CLASSICAL AVERSIVE CONDITIONING INDUCES INCREASED EXPRESSION OF MATURE-BDNF IN THE HIPPOCAMPUS AND AMYGDALA OF PIGEONS R. S. FARIA, a * C. R. SARTORI, b F. CANOVA a AND E. A. M. FERRARI a a Laboratory of Neural Systems and Behavior, Department of Structural and Functional Biology, Institute of Biology, University of Campinas – Unicamp, Cidade Universita ´ria Zeferino Vaz, Rua Monteiro Lobato, 255, P.O. Box 6109, Campinas, SP 13083-970, Brazil b Laboratory of Neurobiology of Pain, Department of Structural and Functional Biology, Institute of Biology, University of Campinas – Unicamp, Cidade Universita ´ria Zeferino Vaz, Rua Monteiro Lobato, 255, P.O. Box 6109, Campinas, SP 13083-970, Brazil Abstract—The expression of brain-derived neurotrophic factor (BDNF), which is found in the pro-BDNF, truncated- BDNF and mature-BDNF isoforms, changes with learning. Mature-BDNF shows a peak of late expression in the hippo- campus that is involved in the persistence of aversive mem- ory in rodents. However, the role of BDNF in the hippocampal synaptic mechanisms involved in the classical conditioning aversive memory in birds still needs clarifica- tion. This study investigated the late expression of BDNF in the hippocampus and amygdala of pigeons trained with tone-shock conditioning and the effects of intra-hippocam- pal infusion of anisomycin (Ani) in these changes. Seven days after implantation of intra-hippocampal microcannu- lae, adult pigeons trained with three tone-shock pairings were assigned to one of three groups: Conditioning and Ani (CondANI), Conditioning and saline vehicle (CondSAL) and Conditioning only (Cond). NAIVE group had no treat- ment or conditioning. Homogenates of tissues from the hip- pocampus and amygdala, obtained 12 h after training, were used to determine the content of mature-BDNF, truncated- BDNF and pro-BDNF using Western blotting. Higher values for mature-BDNF than for truncated- and pro-BDNF content were seen in the hippocampus of Cond and CondSAL birds, but not in the hippocampus of CondANI or NAIVE birds (p < 0.05). The values of mature-BDNF in the amygdala of all the three conditioned groups were higher than those observed for truncated- and pro-BDNF (p < 0.05), which indicates that the activation of this protein in the amygdala was not affected by the infusion of Ani in the hippocampus. The data indicate that the tone-shock conditioning induced the activation of molecular pathways of BDNF in the hippo- campus and amygdala of the pigeons. The decreases in the content of truncated- and pro-BDNF isoforms found in con- ditioned pigeons may suggest cleavage mechanisms induced by the training. Our data confirm previous observa- tions of rodent studies and extend these observations to pigeons, revealing that, in spite of the anatomical differ- ences between the hippocampus of rodents and pigeons, there are functional and molecular mechanisms that are conservative between the species. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: classical aversive conditioning, hippocampus, amygdala, pro-BDNF, truncated-BDNF, mature-BDNF. INTRODUCTION Exposure to aversive and threatening environmental events triggers defense responses and results in the formation of a memory of the aversive experiences that persists over time. Among such experiences, are those that result in classical aversive conditioning (Rescorla, 1968), a form of learning that occurs when an unconditioned aversive stimulus (US, such as a shock) is paired with a discrete neutral stimulus (for example, a tone). Following this pairing, the once neutral stimulus acquires conditioned aversive value and functions as a conditioned stimulus (CS) that can control defensive or fearful responses. Hence, when the animal is exposed to the CS alone, it will exhibit a conditioned fear response (CR; e.g., freezing), which is interpreted as the expression of aversive memory. Many studies have shown that classical aversive conditioning involves the occurrence of complex behavioral processes, which are organized in neural circuits that integrate different brain areas (Anagnostaras et al., 2001; Maren, 2001; Fanselow, 2010). Moreover, the process of classical aversive conditioning involves a sequence of molecular events, including the activation of gene transcription and the synthesis of various proteins which are implicated in the neuronal plasticity underlying the acquisition, consolidation, reconsolidation, persistence and extinction of the aversive memory (Brito et al., 2006, 2011; Bekinschtein et al., 2007; Mizuno et al., 2012). The hippocampus participates in the aversive conditioning of both context and discrete stimuli, whereas the amygdala is indicated to be essentially involved in the conditioning to discrete stimuli (Maren and Baudry, 0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.09.054 * Corresponding author. Tel: +55-19-3521-6200; mobile: +55-19- 96913627. E-mail address: rodolfo_sfaria@yahoo.com.br (R. S. Faria). Abbreviations: Ani, anisomycin; ANOVA, analysis of variance; AP, anteroposterior; BDNF, brain-derived neurotrophic factor; CAU, Careful Exploration; Cond, Conditioning only; CondANI, Conditioning and Ani; CondSAL, Conditioning and saline vehicle; CS, conditioned stimulus; EXP, Exploration; FRE, Freezing; L, lateral; LOC, Locomotion; MOV, Discrete Movements; PRN, Preening; RST, Resting; SEM, standard error of mean; tPA, tissue plasminogen activator; VIG, Vigilance. Neuroscience 255 (2013) 122–133 122