm (bandpass 30 nm)</td>
			<td>To collect intrinsic signals</td>
		</tr>
		<tr>
			<td>Heat filter</td>
			<td>Optical Imaging Inc.</td>
			<td>KG-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. Two-photon rig/imaging</td>
		</tr>
		<tr>
			<td>Two-photon microscope and software</td>
			<td>Prairie Technologies</td>
			<td>&nbsp;</td>
			<td>See Shen et al. 2012 for light path, filters and laser power</td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai XF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x (0.5 NA; 3.5 mm WD)</td>
			<td>Olympus</td>
			<td>UMPLFLN20X</td>
			<td>0.5 NA objective is used only for aligning pipette over the craniotomy (not for two photon imaging)</td>
		</tr>
		<tr>
			<td>20x (1.0 NA; 2.0 mm WD)</td>
			<td>Olympus</td>
			<td>XLUMPLFLN20X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>40x (0.8 NA; 3.3 mm WD)</td>
			<td>Olympus</td>
			<td>LUMPLFLN40X/IR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Newport</td>
			<td>ST-200</td>
			<td>Isolates preparation from external vibrations</td>
		</tr>
		<tr>
			<td>xy stage</td>
			<td>Mike's Machine Co. (Attleboro, MA)</td>
			<td>&nbsp;</td>
			<td>Experimental subject and Sutter micromanipulator placed on xy stage</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">Recipes</td>
					</tr>
					<tr>
						<td>Artificial Cerebro-Spinal Fluid</td>
						<td>NaCl (135 mM), KCl (5.4 mM), MgCl2 (1.0 mM), CaCl2 (1.8 mM), HEPES (5 mM), pH 7.4</td>
					</tr>
					<tr>
						<td>Pipette Solution14</td>
						<td>NaCl (150 mM), KCl (2.5 mM), HEPES (10 mM), pH 7.4</td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

To illustrate the precision of our dye labeling methods, we targeted the smallest micro-domain of any known functional map in the non-rodent neocortex. Sparsely punctuated throughout the orientation map in the primary visual cortex are singularities. These occur at points where all preferred orientations converge such that in false color maps of preferred orientation, the regions around the singularity look like "pinwheels" (Figure 2A-B). One pinwheel per craniotomy is selected for dye labeling (green ci...

Watch this Scientific Journal Video about Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging at JoVE.com

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

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Research

JoVE Journal

Neuroscience

13.6K visualizações.  Medical University of South Carolina. É descrito um método para a marcação de neurónios com corantes fluorescentes funcionais pré-determinados micro-domínios do neocórtex. Primeiro, a imagem do sinal óptico intrínseco é usado para obter um mapa funcional. Em seguida, dois fotões microscopia é utilizado para marcar e neurónios de imagem dentro de um domínio de micro-do mapa.