Physiology& Behavior, Vol. 49, pp. 765-770. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Effects of Nucleus Basolateralis Amygdalae Neurotoxic Lesions on Aversive Conditioning in the Rat CARLO AMBROGI LORENZINI, CORRADO BUCHERELLI, ALDO GIACHETTI, LAURA MUGNAI* AND GIOVANNA TASSONI
Dipartimento di Scienze Fisiologiche, Universitgl degli Studi di Firenze, Viale Morgagni 63, 1-50134, Firenze, Italy *Dipartimento di Biologia Animale e Genetica, Universitd degli Studi di Firenze, Via Romana 17 1-50125, Firenze, Italy R e c e i v e d 14 June 1990
AMBROGI LORENZINI, C., C. BUCHERELLI, A. GIACHETTI, L. MUGNAI AND G. TASSONI. Effects of nucleus basolateralis amygdalae neurotoxic lesions on aversive conditioning in the rat. PHYSIOL BEHAV 49(4) 765-770, 1991.--After bilateral stereotaxic administration of ibotenic acid on the n. basolateralis amygdalae, male adult rats were tested in the light-dark box apparatus to measure the time-course of the acquisition and retention of passive and active avoidance responses. The results show that after the lesions both passive avoidance and active avoidance acquisition were impaired. Passive avoidance responses were retained quite well, while active avoidance responses disappeared quickly. Conditioned freezing was almost completely absent. Thus it appears that the n. basolateralis plays a facilitatory role in all the conditioned responses which were investigated. N. basolateralis amygdalae
Active avoidance
Passive avoidance
Conditioned freezing
coagulation of passing fibres and glial cells as well as the death of neurons. The aim of the present work is to assess the effects of circumscribed lesions of the n. basolateralis amygdalae on the acquisition and retention of passive and active avoidance responses. The lesions will be caused by the stereotaxic administration of the neurotoxin, ibotenic acid. In our investigation we shall use the light-dark box paradigm (1-3) because it enables us to study the time course of acquisition and retention of both responses contemporaneously in the same animal (Rattus norvegicus). Initial freezing [a sign of fear system activation (1,3)] will also be measured in order to ascertain if chemical lesions of the nucleus basolateralis also affect emotionality.
DESTRUCTIVE techniques (6, 24, 26, 31, 35, 38), electrical stimulation (4, 21, 23) and pharmacologically active compounds (13, 15, 20) have all demonstrated that the amygdala plays a facilitatory role in the learning and memory of passive and active avoidance responses. Nonetheless, some reports differ in their conclusions (17, 19, 24, 37). This divergence of opinion may be at least partially due to the fact that, in a large number of the studies, the amygdala was manipulated "in toto." More detailed investigations have shown that the nucleus basolateralis amygdalae may be one of the lymbic structures, together with the nucleus centralis and the nucleus lateraiis (31). In addition, the former is probably most active in mnemonic processes (5, 11, 31, 35) since, between amygdala and prefrontal cortex, the nucleus basolateralis appears to be the most developed connective system (11). Furthermore, it also sends fibres to the n. mediodorsalis thalami which is considered to be an important link in the memory system (5,31). Moreover, there appears to be a positive relationship between the relative size of this nucleus and the degree of encephalization (31). In fact, after demolitive manipulation (10, 12, 18, 28, 29, 33, 34, 38) or electrical stimulation (16,21) there is ample evidence that this nucleus plays a definite role in the acquisition and retention of passive avoidance responses. Less coherent reports exist on the effects of lesions of this nucleus on active avoidance responses: deficits (19, 22, 38), no effects (19,37) or even a better learning performance (17). In most of these studies the n. basolateralis was destroyed by electrocoagulation. It is well known that this technique causes the
METHOD Thirty-two naive male Wistar rats (mean body weight 310 g, Morini, Italy) were employed. The animals were kept singly in stainless steel cages, in a room at a constant temperature of 20---I°C, natural illumination. They received food and water ad lib. The cages were cleaned daily, food troughs and water bottles were refilled when necessary, and always after the testing session. The rats were randomly divided into two groups, the controis (13 subjects) and the group to which ibotenic acid was administered (19 subjects). All rats were anesthetized with intraperitoneally administered ketamine (Ketalar, Parke Davis, 100 mg.kg-~ b.wt.). The animals were placed in a stereotaxic apparatus. The experimental group of subjects (IBO Ss) was injected
765
766
with ibotenic acid (Sigma) using the coordinates + 6 . 2 mm interaural line, + 1.5 mm at the dorsal plane and _+5 with respect to the median line (27). Ibotenic acid was diluted in a phosphate buffer solution (pH 7.4) at a concentration 10 txg.lxl- 1. Of this solution, 0.2 Ixl were injected into each side. Constant velocity injection lasted 3 min, after which the needle of the syringe (S.G.E., type A, 10 pJ) was left in situ for a further 5 min in order to maximize neurotoxin diffusion. The same volume of phosphate buffer solution was administered to control subjects (shamoperated, SO Ss) who underwent the same procedure. Behavioral testing was started 20 days after the operation.
