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5-HT1A agonists disrupt memory of fear conditioning in mice
BIOL PSYCHIATRY 1993;33:247-254
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5-HT1A Agonists Disrupt Memory of Fear Conditioning in Mice David Quarterrnain, Jonathan Clemente, and Anne Shemer
A series of experiments was carried out to analyze the effects of the 5-HT~a agonists tandospirone or buspirone on the retention of fear conditioning in mice. Fear was produced by pairing tone and shock in a conditioned emotional response (CER) paradigm and strength of conditioning was assessed by measuring suppression of drinking in presence of tone. Fear conditioning was disrupted if tandospirone and buspirone were administered before the conditioning sesr;on but not before the test trial. Diazepam disrupted conditioning at both times. Tandospirone did not disrupt performance if conditioning was tested I hr rather than 24 hr after training, suggesting that disrupted memory rather than impaired acquisition was responsible for the deficit. The effect of tandospirone on fear conditioning could be reversed by administration old-amphetamine prior to the retention test, which suggests that information was stored but is inaccessible to normal retrieval cues. Tandospirone and buspirone also retarded extinction, a clear indication that the disruption caused by these drugs is unrelated to their anxiolytic action.
Key Words: 5-HT~A agonists, buspirone, tandospirone, fear conditioning, extinction, anterograde amnesia
Introduction There is considerable evidence that serotonergic neurotransmission is involved in memory processes (for review see O ~ e n ! 982) but the exact nature of this involvement has yet to be specified in detail. An examination of the recent pharmacological literature suggests that manipulations which decrease 5-hydroxytryptamine (5-HT) activity tend to improve retention, whereas those that increase serotonergic activity lead to memory impairment (Airman and Normile 1988). Although many findings are consister~t with this generalization there are sufficient inconsistencies
From the Department of Neurology, New York University Medical Center (DQ, JC) and Pfizer Inc., New York, NY (AS). Address reprint requests to David Quartermaln, Ph.D., Department of Neurology, New York University Medical Center, 550 1st Ave, New York, NY 10016. Received June 5, 1992: revised December 3, 1992. © 1993 Society of Biological Psychiatry
and anomalies to suggest that 5-HT may have a more subtle regulatory influence on memory processing than can be encompassed by a hypothesis based on general alterations in 5-HT transmission. This point of view is reinforced by the identification of multiple receptor subtypes (Peroutka 1987), which indicate that the serotonin system is likely to have more complex behavioral functions than was previously supposed. The newly available drugs that are selective for specific serotonin subsystems may be able to provide analytical tools to elucidate the role that the serotonin system plays in the storage and retrieval of information. Recent research on the 5-HTIA receptor subtype has indicated that this subsystem may have a role in memory processing. These receptors have been identified both as somatodendritic autoreceptors and as presynaptic receptors in the hippocarnpus (Verge et al 1986, Pazos et al 1988). 0006-3223/93/$06.00
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Electrophysiological studies have shown that stimulation of this receptor with selective 5-HTtA agonists produces inhibition of neuronal activity similar to that ob~,:~Ted with serotonin itself (Sprouse and Aghajanian 1987). The high density of this receptor subtype in hippocampus (Pazos 1988) the possible association with growth factors (Whitaker-Azmitia and Azmitia 1990) and regeneration (Rogers et al 1991), and the observation that these receptors are significantly decreased in patients with Alzheimer's disease (Middlemiss et al 1986), all point to the possibility that this system may have a role in memory. The limited research to date supports this conjecture. For example, it has been shown that the direct 5-HTIA agonist 8-OH-DPAT disrupts retention in a passive avoidance task (Carli and Saminin 1992) and impairs spatial learning and working memory in the water maze (Carli et al 1992~. _~uspirone, a partial agonist for the 5-HT~A receptor has been reported to disrupt performance in a spatial navigation task and to impair working memory in the radial arm maze (Rowan et al 1990). Buspirone and other 5-HTtA agonists are either indicated or being developed for therapy of emotional states such as anxiety and depression (Wieland and Lucki 1990). It is therefore important that the role of this receptor in memory processing be carefully evaluated. Although most animal studies show that 5-HT~A agonists when given in acute doses disrupt performance, the basis of the deficit remains unknown. Because behavioral disruption occurs only when the agonists are given before the training session, it is not clear whether the deficit is the result of an acquisition impairment, memory loss, or some combination of both factors. The extent to which the anxiolytic properties of 5-HT~A agonists contribute to the performance deficit is also unclear. The purpose of the present series of experiments was to examine the effects of two 5-HT~A agonists tandospirone and buspirone on the retention of fear conditioning in a behavioral paradigm in which the amnestic and anxiolytic ef~'ccts of the agents could be separately evaluated.
Subjects and Method Subjects Male Swiss Webster mice (Harlan Hsd : ND 4) 6 weeks of age and weighing 25-35 g were the subjects for this study. Animals were housed five per cage with food available ad libitum.
Apparatus Training an~ testing were carried out in two chambers 10 cm long, 10 cm wide, and 20 cm deep. The floors were constructed from two aluminum plates, the bottom 10 cm of which were bent at a 45 ° angle to form a trough with
a 5-mm space between the plates at the bottom. The end walls were made from black Plexiglass. A hole with a 1.5cm diameter was drilled in one end, 2.5 cm above the bottom of the trough. Each chamber was equipped with a graduated drinking tube with a metal spout that projected 5 mm into the chamber. Drinking was detected by standard ddnkometer circuitry. Shock could be delivered to the floor of the chambers through positive and negative leads from a Grason Stadler shocker. Experimental events were scheduled and data recorded using standard programming equipment.
