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Cysteamine-induced depletion of somatostatin produces differential cognitive deficits in rats
42
Brain Research, 482 (1989) 42-48
Elsevier BRE 143(t4
Cysteamine-induced depletion of somatostatin produces differential cognitive deficits in rats Victor J. DeNoble, David J. Hepler and Robert A. Barto E.I. duPont de Nemours & Co., Inc., Medical Products Department, Wilmington, DE 19880-0400 (U.S.A.)
(Accepted 16 August 1988) Key words: Somatostatin; Cysteamine; Behavior; Learning; Memory; Alzheimer's disease
The effects of a variety of doses of systemically administered cysteamine (a somatostatin depletor) were studied on step-through passive avoidance retention, as well as acquisition and performance of a delayed spatial alternation task and a signaled extinction discrimination task in rats. Retention of single trial passive avoidance was significantly reduced by a pretraining (60-min) dose of cysteamine at 50, 100, 150 and 200 mg/kg s.c. This effect was shown to be sensitive to behavioral manipulation; in a second experiment, a retention deficit was found only at the two highest doses tested (150 and 200 mg/kg s.c.) after a second exposure to the footshock. In the operant conditioning studies, biweekly injections (Monday and Wednesday) of cysteamine administered one hour before testing produced no statistically significant changes in acquisition or performance of either the delayed spatial alternation or the signaled discrimination task. The results of these series of experiments suggest that active somatostatin release or chronic somatostatin depletion may selectively affect performance maintained by different behavioral procedures. INTRODUCTION A m o n g the biological markers of post-mortem analysis of Alzheimer's Disease (AD) is a reduction of cortical and hippocampal somatostatin-like immunoactivity 4,13,I6-18. The close correlation of central somatostatin reduction and A D , combined with evidence suggesting a neurotransmitter role for somatostatin t,6,t° has given rise to the suggestion that somatostatin may be involved in cognitive function 3'7'20. Support for this position comes from Haroutunian et al. 7 who demonstrated that cysteamine (2-mercaptoethylamine), a compound that produces a rapid, reversible depletion of central somatostatin 15, produced a significant impairment of passive avoidance (PA) retention when administered (s.c.) immediately after P A acquisition in rats. In a similar experiment, rats given intracerebroventricular (i.c.v,) infusions of cysteamine administered once each day for 4 consecutive days showed a significant deficit in P A retention 3. These two studies have been
interpreted to suggest that depletion of somatostatin results in cognitive deficits. Additional support for this hypothesis comes from studies in which treatment with somatostatin i.c.v, prevented electroconvulsive shock (ECS)-induced retrograde amnesia of a P A response t9'2° and delayed the extinction of an active avoidance response in rats t9'2°. In addition to evaluating the effects of a single administration of different doses of cysteamine on PA retention, Haroutunian et al. 7 studied the time course of cysteamine effects on cortical somatostatin and the non-selective effects of different doses of cysteamine on catecholamines. Using a 150 mg/kg s.c. dose of cysteamine, somatostatin depletion was first observed at 10 min postinjection with maximum depletion occurring within 4 h and returning to near control values within 3 days. Cysteamine in doses ranging from 100 to 250 mg/kg s.c. resulted in depletion of cortical norepinephrine and increased cortical dopamine levels 7. Doses below 100 mg/kg s.c., however, depleted somatostatin without effects on nor-
Correspondence: V.J. DeNoble, E.I. duPont de Nemours & Co., Inc., Medical Products Department, Experimental Station, Bldg. 400, P.O. Box 80400, Wilmington, DE 19880-0400, U.S.A.
0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
43 epinephrine and dopamine. Based on these data, a series of doses of cysteamine were used in the present experiment which were selective for somatostatin depletion or involved alterations in norepinephrine and dopamine levels. In addition, based on the time
plastic floor; the other compartment, made of black plastic, measured 30.5 (L) x 20.3 (H) x 21.5 (W) cm and had a floor made of 4-mm stainless steel rods spaced 1.2 cm apart. A Coulbourn Instruments Grid Floor Shocker was connected to the steel rods and
course of somatostatin depletion 7, the effects of repeated cysteamine administration were also investigated. In the previously mentioned experiments, the effects of altered activity in the somatostatinergic system were evaluated using shock-motivated tasks that measured either single trial avoidance learning or multiple trial active avoidance learning. The 3 experiments described here were designed to: first, replicate previously described impairments of PA retention following cysteamine-induced somatostatin depletion; second, to determine if cysteamine's effects on PA retention can be manipulated by altering acquisition conditions; and third, to characterize the effects of repeated cysteamine administration on acquisition and maintenance of two operant conditioning tasks.
provided a scrambled footshock. The clear compartment was illuminated with a lamp containing a 60-W incandescent bulb placed 60 cm above the floor. The two compartments were separated by a solenoid-operated slide door (Lafayette Instrument Co., Lafayette, IN). A Coulbourn Instruments Electronic Counter recorded acquisition and retention latencies and was activated by the opening or closing of the slide door.
MATERIALS AND METHODS
Experiment 1: passive avoidance retention The effects of cysteamine administration on passive avoidance acquisition were examined as a function of the number of exposures to the unconditioned stimulus (US).
Subjects Experimentally naive male Sprague-Dawley rats (n = 120) 100-125 days old (Charles River Breeding Laboratories, Kingston, NY), and weighing between 150 and 190 g were used. The animals were housed 4 per cage (45.0 L x 20.0 W x 26.0 H cm) with free access to food and water. They were maintained on a 12-h light/dark cycle (lights on 06.00 h) and at room temperature of 22 + 1 °C with a relative humidity of 50 + 10%.
