Atipamezole

For experiments investigating general transduction efficiency three to seven mice were used per serotype and brain region (Figure 1). Animals were deeply anesthetized with a mixture of ketamine and medetomidine (KM; 2.5 mg ketamine-HCl and 0.02 mg medetomidine-HCl/25 g mouse weight) injected intraperitoneally, and positioned in a stereotaxic frame (Kopf Instruments, Tujunga, CA; Stereotaxic System Kopf 1900). A local anaesthetic (lidocaine) was applied subcutaneously before exposure of the skull. Small holes were drilled into the skull and injections were performed unilaterally using a thin glass pipette with 80 nl of virus solution (titer: 9.6 * 10¹¹ viral genomes (VG)/ml in PBS) at a flow rate of 20 nl/min (World Precision Instruments, Sarasota, FL; Nanoliter 2000 Injector). Glass pipettes (World Precision Instruments, Sarasota, FL; Glass Capillaries for Nanoliter 2000; Order# 4878) had been pulled with a long taper and the tip was cut to a diameter of 20-40µm. After the injection, the pipette was left in place for 3 minutes, before being slowly withdrawn. Coordinates for injections were (in mm: caudal, lateral, and ventral to bregma): striatum (0.9, 1.5, 3.2), hippocampus (-1.9, 1.6, 1.6), cortex (-2.9, 4.25, 2.5). After surgery, anesthesia was neutralized with 0.02 ml atipamezole. Mice were monitored daily and intraperitoneal injections of carprofen (0.2 ml of 0.5 mg/ml stock) were applied on the first days after surgery.
For injections of LPS (Escherichia coli 0127:B8, Sigma-Aldrich, Germany; Figure 4A), mice were anesthetized with 1-2 vol% isoflurane in oxygen and two µl of LPS dissolved in saline (5 µg/µl) were infused at a flow rate of 0.2 µl/min into the striatum (coordinates (in mm) relative to bregma: 0.5, 2.0, -3.5). The cannula was left in place for further 5 minutes before being removed.
In the experiments investigating retrograde transport (Figures 5, 6), three mice were unilaterally injected with 250 nl of a 4:1 mixture of rAAV5 solution (titer s.a.) and cholera toxin subunit B-alexa fluor 555 conjugate (Invitrogen, C-22843; 1 mg/ml in PBS) into the hippocampus (same coordinates as above). Surgery, pharmacology, and injection were carried out as above.
When analyzing the time-course of expression (Figure 7 and Figure S4), mice received 80 nl injections into the striatum (titer: 1.01 * 10¹² VG/ml; same coordinates as above). One hemisphere was injected with either a (self-complementing) scGFP/scCherry and the other hemisphere was injected with either a (single strand) ssCherry/ssGFP virus solution. Surgery, pharmacology, and injection as above.

