C-fos mesencephalic nucleus counts in Aggressive, Non-aggressive, and test controls males in a Social Interaction test reared in an enriched or a standard environment

<b>Morphometric and behavioral data are also available</b><b>see link bellow</b><b><br></b><b>Environmental enrichment (EE) protocol </b>During the EE in the brood boxes (8-28 days old), 3 different sets of enrichments were rotated between boxes every 7 days. After reallocation in home cages at 28 days of age, EE was applied in 3 periods with different enrichment sets. Within each period, EE home cages received weekly one of the three enrichment sets in a random order in such a manner that all EE animals were exposed to the 3 enrichment sets. The replacement of the enrichment items was done on a fixed day and time every week, and during this procedure all the animals were removed from the box and placed in a basket. Control animals underwent the same manipulation procedure.<br><b>Social Interaction Test</b> was performed between 118 and 130 days of age. This test is described in detail in Caliva et al (2017, Pout. Sci) including a figure with the schematic representation of the test procedure. Briefly, the SI test consists in encounters between an unfamiliar test adult male and a PC stimulus adult male, in the presence of the test bird’s female cagemate (audience). First, the test male and it’s female cagemate were placed in a central compartment separated by an opaque partition from a PC stimulus male. After 2 min, the test male and the PC stimulus male, remained in the same compartment, while the female cagemate was placed in a nearby compartment at one side of the apparatus, and used as a social audience. Immediately after, the central opaque partition was removed and the test and PC stimulus birds were allowed to interact. Direct interaction lasted a maximum of 10 min. However, if during the interaction a quail received more than 5 consecutive aggressive pecks, showed a clear and continued escaping (retrieval) behavior, and/or showed any sign of physical damage, the interaction was immediately interrupted. A video-camera was positioned 1 m above the apparatus and connected to a computer that allowed constant monitoring and recording during the test while out of the sight of the birds. Using ANY-maze the following aggressive behaviors were recorded: pecks, grabs, mounts, cloacal contacts, threats, chase, and attack with claws. Herein, when grabs, mounts or cloacal contacts were performed by one male towards another male, they were considered as aggressive behaviors. Males that performed more than 5 aggressiveness behaviors were considered aggressive (Ag), and males that did not perform any aggressive behavior towards PC opponent were considered non-aggressive (nonAg). Males exposed to the experimental apparatus without the presence of an opponent (testCON) and non-manipulated males that always remained in the home cage (Naive) with their cagemate were used as control groups. <b>Perfusion, fixation, cut and storage. </b>Sixty minutes’ post SI test, the quails were anaesthetized intra-peritoneal with 30% chloral hydrate and perfused transcardiacally with a bloodwashing solution consisting of 0.8% sucrose, 0.8% NaCl and 0.4% glucose followed by 4% paraformaldehyde in 0.2M borate buffer pH 7.4. The brains were left in the skull overnight at 4 °C, after being removed from the skull and placed in 30% sucrose. The brains were cut either coronally at 50 µm on a freezing microtome and the sections were collected serially in the same fixative solution into five compartments and stored at -24 C in cryoprotection buffer (propylene glycol 30 % sucrose in 0.2 M PB) until required for the immunohistochemical procedures. Naive animals were anaesthetized directly in their home cages.<br><b>c-Fos Immunohistochemistry</b>. For each experimental group eight mesencephalic sections were studied for each animal. The c- Fos label was applied according to the protocol previously described and validated in detail [25, 27,50]. The only exception was that a lower concentration of the anti-Fos primary antibody (SC-52, rabbit IgG, Santa Cruz, in PBST 1: 4000) was used. The specificity of c-Fos anti-body used in the present experiment is well documented in several mammalian and avian species.<br><b>Definitions of neuroanatomical areas. </b>The mesencephalic regions of interest were defined based on neurochemical and functional data previously published by Melleu et al. (2016). Briefly in this study, two levels (intermediate and rostral, A 2.0 and A 3.0 respectively, of stereotaxis of the brain of the quail, in each level, the five neuroanatomical areas of interest were analyzed, the GCt, this is a nucleus that is located at the dorsomedial level of the mesencephalon, dorsally to the nucleus nervi oculomotorii in the intermediate level, and below the posterior commissure at the rostral level. In both anatomical level, the other areas studied were, the nucleus mesencephalicus lateralis pars dorsalis (MLd), above the aqueduct (or tectal ventricle); the nucleus intercollicularis (ICo) which can be further divided into two regions, with respect to their position in relation to the MLd, the medial (ICom) and the lateral (ICol), and surrounding the lateral aqueductal expansion, a different cell layers can also be observed the stratum the stratum griseum periventriculare dorsalis (SGPd).<br> <b>Quantification of the immunohistochemistry. </b>The slides were then taken to an optical microscope (OptiCam Microscopy 0400S B.) coupled with a digital camera (ToupCam SCMOS0300KPA). The quantification of c-Fos-ir cells was performed on photomicrographs of two anatomical levels. The counting areas were positioned in center the regions of interest utilizing anatomical landmarks, principally the lateral expansion of the aqueduct (or tectal ventricle) to the neuroanatomical areas as ICo, MLd and SGPd, and nucleu nervi oculomotorii and posterior commissure to GCt . For each neuroanatomical area, it was measured the millimeters totals of counting zone. In each photomicrograph the total nuclei counted were divided into the total area, to obtain N/mm2 (N= total number of positive nuclei). The area were calculated utilizing the ImageJ software. The photomicrographs were then converted into binary format with a 0.35 threshold relative to the black and white signal level to exclude all faintly, subthreshold stained nuclei .The c-Fos positive nuclei were then manually counted within the square area also with the aid of ImageJ.