Procedure The experiment was carried out over 5 consecutive days. On day 1 animals were weighed and deprived of water. On day 2, mice were given a 5-min adaptation sessie~ in which they were permitted free access to the water tube. On day 3, drinking was recorded in two 5-sec segments. The time required to complete each 5-see period was recorded to the nearest 0.1 see. Mice failing to complete 5 sec of ddn,~ng within 30 sec were discarded from the study. Less than 0.5% were discarded because of failure to adapt to the drinking schedule. Fear conditioning was carded out on day 4. Mice were placed in the chamber with the drinking tube removed and given three pairings of a 10-sec tone (CS) followed by 1-sec duration 0.3 mA foot shock (UCS). Conditioning trials were separated by 60 sec. Mice were given access to water in the home cage for 30 min after each daily session. The test session was conducted on day 5. Following 5 sec of drinking, a pretone timer was activated and the time required to complete an additional 5 sec of drinking was recorded (latency A). Immediately following the completion of drinking the CS was automatically activated and a second timer recorded latency to complete 5-sec of drinking in the presence of the tone (latency B). Mice that failed to complete drinking within 60 sec were assigned the maximum latency as a score. The strength of fear conditioning was expressed as a suppression ratio calculated as A/A + B. A ratio of 0.01 would indicate complete suppression, whereas total absence of suppression would be indicated by a ratio of 0.50.
Drug Administration Tandospirone and buspirone, made fresh for each injection, were dissolved in distilled water and injected in a volume of 10 ml/kg body weight. Diazepam was diluted from ampules (5 mg/ml) using a solution comprising 10% ethyl alcohol and 40% propylene glycol that was injected in a volume of 10 ml/kg.
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Figure 1. Mean suppression ratios and SEMs for mice treated with different doses of tandospirone, buspirone, and diazepam before the conditioning trial.
Figure 2. Mean suppression ratios and SEMs for mice treated with different doses of tandospirone, buspirone, and diazepam before the test session.
Experiment 1: Effects of Pretraining and Pretesting Drug Administration
Bonferroni t-tests revealed that (a) a l-mg/kg dose of buspirone produced a significant increase in mean suppression ratio (t = 4.62 p = <0.001); (b) 2- and 5-mg/kg doses of tandospirone increased suppression ratios relative to the vehicle group (t = 3.61; p = <0.001; and t = 2.93; p = <0.01, respectively); and (c) in the diazepam group significant disraption of suppression was produced only by the highest dose (t = 2.79; p = <0.05). These data show that all of the drugs disrupt the acquisition of fear conditioning when they are administered before the conditioning session.
Experiment 1 analyzed the effects of tandc.~p:,roz,e z.-~d buspirone on both the acquisition and the expression of fear. The drugs were administered to different groups of animals before the conditioning phase, before the testing phase, and before both conditioning and testing. This design permits an evaluation of the effect of the drugs on learning and/or memory processes and also allows an examination of the anticonflict or antianxiety properties of the agents.
Procedure Mice were injected before conditioning with tandospirone (0,2,5, and 10 mg/kg, [the 10-rag dose was dropped after conditioning as the animals refused to drink], buspirone (0,1, and 3 mg/kg), and diazepam (0,1, and 3 mg/kg) either before conditioning, before testing, or before both conditioning and testing. Mice were assigned to experimental conditions and drug and dose groups in a random fashion. Between i0 and i5 mice were allotted to each group. Drugs were injected subcutaneously 30 min before conditioning or testing. The 0 mg/kg groups were injected with the vehicle.
Results P R E T R A I N I N G D R U G A D M I N | S T R A T I O N . T h e s e results are shown in Figure 1. Separate one way analysis of variance (ANOVAs) were computed for each drug condition and the results indicated significant differences among the dose levels for all of the drug groups (tandospirone: F[3,37] = 5.12; p = 0.004; buspirone: F[2,31] = 10.7; p = 0.001; diazepam: F[2,30] = 4.50; p = 0.028).
P R E T E S T I N G D R U G A D M I N I S T R A T I O N . Results of pretest drug treatments are showr,, in Figure 2. Neither tandosph'one (F = 0.34) nor buspirone (F = 1.05) significantly altered suppression ratios when they were injected 30-min before the test session. Diazepam significantly disrupted conditioned suppression when it was injected 30 rain before the test (F[2,31] = 4.9; p = 0.014). P R E C O N D I T I O N I N G AND PRETESTLNG D R U G ADMINIS-
TRA'HON. Results of preconditioning and pretesting drug administration data are shown in Figure 3. Separate one way ANOVAs indicate that the disruption of conditioned suppression induced by preconditioning drug treatment was not attenuated by a second drug treatment before the retention test (tandospirone [2,37] = 12.3; p = <0.001, buspirone F[2,31] = 3.32; p = <0.05, diazepam (F[2,27] = 9.43; p = <0.001). In fact, it appears that a second dose of tandospirone and buspirone reversed the doseeffect curve seen in Figure 1. These results show that the disruption of perfo~'mance indu¢:ed by preconditioning drug administration , ~ l o t be ameliorated by reproducing at testing the drug state that existed at conditioning. This finding rules out state-dependent learning as an explanation for the deficit in conditioned suppression.
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Figure 3. Mean suppression ratios and SEMs for mice treated with different doses of tandospirone, buspirone, and diazepam before both the conditioning and the test sessions.
24 hours Retention test interval
Figure 4. Mean suppression ratios and SEMs for saline- and tandospirone-treated mice tested either 1 or 24 hr after conditioning. Tandospirone and saline were injected 30 rain before the conditioning trial.
Experiment 2: Measurement of Retention l-Elf After Conditioning The results of Experiment 1 show that both tandospirone and buspirone disrupt fear conditioning when they are given before the conditioning trial. Although this paradigm does not pe.,'wht dLrect evaluation of relative strength of conditioning in the drug and control groups, it may be possible to obtain information on whether learning was disrupted by the drug treatment by testing retention shortly after training. It has been demonstrated that memory may be present for several hours after amnestic treatments have been introduced (see Flood and Jarvik 1976). Thus it may be possible to evaluate strength of learning in drug and control mice before the deficit in long-term memory has developed. Accordingly, we tested the effect of one agonist (tandospirone) 1 hr after conditioning and compared performance of this group with a group tested 24 hr following training. Comparable groups were tested following saline treatment.
(F [1,35] = 10.33; p = 0.003). Figure 4 shows that tandospirone-treated mice exhibited unimpaired fear conditioning 1 hr after training, but disrupted performance after 24 hr. On the other hand, saline-treated mice exhibited low suppression ratios at both test intervals. This finding implies that the deficit is the result of disrupted memo,-'), rather than impaired original learning.
