BEHAVIORAL BIOLOGY 24, 349-363 (1978)
A Factor Isolated from Datura stramonium That Affects Learning Retention in Mice A . UNGERER,
F. SCHUBER, AND R. CHAUVIN ~
Laboratoire de Psychophysiologie, 7, rue de l' Universitb, 67000, Strasbourg, Institut de Botanique, 8, rue Goethe, 67000, Strasbourg, and Station d'Ethologie Exp~rimentale, "Les Sources," Mittainville, 78120, Rambouillet, France A water-soluble fraction, devoid of alkaloids, was isolated from Datura stramonium (leaves and seeds). This fraction greatly impaired retention of (i) discriminated avoidance learning, (ii) two-way active avoidance learning, and (iii) a food-reinforced instrumental learning. The retention impairment induced by the Datura extract in these learning tasks was not due to an effect on spontaneous locomotor activity nor to changes in the emotional reactivity of the animals. Preliminary chemical analysis suggests that the active principle of the Datura extract may be a small peptide. It is suggested that this active principle affects some memory processes.
Datura stramonium is a plant belonging to the solanaceae family and which has been known from antiquity for its hallucinogenic properties and its effects on memory. Many cases of poisoning by D. stramonium have been recorded. The symptoms of these poisonings generally are incoordinated movements and behavior, hyperexcitability, and inhibition of the parasympathetic system (Jennings, 1935; Arena, 1954; Gibson, 1961; Rosen and Lechner, 1962). Some cases of complete memory loss following Datura poisoning in adults and adolescents have also been recorded (Mitchell and Mitchell, 1955). These effects have been ascribed to the three alkaloids contained in D. stramonium, atropine, hyoscyamine, and scopolamine. Bose, et al. (1967) have also shown that the total alkaloids of Datura alba greatly impaired the retention of an active avoidance learning task in rats. However, Chauvin, in a preliminary study, observed that the memory impairment following D. stramonium treatment in mice did not seem to be The advice of Prof. CI. Mathis who made the chromatographic analysis of the Datura alkaloids is greatly acknowledged. We thank Dr. Gabriel from the Dausse Laboratory who helped us in preparation of the Datura aqueous extract. We thank Dr. T. Durkin for his help in translating the manuscript and Miss M. Bourgeois for her technical assistance. 349 0091-6773/78/0243 -0349502.00/0 Copyright@ 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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due to the Datura alkaloids. Indeed, the ethanol extracts ofDatura, which contained the Datura alkaloids, did not affect memory retention, while an aqueous extract devoid of those alkaloids greatly impaired learning retention in the same experimental conditions. A study was thus carried out in order to specify the nature of the principle contained in the aqueous extract which produced these impairments. This research has allowed us to isolate a water-soluble fraction which impaired learning retention though devoid of Datura alkaloids as shown by a chromatographic analysis of the alkaloid content of this fraction. The memory effects of this fraction were displayed in various learning tasks in mice: discriminated avoidance learning, two-way active avoidance learning in a Mowrer-Miller cage, and food-reinforced instrumental learning. Analysis of the effects of the Datura extract on locomotor activity and on performance in an open-field test has shown that memory impairment produced by the Datura extract in these learning tasks was not due to an effect on the level of locomotor activity of the animals nor to an action on their emotionality. According to the results obtained from the chemical analysis of the Datura extract, the active principle could be a small peptide.
METHODS Datura Extract Preparation Datura stramonium powder (leaves and seeds) was treated according to the method summarized in Fig. 1. Two extracts were so obtained and tested for behavioral effects. The ethanol extract contained the alkaloids ofDatura, according to chromatographic analysis of this extract. The aqueous extract was devoid of the Datura alkaloids. These alkaloids were eliminated by cold and boiling ethanol treatments. This elimination was confirmed by chromatographic analysis of the aqueous extract. Treatment The ethanol extract, after concentration (Fig. 1), was dissolved in 0.9% NaC1 at the rate of 32 g/liter and was then administered intraperitoneally to mice (0.8 g/kg). This dose produced a deep prostration and a great acceleration of heart rate in animals during the first hours following treatment. Those effects were temporary and had totally disappeared in 24 hr following treatment. The aqueous extract was dissolved in 0.9% NaC1 at the rate of 32 g/liter and was injected intraperitoneally to mice (0.8 g/kg). This extract produced no apparent behavioral effect in the animals except for a slight decrease in mobility for the first few hours following treatment. All control mice received intraperitoneally 25 ml/kg of 0.9% NaC1.
