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Does Induced Recovery from Amnesia Represent a Disinhibition Effect?
Physiology & Behavior, Vol. 60, No. 5, pp. 1375-1378, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights r
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PII S0031-9384(96)00221-1
B
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STEVEN
B. HARROD,l
f MITCHELL
M. METZGER
Kent State University, Received H P B w r a ‘ q w S a r a , the e s b T f a r
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THE presentation of a relatively novel stimulus following extinction can result in at least partial recovery of the original conditioned response. This “disinhibition” effect, first reported by Pavlov (6), was thought to represent the inhibition of inhibitory (extinction) processes. Although early studies of disinhibition utilized Pavlovian conditioning paradigms, this phenomenon has been extended to operant conditioning procedures, as well ( 1,2). Moreover, disinhibition is not limited to the extinction paradigm, as it has been found with a variety of manipulations that lead to suppression of performance (e.g., satiation, unsigrtaled foot shock, and punishment), and it is obtained with various novel stimuli, attesting to the generality of this effect ( 1,2). Interestingly, this description of disinhibition is not unlike a finding that comes out of a very different line of research: recovery of memory following experimentally-induced amnesia. Although reversal of amnesia has been obtained in several ways, of particular concern here are those in which reexposure to the amnestic treatment shortly before testing leads to recovery of the target response (3,8). For example, an early experiment (3) induced retrograde amnesia (RA) for passive avoidance learning by reducing rats’ body temperature (i.e., inducing hypothermia) immediately after fear conditioning. Following a 48-h delay, subjects were recooled
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1996
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Kent, OH 44242 USA
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prior to testing. The r-auks suggested that, when recooled, rats showed avoidance of the fear cues (reversal of amnesia) by avoiding the stimuli paired with shock. These and similar outcomes have been taken as evidence that providing appropriate contextual retrieval cues produces recovery of the memory. Although control conditions ruled out locomotor artifacts as the source of improved test performance, the disinhibition phenomenon suggests an alternative, quite different, interpretation: the recooling may act as a relatively novel stimulus that releases the previously-learned fear response suppressed by the amnestic treatment. More specifically, in this view, amnesia would be analogous to extinction, in that hypothermia treatment suppresses the conditioned fear response. If this is the case, then recooling may be ‘‘releasing’ the fear response, much like a light or a tone in the disinbibition literature. Although evidence exists suggesting that a disinhibition interpretation of recovery from amnesia is unlikely, this alternative explanation cannot be entirely ruled out. For instance, in a study examining recovery of memory from anterograde amnesia (AA), recovery of memory was evident only when subjects’ colonic body temperature at testing matched that of the training temperature; subjects tested at temperatures warmer than the training
s
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HARROD, METZGER AND RICCIO
1376 TABLE 1 MEAN LATENCY (LOG SECONDS) TO ENTER THE BLACK (FEAR) COMPARTMENT AND T’tW’ AT THE 24-H TEST IN EXPERIMENT 1 TTw
Latency Group
C E E
n
Mean
8 9 8
2 1 1
SE
0 0 0
Mean
2 1 1
SE
0 0 0
temperature exhibited AA similar to subjects tested immediately after training (8). Moreover, a similar study demonstrated this effect with a retrograde amnesia paradigm (4). In this study, reversal of retrograde amnesia was observed if rats were tested while slightly hypothermic, whereas no recovery from amnesia was observed if rats were tested at normal or near-normal body temperature. Whether done with a retrograde or anterograde paradigm, the reinstatement of cold-related cues reliably reverses amnesias produced by hypothermia treatment. This stimulus control over the recovery of memory implies that recovery is specific to the original training situation; if the cold stimulus (recooling) were simply acting as a disinhibitor, it is likely that other cold temperatures would disinhibit the behavior, as well. But the problem is that the effectiveness of disinhibiting stimuli may depend on their intensity, as suggested by one of Brimer’s findings ( 1). This leaves open the possibility that other temperatures used in attempting to reverse AA were simply less intense stimuli that did not serve as good disinhibitors (8). Evidence supporting the notion of disinhibition would challenge the interpretations that emphasize memory retrieval as the basis for the alleviation of amnesia by reexposure to the amnestic agent (e.g., 3,7,9). The present study examined the possibility that the hypothermia treatment used to reverse amnesia might also produce recovery of responding following extinction, that is, a disinhibition effect. Except for the use of nonreinforced exposure to the CS, rather than hypothermia, to suppress test performance, the paradigm was essentially the same as that used in many of the studies of reversal of amnesia. EXPERIMENT 1 METHOD
Subjects Thirty 60–100-day-old naive, male Sprague–Dawley rats, purchased from the Hilltop Co. (Scottsdale, PA), served as subjects in the present experiment. All animals were housed individually in hanging wire-mesh cages and were maintained on a 15:09 h light:dark cycle. Food and water were available ad lib. Apparatus The training and testing apparatus was a Plexiglas rectangular box (38 X 18 X 21 cm) divided into two identically sized chambers. One chamber was painted white and fitted with a clear lid, and the walls and lid in the opposite chamber were painted black. A (8 X 10 cm) guillotine doorway divided the apparatus, which allowed the subjects to move freely throughout the two chambers. A 15-W light bulb was suspended from the ceiling 30 cm above the white compartment; no other light source was present in the training-testing room. The floor of the entire apparatus was made up of 2.5 mm stainless-steel rods, evenly spaced 1 cm apart.