Experiment 3: Effect of Tandospirone and Buspirone on Extinction
Twenty mice were injected with tandospirone (5 mg/kg) and 19 with saline 30 min before the conditioning trial. Procedure for conditioning was the same as den~iii,~d in Experiment 1. Ten tandospirone-treated and 9 saline-treated mice were tested 1 hr after conditioning and 10 tandospirone- and 10 saline-treated mice were tested 24 hr after conditioning.
The purpose of Experiment 3 was to determine the effects of tandospirone and buspirone on the extinction of conditioned fear. In extinction, animals are repeatedly exposed to the conditioned stimulus (CS) without the unconditional stimulus (UCS) and they learn to suppress their original conditioned fear response to the CS. As a result there is a marked reduction in the suppressive effects of the CS as indicated by higher suppression ratios. Drugs that have fear- or anxiety-reducing properties might be expected to facilitate extinction. Indeed it has been shown that benzodiazipines speed the extinction of fearomotivated behaviors. (Mason 1983; Goldman 1977; Tenen 1967). If the disruption of fear conditioning by tandospirone and buspirone demonstrated in Experiment 1 was caused by their anxiolytic effects, it might be predicted that they would facilitate extinction of fear. On the other hand, if their disruptive effects resulted from impaired memory, they should impair both acquisition and extinction.
Results
Procedure
Suppression ratios for the four groups are shown in Figure 4. A 2 x 2 ANOVA computed for these data revealed a significant interaction between drug group and test interval
Training and testing were carried out in the apparatus de~c6~d in ~xpe.riment !. Mice were adapted to drink in the chambers on days 1 and 2 and given the standard fear
Procedure
5-HT=^ DisruptFear Conditioning
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Experiment 4: Reversal of Tandospirone Memory Loss by Amphetamine
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Figure 5. Mean suppressionratios 24 hr after a 60-min extinction session. No ext: mice left in home cages; sal-ext: saline injected 30 min before the extinction session; tan-ext: tandospirone (5 mg/kg) before extinction; bus-ext: buspirone (1 mg/kg) before extinction.
conditioning described earlier on day 3. No drugs were administered during fear conditioning. On day 4, mice were given an extinction session in which the CS was presented alone 60 times with an interstimulus interval of 1 rain. Thirty minutes before the session 12 mice were injected with saline, 12 with tandospirone 2 mg/kg, and 12 with l mg/kg buspirone. These drug doses produced the largest effect on suppression in Experiment 1. A control group of 12 mice was not given extinction on day 4. All animals were given l-hr access to water in the home cage after each daily session. The effects of tandospirone and buspirone on extinction were evaluated using the standard test procedure conducted 24 hr after extinction.
Results The results of this experiment are shown in Figure 5. A one-way ANOVA applied to these data showed a significant differeac~ among the four groups (F [3,48] = 8.97; p - <0.001). Post-hoe comparisons using Bonferroni ttests indicated that ~andospirone, buspirone, and the noextinction group were all significantly (p -- <0.05) different from the saline-extinction group (t = 3.99; 4.45 and 4.21 respectively). These results show that buspirone and tandospirone abolish the effects of extinction on fear conditioning. This finding implies that the disruption of fear conditioning demonstrated in Experiment 1 is not caused by alterations in fear motivation or to a decrease in pain sensitivity. The demonstration that tandospirone and buspirone impair both the acquisition and the extinction of fear is more consistent with the notion that these 5-HT]A agonists disl~pt processes involved in the storage and retrieval of the memory of fear conditioning.
The pattern of the results from the first three experiments suggest that tandospirone and buspirone affect fear conditioning by impairing memory of behaviors learned after drug administration. Memory loss caused by pretraining administration of many different forgetting agents can be reversed by pharmacological and behavioral treatments applied before the retention test (Quartermain et al 1988b; Quartermain and Leo 1988). Such findings are usually interpreted as indicating that subjects learned the task but were unable to retrieve the information at the time of testing. The objective of Experiment 4 was to evaluate the memory-impairment hypothesis by attempting to demonstrate that the deficit in fear conditioning can be reversed by treatment with a memory-enhancing agent (d-amphetamine sulphate) administered before the retention test. Amphetamine was employed because it has been shown to alleviate memory loss in animals caused by a wide variety of memory-impairing treatments (Quartermain et al 1988a).
Procedure Forty-eight mice were the subjects for this experiment. Thirty-sb, animals were trained as previously described. Thirty minutes before the conditioning session 12 mice were injected with saline and 24 with tandospirone (5 mg/kg). Thirty minutes before testing, the mice treated with saline and half of the tandospirone group (n = 12) were reinjected with saline, whereas the other half of the tandospirone group was injected with d-amphetamine sulphate (1 mg/kg). In order to evaluate possible nonspecific effects of acute amphetamine on test performance, a group of 12 mice was given sham conditioning and tested after amphetamine treatment. Sham conditioning consisted of 3 CS-UCS pairings using a different CS (clicks of 100 msec duration) and a UCS administered in a different apparatus in a different experimental room. Mice in this group were injected with amphetamine on the test day and tested in the regular training apparatus. We reasoned that if amphetamine improves conditioned suppression by reactivating the memory of the original conditioning, regularly trained animals should show improved performance (lower suppression ratios), whereas sham-trained mice should be unaffected as they had not received fear conditioning in the training apparatus. On the other hand, improved suppreRsion by both sham- and regularly trained animals would indicate that performance was being influenced by nonspecific factors associated with amphetamine treatment.
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Figure 6. Effect of pretest injection of amphetamine (1 mg/kg) on tandospirone-induced amnesia. Tandospirone (5 mg/kg) was administered 30 min before conditioning and amphetamine 30 min before the test session, sal-sal: saline before conditioning and before testing; tan-sal: tandospirone before conditioning, saline before testing; tan-amp: tandospirone before conditioning, amphetamine before testing; tan-amp (sham) was injected with tandospirone and exposed to CS and UCS in a different apparatus in another room and tested in the regular apparatus 30 min after amphetamine. Values are mean suppression ratios and SEMs.