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Each mouse received treatment once only and w a s submitted to only one behavioral test. Discriminated Avoidance Task One hundred and forty-one male Swiss mice were obtained from our laboratory breeding facilities and were 2 to 3 months old at the beginning of the experiment. Mice were trained in a transparent Plexiglas Y-maze whose three alleys (each alley being 5.5 cm high, 4.5 cm wide, and 13 cm long) were set on the median lines of an equilateral triangle. The maze had a grid floor through which shocks (40 V ac, 5 mA) could be applied in any alley. At the end of each alley was a transparent Plexiglas mobile box (5.5 cm high, 4.5 cm wide, and 10 cm long) with an opaque Plexiglas sliding door. This allowed us to transport the animals from the goal alley to the start alley without handling. At the beginning of each trial, the mouse was placed in the start alley and subjected to a conditioning procedure that had two components. The first was an avoidance c o m p o n e n t and the second was a discrimination component. The avoidance c o m p o n e n t involved a temporal discrimination. The animals had to leave the start alley in the first 5 sec; animals that did not make this reponse in the 5-sec delay were held to be making an "avoidance error." The discrimination c o m p o n e n t involved a spatial discrimination. Each mouse was trained to choose the left alley of the maze. The choice of the right alley was held to be a "discrimination e r r o r . " We have termed " a v o i d a n c e c o m p o n e n t " the first c o m p o n e n t of this double conditioning task and "discrimination c o m p o n e n t " the second. The aminal u n d e r w e n t one trial e v e r y minute. W h e n e v e r it made an error, it received brief electric shocks (duration of the shock: 1 sec e v e r y 3 sec) until it entered the left alley. The training session was terminated when the animal reached a criterion of 9 errorless trials out of 10 consecutive trials. The treatment was administered 1 hr, 24 hr, or 7 days after the training session. Retention of the learned task was tested 48 hr following treatment in a second training session (test session) carried out in the same conditions as the learning session. In a second experiment, treatment was administered 2 days before the training session in order to analyze the effects of the aqueous extract on acquisition processes and on performance level. For each session, the number of trials to reach criterion, avoidance errors, and discrimination errors was recorded. Two-Way Active Avoidance Task Twenty-three male Swiss mice, 2 to 3 months old, were trained in a shuttle box. The testing apparatus consisted Of a cage (12 cm high, 12 cm wide, and 24 cm long) with two compartments of the same size separated by an opaque partition with a 5 x 5 cm opening. Each compartment could be lit by a 15-W bulb. The light stimulus was coupled with a sound buzzer.
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The box had a grid floor through which shock (40 V ac, 5 mA) could be applied in either section. Each animal underwent one trial every 30 sec. The double conditioning stimulus (light and sound) was given for 5 sec followed by electric shocks until the animal escaped to the other side of the box. An avoidance response was recorded when the animal moved into the other compartment during the 5 sec of the conditioning stimulus thereby avoiding the shock. The animal was submitted to a first training session. This session was concluded when the animal had made 9 avoidance responses out of 10 consecutive trials. Treatment was given 24 hr after this training session. The retention of animals was tested 3 days after treatment in a second training session, carried out under the same conditions as the first session.
Instrumental Learning Forty-five male Swiss mice were isolated and deprived of food 24 hr before the beginning of the experiment. Then, each day for a period of 45 min, the animals were placed into an apparatus where each lever-pressing response released the opening of a feeder filled with buckweat seeds for 5 sec. Training was continued until each animal made 20 or more leverpressing responses in a single 45-rain session. Animals received treatment 24 hr after they reached this learning criterion. Forty-eight hours following treatment with the aqueous extract the animals were again placed for a period of 45 rain into the apparatus and the number of lever-pressing responses during this session was recorded. During the entire course of the experiment, the animals received 3 g of food per day so that their weight loss did not exceed 10 to 20% of their initial weight.
Measure of Emotional Reactivity (Open-Field Test) According to several authors (Hall, 1934; Archer, 1973), the mobility of animals introduced into a novel environment and the number of boluses emitted during that time are measures of the emotionality level of animals. In order to determine if the Datura extract affected the emotional reactions of animals, 40 male Swiss mice were submitted to an open-field test 48 hr after treatment. Each animal was introduced for a period of 3 rain into a circular enclosure (40 cm in diameter) whose floor was isotropically lit and was divided into nine areas of equal area. The numbers of areas crossed and boluses emitted during the test were recorded.
Measure of Locomotor Activity Thirty-four male Swiss mice were submitted to an actographic test. Forty-eight hours after treatment, each animal was placed for 4 hr into an actographic recorder (18 cm high, 20 cm wide, and 100 cm long) with six transverse light beams. The number of beams crossed by the animal was registered by a pulse counter. This number was recorded every 30 rain.
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Statistics The analysis of variance was used in all the experiments.
RESULTS
Y-maze Learning The aqueous extract, when injected 1 hr after maze learning, deeply impaired retention of this task 48 hr after treatment IF(l,18) = 5.56; P < 0.05]. However, it appeared that the aqueous extract seemed to affect only the retention of the avoidance component and had no effect on the retention of the discrimination component. The treated group significantly differed from the control group for avoidance errors IF (1,18) = 6.01; P < 0.025] but did not differ for discrimination errors (Table 1A). Comparable results were obtained when the aqueous extract was injected 24 hr after learning (Table 1B). In this case, also, the aqueous extract strongly impaired retention of the temporal avoidance component 48 hr after treatment IF (1,40) = 22.63; P < 0.001] but had no effect on retention of the spatial discrimination component. On the other hand, the ethanol extract had no effect on the retention of the Y-maze learning when injected 24 hr after learning. The treated group (E) (N = 10) did not significantly differ from the control group (C) (N = 9) for the number of trials to reach criterion (E:M = 11.80___0.72; C: M = 11.77 ___0.82) nor for the number of discrimination errors (E:M = 0.20 ___0.10; C: M = 0.22 _+ 0.14) or avoidance errors (E:M = 2.00 ___ 0.36; C: M = 1.77 _+ 0.45). These results confirm that the effects of the aqueous extract on retention of the Y-maze learning could not be ascribed to the alkaloids ofDatura. The aqueous extract, when injected 7 days after learning, was without effect on the retention of the avoidance component (Table 1C). Furthermore, the aqueous extract, when injected 2 days before maze learning, did not produce any perturbation in the acquisition of the task (Table ID). According to these results, it appears that the impairment induced by the Datura in the retention of Y-maze learning was not due to an action on performance level nor to a long-term action on acquisition processes. From these data, it appears that the retention of only the avoidance component of Y-maze' learning was affected by the Datura aqueous extract. The retention of both avoidance and discrimination components did not seem to be equally sensitive to treatment. These results are similar to those obtained in the same learning situations with protein synthesis inhibitors (Ungerer, 1975).
