Thionembutal

After a three-day period of adaptation in the animal room, the starting
generation (S0) subjects were submitted to the sociability screening using the
test developed by Bonuti and Morato (8 (link))
in a MOF. The amount of time interacting with another rat was used as the
criterion for selective breeding. The male and female with the highest times of
interaction were put to mate in a separate cage, as was the second
high-sociability male and female. Likewise, the male and female with the lowest
times of interaction and the second lowest were put to mate in separate
cages.
This procedure was repeated when the descendants of S0 (S1) were 60 days old and
again when the descendants of S1 and of the successive generations reached that
age. From generation S3 onward, in order to increase the number of pups, males
were put to mate with two females, following the same socialization criterion.
Animals bred for high sociability were named SOC+ while animals bred for low
sociability were called SOC–. Animals that were not selected for breeding were
kept as a reserve, should any problem occur with the selected animals.
After weaning, the animals of one generation were tested in the MOF. Only then
were the animals of the previous generation killed with an ipbarbiturate overdose injection (Thionembutal, 1 g, Abbott, USA).

Animals were initially deeply anesthetized with the following intravenous anesthetics mixture: Levomepromazine (Neozine; Sanofi-Avensis, São Paulo, São Paulo, Brazil) 0.2–0.4 mg/kg; Midazolan (Dormicum; Roche, São Paulo, São Paulo, Brazil), 0.2–0.4 mg/kg; Ketamine Chloridrate (Vetanarcol; König, São Paulo, São Paulo, Brazil), 10–15 mg/kg. Just before perfusion they received Sodium Heparine 5,000 IU/ml (Roche), 1 ml i.v., to prevent blood coagulation followed by an intravenous lethal dose of Thionembutal (Abbott, Abbott Park, Illinois, USA), 35 mg/kg or higher. ECG was continuously monitored to ensure adequate depth of anesthesia and analgesia. Death was assessed by cessation of ECG activity. They were then transcardially perfused with 0.9% phosphate buffered saline solution (PBS, pH 7.2), 500 ml, followed by 10% formaldehyde in PBS, 2000 ml. The eyes were removed, the retinas were dissected, mounted on a gelatinized slide with the ganglion cells uppermost, exposed to formaldehyde vapor 60°C for 2–3 h, then stained by the method of Nissl. Briefly, the retinas were rehydrated, stained with 0.5% cresyl violet (Merck, Darmstadt, Germany) 50–60°C under microscopic control, dehydrated in graded alcohols, cleared in xylene, and coverslipped.