Retrieval of contextual aversive memory and induction of Zenk expression in the hippocampus of pigeons

Journal Pre-proof Retrieval of contextual aversive memory and induction of Zenk expression in the hippocampus of pigeons Ivana Brito, Luiz Roberto G. Britto, Elenice Aparecida M. Ferrari

PII:

S0361-9230(19)30423-X

DOI:

https://doi.org/10.1016/j.brainresbull.2019.09.013

Reference:

BRB 9781

To appear in:

Brain Research Bulletin

Received Date:

28 May 2019

Revised Date:

8 September 2019

Accepted Date:

27 September 2019

Please cite this article as: Brito I, Britto LRG, Ferrari EAM, Retrieval of contextual aversive memory and induction of Zenk expression in the hippocampus of pigeons, Brain Research Bulletin (2019), doi: https://doi.org/10.1016/j.brainresbull.2019.09.013

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

1 Retrieval of contextual aversive memory and induction of Zenk expression in the hippocampus of pigeons.

Ivana Brito* School of Arts, Sciences and Humanities, São Paulo University, São Paulo, Brazil e-mail: [email protected]

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Luiz Roberto G. Britto Institute of Biomedical Sciences São Paulo University, São Paulo, Brazil e-mail: [email protected]

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Elenice Aparecida M. Ferrari Institute of Biology Campinas University, Campinas, Brazil

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Highlights:

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*Corresponding author Escola de Artes, Ciências e Humanidades da Universidade de São Paulo - USP
Rua Arlindo Bettio, nº. 1000, CEP: 03828-000, Vila Guaraciaba - São Paulo – SP, Brasil.

As in mammals, hippocampus of pigeon is required for the formation and retrieval

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of context-aversive stimulation.

Zenk protein might be a molecular substrate of retrieval of conditioned fear memory in pigeons.

Differential activation in different parts of the avian hippocampus is associated with

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retrieval of fearful memories.

Abstract The hippocampus has a fundamental role in many learning and memory processes, which include the formation and retrieval of context-fear associations, as evidenced by studies in

2 rodents and birds. The present paper has analyzed contextual memory and Zenk expression in the hippocampus of the pigeon after fear conditioning. Pigeons were trained under four conditions: with 3 tone-shock associations (Paired), with shock and tone presented randomly (Unpaired), with exposure to the experimental chamber without stimulation (Control) and with only daily handling (Naive). The testing was conducted 24h after training. All sessions were digitally recorded. The level of freezing expressed by the Paired

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and Unpaired groups differed significantly from that of the control group during both

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training and test sessions. Pigeons from the Paired group revealed a significantly greater

density of Zenk positive nuclei in the ventromedial region of the hippocampus than did the

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Unpaired, Control and Naive groups. These data suggest that Zenk-mediated processes of

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synaptic plasticity in the hippocampus are induced during the retrieval of conditioned fear

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memory in the pigeon.

Keywords: hippocampus, fear conditioning, classical aversive conditioning, freezing

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behavior, Zenk.

Contextual fear memory has an important adaptive function for different organisms and animal models of fear conditioning are widely used for studying the neurobiology of

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learning and memory. Fear conditioning is a form of aversive classical conditioning in which an initially neutral stimulus (CS), such as a tone, is paired with an unconditioned aversive stimulus (US), such as a brief electric shock. After a certain number of pairings, the CS presented alone as well as the experimental context acquire a conditioned aversive value and elicit defensive responses (Ledoux, 1996; White and Salinas, 2003), which is

3 often evaluated by the observation of freezing behavior (Gewirtz et al., 2000; Duvarci and Nader, 2004; Conejo et al., 2007). Many studies have indicated that the hippocampus is required for the formation and retrieval of context-fear associations (Holt and Maren, 1999; Reis et al., 1999; Maren and Holt, 2000; Suzuki et al., 2004; Wiltgen and Tanaka, 2013). The traditional view has been that the retrieval of fear memory to a conditioned context would be disrupted by lesions in

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the hippocampus, whereas a fear memory for a discrete conditioned stimulus would not be

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affected (Kim and Fanselow, 1992). The evidence supporting this view implicated the

ventral hippocampus in the retrieval of memory to an aversive context (Richmond et al.,

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1999; Bast et al., 2001; Zhang et al., 2001). However, further studies have shown that the

1997) and to a tone (Bast et al., 2003).

