Brain Research Bulletin, Vol. 26, pp. 699-706. Q Pergamon Press plc, 1991. Printed in the U.S.A.
0361.9230/91 $3.00 + .oO
Morphine and GABA: Effects on Perception, Escape Response and Long-Term Habituation to a Danger Stimulus in the Crab Chasmagnathus DANIEL
TOMSIC,
HECTOR
MALDONADO’
AND ANA RAKITIN
Laboratorio de Fisiologia de1 Comportamiento Animal, Facultad de Ciencias Exactas y Naturales UBA, Buenos Aires, Argentina Received
5 September
1990
TOMSIC, D., H. MALDONADO AND A. RAKITIN. Morphine and GABA: Effects on perception, escape response and longterm habituafion to a danger stimulus in the crab Chasmagnathus. BRAIN RES BULL 26(5) 699-706, 1991.-Prior results (37) showed that morphine pretreatment reduces the escape response of the crab Chasmqnathus to a shadow passing overhead and prevents the acquisition of a long-term habituation. These results were explained by a reduction in the danger signalled by the stimulus, and to test this hypothesis methods other than morphine injection were used herein to abolish response during training. GABA pretreatment induced a dose-dependent reduction in responsiveness to the danger stimulus, and instances of autotomy were shown with doses larger than 12 kg/g. A response was rarely displayed with a 9 p,g GABAIg dose given 5 min before training, but long-term memory was acquired. In one experiment, both morphine and GABA pretreatment produced similar mild response inhibition during training, but morphine, not GABA impaired long-term habituation. Morphine administered immediately after training had no amnesic effect. These results support the hypothesis that morphine effects may be explained by transient disruption between the stimulus and its danger meaning, ruling out alternative explanations such as response inhibition or amnesia due to either storage or retrieval failure. Habituation
Response-independent
habituation
Morphine
that habituates after repeated stimulation (9). Morphine produced a dose-dependent naloxone-reversible reduction in this response (30). In contrast, doses of morphine with strong inhibitory effect on the escape response (75-100 pg/g) failed to affect the optokinetic response (42), suggesting that the response decrement to a danger stimulus is stimulus-specific, and that morphine acts centrally rather than on visual input and/or motor ability. Two alternative hypotheses may account for the putative central action of morphine on the crab’s escape response to a shadow passing overhead. As previously advanced (30), the morphine effect could result from an interference with the decoding of the danger signal, namely by a reduction in the magnitude of the danger signal associated with the stimulus. An alternative proposal is that morphine raises the response threshold specifically to a danger stimulus. Should the Fist hypothesis be correct, repeated shadow stimulation to preinjected crabs would be expected not to produce long-term (between-session) habituation, since a proper danger stimulation was impaired during training. In contrast, if the second hypothesis is correct, long-term habituation is expected since a meaningful stimulus to H. Maldonado,
Danger stimulus
Crustacea
was present despite a low response level during training. Tomsic and Maldonado (42) demonstrated that morphine pretreatment sharply reduces reactivity during training and impairs the acquisition of long-term habituation, a result that they interpreted as supporting the Fist hypothesis. However, the failure to acquire a long-term habituated response might be accounted for by the impairment of responsiveness itself and not by an impairment in perception. This argument the authors contested on theoretical grounds, recalling that habituation studies support the belief that the stimulus rather than the response is the critical factor in this learning process (4, 11, 13, 34). To decide the issue on experimental grounds, a promising approach would be to compare the morphine effect on long-term habituation to a danger stimulus, with that obtained by using a drug that, without impairing vision, inhibits the escape response regardless of the type of visual stimulus presented, i.e., a drug acting on the efferent limb. For this purpose, GABA appears to be a good candidate, since it is well established as the transmitter substance released by crustacean inhibitory motor axons (5). Accordingly, present experiments are aimed at making such a comparison by testing the effect of GABA pretreatment both on the escape response level to a repeated danger stimulation and on the long-term habituation to the iterated stimulus.
WHEN a passing shadow (a danger stimulus) is presented to the crab Chasmagnathus granulatus, an escape response is elicited
‘Requests for reprints should be addressed taria, (1428) Buenos Aires, Argentina.