Results These results, shown in Figure 6, indicate that the disruption of conditioning produced by tandospirone was reversed by d-amphetamine (tandospirone-saline versus tandospirone-amphetamine t = 2.81;p = 0.033). That this decrease in suppression ratio was not attributable to nonspecific effects of acute amphetamine treatment is shown by the results of the sham-trained group. Sham-trained mice showed significantly higher ratios (less suppression) than regularly trained subjects (t = 3.48; p = 0.002) indicating that amphetamine improved conditioned suppression only in those animals that had been conditioned in the regular training apparatus. These findings suggest that tandospirone causes a memory deficit that can be reversed by amphetamine applied before the retention test.
Discussion The results of this study confirm previous reports that tandospirone, like other 5 - H T I A agonists, can disrupt performance when administered before the training trial. The specific contribution of the present series of experiments is to show that the behavioral disruption is the result of interference with memory processes and not a consequence of impairments of acquisition or alterations in motivational or sensory mechanisms. Both agonists disrupted fear conditioning when given before the conditioning session (Figure 1) but were without effect when they were administered before the test session
once fear had already been established (Figure 2). These results indicate that. the 5-HTIA agonists disrupt the encoding process of the fear but not its expression. This latter finding shows that neither agonist has anxiolytic propelties in the behavioral test used in this study, otherwise fear would have been alleviated at the time of testing. In contrast, diazepam, a benzodiazepine, disrupted fear conditioning when it was given both before conditioning and before testing, an indication of anxiolytic action on the expression of fear. A similar finding has recently been reported to occur following administration of the N-Methyl-D-Aspartate receptor antagonist APV (VL2-amino-5-phosphono valeric acid) (Kim et al i991). These authors suggest that NMDA-dependent LTP (long-term potentiation) may underlie the neural changes occurring in fear conditioning. The present results show that 5-HTIA receptors are also involved in the associative changes underlying fear conditioning. The data in Experiment I show that the disruption of fear conditioning is not the resultof state-dependent learning. Reestablishing the drug state that was present during conditioning by injectinga second dose of the agonist prior to the testsession failsto reverse the behavioral disruption. The resultsof Experiment 3 show that tandospirone and buspirone can also impair the extinction of fear (Figure 5). This finding indicates that the disruptive effects of 5HT~A agonists are apparent in more than one kind of learning and also rules out alterations in fear motivatio~ and pain sensitivity as explanations for their mechanism of action. Other studies that have shown disruption of appetitivelymotivated behaviors by 5-HTIA activation (Winter and Petti 1987) support this view. The most plausible hypothesis to explain the effects of 5-HT~A agonists in this study is that they disrupt memory processes. W h e n pretraining drug administration causes forgetting, the memory loss is usually classifiedas anterograde amnesia (AA). To be an authentic AA, however, it must be shown that the performance deficit is not the result of impaired acquisition. Although there is no direct proof from these experiments that associative processes are normal following drug treatment, the results of Experiment 2, which revealed unimpaired performance I hr after training, provide circumstantial evidence that tandospirone (and presumably also buspirone ) does not impair conditioning. Other evidence from thislaboratory suggests that this is the case. W e have shown (unpublished observations) that mice treated with tandospirone before acquisition exhibit normal rates of learning in a one-way active avoidance task. These results, in conjunction with the demonstration that shock intensities at which mice flinch, run, and vocalize are not altered by tandospirone (unpublished observations), make it reasonable to assume that acquisition processes are not impaired by 5-HT~A ag-
5-HT~^ Dismpt Fear Conditioning
onists. Taken together these findings support, the hypothesis that 5-HTt^ agonists disrupt performance by causing anterograde amnesia. The results of Experiment 4 also support a memory loss interpretation of the deficit in fear conditioning following tandospirone and buspirone treatment. Pretesting amphetamine administration resulted in an almost complete reversal of the deficit. This could not have occlared unless fear conditioning had taken place as the sham-~ained group showed no improvement in conditioned suppression after amphetamine treatment. A memory-impairmen~t hypothesis is also consistent with other data from our l~boratory. We have shown that tandospirone can produce torgetting for both active- and passive-avoidance learning. In the active-avoidance stud~, pretraining doses of tandospirone from 1-10 mg/kg did not alter the number of trials needed to reach a learning criterion of 5/6 avoidances relative to vehicle controls. A 24-hr retention test, however, revealed a robust deficit in groups receiving 2 and 5 mg/kg doses (unpublished observations). Unpublished observations from this laboratory using a single trial step-through passive avoidance task have shown that pretraining doses of 8OH-DPAT and tandospirone produce memory loss. This
amnesia could also be reversed by d-amphetamine injected before the retention test. The anmestic effects of tando,,~pirone were blocked by the 5-HT~A antagonists BMY7378, suggesting that the amnesia was the result of postsynaptic actions of tandospirone. The final profile pre-
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sented by these experiments is of learning that is unimpaired 1 hr after training, that is retrievable when amphetamine is administered, but that is too weak to be present 24-hr posttraining. It is possible that 5-HT~^ receptor activation diminishes the strength of the message encoded or changes it to make it less retrievable from longterm storage. One of the advantages of the "new" generation of anxiolytics/antidepressants of this class is that there are relatively few side effects. The typical cluster of sedative effects and memory impairment associated with the benzodiazepines and the trieyclics have largely been" avoided in ~hese new compounds. It may therefore seem surprising that the animal data provide evidence of memory impairment, particularly as clinical results demonstrate that busprone produces no amnesia when compared with diazepam. (Boulenger et al 1989; Lucki et al 1987), and tandospirone has similarly been reported to have no disruptive effects on cognition (personal communication). A possible reason for the discrepancy between these two sets of data is that drugs are typically administered chronically in the clinical studies and acutely in the animal experiments. It may be that exposure to the drug for a period of time prior to the onset of behavioral testing may eliminate the disruptive effects on cognitive performance. To resolve this issue it will be necessary to design animal experiments that more closely parallel the clinical studies.