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lesions in the dorsal hippocampus disrupt fear conditioning both to a context (Maren et al.,

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Results of investigations into the effects of lesions in the hippocampus have been paralleled by investigations of the mechanisms underlying fear conditioning, which have

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been provided by investigations of the role of NMDA and AMPA glutamate receptors of the hippocampus in aversive learning and memory (for a review see Riedel et al., 2003), and of the signaling cascade triggered by the NMDA receptor activation at the time of learning, which results in protein-dependent synaptic modifications. Signaling cascades

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activated at the time of learning can induce the transcription of immediate early gene such as zenk, which is part of the memory formation cascade and synaptic plasticity underlying different phases of memory (Freeman and Rose, 1995; Ishida et al., 2000; Wallenstein et al., 2001). The zenk gene (also known as zif-268, egr-1 or krox24) belongs to a category of regulatory immediate early genes (IEGs) that activate downstream target genes and are thus

4 considered to trigger the activation of genomic responses in neurons (Christy and Nathans, 1989; Cole et al., 1989; Wisden et al., 1990). Studies have shown that the zenk mRNA and the Zenk protein are upregulated during different forms of associative learning both in rodents and in birds (Mello et al., 1995; Jarvis et al., 1995; Velho et al., 2005; Rossignoli et al., 2017). Such up-regulation of zenk (zif-268), first demonstrated in rats by Hall et al. (2001), was found to occur in specific neuronal populations within the hippocampus during

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the retrieval of contextual fear conditioning. In the pigeon hippocampus (Brito et al., 2006)

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the expression of the Zenk protein in different neuronal populations has only been reported after fear conditioning acquisition, but not after the retrieval of aversive memory.

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The objective of the present study was to analyze the distribution of Zenk-positive

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2. Materials and Methods

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cells in the hippocampus after retrieval of contextual aversive memory in pigeons.

2.1. Subjects

Twenty-three adult male pigeons (Columba livia), 18 months old, weighing an

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average of 300 g each were used. They were housed in individual cages and kept under a 12:12 light dark cycle (lights on at 6:00 a.m.), with free access to food and water. They were distributed in four different groups according to specific behavioral procedures: one group was conditioned with tone-shock pairings (Paired; n=6); a second group was trained with unpaired tones and shocks (Unpaired; n=6); the third was exposed to the context alone with no scheduled stimulation (Control; n=7), and the fourth group, although handled, had

5 no exposure to training or test procedures (Naïve; n=4). The experimental protocol was approved by the Ethics Committee for Animal Experimentation of the Biology Institute, UNICAMP, Brazil (Protocol 834-2). All procedures are in accordance with the guidelines of the National Institutes of Health Laboratory Animal Care and Use Guidelines All experiments have adhered to the National Institutes of Health guide for the care and use of

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laboratory animals (NIH Publications no. 8023, revised 1978).

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2.2. Apparatus

The pigeons were habituated to laboratory conditions in a 60 x 60 x 60 cm chamber,

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illuminated with a white fluorescent lamp with three walls and floor covered with a white

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plastic laminate; the fourth was a one-way mirror. A 30 x 30 x 40 cm wooden chamber, illuminated by a red 25-W incandescent lamp, was used for training and testing in aversive

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classical conditioning. The inner walls and floor of this chamber (referred to as conditioning chamber) were covered with aluminum plates; the front door was a one-way

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mirror. An 80-W loudspeaker was located on a wall in the chamber, 20 cm above the floor and an electrical wire for shock stimulation was available to be connected to electrodes (Azrin, 1959), implanted around the pigeons’ pubic bones, three days before the experiments. A digital video camera was located 1 m away from the front of the

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conditioning chamber. A Berger AS-109 sound stimulator was placed in an adjacent room and connected to a timer for the automatic control of stimulus timing and duration together with a computer and a Foringer source connected to the electrodes’ wire for electrical shock presentation.

2.3. Procedures

6 2.3.1 Adaptation to laboratory conditions After at least 15 days of adaptation to the housing conditions, the pigeons were manipulated, that is, they were removed from their home cages and carried to the laboratory where they were weighed and left inside the habituation chamber for a 40 min period. This procedure, designed for minimizing the stress induced by experimental manipulations and

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enclosure in an experimental chamber, was conducted during a four-day period.

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2.3.2. Training

The Paired group was submitted to a fear conditioning with tone (1000-Hz, 85-dB,

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1s) and shock (10mA, 35ms) pairings presented at the 5th, 10th and 15th minutes of one

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single 20 min session. The Unpaired group was stimulated with non-paired tones and shocks that were presented at random moments, with a minimum inter-stimulus interval of

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1 min and the first stimulus presentation occurring at 3 min of the beginning of the session. Pigeons of the control group were exposed to the training chamber but received no

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programmed stimulus presentation. After the end of the training sessions, all birds were returned to their home cages. The Naive pigeons were taken to the laboratory, weighed and immediately returned back to the home cage, without any exposure to the experimental

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chamber. These training sessions were conducted between 8-9 h a.m.

