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In This Article

  • Summary
  • Abstract
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Protocol

1. Surgical Preparation

  1. 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.
  2. 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.......

Representative Results

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.......

Discussion

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.......

Disclosures

No conflicts of interest declared.

Acknowledgements

This work was supported by grants from the National Eye Institute R01EY017925 and R21EY020985 and funding from the Dana & 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.

....

Materials

NameCompanyCatalog NumberComments
Name of Reagent/MaterialCompanyCatalogue NumberComments
   1. Life support/experiment prep
IsofluraneWebster VetNDC 57319-474-05 
Isoflurane vaporizerMidmarkVIP 3000 
Feedback regulated heating blanketHarvard Apparatus50-7079F 
ECG monitorDigicare BiomedicalLifeWindow Lite 
EEG amplifierA-M Systems1800 
EEG display monitorHewlett Packard78304A 
End tidal CO2 monitorRespironicsNovametrix Capnoguard 1265Optimize ventilation
Carbide drill burrs for drilling boneHenry Scheinfine (0.5 mm tip) and coarse (1.25 mm tip) 
Cement for headplate/chamberDentsply675571, 675572 
Black Powder Tempera PaintSargent Art Inc.22-7185Add to cement to improve light shielding and reduce reflections
Agarose - Type III-ASigmaA9793For minimizing pulsations during intrinsic signal and two-photon imaging
Coverglass: 5 or 8 mm diameter, 0.17 mm thicknessWorld Precision Instruments502040, 502041For minimizing pulsations during imaging, the coverglass may be cut as needed
Brudon curettesGeorge Tiemann105-715-0, 105-715-3Cleaning skull surface
Bone waxEthiconW31GQuickly stop bleeding
Cotton Tipped ApplicatorElectron Microscopy Sciences72308-05Clean and dry bone surface
Dumont #5CO ForcepsFine Science Tools11295-20Grab individual layers of dura or pia
Vannas Spring ScissorsFine Science Tools15000-03Cut dura
GelfoamPfizer09-0396-05To stop bleeding on the dura
Absorption spearsFine Science Tools18105-01Ultra-fast and lint-free wicking of CSF
Blackout materialThorlabsBK5Shield craniotomy
   2. Dye preparation / injection
Dimethyl Sulphoxide (DMSO)SigmaD2650 
PluronicSigmaP2443 
Oregon Green 488 Bapta-1 AMInvitrogenO6807Calcium indicator
Alexa Fluor 594InvitrogenA10438 
Centrifugal filter (0.45 μm pore size)MilliporeUFC30HV00To remove impurities before injection
Glass pipette pullerSutter InstrumentsP97 
Borosilicate glass filamented capillary (1.5 mm outer diameter)World Precision Instruments1B150F-4Dye ejection pipette
MicroloaderEppendorf5242 956 003For loading dye into pipette
MicromanipulatorSutter InstrumentsMP-285To position pipette
Pressure pulse controllerParker HannifinPicoSpritzer IIIFor pressure injection of the dye
Single-cell electroporatorMolecular DevicesAxoporator 800AFor electroporation of the dye
   3. Intrinsic imaging
4x Objective (0.13 NA, 17 mm WD)OlympusUPLFLN4X 
Intrinsic hardware / softwareOptical Imaging Inc.Imager 3001 / VDAQVDAQ software is used for episodic imaging
CCD CameraAdimecAdimec-1000 
Light source power supplyKEPCOATE 15-15M 
Light sourceOptical Imaging Inc.HAL 100Light intensity at the cortical surface is 3-5 mW
Green filter (for vascular image)Optical Imaging Inc.λ = 546 nm (bandpass 30 nm)For reference image of surface vasculature
Red filter (for intrinsic signal)Optical Imaging Inc.λ = 630 nm (bandpass 30 nm)To collect intrinsic signals
Heat filterOptical Imaging Inc.KG-1 
   4. Two-photon rig/imaging
Two-photon microscope and softwarePrairie Technologies See Shen et al. 2012 for light path, filters and laser power
Ti:Sapphire laserSpectra-PhysicsMai Tai XF 
20x (0.5 NA; 3.5 mm WD)OlympusUMPLFLN20X0.5 NA objective is used only for aligning pipette over the craniotomy (not for two photon imaging)
20x (1.0 NA; 2.0 mm WD)OlympusXLUMPLFLN20X 
40x (0.8 NA; 3.3 mm WD)OlympusLUMPLFLN40X/IR 
Air tableNewportST-200Isolates preparation from external vibrations
xy stageMike's Machine Co. (Attleboro, MA) Experimental subject and Sutter micromanipulator placed on xy stage
   
Recipes
Artificial Cerebro-Spinal FluidNaCl (135 mM), KCl (5.4 mM), MgCl2 (1.0 mM), CaCl2 (1.8 mM), HEPES (5 mM), pH 7.4
Pipette Solution14NaCl (150 mM), KCl (2.5 mM), HEPES (10 mM), pH 7.4

References

  1. Blasdel, G. G., Salama, G. Voltage-sensitive dyes reveal a modular organization in monkey striate cortex. Nature. 321, 579-585 (1986).
  2. Grinvald, A., Lieke, E., Frostig, R. D., Gilbert, C. D., Wiesel, T. N.

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