Mapping oxygen concentration in the awake mouse brain.

Experimental procedures Animal preparation and surgery

All animal care and experimentation was performed in accordance with the INSERM Animal Care and Use Committee guidelines. Adult C57BL/6 mice (3–6 months old, 25-–35 g, both males and female, housed in 12-hr light-dark cycle) were used in this study (n = 5 mice for olfactory bulb capillaries, n = 3 mice for cortex capillaries). Chronic glass cranial windows were implanted over the area of interest, either the olfactory bulb or the somatosensory cortex, with great care taken to not disturb the dura mater. The animals received anti-inflammatory and analgesic treatment (Carprofen, one daily 0.15 mg subcutaneous injection, administered pre-surgically and for the three days post-surgery), antibiotics (Ceftrioxone, one daily 0.25 mg subcutaneous injection, administered pre-surgically and for the three days post-surgery) and dexamethasone to reduce cerebral edema (one daily 60 µg subcutaneous injection on the day before surgery, directly before the surgery and the first post-surgical day). Surgical anesthesia was induced with ketamine-xylazine (100 mg and 8 mg per kg body mass, respectively). During surgery, the mice breathed a mixture of air and supplementary oxygen (the final inhaled proportion of oxygen was ~30%) and the body temperature monitored by a rectal probe and maintained at ~36.5°C by a feedback-controlled heating pad. A craniotomy was performed with great care taken not to apply great pressure to the bone and the area was regularly flushed with cool aqueous buffer solution to avoid damage or heating of the underlying tissue. Cover glass (~150 µm thick) was used for the window and sealed in place with photopolymerizable dental cement (Tetric EvoFlow, Ivoclar Vivadent, Schaan, Principality of Liechtenstein), which was also used to form a head-cap in which a titanium head-bar was also embedded. Habituation of mice to head restraint In the weeks preceding surgery, the animals were supplied with a treadmill (Fast-Trac, Bio-Serv, Flemington, New Jersey) in their cages that was similar to that which forms part of the restraint apparatus used during 2PLM recording (see below). In the days that preceded the surgery, the mice were gently habituated to handling, and provided with treats (sugar pellets, Bio-Serv). 2–3 days after the surgery restraint habituation began. The frame used for head restraint during the habituation and imaging consists of a metal frame in which the mouse’s head-bar is secured, and a treadmill wheel (similar to that in the animal’s home cage). This configuration allows the animal limbs and body to move freely while the head is stably fixed. All restraint-habituation sessions were carried out in the set-up that was used for imaging, with the animal kept in the near-dark at all times. Habituation sessions were performed multiple times per day over the course of 2–4 weeks, with the duration increasing from 5 min to >1 hr. Animals were considered ready for use in experiments when they could be easily fixed in the recording apparatus while awake, and their behavior during the sessions consisted of short bouts of locomotion (~30 s) separated by longer periods of stillness (5–15 min) during which measurements were performed. On the days on which recordings were taken, the animals were briefly anesthetized with isoflurane (2%, in air. Total duration of anesthesia 1 hr. Animals were considered ready for use in experiments when they could be easily fixed in the recording apparatus while awake, and their behavior during the sessions consisted of short bouts of locomotion (~30 s) separated by longer periods of stillness (5–15 min) during which measurements were performed. On the days on which recordings were taken, the animals were briefly anesthetized with isoflurane (2%, in air. Total duration of anesthesia