Two-Way Active Avoidance Learning The treated and the control groups did not significantly differ in the acquisition of a two-way active avoidance learning task during the first learning session carried out 24 hr before treatment [number of trials to
**** P 0.051. The Dntura aqueous extract injected 24 hr after the first session impaired the retention of this task 3 days following treatment. The treated animals made significantly more errors (a = 8.00 + 0.61) than the control animals (a = 4.81 & 1.00) during the first 10 trials of the second session [P (1,21) = 4.79; P < 0.051 and took longer to attain the learning criterion [P(1,21) = 6.34; P < 0.0251 (Fig. 2). Instrumental
Learning
The effects of the Datura extract on the retention of instrumental learning were analyzed in two successive experiments carried out in the same experimental conditions. Data obtained in these two experiments have been combined for statistical analysis. The aqueous extract injected 24 hr after acquisition produced a significant decrease in the performance level of animals in the retention test. The treated animals (N = 20) showed a lower level of lever-pressing responses (@ = 54.15 + 4.79) than the control animals (IV = 25) (a = 78.36 ? 3.78) [F(1,43) = 16.51;P < 0.0011. This performance decrease was not due to an action of the Datura extract on food consumption. The aqueous extract produced no decrease in food consumption 48 hr following treatment when the animals had free access
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FIG. 2. Effect of theDaturu aqueous extract on the retention of a conditioned avoidance task. Cumulative records of animals having reached the criterion for each trial during learning sessions, 24 hr before treatment (dark circles) and during retention session, 3 days following treatment (light squares), in the treated group (dotted lines) and in the control group (full lines).
A FACTOR ISOLATED FROM Datura stramonium
357
to food during the 45 rain in the conditioning apparatus. The mean weight of seeds ingested during_ the test did not significantly differ between the treated group (N = 8) (M = 0.72 _+ 0.07) and the control group (N = 9) (M = 0.91 _+ 0.12) [F(1,15) = 1.78; P > 0.05]. Therefore, it does not seem that this performance decrease during the retention test could be explained by Datura interference with motivational factors connected with food ingestion. This performance deficit could possibly be due to a conditioned aversion induced by the Datura extract for the food reinforcement as we have shown with acetoxycycloheximide in the same learning situation (Ungerer et al., 1975). H o w e v e r , such an hypothesis is not in agreement with published data. It should be noted that the Datura extract was injected 24 hr following acquisition and that no example of conditioned aversion generation has been recorded for delays in excess of 12 hr (Rozin, 1969; Taukulis, 1974). Therefore, it appears difficult to assume that the impairment produced by the Datura extract in the retention of an instrumental learning task could be due to an aversion for the food reinforcement associated with the learning situation. Datura Effects on Emotional Reactivity and on Locomotor Activity Performance in the various learning tasks used in this work is likely to be appreciably affected by the emotional level of animals or by their locomotor activity level. Therefore, the retention impairments produced by the Datura extract in these learning tasks could be explained by an action on either of these two behavioral components. In order to test this hypothesis, we have analyzed the effects of the Datura extract on emotional reactivity of animals (open-field) and on locomotor activity. Effects on emotional reactivity in an open-field test. The treated animals (T) did not differ from the control animals (C) either for the number of areas crossed [T: ~V7 = 63.35 _+ 5.38; C: M = 68.05 _+ 7.04; F(1,38) = 0.09; P > 0.05] or for boluses emitted during the test IT: M = 1.70 _+ 0.44; C: M = 2.40 + 0.41; F(1.38) = 1.32; P > 0.05]. Therefore, it appears that the Datura extract, 48 hr following treatment, produced no significant modification in emotional reactivity or in exploratory activity of animals placed in a novel environment. Effects on locomotor activity. The Datura extract produced no decrease in l o c o m o t o r activity, except during the first 30 rain of the test (Fig. 3). During that time, the treated animals (N = 17) crossed the light beams less frequently as compared to controls (N = 17) [F(1,32) = 10.14; P < 0.01]. There was, however, no difference between the treated and the control groups when the total duration of the test was taken into account. These results, in contrast to data of the previous experiment, seem to point to an action of the Datura extract on exploratory activity. The reason for this divergence is not clear and has to be analyzed in other situations involving exploratory activity. Thus the present experiment
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UNGERER, SCHUBER, AND CHAUVIN
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clearly demonstrates that the Datura extract, 48 hr following treatment, does not affect the general activity level of the animals. According to the results of these two behavioral tests, it appears that the effects of the Datura extract on the retention of the various learning tasks used do not seem to be due to an action on the level of emotionality or on general locomotor activity of the animals.
CHEMICAL ANALYSIS The activity of fractions that we obtained during the successive steps of chemical analysis were tested by comparing their effects on the avoidance component retention in Y-maze learning to those of the aqueous extract. The Y-maze learning and the retention test were carried out as previously described. Treatment was given 24 hr after learning. For each fraction, the dose injected was referred to the start weight of aqueous extract; the aqueous extract was injected at dose of 0.8 g/kg. The retention of the animals was tested 48 hr after treatment. The number of avoidance errors made by the animals during the retention test was recorded (Table 2).
Hydrolysis In order to determine if the activity of the aqueous extract could be suppressed by eliminating peptide bonds, acidic and basic hydrolysis of the aqueous extract was carried out. The aqueous extract (640 mg) was dissolved either in 10 ml of 0.1 N HC1 (acidic hydrolysis) or in 10 ml of 1 N NH4OH (basic hydrolysis) and heated to 100°C for 15 hr. The hydrolysates
a M ea n + SE.
