Channelrhodopsin-assisted circuit mapping is a precision technique for functional mapping of long-range neural projections. Here, we show how to use this approach to investigate circuits in the auditory brain stem. In brain slices, axons from multiple sources are often intermixed, making it difficult to isolate individual inputs using electrical stimulation.
By using CRACM, this limitation can be overcome. Finding lambda and consistently defining the stereotaxic coordinate system is challenging as is keeping the duration of the procedure short. Having all steps carefully mapped out will increase your success rate.
Begin by spraying the surgery area with 70%ethanol to sanitize and place sterile towel drapes to cover the surgery area. Remove cage bedding to limit risk of asphyxiation from the recovery cage. Next, put a heating pad under the cage and provide a food and water source.
Next, transfer the animal to a stereotaxic frame and continue anesthesia. Insert a rectal temperature probe and switch on the homeostatic temperature controller. Then, apply ophthalmic ointment to prevent eyes from drying out.
Administer preemptive analgesic. Finally, shave the scalp with electric clippers. Disinfect scalp with three alternating swabs of povidone iodine and 70%ethanol.
Begin surgery by making an incision in the scalp along the midline, starting between the ears and continuing rostral to the eyes, exposing the lambda and bregma sutures. Push skin to the side and remove periosteum from the exposed bone if necessary. Next, mark the lambda suture with a surgical marker, position the tip of the nano-injector so that it is just touching lambda and zero the micromanipulator coordinates.
Use the nano-injector tip and micromanipulator to measure the difference in elevation between the lambda and bregma sutures. Adjust the palette bar height to bring lambda and bregma to within plus or minus 100 micrometer height difference. Map the injection site using the nano-injector tip and micromanipulator coordinate system and mark the site with a surgical marker.
Then, use a micromotor drill with a 0.5 millimeter drill burr to perform a craniotomy over the injection site. To ensure broad transfection of neurons in the target nucleus, use a nano-injector to make injections at various depths in the tissue and in the case of larger brain regions, like the inferior colliculus, make injections over the course of two or more penetrations at different X and Y coordinates. To inject the inferior colliculus, deposit 20 nanoliters of virus in the intervals of 250 micrometers along the Z axis between 2, 250 micrometers and 1, 750 micrometers depth.
For injections in the dorsal cochlear nucleus, deposit 20 nanoliters of virus at a depth of 4, 750 micrometers and 4, 550 micrometers respectively. After injecting at each Z coordinate, wait two to three minutes before moving the injector to the next Z coordinate to allow time for the virus to diffuse away from the injection site, reducing the probability that virus will be sucked up the injection tract when the nano-injector is repositioned. Then, after the last injection, wait three to five minutes before retracting nano-injector from the brain.
When the nano-injector is removed from the brain between penetrations and between animals, eject a small volume of virus from the tip to check that the tip has not clogged. After injections, use sterile PBS to wet the cut edges of the scalp and then gently move the skin back towards the midline. Close the wound with simple interrupted sutures using 6-0 nylon sutures.
Then, apply 0.5 to one milliliter of 2%lidocaine jelly to the wound. Remove the ear bars and temperature probe, turn of isoflurane, and remove the animal from the palette bar and transfer it to the recovery cage. Finally, monitor recovery closely.
Once the animal is fully awake, moving around, and showing no signs of pain or distress, transfer it back into its cage and return the cage to the vivarium. Use standard patch clamp methods to make recordings. Begin by placing the slice in a recording chamber under a fixed stage upright microscope and continuously profuse with artificial cerebrospinal fluid at two milliliters per minute.
Next, patch neurons under visual control using a suitable patch clamp amplifier. Lastly, during wholesale recordings, activate Chronos by delivering brief pulses of 470 nanometer light or ChrimsonR by brief pulses of 580 nanometer light through commercially available LEDs. Results indicated that ChrimsonR injection led to strong expression in the DCN with tdTomato fluorescence visible in cells and fibers.
In the contralateral ICC, fibers strongly labeled with tdTomato were clearly visible after three weeks, demonstrating the long-range trafficking capability of the ChrimsonR-tdTomato construct when injected into auditory brain stem nuclei. Optical activation of ChrimsonR elicted EPSPs in IC VIP neurons, indicating that ChrimsonR is a useful tool for long-range CRACM experiments when the experimental parameters demand the use of red light instead of blue light. However, we found that ChrimsonR was readily activated with blue light, showing the same threshold for blue light activation as Chronos.
For this protocol it is most important to make sure that the stereotaxic coordinates are correct and to confirm that the virus was expressed only in your targeted brain region. By exchanging the Chronos ChrimsonR viroconstruct with a different virus, for example, a cell specific Flex virus, you can answer several anatomical or physiological questions without changing the surgery protocol.