2.3.3. Contextual fear memory Testing On the next day, 24 h following the training, the animals were re-exposed to the

training chamber and tested for conditioning to contextual stimuli. After the 20 min test session the pigeons were transported back to their cages, where they remained for 60 min.

7 The Naive birds were again taken to the laboratory, weighed assessment and returned to the home-cage where they remained for 80 min. All the sessions were video-recorded for posterior behavioral analysis.

2.3.4. Behavioral Analysis Transcription of the behavioral recording was conducted using the EthoLog 2.2

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software (Ethological Transcription Tool, 1995-99) (Ottoni, 2000). The behavior of the

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pigeons was observed by a trained observer who recorded the behavioral sequence

exhibited during the whole session. The subsequent behavioral analyses considered the

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accumulated frequency of behaviors in 8 blocks of 150s intervals.

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The following behaviors were noted (Brito et al., 2006; Reis et al., 1999): - Freezing (FRZ): defined as a characteristic tense and crouched posture where the bird

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displays (a) absence of observable behavior (immobility); (b) flexion of the legs with feet/legs widely separated; and (c) the pectoral region contacting the floor or one wall.

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Additionally, the bird shows (d) limited extension of the neck with the head directed toward a specific point in the environment, (e) flexion of the wings and tail with divergent up or down orientation (not aligned); (f) widely opened eyes and (g) accelerated breathing rate. - Resting (RST): defined as a characteristic relaxed immobility posture identified by (a)

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erect or crouched posture in the center of the chamber or with no contact with the lateral walls; with the bird displaying (b) whole or partial neck retraction; (c) ventral flexion of the neck and inclination of head toward the feet, with no fixed orientation to any specific point of the environment; (d) normal rate of breathing; (e) eyes remain open normally or partially closed.

8 - Discrete movements (MOV): in this category we included all the changes in spatial location of body parts resulting from discrete movements of extension, flexion, adduction, abduction or oscillation of one leg, wing, tail or head. - Pre-exploratory behavior (PRE): reflex responses elicited by the tone or shock stimulus, which precede the exploratory responses, such as startle, body contraction, body shaking. - Exploratory behavior (EXP): responses that result in orientation to particular points or to

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the whole environment, such as neck elongating, alert, head rotation, visual scanning (one

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horizontal slow, point to point, continuous head rotation, 180 degrees wide) and vigilance (sustained visual orientation to an unique point of the environment).

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- Locomotion (LOC): changes in the body spatial location resulting from walking, circling,

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running, flying, jumping.

- Maintenance (MAN): behaviors resulting in vegetative and tonic body adjustments

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2.3.5. Perfusion

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described as swallowing, throwing up, blinking, yawning and preening.

One hour after the end of the test session, the pigeons were anesthetized with ketamine (2 mg/100g, im) and xylazine (5 mg/100g, im) and underwent cardiac perfusion with 0.9% saline and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4).

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The brains were post-fixed in 4% PFA during a 4 h period and subsequently stored in a 30% sucrose solution at 4o C for at least 48h.

2.3.6. Zenk Immunohistochemistry

9 The brains were frozen and cut in 30-m coronal sections on a sliding microtome. The brain sections were first incubated free-floating for 12-16h at 4ºC with a rabbit monoclonal antibody against the Zenk protein (Sc-189; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:1000 in PB containing 0.3% Triton X-100. Tissue was then incubated with a biotinylated goat anti-rabbit serum (Jackson Laboratories, West Grove,

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PA, USA) diluted 1:200 in PB with 0.3% Triton X-100 at 22ºC for 1h. Finally, the sections were incubated for 2h with the avidin-biotin-peroxidase complex (ABC Elite Kit; Vector

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Labs., Burlingame, CA, USA). The reaction with 0.05% 3,3´-diaminobenzidine solution with 3 ml of 0.01% H2O2 during 4-6 min was intensified with 0.05% osmium tetroxide.

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The free-floating sections were washed in PB before each of these procedures. The sections

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were mounted on gelatin-coated glass slides, dehydrated in an ethanol series and

2.3.7. Cell labeling analysis

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coverslipped with Permount (Fisher, Pittsburgh, PA, USA).