White noise was presented throughout the experiment to reduce any potential auditoty disturbances. The hypothermia apparatus was a Forma Scientific (Marietta, OH) water tank, maintained at 3“C. Subjects were immersed via opaque restraining tubes (7.5 cm PVC tubing) containing twelve 1.25-cm holes that allowed water to flow through the restraining apparatus. An aluminum band was attached to the top of the restraining tube, and the band laid across the top of the tank so that the subjects were immersed in the water in a head-up position. Removable Plexiglas caps were applied to the bottom of the tubes to confine the rats within the restraining apparatus. Subjects’ colonic body temperatures were measured with a Fisher Digital Thermometerm (Pittsburgh, PA). The water tanks were located directly outside of the room containing the conditioning apparatus. Procedure Animals were randomly assigned to 1 of 3 groups: the extinction normal (EXT-NORM n = 10), extinction hypothermia (EXT-HYPO n = 10), or the control (CTRL n = 10) group. All animals were handled for 2 min daily, 2 days prior to the beginning of the experiment. Conditioning and extinction. On day 1, all subjects were removed from their home cages and placed facing away from the door on the white side of the training apparatus. After 20s, the door was raised, allowing subjects to cross to the black chamber. Upon crossing, the door was lowered and a 3-s, inescapable, 200 v foot shock was delivered through a matched-impedance shock source. Following foot shock, rats were immediately removed from the black compartment and placed in a holding cage. To insure that all subjects had been conditioned to the black compartment, an immediate test for fear was administered. After 15 rein, all subjects were placed, facing away from the door, in the white chamber. Ten seconds later, the door was raised, giving the rats access to the black chamber. The measures were latency to cross into the black compartment and total time spent in the white (safe) chamber (TTW). A 180-s test was administered and, if a subject exhibited poor learning(i.e., failure to avoid shock-related cues), they were removed from the experiment. This procedure insured that all subjects were equated on the initial acquisition of the task. Following testing, subjects in the EXT-NORM and EXT-HYPO groups were confined to the black chamber and given 4 mitt of extinction training. Animals in the group CTRL were taken to their home cages directly following the immediate test of fear and remained there until testing occurred on day 2. T for disinhibition. On day 2, groups EXT-NORM and CTRL were individually placed in the white chamber facing away from the guillotine door. Ten seconds later, the door was removed from the apparatus, providing access to the black compartment. The EXT-HYPO group was administered hypothermia treatment prior to testing. Subjects were placed in the restraining tubes and immersed to the neck in 3°C water until their colonic body temperatures dropped to 25 f 1“C. Colonic body temperatures were assessed by inserting the temperature probe 3.2 cm into the rats’ rectum. Subjects were then placed in a holding cage until their colonic temperature reached 29–31“C. [Subjects were tested at this temperature because this range of temperature has been reported to yield the highest level of recovery for avoidance memory in rats that have been rendered amnesic by hypothermia treatment (4)]. In a concern that any manipulation might lead to disinhibition, we did not give the EXT-NORM rats any treatment to control for the noncooling experience of the EXT-HYPO rats. For this reason, we did not expose the EXT-NORM rats to the restraining tubes or cooling tank prior to testing. Therefore, the EXT-NORM group served as a baseline control for determining
DISINHIBITION AND RECOVERY FROM AMNESIA if extinction of the fear response was successful. After reaching the appropriate temperature, the rats were tested exactly like the CTRL and EXT-NORM groups. All subjects were given a 600s test during which latency to cross into the black chamber and the total time in the white (safe) chamber (TTW) were measured. RESULTS AND DISCUSSION
The results are presented in Table 1. To help normalize the distributions, all scores were converted to log form. Five animals were dropped from the experiment due to failure to demonstrate passiveavoidance learning on the immediate test. Therefore, the number of subjects was as follows: groups CTRL and EXT-HYPO = 8 each, and EXT-NORM = 9. A l-way ANOVA on the initial training latency revealed no difference in the subjects tendency to cross to the black chamber during training [F(2,22) < 1.0]; thus, all subjects exhibited similar cross-over latencies on the conditioning day. Separate l-way ANOVAs on latency and TfW for the immediate test, which was administered 15 min after training, also revealed no reliable differences; that is, all scores, regardless of