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5-HT1A Agonists Disrupt Memory of Fear Conditioning in Mice David Quarterrnain, Jonathan Clemente, and Anne Shemer
A series of experiments was carried out to analyze the effects of the 5-HT~a agonists tandospirone or buspirone on the retention of fear conditioning in mice. Fear was produced by pairing tone and shock in a conditioned emotional response (CER) paradigm and strength of conditioning was assessed by measuring suppression of drinking in presence of tone. Fear conditioning was disrupted if tandospirone and buspirone were administered before the conditioning sesr;on but not before the test trial. Diazepam disrupted conditioning at both times. Tandospirone did not disrupt performance if conditioning was tested I hr rather than 24 hr after training, suggesting that disrupted memory rather than impaired acquisition was responsible for the deficit. The effect of tandospirone on fear conditioning could be reversed by administration old-amphetamine prior to the retention test, which suggests that information was stored but is inaccessible to normal retrieval cues. Tandospirone and buspirone also retarded extinction, a clear indication that the disruption caused by these drugs is unrelated to their anxiolytic action.
Key Words: 5-HT~A agonists, buspirone, tandospirone, fear conditioning, extinction, anterograde amnesia
Introduction There is considerable evidence that serotonergic neurotransmission is involved in memory processes (for review see O ~ e n ! 982) but the exact nature of this involvement has yet to be specified in detail. An examination of the recent pharmacological literature suggests that manipulations which decrease 5-hydroxytryptamine (5-HT) activity tend to improve retention, whereas those that increase serotonergic activity lead to memory impairment (Airman and Normile 1988). Although many findings are consister~t with this generalization there are sufficient inconsistencies
From the Department of Neurology, New York University Medical Center (DQ, JC) and Pfizer Inc., New York, NY (AS). Address reprint requests to David Quartermaln, Ph.D., Department of Neurology, New York University Medical Center, 550 1st Ave, New York, NY 10016. Received June 5, 1992: revised December 3, 1992. © 1993 Society of Biological Psychiatry
and anomalies to suggest that 5-HT may have a more subtle regulatory influence on memory processing than can be encompassed by a hypothesis based on general alterations in 5-HT transmission. This point of view is reinforced by the identification of multiple receptor subtypes (Peroutka 1987), which indicate that the serotonin system is likely to have more complex behavioral functions than was previously supposed. The newly available drugs that are selective for specific serotonin subsystems may be able to provide analytical tools to elucidate the role that the serotonin system plays in the storage and retrieval of information. Recent research on the 5-HTIA receptor subtype has indicated that this subsystem may have a role in memory processing. These receptors have been identified both as somatodendritic autoreceptors and as presynaptic receptors in the hippocarnpus (Verge et al 1986, Pazos et al 1988). 0006-3223/93/$06.00
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Electrophysiological studies have shown that stimulation of this receptor with selective 5-HTtA agonists produces inhibition of neuronal activity similar to that ob~,:~Ted with serotonin itself (Sprouse and Aghajanian 1987). The high density of this receptor subtype in hippocampus (Pazos 1988) the possible association with growth factors (Whitaker-Azmitia and Azmitia 1990) and regeneration (Rogers et al 1991), and the observation that these receptors are significantly decreased in patients with Alzheimer's disease (Middlemiss et al 1986), all point to the possibility that this system may have a role in memory. The limited research to date supports this conjecture. For example, it has been shown that the direct 5-HTIA agonist 8-OH-DPAT disrupts retention in a passive avoidance task (Carli and Saminin 1992) and impairs spatial learning and working memory in the water maze (Carli et al 1992~. _~uspirone, a partial agonist for the 5-HT~A receptor has been reported to disrupt performance in a spatial navigation task and to impair working memory in the radial arm maze (Rowan et al 1990). Buspirone and other 5-HTtA agonists are either indicated or being developed for therapy of emotional states such as anxiety and depression (Wieland and Lucki 1990). It is therefore important that the role of this receptor in memory processing be carefully evaluated. Although most animal studies show that 5-HT~A agonists when given in acute doses disrupt performance, the basis of the deficit remains unknown. Because behavioral disruption occurs only when the agonists are given before the training session, it is not clear whether the deficit is the result of an acquisition impairment, memory loss, or some combination of both factors. The extent to which the anxiolytic properties of 5-HT~A agonists contribute to the performance deficit is also unclear. The purpose of the present series of experiments was to examine the effects of two 5-HT~A agonists tandospirone and buspirone on the retention of fear conditioning in a behavioral paradigm in which the amnestic and anxiolytic ef~'ccts of the agents could be separately evaluated.
Subjects and Method Subjects Male Swiss Webster mice (Harlan Hsd : ND 4) 6 weeks of age and weighing 25-35 g were the subjects for this study. Animals were housed five per cage with food available ad libitum.
Apparatus Training an~ testing were carried out in two chambers 10 cm long, 10 cm wide, and 20 cm deep. The floors were constructed from two aluminum plates, the bottom 10 cm of which were bent at a 45 ° angle to form a trough with
a 5-mm space between the plates at the bottom. The end walls were made from black Plexiglass. A hole with a 1.5cm diameter was drilled in one end, 2.5 cm above the bottom of the trough. Each chamber was equipped with a graduated drinking tube with a metal spout that projected 5 mm into the chamber. Drinking was detected by standard ddnkometer circuitry. Shock could be delivered to the floor of the chambers through positive and negative leads from a Grason Stadler shocker. Experimental events were scheduled and data recorded using standard programming equipment.
Procedure The experiment was carried out over 5 consecutive days. On day 1 animals were weighed and deprived of water. On day 2, mice were given a 5-min adaptation sessie~ in which they were permitted free access to the water tube. On day 3, drinking was recorded in two 5-sec segments. The time required to complete each 5-see period was recorded to the nearest 0.1 see. Mice failing to complete 5 sec of ddn,~ng within 30 sec were discarded from the study. Less than 0.5% were discarded because of failure to adapt to the drinking schedule. Fear conditioning was carded out on day 4. Mice were placed in the chamber with the drinking tube removed and given three pairings of a 10-sec tone (CS) followed by 1-sec duration 0.3 mA foot shock (UCS). Conditioning trials were separated by 60 sec. Mice were given access to water in the home cage for 30 min after each daily session. The test session was conducted on day 5. Following 5 sec of drinking, a pretone timer was activated and the time required to complete an additional 5 sec of drinking was recorded (latency A). Immediately following the completion of drinking the CS was automatically activated and a second timer recorded latency to complete 5-sec of drinking in the presence of the tone (latency B). Mice that failed to complete drinking within 60 sec were assigned the maximum latency as a score. The strength of fear conditioning was expressed as a suppression ratio calculated as A/A + B. A ratio of 0.01 would indicate complete suppression, whereas total absence of suppression would be indicated by a ratio of 0.50.