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The quantification of immunoreactivity of neuronal nuclei within the hippocampus was conducted using the Image software (NIH, USA) (Rasband and Bright, 1995). Sections from both hemispheres were examined under a light microscope with a minimum of 4 sections from each brain being examined. The approximate coordinates are indicated in

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schematic representation of these sections (Karten and Hodos, 1967). The whole hippocampus was first inspected and the immunoreactive nuclei were counted at three different locations in each brain section: the dorsal, ventro-medial and ventro-lateral regions. The sample contours of the three regions of interest were manually performed following anatomic orientations. The dorsal region (Hp-D; see schema in Figure 1) was

10 defined as an area located between two virtual transverse lines: one crossing the hippocampus from the bend point of the lateral ventricle to the dorsal limit of the telencephalon and the other traced above the “V” area (inferior limit) (Hough et al., 2002; Kahn et al., 2003). The ventral region (Hp-V, see Figure 1) was considered as an area extending from the Hp-D and the intersection of the V – cell columns, which are clearly discernible in Nissl-stained material (Krebs et al., 1991). A straight line departing from the

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intersection of the two V – cell columns and extending to center of the lower limit of Hp-D

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separated the Hp-Vm and the Hp-Vl, with the former extending to the medial sagittal line

and the latter extending to the border of the lateral ventricle. The numbers of Zenk-positive

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nuclei were counted in the entire selected region. Although the sizes of the samples

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(volume) may present small variations, once they were manually delimited, the quantification of the immunoreactive cells by relationship between the total number of

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Zenk-positive cells per mm2 (density) equates the regions.

A threshold for stained cell counting was defined on the basis of background

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staining, with cell nuclei exhibiting optical density at least 3 times greater than the threshold. A size limit was established to exclude objects that were too small to be considered Zenk-positive nuclei (e.g. peroxidase-stained red blood cells or immunoperoxidase reaction artefacts) as well as to avoid situations in which a single Zenk-

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positive nucleus is quantified as multiple nuclei. As mentioned above, the results of this counting procedure were expressed as the density of Zenk-positive nuclei (Brito et al., 2006).

2.3.8. Statistical Analysis

11 Between-group differences related to the occurrence of each one of the behavioral categories were analyzed with a two-way ANOVA, with numbers of responses in each category (discrete movements, pre-exploratory, exploratory, locomotion, maintenance, relaxed immobility and freezing) as the dependent variable and group as factor. Freezing behavior in all the three groups was also analyzed with ANOVA; the number of instances of freezing served as the dependent variable, while the group (Paired, Unpaired and

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Control), session (training and testing) were the independent variables and time-intervals

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served as repeated measures. All the post-hoc multiple comparisons tests were conducted with the Tukey-Kramer test.

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The density of Zenk-positive nuclei computed for each of the three regions of the

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hippocampus was compared by a two-way ANOVA, considering group and hippocampal regions (HpD, HpVm e HpVl) as independent variables and density of Zenk-positive nuclei

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as the dependent variable. The post hoc analyses were performed with the Tukey-Kramer’s

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test.

INSERT FIGURE 1

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3. Results

3.1. Effect of tone-shock associations on freezing during training and test of contextual aversive memory. Figure 2 presents the mean percentages and standard error of the mean (s.e.m.) of the different types of behavior that were recorded during the sessions of training and contextual testing session of the pigeons in the three groups, Paired, Unpaired and Control.

12 For each category, percentages were calculated on the basis of the total number of instances recorded for each type of behavior during that session (all categories considered as 100%). During the training, the three groups exhibited the same types of behavior, which indicate behavior similarity among the groups. During the testing, the behavior diverged among the groups and the Paired group showed more freezing than did either the Unpaired or the Control groups. The significance of this difference was revealed by the analysis with

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ANOVA as a significant effect of group for the occurrence of freezing (F2, 16 = 18.35; p <

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0.0001), locomotion (F1,16 = 4.73; p < 0.05), maintenance (F1, 16 = 5.43; p < 0.05), and rest

(F1,16 = 37.53; p < 0.0001). Significant differences between the training and testing sessions

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were confirmed for freezing (F1,16 = 6.09; p < 0.05) and locomotion (F1, 16 = 45.58; p <

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0.0001), and significant group x session interactions were seen for freezing (F2,2 = 17.19; p < .001), locomotion (F2, 2 = 5.06; p < 0.05) and resting (F2, 2= 5.32; p < 0.05). Post hoc

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analysis with the Tukey-Kramer test indicated that the Paired group expressed more freezing during the testing to the context than did Unpaired and Control groups (p<0.05),

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while the Unpaired group exhibited more exploration; the Control group showed more resting and maintenance behaviors as compared to the Paired and Unpaired groups, for both sessions (p < 0.05).