GABA
Laboratorio
699
Fisiologia
Comportamiento,
Depto Biologia,
Pabellon 2, Ciudad Universi-
700
TOMSIC, MALDONADO
GENERAL METHOD Animals The animals were adult male Chasmagnathus crabs, 2.8-3.0 cm across the carapace, collected from water less than 1 m deep in the rias (narrow coastal inlets) of San Clemente de1 Tuyu, Argentina, and transported to the laboratory, where they were lodged in plastic tanks (35 x 48 X 27 cm) filled to 2 cm depth with water without aeration, at a density of 35 crabs per tank. Water used in tanks and other containers during experiments was collected from the same place animals were captured (salinity lo-14%0, pH 7.4-7.6). The holding room was maintained on a 12-h light-dark cycle (lights on 0700-1900 h). Animals were fed rabbit pellets (Nutrientes SA) every 3 days and after feeding the water was changed. Temperature of both holding and experimental rooms as well as the alley between them was maintained within a range of 19-24°C. Experiments were conducted during daylight between the 2nd and 6th day after animals’ arrival, except when the response level failed to reach the acceptance criterion (see below) since in such case animals were kept for a previous adaptation period (3 or 4 days) in their home tanks. Each crab was used in only one experiment. Mean crab weight was determined as described elsewhere (9) (17.3 g, SE 0.2, n = 60) and absolute drug doses calculated according to this mean. Chasmagnuthus can be captured during the whole year except for the few coldest winter days (water temperature below 10°C). The level of response to a passing shadow is usually lower in animals coming from capture efforts corresponding to winter or the beginning of spring. Experiments of the present study were performed between October and May (i.e., late spring, summer and fall). Apparatus The apparatus used is described in detail elsewhere (39). Briefly, the experimental unit was the uctometer: a bowl-shaped plastic container with steep concave walls and a circular central flat floor 10 cm in diameter, covered to a depth of 0.5 cm with water. The crab was lodged in the container which was suspended by three strings from an upper wooden framework (23 x 23 x 30 cm) and illuminated by a 10-W lamp placed 30 cm above the animal. An opaque rectangle screen (25 x 13 cm) could be moved horizontally across the upper border of the framework by a motor at an angular speed which allowed it to cover the entire opening in 2.3 s, thereby projecting a shadow on the crab. Screen displacements provoked a crab’s running response and consequently container oscillations. A stylus was centrally cemented to the bottom of the container and connected to a piezoelectric transducer. Container oscillations induced, through the transducer, electrical signals proportional to the velocity of the oscillations (10). Such signals were amplified, integrated during the recording time (9 s) and translated into numerical units ranging from zero to 1020, before being processed by computer. Thus the scores were correlated proportionally to the velocity and number of the container oscillations recorded during 9 s. The amplification of the voltage changes was kept at such a gain that scores remained below 1020. The same activity unit and amplification gain has been used in all recent experiments at our laboratory [e.g., (28, 29, 40, 42)]. The experimental room had 40 actometers, isolated from each other by lateral partitions and a frontal wall. In order to avoid unobserved malfunctioning the actometers were periodically calibrated against one another by throwing a small lead ball from the upper border of the framework to the center of the
AND RAKITIN
container and recording the score for 9 s. A noticeable mity of scores was obtained (e.g.. a mean of 513 i9.48 40 actometers).
