Classical aversive conditioning induces increased expression of mature-BDNF in the hippocampus and amygdala of pigeons.

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 hippocampus that is involved in the persistence of aversive memory in rodents. However, the role of BDNF in the hippocampal synaptic mechanisms involved in the classical conditioning aversive memory in birds still needs clarification. 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-hippocampal infusion of anisomycin (Ani) in these changes. Seven days after implantation of intra-hippocampal microcannulae, 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 treatment or conditioning. Homogenates of tissues from the hippocampus and amygdala, obtained 12h 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 hippocampus and amygdala of the pigeons. The decreases in the content of truncated- and pro-BDNF isoforms found in conditioned pigeons may suggest cleavage mechanisms induced by the training. Our data confirm previous observations of rodent studies and extend these observations to pigeons, revealing that, in spite of the anatomical differences between the hippocampus of rodents and pigeons, there are functional and molecular mechanisms that are conservative between the species.

Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation

In this study, we evaluated the expression of the Zenk protein within the nucleus taeniae of the pigeon’s amygdala (TnA) after training in a classical aversive conditioning, in order to improve our understanding of its functional role in birds. Thirty-two 18-month-old adult male pigeons (Columba livia), weighing on average 350 g, were trained under different conditions: with tone-shock associations (experimental group; EG); with shock-alone presentations (shock group; SG); with tone-alone presentations (tone group; TG); with exposure to the training chamber without stimulation (context group; CG), and with daily handling (naive group; NG). The number of immunoreactive nuclei was counted in the whole TnA region and is reported as density of Zenk-positive nuclei. This density of Zenk-positive cells in the TnA was significantly greater for the EG, SG and TG than for the CG and NG (P < 0.05). The data indicate an expression of Zenk in the TnA that was driven by experience, supporting the role of this brain area as a critical element for neural processing of aversive stimuli as well as meaningful novel stimuli.

Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation.

In this study, we evaluated the expression of the Zenk protein within the nucleus taeniae of the pigeon's amygdala (TnA) after training in a classical aversive conditioning, in order to improve our understanding of its functional role in birds. Thirty-two 18-month-old adult male pigeons (Columba livia), weighing on average 350 g, were trained under different conditions: with tone-shock associations (experimental group; EG); with shock-alone presentations (shock group; SG); with tone-alone presentations (tone group; TG); with exposure to the training chamber without stimulation (context group; CG), and with daily handling (naive group; NG). The number of immunoreactive nuclei was counted in the whole TnA region and is reported as density of Zenk-positive nuclei. This density of Zenk-positive cells in the TnA was significantly greater for the EG, SG and TG than for the CG and NG (P < 0.05). The data indicate an expression of Zenk in the TnA that was driven by experience, supporting the role of this brain area as a critical element for neural processing of aversive stimuli as well as meaningful novel stimuli.

Glutamatergic neurotransmission mediated by NMDA receptors in the inferior colliculus can modulate haloperidol-induced catalepsy.

The inferior colliculus (IC) is primarily involved in the processing of auditory information, but it is distinguished from other auditory nuclei in the brainstem by its connections with structures of the motor system. Functional evidence relating the IC to motor behavior derives from experiments showing that activation of the IC by electrical stimulation or excitatory amino acid microinjection causes freezing, escape-like behavior, and immobility. However, the nature of this immobility is still unclear. The present study examined the influence of excitatory amino acid-mediated mechanisms in the IC on the catalepsy induced by the dopamine receptor blocker haloperidol administered systemically (1 or 0.5 mg/kg) in rats. Haloperidol-induced catalepsy was challenged with prior intracollicular microinjections of glutamate NMDA receptor antagonists, MK-801 (15 or 30 mmol/0.5 microl) and AP7 (10 or 20 nmol/0.5 microl), or of the NMDA receptor agonist N-methyl-d-aspartate (NMDA, 20 or 30 nmol/0.5 microl). The results showed that intracollicular microinjection of MK-801 and AP7 previous to systemic injections of haloperidol significantly attenuated the catalepsy, as indicated by a reduced latency to step down from a horizontal bar. Accordingly, intracollicular microinjection of NMDA increased the latency to step down the bar. These findings suggest that glutamate-mediated mechanisms in the neural circuits at the IC level influence haloperidol-induced catalepsy and participate in the regulation of motor activity.