group, were very high, implying that fear conditioning was successfid [Latency: F(2,22) < 1.0; and TTW: F’(2,22) < 1.0]. To further assess whether or not subjects acquired fear to the black compartment, a dependent groups t-test was used to compare scores collapsed across all groups for both the initial training latency and immediate latency following training. This comparison revealed that immediate test latencies were reliably longer than training latencies, indicating that subjects in each of the 3 groups acquired fear to the black compartment (t= 8.56, p < 0.01). Separate one-way ANOVAs on the 24-h latency and TTW scores indicated a significant effect for both measures [F(2,22) = 8.941 for latency, p <0.01 and F(2,22) = 5.867 for TTW, p < 0.01]. Planned t-tests (one tailed) on the latency scores revealed that the EXT-NORM and EXT-HYPO groups differed significantly from the group CTRL (t = 2.37, p < 0.05; t = 3.85, p < respectively). Furthermore, although these 2 groups differed from one another (t = 2.29, p < 0.05), the mean of the EXT-HYPO group (M = 1.214) was significantly lower than that of the EXT-NORM group (M = 1.833); that is, subjects receiving hypothermia crossed to the black chamber faster than rats receiving extinction alone. This result is opposite to what would be expected if hypothermia were acting as a disinhibitor. Planned t-tests (one-tailed) for the 24-h TTW scores revealed that the EXT-NORM and EXT-HYPO groups were not significantly different from one another (t = 1.75, 0.05 < p < 0.10) but both extinction groups differed significantly from group CTRL (EXT-NORM vs. CTRL; t= 2.23, p < and EXTHYPO vs. CTRL; t= 3.07, p < 0.01). Thus, following the extinction treatment, the rats spent an extended amount of time on the black side, evidencing reduced fear of the black compartment. However, neither the latency nor TTW data suggest that hypothermia treatment prior to testing results in recovery of the avoidance (fear) to the black compartment.
1377 served as subjects in passive-avoidance conditioning and conditioned taste-aversion learning prior to the present study. Subjects were housed in groups of 4 and were ear-punched for identification. A
The trainingltesting and hypothermia apparatus were identical to that of Experiment 1.
Random assignment and group designation were identicaI to that of Experiment 1. The groups were the control (CTRL n = 10), extinction/normal (EXT-NORM n = 10), and the extinction/hypothermia (EXT-HYPO n = 10) groups. Conditioning. On day 1, all rats received a punishment/fear conditioning session (modified passive-avoidance training). Subjects were taken from their home cages and placed in the white chamber facing away from the guillotine door. After 2 rein, the door was raised, allowing access to the black chamber. Upon crossing, the door was closed, confining the subjects to the black compartment. Then 6 3-s, 250 v foot shocks were delivered at 10,30,40, 70,90, and 100s. Twenty seconds after the last shock, the door was raised and the rats were allowed to cross back into the white chamber. After 2 min had elapsed, the door was raised and an identical conditioning procedure was repeated. If subjects remained in the white compartment for more than 10s, they were gently pushed through the doorway by the experimenter. Following conditioning, all subjects were removed from the white chamber and taken back to the home cage. Additionally, in this experiment, we did not administer an immediate test for acquisition of fear conditioning as in Experiment 1. Extinction. On the second day, the 2 extinction groups were taken individually from their home cages and placed directly into the black chamber for 12 min with the door closed. No shocks were presented during this period. Following the extinction exposure, all subjects were taken back to their home cages. Test for disinhibition. Hypothermia treatment was administered immediately prior to the test for disinhibition, and this occurred 24 h after extinction (day 3). As in Experiment 1, rats in the EXT-HYPO group were administered hypothermia treatment until their temperatures reached 25”C, and were then tested after they had rewarmed to 29–31°C. All 3 groups were administered a 600-s test identical to that used in Experiment 1. Again, retention of fear was indexed in terms of latencies to cross and the amount of time spent in the white (safe) chamber (TTW). RESULTS AND DISCUSSION
Again, all data were converted to log form to help normalize the distributions, and are presented in Table 2. A l-way ANOVA
EXPERIMENT 2
TABLE 2
Although the training procedure in Experiment 1 was the same as that used in many RA studies, other paradigms have also been employed, particularly in reseamh on anterograde amnesia (8). Accordingly, using a slightly different task, our training procedure in Experiment 2 utilized a modified passive avoidance procedure that was followed by relatively long ( 12-min) extinction training.