Procedure Passive avoidance training began by placing the rat into the clear compartment and following a 10-s delay, the slide door was raised. Once the rat moved completely into the dark compartment (all 4 paws on the shock grid floor), the slide door was lowered and after a 10-s delay, a 1.0-mA shock was applied to the grid floor for 3 s (single-shock procedure). In some experiments, the rats were given a second 3-s shock 10 s after the first one (double shock procedure). The rats were immediately removed from the dark compartment after receiving the final shock and returned to their home cages. Rats not entering the dark compartment within 90 s were removed from the study. A retention test was performed 24 h later. It proceeded in the same manner as the training session except no shock was applied to the grid floor when the rats entered the dark compartment. During the retention test, the rats were provided access to the dark compartment for 300 s. Cysteamine at 0, 50, 100, 150 and 200 mg/kg s.c. was administered one hour prior to the training session. The number of rats that entered the dark compartment within 300 s was determined for each drug group and compared to that of the vehicle-treated group using the 2"2statistic.
Experiment 2: delayed spatial alternation (DSA ) Apparatus The experimental sessions were conducted in a two-compartment passive avoidance box: one compartment, made of clear plastic, measured 21 (L) x 24.5 (H) × 17 (W) cm and had a perforated, clear
To determine the generality of the cysteamine-induced PA deficit, the effects of chronic somatostatin depletion on a repetitive learning test were evaluated.
44
Subjects Fourteen experimentally naive male SpragueDawley rats 90-120 days old and weighing between 250 and 290 g were used. Each rat was individually housed and was allowed food continuously for 3 weeks, during which time body weights were recorded daily. The mean weights were calculated from the last 3 days of the 3-week period, after which the rats were reduced to and maintained at 85% of their free feeding weights. These weights were adjusted every two weeks to control for normal growth.
Apparatus Twelve identical Coulbourn modular operant chamber cages placed in sound-attenuating cubicles were used. Located at one end of the operant chamber was a pellet receptacle, two cue lights, a speaker, a house light, and two response levers; one lever was positioned to the right, and the other to the left on the test panel with a cue light located immediately above each lever. White noise was present, and an exhaust fan provided ventilation. The DSA acquisition procedure consisted of two parts: preliminary training sessions and alternation training sessions. Sessions lasted for 100 reinforced trials, and the rats received daily training sessions, 5 days each week.
Procedure Preliminary training. Each rat was trained to leverpress for a 45-rag food pellet (BioServe Dustless Precision Pellets) with one of the two levers in the operant chamber active. Half of the animals received initial training on the right lever and the other half received training on the left lever. When 100 food pellets were obtained under a Fixed Ratio 5 schedule (FR 5) during two consecutive sessions, the position of the active and inactive levers was reversed. After 100 reinforcements were obtained on the newly positioned lever, during two consecutive sessions, both levers were made active in the operant chamber and the animal proceeded with alternation training. Alternation training. Each rat was required to press alternate levers on successive trials with an intertrial interval (ITI) of I s. During the ITI, the house light was extinguished. When the ITI ended, the house light was re-illuminated. In the first 3 training sessions a cue light was illuminated over the correct lever to establish the alternation behavior after
which the cue light was removed from all further experimental sessions. Completion of the FR 5 on the correct lever terminated the trial and produced a food reinforcement. Presses on either lever during the ITI reset the interval to zero. The effect of lengthening the ITI duration was then examined by increasing the delay between trials. ITI intervals of 1, 2, 4 and 8 s were tested in ascending order and each ITI remained constant within each session. Biweekly (Monday and Wednesday), the rats were dosed with vehicle (n = 4), 50 or 150 mg/kg s.c. of cysteamine (n = 5 per dose) 60 rain before testing. The mean percent correct bar presses across delays as a function of the treatment condition was analyzed using a repeated-measures analysis of variance.
Experiment 3: signaled extinction discrimination (SED) Chronic cysteamine administration was evaluated for its ability to disrupt the DSA task in which correct responding is dependent upon spatial memory. In this study, a non-spatial memory task was used and the effects of chronic dosing of cysteamine on the acquisition and reversal of a signaled extinction discrimination (SED) test were evaluated.
Subjects and Apparatus Twelve experimentally naive male Sprague-Dawley rats were maintained under conditions previously described in the DSA task, and tested in the same apparatus as that used in the previous experiment.
Procedure All rats were trained to lever-press under an FR 10 schedule for food presentation on the left lever for 4 consecutive days. The rats were then assigned to one of 3 drug treatment groups and the schedule controlling reinforcement was changed to an SED. Under the SED schedule, the house light served as a discriminative stimulus (SD) and signaled the availability of 10 food pellets, each delivered after completion of an FR 10. After completion of this sequence, the house light was extinguished, signaling the onset of a 60-s period in which responses were not reinforced (extinction period). At the end of the 60-s period, the house light was illuminated and the FR 10 food avail-
45
CYSTEAMINE IMPAIRED DOUBLE-SHOCK PASSIVE AVOIDANCE
CYSTEAMINE IMPAIRED SINGLE-SHOCK PASSIVE AVOIDANCE
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Fig. 1. Effects of cysteamine administration 60 min before training on a single footshock passive avoidance retention task. The median retention latency in seconds (300 s max) to enter the dark compartment is shown as a function of drug dose. Each bar is a median obtained from 12 rats. Cysteamine significantly impaired retention latencies at 50, 100, 150 and 200 mg/kg s.c. doses, compared to saline-treated controls (** P < 0.01, Z2-test). ability component was again in effect. The two conditions were repeated 10 times providing 10 food availability periods and 10 extinction periods. After 13 sessions of SED acquisition, the conditions signaling food and extinction periods were reversed (i.e. house light on for extinction, house light off for food availability). Biweekly (Monday and Wednesday), the rats were dosed with vehicle (n = 4), 50 or 150 mg/kg s.c. of cysteamine (n = 4 per dose) 60 min before testing. For each test session, the ratio of correct (presses during the food access periods) to incorrect responses (presses during the extinction periods) was calculated and analyzed with a repeated-measures analysis of variance.
