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Effects of post-training bicuculline and muscimol on retention: Lack of state dependency
BEHAVIORALAND NEURALBIOLOGY54, 156--164 (1990)
Effects of Post-training Bicuculline and Muscimol on Retention" Lack of State Dependency CLAUDIO CASTELLANO Istituto di Psicobiologia e Psicofarmacologia del CNR via Reno 1, 00198 Rome, Italy AND JAMES L. MCGAUGH 1 Center for the Neurobiology of Learning and Memory, and Department of Psychobiology, University of California at Irvine, lrvine, California 92717 Immediate post-training intraperitoneal injections of the GABA antagonist bicuculline (0.25 or 0.5 mg/kg) or of the GABA agonist muscimol (1.0 or 2.0 mg/kg) improved and impaired, respectively, retention of CD1 mice tested 24 h after training in a one-trial inhibitory avoidance task. Administration of bicuculline or muscimol prior to the retention test did not modify retention latencies of mice that had received either saline or the same drug immediately after training. These findings indicate that the effects of post-training administration of bicuculline and muscinol on retention are not state dependent and, thus, argue against a general state-dependency interpretation of the effects of post-training treatments affecting retention. The findings are consistent with previous evidence indicating that GABAergic drugs affect retention through influences on memory storage processes. © 1990AcademicPress, Inc. T h e r e is e x t e n s i v e e v i d e n c e i n d i c a t i n g t h a t the a d m i n i s t r a t i o n o f the G A B A e r g i c a n t a g o n i s t b i c u c u l l i n e i m m e d i a t e l y after t r a i n i n g i m p r o v e s r e t e n t i o n o f r o d e n t s t r a i n e d a n d t e s t e d in a v a r i e t y o f l e a r n i n g t a s k s , including active avoidance, inhibitory avoidance, and discrimination l e a r n i n g t a s k s ( B r i o n i & M c G a u g h , 1988; C a s t e l l a n o & P a v o n e , 1988; Y o n k o v & G e o r g i e v , 1985). It s e e m s c l e a r t h a t the m e m o r y - e n h a n c i n g effects of b i c u c u l l i n e are d u e to i n f l u e n c e s o n c e n t r a l G A B A e r g i c s y s t e m s as i n t r a p e r i t o n e a l i n j e c t i o n s o f b i c u c u l l i n e m e t h i o d i d e , w h i c h d o e s n o t r e a d i l y pass the b l o o d - b r a i n b a r r i e r , are i n e f f e c t i v e ( B r i o n i & M c G a u g h , i Supported by Research Grant MH12526 from NIMH and NIDA and ONR Contract N00014 87 K 0518 (to LL.M.). Requests for reprints should be addressed to Dr. C. Castellano. 156 0163-1047/90 $3.00 Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin anyformreserved.
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1988). In addition, numerous studies have reported that retention is also enhanced by post-training administration of the GABAergic antagonist picrotoxin (Bovet, McGaugh, & Oliverio, 1966; Breen & McGaugh, 1961; Brioni & McGaugh, 1988; Castellano & Pavone, 1988; Grecksch & Matthies, 1981). Other recent findings indicate that retention is impaired by post-training administration of the GABAergic agonist muscimol (Castellano & Pavone, 1988). As has been found with other drugs (McGaugh, 1973) as well as hormones (McGaugh, 1983), the effects of GABAergic antagonists and agonists on retention are time-dependent. That is, the effects are greatest when the injections are administered shortly following training. Thus, such findings have generally been interpreted as supporting the view that the drugs affect retention by modulating activity in brain systems involved in memory storage (McGaugh, 1989)o There is, however, evidence suggesting that, under some conditions, the memory impairment produced by post-training treatments may be due to state dependency. That is, it is suggested that the impaired retention may be due to differences in brain states at the time of storage and retrieval of the information (Izquierdo, 1984). For example, the memory-impairing effects found with post-training administration of a variety of treatments, including/3-endorphin, enkephalins, or high doses of epinephrine, are attenuated if the same treatments are administered both post-training and prior to testing (Introini, 1984; Izquierdo, 1980; Izquierdo & Dias, 1987; Izquierdo & McGaugh, 1985, 1987). Such findings have raised the question of whether state dependency is a special case of amnesia induced by post-training treatments or whether all retrograde amnesia is due to state dependency. The view that state dependency is a special case is supported by our recent finding that the retrograde amnesia induced by post-training morphine (Castellano, 1975; Castellano, Pavone, & Puglisi-Allegra, 1984; Izquierdo, 1979; Messing et al., 1979) is not state-dependent: the amnesia for an inhibitory avoidance response is not attenuated by morphine administered prior to the retention test (Castellano & McGaugh, 1989b). Such findings are consistent with the view that morphine impairs retention by modulating memory storage processes rather than by inducing state dependency. In other recent research we have obtained evidence indicating that the retention-enhancing effects of post-training injections of the GABAergic agonist picrotoxin are not based on state dependency: Injections of picrotoxin administered prior to the retention test in an inhibitory avoidance task do not influence the retention performance of animals given either saline or picrotoxin post-training (Castellano & McGaugh, 1989a). As a further examination of this issue, the present experiments examined the effects of post-training and pretest administration of bicuculline and muscimol to determine whether their retention-enhancing and -impairing effects, respectively, involve state dependency.
