The overall goal of this experiment is to map odor evoked activities at the surface of the olfactory bulb of mice using intrinsic optical signals and flavoprotein autofluorescent signals or FAS imaging after having anesthetized the mouse and expose the skull. The bone above the olfactory bulb is thinned. A bone flap is drawn using the tip of a scalpel blade and then removed a chamber is made of dental cement and surrounds the exposed surface of the olfactory bulb.
It is filled with aros and a cover glass is placed on top of this preparation. Olfactory maps are acquired sequentially in the same mouse using intrinsic optical signals and FAS imaging, Imaging other evoked activities in the mouse olfactory bulb using optical reflectance and autofluorescent signals can help answer key questions concerning the special coating of orders in the olfactory bulb. The main advantage of this technique over existing methods such as two doxy glucose imaging or FMRI is to make it possible to record order evok activities at a special resolution of a single glamor list.
Generally, individuals new to these methods will have difficulties in the beginning of their work because both surgical and instrumental skills are slow to acquire. Visual demonstration of this method is critical to draw attention on the crucial steps of the surgical and imaging procedures. Pinch the hind paw of the mouse to confirm anesthesia and remove the hair from the scalp using clippers.
Wipe any residual hair off the skin with sterile gauze pads soaked in distilled water. Place the mouse in the stereotaxic frame such that the snout is in the same plane as the back of the head. In order to position the olfactory bulb horizontally, firmly secure the ear and nose bars in order to prevent movement.
Apply ophthalmic ointment to the eyes to prevent drying during imaging, and then disinfect the scalp area with Betadine using sterile scissors. Make an incision at the back of the head between the ears, then cut in both sides towards the base of the ear and in the anterior posterior direction towards the forehead along the eyelids. Finish removing the scalp by cutting the skin on the snout.
Using the nose bar as a guide. Position the animal under a stereo microscope and use a cotton swab soaked with saline to gently detach the periosteum on the top of the skull. Use a pair of forceps to remove the remaining tissue and scrape the surface of the skull with a scalpel to ensure a clean preparation in order to keep the olfactory bulb area moist throughout the experiment.
Place a piece of absorbable gelatin, sponge soaked with distilled water on the bone above the olfactory bulb located between the eyes. Remove the gelatin sponge and start by gently scraping the bone with a number 10 scalpel blade. Keep a constant angle of 45 degrees between the blade and the bone and move the blade from the eyelids to the sagittal side of the bulb area.
Being careful not to apply vertical pressure on the bone or scratch the bone above the venous sinus during the thinning process. Stop every five minutes and place a hydrated gelatin sponge on the bone to cool down the preparation. Swab the skull with the sponge to remove bone, dust and wipe the tip of the scalpel periodically to keep it clean and keep it sharp.
Continue until the spongy bone layer or trabecula is visible. When the fine vasculature of the olfactory bulb is visible, stop scratching the bone and use the tip of a number 11 scalpel to draw a rectangular area and closing the olfactory bulb. Keeping the window within the boundary of the venous sinuses continue to scratch the bone within the rectangular area.
Be extra cautious of the depth of the scalpel tip to avoid touching the dura surface. In order to get a sense of the thickness of the remaining bone, push it gently with the tip of a para of forceps. If the bone folds under pressure, move to the next step.
Add a drop of saline and use the tip of the scalpel oriented horizontally to lift up the bone flap. Carefully remove the flap using forceps to avoid tearing off the remaining bone. Once the olfactory bulb surface is exposed, check for the absence of any bleeding or blood vessels.Anastomosis.
Wipe the area with a gelatin sponge soaked in saline. In order to keep the olfactory bulb moist, apply poly acrylate dental cement using a 29 gauge syringe to form a well on the bone around the cranial window. Place a drop of low melting point aros over the dura and place sterile cover glass over the cranial window for imaging.
Place the stereotaxic frame under the stereo microscope with the cranial window centered in the field of view focus to bring the capillaries into view. Switch to 580 nanometer green light and capture an anatomic control image to check the state of the preparation during imaging. Then proceed to capture the stimulation trials using either intrinsic optical signal imaging under 630 nanometers illumination or flavoprotein autofluorescent signal imaging Under 480 nanometers illumination, A standard imaging session includes a baseline of five to 10 seconds where only air is delivered, followed by the odor stimulation for three to 10 seconds depending on the chosen odor concentration.
And finally, 70 to 82 seconds of air delivery is recorded for baseline recovery. Here, the vasculature of the olfactory bulb is imaged through the cranial window, followed by the results of a stimulation trial using h sinal as the odorant captured using both iOS and FAS imaging. Using iOS imaging, odor evoked activity is shown as dark areas of absorbence areas activated by AL are indicated by the white arrows, and active areas can be seen in both single and averaged multiple trials.
Switching to FAS imaging in the same mouse OD evoked activity is now shown as white areas of autofluorescence emission indicated by the black arrows, and can also be seen in both single and averaged multiple trials. Notice that both the black zones of absorption seen using iOS imaging and the white areas of autofluorescence emission seen using FAS imaging overlap. This spatial match confirms that the two techniques allow the visualization of the same olfactory glomeruli Once master imaging order evoked activities using optical reflectance and autofluorescence signals can be done in one hour for surgery and a couple of hours for image acquisition.
The main drawbacks of using these optical imaging methods are the fact that they cannot be applied to visualize olfactory maps in a way, free moving mice. And due to the limited penetration of photons into the biological tissues, they do not give access to the ventral olfactory glomeruli after its development in the late nineties, recording intrinsic signals in the olfactory system. Perhaps a way for researchers in the field of optical imaging to explore the characteristics of the special coating of od.
In olfactory bulb flavoprotein autofluorescence imaging is a promising technique to give new insights in the special coating of OD using endogenous optical signals.