Drug preparation and administration Cysteamine (Sigma Chemical Co.) was administered s.c. in a volume of 1 ml/kg of body weight at doses of 0, 50, 100, 150 and 200 mg/kg. All doses were calculated as the salt. Cysteamine solutions were adjusted to a p H of 7.2 + 0.2 by the addition of 1 N N a O H . Saline served as the vehicle and all drug solutions were prepared immediately prior to use. RESULTS
Immediately following cysteamine injections, and
Saline
50 100 150 200 Dose of Cystearnlne (mg/kg s.c.)
Fig. 2. Summary of behavioral data from the double footshock
passive avoidance task. The median retention latency in seconds (300 s max) to enter the dark compartment is shown as a function of drug dose. Each bar is a median obtained from 12 rats. Cysteamine significantly impaired retention latencies at 150 and 200 mg/kg s.c. doses, compared to saline-treated controls (** P < 0.01, ~-test). continuing for a maximum of 20 min, the rats demonstrated unusual locomotor behavior characterized by backwards walking and mild hyperactivity. These behavioral effects were dose-dependent since, at the lowest dose of 50 mg/kg, these behaviors were only marginally perceptible. As the dose increased towards the highest doses of 150 and 200 mg/kg, these behaviors became quite profound. Within 40 min of the injection, however, all rats appeared completely normal. The highest doses of cysteamine (150 and 200 mg/kg) also produced visible tissue irritation characterized by redness and swelling at the injection site. No other effects were observed and all rats were healthy at the completion of their respective studies.
Passive avoidance retention The median latency to enter the dark compartment of the PA apparatus on the training day was 13.9 and 14.3 s for vehicle- and cysteamine-treated groups, respectively. In addition, none of the rats reached the training day cut-off of 90 s, suggesting that cysteamine did not produce a motoric impairment when administered 60 min before training. During the retention tests 24 h later, median entry latencies for the rats treated with saline reached the maximum of 300 s. Cysteamine, at all doses tested, significantly decreased the entry latencies from 300 s (saline) to 51, 34, 165 and 30 s (for the 50, 100, 150 and 200 mg/kg s.c. dosed groups, respectively, P < 0.01 [Fig. 1]).
46 CYSTEAMINE DID NOT IMPAIR DELAYED SPATIAL ALTERNATION I O0
CYSTEAMINE
Saline 50 mg/kg s . c 150 mg/kg s.c
• 90
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Fig. 3. The mean percent correct lever presses is shown as a function of test sessions for the delayed spatial alternation task. The rats were dosed biweekly with cysteamine or vehicle 60 rain before testing. Each point is a mean obtained from 5 rats for the cysteamine treatment groups or 4 rats for the vehicle group. A cue light appeared over the correct lever for the first 3 test sessions only. The intertrial interval was increased from 1 to 2, 4 and 8 s, respectively. There were no significant differences in performance between the rats dosed with 50 or 150 mg/kg s.c. cysteamine and saline, P > 0.05. The cysteamine-induced decrease in retention latency was shown to be sensitive to behavioral manipulation since, using the double shock procedure, cysteamine p r o d u c e d retention deficits only at the highest doses (125 and 20 s, respectively, at 150 and 200 mg/kg s.c.), whereas lower doses (50 and 100 mg/kg s.c.) that were effective in producing amnesia in the single shock p r o c e d u r e did not p r o d u c e a retention deficit in the double shock p r o c e d u r e (Fig. 2).
Delayed spatial alternation W i t h o u t vehicle or cysteamine p r e t r e a t m e n t , all animals acquired and maintained lever pressing under the F R 5 schedule of food presentation during the preliminary training sessions. During the first 3 sessions of the alternation testing procedure, when the correct lever was signaled by a cue light, the mean percent correct responding increased from below 50% to 85% for all groups (Fig. 3). R e m o v a l of the cue light p r o d u c e d a small decrease in correct responding for all groups. However, across all ITIs (1, 2, 4 and 8 s, respectively) there were no significant differences in the performance of D S A task between saline and cysteamine treated groups (P > 0.05).
Signaled extinction discrimination Prior to vehicle or cysteamine p r e t r e a t m e n t , all
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Reversal
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Fig. 4. The mean ratio of correct to incorrect lever presses is shown as a function of test sessions for the signaled extinction discrimination tests. The rats were dosed biweekly with cysteamine or vehicle 60 min before testing. Each point is a mean obtained from 4 rats. After the thirteenth test session, cues signaling the extinction and reinforced components were reversed. There were no significant differences in performance between the rats dosed with 50 or 150 mg/kg s.c. cysteamine and saline during either the acquisition (Sessions 1-13) or reversal phase (Sessions 14-23), P > 0.05.
