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

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 orientation^1-4, direction^5-7, ocular dominance^8,9 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 cortex^10,11. 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 bodies^6,14. 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 published^9,15.
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 applicators and cotton gauze. Apply bone wax (as needed) to the skull, to stop occasional bleeding through small emissary veins.
3. Attach a t....

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.......

Materials

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