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Neuroscience

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

Published: December 12th, 2012

DOI:

10.3791/50025

1Department of Neuroscience, Medical University of South Carolina

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, 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.

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.......

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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.......

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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.......

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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.

....

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

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