E. D E A E and S ep had ex G-10
A 2.4 _+ 0.49
II 1.95 + 0.31 B 0.66 _+ 0.23
III 1.3 -+ 0.33
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A 1.54 _+ 0.43
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I 2.31 _+ 0.30
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D. D E A E and S e p h a d e x G-25
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A. H y d r o l y s i s
Avoidance errors NaC1 0.9 _+ 0.23 NaC1 0.9 +- 0.15 NaCI 1.09 _+ 0.34 NaC1 l . l _+ 0.31 NaCI 0.60 + 0.15
Datura 2.55 -+ 0.58 Datura 2.45 +- 0.51 Datura 3.18 -+_+0.68 Datura 3.1 -+ 0.50 Datura 2.11 +_ 0.38
F (3,34) = 7.38
F (4,67) = 3.77
F (3,39) = 4.772
F (3,42) = 6.11
F (3,34) - 2.87
F
TABLE 2 Effect of the F r a c t i o n s O b t a i n e d duri ng the S u c c e s s i v e Steps of C h e m i c a l A n a l y s i s on the R e t e n t i o n of Y - M a z e L e a r n i n g
< 0.001
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= 0.05
P
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UNGERER, SCHUBER, AND CHAUVIN
were then evaporated and dissolved in 20 ml of 0.9% NaCI. The animals treated with hydrolysates did not significantly differ from the NaC1 controls [F(1,34) = 1.05; P > 0.05] and made fewer errors compared to the animals treated with the aqueous extract [F(1,34) = 5.21; P < 0.05] (Table 2A). These results showed that the activity of the aqueous extract was suppressed by hydrolysis and that consequently, the active principle of this extract could be due to a peptide or a small protein. Dialysis The aqueous extract (480 mg dissolved in 10 ml of distilled water) was dialyzed against two changes of 10 vol of distilled water at 4°C for 24 hr each. The nondiffusible material (NDM) and the dialysate were then evaporated and dissolved in 0.9% NaC1. The dialysate impaired learning retention to the same extent as the aqueous extract [F(1,42) = 1.68; P > 0.05], whereas the nondiffusible material was without effect (NDM vs NaCI: F(1,42) = 0.06; P > 0.05) (Table 2B). These results indicate that the active principle has a molecular weight not greater than 20,000. Chromatography on DEAE-Cellulose and on Sephadex G-25 and G-IO The aqueous extract (480 mg) was dialyzed against distilled water; the dialysate, after freeze-drying, was dissolved in 0.05 M ammonium bicarbonate and layered onto a DEAE-cellulose column (1.6 x 35 cm). The column was eluted by a discontinuous gradient of 0.05 M (220 ml) and 0.3 M (270 ml) ammonium bicarbonate. Fractions collected were freeze-dried and then dissolved in 0.9% NaC1. Activity of the aqueous extract was concentrated in Fraction B (eluted at 0.3 M concentration), whereas Fraction A (eluted at 0.05 M concentration) was without effect on learning retention [Fraction A vs Datura:F(1,39) = 8.05; P < 0.01; Fraction B v s Datura: F(1,39) = 2.01; P > 0.05] (Table 2C). According to these results, the polarity of the active principle would be acidic. Fraction B, after evaporation and salt elimination, was dissolved in 0.025 M ammonium bicarbonate and layered onto a Sephadex G-25 Fine column (1.6 x 110 cm; void volume = 121 ml; flow rate = 12 ml/hr). Three fractions were collected, freeze-dried, and then dissolved in 0.9% NaCI: Fraction I (elution volume from 121 to 193 ml), Fraction II (elution volume from 193 to 229 ml), and Fraction III (elution volume from 229 to 380 ml). The extract activity was concentrated in Fraction I (Fraction I vs Datura: F(1,67) = 2.32; Fraction H vs Datura: F(1,67) = 4.87; Fraction III vs Datura: F(1,67) = 8.95; for o~-- .05 F(1,67) = 4.00] (Table 2D). According to these results, the molecular weight of the active principle is about 2000. Fraction I, after evaporation and salt elimination, was dissolved in 5 mM ammonium bicarbonate and layered onto a Sephadex G-10 column (1.6 x 50 cm; void volume = 43 ml; flow rate = 10 ml/hr). T w o fractions were collected, freeze-dried, and then dissolved in 0.9% NaCI: Fraction A
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corresponding to the void volume of the column and Fraction B corresponding to the bed volume (90 ml). The extract activity was concentrated in Fraction A (Fraction A vs Datura: [F(1,34) = 0.34; P > 0.05], whereas Fraction B was without effect (Fraction B vs NaCI: F(1,34) = 0.02; P > 0.05] (Table 2E). Consequently, it appears that the active principle is not retained by the Sephadex G-10 column. These results indicate that the active principle has a molecular weight greater than 700, which rules out the possibility that it may be an amino acid. Results of chemical analysis show that the active principle of the aqueous extract was hydrolyzable, dialyzable, and that its molecular weight is approximatively 2000. The Fraction A that we obtained after Sephadex G-10 chromatography was hydrolyzed by 6 N HC1 for 24 hr at 100°C. Chromatographic analysis revealed the presence of the following amino acids in the hydrolysate: Glu, Asp, Leu, Lys, Gly, and Ala.