Apparatus The apparatus consisted of a light-dark box, made of a light chamber (30 x 21 x 15 cm) of white opaque plastic with a transparent lid, and a dark chamber (30 x 21 x 15 cm) with five sides of dark opaque plastic. The floor of both chambers was made of 2-mm stainless steel rods spaced 1 cm; the floor of the dark chamber could be electrified. The chambers were connected by a guillotine door (6 x 8 cm). The apparatus was placed in an acoustically insulated room which was kept at a constant temperature of 2 0 - I ° C . Illumination inside the light chamber was 60 lux. Every day, the animals were manually placed, one by one, inside the apparatus. Latency values were taken when the animals had placed all 4 paws in the appropriate chamber.
Procedure The experiment consisted of a total of 20 trials. It began with two cycles of 7 consecutive single daily trials. After a 3-week interval, there was a third cycle of 6 consecutive single daily trials. All trials started at 9:00 a.m. During Cycle 1 (Acquisition), the Ss were placed inside the light chamber of the apparatus, with the connecting door open. Once the animals had spontaneously gone inside the dark chamber, or had been placed inside it (when step-through latency duration exceeded 180 s), the connecting door was closed. In the dark chamber the Ss received an inescapable footshock (0.8 mA, 2 s). This gave rise to appreciable motor reactions; only seldom did vocalization occur. Following the footshock, the connecting door was opened, and if the animal was still inside the dark chamber 10 s later, then a second footshock was administered (0.8 mA, 5 s). Exit latency was measured up to 15 s. Exits from the dark chamber with latencies of less than 10 s were considered active avoidance responses. During Cycles 2 (Retention 1, 7 trials) and 3 (Retention 2, 6 trials) no shocks were administered, and the trials were divided into two phases. The first phase which started at 9:00 a.m. was to assess the retention of the passive avoidance response. The Ss were placed directly into the light chamber with the connecting door open, and step-through latency was measured up to 180 s. As soon as the Ss went inside the dark chamber they were returned to their home cage. The second phase (11:00 a.m.) was to assess the retention of the active avoidance response. The Ss were placed directly inside the dark chamber, with the connecting door open and exit latencies were taken up to 15 s. Exits from the dark chamber with latencies of less than 10 s were considered as active avoidance responses. In all trials, the duration of initial freezing in the light chamber was measured. (Freezing is defined as motor inhibition resulting in immobility, which ends when voluntary movements are performed.) Initial freezing was that exhibited by the Ss immediately after having been placed inside the apparatus. Passive avoidance (step-through latency) and freezing data were grouped as
AMBROGI LORENZINI ET AL.
