DAY-NIGHT VARIATION IN THE AVERSIVE CLASSICAL CONDITIONING AND ZENK PROTEIN EXPRESSION IN THE HIPPOCAMPUS OF PIGEONS (C. livid)

*Corresponding author e-mail: fernando.canova@gmail.com Introduction: Many studies suggest that these behavioral processes are affected by the circadian timing system. The procedures of classical aversive conditioning and analysis of the conditioned freezing response are useful for the study of behavior and the underlying neural mechanisms. Previous studies showed the induction of Zenk expression in the hippocampus of pigeons after training in classical aversive conditioning. Methods: This study investigated day - night variations in the acquisition and retrieval of aversive classical conditioning and in the expression of Zenk protein in the hippocampus of pigeons. The Experiment I evaluated the conditioning to the context in pigeons attributed to conditioned (COND), control (CC) or naive groups (N). The Experiment II investigated the aversive conditioning to the tone in groups of pigeons that received tone-shock pairing (PS), unpaired tone and shock (NPS) or tone alone (TS). In both experiments the photoperiod was used with pulses of light (15 min) at ZT00 and ZT12 and the sessions conducted at ZT02 and ZT14. The results of the Experiment I indicated between groups differences in the occurrence of freezing ( p <0.05). The analysis of the Zenk-positive cells in the hippocampus showed a significant increase in the HPV of the COND ZT02 group compared with the other groups ( p <0.05), indicating a significant time difference when compared to COND ZT14 ( p <0.05). Higher density of Zenk-positive cells was found in the HpVM of COND ZT02 group compared to HpVL ( p <0.001) and to HpVM of other groups ( p <0.05). Experiment II data showed higher occurrence of exploratory risk assessment in PS and NPS groups (p>0.05) as compared to TS (P<0.5). The occurrence of freezing in the PS groups was different from TS groups (p <0,05). Higher Zenkpositive cells densities in HpV than in HpD were observed in the animals trained with paired or unpaired tone and shock stimulation (p <0.05). These PS and NPS groups showed no significant differences in the density of labeling of Zenk-positive nuclei in HpVL as compared to HpVM (p>0.05). Results and Conclusion: Data from both experiments showed different behavioral patterns during the exposure to the conditioned aversive context and to the conditioned aversive tone. The variations in Zenk expression indicate differential activation of the hippocampal regions during retrieval of aversive memory. The day-night variation in the conditioned freezing to the context as well as in the Zenk expression in the hippocampus suggests that these processes may be modulated by the circadian timing system. Support: FAPESP fellowship, Graduate Program in Functional and Molecular Biology; CNPq. Publication History Article published online: 16 June 2026 © 2009. Brazilian Sleep Academy. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/) Thieme Revinter Publicações Ltda. Rua Rego Freitas, 175, loja 1, República, São Paulo, SP, CEP 01220-010, Brazil