DISCUSSION The Datura aqueous extract greatly impaired retention in the three learning situations used in this study. According to the results of control experiments, this impairment does not seem to be due to an action on the performance level of animals nor to an action on acquisition processes. Furthermore, the Datura extract had no effect on general locomotor activity nor on emotional reactivity of animals, as shown by actographic and open-field test results. In some experimental conditions, the Datura extract seemed to affect exploratory activity of animals. However, it appears difficult to ascribe the retention deficit produced by the Datura extract to such an action. Indeed, it is important to emphasize that the Datura extract injected 48 hr before maze learning did not disturb acquisition, though the animal was placed in a novel situation. However, the Datura extract greatly impaired performance in the retention tests, even though this situation then did not involve any novel component for the animal. Nevertheless, it should be emphasized that the Datura extract could affect some motivational processes and thus interfere with performance during retention testing. Further experiments are necessary to verify this hypothesis. However, if we consider all our results, these are in agreement with an interpretation of the Datura effects on learning retention in terms of an action on memory processes. Such an interpretation leads to several considerations. First, in none of the three learning tasks used, did the Datura extract erase all memory of the learned task. Second, the retention of a learned task is affected by the Datura extract for a relatively long period of time (24 hr), if we compare this time to the maximum effectiveness delays of other drugs or treatments that affect memory retention (Soumireu-Mourat, 1976). Nevertheless, 24-hr effectiveness delays have been observed with various treatments, especially with scopolamine (Wiener and Deutsch, 1968), physostigmine (Lewis and Bregman, 1972;
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Puerto et al., 1976), pentylenetetrazol (Pearlman et al., 1961), KC1 (Avis and Carlton, 1968; Buresova and Bures, 1971), and puromycin (Flexner et al., 1963; Ungerer, 1969). Treatments which impair memory with such long delays would not be likely to affect memory formation processes, but rather, would interfere either with memory recall or memory expression or would accelerate forgetting. The Datura extract, in view of its long gradient of effectiveness, could affect, as these other treatments, the same processes. However, according to the fact that the Datura effects are time dependent, we cannot rule out the possibility that the Datura extract could affect some slow memory formation processes (McGaugh, 1966). Finally, it should be emphasized that the Datura extract has long-lasting effects on memory retention since these effects were still observed 48 hr following treatment. The mechanisms by which the Datura extract impairs memory processes remains unknown. The presence of several amino acids in the active fraction obtained after DEAE-cellulose and gel-Sephadex filtration point to the possibility that the active principle of the Datura extract could be a small peptide. In agreement with this hypothesis, it should be noted that several studies in the last decade have shown the role of peptides in some behavioral processes. This role is not related to a hormonal action but seems to be due to either stimulating or inhibiting effects on cerebral activity (Iversen, 1974). It should likewise be recalled that peptide analogs of ACTH and vasopressin (ACTHI.~07-L-Phe, desglycinamide vasopressin) facilitated learning and delayed extinction of avoidance learning in rats independently of all endocrine effects (De Wied, 1973). These peptides appear to have a specific effect on cerebral activity. Indeed, when D-Phe was substituted to L-Phe in position 7 of ACTHI_10, the peptide so obtained delayed acquisition and increased the rate of extinction and, therefore, had behavioral effects opposite to those of ACTH~_~07-L-Phe. According to Schottman et al. (1972) the behavioral effects of these two peptides could be related to their action on cerebral protein synthesis. In fact, ACTH~_107-L-Phe had a stimulating effect on leucine incorporation in macromolecules of the brain stem, whereas ACTH~_~07-D-Phe caused a decrease in leucine incorporation. However, such an action cannot be ascribed in the case of the Datura extract, as shown by the lack of effects on valine incorporation in cerebral proteins (Ungerer, 1975). At present, analysis of the aqueous extract is being pursued to isolate and to specify the chemical nature of the active principle. This study will allow us to determine whether this active principle presents some analogies to the behaviorally active peptides discussed above. REFERENCES Archer, J. (1973). Tests for emotionality in rats and mice: A review. Anita. Behav. 21, 205-235. Arena, J. M. (1954). Poisoning in infants and children: I. Accidents and emergencies. Pediat. Clin. North Amer. 1:777.