Drug Administration Tandospirone and buspirone, made fresh for each injection, were dissolved in distilled water and injected in a volume of 10 ml/kg body weight. Diazepam was diluted from ampules (5 mg/ml) using a solution comprising 10% ethyl alcohol and 40% propylene glycol that was injected in a volume of 10 ml/kg.
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Figure 1. Mean suppression ratios and SEMs for mice treated with different doses of tandospirone, buspirone, and diazepam before the conditioning trial.
Figure 2. Mean suppression ratios and SEMs for mice treated with different doses of tandospirone, buspirone, and diazepam before the test session.
Experiment 1: Effects of Pretraining and Pretesting Drug Administration
Bonferroni t-tests revealed that (a) a l-mg/kg dose of buspirone produced a significant increase in mean suppression ratio (t = 4.62 p = <0.001); (b) 2- and 5-mg/kg doses of tandospirone increased suppression ratios relative to the vehicle group (t = 3.61; p = <0.001; and t = 2.93; p = <0.01, respectively); and (c) in the diazepam group significant disraption of suppression was produced only by the highest dose (t = 2.79; p = <0.05). These data show that all of the drugs disrupt the acquisition of fear conditioning when they are administered before the conditioning session.
Experiment 1 analyzed the effects of tandc.~p:,roz,e z.-~d buspirone on both the acquisition and the expression of fear. The drugs were administered to different groups of animals before the conditioning phase, before the testing phase, and before both conditioning and testing. This design permits an evaluation of the effect of the drugs on learning and/or memory processes and also allows an examination of the anticonflict or antianxiety properties of the agents.
Procedure Mice were injected before conditioning with tandospirone (0,2,5, and 10 mg/kg, [the 10-rag dose was dropped after conditioning as the animals refused to drink], buspirone (0,1, and 3 mg/kg), and diazepam (0,1, and 3 mg/kg) either before conditioning, before testing, or before both conditioning and testing. Mice were assigned to experimental conditions and drug and dose groups in a random fashion. Between i0 and i5 mice were allotted to each group. Drugs were injected subcutaneously 30 min before conditioning or testing. The 0 mg/kg groups were injected with the vehicle.
Results P R E T R A I N I N G D R U G A D M I N | S T R A T I O N . T h e s e results are shown in Figure 1. Separate one way analysis of variance (ANOVAs) were computed for each drug condition and the results indicated significant differences among the dose levels for all of the drug groups (tandospirone: F[3,37] = 5.12; p = 0.004; buspirone: F[2,31] = 10.7; p = 0.001; diazepam: F[2,30] = 4.50; p = 0.028).
P R E T E S T I N G D R U G A D M I N I S T R A T I O N . Results of pretest drug treatments are showr,, in Figure 2. Neither tandosph'one (F = 0.34) nor buspirone (F = 1.05) significantly altered suppression ratios when they were injected 30-min before the test session. Diazepam significantly disrupted conditioned suppression when it was injected 30 rain before the test (F[2,31] = 4.9; p = 0.014). P R E C O N D I T I O N I N G AND PRETESTLNG D R U G ADMINIS-
TRA'HON. Results of preconditioning and pretesting drug administration data are shown in Figure 3. Separate one way ANOVAs indicate that the disruption of conditioned suppression induced by preconditioning drug treatment was not attenuated by a second drug treatment before the retention test (tandospirone [2,37] = 12.3; p = <0.001, buspirone F[2,31] = 3.32; p = <0.05, diazepam (F[2,27] = 9.43; p = <0.001). In fact, it appears that a second dose of tandospirone and buspirone reversed the doseeffect curve seen in Figure 1. These results show that the disruption of perfo~'mance indu¢:ed by preconditioning drug administration , ~ l o t be ameliorated by reproducing at testing the drug state that existed at conditioning. This finding rules out state-dependent learning as an explanation for the deficit in conditioned suppression.
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Figure 3. Mean suppression ratios and SEMs for mice treated with different doses of tandospirone, buspirone, and diazepam before both the conditioning and the test sessions.
24 hours Retention test interval
Figure 4. Mean suppression ratios and SEMs for saline- and tandospirone-treated mice tested either 1 or 24 hr after conditioning. Tandospirone and saline were injected 30 rain before the conditioning trial.
Experiment 2: Measurement of Retention l-Elf After Conditioning The results of Experiment 1 show that both tandospirone and buspirone disrupt fear conditioning when they are given before the conditioning trial. Although this paradigm does not pe.,'wht dLrect evaluation of relative strength of conditioning in the drug and control groups, it may be possible to obtain information on whether learning was disrupted by the drug treatment by testing retention shortly after training. It has been demonstrated that memory may be present for several hours after amnestic treatments have been introduced (see Flood and Jarvik 1976). Thus it may be possible to evaluate strength of learning in drug and control mice before the deficit in long-term memory has developed. Accordingly, we tested the effect of one agonist (tandospirone) 1 hr after conditioning and compared performance of this group with a group tested 24 hr following training. Comparable groups were tested following saline treatment.
(F [1,35] = 10.33; p = 0.003). Figure 4 shows that tandospirone-treated mice exhibited unimpaired fear conditioning 1 hr after training, but disrupted performance after 24 hr. On the other hand, saline-treated mice exhibited low suppression ratios at both test intervals. This finding implies that the deficit is the result of disrupted memo,-'), rather than impaired original learning.
Experiment 3: Effect of Tandospirone and Buspirone on Extinction
Twenty mice were injected with tandospirone (5 mg/kg) and 19 with saline 30 min before the conditioning trial. Procedure for conditioning was the same as den~iii,~d in Experiment 1. Ten tandospirone-treated and 9 saline-treated mice were tested 1 hr after conditioning and 10 tandospirone- and 10 saline-treated mice were tested 24 hr after conditioning.