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METHODS
Subjects Male CD1 mice (River Laboratories, Como, Italy) weighing approximately 25 g were caged in groups of eight with food and water available ad libitum and maintained on a 12-h light-dark cycle (lights on at 07:00) at a constant temperature of 21°C for 2 weeks prior to the experiments.
Apparatus and Procedures The step-through inhibitory avoidance apparatus, similar to that previously described by Castellano, Pavone, and Puglisi-Allegra (1984), consisted of a 20 x 20 x 20-cm Lucite box with black walls and a grid floor. A platform (12 cm long, 7.5 cm wide) extended from a small door (4 x 3 cm) in the front of the box. When the animal entered the box with all four feet the step-through latency was recorded, the entry was closed with a sliding door, and a footshock (0.7 mA, 1.0 s, 50 Hz) was delivered. The mouse was then immediately returned to its home cage. For the retention test 24 h later the mouse was placed on the platform as in the training session and the step-through latency (maximum of 240 s) was recorded. In all experiments each group consisted of 16 mice. Mann-Whitney U tests (two-tailed probabilities) were used for all statistical tests. Muscimol (Sigma) was dissolved in physiological saline (0.9% NaCI); bicuculline (Sigma) was first dissolved in a few drops of 0.1 N HC1, after which the final volume was made up with saline. The pH of the solution was 6.5. Control groups were injected with physiological saline. The injections were administered ip in a volume of 10.0 ml/kg. The drug doses were selected on the basis of previous experiments (Brioni & McGaugh, 1988; Castellano & Pavone, 1988). EXPERIMENT 1 In the first set of experiments, different groups of mice received injections of saline, bicuculline (0.25 or 0.5 mg/kg), or muscimol (1.0 or 2.0 mg/kg) immediately after training and saline 3 rain prior to the retention test 24 h later. These conditions were included in order to replicate previous findings of enhancement and impairment of retention with bicucuUine and muscimol, respectively, and to serve as controls for subsequent experiments examining the effects of injections of these drugs prior to testing. An additional group of mice was injected with the bicuculline vehicle only, immediately after training and 3 rain before the retention test in order to determine possible effects of the vehicle solution. Other groups of mice were injected with the highest doses of either bicuculline (0.5 mg/kg) or muscimol (2.0 mg/kg) 30, 10, or 3 rain
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BICUCULLINE, MUSCIMOL, AND MEMORY TABLE 1 Effects of Bicuculline and Muscimol on Step-Through Latencies Retention latencies (s) Treatment Immediate post-training Saline Bicuculline 0.25 mg/kg 0.50 Muscimol 1.0 2.0
3-min pretest
Median
(IQ)
Saline
100.5
(63.0-123.5)
Saline Saline
173.2 240.0
(131.0-207.0)* (218.0-240.0) *'+
Saline Saline
49.3 10.5
(24.5-63.5)* 7.0-19.5) *'+
Initial step-through response latencies (s) Median Prior to training Saline 3 min I0 30 Bicuculline (0.5 mg/kg) 3 min 10 3O Muscimol (2.0 mg/kg) 3 min 10 3O
(IQ)
9.0 9.5 9.0
(7.5-10.0) (8.5-10.0) (7.0-10.0)
8.0 9.0 8.5
(6.5-9.0) (7.5-10.5) (8.0-9.5)
7.5 8.5 6.5
(5.0-10.0) (7.5-9.5) (5.0-10.5)
* p < .002 vs saline controls (Mann-Whitney U tests). N = 16 per group; + p < .002 vs lower dose (Mann-Whitney U tests). N = 16 per group. p r i o r to t r a i n i n g in o r d e r to d e t e r m i n e w h e t h e r t h e d r u g s a f f e c t e d r e s p o n s e l a t e n c i e s at t h e s e t i m e s f o l l o w i n g t h e i n j e c t i o n s . T h e r e t e n t i o n l a t e n c i e s o f t h e saline a n d b i c u c u l l i n e v e h i c l e c o n t r o l g r o u p s w e r e h i g h l y similar. T h e m e d i a n s a n d i n t e r q u a r t i l e r a n g e s (IQ) w e r e saline, 100.5 (63.0-123.5) a n d b i c u c u l l i n e v e h i c l e , 99.5 (71.0-120.0). A s s h o w n in T a b l e 1, t h e r e t e n t i o n l a t e n c i e s o f b o t h b i c u c u l l i n e g r o u p s w e r e significantly h i g h e r t h a n t h o s e o f t h e c o n t r o l s . T h e e f f e c t s o f bic u c u l l i n e w e r e d o s e - d e p e n d e n t : T h e r e t e n t i o n l a t e n c i e s o f t h e 0.5 m g / k g b i c u c u l l i n e g r o u p w e r e s i g n i f i c a n t l y h i g h e r t h a n t h o s e o f t h e 0.25 m g / k g g r o u p . T h e r e t e n t i o n - i m p a i r i n g effects o f m u s c i m o l w e r e a l s o d o s e - d e p e n d e n t : T h e r e t e n t i o n l a t e n c i e s o f b o t h m u s c i m o l g r o u p s w e r e signific a n t l y l o w e r t h a n t h o s e o f t h e saline c o n t r o l s , a n d t h o s e o f m i c e g i v e n t h e 2.0 m g / k g d o s e w e r e s i g n i f i c a n t l y l o w e r t h a n t h o s e g i v e n t h e 0.5
