Preparation for intrinsic optical imaging

The head of the mouse is carefully shaved and cleaned with a tissue sprayed with 70% ethanol solution. The mouse is then moved under the surgery optical microscope (Leica) and held in place by a nose-clamp holder and ear-bars. The skin on top of the skull is opened with surgical scissors exposing an area extending from the right temporal muscle to approximately 5 mm left of the sagittal suture and approximately 5 mm caudal to the lambda and 5 mm rostral to the bregma. After removing the periosteum and cleaning the surface of the skull the exposed region is dried by a gentle blow of compressed air. The region corresponding to the barrel cortex is identied (1.5 mm caudal to the bregma and 3.5 mm lateral). A custom made plastic ring of 7 mm diameter is centered on the identied region and two small drops of super-glue are used to keep it in place. The external side of the plastic ring is then covered with vaseline and the cavity is lled with cortex buer to allow better transcranial visibility. A square of approximately 1 cm of side is cut from a cover glass to close the upper part of the plastic ring in order to avoid cortex buer evaporation. It is important to remove all the bubbles from the solution since they may interfere with imaging. Finally the whisker of interest on the contralateral side, which in our case was C2, is painted

with nail polish to be easily identied.

3.4.2.3 Acquisition of Intrinsic optical signal images

The mouse was transfered on the intrinsic optical imaging setup and kept still with a nose clamp. The holder was then rotated in order to have the part of the skull corresponding to the barrel cortex as at as possible. The camera was adjusted and focused to obtain a eld of view as homogeneously sharp as possible. The focus of the camera was then lowered by 300µm, corresponding to low L2/3 and L4 cells. The two optic ber light sources where positioned as to have an equally distributed illumination of the surface. Finally the selected whisker was inserted into a glass capillary that was attached to a piezoelectric actuator (PL-140.11 bender controlled by a E-650 driver; Physik Instrumente) triggered by a pulse stimulator (Master-8, A.M.P.I.). Images were recorded by a VDAQ Imager 3001 system (Optical Imaging) equipped with a low read noise charge-coupled device (CCD) camera (1M pixel, 50 fps, 10 bit) using a stable halogen light source (100 W) in combination with a light guide system and a 700 nm interference lter (30 nm bandwidth) Accolla et al. (2007). Typically, 9 trials where recorded with 20s intervals:

the rst one as a control and lacking the actual whisker stimulation and the eight others with whisker stimulation. A trial consists of a total of 50 images. The rst 10 images, spanning 1s, were used as a baseline period. This interval is then followed by 12 images, spanning 1.125 s, where the whisker is stimulated. Finally the post stimulus period ranges from image 23 to image

Figure 3.5: Thresholded intrinsic optical imaging signal overlapped to a tran-scranial recorded blood vessel pattern image. Scale bar 0.5 mm.

50. The visible response was usually observed in the range of frames 20 to 40.

At the end of the imaging session, an image of the brain's surface with the visible blood vessels pattern was taken using green light (546 nm interference lter). Intrinsic optical signal Images where imported in GIMP (GNU Image Manipulation Program) where after applying a Gaussian Blur (radius = 10) and an arbitrary threshold where overlapped to the blood vessels pattern to determine the location of the center of the selected barrel (Fig. 3.5).

3.4.2.4 Craniotomy

The mouse is then transferred back to the surgery setup. 20µl of dexametha-son are delivered by IM injection. The glass coverslip and the plastic ring are gently removed. The surface of the skull is cleaned from the remnant of super glue and vaseline with a q-tip. The center of the barrel is labeled on the skull with a surgical pencil and the regions of the skull which will not host the craniotomy are delicately scraped with the tips of surgical forceps

to remove the remaining dried periostium and increase the roughness of the surface. The temporal muscle is then circumspectly detached from the skull avoiding bleeding. A plastic ring similar to the one used for the intrinsic op-tical imaging is placed on top of the skull and centered on the barrel mark.

The bottom part of the ring is glued to the skull. A thin layer of super glue is also applied on the remaining portion of exposed skull outside the ring and the space left after detaching the temporal muscle. Before the super glue has completely dried, dental acrylic is applied everywhere excepted inside the plastic ring. The ring along with the dental cement forms a well which is need for the imaging phase to have enough liquid between the objective and surface of the brain. A craniotomy of slightly more than 3 mm is performed using a dental drill. When the groove created by the drill is deep enough the remaining island of bone will shift downward if carefully pressed with surgical tweezers and the bone can be removed pulling with a syringe needle (30 G). The craniotomy is covered with gelfoam soaked in cortex buer to stop any eventual bleeding and to protect the brain while the DNA mix is prepared.