uniforfor the
Experimental Procedure A stimulation session comprised 15 or 3 trials given with 180-s intertrial intervals and was preceded by 30 min of adaptation in the actometer. Each trial lasted 9 s and consisted of passing the screen 4 times over the actometer, recording the crab’s activity during the entire trial time. Short-term experiments included a single 15-trial session, and long-term experiments two sessions, a first one of 15 trials and a second one of 3 trials, separated by a rest interval. When 2 sessions were run, crabs were individually housed in plastic containers both for 24 h before the first session and for the rest interval. The water in the plastic containers was changed daily and that in the actometers just before each experiment. Throughout this paper, either the single session of a short-term experiment or the first session of a long-term experiment is called training session, whereas the second session is termed testing session. A computer was employed to program trial sequences, trial duration and intertrial intervals, as well as to monitor experimental events. Since the number of actometers was insufficient to run all groups of each experiment simultaneously, replications during the same day were necessary. An equal number of crabs per group was used in each replication, but animals of a same group were placed in different actometers each time. Thus any potential effect of time of day and/or between-actometer differences was offset. The crab’s baseline responsiveness to the passing shadow proved remarkably consistent up to 10 days after arrival, but on occasion animals coming from different capture efforts presented differences in response level. Therefore, only crabs belonging to a same capture were used in each experiment, and in addition, an acceptance test aimed at ensuring a minimum level of responsiveness was employed. During the 2nd day after arrival and prior to any experimental use, one group of 40 crabs was given a 3-trial session. If the mean response obtained by averaging accumulated scores for the 3-trial session proved less than 1000, a second acceptance test was done 3-4 days later and if scores again failed to meet the criterion, no experiments were performed with crabs coming from such capture effort. Before animals were placed in the actometers or in the rest containers to start an experiment, they underwent a selection test: each crab was turned on its back and only animals that immediately returned to their normal position were used. The rationale behind this selection is that crabs with a slow righting reaction show a low responsiveness to a large diversity of stimuli, and at a later time, they usually present unhealthy symptoms. No more than 10% of tested crabs were eliminated. Injections Animals were injected by means of a Hamilton syringe through the right side of the cephalothoracic-abdominal membrane, i.e., the right metabranchial region. A small rubber ring placed in the needle 4 mm from the tip acted as a stop, ensuring the injected solution was released roughly at the center of the pericardial sac. Unlike the method used in other work at our laboratory, where injections were given through a needle chronically implanted in the carapace (9,30), the present technique allows animals to be readily moved from the actometers to be housed in individual containers during the intersession interval.
MORPHINE AND GABA: DANGER STIMULUS
IN CRAB
701
Injections consisted of 100 pl of the vehicle (NaCl, 1.6 %) or a drug solution. Morphine-HCl was purchased from SaporitiArgentina and gamma-aminobutyric acid from Sigma Chemicals. USA. Statistics The data from each experiment were analyzed with a oneway analysis of variance (ANOVA) followed by Duncan multiple range tests. Description of the Escape Response in the Actometer The escape response in the actometer consists of the crab starting to run in an attempt to move away from the passing shadow. However, since the steep concavity of the walls prevents the animal from climbing up, each running effort is confined to the flat center of the container, in such a way that the escape response during a single trial looks like a series of flights from the center toward the base of the walls. The extension and speed of each flight as well as its frequency vanish over each 9-s trial, and the total values per trial of these parameters also decrease over training. It is apparent that the lower the strength of an escape response, the lower the velocity and number of container oscillations during the 9-s trial. Therefore, a roughly linear correlation between response and score is expected since, as above mentioned, the electrical signal produced by the piezoelectric transducer is in turn proportional to the velocity and number of oscillations (10). This prediction is borne out by results from a test often performed in our laboratory in which the intensity of the escape response is scored by an observer as belonging to one of three categories (low, medium and high) and then compared with the score actually recorded. Values higher than 700 corresponded to responses classified as high responses; lower than 300 to low responses; and between 200 and 800, though with a noticeable preponderance of 400-600, to medium responses. The test showed higher interobserver reliability. Container movements caused by crab’s response to stimulation last less than 9 s, namely, less than the trial time. A spontaneous exploratory behavior consisting of slow displacements occurs mainly during the first minutes of the adaptation time and sporadically during intertrial intervals. Indeed, during most of the intertrial intervals the crab remains motionless at the center of the actometer. Thus no escape response is displayed either during the adaptation time or during intertrial intervals, and vice versa, no exploratory activity is shown during the trial time. During both training and testing session, no changes in behavior other than those related with the strength of the escape response, are observed.