160
CASTELLANO AND MCGAUGH TABLE 2 EffectsofBicucullineand Muscimolon ~mningStep-Through L~encies
Drug and time of injection prior to training Saline 3 rain 10 30 Bicuculline (0.5 mg/kg) 3 rnin 10 30 Muscimol (2.0 mg/kg) 3 min 10 30
Initial step-through response latency Median
(IQ)
9.0 9.5 9.0
(7.5-10.0) (8.5-10.0) (7.0-10.0)
8.0 9.0 8.5
(6.5-9.0) (7.5-10.5) (8.0-9.5)
7.5 8.5 6.5
(5.0-10.0) (7.5-9.5) (5.0-10.5)
mg/kg dose. Further, as can be seen in Table 2, neither drug affected initial step-through response latencies when administered 30, 10, or 3 rain prior to the training trial. EXPERIMENT 2 Experiment 2 examined the effects of bicuculline or muscimol administered prior to the retention test. Mice were trained on the inhibitory avoidance task and given post-training injections of saline, bicuculline (0.25 or 0.5 mg/kg), or muscimol (1.0 or 2.0 mg/kg). A second injection of saline, bicuculline, or muscimol was administered at one of several intervals (3, 10, or 30 min) prior to the 24-h retention test. As shown in Tables 3 and 4, bicuculline and muscimol, administered at the three time intervals prior to retention testing, did not affect the retention performance of mice given saline injections post-training. Further, in all groups given post-training bicuculline, or muscimol, the retention latencies of mice given the same drugs (in either the same or a different dose) prior to the retention test were highly comparable to those of mice (shown in Table 1) given drugs post-training and saline prior to the retention test. As in Experiment 1, the effects of post-training bicuculline and muscimol were dose-dependent. The latencies of groups given the lower dose of the two drugs post-training differed significantly from those of the saline controls as well as those of the groups given the higher dose. DISCUSSION The findings of the present experiments provide additional evidence that, in mice, post-training administration of the GABAergic antagonist
161
BICUCULLINE, MUSCIMOL, AND MEMORY TABLE 3 Effects of Bicuculline Administered Post-training and prior to Retention Test Treatment
Post-training
Prior to retention test (mg/kg)
Retention latencies Median
(IQ)
Saline Saline
Saline--10 min Saline--30 min
84.5 100.3
(66.0-112.5) (50.0-130.5)
Saline Saline Saline Saline
Bicuculline Bicuculline Bicuculline Bicuculline
(0.25)---3 min (0.50)--3 min (0.50)--10 min (0.50)--30 min
99.6 106.0 94.4 98.7
(60.0-130.2) (70.5-128.5) (62.5-139.6) (57.5--134.5)
Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline
(0.25)----3 min (0.50)--3 min (0.25)--3 min (0.50)--3 min (0.50)--10 min (0.50)--30 min
159.5 170.5 240.0 240.0 240.0 240.0
(114.5-187.0) *'+ (135.0-200.0) *'+ (211.0-240.0)* (214.5-240.0)* (187.0-240.0)* (193.5-240.0)*
Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline
(0.25) (0.25) (0.50) (0.50) (0.50) (0.50)
* p < .002 vs all groups given saline post-training; + p < .002 vs all groups given 0.50 bicuculline (Mann-Whitney U tests). N = 16 per group.
TABLE 4 Effects of Muscimol Administered Post-Training and prior to Retention Test Treatment
Post-training
Prior to retention test (mg/kg)
Retention latencies (s) Median
(IQ)
Saline Saline
Saline--10 rain Saline--30 rain
84.5 100.3
(66.0-112.5) (50.0-130.5)
Saline Saline Saline Saline
Muscimol Muscimol Muscimol Muscimol
(1.0)--3 min (2.0)--3 rain (2.0)--10 min (2.0)--30 min
94.6 104.7 103.5 100.5
(66.0-150.0) (71.0-146.0) (56.5-168.5) (54.5-119.0)
Muscimol Muscimol Muscimol Muscimol Muscimol Muscimol
(1.0)--3 min (2.0)--3 min (1.0)--3 min (2.0)--3 min (2.0)--10 min (2.0)---30 min
53.5 51.5 8.5 9.5 17.5 14.3
Muscimol Muscimol Muscimol Muscimol Muscimol Muscimol
(1.0) (1.0) (2.0) (2.0) (2.0) (2.0)
(35.5-69.5) *'+ (35.0-77.0) *.+ (6.0-21.0)* (6.5-15.5)* (10.0-41.0)* (5.5-26.0)*
* p < .002 vs all groups given saline post-training; + p < .002 vs all groups given 2.0 Muscimol post-training (Mann-Whitney U tests). N = 16 per group.