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INSERT FIGURE 2

Figure 3 shows the response curves of freezing behavior during the training and

testing sessions for the Paired, Unpaired and Control groups. Each point presents freezing data for a pool of five blocks of 30-s time sampling intervals. During the first two blocks in the training session (corresponding to 5 min preceding the first tone-shock pairing), the

13 Paired and Unpaired groups present a lower expression of freezing than they did in the other six blocks. During the testing to the context, the pigeons in the Paired group expressed higher levels of freezing, which were maintained throughout the entire session; the level of freezing of the Unpaired and Control group was much lower. Statistical analysis with an ANOVA test confirmed a significant effect of group (F2, 16 = 112.41; p < 0.0001), session (F1, 16 = 14.07; p < 0.01) and of time-intervals (F1, 7 =10.64; p < 0.0001).

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There were significant interactions between group and session (F2, 2 = 42.46; p < 0.0001),

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between group and block of time intervals (F2, 16 = 10.64; p < 0.0001), between session x block of time sampling intervals (F1, 7 = 40.32; p < 0.0001), as well as between group x

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session x block (F14,98 = 5.13; p < 0.0001). Post hoc analysis with the Tukey-Kramer test

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confirmed significant differences for blocks, with the occurrence of freezing in the first two blocks significantly lower than in freezing in the other blocks of time sampling intervals

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during the training session (p < 0.05). The differences for groups were confirmed for the training and the testing session, revealing that the Control group was significantly different

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from the other two groups in both sessions (p < 0.05); the Paired group was significantly different from both Unpaired and Control during the session of testing to the context (p < 0.05).

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INSERT FIGURE 3

3.2. Analysis of density of Zenk-positive cell nuclei during retrieval of contextual aversive memory The testing for contextual memory induced greater density of Zenk-positive cell nuclei in the hippocampus of the pigeons in the Paired group than in those of the Unpaired,

14 Control and Naïve groups (F3, 19 = 10.02, p < 0.0001; Figure 4). Moreover, Post hoc TukeyKramer’s test confirmed that Unpaired groups had significantly higher densities of Zenkpositive nuclei than did Control and Naïve groups (p < 0.05). The greatest density of Zenkpositive cells was found in the ventral region of the Paired group as compared the other groups, Unpaired, Control and Naïve (Figure 5). The differences in the density of Zenkpositive nuclei found in the ventral and dorsal regions of the hippocampus were statistically

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significant, as revealed by a two-way ANOVA test as a significant effect of group (F3,19 =

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12.79; p < 0.001) and region (F1,19 = 19,31; p < 0.001), as well as a significant interaction

between group and region (F3,3 = 14,47; p < 0.0001). Post hoc testing (Tukey-Kramer’s test)

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confirmed that both the Paired and the Unpaired groups had significantly higher densities of

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Zenk-positive nuclei in both dorsal and ventral regions than did Control and Naïve groups (p < 0.05); moreover, the Zenk-positive nuclei density observed in the ventral region of the

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hippocampus of the Paired group was greater than that of the other groups, Unpaired,

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Control and Naïve (p < 0.05) (see Figures 4 e 5).

INSERT FIGURE 4

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INSERT FIGURE 5

Comparisons of Zenk-positive nuclei in the dorsal and the two ventral sub-regions,

ventro-medial (HpVm) and ventro-lateral (HpVl) (see Figure 6) showed greater densities of Zenk labeling in the Hp-Vm of the Paired group than in the ventro-lateral and dorsal

hippocampus. Statistical comparisons with ANOVA indicated a significant effect for group (F3,19 = 14.19; p < 0.0001), and region (F2,19 = 34.11; p < 0.0001) as well as a significant

15 interaction between group and region (F3,6 = 2.78; p < 0.05). Post hoc comparisons with the Tukey-Kramer test confirmed significant differences between the Zenk density in the ventro-medial hippocampus for the Paired group and for the other groups, Unpaired, Control and Naïve (p < 0.05). No differences in Zenk density were observed in the dorsal region between the Paired and Unpaired groups (p > 0.05) or between the Control and Naïve group (p > 0.05). Moreover, there was no difference between the dorsal and ventro-

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medial regions for the Paired group (p > 0.05). A clearly different pattern in the number

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and distribution of Zenk-positive nuclei is readily observed in the photomicrographs of

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frontal sections of the hippocampus of the four groups of pigeons (see Figure 7).

