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1Department of Neuroscience, Medical University of South Carolina
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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.
Keywords: Neuroscience, Issue 70, Molecular Biology, Cellular Biology, Anatomy, Physiology, Two-photon imaging, non-rodent, cortical maps, functional architecture, orientation pinwheel singularity, optical imaging, calcium-sensitive dye, bulk loading, single-cell electroporation
O'Herron, P., Shen, Z., Lu, Z., Schramm, A. E., Levy, M., Kara, P. Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging. J. Vis. Exp. (70), e50025, doi:10.3791/50025 (2012).
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
2. Intrinsic Signal Optical Imaging
3. Bulk Loading of Fluorescent Calcium Indicators
4. Single-cell Electroporation of Fluorescent Dyes
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 circle in Figure 2C), being sure to avoid large vessels and arterioles in particular15. With bulk-loading of OGB-1 AM, tracking changes in neuronal calcium indicator fluorescence to a sequence of oriented grating visual stimuli reveals single-cell resolution functional maps around pinwheel singularities (Figure 4). With single-cell electroporation, the spatial organization of dendrites and axons from a single neuron located at a pinwheel singularity is determined in vivo (Figure 5).
Figure 1. Intrinsic signal optical imaging setup. (A) The animal is positioned on the xy stage and intrinsic signal imaging is performed via a CCD camera mounted on the two-photon microscope. The light source and computer used to control the imaging session are brought over to the setup for intrinsic imaging and moved away before the two-photon imaging phase of the experiment begins. (B) Close up view of the 4x objective and the 630 nm illumination through liquid light guides. Either two light guides attached to metal posts (left), or a single illuminating ring attached to directly the objective (right) can be used. Blackout material used to shield the objective from external sources of illumination, e.g., visual stimulus display, during optical imaging sessions, is not shown. (C) Schematic of the microscope and light path in the conventional two-photon imaging configuration. See Supplementary Fig. 20 in Shen et al. 201215 for a description of the complete two-photon light path. The retractable mirror is positioned to deflect the light from the laser through the dichroic and onto the cortex. The primary dichroic, which passes the laser beam and reflects the emitted light from the cortex to the PMT's, is moved into the light path by turning the filter turret. (D) For intrinsic imaging the mirror is retracted and the filter turret is turned to move the primary dichroic out of the way, allowing a direct light path from the cortex to the CCD camera. Click here to view larger figure.
Figure 2. Using functional maps obtained with intrinsic signal imaging to select a micro-domain for labeling neurons with fluorescent dyes. (A) Orientation map from cat primary visual cortex acquired via intrinsic signal optical imaging. The color of each pixel represents the preferred orientation at that location. Potential orientation pinwheel singularities suitable for labeling neurons with fluorescent dyes are shown within black circles. (B) The map of cortical surface blood vessels overlaid with the orientation map. (C) Pinwheels within red circles are near large blood vessels or arterioles and so the pinwheel site within the green circle is chosen as the target for dye labeling. The dashed rectangle corresponds to the region shown in Figure 3B. Scale bar in (A) 500 μm.
Figure 3. Optimizing the pipette placement for loading neurons with fluorescent dyes. (A) Schematic side-view of the recording chamber shows that the dye loading site to be targeted in visual cortex (red-and-white bull's eye) is ~200 μm below the cortical surface. The xy coordinate of the target corresponds to the location of an orientation pinwheel singularity determined from intrinsic signal imaging. The pipette will enter the cortex at a 30° angle. If the cortical surface is parallel to the xy stage surface, the pipette tip will move laterally approximately 350 μm before reaching the pinwheel singularity. (B) Top view of recording chamber shows the cortical surface vasculature and the pipette entry position (red star) that is 350 μm from the pinwheel singularity. (C) The xy stage position is adjusted such that the pipette entry position is centered under the 20x or 40x objective (3.3-3.5 mm WD). (D) The objective is raised ~2 mm. (E) The pipette is brought over the craniotomy and its tip is positioned directly above the entry position on the cortical surface using green epi-fluorescent light. (F) The pipette and objective are lowered concurrently using bright-field illumination until the cortical surface blood vessels come into focus. (G) The objective and ACSF are removed and a drop of 3% agarose is applied over the craniotomy. (H) Two-photon visualization with a 40x (0.8 NA, 3.3 mm WD) objective is used to follow the pipette down to the chosen cortical depth and inject the dye. After confirming that neurons are successfully labeled with fluorescent dye, typically, a higher NA 20x objective (1.0 NA, 2.0 mm WD) is used to collect two-photon t- and z-series data. Schematics shown in A, C-H are not drawn to scale.
Figure 4. Correspondence of orientation preference in two-photon and intrinsic signal imaging functional maps. (A) Two-photon imaged area showing cell bodies labeled with OGB-1 AM in visual cortex layer 2/3. (B) Orientation preference of individual neuronal cell bodies from the same site shown in (A). Around the pinwheel singularity that was centered in the imaged region is an organized map of preferred stimulus orientation. (C) The orientation map obtained with intrinsic signal imaging. The black square corresponds to the region shown in (B). These data were obtained from the same experiment shown in Figures 2A-C and 3B. Scale bar in (A-B) 100 μm and (C) 500 μm.
