Experienced dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters. One of the well-established experienced dependent genes is activity regulated cytoskeletal associated protein or arc.
The transcription of ARC is rapidly and highly induced by intensified neuronal activity To enable the examination of arc expression in live neurons by two photo microscopy, we make use of a knockin mouse line in which A GFP reporter is placed under the control of the endogenous arc promoter. This protocol describes the surgical preparations and imaging procedures for tracking experienced dependent arc GFP expression patterns in neuronal ensembles in the live animal. First, we'll describe the animal preparation needed for this technique.
An arc GFP animal will undergo cranial window surgery and recover from surgery for approximately two weeks. The cranial window quality is then checked and if neurons can be imaged through it, the animal is placed in a qua environment until the beginning of behavioral and two photon imaging experiments. These animals are then repeatedly imaged by two photo microscopy.
After desired behavioral paradigms are completed over the course of several days, clean all tools in a hot bead sterilizer. Before aseptic surgery, clean the surgery site with 70%ethanol and put down clean drop cloths. Anesthetize the animal with 1.2%Everton solution given at 0.02 milliliters per gram intraperitoneal.
Check the anesthesia level using tail or toe pinches to ensure full sedation. Cover the animal's eyes with sterile ophthalmic ointment and inject Dexamethasone 0.2 milligrams per kilogram and carprofen five milligrams per kilogram subcutaneously. Shave the hair over the skull between the ears and sterilize the skin with Betadine scrub and three alternating swabs of 70%Ethanol mount the animal in a stereotaxic surgery stage with ear bars with a water circulation heating pad below the animal set at 37 degrees.
Regularly check the animal's anesthesia levels and supplement the original anesthetic dose as needed. Inject 200 microliters of 0.5%Marcaine under the scalp skin to numb the area, incise the skin, and remove the skin flap over the skull. Remove the periosteum and dry the area with clean cotton swabs.
Use a high speed dental drill with a 0.5 millimeter burr to gently outline a three to five millimeter diameter circle over the brain region of interest depending on your needs. Periodically wet the drilling site with sterile 0.9%saline and clear away bone dust with clean cotton swabs. If the bone bleeds, use gel foam pre soaked with sterile saline to blot the bleed and wait for it to stop.
When the last bone layer is reached, lift and remove the bone island with fine tipped forceps. You may encounter dural attachments to the bottom shelf of the bone. If your window crosses of bone suture, these should be gently removed.
As the bone flap is lifted, roll moist and gel foam gently over the exposed dura to clean its surface and wait for any dural bleeding to stop. If your region of interest is under a location with a dura is particularly thick, removing the dura above it may be necessary. If this is the case, gently separate the dura from the PIA below with very fine forceps.
Make a small incision in the lifted dura with another pair of fine forceps. Then grasp the cut edges and gently split the dura. The dura is thin but strong, so be careful not to slice the brain with the intact edges of the dura.
As you work back and away from your region of interest, rinse the area liberally with sterile A CSF or sterile saline. If the surrounding skull curves significantly around the exposed dura or pia, use quick cell adhesive to fill in spaces that would not make close contact with the window cover slip in regions that will not be imaged. Then lay a sterile glass cover slip gently over the dura or pia.
Use Sano acrylic gel to cover the elastomer adhesive and the edges of the glass. Cover slip. Cover the entire exposed skull with Sano cyanoacrylate gel or a crazy glue and dental cement mixture.
Embed a custom made metal head fixation bar at the opposite end of the skull. Return the animal to a warm recovery chamber after an intraperitoneal injection of ketoprofen five milligrams per kilogram for pain management. Continue the the analgesic for two days post-op.
After two weeks of post-operative recovery, anesthetize the animal with ISO fluorine. 5%for induction, 1.5%for maintenance. Mount it in a custom made microscope stage with a head fixation frame and check the cranial window for optical clarity.
Under illumination with blue light brain surface blood vessel clarity is highly indicative of cranial window quality. If their edges are sharply defined, the window is likely to be usable. Before beginning a behavioral protocol, the animal should be kept in a consistent home cage environment to minimize day-to-day variation.
In baseline arc, GFP expression levels commence behavioral training or environmental stimulation paradigms depending on the brain region of interest. For example, animals can be exposed to different visual environments over consecutive days to image distinctive populations of neurons in the visual cortex arc, GFP fluorescence in neurons typically reaches its peak level two hours after stimulation. The experimental timeline may be optimized to facilitate the detection of ARC GFP expression in neurons at their peak fluorescence levels.
After a behavioral training session or environmental stimulation is completed, anesthetize the animal with ISO fluorine 5%for induction, 1.5%for maintenance. Mount the animal in the custom made microscope stage with head fixation frame. Under the two photon microscope, the animal's head position is fixed to the head fixation frame that connects directly to the stage using the implanted metal bar on the skull.
ISO fluorine and oxygen is continuously supplied to the mouse through a nose cone. Body temperature is maintained using a heating pad. Ensure that the microscope detectors are protected from ambient light.
By conducting two photon laser scanning in a dark room, use a 20 x or 25 x 1.05 numerical aperture water immersion lens for imaging. First, under epi fluorescence illumination, acquire an image of the surface blood vessel patterns over the brain region of interest with a CCD camera. For future image alignment, start two photon laser scanning to acquire a 3D image stack.
The excitation wavelength of the two photon laser is set at 920 nanometers and the power is set at approximately 50. Milliwatts emitted fluorescence is simultaneously detected in both a green and a red channel arc. GFP fluorescence only appears in the green channel, whereas tissue autofluorescence appears in both channels.
Typical image stacks have dimensions of approximately 320 by 300, 20 by 100 micrometers, a horizontal resolution of 0.5 micrometers per pixel, and a vertical resolution of three micrometers per pixel. After acquiring the image stack, return the animal to its home cage. Do not disturb the animal until the next behavioral and imaging session.
Repeat the behavior and imaging procedure across days as desired. Use the previously acquired brain surface blood vessel image to orient back to the same imaging location. The strength of this technique is that it allows for the examination of arc Gene expression changes in the same set of neurons over multiple days.
In the live animal. Standard HISTOCHEMICAL methods can achieve single cell resolution, but cannot track gene expression changes in the same neurons over multiple days. Other imaging techniques such as magnetic resonance or nuclear imaging methods lack the resolution to image individual neurons.
As such, no other existing methods to measure gene expression changes can match both the spatial resolution and the temporal coverage of the imaging method described here. A critical step in this procedure is the cranial window surgery. It should be performed with extra care to avoid trauma to the brain or leaving blood beneath the glass cover slip.
The dura can be removed if necessary. Keep in mind that the maximum imaging depth in the brain using this procedure will depend on the cranial window clarity, the microscope setup, and the brightness of the fluorescent reporters. This in vivo imaging protocol is an efficient and versatile method to obtain information about neuroplasticity related molecular dynamics in individual neurons in response to various behavioral experiences, and is likely to be generally applicable to transgenic mice carrying fluorescent reporters for other experienced regulated genes.