animals initiated and maintained responding under the F R 10 schedule of food presentations. Responding during extinction periods during the first 5 sessions was initially greater than or equal to the number of responses that occurred during the food access periods (Fig. 4; sessions 1-5). However, as training continued, the number of responses during the extinction periods fell to less than half of those that occurred during the food access periods, demonstrating the acquisition of the lever press discrimination. Reversal of the cues signaling extinction/food periods p r o d u c e d an immediate increase in extinction responding which decreased only slightly over the next 10 sessions (Fig. 4). There were no significant differences in the acquisition of the S E D task, or the reversal, between saline- and cysteamine-treated groups ( e > 0.05). DISCUSSION The results of the 3 experiments taken together demonstrate that systemic administration of cysteamine leads to a dose-dependent deficit in P A retention but does not effect the acquisition or performance of either the D S A or a SED task. The effect of
47 post-training administration of cysteamine on PA retention has been previously demonstrated and correlated with doses that selectively deplete cortical somatostatin 7. In the present study, similar doses (50-200 mg/kg s.c.) impaired PA retention when administered one hour prior to the training session in the single shock procedure. This effect, however, was not dose-related with the lowest and highest dose producing a retention deficit (Fig. 1). Nevertheless, the results of the single shock PA experiment support the notion that somatostatin has a role in the mechanisms which underlie learning and memory. However, the results of the double shock PA procedure, combined with the failure of biweekly injections of 50 and 150 mg of cysteamine to produce a disruption of the acquisition or performance of both the DSA and SED tasks, suggest that the role of somatostatin may be more limited than previously thought. The cysteamine-induced disruption of PA retention was shown to be sensitive to behavioral manipulation since the use of a double footshock shifted the dose-effect curve to the right (Fig. 1 vs Fig. 2). Doses of 50 and 100 mg/kg s.c. of cysteamine have been used in other studies 7,14As and are shown to be potent depletors of cortical somatostatin and result in a PA deficit. However, by doubling the exposure to the noxious stimulus (UCS) and strengthening the association between UCS and the conditioned stimulus in the present experiment, the behavioral effects of cysteamine administration were attenuated. A possible biochemical explanation of these findings is that the low doses of cysteamine (50 and 100 mg/kg s.c.) have selective effects on cortical somatostatin, while the higher doses (150 and 200 mg/kg s.c.) deplete somatostatin but also induce a decrease in cortical norepinephrine and increase cortical dopamine levels7; therefore, the behavioral deficit in the second PA experiment may result from both cysteamine-induced changes in cortical catecholamines as well as a decrease in somatostatin. The results may not be generalizable to other pharmacological treatments that produce amnesia since, using an identical procedure, the amnestic effects of scopolamine on PA retention, at doses ranging from 0.5 to 2.0 mg/kg s.c., were not affected by exposing the animal to a double footshock (Strek and DeNoble, unpublished observation). These data suggest that the effects of cysteamine PA behavior are more subject to situational
variables than the amnesia produced by anticholinergic drugs. Spatial alternation tasks have been shown to be sensitive to both pharmacological and physiological intervention. For example, scopolamine impairs the accuracy of performance of a variable-interval spatial alternation task 8;9. Similarly, (+)-amphetamine administration produces a disruption of both delayed spatial alternation and delayed-match-to-sample performance in rats 11. Using a different approach, it has been shown that hippocampal lesions disrupt single alternation behavior in rats 21. The SED task has been similarly shown to be very sensitive to pharmacologic manipulation 5. All of these studies demonstrate that alternation and discrimination behavior is very sensitive to pharmacological and physiological manipulation. Therefore, the failure of cysteamine to alter acquisition or performance in these tests cannot be attributed to a lack of sensitivity of the behavioral task, but instead suggests that cysteamine-induced changes in central nervous system function does not alter the associative processes underlying complex operant conditioning tests. One possible explanation for the differential effects of cysteamine on single trial learning vs multiple trial operant conditioning comes from Haroutunian et al. 7. These authors have suggested that the cysteamine-induced depletion of somatostatin may be less critical to the learning and memory process than the acute somatostatin-releasing properties which occur 15-60 min after the cysteamine injection (150 mg/kg s.c.) 7. If this interpretation is correct, then the results of the present study with passive avoidance would result from the active release of cortical somatostatin, and the failure of cysteamine to alter DSA or SED could be interpreted as a result of the chronic administration which would maintain decreased cortical levels of somatostatin without sustained somatostatin release. Additional experiments will be required to precisely delineate the role of somatostatin release and subsequent depletion on different types of cognitive processes. ACKNOWLEDGEMENTS The authors thank Dr. W.K. Schmidt and Ms. L.C. Johnson for critically reviewing this manuscript and Mrs. D.L. Gorman for her typing assistance.
48 REFERENCES 1 Bakhit, C., Benoit, R. and Bloom, F.E., Effects of cysteamine on pro-somatostatin-related peptides, Regul. Pept., 6 (1983) 169-177. 2 Bakhit, C. and Swerdlow, N.R., Behavioral changes following central injection of cysteamine in rats, Soc. Neurosci. Abstr., 10 (1984) 182. 3 Bakhit, C. and Swerdlow, N.R., Behavioral changes following central injection of cysteamine in rats, Brain Research, 365 (1986) 159-163. 4 Beal, M.F., Mazurek, M.F., Tran, V.T., Chattha, G., Bird, E.D. and Martin, J.B., Reduced numbers of somatostatin receptors in the cerebral cortex in Alzheimer's disease, Science, 229 (1985) 289-291. 5 Cook, L., Effects of drugs on operant conditioning. In H. Steinberg, A.V.S. de Reuck and J. Knight (Eds.), Ciba Foundation Symposium jointly with the Co-ordinating Committee for Symposia on Drug Action on Animal Behaviour and Drug Action, 1964, pp. 23-40. 6 Epelbaum, J., Brazeau, P., Tsang, D., Brower, J. and Martin, J.B., Subcellular distribution of radioimmunoassay somatostatin in rat brain, Brain Research, 126 (1977) 309-323. 7 Haroutunian, V., Mantin, R., Campbell, G.A., Tsuboyama, G.K. and Davis K.L., Cysteamine-induced depletion of central somatostatin-like immunoactivity: effects on behavior, learning, memory and brain neurochemistry, Brain Research, 403 (1987) 234-242. 8 Heise, G.A., Conner, R. and Martin, R.A., Effects of scopolamine on variable intertrial interval spatial alternation and memory in the rat, Psychopharmacology, 49 (1976) 131-137. 9 Heise, G.A., Hrabrich, B., Lilie, N.L. and Martin, R.A., Scopolamine effects on delayed spatial alternation in the rat. Pharmacol. Biochem. Behav., 3 (1975) 993-1002. 10 Iversen, L.L., Iversen, S.D., Bloom, F.E., Douglas, C., Brown, M. and Vale, M., Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature (Lond.), 273 (1978) 161-164. 11 Kesner, R.P., Bierley, R.A. and Pebbles, P., Short-term