follows: days 2-8 for acquisition, days 9-14 and 36-41 for retention. Active avoidance data (exit latency and active avoidance responses) were grouped as follows: days 1-7 for acquisition, days 8-14 and 36--41 for retention. This was because step-through latencies and freezing durations of day 1 were measured before footshocks had been administered (preshock), while exit latencies were measured after the inescapable footshocks. Similarly the step-through latencies and freezing duration of day 8 were measured before the footshock omission while the exit latencies of the same day were measured after footshock omission. Some days after the end of Cycle 3, pain threshold was measured in all animals in order to ascertain whether the ibotenic lesion of the n. basolateralis had somehow influenced nociception. Pain threshold measurements were performed after the experiment to avoid any possible behavioral influence of the testing procedures. All Ss were tested with the hot-plate apparatus. This consisted of a flat-bottomed stainless steel container (28.5 x 36 x 15.5 cm) which was placed in a constant temperature water bath (56°C) (KW Mod W.82). The water was continuously stirred in order to keep the temperature of the stainless steel surfaces constant. Once they had been placed inside the container, the rats were removed as soon as they gave signs that pain threshold had been reached (e.g., paw-licking or jumping). Cut-off time was 45 s, since any longer times inside the apparatus could cause tissue damage. Human observers measured the time interval between the placement of the animals on the bottom of the container and the licking or jumping action. Statistical analysis was performed on the grouped data: ANOVA was used for all temporal parameters (step-through and exit latency values, freezing duration, licking or jumping latency values). The ×2 test for active avoidance responses was also used.
Histology The brains were dissected and the appropriate portions were embedded in paraplast (Fisher). Sections 10 Ixm thick were serially made. Sections were stained overnight with toluidine blue (1:10,000). The sections were examined by two independent observers who, unaware of the behavioral results, decided which of the animals would be retained for statistical analysis and which would be discarded. RESULTS
Histology Bilateral amygdala lesions which included destruction of the n. basolateralis were confirmed in all animals. Reconstruction diagrams of the minimum (darkened) and maximum (striped) extent of the amygdala lesions are shown in Fig. 1, on serial plates from the atlas of Paxinos and Watson (27). No cavities were observed within the lesioned areas.
Preshock Step-Through Latency As shown in Fig. 2, on Trial 1 both groups of Ss (IBO and SO) exhibited very low step-through latency values. Indeed, there were no significant differences between the latency values of the two groups of Ss, F(1,30)=0.31, N.S.
Passive Avoidance As shown in Fig. 2, there were significant differences in stepthrough latency values between IBO and SO Ss. In all trials, the
N. BASOLATERALIS AND AVOIDANCE RESPONSES
767
ACQUISITION
RETENTION
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FIG. 2. Passive avoidance (step-through latency). O: IBO Ss; C): s o Ss. Symbols indicate trial mean values.
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0031-9384/91 $3.00 + .00
Effects of Nucleus Basolateralis Amygdalae Neurotoxic Lesions on Aversive Conditioning in the Rat CARLO AMBROGI LORENZINI, CORRADO BUCHERELLI, ALDO GIACHETTI, LAURA MUGNAI* AND GIOVANNA TASSONI
Dipartimento di Scienze Fisiologiche, Universitgl degli Studi di Firenze, Viale Morgagni 63, 1-50134, Firenze, Italy *Dipartimento di Biologia Animale e Genetica, Universitd degli Studi di Firenze, Via Romana 17 1-50125, Firenze, Italy R e c e i v e d 14 June 1990
AMBROGI LORENZINI, C., C. BUCHERELLI, A. GIACHETTI, L. MUGNAI AND G. TASSONI. Effects of nucleus basolateralis amygdalae neurotoxic lesions on aversive conditioning in the rat. PHYSIOL BEHAV 49(4) 765-770, 1991.--After bilateral stereotaxic administration of ibotenic acid on the n. basolateralis amygdalae, male adult rats were tested in the light-dark box apparatus to measure the time-course of the acquisition and retention of passive and active avoidance responses. The results show that after the lesions both passive avoidance and active avoidance acquisition were impaired. Passive avoidance responses were retained quite well, while active avoidance responses disappeared quickly. Conditioned freezing was almost completely absent. Thus it appears that the n. basolateralis plays a facilitatory role in all the conditioned responses which were investigated. N. basolateralis amygdalae
Active avoidance
Passive avoidance
Conditioned freezing
coagulation of passing fibres and glial cells as well as the death of neurons. The aim of the present work is to assess the effects of circumscribed lesions of the n. basolateralis amygdalae on the acquisition and retention of passive and active avoidance responses. The lesions will be caused by the stereotaxic administration of the neurotoxin, ibotenic acid. In our investigation we shall use the light-dark box paradigm (1-3) because it enables us to study the time course of acquisition and retention of both responses contemporaneously in the same animal (Rattus norvegicus). Initial freezing [a sign of fear system activation (1,3)] will also be measured in order to ascertain if chemical lesions of the nucleus basolateralis also affect emotionality.