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INSERT FIGURE 7

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INSERT FIGURE 6

4. Discussion

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The present study has shown that training pigeons with tone-shock pairings results in

the expression of a robust aversive memory, as indicated by the occurrence of intense and prolonged freezing throughout the entire testing session. Moreover, the retrieval of the contextual aversive memory triggered an enhanced expression of the Zenk protein in the hippocampus of these pigeons. However, pigeons that were trained with tone and shock presented in a random sequence also had elevated Zenk expression in the hippocampus. The

16 differences in the density of Zenk–positive nuclei between the groups indicate that the type of training affected the activation of Zenk in the hippocampus. In addition, the data have shown that the level of Zenk expression differs according to the hippocampus region, with the Hp-Vm area of pigeons receiving paired aversive stimuli showing the greatest density of Zenk-positive nuclei as compared to the other groups, Unpaired, Control and Naïve, although no between groups differences were seen in the Hp-Vl area. Both groups of

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pigeons receiving paired or random tones and shocks revealed significantly greater Zenk

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expression than did the control group. Together, these data indicate that Zenk is activated in the context of the retrieval of aversive memories. Also, they are directly related to previous

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data showing that training in classical aversive conditioning induces Zenk expression in the

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hippocampus of pigeons (Brito et al., 2006). These observations corroborate previous data on rodents by Hall et al. (2001) that reported up regulation of zif/268 (zenk) expression in

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rats during the retrieval of recent aversive memory to the context. These data are also consistent with other studies in birds and mammals indicating the striking functional

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importance of the hippocampus in fear conditioning (Bast et al., 2001, 2003; Kim and Fanselow, 1992; Reis et al., 1999).

The data on the occurrence of freezing behavior showed that the first presentation of an unconditioned aversive stimulus, whether in association with the tone (Paired group) or

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not (Unpaired group) induced an increase of freezing behavior that was sustained until the end of the session. On the other hand, the Control group showed decrease in freezing during the exposure to the experimental chamber. Both Paired and Unpaired groups presented greater levels of freezing during the training than did the Control group. However, during the testing of reaction to the context the Paired and Unpaired groups differed in the behavior expressed, with Paired pigeons showing greater and prolonged freezing behavior throughout

17 the whole session. The pigeons in the Unpaired and Control groups, however, displayed varied behaviors and less frequent freezing. The results from the training and testing sessions of the Paired group are in agreement with those in the literature regarding unconditioned responses to shock stimulation as well as conditioned responses to a dangerous context (Cruz et al., 1993; Reis et al., 1999). Indeed, the presentation of an aversive stimulus, such as a shock, elicits robust

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startling, jumping, vocalization, intense and varied types of locomotion – including flying,

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which are unconditioned responses to the shock. Such responses are in general followed

immediately by exploration and, if they are unable to move to another place, the animals

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display profound immobility, known as freezing behavior. Therefore, the experimental

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context where aversive conditioning takes place acquires conditioned aversive properties and becomes able to control conditioned freezing response, even in the absence of other

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aversive stimuli (Bast et al., 2001; Fanselow, 2000). Therefore, the expression of a conditioned freezing is interpreted as the retrieval of the memory of the aversive stimulation

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experienced 24 h before. Many studies in the literature refer to conditioned freezing as expression of conditioned fear of that context, since freezing is part of the defense repertoire of an organism (Shuhama et al., 2007). The testing of response to the context involves the re-exposure to an environment

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which may have acquired a conditioned aversive function since it is the same environment where the aversive stimulation was experienced. The fact that pigeons in the Unpaired group revealed a lower level of freezing during this test than did the Paired group indicates that exposure to unpaired tone and shock stimulations did not result in strong conditioning and learning. However, the relatively high level of freezing expressed during the training session by the pigeons in the Unpaired group may be interpreted as evidence of some degree of

18 contextual conditioning resulting from the association between the shock and the context. If this is true, the low level of freezing that occurred during the testing session of the Unpaired group may be indicative of extinction or impairment of such a conditioned response. However, the unpaired pigeons engaged in various types of behavior, such as flying, locomotion, jumping and wall pecking behaviors, all of which might be interpreted as attempts to escape from a situation with aversive characteristics. Moreover, the unpaired

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group exhibited more exploration during the testing to the context than did paired and

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Control groups, which may represent the modification on their fear response to context if we consider the effectiveness of conditioning. Despite the unpaired group showing no test

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freezing to the context, the joint analysis of the behavioral data and the Zenk expression to

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paired and unpaired groups led to an initial discussion on the selective participation of the pigeon dorsal hippocampus in aversive context conditioning, developed further.

