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

The overall goal of this procedure is to precisely target micro domains in a functional map to study the structure and function of neurons at high resolution. This is accomplished by first performing intrinsic signal imaging over a large area of cortex to obtain a map of the stimulus feature of interest. The second step is to determine the precise location in the map that is to be targeted for high resolution to photon imaging.

Next, after carefully positioning the pipette loaded with fluorescent dye neurons are labeled with the fluorescent marker. Finally, high resolution to photon images of neuronal responses or structures are collected. Ultimately, an intrinsic signal feature map can be used to find a region of interest, such as an orientation pinwheel singularity that can then be targeted for study at single neuron resolution.

The main advantage of this protocol is that one can image the cortex at different spatial scales using the same microscope setup with only a few quick adjustments to the system. This allows one to combine these techniques to use the course resolution intrinsic signal map as a guide for choosing a region of neurons to label with fluorescent probes for high resolution imaging. The animal has been anesthetized according to an institutional animal care induced committee approved protocol, and the skull has been exposed and cleaned.

Dental cement mix has been used to attach a stainless steel head plate with a rectangular opening in the center to the skull. A metal chamber on top of the head plate serves as a reservoir for imaging with the water immersion objective lens. The head plate has been secured on an XY stage using metal posts.

Use a dental drill to thin a large area of bone within the rectangular opening of the head plate. Applying bone waxes needed to stop occasional bleeding. The thin area must extend while beyond the boundaries of the planned craniotomy.

Then drill a two by two millimeter square outline in the bone for the craniotomy. Rinse the chamber periodically with fresh artificial cerebral spinal fluid to remove bone shards and minimize heat dissipation to the underlying cortex. Now, lift the bone with a number three forceps.

Use a number five forceps and Vana spring scissors to remove the upper layers of the dura, leaving the bottom layer intact. The final layer of dura is transparent and peel vessels should be clearly visible through it. Apply a drop of warm 2%aeros dissolved in A CSF and immediately place a cover glass over the craniotomy.

First, a gel foam soaked with A CSF around the outside of the cover glass to prevent the agros from drying during intrinsic signal imaging. Then attach the intrinsic signal imaging CCG camera to the standard C mount port on top of the two photo microscope and place an illuminating light source on the air table near the XY stage position and secure the light guide over the craniotomy. Retract the primary dichroic and the mirror below the seamount port so that the reflected red light needed for intrinsic signals will pass directly from the craniotomy to the CCD camera.

Insert an infrared filter into the light path to prevent the cortex from heating. Then place a filter that passes green light and record a digital reference image of the cortical surface blood vessels. Next, move the Z focus to 500 microns below the cortical surface.

Replace the green filter with a red filter to illuminate the cortex for measuring intrinsic signals. Ensure that the cortex is uniformly illuminated. Shield the craniotomy from the light produced by the visual stimulus display and present a sequence of visual stimuli and record reflectance data images to generate an orientation map Within 10 minutes, collect data corresponding to four repetitions of a sequence of eight stimuli with four orientations and two directions for each orientation.

Now analyze the intrinsic signal data to generate a false orientation color map as seen here. Then select potential target regions for two photon labeling of neurons. For example, orientation pinwheel singularities points in the map where all preferred orientations converge.

Next, overlay the orientation map and the image of the cortical surface vasculature and use the layout of functional domains and the location of large surface vessels to select a target, avoiding locations with large blood vessels and arterials. Prepare the fluorescent D solutions and pull a long taper pipette according to instructions in the written protocol. Load five microliters of the dye mixture into the pipette.

Then remove the final layer of dura to facilitate pipette insertion and obtain the highest quality two photon images as illustrated by this diagram. The pipette must be driven into the cortex at a 30 degree angle to pass between the chamber and objective. Therefore, the cortical surface location of the pipette entry position will not be directly over the targeted region of interest.

For example, to label a target region 200 microns below the cortical surface, the pipette entry position on the cortical surface will be approximately 350 microns lateral to the target position. Move the XY stage to center the cortical surface entry position under the 20 x or 40 x objective. Once the entry position is centered under the objective, raise the Z position of the objective by two millimeters.

Then switch on the green epi fluorescence light, and visualize the red Alexa dye in the pipette and move the pipette until the tip is above the cortical surface entry position. While viewing the pipette tip through the oculars of the microscope with brightfield illumination, lower the pipette until the tip reaches the desired entry position on the cortical surface. Next, remove the objective and use lint-free absorption spears to wick excess cerebral spinal fluid from the craniotomy and dry the adjacent bone.

Then apply a drop of warm 3%arose, dissolved in A CSF to stabilize the cortex. Once the aros has solidified, insert a 40 x objective and refill the chamber with a CSF. Next, use the diagonal axis of the micro manipulator to move the pipette until the tip reaches the desired depth in the cortex.

Occasional pressure ejection puffs of the DI mixture will reduce the likelihood that the pipette tip will clog when the pipette reaches layer two, three small puffs of the DI mixture will illuminate the extra cellular space and reveal a dense assembly of circular cell bodies as dark shadows for loading neuronal bodies in a sphere of cortex 300 to 600 microns in diameter. Inject the Dix mixture into the extracellular space using 30 to 90 pulses. Each pulse one second, two to 10 PSI.

After the injection is complete, wait a few minutes, then remove the pipette and objective. The fluorescent calcium indicator will be fully taken up by neurons in one hour. Finally, remove the aros used for the downloading and rinse the recording chamber.

Put a small drop of two to 3%arose on the surface and quickly place a cover glass over the craniotomy. Insert a high NA 20 x objective into the objective holder and recenter the labeled cortical region under the objective. Using the XY stage shield the craniotomy from extraneous light sources and collect high resolution images of the labeled neurons in vivo.

Alternatively, single cell electroporation can be used for studying dendritic morphology or functional imaging of dendrites. In this methodology, the DI are prepared according to the written protocol. Then at the appropriate point in the procedure, the pipette tip is positioned adjacent to a neuronal cell body membrane and an axo is used to apply one to three pulses.

The immediate filling of the neuron with dye is readily visualized with two photon microscopy. This image shows a two photon imaged area showing cell bodies labeled with the fluorescent calcium indicator OGB 1:00 AM in visual cortex layer two three, the scale bar represents 100 microns. This image shows the orientation preference of individual neuronal cell bodies from the same site shown in the previous image.

An organized map of preferred stimulus orientation is seen around the pinwheel singularity that was centered in the imaged region. Here we see the orientation map obtained with intrinsic signal imaging. The black square corresponds to the region shown in the previous two photon image.

This image shows the dendrites and cell body of a single neuron overlaid with the orientation map. The neuron was electroporated with Alexa floor 5 94 under two photon guidance. As Z stack was collected in vivo and dendritic processes were traced offline using neuro lucid as software.

The orientation map was obtained using intrinsic signal imaging before single cell electroporation. The scale bar represents 100 microns. This 10 micron Z projection from cortical layer one shows apical dendrites.

The red arrows show spines along dendrites while these 10 micron Z projections from cortical layer two three show basal dendrites with spines indicated by red arrows and axons indicated by white arrows. This method is useful for studying functional maps in the cortex at different spatial scales and for precisely targeting neurons in a specific domain of interest. Within the map, we have demonstrated the application of the method for examining orientation selective maps, but it can be extended to other visual feature maps such as retina, toppy, ocular dominance, and direction, or to other sensory modalities that have functionally organized maps such as those that encode somato sensation or audition.