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CASTELLANO AND MCGAUGH
enhances retention of an inhibitory avoidance response while retention is impaired by post-training injections of the GABAergic agonist muscimol (Brioni & McGaugh, 1988; Castellano & Pavone, 1988). Further, the fact that the drugs were administered immediately after training, 24 h prior to the retention test, as well as the finding that administration of these drugs prior to training did not affect the training response latencies clearly argues against any interpretation suggesting that the effects may be due to nonspecific drug influences on response latencies in this task. In previous studies of the effect of bicuculline and muscimol we have found that these drugs do not affect retention if administered 2 h post-training and, when administered immediately after the initial trial, do not affect the 24-h test latencies of unshocked controls (Brioni & McGaugh, 1988; Castellano & Pavone, 1988). All of these findings are consistent with the interpretation that the effects of GABAergic drugs on retention performance are due to modulatory influences on posttraining memory storage processes (McGaugh, 1973; 1989). The findings of other recent experiments from our laboratories suggest that post-training administration of GABAergic drugs may influence retention through influences involving the amygdaloid complex. Intraamygdala injections of GABAergic drugs produce dose-dependent and time-dependent effects on retention of an inhibitory avoidance response comparable to those found when the drugs are injected systemically (Brioni, Nagahara & McGaugh, 1989; Castellano, Brioni, Nagahara, & McGaugh, 1989). The main finding that emerges from the present experiments is that the retention-enhancing effects of post-training administration of bicuculline, and the retention-impairing effects of post-training administration of muscimol, are not based on state dependency. The findings clearly indicate that administration of bicuculline or muscimol 30, 10, or 3 rain prior to the retention test did not affect the retention latencies of animals which were given either saline or the same drugs immediately after training. These results are consistent with our previous findings that the retention enhancement produced by post-training injections of the GABAergic antagonist picrotoxin as well as the retrograde amnesia produced by the opiate agonists dynorphin and morphine are not based on state dependency (Castellano & McGaugh, 1989b; Introini-Collison, Cahill, Baratti, & McGaugh, 1989). Thus, these findings argue against the general hypothesis that retention performance reflects the degree to which the brain state at the time of the retention test is congruent with that which normally occurs, or is induced, following training. Our findings suggest that subsequent experiments examining post-training state-dependent effects should attempt to elucidate the special conditions which appear to be required for inducing state dependency. Clearly, the conditions do not
BICUCULLINE, MUSCIMOL, AND MEMORY i n c l u d e t h o s e t h a t w e h a v e u s e d in o u r r e c e n t e x p e r i m e n t s this i s s u e .
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REFERENCES Breen, R. A., & McGaugh, J. L. (1961). Facilitation of maze learning with post-trial injections of picrotoxin. Journal of Comparative and Physiological Psychology, 54, 498-501. Bovet, D., McGaugh, J. L., & Oliverio, A. (1966). Effects of post-trial administration of drugs on avoidance learning of mice. Life Sciences, 5, 1309-1315. Brioni, J. D., & McGaugh, J. L. (1988). Post-training administration of GABAergic antagonists enhances retention of aversively motivated tasks. Psychopharmacology, 96, 505-510. Brioni, J. D., Nagahara, A. H., & McGaugh, J. L. (1989). Involvement of the amygdala GABAergic system in the modulation of memory storage. Brain Research, 487, 105112. Castellano, C. (1975). Effects of morphine and heroin on discrimination learning and consolidation in mice. Psychopharmacologia, 42, 235-242. Castellano, C., & McGaugh, J. L. (1989a). Retention enhancement with post-training picrotoxin: Lack of state dependency. Behavioral and Neural Biology, 51, 165-170. Castellano, C., & McGaugh, J. L. (1989b). Effect of morphine on one-trial inhibitory avoidance in mice: Lack of state-dependency. Psychobiology, 17, 89-92. Castellano, C., & Pavone, F. (1988). Effects of ethanol on passive avoidance behavior in the mouse: Involvement of GABAergic mechanisms. Pharmacology, Biochemistry and Behavior, 29, 321-324. Castellano, C., Pavone, F., & Puglisi-Allegra, S. (1984). Morphine and memory in DBA/2 mice: Effects of stress and of prior experience. Behavioural Brain Research, 11, 3I0. Castellano, C., Brioni, J. D., Nagahara, A. H., & McGaugh, J. L. (1989). Posttraining systemic and intra-amygdala administration of the GABA-b agonist baclofen impair retention. Behavioral and Neural Biology, 52, 170-179. Grecksch, G., & Matthies, H. (1981). Differential effects of intrahippocampally