Figure 5. In vivo morphology of a single neuron at an orientation pinwheel singularity. (A) The dendrites and cell body of a single neuron overlaid with the orientation map from the same visual cortical site. The neuron was electroporated with Alexa Fluor 594 under two-photon guidance, a z-stack was collected in vivo, and dendritic processes were traced offline using Neurolucida software. The orientation map was obtained using intrinsic signal imaging before single-cell electroporation (B) Average intensity 10-μm-z-projection from cortical layer 1 shows apical dendrites. (C-D) Average intensity 10-μm-z-projections from cortical layer 2/3 show basal dendrites and axons. Red arrows show spines along dendrites and white arrows point to individual axons. Scale bars in (A-D) 100 μm.
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 application of clinically therapeutic drugs18.
No maps for stimulus orientation, direction, ocular dominance, or binocular disparity have been reported in the visual cortex of adult wild-type rodents6,19-21. However, the ease of loading neurons with fluorescent dyes and attaining imaging stability, together with the powerful genetic tools available for mice has resulted in the vast majority of studies using two-photon imaging of the cerebral cortex being performed in rodents instead of ferrets, cats, and monkeys.
High-resolution two-photon imaging of neurons in non-rodent mammals can be very challenging because of long surgery times, thicker bone and dura, and extra steps needed to keep respiratory and cardiovascular mediated pulsations of the brain to a minimum15. Transitions from surgery to intrinsic signal imaging and intrinsic to two-photon imaging need to be efficient, requiring timely preparation of reagents, dyes and the execution of micro-surgical procedures. Using the two-photon microscope for intrinsic signal imaging obviates the need for much of the equipment used in conventional intrinsic signal optical imaging rigs22 and reduces the time required to setup intrinsic imaging and transition to subsequent two-photon imaging.
Our approach is specifically designed to provide high-resolution two-photon imaging in pre-determined functional domains. This provides minimal cross talk of functional signals between neuronal cell bodies, and individual dendritic spines and axons can be resolved in vivo. Using a high numerical aperture (NA = 1.0) objective and filling the back aperture of the objective with beam expansion optics (see Supplementary Fig. 20 in Shen et al., 201215) are critical for high-resolution imaging. Depending on the speed of imaging in XY and/or Z, respiratory and cardiovascular pulsations may reduce the effective imaging resolution no matter how good the NA and optical light path are. By limiting the craniotomy size in non-rodents to 2 x 2 mm, using the relatively higher concentration of agarose with a coverglass, and performing a pneumothorax and lumbar suspension when necessary15, brain pulsations are limited to 1-2 μm. Minimizing respiratory and cardiovascular induced pulsations of the neocortex to 1-2 μm during bulk loading of calcium indicators also ensures that the cortex forms a tight seal around the inserted pipette and that no dye tracks up along the shank of pipette to the cortical surface. Selecting dye injection and imaging sites away from large arterioles is also critical for high quality functional imaging because relatively fast sensory-evoked hyperemia (hemodynamic) artifacts will be excluded15.
At the craniotomy site, leaving the final layer of dura intact throughout the intrinsic signal imaging and fluorescent dye preparation phases - thus removing it just before the pipette insertion into the cortex for dye loading - increases the success rate and quality of neuronal labeling in the following ways: (1) keeps the cortical surface clean and/or prevents dural tissue regrowth so that the pipette tip is less likely to get clogged and the transparency of the tissue is maintained, (2) avoids having the agarose used for intrinsic imaging touch the cortical surface blood vessels which may get damaged when the agarose is removed, (3) prevents the agarose used during intrinsic imaging from entering between the dura and cortex underneath the skull around the edges of the craniotomy. Agarose in these regions will increase the distance between the coverglass and the cortical surface which makes the insertion of the pipette more difficult, leads to compression of the cortical tissue and reduces the transparency for imaging.
When preparing the AM fluorescent dyes for bulk loading, the 20% Pluronic/DMSO mixture should never be used beyond 1 week from its initial preparation - old Pluronic/DMSO quenches fluorescence. For optimal bulk loading of many adjacent cell bodies with a functional indicator, e.g., OGB-1 AM, the dye mixture is stored on ice, loaded into the pipette as needed but always injected into the brain within 2 hr of its preparation.
During the bulk loading phase of the experiment, repeated 1 sec pulses of pressure rather than a continuous injection of 1 min allows for iterative calibration of ejection parameters, which ensures that excessive dye or pressure is not applied. Because of the potential tissue damage from multiple pipette insertions needed to label very large regions of tissue and potential toxicity from large volumes of Pluronic/DMSO from such a series of many injections, we prefer one 300-600 μm dye injection site per 2 x 2 mm craniotomy. Compared to blind injections14, two-photon visually guided navigation around blood vessels, use of negative contrast of neuronal cell bodies to determine lamina position, and gauging the spread of the dye upon pressure ejection as an index of the volume of cells that will be labeled with OGB-1 AM, improves the quality of dye loading in non-rodent preparations.
The protocol we describe here leads to successful labeling of neurons with fluorescent dyes in targeted micro-domains in every animal attempted. For bulk loading with OGB-1 AM, we consistently obtain visually-evoked responses in 75-100% of simultaneously imaged neurons9,15 and the technique is sensitive to the detection of single action potentials in vivo23,24.
No conflicts of interest declared.
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.
|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|
|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|
|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|
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