All captures were approved by the Swedish Ethical Committee on Animal Research (application numbers C212/9 and C47/9) and the Swedish Environmental Protection Agency. Fieldwork was carried out in Dalarna, Sweden during February-March (winter) and in June (summer) 2010 and 2011. We selected six female and six male hibernating brown bears, two to four years old, previously fitted with global positioning system (GPS) collars and very high frequency (VHF) abdominal implants. One female was anesthetized during both years. We only anesthetized subadults to reduce the chance of encountering females with cubs in the dens and to avoid older animals, considered to pose greater risk to the capture team. Snow depth ranged from 80–120 cm with temperatures ranging from −15°C to +1°C.
We located bears using GPS and VHF radio collars/implants (Figure S1 and S2). All dens were between 5 and 20 km from plowed roads, so we used snowmobiles to transport equipment and the field team to within 200–800 m of the den. Once we had located the den entrance and removed the snow (Figure S3), a metal grate was placed over the entrance. Two field personnel held the grate over the entrance using their own body weight and were assisted by up to three more people if necessary to keep the bear in the den. Anesthetic agents were administered by remote darting through the grate (Figure S4) using a flashlight and CO₂ powered rifle (Dan-Inject®, Børkop, Denmark) fired from 0.3–3.5 meters distance. Darts were 3 ml with a 2.0×30 mm barbed needle (Dan-Inject®). The bears were anesthetized with a total dose of 0.6–2.5 mg of medetomidine (Domitor® 1 mg/ml, and Zalopine®,10 mg/ml, Orion Pharma Animal Health, Turku, Finland) and 31–125 mg tiletamine-zolazepam (Zoletil®, 500 mg/vial, Virbac, Carros, France). A second dart with a full dose was administered if the bear was mobile after 10 minutes. In four bears, 75–100 mg ketamine (Narketan 10®, 100 mg/ml, Chassot, Dublin, Ireland) was hand-injected before handling and for six immobilizations; 37–75 mg of ketamine was included in the initial dart.
Once anesthetized, we took each of the bears out of the den (Figure S5) and placed them on an insulated blanket. We measured temperature, heart rate, and respiratory rate in all bears. We were unable to obtain pulse oximetry readings with a veterinary sensor clip placed on the tongue, lip, ears, or vulva were from the first four bears during February, so we abandoned this for the remaining bears. Blood samples from the femoral artery were collected anerobically in pre-heparinized syringes from ten bears at 15–25 and 65–75 minutes from darting. The samples were immediately analyzed in a portable analyzer (iSTAT 1® Portable Clinical Analyzer, Abbott Laboratories, Abbott Park IL, 60064-6048, USA) with the bear captured both years only sampled during the second year. Blood gas samples and pH were corrected to rectal temperature. Intranasal oxygen was provided from a portable oxygen cylinder to eight bears via a nasal line inserted 10 cm into one nostril with an oxygen flow rate of 0.5–2.0 liters per minute after the first arterial sample was collected.
After sampling, we placed the bears back into the dens and antagonized the effects of medetomidine with atipamezole (Antisedan®, 5 mg/ml, Orion Pharma Animal Health, Turku, Finland) given intramuscularly at 5 mg per mg of medetomidine. We covered the entrance with branches and snow and the bears were left to recover undisturbed.
In June we recaptured bears by darting from a helicopter as previously described [16] (link). Ten bears were captured with 5 mg medetomidine combined with 250 mg zolazepam-tiletamine and one was darted twice for a total of 10 mg medetomidine and 500 mg zolazepam-tiletamine. Two smaller bears (22 and 28 kg) were immobilized with 2.5 mg medetomidine and 125 mg zolazepam-tiletamine. Sampling was conducted as described for February bears, except that a narrower time range was selected for each arterial sample (20–30 minutes and 60–65 minutes from darting).
Hypoxemia was defined as mild (PaO₂ 60–80 mmHg), marked (PaO₂ 40–60 mmHg), or severe (PaO₂<40 mmHg). Acidemia was defined as a pH <7.35, and acidemia was considered marked if pH <7.25. Hypocapnia was defined as a PaCO₂<35 mmHg and hypercapnia was defined as mild (PaCO₂ 45–60 mmHg) or marked (PaCO₂>60 mmHg). A paired two-tailed t-test was used to compare the first and second sample at both winter and summer captures, and between winter and summer for both the first and second samples. Bears not receiving oxygen were excluded from comparisons that included a second sample for the variables with direct relation to oxygen (PaO_2, PaCO_2, SaO₂, HCO₃ and pH).

On the day of the study, a cephalic venous catheter was placed in each dog. The CRI dosages in this study were derived from our pilot study results. The dexmedetomidine CRI solution was made by mixing 0.2 mL of dexmedetomidine (Dexdomitor^®, Zoetis Inc., Kalamazoo, MI, USA) with 9.8 mL of physiological saline, resulting in a total volume of 10 mL at a concentration of 10 μg/mL. It was administered using a Medfusion^® syringe pump (Model 3500 V6, Smiths Medical, St. Paul, MN, USA).
For the atipamezole CRI solution, the total amount needed was calculated at 50 µg/kg. The calculated volume was withdrawn from the original drug solution (Antisedan^®, Zoetis Inc., Kalamazoo, MI, USA) and then diluted with sterile sodium chloride 0.9% to achieve a total volume of 5ml. This volume was delivered using the Medfusion^® syringe pump (Model 3500 V6, Smiths Medical, St. Paul, MN, USA) at the rate specified in the study. Both infusions were administered through an IV extension set directly attached to the preplaced IV catheter.
The awake baseline of the vital signs (see below) and EEG were recorded before the sedation started. Thereafter, the sedation procedure commenced with an initial intravenous dose of 10 μg/kg (or 60 μg/kg/h) dexmedetomidine administered over 10 min. This sedation was maintained through three decremental CRI doses, each lasting 15 min (Table 1). All infusions were controlled using a syringe pump, with dosages sequentially decreasing as follows: 3 μg/kg/h, 2 μg/kg/h, and finally, 1 μg/kg/h. The complete duration of the study when dexmedetomidine was administered was 55 min. Following the last dexmedetomidine CRI administration, the dogs promptly received atipamezole treatment through an intravenous dose of 50 μg/kg administered over 5 min (or 600 μg/kg/h).
The treatment protocol and timeline details are provided in Table 1, in addition to the continuous collection of EEG data every 2 s by the EEG monitor throughout the entire experiment. Analgesic properties were assessed (see below) by application of noxious stimuli (NOX) as indicated in Table 1. Furthermore, cardiorespiratory assessments were conducted at intervals of every 3 min within each phase of the experiment.