memory: the role of d-amphetamine, Pharmacol. Biochem. Behav., 15 (1981) 673-676. 12 Matejcek, M. and Devos, J.E., Selected methods of quantitative EEG analysis and their applications in psychotropic drug research. In P. Kellaway and I. Petersen Eds.), Quantitative Analytic Studies in Epilepsy, Raven, New York, 1976, pp. 183-205. 13 Olpe, H.R., Balcar, V.J., Bittiger, H. and Sieber, P., Central action of somatostatin, Eur. J. Pharmacol., 63 (1980) 127-133. 14 Palkovitis, M., Brownstein, M.J., Eiden, L.E., Beinfeld, M.C., Russell, J., Arimura, A. and Szabo, S., Selective depletion of somatostatin in rat brain by cysteamine, Brain Research, 240 (1982) 178-180. 15 Sagar, S.M., Landry, D., Millard, W.J., Badger, T.M., Arnold, M.A. and Martin, J.B., Depletion of somatostatinlike immunoreactivity in the rat central nervous system by cysteamine, J. Neurosci., 2 (1982) 225-231. 16 Sorensen, K.V., Somatostatin: localization and distribution in the cortex and subcortical white matter of human brain, Neuroscience, 7 (1982) 1227-1232. 17 Tamminga, C.A., Foster, N.L., Fedio, P., Bird, E.D. and Chase, T.N., Alzheimer's disease: low cerebral somatostatin levels correlate with impaired cognitive function and cortical metabolism, Neurology, 37 (1987) 161-165. 18 Vale, W., Ling, N., Rivier, J., Villarreal, J., Rivier, C., Douglas, C. and Brown, M., Anatomic and phylogenetic distribution of somatostatin, Metabolism, Suppl. 1, 25 (1976) 1491-1494. 19 Vescsei, L., Bollok, I. and Telegdy, G., Intracerebroventricular somatostatin attenuates electroconvulsive shockinduced amnesia in rats, Peptides, 4 (1983) 293-295. 20 Vescsei, L., Kiraly, C., Bollok, I., Nagy, A., Varga, J., Penke, B. and Telegdy, G., Comparative studies with somatostatin and cysteamine in different behavioral tests in rats, Pharmacol. Biochem, Behav., 21 (1984) 833-837. 21 Walker, D.W. and Means, L.W, Single-alternation performance in rats with hippocampal lesions: disruption by an irrelevant task interposed during the intertrial interval, Behay. Biol., 9 (1973) 93-104.
Brain Research, 482 (1989) 42-48
Elsevier BRE 143(t4
Cysteamine-induced depletion of somatostatin produces differential cognitive deficits in rats Victor J. DeNoble, David J. Hepler and Robert A. Barto E.I. duPont de Nemours & Co., Inc., Medical Products Department, Wilmington, DE 19880-0400 (U.S.A.)
(Accepted 16 August 1988) Key words: Somatostatin; Cysteamine; Behavior; Learning; Memory; Alzheimer's disease
The effects of a variety of doses of systemically administered cysteamine (a somatostatin depletor) were studied on step-through passive avoidance retention, as well as acquisition and performance of a delayed spatial alternation task and a signaled extinction discrimination task in rats. Retention of single trial passive avoidance was significantly reduced by a pretraining (60-min) dose of cysteamine at 50, 100, 150 and 200 mg/kg s.c. This effect was shown to be sensitive to behavioral manipulation; in a second experiment, a retention deficit was found only at the two highest doses tested (150 and 200 mg/kg s.c.) after a second exposure to the footshock. In the operant conditioning studies, biweekly injections (Monday and Wednesday) of cysteamine administered one hour before testing produced no statistically significant changes in acquisition or performance of either the delayed spatial alternation or the signaled discrimination task. The results of these series of experiments suggest that active somatostatin release or chronic somatostatin depletion may selectively affect performance maintained by different behavioral procedures. INTRODUCTION A m o n g the biological markers of post-mortem analysis of Alzheimer's Disease (AD) is a reduction of cortical and hippocampal somatostatin-like immunoactivity 4,13,I6-18. The close correlation of central somatostatin reduction and A D , combined with evidence suggesting a neurotransmitter role for somatostatin t,6,t° has given rise to the suggestion that somatostatin may be involved in cognitive function 3'7'20. Support for this position comes from Haroutunian et al. 7 who demonstrated that cysteamine (2-mercaptoethylamine), a compound that produces a rapid, reversible depletion of central somatostatin 15, produced a significant impairment of passive avoidance (PA) retention when administered (s.c.) immediately after P A acquisition in rats. In a similar experiment, rats given intracerebroventricular (i.c.v,) infusions of cysteamine administered once each day for 4 consecutive days showed a significant deficit in P A retention 3. These two studies have been
interpreted to suggest that depletion of somatostatin results in cognitive deficits. Additional support for this hypothesis comes from studies in which treatment with somatostatin i.c.v, prevented electroconvulsive shock (ECS)-induced retrograde amnesia of a P A response t9'2° and delayed the extinction of an active avoidance response in rats t9'2°. In addition to evaluating the effects of a single administration of different doses of cysteamine on PA retention, Haroutunian et al. 7 studied the time course of cysteamine effects on cortical somatostatin and the non-selective effects of different doses of cysteamine on catecholamines. Using a 150 mg/kg s.c. dose of cysteamine, somatostatin depletion was first observed at 10 min postinjection with maximum depletion occurring within 4 h and returning to near control values within 3 days. Cysteamine in doses ranging from 100 to 250 mg/kg s.c. resulted in depletion of cortical norepinephrine and increased cortical dopamine levels 7. Doses below 100 mg/kg s.c., however, depleted somatostatin without effects on nor-
Correspondence: V.J. DeNoble, E.I. duPont de Nemours & Co., Inc., Medical Products Department, Experimental Station, Bldg. 400, P.O. Box 80400, Wilmington, DE 19880-0400, U.S.A.