MEAN LATENCY (LOG SECONDS) TO ENTER THE BLACK (FEAR) COMPARTMENT AND TTW AT THE 48-H TEST (DAY 3) IN EXPERIMENT 2 Latency Group
n
TTw
Mean
SE
Mean
SE
METHOD
Subjects Thirty 100–150-day-old male, Sprague–Dawley rats, purchased from the Hilltop Co., served as subjects. The animals also
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HARROD, METZGER AND RICCIO
on the TTW scores indicated that there were reliable differences among the groups on the test for fear to the black chamber [F(2,27) = 4.141, p < 0.05]. Planned t-tests (one-tailed) for the TTW data revealed that the EXT-NORM and EXT-HYPO groups were significantly lower than group CTRL (t = 1.73, p < 0.05; t = 2.72, p < 0.01, respectively]; neither group displayed fear to the black chamber. Although the EXT-HYPO group demonstrated lower TTW scores than the EXT-NORM group, they were not significantly different (t = 1.36, p > 0.05). Contrary to the results from the TTW scores, an ANOVA on the latency scores indicated that there were no reliabIe differences among the groups on the test day [F(2,27) = 1.644, p > 0.05]. One reason for this discrepancy between TTW and latency scores in this experiment is that the TTW measure may be a more sensitive indicator of fear with this paradigm. On the retention test, subjects will occasionally cross over to the black (shock) chamber relatively quickly, but then return to the white (safe) chamber and remain there for the duration of the test. This results in relatively short latency scores and high TTW scores. It is clear from these data that the induction of hypothermia prior to testing does not result in recovery of avoidance of the black compartment. Thus, there is no evidence that hypothermia disinhibited a conditioned fear response in this modified passiveavoidance procedure. One concern for using subjects that had already received passive-avoidance training (previous to this experiment) is that positive transfer may have occurred between the 2 training situations. However, given the similar results of Experiments 1 and 2, there seems little reason to believe that the training received by subjects prior to Experiment 2 confounded the outcome. GENERAL DISCUSSION The contextual retrieval cue explanation for the recovery of memory following experimentally-induced amnesia, in studies that used hypothermia or hyperthermia, was reevaluated (3,5,8). The present study questioned whether or not recovery of the target response following reexposure to the amnestic treatment reflects a disinhibition effect. Thus, the effects of hypothermia on performance were examined following an extinction exposure in the standard passive-avoidance task (Experiment 1) and a modified passive-avoidance procedure (Experiment 2). The present findings do not support the notion that hypothermia acts as a disinhibitor of a previously-conditioned and extinguished fear response. In both experiments, rats receiving hy-
pothermia shortly prior to testing actually exhibited shorter TTW scores than rats receiving extinction only. Because the procedures closely parallel those used to produce reversal of amnesia, it is not likely that the recovery of memory reported with reexposure to the amnestic agent (e.g., 3,8) can be accounted for by a disinhibition of the conditioned fear response. The contextual cues hypothesis of memory retrieval states that optimal retrieval of the memory for a training situation occurs when subjects are tested in the same contextual state as that of training. Therefore, it might be argued that one reason why hypothermia did not act as a disinhibitor in the current experiments is because animals were tested in an altered (recooled) state. However, there is evidence that indicates that when rats are given hypothermia treatment shortly after passive-avoidance training to induce RA, the reversal of amnesia does not occur when animals are recooled, but allowed to rewarm to normal body temperature prior to testing (4). Additionally, these experiments were aimed to address whether or not recooling acts as a disinhibitor in recovery of amnesia procedures. Because amnesia-recovery studies typically test subjects in an altered (recooIed) state, we attempted to parallel those studies as closely as possible. In addition to the methodological implications of this “negative result,” the findings are interesting with respect to disinhibition processes: Why did hypothermia, a novel and intense stimulus, fail to disinhibit an extinguished conditioned response? Unlike the present experiment, traditional disinhibition studies have generally presented a punctate auditory or visual stimulus ( 1,2). The hypothermia-inducing agent (cold water) is a tactile stimulus, requiring that the subject be exposed to the stimulus for 5 to 10 min to reach hypothermia criterion. Although it may now be interesting to examine whether or not traditional disinhibitors would have worked to disinhibit the fear response within the present paradigm, that issue is quite different from our original goal of mimicking the major features of experiments inducing recovery from amnesia by reexposure to the amnestic agent. Overall, this study provides no evidence that hypothermia acts as a disinhibitor and therefore reduces the plausibility of one potential challenge to the retrieval cue interpretation of the recovery of memory following amnesia. ACKNOWLEDGEMENTS The r
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REFERENCES 1. Bnmer, C. J. Disinhibitionof an operant response. Learn. Motiv. 1:346–371;1970. B
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W af L B R b t 1 F
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3
1 H
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P A
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hyperthermia:An unusuatlyprofound,yet reversible,memoryloss. B P D R b 9 R b i S t
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1 B
0
$
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PII S0031-9384(96)00221-1
B
D
I
C
O
R aD
STEVEN
B. HARROD,l
f MITCHELL
M. METZGER
Kent State University, Received H P B w r a ‘ q w S a r a , the e s b T f a r
M 6
R