The purpose of Experiment 3 was to determine the effects of tandospirone and buspirone on the extinction of conditioned fear. In extinction, animals are repeatedly exposed to the conditioned stimulus (CS) without the unconditional stimulus (UCS) and they learn to suppress their original conditioned fear response to the CS. As a result there is a marked reduction in the suppressive effects of the CS as indicated by higher suppression ratios. Drugs that have fear- or anxiety-reducing properties might be expected to facilitate extinction. Indeed it has been shown that benzodiazipines speed the extinction of fearomotivated behaviors. (Mason 1983; Goldman 1977; Tenen 1967). If the disruption of fear conditioning by tandospirone and buspirone demonstrated in Experiment 1 was caused by their anxiolytic effects, it might be predicted that they would facilitate extinction of fear. On the other hand, if their disruptive effects resulted from impaired memory, they should impair both acquisition and extinction.
Results
Procedure
Suppression ratios for the four groups are shown in Figure 4. A 2 x 2 ANOVA computed for these data revealed a significant interaction between drug group and test interval
Training and testing were carried out in the apparatus de~c6~d in ~xpe.riment !. Mice were adapted to drink in the chambers on days 1 and 2 and given the standard fear
Procedure
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Experiment 4: Reversal of Tandospirone Memory Loss by Amphetamine
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~
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Figure 5. Mean suppressionratios 24 hr after a 60-min extinction session. No ext: mice left in home cages; sal-ext: saline injected 30 min before the extinction session; tan-ext: tandospirone (5 mg/kg) before extinction; bus-ext: buspirone (1 mg/kg) before extinction.
conditioning described earlier on day 3. No drugs were administered during fear conditioning. On day 4, mice were given an extinction session in which the CS was presented alone 60 times with an interstimulus interval of 1 rain. Thirty minutes before the session 12 mice were injected with saline, 12 with tandospirone 2 mg/kg, and 12 with l mg/kg buspirone. These drug doses produced the largest effect on suppression in Experiment 1. A control group of 12 mice was not given extinction on day 4. All animals were given l-hr access to water in the home cage after each daily session. The effects of tandospirone and buspirone on extinction were evaluated using the standard test procedure conducted 24 hr after extinction.
Results The results of this experiment are shown in Figure 5. A one-way ANOVA applied to these data showed a significant differeac~ among the four groups (F [3,48] = 8.97; p - <0.001). Post-hoe comparisons using Bonferroni ttests indicated that ~andospirone, buspirone, and the noextinction group were all significantly (p -- <0.05) different from the saline-extinction group (t = 3.99; 4.45 and 4.21 respectively). These results show that buspirone and tandospirone abolish the effects of extinction on fear conditioning. This finding implies that the disruption of fear conditioning demonstrated in Experiment 1 is not caused by alterations in fear motivation or to a decrease in pain sensitivity. The demonstration that tandospirone and buspirone impair both the acquisition and the extinction of fear is more consistent with the notion that these 5-HT]A agonists disl~pt processes involved in the storage and retrieval of the memory of fear conditioning.
The pattern of the results from the first three experiments suggest that tandospirone and buspirone affect fear conditioning by impairing memory of behaviors learned after drug administration. Memory loss caused by pretraining administration of many different forgetting agents can be reversed by pharmacological and behavioral treatments applied before the retention test (Quartermain et al 1988b; Quartermain and Leo 1988). Such findings are usually interpreted as indicating that subjects learned the task but were unable to retrieve the information at the time of testing. The objective of Experiment 4 was to evaluate the memory-impairment hypothesis by attempting to demonstrate that the deficit in fear conditioning can be reversed by treatment with a memory-enhancing agent (d-amphetamine sulphate) administered before the retention test. Amphetamine was employed because it has been shown to alleviate memory loss in animals caused by a wide variety of memory-impairing treatments (Quartermain et al 1988a).
Procedure Forty-eight mice were the subjects for this experiment. Thirty-sb, animals were trained as previously described. Thirty minutes before the conditioning session 12 mice were injected with saline and 24 with tandospirone (5 mg/kg). Thirty minutes before testing, the mice treated with saline and half of the tandospirone group (n = 12) were reinjected with saline, whereas the other half of the tandospirone group was injected with d-amphetamine sulphate (1 mg/kg). In order to evaluate possible nonspecific effects of acute amphetamine on test performance, a group of 12 mice was given sham conditioning and tested after amphetamine treatment. Sham conditioning consisted of 3 CS-UCS pairings using a different CS (clicks of 100 msec duration) and a UCS administered in a different apparatus in a different experimental room. Mice in this group were injected with amphetamine on the test day and tested in the regular training apparatus. We reasoned that if amphetamine improves conditioned suppression by reactivating the memory of the original conditioning, regularly trained animals should show improved performance (lower suppression ratios), whereas sham-trained mice should be unaffected as they had not received fear conditioning in the training apparatus. On the other hand, improved suppreRsion by both sham- and regularly trained animals would indicate that performance was being influenced by nonspecific factors associated with amphetamine treatment.
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•~
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~
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Figure 6. Effect of pretest injection of amphetamine (1 mg/kg) on tandospirone-induced amnesia. Tandospirone (5 mg/kg) was administered 30 min before conditioning and amphetamine 30 min before the test session, sal-sal: saline before conditioning and before testing; tan-sal: tandospirone before conditioning, saline before testing; tan-amp: tandospirone before conditioning, amphetamine before testing; tan-amp (sham) was injected with tandospirone and exposed to CS and UCS in a different apparatus in another room and tested in the regular apparatus 30 min after amphetamine. Values are mean suppression ratios and SEMs.
Results These results, shown in Figure 6, indicate that the disruption of conditioning produced by tandospirone was reversed by d-amphetamine (tandospirone-saline versus tandospirone-amphetamine t = 2.81;p = 0.033). That this decrease in suppression ratio was not attributable to nonspecific effects of acute amphetamine treatment is shown by the results of the sham-trained group. Sham-trained mice showed significantly higher ratios (less suppression) than regularly trained subjects (t = 3.48; p = 0.002) indicating that amphetamine improved conditioned suppression only in those animals that had been conditioned in the regular training apparatus. These findings suggest that tandospirone causes a memory deficit that can be reversed by amphetamine applied before the retention test.