0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
43 epinephrine and dopamine. Based on these data, a series of doses of cysteamine were used in the present experiment which were selective for somatostatin depletion or involved alterations in norepinephrine and dopamine levels. In addition, based on the time
plastic floor; the other compartment, made of black plastic, measured 30.5 (L) x 20.3 (H) x 21.5 (W) cm and had a floor made of 4-mm stainless steel rods spaced 1.2 cm apart. A Coulbourn Instruments Grid Floor Shocker was connected to the steel rods and
course of somatostatin depletion 7, the effects of repeated cysteamine administration were also investigated. In the previously mentioned experiments, the effects of altered activity in the somatostatinergic system were evaluated using shock-motivated tasks that measured either single trial avoidance learning or multiple trial active avoidance learning. The 3 experiments described here were designed to: first, replicate previously described impairments of PA retention following cysteamine-induced somatostatin depletion; second, to determine if cysteamine's effects on PA retention can be manipulated by altering acquisition conditions; and third, to characterize the effects of repeated cysteamine administration on acquisition and maintenance of two operant conditioning tasks.
provided a scrambled footshock. The clear compartment was illuminated with a lamp containing a 60-W incandescent bulb placed 60 cm above the floor. The two compartments were separated by a solenoid-operated slide door (Lafayette Instrument Co., Lafayette, IN). A Coulbourn Instruments Electronic Counter recorded acquisition and retention latencies and was activated by the opening or closing of the slide door.
MATERIALS AND METHODS
Experiment 1: passive avoidance retention The effects of cysteamine administration on passive avoidance acquisition were examined as a function of the number of exposures to the unconditioned stimulus (US).
Subjects Experimentally naive male Sprague-Dawley rats (n = 120) 100-125 days old (Charles River Breeding Laboratories, Kingston, NY), and weighing between 150 and 190 g were used. The animals were housed 4 per cage (45.0 L x 20.0 W x 26.0 H cm) with free access to food and water. They were maintained on a 12-h light/dark cycle (lights on 06.00 h) and at room temperature of 22 + 1 °C with a relative humidity of 50 + 10%.
Procedure Passive avoidance training began by placing the rat into the clear compartment and following a 10-s delay, the slide door was raised. Once the rat moved completely into the dark compartment (all 4 paws on the shock grid floor), the slide door was lowered and after a 10-s delay, a 1.0-mA shock was applied to the grid floor for 3 s (single-shock procedure). In some experiments, the rats were given a second 3-s shock 10 s after the first one (double shock procedure). The rats were immediately removed from the dark compartment after receiving the final shock and returned to their home cages. Rats not entering the dark compartment within 90 s were removed from the study. A retention test was performed 24 h later. It proceeded in the same manner as the training session except no shock was applied to the grid floor when the rats entered the dark compartment. During the retention test, the rats were provided access to the dark compartment for 300 s. Cysteamine at 0, 50, 100, 150 and 200 mg/kg s.c. was administered one hour prior to the training session. The number of rats that entered the dark compartment within 300 s was determined for each drug group and compared to that of the vehicle-treated group using the 2"2statistic.
Experiment 2: delayed spatial alternation (DSA ) Apparatus The experimental sessions were conducted in a two-compartment passive avoidance box: one compartment, made of clear plastic, measured 21 (L) x 24.5 (H) × 17 (W) cm and had a perforated, clear
To determine the generality of the cysteamine-induced PA deficit, the effects of chronic somatostatin depletion on a repetitive learning test were evaluated.
44
Subjects Fourteen experimentally naive male SpragueDawley rats 90-120 days old and weighing between 250 and 290 g were used. Each rat was individually housed and was allowed food continuously for 3 weeks, during which time body weights were recorded daily. The mean weights were calculated from the last 3 days of the 3-week period, after which the rats were reduced to and maintained at 85% of their free feeding weights. These weights were adjusted every two weeks to control for normal growth.
Apparatus Twelve identical Coulbourn modular operant chamber cages placed in sound-attenuating cubicles were used. Located at one end of the operant chamber was a pellet receptacle, two cue lights, a speaker, a house light, and two response levers; one lever was positioned to the right, and the other to the left on the test panel with a cue light located immediately above each lever. White noise was present, and an exhaust fan provided ventilation. The DSA acquisition procedure consisted of two parts: preliminary training sessions and alternation training sessions. Sessions lasted for 100 reinforced trials, and the rats received daily training sessions, 5 days each week.
Procedure Preliminary training. Each rat was trained to leverpress for a 45-rag food pellet (BioServe Dustless Precision Pellets) with one of the two levers in the operant chamber active. Half of the animals received initial training on the right lever and the other half received training on the left lever. When 100 food pellets were obtained under a Fixed Ratio 5 schedule (FR 5) during two consecutive sessions, the position of the active and inactive levers was reversed. After 100 reinforcements were obtained on the newly positioned lever, during two consecutive sessions, both levers were made active in the operant chamber and the animal proceeded with alternation training. Alternation training. Each rat was required to press alternate levers on successive trials with an intertrial interval (ITI) of I s. During the ITI, the house light was extinguished. When the ITI ended, the house light was re-illuminated. In the first 3 training sessions a cue light was illuminated over the correct lever to establish the alternation behavior after
which the cue light was removed from all further experimental sessions. Completion of the FR 5 on the correct lever terminated the trial and produced a food reinforcement. Presses on either lever during the ITI reset the interval to zero. The effect of lengthening the ITI duration was then examined by increasing the delay between trials. ITI intervals of 1, 2, 4 and 8 s were tested in ascending order and each ITI remained constant within each session. Biweekly (Monday and Wednesday), the rats were dosed with vehicle (n = 4), 50 or 150 mg/kg s.c. of cysteamine (n = 5 per dose) 60 rain before testing. The mean percent correct bar presses across delays as a function of the treatment condition was analyzed using a repeated-measures analysis of variance.