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Avis, H. A., and Carlton, P. L. (1968). Retrograde amnesia produced by hippocampal spreading depression. Science 161, 73-75. Bose, 13. G., Matin, M. A., Vijayvargiya, R., and Lahiri, M. (1967). Effect of solaneceous alkaloids on the conditioned avoidance response in trained animals. Curr. Sci. India 36, 433-434. Buresova, O., Bures, J. (1971). The effect of prolonged cortical spreading depression on consolidation of visual engrams in rats. Psychopharmacologia (Berlin) 20, 57-65. De Wied, D. (1973). Peptides and behavior. In H. P. Zippel (Ed.), "Memory and Transfer of Information," pp. 373-389. New York: Plenum Press. Flexner, J. B., Flexner, L. B., and Stellar, E. (1963). Memory in mice as affected by intracerebral puromycin. Science 141, 57-59. Gibson, R. K. (1961). Jimson weed poisoning in children. J. Indiana S. M. Ass. 54, 1018-1020. Hall, C. S. (1934). Emotional behavior in the rat: Defecation and urination as measures of individual differences in emotionality. J. Comp. Psychol. 18, 385-403. Iversen, L. L. (1974). Neuropharmacology of peptides. Nature (London) 252, 630. Jennings, R. E. (1935). Stramonium poisoning: A review of the literature and a report of two cases. J. Pediat. 6, 657. Lewis, D. J., and Bregman, N. J. (1972). The cholinergic system, amnesia and memory. Physiol, Behav. 8, 511-514. McGaugh, J. L. (1966). Time-dependent processes in memory storage. Science 153, 13511358. Mitchell, J. E., and Mitchell, F. N. (1955). Jimson weed (Datura stramonium) poisoning in childhood. J. Pediat. 47, 227. Pearlman, C. A., Jr., Sharpless, S. K., and Jarvik, M. E. (1961). Retrograde amnesia produced by anesthetics and convulsant agents. J. Comp. Physiol. Psychol. 54, 109112. Puerto, A, Molina, F., Rogers, J., and Moss, M. E. (1976). Physostigmine-induced amnesia for an escape response 12 to 72 hours after training. Behav. Biol. 16, 85-90. Rosen, C. S., and Lechner, M. (1962). Jimson weed intoxication. N. Engl. J. Med. 267, 448. Rozin, T. (1969). Central or peripheral mediation of learning with long CS-US intervals in the feeding system. J. Comp. Physiol. Psychol. 67, 421-429. Schottman, P., Gispen, W. H.~ and De Wied, D. (1972). Effects of ACTH analogues on macromolecule metabolism in the brain stem of hypophysectomized rats. Brain Res. 46, 349-362. Soumireu-Mourat, B. (1976). "'La Phase Labile des Processus Mn~siques chez la Souris: Etude de Diff6rents Traitements Amn~siants et de leurs Gradients d'Efficacit6." Th~se Doctorat d'Etat, Univ. Bordeaux, p. 294. Taukulis, H. K. (1974). Odor aversions produced over long CS-US delays. Behav. Biol. 10, 505-510. Ungerer, A. (1969). Effets compar6s de la puromycine et du Datura stramonium sur la r6tention d'un apprentissage instrumental chez la souris. C. R. Acad. Sci. 269 (D), 910-913. Ungerer, A. (1976). "Analyse des D6ficits Mn6siques Induits par les Inhibiteurs de la Synth~se Prot~ique chez la Souris." Th~se Doctorat d'Etat, Univ. Strasbourg, p. 230. Ungerer, A., Marchi, D., Ropartz, P., and Weil, J. H. (1975). Aversive effects and retention impairment induced by acetoxycycloheximide in an instrumental task. Physiol. Behav. 15, 55-62. Wiener, N., and Deutsch, J, A. (1968). Temporal aspects of anticholinergic and anticholinesterase-induced amnesia for an appetitive habit. J. Comp. Physiol. Psychol. 66, 613-617.
A Factor Isolated from Datura stramonium That Affects Learning Retention in Mice A . UNGERER,
F. SCHUBER, AND R. CHAUVIN ~
Laboratoire de Psychophysiologie, 7, rue de l' Universitb, 67000, Strasbourg, Institut de Botanique, 8, rue Goethe, 67000, Strasbourg, and Station d'Ethologie Exp~rimentale, "Les Sources," Mittainville, 78120, Rambouillet, France A water-soluble fraction, devoid of alkaloids, was isolated from Datura stramonium (leaves and seeds). This fraction greatly impaired retention of (i) discriminated avoidance learning, (ii) two-way active avoidance learning, and (iii) a food-reinforced instrumental learning. The retention impairment induced by the Datura extract in these learning tasks was not due to an effect on spontaneous locomotor activity nor to changes in the emotional reactivity of the animals. Preliminary chemical analysis suggests that the active principle of the Datura extract may be a small peptide. It is suggested that this active principle affects some memory processes.
Datura stramonium is a plant belonging to the solanaceae family and which has been known from antiquity for its hallucinogenic properties and its effects on memory. Many cases of poisoning by D. stramonium have been recorded. The symptoms of these poisonings generally are incoordinated movements and behavior, hyperexcitability, and inhibition of the parasympathetic system (Jennings, 1935; Arena, 1954; Gibson, 1961; Rosen and Lechner, 1962). Some cases of complete memory loss following Datura poisoning in adults and adolescents have also been recorded (Mitchell and Mitchell, 1955). These effects have been ascribed to the three alkaloids contained in D. stramonium, atropine, hyoscyamine, and scopolamine. Bose, et al. (1967) have also shown that the total alkaloids of Datura alba greatly impaired the retention of an active avoidance learning task in rats. However, Chauvin, in a preliminary study, observed that the memory impairment following D. stramonium treatment in mice did not seem to be The advice of Prof. CI. Mathis who made the chromatographic analysis of the Datura alkaloids is greatly acknowledged. We thank Dr. Gabriel from the Dausse Laboratory who helped us in preparation of the Datura aqueous extract. We thank Dr. T. Durkin for his help in translating the manuscript and Miss M. Bourgeois for her technical assistance. 349 0091-6773/78/0243 -0349502.00/0 Copyright@ 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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due to the Datura alkaloids. Indeed, the ethanol extracts ofDatura, which contained the Datura alkaloids, did not affect memory retention, while an aqueous extract devoid of those alkaloids greatly impaired learning retention in the same experimental conditions. A study was thus carried out in order to specify the nature of the principle contained in the aqueous extract which produced these impairments. This research has allowed us to isolate a water-soluble fraction which impaired learning retention though devoid of Datura alkaloids as shown by a chromatographic analysis of the alkaloid content of this fraction. The memory effects of this fraction were displayed in various learning tasks in mice: discriminated avoidance learning, two-way active avoidance learning in a Mowrer-Miller cage, and food-reinforced instrumental learning. Analysis of the effects of the Datura extract on locomotor activity and on performance in an open-field test has shown that memory impairment produced by the Datura extract in these learning tasks was not due to an effect on the level of locomotor activity of the animals nor to an action on their emotionality. According to the results obtained from the chemical analysis of the Datura extract, the active principle could be a small peptide.