0.0
0.06
0.6
60.0
1. GABA administration produces a dose-dependent reduction in responsiveness to a danger stimulus. Ordinates: average of accumulated FIG.
scores pe.r animal for 15 trials. Asterisks stand for significant differences between the saline control group and each one of the other groups (Duncan test) *p
0361.9230/91 $3.00 + .oO
Morphine and GABA: Effects on Perception, Escape Response and Long-Term Habituation to a Danger Stimulus in the Crab Chasmagnathus DANIEL
TOMSIC,
HECTOR
MALDONADO’
AND ANA RAKITIN
Laboratorio de Fisiologia de1 Comportamiento Animal, Facultad de Ciencias Exactas y Naturales UBA, Buenos Aires, Argentina Received
5 September
1990
TOMSIC, D., H. MALDONADO AND A. RAKITIN. Morphine and GABA: Effects on perception, escape response and longterm habituafion to a danger stimulus in the crab Chasmagnathus. BRAIN RES BULL 26(5) 699-706, 1991.-Prior results (37) showed that morphine pretreatment reduces the escape response of the crab Chasmqnathus to a shadow passing overhead and prevents the acquisition of a long-term habituation. These results were explained by a reduction in the danger signalled by the stimulus, and to test this hypothesis methods other than morphine injection were used herein to abolish response during training. GABA pretreatment induced a dose-dependent reduction in responsiveness to the danger stimulus, and instances of autotomy were shown with doses larger than 12 kg/g. A response was rarely displayed with a 9 p,g GABAIg dose given 5 min before training, but long-term memory was acquired. In one experiment, both morphine and GABA pretreatment produced similar mild response inhibition during training, but morphine, not GABA impaired long-term habituation. Morphine administered immediately after training had no amnesic effect. These results support the hypothesis that morphine effects may be explained by transient disruption between the stimulus and its danger meaning, ruling out alternative explanations such as response inhibition or amnesia due to either storage or retrieval failure. Habituation
Response-independent
habituation
Morphine
that habituates after repeated stimulation (9). Morphine produced a dose-dependent naloxone-reversible reduction in this response (30). In contrast, doses of morphine with strong inhibitory effect on the escape response (75-100 pg/g) failed to affect the optokinetic response (42), suggesting that the response decrement to a danger stimulus is stimulus-specific, and that morphine acts centrally rather than on visual input and/or motor ability. Two alternative hypotheses may account for the putative central action of morphine on the crab’s escape response to a shadow passing overhead. As previously advanced (30), the morphine effect could result from an interference with the decoding of the danger signal, namely by a reduction in the magnitude of the danger signal associated with the stimulus. An alternative proposal is that morphine raises the response threshold specifically to a danger stimulus. Should the Fist hypothesis be correct, repeated shadow stimulation to preinjected crabs would be expected not to produce long-term (between-session) habituation, since a proper danger stimulation was impaired during training. In contrast, if the second hypothesis is correct, long-term habituation is expected since a meaningful stimulus to H. Maldonado,
Danger stimulus
Crustacea
was present despite a low response level during training. Tomsic and Maldonado (42) demonstrated that morphine pretreatment sharply reduces reactivity during training and impairs the acquisition of long-term habituation, a result that they interpreted as supporting the Fist hypothesis. However, the failure to acquire a long-term habituated response might be accounted for by the impairment of responsiveness itself and not by an impairment in perception. This argument the authors contested on theoretical grounds, recalling that habituation studies support the belief that the stimulus rather than the response is the critical factor in this learning process (4, 11, 13, 34). To decide the issue on experimental grounds, a promising approach would be to compare the morphine effect on long-term habituation to a danger stimulus, with that obtained by using a drug that, without impairing vision, inhibits the escape response regardless of the type of visual stimulus presented, i.e., a drug acting on the efferent limb. For this purpose, GABA appears to be a good candidate, since it is well established as the transmitter substance released by crustacean inhibitory motor axons (5). Accordingly, present experiments are aimed at making such a comparison by testing the effect of GABA pretreatment both on the escape response level to a repeated danger stimulation and on the long-term habituation to the iterated stimulus.
WHEN a passing shadow (a danger stimulus) is presented to the crab Chasmagnathus granulatus, an escape response is elicited
‘Requests for reprints should be addressed taria, (1428) Buenos Aires, Argentina.