or systemically applied picrotoxin on memory consolidation in rats. Pharmacology, Biochemistry and Behavior, 14, 613-616. Introini, I. B. (1984). Participation de peptidos opioides endogenos en el proceso do consolidacion de la memoria. Su possible interaction con otros sistemas neuronales. Doctoral dissertation, Facultad de Farmacia y Bioquimica, University of Buenos Aires. Introini-Collison, I. B., Cahill, L., Baratti, C. M., & McGaugh, J. L. (1987). Dynorphin induces task-specific impairment of memory. Psychobiology, 15, 171-174. Izquierdo, I. (1979). Effect of naloxone and morphine on various forms of memory in the rat: Possible role of endogenous opiate mechanisms in memory consolidation. Psychopharmacology, 66, 199-203. Izquierdo, I. (1980). Effect of/~-endorphin and naloxone on acquisition, memory and retrieval of shuttle-avoidance and habituation learning in rats. Psychopharmacology, 69, 111-115. Izquierdo, I. (1984). Endogenous state dependency: Memory depends on the relation between the neurohumoral and hormonal states present after training and at the time of testing. In G. Lynch, J. L. McGaugh, & N. M. Weinberger (Eds.), Neurobiology of learning and memory (pp. 333-350). New York: Guilford. Izquierdo, I., & Dias, R. D. (1983). Memory modulation by the administration of ACTH, adrenaline, or/3-endorphin after training and prior to testing in an inhibitory avoidance task in rats. Brazilian Journal of Medical and Biological Research, 15, 54-64. Izquierdo, I., & McGaugh, J. L. (1985). Effect of novel experience on retention of inhibitory
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avoidance behavior in mice: The influence of previous exposure to the same or another experience. Behavioral and Neural Biology, 47, 109-115. Izquierdo, I., & McGaugh, J. L. (1987). Retention impairment by post-training epinephrine: Role of state dependency and of endogenous opioid mechanisms. Behavioral Neuroscience, 101, 778-781. McGaugh, J. L. (1973). Drug facilitation of learning and memory. Annual Review of Pharmacology, 13, 229-241. McGaugh, J. L. (1983). Hormonal influences on memory. Annual Review of Psychology, 34, 297-323. McGaugh, J. L. (1989). Involvement of hormonal and neuromodulatory systems in the regulation of memory storage. Annual Review of Neuroscience, 12, 255-287. Messing, R. B., Jensen, R. A., Martinez, J. L., Jr., Spiehler, V. R., Vasquez, B. J., Soumireu-Mourat, B., Liang, K. C., & McGaugh, J. L. (1978). Naloxone enhancement of memory. Behavioral and Neural Biology, 27, 226-275.
Effects of Post-training Bicuculline and Muscimol on Retention" Lack of State Dependency CLAUDIO CASTELLANO Istituto di Psicobiologia e Psicofarmacologia del CNR via Reno 1, 00198 Rome, Italy AND JAMES L. MCGAUGH 1 Center for the Neurobiology of Learning and Memory, and Department of Psychobiology, University of California at Irvine, lrvine, California 92717 Immediate post-training intraperitoneal injections of the GABA antagonist bicuculline (0.25 or 0.5 mg/kg) or of the GABA agonist muscimol (1.0 or 2.0 mg/kg) improved and impaired, respectively, retention of CD1 mice tested 24 h after training in a one-trial inhibitory avoidance task. Administration of bicuculline or muscimol prior to the retention test did not modify retention latencies of mice that had received either saline or the same drug immediately after training. These findings indicate that the effects of post-training administration of bicuculline and muscinol on retention are not state dependent and, thus, argue against a general state-dependency interpretation of the effects of post-training treatments affecting retention. The findings are consistent with previous evidence indicating that GABAergic drugs affect retention through influences on memory storage processes. © 1990AcademicPress, Inc. T h e r e is e x t e n s i v e e v i d e n c e i n d i c a t i n g t h a t the a d m i n i s t r a t i o n o f the G A B A e r g i c a n t a g o n i s t b i c u c u l l i n e i m m e d i a t e l y after t r a i n i n g i m p r o v e s r e t e n t i o n o f r o d e n t s t r a i n e d a n d t e s t e d in a v a r i e t y o f l e a r n i n g t a s k s , including active avoidance, inhibitory avoidance, and discrimination l e a r n i n g t a s k s ( B r i o n i & M c G a u g h , 1988; C a s t e l l a n o & P a v o n e , 1988; Y o n k o v & G e o r g i e v , 1985). It s e e m s c l e a r t h a t the m e m o r y - e n h a n c i n g effects of b i c u c u l l i n e are d u e to i n f l u e n c e s o n c e n t r a l G A B A e r g i c s y s t e m s as i n t r a p e r i t o n e a l i n j e c t i o n s o f b i c u c u l l i n e m e t h i o d i d e , w h i c h d o e s n o t r e a d i l y pass the b l o o d - b r a i n b a r r i e r , are i n e f f e c t i v e ( B r i o n i & M c G a u g h , i Supported by Research Grant MH12526 from NIMH and NIDA and ONR Contract N00014 87 K 0518 (to LL.M.). Requests for reprints should be addressed to Dr. C. Castellano. 156 0163-1047/90 $3.00 Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin anyformreserved.