Immunohistochemical distribution of AMPA-type label in the pigeon (C. livia) hippocampus.

The avian hippocampal formation (HF) is reported to have a role equivalent to that of the mammalian hippocampus, which may involve the glutamatergic system as well. In the present paper we offer evidence of the occurrence and distribution of the subunits composing AMPA-type glutamate receptors on neurons in the hippocampus region of the pigeon brain. The experiment analyzed the immunolabeling of glutamate receptor (GluR)(1)(,) GluR(4,) and GluR(2/3) receptor subunits in adult pigeons and found consistent evidence that neurons located in the five main areas of the avian HF have these AMPA-type subunits, but their incidence varies according to position and neuro-type. About 20%-35% of the irregular and 35%-70% of the triangular neurons on the lateral and medial "V" arms contain GluR(1) and GluR(2/3), while GluR(4) was found only at rounded neurons. The majority of the triangular neurons (over 90%) and about half of the irregular neurons in the medial area contain GluR(1) and GluR(2/3,) whereas the rounded neurons contain primarily GluR(4) (95%). Labeling revealed GluR(1) (40%-60%) and GluR(2/3) (30%) in the dorsomedial and lateral areas but only in irregular neurons, while 60%-80% of the rounded neurons synthesize GluR(4). While triangular and irregular neurons appear to match the description of projecting neurons, rounded ones seem to participate in local circuits. A discussion of the functional significance of the avian HF concentrates on a postulation of the "V" arms as equivalent to the dentate gyrus and the dorsomedial area being similar to the Ammon's horn.

Classical tone-shock conditioning induces Zenk expression in the pigeon (Columba livia) hippocampus.

The hippocampus is involved in fear conditioning, although the molecular events underlying this function are still under investigation. The authors analyzed the expression of the Zenk proto-oncogene product within the pigeon (Columba livia) hippocampus after training with a classical aversive conditioning protocol using tone-shock associations. Control groups were trained with shock or tone alone or were only exposed to the experimental chamber and manipulated. Experimental pigeons showed significant increases of Zenk expression in the ventromedial region of the hippocampus, whereas both the experimental and shock groups had increased Zenk expression in the dorsal region. The expression of Zenk in specific neuronal populations within the pigeon hippocampus may be indicative of plasticity-associated aversive classical conditioning.

Operant discriminative learning and evidence of subtelencephalic plastic changes after long-term detelencephalation in pigeons.

We analyzed operant discrimination in detelencephalated pigeons and neuroanatomical substrates after long-term detelencephalation. In Experiment I, experimental pigeons with massive telencephalic ablation and control pigeons were conditioned to key peck for food. Successive discrimination was made under alternating red (variable-ratio reinforcement) and yellow (extinction) lights in one key of the chamber. These relations were interchanged during reversal discrimination. The sessions were run until steady-state rates were achieved. Experiment II analyzed the morphology of the nucleus rotundus and optic tectum in long-term detelencephalated and control birds, using a Klüver-Barrera staining and image analyzer system. Detelencephalated birds had more training sessions for response shaping and steady-state behavior (p < 0.001), higher red key peck rates during discrimination (p < 0.01), and reversal discrimination indexes around 0.50. Morphometric analysis revealed a decreased number of neurons and increased vascularity, associated with increases in the perimeter (p < 0.001) in the nucleus rotundus. In the optic tectum, increases in the perimeter (p < 0.05) associated with disorganization in the layers arrangement were seen. The data indicate that telencephalic systems might have an essential function in reversal operant discrimination learning. The structural characteristics of subtelencephalic systems after long-term detelencephalation evidence plastic changes that might be related to functional mechanisms of learning and neural plasticity in pigeons.

Locomotor response to an open field during C57BL/6J active and inactive phases: differences dependent on conditions of illumination.