1 w
s
1 v a r t E b
p h r t
r p
2f e t f 4 m S w r p n t m a a
s t s
D
A
M
25 January D
i
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t w
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passive-avoidanceprocedureand lengthening e
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THE presentation of a relatively novel stimulus following extinction can result in at least partial recovery of the original conditioned response. This “disinhibition” effect, first reported by Pavlov (6), was thought to represent the inhibition of inhibitory (extinction) processes. Although early studies of disinhibition utilized Pavlovian conditioning paradigms, this phenomenon has been extended to operant conditioning procedures, as well ( 1,2). Moreover, disinhibition is not limited to the extinction paradigm, as it has been found with a variety of manipulations that lead to suppression of performance (e.g., satiation, unsigrtaled foot shock, and punishment), and it is obtained with various novel stimuli, attesting to the generality of this effect ( 1,2). Interestingly, this description of disinhibition is not unlike a finding that comes out of a very different line of research: recovery of memory following experimentally-induced amnesia. Although reversal of amnesia has been obtained in several ways, of particular concern here are those in which reexposure to the amnestic treatment shortly before testing leads to recovery of the target response (3,8). For example, an early experiment (3) induced retrograde amnesia (RA) for passive avoidance learning by reducing rats’ body temperature (i.e., inducing hypothermia) immediately after fear conditioning. Following a 48-h delay, subjects were recooled
‘
s
r
r C
r
a f a
d a 4 u
r
a
a
r c
b
r
C. RICCIO
1996
p
d 1i
o s
AND DAVID
r f
T (
e R
Kent, OH 44242 USA
r w i
An Ei
prior to testing. The r-auks suggested that, when recooled, rats showed avoidance of the fear cues (reversal of amnesia) by avoiding the stimuli paired with shock. These and similar outcomes have been taken as evidence that providing appropriate contextual retrieval cues produces recovery of the memory. Although control conditions ruled out locomotor artifacts as the source of improved test performance, the disinhibition phenomenon suggests an alternative, quite different, interpretation: the recooling may act as a relatively novel stimulus that releases the previously-learned fear response suppressed by the amnestic treatment. More specifically, in this view, amnesia would be analogous to extinction, in that hypothermia treatment suppresses the conditioned fear response. If this is the case, then recooling may be ‘‘releasing’ the fear response, much like a light or a tone in the disinbibition literature. Although evidence exists suggesting that a disinhibition interpretation of recovery from amnesia is unlikely, this alternative explanation cannot be entirely ruled out. For instance, in a study examining recovery of memory from anterograde amnesia (AA), recovery of memory was evident only when subjects’ colonic body temperature at testing matched that of the training temperature; subjects tested at temperatures warmer than the training
s
k
1375
HARROD, METZGER AND RICCIO
1376 TABLE 1 MEAN LATENCY (LOG SECONDS) TO ENTER THE BLACK (FEAR) COMPARTMENT AND T’tW’ AT THE 24-H TEST IN EXPERIMENT 1 TTw
Latency Group
C E E
n
Mean
8 9 8
2 1 1
SE
0 0 0
Mean
2 1 1
SE
0 0 0
temperature exhibited AA similar to subjects tested immediately after training (8). Moreover, a similar study demonstrated this effect with a retrograde amnesia paradigm (4). In this study, reversal of retrograde amnesia was observed if rats were tested while slightly hypothermic, whereas no recovery from amnesia was observed if rats were tested at normal or near-normal body temperature. Whether done with a retrograde or anterograde paradigm, the reinstatement of cold-related cues reliably reverses amnesias produced by hypothermia treatment. This stimulus control over the recovery of memory implies that recovery is specific to the original training situation; if the cold stimulus (recooling) were simply acting as a disinhibitor, it is likely that other cold temperatures would disinhibit the behavior, as well. But the problem is that the effectiveness of disinhibiting stimuli may depend on their intensity, as suggested by one of Brimer’s findings ( 1). This leaves open the possibility that other temperatures used in attempting to reverse AA were simply less intense stimuli that did not serve as good disinhibitors (8). Evidence supporting the notion of disinhibition would challenge the interpretations that emphasize memory retrieval as the basis for the alleviation of amnesia by reexposure to the amnestic agent (e.g., 3,7,9). The present study examined the possibility that the hypothermia treatment used to reverse amnesia might also produce recovery of responding following extinction, that is, a disinhibition effect. Except for the use of nonreinforced exposure to the CS, rather than hypothermia, to suppress test performance, the paradigm was essentially the same as that used in many of the studies of reversal of amnesia. EXPERIMENT 1 METHOD
Subjects Thirty 60–100-day-old naive, male Sprague–Dawley rats, purchased from the Hilltop Co. (Scottsdale, PA), served as subjects in the present experiment. All animals were housed individually in hanging wire-mesh cages and were maintained on a 15:09 h light:dark cycle. Food and water were available ad lib. Apparatus The training and testing apparatus was a Plexiglas rectangular box (38 X 18 X 21 cm) divided into two identically sized chambers. One chamber was painted white and fitted with a clear lid, and the walls and lid in the opposite chamber were painted black. A (8 X 10 cm) guillotine doorway divided the apparatus, which allowed the subjects to move freely throughout the two chambers. A 15-W light bulb was suspended from the ceiling 30 cm above the white compartment; no other light source was present in the training-testing room. The floor of the entire apparatus was made up of 2.5 mm stainless-steel rods, evenly spaced 1 cm apart.