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157
1988). In addition, numerous studies have reported that retention is also enhanced by post-training administration of the GABAergic antagonist picrotoxin (Bovet, McGaugh, & Oliverio, 1966; Breen & McGaugh, 1961; Brioni & McGaugh, 1988; Castellano & Pavone, 1988; Grecksch & Matthies, 1981). Other recent findings indicate that retention is impaired by post-training administration of the GABAergic agonist muscimol (Castellano & Pavone, 1988). As has been found with other drugs (McGaugh, 1973) as well as hormones (McGaugh, 1983), the effects of GABAergic antagonists and agonists on retention are time-dependent. That is, the effects are greatest when the injections are administered shortly following training. Thus, such findings have generally been interpreted as supporting the view that the drugs affect retention by modulating activity in brain systems involved in memory storage (McGaugh, 1989)o There is, however, evidence suggesting that, under some conditions, the memory impairment produced by post-training treatments may be due to state dependency. That is, it is suggested that the impaired retention may be due to differences in brain states at the time of storage and retrieval of the information (Izquierdo, 1984). For example, the memory-impairing effects found with post-training administration of a variety of treatments, including/3-endorphin, enkephalins, or high doses of epinephrine, are attenuated if the same treatments are administered both post-training and prior to testing (Introini, 1984; Izquierdo, 1980; Izquierdo & Dias, 1987; Izquierdo & McGaugh, 1985, 1987). Such findings have raised the question of whether state dependency is a special case of amnesia induced by post-training treatments or whether all retrograde amnesia is due to state dependency. The view that state dependency is a special case is supported by our recent finding that the retrograde amnesia induced by post-training morphine (Castellano, 1975; Castellano, Pavone, & Puglisi-Allegra, 1984; Izquierdo, 1979; Messing et al., 1979) is not state-dependent: the amnesia for an inhibitory avoidance response is not attenuated by morphine administered prior to the retention test (Castellano & McGaugh, 1989b). Such findings are consistent with the view that morphine impairs retention by modulating memory storage processes rather than by inducing state dependency. In other recent research we have obtained evidence indicating that the retention-enhancing effects of post-training injections of the GABAergic agonist picrotoxin are not based on state dependency: Injections of picrotoxin administered prior to the retention test in an inhibitory avoidance task do not influence the retention performance of animals given either saline or picrotoxin post-training (Castellano & McGaugh, 1989a). As a further examination of this issue, the present experiments examined the effects of post-training and pretest administration of bicuculline and muscimol to determine whether their retention-enhancing and -impairing effects, respectively, involve state dependency.
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METHODS
Subjects Male CD1 mice (River Laboratories, Como, Italy) weighing approximately 25 g were caged in groups of eight with food and water available ad libitum and maintained on a 12-h light-dark cycle (lights on at 07:00) at a constant temperature of 21°C for 2 weeks prior to the experiments.
Apparatus and Procedures The step-through inhibitory avoidance apparatus, similar to that previously described by Castellano, Pavone, and Puglisi-Allegra (1984), consisted of a 20 x 20 x 20-cm Lucite box with black walls and a grid floor. A platform (12 cm long, 7.5 cm wide) extended from a small door (4 x 3 cm) in the front of the box. When the animal entered the box with all four feet the step-through latency was recorded, the entry was closed with a sliding door, and a footshock (0.7 mA, 1.0 s, 50 Hz) was delivered. The mouse was then immediately returned to its home cage. For the retention test 24 h later the mouse was placed on the platform as in the training session and the step-through latency (maximum of 240 s) was recorded. In all experiments each group consisted of 16 mice. Mann-Whitney U tests (two-tailed probabilities) were used for all statistical tests. Muscimol (Sigma) was dissolved in physiological saline (0.9% NaCI); bicuculline (Sigma) was first dissolved in a few drops of 0.1 N HC1, after which the final volume was made up with saline. The pH of the solution was 6.5. Control groups were injected with physiological saline. The injections were administered ip in a volume of 10.0 ml/kg. The drug doses were selected on the basis of previous experiments (Brioni & McGaugh, 1988; Castellano & Pavone, 1988). EXPERIMENT 1 In the first set of experiments, different groups of mice received injections of saline, bicuculline (0.25 or 0.5 mg/kg), or muscimol (1.0 or 2.0 mg/kg) immediately after training and saline 3 rain prior to the retention test 24 h later. These conditions were included in order to replicate previous findings of enhancement and impairment of retention with bicucuUine and muscimol, respectively, and to serve as controls for subsequent experiments examining the effects of injections of these drugs prior to testing. An additional group of mice was injected with the bicuculline vehicle only, immediately after training and 3 rain before the retention test in order to determine possible effects of the vehicle solution. Other groups of mice were injected with the highest doses of either bicuculline (0.5 mg/kg) or muscimol (2.0 mg/kg) 30, 10, or 3 rain