DESTRUCTIVE techniques (6, 24, 26, 31, 35, 38), electrical stimulation (4, 21, 23) and pharmacologically active compounds (13, 15, 20) have all demonstrated that the amygdala plays a facilitatory role in the learning and memory of passive and active avoidance responses. Nonetheless, some reports differ in their conclusions (17, 19, 24, 37). This divergence of opinion may be at least partially due to the fact that, in a large number of the studies, the amygdala was manipulated "in toto." More detailed investigations have shown that the nucleus basolateralis amygdalae may be one of the lymbic structures, together with the nucleus centralis and the nucleus lateraiis (31). In addition, the former is probably most active in mnemonic processes (5, 11, 31, 35) since, between amygdala and prefrontal cortex, the nucleus basolateralis appears to be the most developed connective system (11). Furthermore, it also sends fibres to the n. mediodorsalis thalami which is considered to be an important link in the memory system (5,31). Moreover, there appears to be a positive relationship between the relative size of this nucleus and the degree of encephalization (31). In fact, after demolitive manipulation (10, 12, 18, 28, 29, 33, 34, 38) or electrical stimulation (16,21) there is ample evidence that this nucleus plays a definite role in the acquisition and retention of passive avoidance responses. Less coherent reports exist on the effects of lesions of this nucleus on active avoidance responses: deficits (19, 22, 38), no effects (19,37) or even a better learning performance (17). In most of these studies the n. basolateralis was destroyed by electrocoagulation. It is well known that this technique causes the
METHOD Thirty-two naive male Wistar rats (mean body weight 310 g, Morini, Italy) were employed. The animals were kept singly in stainless steel cages, in a room at a constant temperature of 20---I°C, natural illumination. They received food and water ad lib. The cages were cleaned daily, food troughs and water bottles were refilled when necessary, and always after the testing session. The rats were randomly divided into two groups, the controis (13 subjects) and the group to which ibotenic acid was administered (19 subjects). All rats were anesthetized with intraperitoneally administered ketamine (Ketalar, Parke Davis, 100 mg.kg-~ b.wt.). The animals were placed in a stereotaxic apparatus. The experimental group of subjects (IBO Ss) was injected
765
766
with ibotenic acid (Sigma) using the coordinates + 6 . 2 mm interaural line, + 1.5 mm at the dorsal plane and _+5 with respect to the median line (27). Ibotenic acid was diluted in a phosphate buffer solution (pH 7.4) at a concentration 10 txg.lxl- 1. Of this solution, 0.2 Ixl were injected into each side. Constant velocity injection lasted 3 min, after which the needle of the syringe (S.G.E., type A, 10 pJ) was left in situ for a further 5 min in order to maximize neurotoxin diffusion. The same volume of phosphate buffer solution was administered to control subjects (shamoperated, SO Ss) who underwent the same procedure. Behavioral testing was started 20 days after the operation.
Apparatus The apparatus consisted of a light-dark box, made of a light chamber (30 x 21 x 15 cm) of white opaque plastic with a transparent lid, and a dark chamber (30 x 21 x 15 cm) with five sides of dark opaque plastic. The floor of both chambers was made of 2-mm stainless steel rods spaced 1 cm; the floor of the dark chamber could be electrified. The chambers were connected by a guillotine door (6 x 8 cm). The apparatus was placed in an acoustically insulated room which was kept at a constant temperature of 2 0 - I ° C . Illumination inside the light chamber was 60 lux. Every day, the animals were manually placed, one by one, inside the apparatus. Latency values were taken when the animals had placed all 4 paws in the appropriate chamber.