Discussion The results of this study confirm previous reports that tandospirone, like other 5 - H T I A agonists, can disrupt performance when administered before the training trial. The specific contribution of the present series of experiments is to show that the behavioral disruption is the result of interference with memory processes and not a consequence of impairments of acquisition or alterations in motivational or sensory mechanisms. Both agonists disrupted fear conditioning when given before the conditioning session (Figure 1) but were without effect when they were administered before the test session
once fear had already been established (Figure 2). These results indicate that. the 5-HTIA agonists disrupt the encoding process of the fear but not its expression. This latter finding shows that neither agonist has anxiolytic propelties in the behavioral test used in this study, otherwise fear would have been alleviated at the time of testing. In contrast, diazepam, a benzodiazepine, disrupted fear conditioning when it was given both before conditioning and before testing, an indication of anxiolytic action on the expression of fear. A similar finding has recently been reported to occur following administration of the N-Methyl-D-Aspartate receptor antagonist APV (VL2-amino-5-phosphono valeric acid) (Kim et al i991). These authors suggest that NMDA-dependent LTP (long-term potentiation) may underlie the neural changes occurring in fear conditioning. The present results show that 5-HTIA receptors are also involved in the associative changes underlying fear conditioning. The data in Experiment I show that the disruption of fear conditioning is not the resultof state-dependent learning. Reestablishing the drug state that was present during conditioning by injectinga second dose of the agonist prior to the testsession failsto reverse the behavioral disruption. The resultsof Experiment 3 show that tandospirone and buspirone can also impair the extinction of fear (Figure 5). This finding indicates that the disruptive effects of 5HT~A agonists are apparent in more than one kind of learning and also rules out alterations in fear motivatio~ and pain sensitivity as explanations for their mechanism of action. Other studies that have shown disruption of appetitivelymotivated behaviors by 5-HTIA activation (Winter and Petti 1987) support this view. The most plausible hypothesis to explain the effects of 5-HT~A agonists in this study is that they disrupt memory processes. W h e n pretraining drug administration causes forgetting, the memory loss is usually classifiedas anterograde amnesia (AA). To be an authentic AA, however, it must be shown that the performance deficit is not the result of impaired acquisition. Although there is no direct proof from these experiments that associative processes are normal following drug treatment, the results of Experiment 2, which revealed unimpaired performance I hr after training, provide circumstantial evidence that tandospirone (and presumably also buspirone ) does not impair conditioning. Other evidence from thislaboratory suggests that this is the case. W e have shown (unpublished observations) that mice treated with tandospirone before acquisition exhibit normal rates of learning in a one-way active avoidance task. These results, in conjunction with the demonstration that shock intensities at which mice flinch, run, and vocalize are not altered by tandospirone (unpublished observations), make it reasonable to assume that acquisition processes are not impaired by 5-HT~A ag-
5-HT~^ Dismpt Fear Conditioning
onists. Taken together these findings support, the hypothesis that 5-HTt^ agonists disrupt performance by causing anterograde amnesia. The results of Experiment 4 also support a memory loss interpretation of the deficit in fear conditioning following tandospirone and buspirone treatment. Pretesting amphetamine administration resulted in an almost complete reversal of the deficit. This could not have occlared unless fear conditioning had taken place as the sham-~ained group showed no improvement in conditioned suppression after amphetamine treatment. A memory-impairmen~t hypothesis is also consistent with other data from our l~boratory. We have shown that tandospirone can produce torgetting for both active- and passive-avoidance learning. In the active-avoidance stud~, pretraining doses of tandospirone from 1-10 mg/kg did not alter the number of trials needed to reach a learning criterion of 5/6 avoidances relative to vehicle controls. A 24-hr retention test, however, revealed a robust deficit in groups receiving 2 and 5 mg/kg doses (unpublished observations). Unpublished observations from this laboratory using a single trial step-through passive avoidance task have shown that pretraining doses of 8OH-DPAT and tandospirone produce memory loss. This
amnesia could also be reversed by d-amphetamine injected before the retention test. The anmestic effects of tando,,~pirone were blocked by the 5-HT~A antagonists BMY7378, suggesting that the amnesia was the result of postsynaptic actions of tandospirone. The final profile pre-
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sented by these experiments is of learning that is unimpaired 1 hr after training, that is retrievable when amphetamine is administered, but that is too weak to be present 24-hr posttraining. It is possible that 5-HT~^ receptor activation diminishes the strength of the message encoded or changes it to make it less retrievable from longterm storage. One of the advantages of the "new" generation of anxiolytics/antidepressants of this class is that there are relatively few side effects. The typical cluster of sedative effects and memory impairment associated with the benzodiazepines and the trieyclics have largely been" avoided in ~hese new compounds. It may therefore seem surprising that the animal data provide evidence of memory impairment, particularly as clinical results demonstrate that busprone produces no amnesia when compared with diazepam. (Boulenger et al 1989; Lucki et al 1987), and tandospirone has similarly been reported to have no disruptive effects on cognition (personal communication). A possible reason for the discrepancy between these two sets of data is that drugs are typically administered chronically in the clinical studies and acutely in the animal experiments. It may be that exposure to the drug for a period of time prior to the onset of behavioral testing may eliminate the disruptive effects on cognitive performance. To resolve this issue it will be necessary to design animal experiments that more closely parallel the clinical studies.
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Quartermain D, Judge ME, Leo P (1988b): Attenuation of forgetting by pharmacological stimulation of aminergic neurotransmitter systems. Pharmacol Biochem Behav 30:77-81. Quartermain D, Leo P (1988): Alleviation of scopolamine amnesia by different retrieval enhancing treatments. Pharmacal Biochem Behav 30:1093-1096. Rogers C, Shemer A, Azmitia EC, Whitaker-Azmitia PM (1991): Effect of tandospirone (SM 3997) on production of growth factors from primary cultures of astroglial cells derived from different brain regions. Soc Neurosci Abstr #39.15. Rowan MJ, Cullen WK, Moulton B (1990); Buspirone impairment of performance of passive avoidance and spatial learning tasks in the rat. Psychopharmacology 100(3):393-398 Sprouse JS, Aghajanian GK (1987): Electrophysiological responses of serotonergic dorsal raphe neurons to 5-HTIa and 5-HTIb agonists. Synapse 1:3-9. Tenen SS (1967): Recovery time as a measure of CER strength:
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