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BICUCULLINE, MUSCIMOL, AND MEMORY TABLE 1 Effects of Bicuculline and Muscimol on Step-Through Latencies Retention latencies (s) Treatment Immediate post-training Saline Bicuculline 0.25 mg/kg 0.50 Muscimol 1.0 2.0
3-min pretest
Median
(IQ)
Saline
100.5
(63.0-123.5)
Saline Saline
173.2 240.0
(131.0-207.0)* (218.0-240.0) *'+
Saline Saline
49.3 10.5
(24.5-63.5)* 7.0-19.5) *'+
Initial step-through response latencies (s) Median Prior to training Saline 3 min I0 30 Bicuculline (0.5 mg/kg) 3 min 10 3O Muscimol (2.0 mg/kg) 3 min 10 3O
(IQ)
9.0 9.5 9.0
(7.5-10.0) (8.5-10.0) (7.0-10.0)
8.0 9.0 8.5
(6.5-9.0) (7.5-10.5) (8.0-9.5)
7.5 8.5 6.5
(5.0-10.0) (7.5-9.5) (5.0-10.5)
* p < .002 vs saline controls (Mann-Whitney U tests). N = 16 per group; + p < .002 vs lower dose (Mann-Whitney U tests). N = 16 per group. p r i o r to t r a i n i n g in o r d e r to d e t e r m i n e w h e t h e r t h e d r u g s a f f e c t e d r e s p o n s e l a t e n c i e s at t h e s e t i m e s f o l l o w i n g t h e i n j e c t i o n s . T h e r e t e n t i o n l a t e n c i e s o f t h e saline a n d b i c u c u l l i n e v e h i c l e c o n t r o l g r o u p s w e r e h i g h l y similar. T h e m e d i a n s a n d i n t e r q u a r t i l e r a n g e s (IQ) w e r e saline, 100.5 (63.0-123.5) a n d b i c u c u l l i n e v e h i c l e , 99.5 (71.0-120.0). A s s h o w n in T a b l e 1, t h e r e t e n t i o n l a t e n c i e s o f b o t h b i c u c u l l i n e g r o u p s w e r e significantly h i g h e r t h a n t h o s e o f t h e c o n t r o l s . T h e e f f e c t s o f bic u c u l l i n e w e r e d o s e - d e p e n d e n t : T h e r e t e n t i o n l a t e n c i e s o f t h e 0.5 m g / k g b i c u c u l l i n e g r o u p w e r e s i g n i f i c a n t l y h i g h e r t h a n t h o s e o f t h e 0.25 m g / k g g r o u p . T h e r e t e n t i o n - i m p a i r i n g effects o f m u s c i m o l w e r e a l s o d o s e - d e p e n d e n t : T h e r e t e n t i o n l a t e n c i e s o f b o t h m u s c i m o l g r o u p s w e r e signific a n t l y l o w e r t h a n t h o s e o f t h e saline c o n t r o l s , a n d t h o s e o f m i c e g i v e n t h e 2.0 m g / k g d o s e w e r e s i g n i f i c a n t l y l o w e r t h a n t h o s e g i v e n t h e 0.5
160
CASTELLANO AND MCGAUGH TABLE 2 EffectsofBicucullineand Muscimolon ~mningStep-Through L~encies
Drug and time of injection prior to training Saline 3 rain 10 30 Bicuculline (0.5 mg/kg) 3 rnin 10 30 Muscimol (2.0 mg/kg) 3 min 10 30
Initial step-through response latency Median
(IQ)
9.0 9.5 9.0
(7.5-10.0) (8.5-10.0) (7.0-10.0)
8.0 9.0 8.5
(6.5-9.0) (7.5-10.5) (8.0-9.5)
7.5 8.5 6.5
(5.0-10.0) (7.5-9.5) (5.0-10.5)
mg/kg dose. Further, as can be seen in Table 2, neither drug affected initial step-through response latencies when administered 30, 10, or 3 rain prior to the training trial. EXPERIMENT 2 Experiment 2 examined the effects of bicuculline or muscimol administered prior to the retention test. Mice were trained on the inhibitory avoidance task and given post-training injections of saline, bicuculline (0.25 or 0.5 mg/kg), or muscimol (1.0 or 2.0 mg/kg). A second injection of saline, bicuculline, or muscimol was administered at one of several intervals (3, 10, or 30 min) prior to the 24-h retention test. As shown in Tables 3 and 4, bicuculline and muscimol, administered at the three time intervals prior to retention testing, did not affect the retention performance of mice given saline injections post-training. Further, in all groups given post-training bicuculline, or muscimol, the retention latencies of mice given the same drugs (in either the same or a different dose) prior to the retention test were highly comparable to those of mice (shown in Table 1) given drugs post-training and saline prior to the retention test. As in Experiment 1, the effects of post-training bicuculline and muscimol were dose-dependent. The latencies of groups given the lower dose of the two drugs post-training differed significantly from those of the saline controls as well as those of the groups given the higher dose. DISCUSSION The findings of the present experiments provide additional evidence that, in mice, post-training administration of the GABAergic antagonist
161
BICUCULLINE, MUSCIMOL, AND MEMORY TABLE 3 Effects of Bicuculline Administered Post-training and prior to Retention Test Treatment
Post-training
Prior to retention test (mg/kg)
Retention latencies Median
(IQ)
Saline Saline
Saline--10 min Saline--30 min
84.5 100.3
(66.0-112.5) (50.0-130.5)
Saline Saline Saline Saline
Bicuculline Bicuculline Bicuculline Bicuculline
(0.25)---3 min (0.50)--3 min (0.50)--10 min (0.50)--30 min
99.6 106.0 94.4 98.7
(60.0-130.2) (70.5-128.5) (62.5-139.6) (57.5--134.5)
Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline
(0.25)----3 min (0.50)--3 min (0.25)--3 min (0.50)--3 min (0.50)--10 min (0.50)--30 min
159.5 170.5 240.0 240.0 240.0 240.0
(114.5-187.0) *'+ (135.0-200.0) *'+ (211.0-240.0)* (214.5-240.0)* (187.0-240.0)* (193.5-240.0)*
Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline Bicuculline
(0.25) (0.25) (0.50) (0.50) (0.50) (0.50)
* p < .002 vs all groups given saline post-training; + p < .002 vs all groups given 0.50 bicuculline (Mann-Whitney U tests). N = 16 per group.