Time of day has proven to be a source of variability in diverse behavioral measures. Knowledge of the pattern of this temporal effect as well as its origin (exogenous or endogenous) is essential for a precise description of any behavior. This study analyzed the effect of the external light-dark cycle and the internal rest-activity rhythm on the response of C57BL/6J mice to a novel environment. In a first experiment, animals maintained in a 12:12-h light-dark cycle were tested in an open field at six different times of day. A diurnal rhythm of ambulation in the open field was observed with greater levels of activity exhibited by those groups tested at night. Long-term and short-term behavioral habituation to spatial novelty were also affected by phase of the light-dark cycle. A second experiment was designed to control for any direct effect of the light-dark cycle by keeping the animals in dim green light where entrainment was maintained by a skeleton photoperiod (two 15-min bright-light pulses separated by 12 hours of green, dim light). This second group of animals was tested at two different circadian phases under the same conditions of illumination. One group was tested during the subjective night and another group during the subjective day, i.e., 2 or 14 h after the onset of the active phase, as assessed by wheel-running behavior. No effect of circadian phase on ambulation or habituation of this response to the open field was observed in these animals. Taken together, these results suggest that spatial novelty is equally arousing regardless of circadian phase and that the conditions of illumination can dramatically alter the response to a novel environment.

Role of the hippocampus in contextual memory after classical aversive conditioning in pigeons (C. livia)

We investigated the effects of hippocampal lesions with ibotenic acid (IBO) on the memory of the sound-context-shock association during reexposure to the conditioning context. Twenty-nine adult pigeons were assigned to a non-lesioned control group (CG, N = 7), a sham-lesioned group (SG, N = 7), a hippocampus-lesioned experimental group (EG, N = 7), and to an unpaired nonlesioned group (tone-alone exposure) (NG, N = 8). All pigeons were submitted to a 20-min session in the conditioning chamber with three associations of sound (1000 Hz, 85 dB, 1 s) and shock (10 mA, 1 s). Experimental and sham lesions were performed 24 h later (EG and SG) when EG birds received three bilateral injections (anteroposterior (A), 4.5, 5.25 and 7.0) of IBO (1 µl and 1 µg/µl) and SG received one bilateral injection (A, 5.25) of PBS. The animals were reexposed to the training context 5 days after the lesion. Behavior was videotaped for 20 min and analyzed at 30-s intervals. A significantly higher percent rating of immobility was observed for CG (median, 95.1; range, 79.2 to 100.0) and SG (median, 90.0; range, 69.6 to 95.0) compared to EG (median, 11.62; range, 3.83 to 50.1) and NG (median, 7.33; range, 6.2 to 28.1) (P<0.001) in the training context. These results suggest impairment of contextual fear in birds who received lesions one day after conditioning and a role for the hippocampus in the modulation of emotional aversive memories in pigeons

Role of the hippocampus in contextual memory after classical aversive conditioning in pigeons (C. livia).

We investigated the effects of hippocampal lesions with ibotenic acid (IBO) on the memory of the sound-context-shock association during reexposure to the conditioning context. Twenty-nine adult pigeons were assigned to a non-lesioned control group (CG, N = 7), a sham-lesioned group (SG, N = 7), a hippocampus-lesioned experimental group (EG, N = 7), and to an unpaired nonlesioned group (tone-alone exposure) (NG, N = 8). All pigeons were submitted to a 20-min session in the conditioning chamber with three associations of sound (1000 Hz, 85 dB, 1 s) and shock (10 mA, 1 s). Experimental and sham lesions were performed 24 h later (EG and SG) when EG birds received three bilateral injections (anteroposterior (A), 4.5, 5.25 and 7.0) of IBO (1 microl and 1 microg/microl) and SG received one bilateral injection (A, 5.25) of PBS. The animals were reexposed to the training context 5 days after the lesion. Behavior was videotaped for 20 min and analyzed at 30-s intervals. A significantly higher percent rating of immobility was observed for CG (median, 95.1; range, 79.2 to 100.0) and SG (median, 90.0; range, 69.6 to 95.0) compared to EG (median, 11.62; range, 3.83 to 50.1) and NG (median, 7.33; range, 6.2 to 28.1) (P<0.001) in the training context. These results suggest impairment of contextual fear in birds who received lesions one day after conditioning and a role for the hippocampus in the modulation of emotional aversive memories in pigeons.

The functional value of sound and exploratory behaviour in detelencephalated pigeons.