METHODS Datura Extract Preparation Datura stramonium powder (leaves and seeds) was treated according to the method summarized in Fig. 1. Two extracts were so obtained and tested for behavioral effects. The ethanol extract contained the alkaloids ofDatura, according to chromatographic analysis of this extract. The aqueous extract was devoid of the Datura alkaloids. These alkaloids were eliminated by cold and boiling ethanol treatments. This elimination was confirmed by chromatographic analysis of the aqueous extract. Treatment The ethanol extract, after concentration (Fig. 1), was dissolved in 0.9% NaC1 at the rate of 32 g/liter and was then administered intraperitoneally to mice (0.8 g/kg). This dose produced a deep prostration and a great acceleration of heart rate in animals during the first hours following treatment. Those effects were temporary and had totally disappeared in 24 hr following treatment. The aqueous extract was dissolved in 0.9% NaC1 at the rate of 32 g/liter and was injected intraperitoneally to mice (0.8 g/kg). This extract produced no apparent behavioral effect in the animals except for a slight decrease in mobility for the first few hours following treatment. All control mice received intraperitoneally 25 ml/kg of 0.9% NaC1.
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352
UNGERER, SCHUBER, AND CHAUVIN
Each mouse received treatment once only and w a s submitted to only one behavioral test. Discriminated Avoidance Task One hundred and forty-one male Swiss mice were obtained from our laboratory breeding facilities and were 2 to 3 months old at the beginning of the experiment. Mice were trained in a transparent Plexiglas Y-maze whose three alleys (each alley being 5.5 cm high, 4.5 cm wide, and 13 cm long) were set on the median lines of an equilateral triangle. The maze had a grid floor through which shocks (40 V ac, 5 mA) could be applied in any alley. At the end of each alley was a transparent Plexiglas mobile box (5.5 cm high, 4.5 cm wide, and 10 cm long) with an opaque Plexiglas sliding door. This allowed us to transport the animals from the goal alley to the start alley without handling. At the beginning of each trial, the mouse was placed in the start alley and subjected to a conditioning procedure that had two components. The first was an avoidance c o m p o n e n t and the second was a discrimination component. The avoidance c o m p o n e n t involved a temporal discrimination. The animals had to leave the start alley in the first 5 sec; animals that did not make this reponse in the 5-sec delay were held to be making an "avoidance error." The discrimination c o m p o n e n t involved a spatial discrimination. Each mouse was trained to choose the left alley of the maze. The choice of the right alley was held to be a "discrimination e r r o r . " We have termed " a v o i d a n c e c o m p o n e n t " the first c o m p o n e n t of this double conditioning task and "discrimination c o m p o n e n t " the second. The aminal u n d e r w e n t one trial e v e r y minute. W h e n e v e r it made an error, it received brief electric shocks (duration of the shock: 1 sec e v e r y 3 sec) until it entered the left alley. The training session was terminated when the animal reached a criterion of 9 errorless trials out of 10 consecutive trials. The treatment was administered 1 hr, 24 hr, or 7 days after the training session. Retention of the learned task was tested 48 hr following treatment in a second training session (test session) carried out in the same conditions as the learning session. In a second experiment, treatment was administered 2 days before the training session in order to analyze the effects of the aqueous extract on acquisition processes and on performance level. For each session, the number of trials to reach criterion, avoidance errors, and discrimination errors was recorded. Two-Way Active Avoidance Task Twenty-three male Swiss mice, 2 to 3 months old, were trained in a shuttle box. The testing apparatus consisted Of a cage (12 cm high, 12 cm wide, and 24 cm long) with two compartments of the same size separated by an opaque partition with a 5 x 5 cm opening. Each compartment could be lit by a 15-W bulb. The light stimulus was coupled with a sound buzzer.
A FACTOR ISOLATED FROM Datura stramonium
353
The box had a grid floor through which shock (40 V ac, 5 mA) could be applied in either section. Each animal underwent one trial every 30 sec. The double conditioning stimulus (light and sound) was given for 5 sec followed by electric shocks until the animal escaped to the other side of the box. An avoidance response was recorded when the animal moved into the other compartment during the 5 sec of the conditioning stimulus thereby avoiding the shock. The animal was submitted to a first training session. This session was concluded when the animal had made 9 avoidance responses out of 10 consecutive trials. Treatment was given 24 hr after this training session. The retention of animals was tested 3 days after treatment in a second training session, carried out under the same conditions as the first session.
Instrumental Learning Forty-five male Swiss mice were isolated and deprived of food 24 hr before the beginning of the experiment. Then, each day for a period of 45 min, the animals were placed into an apparatus where each lever-pressing response released the opening of a feeder filled with buckweat seeds for 5 sec. Training was continued until each animal made 20 or more leverpressing responses in a single 45-rain session. Animals received treatment 24 hr after they reached this learning criterion. Forty-eight hours following treatment with the aqueous extract the animals were again placed for a period of 45 rain into the apparatus and the number of lever-pressing responses during this session was recorded. During the entire course of the experiment, the animals received 3 g of food per day so that their weight loss did not exceed 10 to 20% of their initial weight.
Measure of Emotional Reactivity (Open-Field Test) According to several authors (Hall, 1934; Archer, 1973), the mobility of animals introduced into a novel environment and the number of boluses emitted during that time are measures of the emotionality level of animals. In order to determine if the Datura extract affected the emotional reactions of animals, 40 male Swiss mice were submitted to an open-field test 48 hr after treatment. Each animal was introduced for a period of 3 rain into a circular enclosure (40 cm in diameter) whose floor was isotropically lit and was divided into nine areas of equal area. The numbers of areas crossed and boluses emitted during the test were recorded.