GABA
Laboratorio
699
Fisiologia
Comportamiento,
Depto Biologia,
Pabellon 2, Ciudad Universi-
700
TOMSIC, MALDONADO
GENERAL METHOD Animals The animals were adult male Chasmagnathus crabs, 2.8-3.0 cm across the carapace, collected from water less than 1 m deep in the rias (narrow coastal inlets) of San Clemente de1 Tuyu, Argentina, and transported to the laboratory, where they were lodged in plastic tanks (35 x 48 X 27 cm) filled to 2 cm depth with water without aeration, at a density of 35 crabs per tank. Water used in tanks and other containers during experiments was collected from the same place animals were captured (salinity lo-14%0, pH 7.4-7.6). The holding room was maintained on a 12-h light-dark cycle (lights on 0700-1900 h). Animals were fed rabbit pellets (Nutrientes SA) every 3 days and after feeding the water was changed. Temperature of both holding and experimental rooms as well as the alley between them was maintained within a range of 19-24°C. Experiments were conducted during daylight between the 2nd and 6th day after animals’ arrival, except when the response level failed to reach the acceptance criterion (see below) since in such case animals were kept for a previous adaptation period (3 or 4 days) in their home tanks. Each crab was used in only one experiment. Mean crab weight was determined as described elsewhere (9) (17.3 g, SE 0.2, n = 60) and absolute drug doses calculated according to this mean. Chasmagnuthus can be captured during the whole year except for the few coldest winter days (water temperature below 10°C). The level of response to a passing shadow is usually lower in animals coming from capture efforts corresponding to winter or the beginning of spring. Experiments of the present study were performed between October and May (i.e., late spring, summer and fall). Apparatus The apparatus used is described in detail elsewhere (39). Briefly, the experimental unit was the uctometer: a bowl-shaped plastic container with steep concave walls and a circular central flat floor 10 cm in diameter, covered to a depth of 0.5 cm with water. The crab was lodged in the container which was suspended by three strings from an upper wooden framework (23 x 23 x 30 cm) and illuminated by a 10-W lamp placed 30 cm above the animal. An opaque rectangle screen (25 x 13 cm) could be moved horizontally across the upper border of the framework by a motor at an angular speed which allowed it to cover the entire opening in 2.3 s, thereby projecting a shadow on the crab. Screen displacements provoked a crab’s running response and consequently container oscillations. A stylus was centrally cemented to the bottom of the container and connected to a piezoelectric transducer. Container oscillations induced, through the transducer, electrical signals proportional to the velocity of the oscillations (10). Such signals were amplified, integrated during the recording time (9 s) and translated into numerical units ranging from zero to 1020, before being processed by computer. Thus the scores were correlated proportionally to the velocity and number of the container oscillations recorded during 9 s. The amplification of the voltage changes was kept at such a gain that scores remained below 1020. The same activity unit and amplification gain has been used in all recent experiments at our laboratory [e.g., (28, 29, 40, 42)]. The experimental room had 40 actometers, isolated from each other by lateral partitions and a frontal wall. In order to avoid unobserved malfunctioning the actometers were periodically calibrated against one another by throwing a small lead ball from the upper border of the framework to the center of the
AND RAKITIN
container and recording the score for 9 s. A noticeable mity of scores was obtained (e.g.. a mean of 513 i9.48 40 actometers).