Exploratory behaviour in response to a sound previously paired to a shock was analyzed before and after massive telencephalic lesions. Six pigeons were submitted to sound shock associations, habituation tests, telencephalic lesion and habituation retesting. Eighteen birds divided into three sham-lesioned groups received sound shock pairing (Control 1), pre-exposure to sound stimulation (Control 2) or pre-exposure to electric shocks (Control 3) before the behavioural tests. The pre-surgery tests consisted of 25 1 s pulses of sound A (1000 Hz, 83 dB) or B (500 Hz, 85 dB), every 30 s. Retests took place 10 days after surgery using the same sequence of procedures. The behaviour exhibited immediately after each sound stimulation was recorded manually and on video during the sessions. Analysis of variance showed an effect of group {F(3, 20)= 13.37, p < 0.0001) suggesting that pre-exposure to tone-shock association affected the exploration in response to the sound presented in another context. The Experimental and Control 1 birds showed no robust decrease in responses during the pre-surgery sessions. Post-surgery exploration data for detelencephalated birds showed a lower level of responding (p < 0.05). These data suggest a retardation in the typical reduction of responses to recurrent stimulation and support other evidences of the potentiating effect of sound shock association on responding. They are also indicative of telencephalic modulation of exploratory behaviour and strengthen the arguments for the subtelencephalic storage of associative information concerning the functional value of acoustic stimulation.

Automated measurement of mouse freezing behavior and its use for quantitative trait locus analysis of contextual fear conditioning in (BALB/cJ x C57BL/6J)F2 mice.

The most commonly measured mouse behavior in fear conditioning tests is freezing. A technical limitation, particularly for genetic studies, is the method of direct observation used for quantifying this response, with the potential for bias or inconsistencies. We report the use of a computerized method based on latency between photobeam interruption measures as a reliable scoring criterion in mice. The different computer measures obtained during contextual fear conditioning tests showed high correlations with hand-scored freezing; r values ranged from 0.87 to 0.94. Previously reported strain differences between C57BL/6J and DBA/2J in context-dependent fear conditioning were also detected by the computer-based system. In addition, the use of computer-scored freezing of 199 (BALB/cJ x C57BL/6J)F2 mice enabled us to detect a suggestive gender-dependent chromosomal locus for contextual fear conditioning on distal chromosome 8 by QTL analysis. Automation of freeze scoring would significantly increase efficiency and reliability of this learning and memory test.

Habituation to sound during morning and night sessions in pigeons (Columba livia).

Habituation to acoustic stimulation was analyzed through night and morning sessions in pigeons submitted to a 12:12 h light-dark (LD) cycle. We demonstrate a lack of habituation in nocturnal sessions as compared to typical habituation curves in morning sessions. Temporal organization of the habituation process raises issues concerning the biological meaning of sound in adaptation to the environment and/or the possible interaction of well known endogenous physiological rhythms with learning. A second experiment was performed to establish whether the LD cycle was responsible for the night/day difference observed in the first experiment. In this experiment, the animals were subjected to two days of continuous light (LL) before the learning sessions. No difference in habituation according to time of day was observed in these conditions suggesting a direct effect of the LD cycle in the habituation to sound.

Operant discrimination learning in detelencephalated pigeons (Columba livia).

Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into three groups: non-lesioned (N = 6), sham-lesioned (N = 5) and telencephalon lesioned (N = 10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80% of the ad libitum weight. Successive discrimination was programmed by the alteration of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with variable-ratio reinforcement; the yellow key was correlated with extinction. Session were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning by detelencephalated birds. Response shaping and steady-state rates required a larger number of sessions for lesioned pigeons (P < 0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components as compared to the non-lesioned (red: 16.51 +/- 2.0; yellow: 2.02 +/- 0.64) and sham-lesioned (red: 22.84 +/- 1.77; yellow: 4.72 +/- 1.99) groups (P < 0.05). These data show that telencephalic systems are not essential for operant discrimination learning but play a role in the modulation of discriminative behavior. Subtelencephalic systems appear to be functionally important for the organization and storage of learning.

Operant discrimination learning in detelencephalated pigeons (Columba livia)

Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into the three groups: non-lesioned (N=6), sham-lesioned (N=5) and telencephalon lesioned (N=10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80 percent of the ad libitum weight. Successive discrimination was programmed by the alternation of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with extinction. Sessions were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning required a larger number of sessions for lesioned pigeons (P<0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components...

Habituation to sound stimulation in detelencephalated pigeons (Columba livia).

