Twenty-one naive adult male Wistar rats were individually housed and maintained in a 12-h-light-dark cycle (8:00–20:00 h). Food was available ad libitum but access to water was restricted to the daily experimental 15-min drinking sessions at 10:00 h and to a daily additional 20-min rehydration session at 16:00 h. Water Baseline (BL) was recorded during the morning sessions in which the rats were handled for 3–5 min after BL1, BL3 and BL5 in order to avoid stress. After the water intake baseline, the animals were randomly assigned to one of the following groups: Novel (n = 8), Familiar (n = 5) and Control (n = 8). A cider vinegar solution (3%) was available during one (Novel) or two (Familiar) experimental sessions. The control group drank water throughout the experiment.

Ninety minutes after the experimental session each animal was sacrificed, the brain was removed and quickly frozen in isopentane (2-methylbutane; Sigma-Aldrich, Germany) to be stored at − 80 ºC. Brain coronal Sections (20 µm) were cut in a cryostat (Microm, HM505E, Germany). From each brain, sixty sections were taken in order to assess CCO in BLA, Cpu and Pir. The stereotaxic coordinates of the brain areas assessed according to the Paxinos and Watson atlas [18] are shown in Fig. 1.

Anatomical location of Regions of Interest. BLA = Basolateral amygdala (− 2.52 mm), Cpu = Caudate/Putamen (− 0.12 mm), Pir = Piriform cortex (− 2.52 mm). Anteroposterior coordinates according to bregma [17]. All coordinates were taken from coronal brain slices

The procedures were approved by the University of Granada Ethics Committee for Animal Research and by the Regional Ministry of Agriculture, Fisheries and Rural Development of Andalusia (17-02-15-195), following the ARRIVE guidelines and in accordance with the EU Directive 2010/63/EU for animal experiments.

The procedure for quantitative CCO histochemistry has been described elsewhere [13, 19]. In brief, after obtaining sets of tissue homogenate standards from the Wistar rat at different thicknesses (10, 30, 50 and 70 µm) in order to quantify the enzymatic activity, both sections and standards were incubated for 5 min in 0.1 M phosphate buffer (7.6 pH) with 10% sucrose (w/v) and 5% glutaraldehyde (v/v) (Merck, Germany). The slides were then rinsed 3 × in a 0.01 M phosphate buffer (7.6 pH) with 10% sucrose (w/v) and 0.05 M Tris buffer (7.6 pH) with 275 mg/L hexahydrated cobalt chloride, 10% sucrose (w/v), and 0.5% dimethylsulfoxide (v/v) for 10 min. This was followed by 1 × 0.01 M phosphate buffer (7.6 pH). Then, the sections and standards were incubated in 0.0075% cytochrome-c (w/v), 0.002% catalase (v/v), 5% sucrose (w/v), 0.25% dimethylsulfoxide (v/v) and 0.05% diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Madrid, Spain). Both sections and standards were incubated for 5 min in 0.1 phosphate buffer (7.6 pH), at 37 ºC for 1 h. The reaction was stopped by fixing the tissue in buffered 4% (v/v) formalin 30 min RT. Finally, the slides were dehydrated, cleared with xylol and cover-slipped with Entellan (Merck, Germany). The intensity of the CCO staining was quantified through an optic densitometry analysis using a computerized image analysis system (MCID Elite, Interfocus Imaging Ltd., United Kingdom). The mean optical density (OD) of each region was measured in the right hemisphere using three consecutive sections. In each section, four non-overlapping readings were taken using a square-shaped sampling window that was adjusted for each region size, taking a total of 12 measurements per region and subject. These regions were averaged to obtain one mean per region for each animal. Then, OD values were converted to CCO activity units determined through the enzymatic activity of the standards measured spectrometrically.

Flavor neophobia and AN were assessed using a two-factor mixed ANOVA design that includes a between-groups factor Group with 2 levels (Control group drinking water; Familiar group drinking vinegar twice) and a within-subjects factor Days with 3 levels (Water baseline, first vinegar exposure, second vinegar exposure), being the rat ID the random factor (animals were randomly assigned to groups). Post-hoc Bonferroni tests were applied.

Given the need to sacrifice the animals for assessing brain CCO activity, an additional Novel group was required. Thus, a two-factor mixed ANOVA design was applied including a between-groups factor Group with 3 levels (Control group drinking water; Novel group drinking vinegar once, Familiar group drinking vinegar twice) and a within-subjects factor Region of Interest (ROI) with 3 levels (BLA, Cpu, Pir). Again, since animals were randomly assigned to the groups, rat ID was the random factor. Post-hoc comparisons were performed with post-hoc Bonferroni tests.