TABLE 4 Effects of Muscimol Administered Post-Training and prior to Retention Test Treatment
Post-training
Prior to retention test (mg/kg)
Retention latencies (s) Median
(IQ)
Saline Saline
Saline--10 rain Saline--30 rain
84.5 100.3
(66.0-112.5) (50.0-130.5)
Saline Saline Saline Saline
Muscimol Muscimol Muscimol Muscimol
(1.0)--3 min (2.0)--3 rain (2.0)--10 min (2.0)--30 min
94.6 104.7 103.5 100.5
(66.0-150.0) (71.0-146.0) (56.5-168.5) (54.5-119.0)
Muscimol Muscimol Muscimol Muscimol Muscimol Muscimol
(1.0)--3 min (2.0)--3 min (1.0)--3 min (2.0)--3 min (2.0)--10 min (2.0)---30 min
53.5 51.5 8.5 9.5 17.5 14.3
Muscimol Muscimol Muscimol Muscimol Muscimol Muscimol
(1.0) (1.0) (2.0) (2.0) (2.0) (2.0)
(35.5-69.5) *'+ (35.0-77.0) *.+ (6.0-21.0)* (6.5-15.5)* (10.0-41.0)* (5.5-26.0)*
* p < .002 vs all groups given saline post-training; + p < .002 vs all groups given 2.0 Muscimol post-training (Mann-Whitney U tests). N = 16 per group.
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CASTELLANO AND MCGAUGH
enhances retention of an inhibitory avoidance response while retention is impaired by post-training injections of the GABAergic agonist muscimol (Brioni & McGaugh, 1988; Castellano & Pavone, 1988). Further, the fact that the drugs were administered immediately after training, 24 h prior to the retention test, as well as the finding that administration of these drugs prior to training did not affect the training response latencies clearly argues against any interpretation suggesting that the effects may be due to nonspecific drug influences on response latencies in this task. In previous studies of the effect of bicuculline and muscimol we have found that these drugs do not affect retention if administered 2 h post-training and, when administered immediately after the initial trial, do not affect the 24-h test latencies of unshocked controls (Brioni & McGaugh, 1988; Castellano & Pavone, 1988). All of these findings are consistent with the interpretation that the effects of GABAergic drugs on retention performance are due to modulatory influences on posttraining memory storage processes (McGaugh, 1973; 1989). The findings of other recent experiments from our laboratories suggest that post-training administration of GABAergic drugs may influence retention through influences involving the amygdaloid complex. Intraamygdala injections of GABAergic drugs produce dose-dependent and time-dependent effects on retention of an inhibitory avoidance response comparable to those found when the drugs are injected systemically (Brioni, Nagahara & McGaugh, 1989; Castellano, Brioni, Nagahara, & McGaugh, 1989). The main finding that emerges from the present experiments is that the retention-enhancing effects of post-training administration of bicuculline, and the retention-impairing effects of post-training administration of muscimol, are not based on state dependency. The findings clearly indicate that administration of bicuculline or muscimol 30, 10, or 3 rain prior to the retention test did not affect the retention latencies of animals which were given either saline or the same drugs immediately after training. These results are consistent with our previous findings that the retention enhancement produced by post-training injections of the GABAergic antagonist picrotoxin as well as the retrograde amnesia produced by the opiate agonists dynorphin and morphine are not based on state dependency (Castellano & McGaugh, 1989b; Introini-Collison, Cahill, Baratti, & McGaugh, 1989). Thus, these findings argue against the general hypothesis that retention performance reflects the degree to which the brain state at the time of the retention test is congruent with that which normally occurs, or is induced, following training. Our findings suggest that subsequent experiments examining post-training state-dependent effects should attempt to elucidate the special conditions which appear to be required for inducing state dependency. Clearly, the conditions do not
BICUCULLINE, MUSCIMOL, AND MEMORY i n c l u d e t h o s e t h a t w e h a v e u s e d in o u r r e c e n t e x p e r i m e n t s this i s s u e .
163 examining
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