White noise was presented throughout the experiment to reduce any potential auditoty disturbances. The hypothermia apparatus was a Forma Scientific (Marietta, OH) water tank, maintained at 3“C. Subjects were immersed via opaque restraining tubes (7.5 cm PVC tubing) containing twelve 1.25-cm holes that allowed water to flow through the restraining apparatus. An aluminum band was attached to the top of the restraining tube, and the band laid across the top of the tank so that the subjects were immersed in the water in a head-up position. Removable Plexiglas caps were applied to the bottom of the tubes to confine the rats within the restraining apparatus. Subjects’ colonic body temperatures were measured with a Fisher Digital Thermometerm (Pittsburgh, PA). The water tanks were located directly outside of the room containing the conditioning apparatus. Procedure Animals were randomly assigned to 1 of 3 groups: the extinction normal (EXT-NORM n = 10), extinction hypothermia (EXT-HYPO n = 10), or the control (CTRL n = 10) group. All animals were handled for 2 min daily, 2 days prior to the beginning of the experiment. Conditioning and extinction. On day 1, all subjects were removed from their home cages and placed facing away from the door on the white side of the training apparatus. After 20s, the door was raised, allowing subjects to cross to the black chamber. Upon crossing, the door was lowered and a 3-s, inescapable, 200 v foot shock was delivered through a matched-impedance shock source. Following foot shock, rats were immediately removed from the black compartment and placed in a holding cage. To insure that all subjects had been conditioned to the black compartment, an immediate test for fear was administered. After 15 rein, all subjects were placed, facing away from the door, in the white chamber. Ten seconds later, the door was raised, giving the rats access to the black chamber. The measures were latency to cross into the black compartment and total time spent in the white (safe) chamber (TTW). A 180-s test was administered and, if a subject exhibited poor learning(i.e., failure to avoid shock-related cues), they were removed from the experiment. This procedure insured that all subjects were equated on the initial acquisition of the task. Following testing, subjects in the EXT-NORM and EXT-HYPO groups were confined to the black chamber and given 4 mitt of extinction training. Animals in the group CTRL were taken to their home cages directly following the immediate test of fear and remained there until testing occurred on day 2. T for disinhibition. On day 2, groups EXT-NORM and CTRL were individually placed in the white chamber facing away from the guillotine door. Ten seconds later, the door was removed from the apparatus, providing access to the black compartment. The EXT-HYPO group was administered hypothermia treatment prior to testing. Subjects were placed in the restraining tubes and immersed to the neck in 3°C water until their colonic body temperatures dropped to 25 f 1“C. Colonic body temperatures were assessed by inserting the temperature probe 3.2 cm into the rats’ rectum. Subjects were then placed in a holding cage until their colonic temperature reached 29–31“C. [Subjects were tested at this temperature because this range of temperature has been reported to yield the highest level of recovery for avoidance memory in rats that have been rendered amnesic by hypothermia treatment (4)]. In a concern that any manipulation might lead to disinhibition, we did not give the EXT-NORM rats any treatment to control for the noncooling experience of the EXT-HYPO rats. For this reason, we did not expose the EXT-NORM rats to the restraining tubes or cooling tank prior to testing. Therefore, the EXT-NORM group served as a baseline control for determining
DISINHIBITION AND RECOVERY FROM AMNESIA if extinction of the fear response was successful. After reaching the appropriate temperature, the rats were tested exactly like the CTRL and EXT-NORM groups. All subjects were given a 600s test during which latency to cross into the black chamber and the total time in the white (safe) chamber (TTW) were measured. RESULTS AND DISCUSSION
The results are presented in Table 1. To help normalize the distributions, all scores were converted to log form. Five animals were dropped from the experiment due to failure to demonstrate passiveavoidance learning on the immediate test. Therefore, the number of subjects was as follows: groups CTRL and EXT-HYPO = 8 each, and EXT-NORM = 9. A l-way ANOVA on the initial training latency revealed no difference in the subjects tendency to cross to the black chamber during training [F(2,22) < 1.0]; thus, all subjects exhibited similar cross-over latencies on the conditioning day. Separate l-way ANOVAs on latency and TfW for the immediate test, which was administered 15 min after training, also revealed no reliable differences; that is, all scores, regardless of