Habituation to sound stimulation was analyzed in terms of the functional role of the telencephalon in learning. Sixteen pigeons were exposed to 1000-Hz, 83-dB, 1-s sound (stimulus A) at 30-s intervals until there was habituation of the exploratory and pre-exploratory responses. The learning criterion was 10 trials without the occurrence of these responses. Twenty-four hours after habituation to stimulus A the birds were tested with a 500-Hz, 85-dB, 1-s sound (stimulus B). On the day following habituation to stimulus B, the birds of the experimental group (N = 8) suffered ablation of the telencephalon and the birds of the control group (N = 8) had sham surgery. Retesting with the same sequence of procedures was carried out 10 days after surgery. In the POST-lesion situation there was a decrease of the number of habituation trials to stimulus A (P less than 0.01) and to stimulus B (P less than 0.05) by experimental pigeons compared to the PRE-lesions situation. The data suggest an interaction of a facilitatory effect of the lesion and long-term learning effects.

Effects of massive telencephalic lesions on the organization of behavior in pigeons (Columba livia).

The effects of massive lesions of the telencephalon on the behavioral repertoire of captive pigeons were investigated. The behavior of four birds with an intact telencephalon was compared to that of four birds submitted to ablation of telencephalic structures. Behavioral audio recording was done according to previously defined categories in three daily sessions for fifteen days. Experimental birds were followed up for one month. Immediately after the lesion, operated birds showed decreases in the occurrence of different behaviors such as coordinated movements, feeding, interaction, preening, maintenance, exploration and vocalization and increases in locomotion and discrete movements of the body when compared to the control birds (P less than 0.05). Recovery of exploration (P less than 0.05), feeding and localization was observed during the follow-up period. These data are interpreted as suggestive of a functional role of the telencephalon in the organization of behavior and a long-term recovery of behavior after detelencephalation.

Habituation to sound stimulation in detelencephalated pigeons (Columbia livia)

Habituation to sound stimulation was analyzed in terms of the functional role of the telencephalon in learning. Sixteen pigeons were exposed to 1000-Hz, 83-dB, 1-s sound (stimulus A) at 30-s intervals until there was habituation of the exploratory and pre-exploratory responses. The learning criterion was 10 trials without the occurrence of these responses. Twenty-four hours after habituation to stimulus A the birds were tested with a 500-Hz, 85-dB, 1-s sound (stimulus B). On the day following habituation to stimulus B, the birds of the experimental group (N=8) suffered ablation of the telencephalon and the birds of the control group (N=8) had sham surgery. Retesting with the same sequence of procedures was carried out 10 days after surgery. in the POST-lesion situation there was a decrease of the number of habituation trials to stimulus A (P<0.01) and to stimulus B (P<0.05) by experimental pigeons compared to the PRE-lesion situation. The data suggest an interaction of a facilitatory effect of the lesion and long-term learning effects

Effects of massive telencephalic lesions on the organization of behavior in pigeons (Columba livia)

The effect of massive lesions of the telencephalon on the repertoire of captive pigeons were investigated. The behavior of four birds with an intact telencephalon was compared to that of four birds submitted to ablation of telencephalic structures. Behavioral audio recording was done according to previously defined categories in three daily sessions for fifteen days. Experimental birds were followed up for month. Immediately after the lesion, operated birds showed decreases in the occurrence of different behaviors such as coording movements, feeding, interaction, preening, maintenance, exploration and vocalization and increases in locomotion and discrete movements of the body when compared to the control birds (P<0.05). Recovery of exploration (P<0.05), feeding and localization was during the follow-up period. These data are interpreted as suggestive of a functional role of the telencephalon in the organization of behavior and a long-term recovery of behavior after detelencephalation

Key pecking operant conditioning in detelencephalated pigeons (Columba livia).

The present paper analyzed the acquisition and maintenance of key pecking reinforced with food by pigeons with massive ablation of telencephalon structures (detelencephalated pigeon). The subjects were adult pigeons maintained at 80% of their free feeding weight. Expt. I analyzed the acquisition of key pecking by 3 previously detelencephalated pigeons and 3 normal controls. Expt. II investigated the effects of detelencephalation on the retention of learning through comparisons of pre- and postsurgery key peck rates in schedules of continuous reinforcement. In both experiments the duration of reinforcement was manipulated (20 s and 10 s). The results showed that detelencephalated pigeons are able to learn an operant response and to retain it after telencephalic ablation. Variability in key pecking rate was greater when the lesion preceded conditioning. The data suggest that the telencephalic systems participate in the organization of the key pecking behaviour patterns under this schedule of reinforcement but are not essential for this kind of learning. It is also suggested that subtelencephalic systems participate in the retention of learning.