The aim of this method is to identify the functional connectivity of long-range inputs from distant brain regions using photostimulation in brain slices. The stereotaxic targeting of a specific brain region for various mediated expression of light sensitive ion channels allows the selective stimulation of axons coming from that region with light. Demonstrating the procedure will be Louis Richevaux, a PhD student from my group.
Before the surgery, verify that the animal is well anesthetized with a toe pinch. Gently pull out its tongue to facilitate breathing. Then, shave the cranial hair.
Inject 20 microliters of lidocaine hydrochloride under the skin of the head for local anesthesia and wait five minutes. To expose the skull, make a cut on the scalp. Then, place the animal in a stereotaxic frame.
Insert the ear bars and rest them on the bones slightly rostral to the ears and pull down the skin to create good access to the skull. Tighten the ear bars and install the nose piece. Maintain the animal's body horizontally and at the level of the head using a height-adjusted support.
Place a heating pad under the animal to keep it at physiological temperature. Then clean the skull by applying 0.9%sodium chloride with a cotton swab to remove soft tissue. Adjust the skull so that the bregma lambda access is leveled.
Then, locate the injection site on the skull according to the posterior and medial coordinates and position the injection needle above it. Mark the skull with a disposable needle. Subsequently, move the injection needle upward by four centimeters.
Using a 0.5 millimeter burr, create a one millimeter diameter craniotomy on the mark at one half of the maximum speed. Swab with a tissue if any bleeding occurs. Next, empty the water contained in the Hamilton syringe for storage by completely ejecting it with a pump.
Only the needle is filled with water. Thaw the viral solution to be injected on ice, then, briefly remove it from the ice and obtain 700 nanoliters of the solution with a micropipette. Next, deposit a drop on a piece of paraffin film.
Place the paraffin film on top of the craniotomy. Plunge the needle in the drop of viral solution without changing the anteroposterior and lateral position. Afterward, use the withdraw function of the pump to fill the syringe with about 500 nanoliters of the viral solution on the paraffin film.
Perform this procedure under the stereoscope, watch the drop disappearing, and make sure not to aspirate any air. Make sure the syringe has been filled correctly. Test the ejection system by driving down the plunger to test eject a small drop of liquid.
Subsequently, insert the needle into the brain to the chosen depth. Push the run button for injection. Then, slowly withdraw the needle over three to five minutes.
Immediately wash the needle in clean distilled water by filling emptying it several times in order to avoid clogging. Afterward, remove the animal from the stereotaxic frame. Suture the skin and make three or four stitches tied with 2-1-1 standard surgical knots.
To extract the brain, make a cut on the skin from the neck to nose, then section the last vertebrae from the skull with scissors. Retract the skin and cut the skull along the midline from caudal to rostral direction. Carefully remove the parietal bone and the caudal part of the frontal bone.
Extract the brain with a small rounded spatula by inserting it between the brain and the cranial floor, sectioning the olfactory bulb, optic nerve, and other cranial nerves and cerebellum. Gently submerge the brain in ice cold cutting solution. Subsequently, transfer the brain to a filter paper and gently dry the cortical surface.
Glue the brain cortex to the specimen holder of a vibratome with the caudal side facing the blade in order to cut horizontal brain slices. Next, fill the cutting chamber with ice cold oxygenated cutting solution so the brain is fully immerged. Section 300 micrometer thick slices with the vibratome at a speed of 07 millimeters per second at one millimeter amplitude.
At this stage, it is recommended to briefly check the Chronos-GFP expression in the thalamus using a fluorescent flashlight and corresponding filter glasses. To perform whole-cell patch-clamp recording, gently transfer a brain slice containing the hippocampal complex to the recording chamber. Continuously perfuse the recording chamber with 34 degrees Celsius ACSF bubbled with carbogen at two to three milliliters per minute.
Briefly examine Chronos-GFP expression in axon terminals in the region of interest with blue LED illumination at 4X magnification. Change to a 63X immersion objective and adjust the focus. Check for axons expressing Chronos-GFP and choose a pyramidal neuron for patch recording.
Next, fill a pipette with potassium gluconate based internal solution. Mount it in the pipette holder on the head stage. Patch the cell in voltage clamp configuration.
Approach the identified neuron and delicately press the pipette tip onto the soma. The positive pressure should produce a dimple on the membrane surface. Then, release the pressure to create a gigoOm seal.
Once sealed, set the holding voltage to minus 65 millivolts. Break the membrane with a sharp pulse of negative pressure. Record the responses of the neuron to hyperpolarizing and depolarizing current steps in whole-cell current clamp mode.
Record in current or voltage clamp postsynaptic responses to 475 nanometer LED whole-field stimulation of afferent fibers expressing Chronos. Stimulate with trains of ten stimulations of two millisecond duration at 20 hertz. The activity of presubicular neurons in layer three in response to hyperpolarizing and depolarizing current steps was recorded in the whole-cell patch-clamp configuration.
Stimulating ADN axon terminals expressing Chronos-GFP elicited excitatory post-synaptic potentials in presubicular layer three principal cells in current clamp mode. Depending on light intensity, the EPSPs could reach action potential threshold. Post-synaptic responses were also observed in voltage clamp mode, as excitatory post-synaptic currents were elicited.
Shown here is a layer three pyramidal neuron surrounded by thalamic axons expressing Chronos-GFP in presubicular superficial layers with DAPI staining imaged with an epifluorescence microscope. And this image was taken at a higher magnification with a confocal microscope. Careful leveling of the bregma along the axis in performing pilot experiments with a fluorescent tracer are crucial to improve the precision of the stereotaxic injection.
This procedure can also be used to investigate converging projections from different areas by injecting at two generative scene sensitive to two different wavelengths. The development of this technique has allowed precise anatomical and functional circuit analysis between distant brain regions in a cell type specific manner.
