Monoamine brain content of mainland and island populations of Podarcis siculus
Description
In accordance with the permit of the Ministry of Environment and Energy (Class: AP /L612-07 I 18-481 65; No: 517-07-l-l-l-18-4), and the permit of the Ethical committee of the Faculty of Science, University of Zagreb, Croatia (No: 251-58-10617-18-16), animals (10 males from each location) were captured by noosing, and transported to the laboratory in cloth bags. Since the population on islet Pijavica is strictly protected, this was the maximum number of animals we were permitted to collect. In the laboratory, animals were maintained in individual terraria (40x30cm) with peat as a substrate, fed every two days with crickets (Acheta domestica), that were supplemented with calcium and vitamins, with water available ad libitum. The light cycle was the same as the natural cycle (14:10 h, light: dark). Room temperature varied between 22°C during the night and 30° C during the day, to simulate natural day-night temperature variation. Lizards were allowed to habituate to laboratory conditions for one week before being subjected to behavioral testing. Brain samples were collected two weeks after the last behavioral test (beginning of July 2018). The experiments were conducted in accordance with the Directive of the European Parliament and of the Council (2010/63/EU) and the Croatian Animal Protection Law (“Narodne Novine”, 102/17 and 32/19). All efforts were made to reduce the number of animals used and to ensure animal welfare.
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Neurochemical measurements Brain tissue collection After behavior trials were completed, brain samples from both lizard populations were collected on two consecutive days between 10:00 and 12:00 h. Each animal was brought to the laboratory, located adjacent to the animal room, in its own terrarium and immediately put in a Falcone tube with 5% isoflurane. Anesthetized animal was decapitated, and the whole brain was quickly isolated, weight, put on dry ice and stored at -80C until further processing. The average lizard handling time before decapitation was 5.05 ± 1.98 minutes, while the average brain handling time before freezing was 3.85 ± 1.04 minutes. Neither of the handling times significantly differed between the two populations (p=0.135 for lizard handling, and p=0.136 for brain handling). ELISA Brain samples were homogenized in 4 volumes (w/v) of 0.01 N hydrochloric acid solution containing 1 mM EDTA and 4 mM Na2S2O5 using an ultrasound homogenizer (Bandelin Sonopuls). Tissue homogenates were then centrifuged at 24,000 x g for 20 min at 4°C, and aliquots of the clear supernatant were used for the measurements. 5-HT concentration was determined using the Serotonin Research ELISA kit, and DA, NA and ADR concentrations using the 3-CAT Research ELISA (Demeditec Diagnostics GmbH, Germany), according to the kit instructions. Samples were assayed in duplicate in a single assay (one for 5-HT and one for the three catecholamines), after determining optimal sample concentrations for each assay. Intra-assay coefficients of variations (M ± SEM) were 3.25±0.42% for DA, 3.48±0.41% for NA, 3.64±0.51% for ADR, and 4.88±0.54% for 5-HT. A calibration curve was drawn based on the absorbance measured at 450 nm on a microplate reader (Bio-Rad, Germany) and known concentrations of the standard solutions. Concentration values of samples were obtained by interpolating them to the calibration curve, using 4-parameters non-linear regression curve fitting. Results were expressed in pg of 5-HT/DA/NA/ADR per mg of wet brain tissue.