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The induction of Zenk expression in the hippocampus of pigeons during retrieval of contextual memory is directly related to an extensive literature on the role of the

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hippocampus in memory processes in mammals and birds (Bast et al., 2001a; Bast et al., 2001b; Bast et al., 2003; Eichenbaum et al., 1992; Zhang et al., 2001). The data of the present study show that retrieval of the contextual aversive memory induced Zenk expression in the hippocampus, which was more evidenced in the Paired and Unpaired

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groups, which had experienced the shock, than in the Control and Naive groups. An analysis of the distribution of Zenk-positive nuclei in each region of the hippocampus reveals that both Paired and Unpaired groups had a higher density of immunolabeled cells in the Hp-D and Hp-V regions than did the Control and Naïve groups. One key observation of the present study is that tone-shock conditioning induced a significantly greater labeling of Zenk-positive cells in the HpVm, which was clearly distinct in the Paired group. This shows

19 that the retrieval of aversive contextual memory was accompanied by a differential regional activation of Zenk expression. These observations are in line with the report by Hall et al. (2001) in the same sense that, in response to aversive context retrieval testing in rats a large expression of zenk was observed in the CA1 but not in the dentate gyrus of the hippocampus, when the animals were exposed to contextual conditioning. Moreover, some studies do not identify the dorsal hippocampus as an essential neural structure in all steps to

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the developing contextual aversive memory in rodents (Conejo er al, 2007; Gewirtz et all,

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2000). Lee and Kesner (2004) show that hippocampal subregions participate in different moments of the aversive conditioning once the role of CA3 was minimal in retrieving

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contextual memory after a long time period, despite being critically involved in the initial

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acquisition of the conditioning together with CA1 and dentate gyrus. The selective participation of the hippocampal regions may also be related to the

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time of exposure to the aversive context because differences in the c-fos expression of CA1, CA3 and amygdala were found between a short exposure versus a long exposure to the

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conditioning context fear (Perez er al., 2005). Results similar to the ones obtained in this study were presented by Conejo (2007), where there was no difference detected, between conditioned and non-conditioned groups, in c-fos expression in the dorsal subregions CA1 and CA3 of rats after exposure to the tone and to the conditioned context. Although the

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direct participation of these structures in the expression of the aversive conditioning, their participation in the initial stages of the conditioning could not be discarded, according to the authors. The data of these works taken in conjunction with ours indicate that, despite the birds following a vastly independent evolution of superior cognitive abilities (Bingman and Sharp, 2004), they seem to have developed a network organization in hippocampal formation comparable to that of mammals. Moreover, the induction of Zenk expression in

20 the hippocampus of pigeons during retrieval of contextual conditioning supports the notion that the aversive contextual memory in the pigeon, as well as in rodents, depends on mechanisms mediated by glutamate in the hippocampus. The regional subdivision into dorsal and ventral hippocampus is supported by a general set of connections among different regions of hippocampal formation as well as by neurochemical organization, cellular profiles and electrophysiological pattern of neuronal

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activity (Hough et al., 2002; Kahn et al., 2003; Krebs et al., 1991). Several studies have

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described the intrinsic and extrinsic connectivity of the pigeon hippocampus and their

relation to the functional organization (Erichsen et al., 1991; Hough et al., 2002; Kahn et al.,

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2003; Krebs et al., 1991). Accordingly, the selective up-regulation of IEG zenk in neurons of

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the HpVm region suggests that they may be particularly involved in the retrieval of memories related to aversive contingencies between stimuli. Moreover, a high density of

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neurons in the HpD region was identified by immunolabeling for Zenk. This population of Zenk-positive cells is presumably related with the processing of stimulation provided by

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both tone and shock stimuli. Together, these observations suggest that the retrieval of the memory of experiences with paired stimuli may involve the activation of different circuits than do the unpaired ones. These data are directly related to those provided by a previous report by Brito et al. (2006) showing that a selective increase of Zenk-positive nuclei in the

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Hp-Vm was induced by the training with contingent tone-shock presentations. Besides, training with tone and shock either paired, unpaired or presented alone induced increases in Zenk density in the Hp-D. This shows that our data are also related to the selective activation of neuronal populations reported for the rodent hippocampus after classical aversive conditioning (Hall et al., 2001).

21 Both the Hp-D and Hp-Vm constitue a circuit in the avian hippocampus that may be compared to the well-Known trisynaptic circuit of the mammalian hippocampus (Kahn et al., 2003). The avian HpD receives input from the medial septum and the terminals of medial tract fibers may connect it to other structures related to the processing of both tone and shock stimulations (Benowitz and Karten, 1976; Bingman et al., 1994; Bouille et al., 1977; Casini et al., 1986; Shimizu and Karten, 1990; Shimizu et al., 2004). Therefore, the

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neuronal activation of Hp-D, in both hemispheres, is involved with the processing of input

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arriving in the hippocampus and may be related to information processing during fear

conditioning. In the present study, the elevated induction of Zenk in to the Hp-D of pigeons

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which received both tones and shock stimulation during training seems to support this

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interpretation. The selective increase of Zenk-positive nuclei in the Hp-Vm can be related to the fact that this region, on the other hand, is functionally related ipsilaterally to the Hp-D

