Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Virally delivered optogenetic constructs permit detailed electrophysiological characterization of the physiology and plasticity of specific synapses in acute brain slices. The primary advantages of activating synapses optogenetically is the ability to study long-range pathways, and the selective stimulation of axons which are not anatomically separated. Demonstrating the procedure will be Dr.Lisa Kinnavane, a research associate from our laboratory.

To begin, load a Hamilton syringe into a microinjection syringe pump attached to a movable arm mounted to a stereotaxic frame. Then place a five-microliter aliquot of the virus in a microcentrifuge. Spin the tube for a few seconds and pipette two microliters of the viral preparation into the tube’s lid.

To fill the syringe with the viral preparation, view the needle tip with a surgical microscope. Then manually place the bolus of the virus at the tip of the needle, and withdraw the syringe plunger using the pump controls. Set the pump injection volume to 300 nanoliters and flow rate to 100 nanoliters per minute.

Run the pump and confirm proper flow by observing the droplet of the virus at the needle tip. Absorb the virus on a cotton bud, and clean the needle with 70%ethanol. Next, using the adjuster screws on the stereotaxic frame, navigate the needle tip to the bregma, and take note of the stereotaxic measurements observed on the three vernier scales on the frame.

Add, or subtract these distances from bregma coordinates. Make a burr hole at the skull surface using a micro-drill mounted to the stereotaxic arm. Insert the needle into the brain at the predetermined dorsoventral coordinate, and infuse a predetermined volume of the viral preparation.

After infusion, leave the needle in situ for 10 minutes to allow for the diffusion of the bolus. Then remove the needle, and run the pump to ensure that the needle is not blocked. Two weeks after the viral injection, euthanize the rat, rapidly dissect the entire brain, and transfer it into the sucrose cutting solution.

Using a metal teaspoon, pick up the brain, discard the excess solution, and place it onto a filter paper. Using a scalpel, remove the cerebellum, and cut the cerebrum in the coronal plane approximately halfway along its length. The posterior half is the LEC tissue block.

Return the tissue block and the remaining brain to the sucrose cutting solution. Next, place a drop of cyanoacrylate glue onto a vibratome stage, and spread it into a thin layer. Then using a teaspoon, pick the LEC tissue block, and transfer it onto the glue patch, such that the anterior coronal cut has adhered.

Install the stage into the vibratome tissue chamber, and quickly pour sufficient sucrose cutting solution to submerge the tissue. After orienting the ventral surface of the tissue block toward the blade, cut 350-micrometer thick slices from ventral to dorsal using a slow blade advancement speed. Typically, seven slices can be obtained per hemisphere.

Transfer the slices to the slice collection chamber. Then place the collection chamber in a 34 degree Celsius water bath for one hour before returning to room temperature. Place the remainder of the brain in paraformaldehyde for 48 hours.

For target cell identification, place the slice into the recording chamber, and immobilize it using a slice anchor. Under low magnification, using infrared illumination, navigate to the LEC layer five. Then change to the high magnification water immersion objective, and identify the pyramidal neurons.

Mark the position of the cell on the monitor with tape. Next, to form a whole cell patch clamp, fabricate a borosilicate glass micro-pipette, and fill it with intracellular recording solution. Place the filled micro-pipette in the electrode holder and apply positive pressure by blowing hard into a mouthpiece connected to the electrode holder side port.

Raise the microscope objective such that a meniscus forms and insert the electrode into the meniscus until it can be seen on the microscope. Open the Seal Test window, and determine whether the pipette resistance is three to five megaohms. Then touch the identified cell with a pipette tip, resulting in an indentation in the cell membrane.

Next, apply negative pressure by suction at the mouthpiece, increasing pipette resistance. Continue applying negative pressure gradually until the cell membrane ruptures, resulting in whole cell capacitance transients. To enter the current clamp configuration, use a mounted LED directed into the microscope light path with filter cubes and appropriate optics, and apply light pulses to the slice via the 40X objective.

Deliver trains of multiple light pulses at five hertz, 10 hertz, and 20 hertz to investigate the presynaptic release properties. To allow biocytin to fill the neuron, wait for at least 15 minutes after entering the whole cell configuration. In voltage clamp, monitor the membrane capacitance and input resistance.

Then slowly withdraw the pipette along the approach angle away from the cell’s soma, observing the slow disappearance of capacitance transients and membrane current, indicating the resealing of the cell membrane and formation of an outside-out patch at the pipette tip. Place the slice into paraformaldehyde in a 24-well plate and incubate overnight. Using OCT medium, attach a tissue block to the cryostat specimen disc.

To freeze the tissue, place isopentane in an appropriate container and submerge the specimen disc, ensuring the tissue is above the level of the isopentane. Then lower the container of isopentane into liquid nitrogen, and allow the tissue to freeze. Once the tissue is completely frozen, leave the tissue block in the cryostat chamber at minus 20 degrees Celsius for 30 minutes to allow the temperature of the block to equilibrate.

After equilibration, cut 40-micrometer thick sections in the cryostat, and use a fine paintbrush to guide the sections off the blade. Adhere these frozen sections to a room temperature poly-L-lysine-coated glass microscope slide by touching the slide to the sections. After adding 150 microliters of mounting medium to each slide, apply the cover slip, and remove any air bubbles by gently pressing on the cover slip.

Cover the slides to protect against photobleaching, and let them air dry for 12 hours. Then using a fluorescence microscope, examine the location of the viral injection site. For biocytin staining, incubate the slices in 3%hydrogen peroxide in PBS for 30 minutes to block any endogenous peroxidase activity.

Then wash the brain slices with PBS until no further oxygen bubbles are visible. Next, incubate the slices for three hours in 1%avidin-biotinylated HRP complex solution in PBS containing 0.1%Triton X-100. After six PBS washes, incubate each slice in DAB solution until the biocytin staining of neuronal structures becomes visible.

Stop the reaction by transferring the slices in cold PBS. Then mount the slices onto the glass microscope slides using a brush. After removing excess PBS, add mounting medium.

Cover the slices using cover slips, and let them air dry, as demonstrated earlier. A healthy pyramidal cell was located and patched. If post-synaptic cell identification is required, a cell expressing the fluorescent marker should be localized using widefield optics.

Single light pulses of two milliseconds resulted in simple waveform optogenetic excitatory post-synaptic potentials. Short-term plasticity of the synapse is examined by applying five, 10, and 20 hertz trains of light stimulation. While investigating long-term plasticity, adding cholinergic agonist carbachol to the circulating aCSF for 10 minutes caused a long-term depression that was still evident 40 minutes after removal of the ligand.

The length of the injection site was examined histologically. The fluorescent reporter mCherry was localized to the deeper layers of the prelimbic and infralimbic cortex. Further, staining a biocytin-filled cell confirmed its location and morphology.

The critical steps of this protocol are accurate transduction of the afferent brain region, which can be verified post hoc, and rapid dissection of the brain when preparing acute slices. This procedure is easily combined with fluorescent labeling of an individual cell type, such as a particular interneuron sub-class. To do this, we’d use a transgenic animal that expresses a recombinase in that particular cell type.

Virally delivered optogenetic constructs have allowed rapid advancements in the fields of systems and circuit neuroscience.