Experiment 3: signaled extinction discrimination (SED) Chronic cysteamine administration was evaluated for its ability to disrupt the DSA task in which correct responding is dependent upon spatial memory. In this study, a non-spatial memory task was used and the effects of chronic dosing of cysteamine on the acquisition and reversal of a signaled extinction discrimination (SED) test were evaluated.
Subjects and Apparatus Twelve experimentally naive male Sprague-Dawley rats were maintained under conditions previously described in the DSA task, and tested in the same apparatus as that used in the previous experiment.
Procedure All rats were trained to lever-press under an FR 10 schedule for food presentation on the left lever for 4 consecutive days. The rats were then assigned to one of 3 drug treatment groups and the schedule controlling reinforcement was changed to an SED. Under the SED schedule, the house light served as a discriminative stimulus (SD) and signaled the availability of 10 food pellets, each delivered after completion of an FR 10. After completion of this sequence, the house light was extinguished, signaling the onset of a 60-s period in which responses were not reinforced (extinction period). At the end of the 60-s period, the house light was illuminated and the FR 10 food avail-
45
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Fig. 1. Effects of cysteamine administration 60 min before training on a single footshock passive avoidance retention task. The median retention latency in seconds (300 s max) to enter the dark compartment is shown as a function of drug dose. Each bar is a median obtained from 12 rats. Cysteamine significantly impaired retention latencies at 50, 100, 150 and 200 mg/kg s.c. doses, compared to saline-treated controls (** P < 0.01, Z2-test). ability component was again in effect. The two conditions were repeated 10 times providing 10 food availability periods and 10 extinction periods. After 13 sessions of SED acquisition, the conditions signaling food and extinction periods were reversed (i.e. house light on for extinction, house light off for food availability). Biweekly (Monday and Wednesday), the rats were dosed with vehicle (n = 4), 50 or 150 mg/kg s.c. of cysteamine (n = 4 per dose) 60 min before testing. For each test session, the ratio of correct (presses during the food access periods) to incorrect responses (presses during the extinction periods) was calculated and analyzed with a repeated-measures analysis of variance.
Drug preparation and administration Cysteamine (Sigma Chemical Co.) was administered s.c. in a volume of 1 ml/kg of body weight at doses of 0, 50, 100, 150 and 200 mg/kg. All doses were calculated as the salt. Cysteamine solutions were adjusted to a p H of 7.2 + 0.2 by the addition of 1 N N a O H . Saline served as the vehicle and all drug solutions were prepared immediately prior to use. RESULTS
Immediately following cysteamine injections, and
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Fig. 2. Summary of behavioral data from the double footshock
passive avoidance task. The median retention latency in seconds (300 s max) to enter the dark compartment is shown as a function of drug dose. Each bar is a median obtained from 12 rats. Cysteamine significantly impaired retention latencies at 150 and 200 mg/kg s.c. doses, compared to saline-treated controls (** P < 0.01, ~-test). continuing for a maximum of 20 min, the rats demonstrated unusual locomotor behavior characterized by backwards walking and mild hyperactivity. These behavioral effects were dose-dependent since, at the lowest dose of 50 mg/kg, these behaviors were only marginally perceptible. As the dose increased towards the highest doses of 150 and 200 mg/kg, these behaviors became quite profound. Within 40 min of the injection, however, all rats appeared completely normal. The highest doses of cysteamine (150 and 200 mg/kg) also produced visible tissue irritation characterized by redness and swelling at the injection site. No other effects were observed and all rats were healthy at the completion of their respective studies.
Passive avoidance retention The median latency to enter the dark compartment of the PA apparatus on the training day was 13.9 and 14.3 s for vehicle- and cysteamine-treated groups, respectively. In addition, none of the rats reached the training day cut-off of 90 s, suggesting that cysteamine did not produce a motoric impairment when administered 60 min before training. During the retention tests 24 h later, median entry latencies for the rats treated with saline reached the maximum of 300 s. Cysteamine, at all doses tested, significantly decreased the entry latencies from 300 s (saline) to 51, 34, 165 and 30 s (for the 50, 100, 150 and 200 mg/kg s.c. dosed groups, respectively, P < 0.01 [Fig. 1]).
46 CYSTEAMINE DID NOT IMPAIR DELAYED SPATIAL ALTERNATION I O0
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Fig. 3. The mean percent correct lever presses is shown as a function of test sessions for the delayed spatial alternation task. The rats were dosed biweekly with cysteamine or vehicle 60 rain before testing. Each point is a mean obtained from 5 rats for the cysteamine treatment groups or 4 rats for the vehicle group. A cue light appeared over the correct lever for the first 3 test sessions only. The intertrial interval was increased from 1 to 2, 4 and 8 s, respectively. There were no significant differences in performance between the rats dosed with 50 or 150 mg/kg s.c. cysteamine and saline, P > 0.05. The cysteamine-induced decrease in retention latency was shown to be sensitive to behavioral manipulation since, using the double shock procedure, cysteamine p r o d u c e d retention deficits only at the highest doses (125 and 20 s, respectively, at 150 and 200 mg/kg s.c.), whereas lower doses (50 and 100 mg/kg s.c.) that were effective in producing amnesia in the single shock p r o c e d u r e did not p r o d u c e a retention deficit in the double shock p r o c e d u r e (Fig. 2).