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and to Hp-Vl, but has also reciprocal contralateral connections with the Hp-Vm. Thus, the selective increase of Zenk-positive nuclei in this region may be due to the fact that it

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receives, processes and integrates inputs arising from both hemispheres. This hypothesis may also be supported by the fact that the Hp-Vl region of the hippocampus, which is characterized by intrinsic ipsilateral connections (Atoji and Wild, 2004; Kahn et al., 2003), expressed the lowest neuronal activation, independently of the training protocol. Taken

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together, the results of this study corroborate previous data of the literature about classical aversive conditioning in pigeons and rodents, suggesting differential activation of extra- and intrahippocampal circuits during acquisition and retrieval of contextual aversive memory. In summary, the results of the present study have shown that exposure to a conditioned aversive context correlates with the retrieval of contextual memory, as indicated by the elevated occurrence of freezing when exposed to that context, as well as with

22 increases of activation of the IEG zenk in the hippocampus, as indicated by the large number of Zenk-positive nuclei in the HpVm and HpD. These observations extend to birds the widely held view that the hippocampus of mammals is required for the formation and retrieval of context-aversive stimulation (Maren and Fanselow, 1996). Moreover, they are consistent with the view that the IEG zenk might be a molecular substrate of retrieval of

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conditioned fear memory.

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Declarations of interest

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The authors declare no conflict of interests.

Funding

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This research was supported by research grants from CNPq, FAPESP and FAEPEXUNICAMP. B. V. The authors would like to acknowledge the technical assistance of

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Adilson da Sila Alves and Washington Luiz. L. Gomes.

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Figure 1. Schemes of frontal sections of the pigeon brain showing the hipocampal subdivisions (Karten and Hodos, 1967) analysed in this study. Dorsal hippocampus (Hp-D);

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medial hippocampus (Hp-Vm); lateral hippocampus (Hp-Vl); arcopallium (A); nidopallium (N); hyperpallium (H); ectostriatum (E); ventriculus (V) (Reiner et al., 2004). The numbers on the top of each schematic indicate the stereotaxic plane. From a stereotaxic Atlas of the

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Brain of the Pigeon, by H. J. Karten and W. Hodos, pp. 74, 84, and 92. Copyright 1967 by

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Johns Hopkins University Press.

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Figure 2. Mean percentage of behaviors in each category during the training with toneshock conditioning and the test to the context, for Paired, Unpaired and Control groups (means ± s.e.m.). Categories are: MOV= discrete movements; Pre = pre-exploratory; EXP = exploratory; MAN = maintenance behavior; LOC= locomotion; RST = rest; FRZ =

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freezing).

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Figure 3. Freezing behavior recorded during the training with tone-shock conditioning and the test to the context. Mean values of freezing (± s.e.m.) are plotted across blocks of five 30-s time intervals (150 s) for the Paired, the Unpaired and Control groups.

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Figure 4. Average of the density of zenk-labeled nuclei in the hippocampus of pigeons from Paired, Unpaired, Control and Naïve groups; * p < 0.05 in relation to the Control, Naïve, and Paired groups; ** p < 0.05 in relation to the Control, Naïve, and Unpaired groups (one-

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way ANOVA followed by Tukey-Kramer Multiple-Comparison test).

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Figure 5. Mean (

-immunoreactive nuclei in the ventral

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hippocampus, (Hp-V) and the dorsal hippocampus (Hp-D). Paired and the Unpaired groups had significantly higher density of Zenk-positive cells in the Hp-D and Hp-V as compared to the Control and the Naïve groups (p < 0.05). Zenk-positive cell density in the Hp-V of

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the Paired group was significantly different from all the other groups, Unpaired, Control

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and Naïve (p < 0.05) (ANOVA two-way with Tukey-Kramer Multiple-Comparison tests).

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Figure 6

-immunoreactive nuclei in the Hp-Vm, Hp-Vl,

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and Hp-D in each group of pigeons. Hp-Vm of the Paired group significantly differed from the other groups, Unpaired, Control and Naïve (p < 0.05). No differences were detected

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between the cell labeling in the Hp-D region of Paired as compared to the Unpaired groups as well as in the Hp-D of the Control as compared to the Naïve group (p > 0.05); HpVm

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and HpD regions of the Paired group showed no statistical differences (p > 0.05).

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Figure 7. Digital images of frontal sections of the hippocampus of birds from the Paired, Unpaired, Control and Naive groups (NG). The scheme at the top left, first image, indicates the hippocampal subregions analyzed. Dorsal hippocampus (Hp-D); medial hippocampus (Hp-Vm); lateral hippocampus (Hp-Vl).