uniforfor the
Experimental Procedure A stimulation session comprised 15 or 3 trials given with 180-s intertrial intervals and was preceded by 30 min of adaptation in the actometer. Each trial lasted 9 s and consisted of passing the screen 4 times over the actometer, recording the crab’s activity during the entire trial time. Short-term experiments included a single 15-trial session, and long-term experiments two sessions, a first one of 15 trials and a second one of 3 trials, separated by a rest interval. When 2 sessions were run, crabs were individually housed in plastic containers both for 24 h before the first session and for the rest interval. The water in the plastic containers was changed daily and that in the actometers just before each experiment. Throughout this paper, either the single session of a short-term experiment or the first session of a long-term experiment is called training session, whereas the second session is termed testing session. A computer was employed to program trial sequences, trial duration and intertrial intervals, as well as to monitor experimental events. Since the number of actometers was insufficient to run all groups of each experiment simultaneously, replications during the same day were necessary. An equal number of crabs per group was used in each replication, but animals of a same group were placed in different actometers each time. Thus any potential effect of time of day and/or between-actometer differences was offset. The crab’s baseline responsiveness to the passing shadow proved remarkably consistent up to 10 days after arrival, but on occasion animals coming from different capture efforts presented differences in response level. Therefore, only crabs belonging to a same capture were used in each experiment, and in addition, an acceptance test aimed at ensuring a minimum level of responsiveness was employed. During the 2nd day after arrival and prior to any experimental use, one group of 40 crabs was given a 3-trial session. If the mean response obtained by averaging accumulated scores for the 3-trial session proved less than 1000, a second acceptance test was done 3-4 days later and if scores again failed to meet the criterion, no experiments were performed with crabs coming from such capture effort. Before animals were placed in the actometers or in the rest containers to start an experiment, they underwent a selection test: each crab was turned on its back and only animals that immediately returned to their normal position were used. The rationale behind this selection is that crabs with a slow righting reaction show a low responsiveness to a large diversity of stimuli, and at a later time, they usually present unhealthy symptoms. No more than 10% of tested crabs were eliminated. Injections Animals were injected by means of a Hamilton syringe through the right side of the cephalothoracic-abdominal membrane, i.e., the right metabranchial region. A small rubber ring placed in the needle 4 mm from the tip acted as a stop, ensuring the injected solution was released roughly at the center of the pericardial sac. Unlike the method used in other work at our laboratory, where injections were given through a needle chronically implanted in the carapace (9,30), the present technique allows animals to be readily moved from the actometers to be housed in individual containers during the intersession interval.
MORPHINE AND GABA: DANGER STIMULUS
IN CRAB
701
Injections consisted of 100 pl of the vehicle (NaCl, 1.6 %) or a drug solution. Morphine-HCl was purchased from SaporitiArgentina and gamma-aminobutyric acid from Sigma Chemicals. USA. Statistics The data from each experiment were analyzed with a oneway analysis of variance (ANOVA) followed by Duncan multiple range tests. Description of the Escape Response in the Actometer The escape response in the actometer consists of the crab starting to run in an attempt to move away from the passing shadow. However, since the steep concavity of the walls prevents the animal from climbing up, each running effort is confined to the flat center of the container, in such a way that the escape response during a single trial looks like a series of flights from the center toward the base of the walls. The extension and speed of each flight as well as its frequency vanish over each 9-s trial, and the total values per trial of these parameters also decrease over training. It is apparent that the lower the strength of an escape response, the lower the velocity and number of container oscillations during the 9-s trial. Therefore, a roughly linear correlation between response and score is expected since, as above mentioned, the electrical signal produced by the piezoelectric transducer is in turn proportional to the velocity and number of oscillations (10). This prediction is borne out by results from a test often performed in our laboratory in which the intensity of the escape response is scored by an observer as belonging to one of three categories (low, medium and high) and then compared with the score actually recorded. Values higher than 700 corresponded to responses classified as high responses; lower than 300 to low responses; and between 200 and 800, though with a noticeable preponderance of 400-600, to medium responses. The test showed higher interobserver reliability. Container movements caused by crab’s response to stimulation last less than 9 s, namely, less than the trial time. A spontaneous exploratory behavior consisting of slow displacements occurs mainly during the first minutes of the adaptation time and sporadically during intertrial intervals. Indeed, during most of the intertrial intervals the crab remains motionless at the center of the actometer. Thus no escape response is displayed either during the adaptation time or during intertrial intervals, and vice versa, no exploratory activity is shown during the trial time. During both training and testing session, no changes in behavior other than those related with the strength of the escape response, are observed.
0.0
0.06
0.6
60.0
1. GABA administration produces a dose-dependent reduction in responsiveness to a danger stimulus. Ordinates: average of accumulated FIG.
scores pe.r animal for 15 trials. Asterisks stand for significant differences between the saline control group and each one of the other groups (Duncan test) *p