group, were very high, implying that fear conditioning was successfid [Latency: F(2,22) < 1.0; and TTW: F’(2,22) < 1.0]. To further assess whether or not subjects acquired fear to the black compartment, a dependent groups t-test was used to compare scores collapsed across all groups for both the initial training latency and immediate latency following training. This comparison revealed that immediate test latencies were reliably longer than training latencies, indicating that subjects in each of the 3 groups acquired fear to the black compartment (t= 8.56, p < 0.01). Separate one-way ANOVAs on the 24-h latency and TTW scores indicated a significant effect for both measures [F(2,22) = 8.941 for latency, p <0.01 and F(2,22) = 5.867 for TTW, p < 0.01]. Planned t-tests (one tailed) on the latency scores revealed that the EXT-NORM and EXT-HYPO groups differed significantly from the group CTRL (t = 2.37, p < 0.05; t = 3.85, p < respectively). Furthermore, although these 2 groups differed from one another (t = 2.29, p < 0.05), the mean of the EXT-HYPO group (M = 1.214) was significantly lower than that of the EXT-NORM group (M = 1.833); that is, subjects receiving hypothermia crossed to the black chamber faster than rats receiving extinction alone. This result is opposite to what would be expected if hypothermia were acting as a disinhibitor. Planned t-tests (one-tailed) for the 24-h TTW scores revealed that the EXT-NORM and EXT-HYPO groups were not significantly different from one another (t = 1.75, 0.05 < p < 0.10) but both extinction groups differed significantly from group CTRL (EXT-NORM vs. CTRL; t= 2.23, p < and EXTHYPO vs. CTRL; t= 3.07, p < 0.01). Thus, following the extinction treatment, the rats spent an extended amount of time on the black side, evidencing reduced fear of the black compartment. However, neither the latency nor TTW data suggest that hypothermia treatment prior to testing results in recovery of the avoidance (fear) to the black compartment.
1377 served as subjects in passive-avoidance conditioning and conditioned taste-aversion learning prior to the present study. Subjects were housed in groups of 4 and were ear-punched for identification. A
The trainingltesting and hypothermia apparatus were identical to that of Experiment 1.
Random assignment and group designation were identicaI to that of Experiment 1. The groups were the control (CTRL n = 10), extinction/normal (EXT-NORM n = 10), and the extinction/hypothermia (EXT-HYPO n = 10) groups. Conditioning. On day 1, all rats received a punishment/fear conditioning session (modified passive-avoidance training). Subjects were taken from their home cages and placed in the white chamber facing away from the guillotine door. After 2 rein, the door was raised, allowing access to the black chamber. Upon crossing, the door was closed, confining the subjects to the black compartment. Then 6 3-s, 250 v foot shocks were delivered at 10,30,40, 70,90, and 100s. Twenty seconds after the last shock, the door was raised and the rats were allowed to cross back into the white chamber. After 2 min had elapsed, the door was raised and an identical conditioning procedure was repeated. If subjects remained in the white compartment for more than 10s, they were gently pushed through the doorway by the experimenter. Following conditioning, all subjects were removed from the white chamber and taken back to the home cage. Additionally, in this experiment, we did not administer an immediate test for acquisition of fear conditioning as in Experiment 1. Extinction. On the second day, the 2 extinction groups were taken individually from their home cages and placed directly into the black chamber for 12 min with the door closed. No shocks were presented during this period. Following the extinction exposure, all subjects were taken back to their home cages. Test for disinhibition. Hypothermia treatment was administered immediately prior to the test for disinhibition, and this occurred 24 h after extinction (day 3). As in Experiment 1, rats in the EXT-HYPO group were administered hypothermia treatment until their temperatures reached 25”C, and were then tested after they had rewarmed to 29–31°C. All 3 groups were administered a 600-s test identical to that used in Experiment 1. Again, retention of fear was indexed in terms of latencies to cross and the amount of time spent in the white (safe) chamber (TTW). RESULTS AND DISCUSSION
Again, all data were converted to log form to help normalize the distributions, and are presented in Table 2. A l-way ANOVA
EXPERIMENT 2
TABLE 2
Although the training procedure in Experiment 1 was the same as that used in many RA studies, other paradigms have also been employed, particularly in reseamh on anterograde amnesia (8). Accordingly, using a slightly different task, our training procedure in Experiment 2 utilized a modified passive avoidance procedure that was followed by relatively long ( 12-min) extinction training.