Procedure The experiment consisted of a total of 20 trials. It began with two cycles of 7 consecutive single daily trials. After a 3-week interval, there was a third cycle of 6 consecutive single daily trials. All trials started at 9:00 a.m. During Cycle 1 (Acquisition), the Ss were placed inside the light chamber of the apparatus, with the connecting door open. Once the animals had spontaneously gone inside the dark chamber, or had been placed inside it (when step-through latency duration exceeded 180 s), the connecting door was closed. In the dark chamber the Ss received an inescapable footshock (0.8 mA, 2 s). This gave rise to appreciable motor reactions; only seldom did vocalization occur. Following the footshock, the connecting door was opened, and if the animal was still inside the dark chamber 10 s later, then a second footshock was administered (0.8 mA, 5 s). Exit latency was measured up to 15 s. Exits from the dark chamber with latencies of less than 10 s were considered active avoidance responses. During Cycles 2 (Retention 1, 7 trials) and 3 (Retention 2, 6 trials) no shocks were administered, and the trials were divided into two phases. The first phase which started at 9:00 a.m. was to assess the retention of the passive avoidance response. The Ss were placed directly into the light chamber with the connecting door open, and step-through latency was measured up to 180 s. As soon as the Ss went inside the dark chamber they were returned to their home cage. The second phase (11:00 a.m.) was to assess the retention of the active avoidance response. The Ss were placed directly inside the dark chamber, with the connecting door open and exit latencies were taken up to 15 s. Exits from the dark chamber with latencies of less than 10 s were considered as active avoidance responses. In all trials, the duration of initial freezing in the light chamber was measured. (Freezing is defined as motor inhibition resulting in immobility, which ends when voluntary movements are performed.) Initial freezing was that exhibited by the Ss immediately after having been placed inside the apparatus. Passive avoidance (step-through latency) and freezing data were grouped as
AMBROGI LORENZINI ET AL.
follows: days 2-8 for acquisition, days 9-14 and 36-41 for retention. Active avoidance data (exit latency and active avoidance responses) were grouped as follows: days 1-7 for acquisition, days 8-14 and 36--41 for retention. This was because step-through latencies and freezing durations of day 1 were measured before footshocks had been administered (preshock), while exit latencies were measured after the inescapable footshocks. Similarly the step-through latencies and freezing duration of day 8 were measured before the footshock omission while the exit latencies of the same day were measured after footshock omission. Some days after the end of Cycle 3, pain threshold was measured in all animals in order to ascertain whether the ibotenic lesion of the n. basolateralis had somehow influenced nociception. Pain threshold measurements were performed after the experiment to avoid any possible behavioral influence of the testing procedures. All Ss were tested with the hot-plate apparatus. This consisted of a flat-bottomed stainless steel container (28.5 x 36 x 15.5 cm) which was placed in a constant temperature water bath (56°C) (KW Mod W.82). The water was continuously stirred in order to keep the temperature of the stainless steel surfaces constant. Once they had been placed inside the container, the rats were removed as soon as they gave signs that pain threshold had been reached (e.g., paw-licking or jumping). Cut-off time was 45 s, since any longer times inside the apparatus could cause tissue damage. Human observers measured the time interval between the placement of the animals on the bottom of the container and the licking or jumping action. Statistical analysis was performed on the grouped data: ANOVA was used for all temporal parameters (step-through and exit latency values, freezing duration, licking or jumping latency values). The ×2 test for active avoidance responses was also used.
Histology The brains were dissected and the appropriate portions were embedded in paraplast (Fisher). Sections 10 Ixm thick were serially made. Sections were stained overnight with toluidine blue (1:10,000). The sections were examined by two independent observers who, unaware of the behavioral results, decided which of the animals would be retained for statistical analysis and which would be discarded. RESULTS
Histology Bilateral amygdala lesions which included destruction of the n. basolateralis were confirmed in all animals. Reconstruction diagrams of the minimum (darkened) and maximum (striped) extent of the amygdala lesions are shown in Fig. 1, on serial plates from the atlas of Paxinos and Watson (27). No cavities were observed within the lesioned areas.
Preshock Step-Through Latency As shown in Fig. 2, on Trial 1 both groups of Ss (IBO and SO) exhibited very low step-through latency values. Indeed, there were no significant differences between the latency values of the two groups of Ss, F(1,30)=0.31, N.S.
Passive Avoidance As shown in Fig. 2, there were significant differences in stepthrough latency values between IBO and SO Ss. In all trials, the
N. BASOLATERALIS AND AVOIDANCE RESPONSES
767
ACQUISITION
RETENTION
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FIG. 2. Passive avoidance (step-through latency). O: IBO Ss; C): s o Ss. Symbols indicate trial mean values.
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