Delayed spatial alternation W i t h o u t vehicle or cysteamine p r e t r e a t m e n t , all animals acquired and maintained lever pressing under the F R 5 schedule of food presentation during the preliminary training sessions. During the first 3 sessions of the alternation testing procedure, when the correct lever was signaled by a cue light, the mean percent correct responding increased from below 50% to 85% for all groups (Fig. 3). R e m o v a l of the cue light p r o d u c e d a small decrease in correct responding for all groups. However, across all ITIs (1, 2, 4 and 8 s, respectively) there were no significant differences in the performance of D S A task between saline and cysteamine treated groups (P > 0.05).
Signaled extinction discrimination Prior to vehicle or cysteamine p r e t r e a t m e n t , all
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Fig. 4. The mean ratio of correct to incorrect lever presses is shown as a function of test sessions for the signaled extinction discrimination tests. The rats were dosed biweekly with cysteamine or vehicle 60 min before testing. Each point is a mean obtained from 4 rats. After the thirteenth test session, cues signaling the extinction and reinforced components were reversed. There were no significant differences in performance between the rats dosed with 50 or 150 mg/kg s.c. cysteamine and saline during either the acquisition (Sessions 1-13) or reversal phase (Sessions 14-23), P > 0.05.
animals initiated and maintained responding under the F R 10 schedule of food presentations. Responding during extinction periods during the first 5 sessions was initially greater than or equal to the number of responses that occurred during the food access periods (Fig. 4; sessions 1-5). However, as training continued, the number of responses during the extinction periods fell to less than half of those that occurred during the food access periods, demonstrating the acquisition of the lever press discrimination. Reversal of the cues signaling extinction/food periods p r o d u c e d an immediate increase in extinction responding which decreased only slightly over the next 10 sessions (Fig. 4). There were no significant differences in the acquisition of the S E D task, or the reversal, between saline- and cysteamine-treated groups ( e > 0.05). DISCUSSION The results of the 3 experiments taken together demonstrate that systemic administration of cysteamine leads to a dose-dependent deficit in P A retention but does not effect the acquisition or performance of either the D S A or a SED task. The effect of
47 post-training administration of cysteamine on PA retention has been previously demonstrated and correlated with doses that selectively deplete cortical somatostatin 7. In the present study, similar doses (50-200 mg/kg s.c.) impaired PA retention when administered one hour prior to the training session in the single shock procedure. This effect, however, was not dose-related with the lowest and highest dose producing a retention deficit (Fig. 1). Nevertheless, the results of the single shock PA experiment support the notion that somatostatin has a role in the mechanisms which underlie learning and memory. However, the results of the double shock PA procedure, combined with the failure of biweekly injections of 50 and 150 mg of cysteamine to produce a disruption of the acquisition or performance of both the DSA and SED tasks, suggest that the role of somatostatin may be more limited than previously thought. The cysteamine-induced disruption of PA retention was shown to be sensitive to behavioral manipulation since the use of a double footshock shifted the dose-effect curve to the right (Fig. 1 vs Fig. 2). Doses of 50 and 100 mg/kg s.c. of cysteamine have been used in other studies 7,14As and are shown to be potent depletors of cortical somatostatin and result in a PA deficit. However, by doubling the exposure to the noxious stimulus (UCS) and strengthening the association between UCS and the conditioned stimulus in the present experiment, the behavioral effects of cysteamine administration were attenuated. A possible biochemical explanation of these findings is that the low doses of cysteamine (50 and 100 mg/kg s.c.) have selective effects on cortical somatostatin, while the higher doses (150 and 200 mg/kg s.c.) deplete somatostatin but also induce a decrease in cortical norepinephrine and increase cortical dopamine levels7; therefore, the behavioral deficit in the second PA experiment may result from both cysteamine-induced changes in cortical catecholamines as well as a decrease in somatostatin. The results may not be generalizable to other pharmacological treatments that produce amnesia since, using an identical procedure, the amnestic effects of scopolamine on PA retention, at doses ranging from 0.5 to 2.0 mg/kg s.c., were not affected by exposing the animal to a double footshock (Strek and DeNoble, unpublished observation). These data suggest that the effects of cysteamine PA behavior are more subject to situational
variables than the amnesia produced by anticholinergic drugs. Spatial alternation tasks have been shown to be sensitive to both pharmacological and physiological intervention. For example, scopolamine impairs the accuracy of performance of a variable-interval spatial alternation task 8;9. Similarly, (+)-amphetamine administration produces a disruption of both delayed spatial alternation and delayed-match-to-sample performance in rats 11. Using a different approach, it has been shown that hippocampal lesions disrupt single alternation behavior in rats 21. The SED task has been similarly shown to be very sensitive to pharmacologic manipulation 5. All of these studies demonstrate that alternation and discrimination behavior is very sensitive to pharmacological and physiological manipulation. Therefore, the failure of cysteamine to alter acquisition or performance in these tests cannot be attributed to a lack of sensitivity of the behavioral task, but instead suggests that cysteamine-induced changes in central nervous system function does not alter the associative processes underlying complex operant conditioning tests. One possible explanation for the differential effects of cysteamine on single trial learning vs multiple trial operant conditioning comes from Haroutunian et al. 7. These authors have suggested that the cysteamine-induced depletion of somatostatin may be less critical to the learning and memory process than the acute somatostatin-releasing properties which occur 15-60 min after the cysteamine injection (150 mg/kg s.c.) 7. If this interpretation is correct, then the results of the present study with passive avoidance would result from the active release of cortical somatostatin, and the failure of cysteamine to alter DSA or SED could be interpreted as a result of the chronic administration which would maintain decreased cortical levels of somatostatin without sustained somatostatin release. Additional experiments will be required to precisely delineate the role of somatostatin release and subsequent depletion on different types of cognitive processes. ACKNOWLEDGEMENTS The authors thank Dr. W.K. Schmidt and Ms. L.C. Johnson for critically reviewing this manuscript and Mrs. D.L. Gorman for her typing assistance.
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