MEAN LATENCY (LOG SECONDS) TO ENTER THE BLACK (FEAR) COMPARTMENT AND TTW AT THE 48-H TEST (DAY 3) IN EXPERIMENT 2 Latency Group
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Subjects Thirty 100–150-day-old male, Sprague–Dawley rats, purchased from the Hilltop Co., served as subjects. The animals also
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on the TTW scores indicated that there were reliable differences among the groups on the test for fear to the black chamber [F(2,27) = 4.141, p < 0.05]. Planned t-tests (one-tailed) for the TTW data revealed that the EXT-NORM and EXT-HYPO groups were significantly lower than group CTRL (t = 1.73, p < 0.05; t = 2.72, p < 0.01, respectively]; neither group displayed fear to the black chamber. Although the EXT-HYPO group demonstrated lower TTW scores than the EXT-NORM group, they were not significantly different (t = 1.36, p > 0.05). Contrary to the results from the TTW scores, an ANOVA on the latency scores indicated that there were no reliabIe differences among the groups on the test day [F(2,27) = 1.644, p > 0.05]. One reason for this discrepancy between TTW and latency scores in this experiment is that the TTW measure may be a more sensitive indicator of fear with this paradigm. On the retention test, subjects will occasionally cross over to the black (shock) chamber relatively quickly, but then return to the white (safe) chamber and remain there for the duration of the test. This results in relatively short latency scores and high TTW scores. It is clear from these data that the induction of hypothermia prior to testing does not result in recovery of avoidance of the black compartment. Thus, there is no evidence that hypothermia disinhibited a conditioned fear response in this modified passiveavoidance procedure. One concern for using subjects that had already received passive-avoidance training (previous to this experiment) is that positive transfer may have occurred between the 2 training situations. However, given the similar results of Experiments 1 and 2, there seems little reason to believe that the training received by subjects prior to Experiment 2 confounded the outcome. GENERAL DISCUSSION The contextual retrieval cue explanation for the recovery of memory following experimentally-induced amnesia, in studies that used hypothermia or hyperthermia, was reevaluated (3,5,8). The present study questioned whether or not recovery of the target response following reexposure to the amnestic treatment reflects a disinhibition effect. Thus, the effects of hypothermia on performance were examined following an extinction exposure in the standard passive-avoidance task (Experiment 1) and a modified passive-avoidance procedure (Experiment 2). The present findings do not support the notion that hypothermia acts as a disinhibitor of a previously-conditioned and extinguished fear response. In both experiments, rats receiving hy-
pothermia shortly prior to testing actually exhibited shorter TTW scores than rats receiving extinction only. Because the procedures closely parallel those used to produce reversal of amnesia, it is not likely that the recovery of memory reported with reexposure to the amnestic agent (e.g., 3,8) can be accounted for by a disinhibition of the conditioned fear response. The contextual cues hypothesis of memory retrieval states that optimal retrieval of the memory for a training situation occurs when subjects are tested in the same contextual state as that of training. Therefore, it might be argued that one reason why hypothermia did not act as a disinhibitor in the current experiments is because animals were tested in an altered (recooled) state. However, there is evidence that indicates that when rats are given hypothermia treatment shortly after passive-avoidance training to induce RA, the reversal of amnesia does not occur when animals are recooled, but allowed to rewarm to normal body temperature prior to testing (4). Additionally, these experiments were aimed to address whether or not recooling acts as a disinhibitor in recovery of amnesia procedures. Because amnesia-recovery studies typically test subjects in an altered (recooIed) state, we attempted to parallel those studies as closely as possible. In addition to the methodological implications of this “negative result,” the findings are interesting with respect to disinhibition processes: Why did hypothermia, a novel and intense stimulus, fail to disinhibit an extinguished conditioned response? Unlike the present experiment, traditional disinhibition studies have generally presented a punctate auditory or visual stimulus ( 1,2). The hypothermia-inducing agent (cold water) is a tactile stimulus, requiring that the subject be exposed to the stimulus for 5 to 10 min to reach hypothermia criterion. Although it may now be interesting to examine whether or not traditional disinhibitors would have worked to disinhibit the fear response within the present paradigm, that issue is quite different from our original goal of mimicking the major features of experiments inducing recovery from amnesia by reexposure to the amnestic agent. Overall, this study provides no evidence that hypothermia acts as a disinhibitor and therefore reduces the plausibility of one potential challenge to the retrieval cue interpretation of the recovery of memory following amnesia. ACKNOWLEDGEMENTS The r
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REFERENCES 1. Bnmer, C. J. Disinhibitionof an operant response. Learn. Motiv. 1:346–371;1970. B
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hyperthermia:An unusuatlyprofound,yet reversible,memoryloss. B P D R b 9 R b i S t
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