Bruno Cozzi

The amygdaloid complex, also known as the amygdala, is a heterogeneous group of distinct nuclear and cortical pallial and subpallial structures. The amygdala plays an important role in several complex functions including emotional behavior and learning. The expression of calcium‐binding proteins and peptides in GABAergic neurons located in the pallial and subpallial amygdala is not uniform and is sometimes restricted to specific groups of cells. Vasoactive intestinal polypeptide (VIP) is present in specific subpopulations of GABAergic cells in the amygdala. VIP immunoreactivity has been observed in somatodendritic and axonal profiles of the rat basolateral and central amygdala. However, a comprehensive analysis of the distribution of VIP immunoreactivity in the various pallial and subpallial structures is currently lacking. The present study used immunohistochemical and morphometric techniques to analyze the distribution and the neuronal localization of VIP immunoreactivity in the rat pallial and subpallial amygdala. In the pallial amygdala, VIP‐IR neurons are local inhibitory interneurons that presumably directly and indirectly regulate the activity of excitatory pyramidal neurons. In the subpallial amygdala, VIP immunoreactivity is expressed in several inhibitory cell types, presumably acting as projection or local interneurons. The distribution of VIP immunoreactivity is non‐homogeneous throughout the different areas of the amygdaloid complex, suggesting a distinct influence of this neuropeptide on local neuronal circuits and, consequently, on the cognitive, emotional, behavioral and endocrine activities mediated by the amygdala.

Infrared thermography (IRT) has been recently applied to measure lacrimal caruncle temperature non-invasively since this region is related to the sympathetic response, and it seems a promising technique that is able to infer negative emotions in sheep (e.g., fear). However, the scientific literature so far is limited in understanding whether a caruncle’s temperature changes also in response to positive emotional states in sheep. Through classical conditioning, we aimed to assess how a positive or a negative event affects the physiological (lacrimal caruncle temperature measured with IRT and cortisol levels) and behavioral responses of sheep (ear position). Fourteen ewes from the same flock were randomly assigned to two treatment groups: positive (n = 7) and negative (n = 7). Each group was then trained through classical conditioning to associate a neutral auditory (ring bell) stimulus to an oncoming event: for the positive group, the presence of a food reward (maize grains), while for the negative one, the opening of an umbrella. After three weeks of training, before (at rest) and after (post-treatment), lacrimal caruncle temperature was non-invasively measured via IRT, and saliva samples were gently collected to measure cortisol levels. During treatment, sheep behavior was videorecorded and then analyzed using a focal animal sampling technique. At rest, the eye’s lacrimal caruncle temperature was similar in both groups, while post-treatment, a significant increase was shown only in the negative group (t-test; p = 0.017). In the anticipation phase, sheep in the positive group kept their ears forward longer compared to those in the negative one (Mann–Whitney; p < 0.014), 8.3 ± 2.1 s and 5.2 ± 4.2 s, respectively. The behavioral response observed reflects a learnt association between a neutral stimulus and events with different emotional valence. Cortisol concentration slightly increased in both groups post-treatment. Our results confirm that IRT is a non-invasive technique that can be useful when applied to assess how positive and negative events may affect the physiological response in sheep.