Measure of Locomotor Activity Thirty-four male Swiss mice were submitted to an actographic test. Forty-eight hours after treatment, each animal was placed for 4 hr into an actographic recorder (18 cm high, 20 cm wide, and 100 cm long) with six transverse light beams. The number of beams crossed by the animal was registered by a pulse counter. This number was recorded every 30 rain.
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UNGERER, SCHUBER, AND CHAUVIN
Statistics The analysis of variance was used in all the experiments.
RESULTS
Y-maze Learning The aqueous extract, when injected 1 hr after maze learning, deeply impaired retention of this task 48 hr after treatment IF(l,18) = 5.56; P < 0.05]. However, it appeared that the aqueous extract seemed to affect only the retention of the avoidance component and had no effect on the retention of the discrimination component. The treated group significantly differed from the control group for avoidance errors IF (1,18) = 6.01; P < 0.025] but did not differ for discrimination errors (Table 1A). Comparable results were obtained when the aqueous extract was injected 24 hr after learning (Table 1B). In this case, also, the aqueous extract strongly impaired retention of the temporal avoidance component 48 hr after treatment IF (1,40) = 22.63; P < 0.001] but had no effect on retention of the spatial discrimination component. On the other hand, the ethanol extract had no effect on the retention of the Y-maze learning when injected 24 hr after learning. The treated group (E) (N = 10) did not significantly differ from the control group (C) (N = 9) for the number of trials to reach criterion (E:M = 11.80___0.72; C: M = 11.77 ___0.82) nor for the number of discrimination errors (E:M = 0.20 ___0.10; C: M = 0.22 _+ 0.14) or avoidance errors (E:M = 2.00 ___ 0.36; C: M = 1.77 _+ 0.45). These results confirm that the effects of the aqueous extract on retention of the Y-maze learning could not be ascribed to the alkaloids ofDatura. The aqueous extract, when injected 7 days after learning, was without effect on the retention of the avoidance component (Table 1C). Furthermore, the aqueous extract, when injected 2 days before maze learning, did not produce any perturbation in the acquisition of the task (Table ID). According to these results, it appears that the impairment induced by the Datura in the retention of Y-maze learning was not due to an action on performance level nor to a long-term action on acquisition processes. From these data, it appears that the retention of only the avoidance component of Y-maze' learning was affected by the Datura aqueous extract. The retention of both avoidance and discrimination components did not seem to be equally sensitive to treatment. These results are similar to those obtained in the same learning situations with protein synthesis inhibitors (Ungerer, 1975).
Two-Way Active Avoidance Learning The treated and the control groups did not significantly differ in the acquisition of a two-way active avoidance learning task during the first learning session carried out 24 hr before treatment [number of trials to
**** P 0.051. The Dntura aqueous extract injected 24 hr after the first session impaired the retention of this task 3 days following treatment. The treated animals made significantly more errors (a = 8.00 + 0.61) than the control animals (a = 4.81 & 1.00) during the first 10 trials of the second session [P (1,21) = 4.79; P < 0.051 and took longer to attain the learning criterion [P(1,21) = 6.34; P < 0.0251 (Fig. 2). Instrumental
Learning
The effects of the Datura extract on the retention of instrumental learning were analyzed in two successive experiments carried out in the same experimental conditions. Data obtained in these two experiments have been combined for statistical analysis. The aqueous extract injected 24 hr after acquisition produced a significant decrease in the performance level of animals in the retention test. The treated animals (N = 20) showed a lower level of lever-pressing responses (@ = 54.15 + 4.79) than the control animals (IV = 25) (a = 78.36 ? 3.78) [F(1,43) = 16.51;P < 0.0011. This performance decrease was not due to an action of the Datura extract on food consumption. The aqueous extract produced no decrease in food consumption 48 hr following treatment when the animals had free access
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FIG. 2. Effect of theDaturu aqueous extract on the retention of a conditioned avoidance task. Cumulative records of animals having reached the criterion for each trial during learning sessions, 24 hr before treatment (dark circles) and during retention session, 3 days following treatment (light squares), in the treated group (dotted lines) and in the control group (full lines).
A FACTOR ISOLATED FROM Datura stramonium
357
to food during the 45 rain in the conditioning apparatus. The mean weight of seeds ingested during_ the test did not significantly differ between the treated group (N = 8) (M = 0.72 _+ 0.07) and the control group (N = 9) (M = 0.91 _+ 0.12) [F(1,15) = 1.78; P > 0.05]. Therefore, it does not seem that this performance decrease during the retention test could be explained by Datura interference with motivational factors connected with food ingestion. This performance deficit could possibly be due to a conditioned aversion induced by the Datura extract for the food reinforcement as we have shown with acetoxycycloheximide in the same learning situation (Ungerer et al., 1975). H o w e v e r , such an hypothesis is not in agreement with published data. It should be noted that the Datura extract was injected 24 hr following acquisition and that no example of conditioned aversion generation has been recorded for delays in excess of 12 hr (Rozin, 1969; Taukulis, 1974). Therefore, it appears difficult to assume that the impairment produced by the Datura extract in the retention of an instrumental learning task could be due to an aversion for the food reinforcement associated with the learning situation. Datura Effects on Emotional Reactivity and on Locomotor Activity Performance in the various learning tasks used in this work is likely to be appreciably affected by the emotional level of animals or by their locomotor activity level. Therefore, the retention impairments produced by the Datura extract in these learning tasks could be explained by an action on either of these two behavioral components. In order to test this hypothesis, we have analyzed the effects of the Datura extract on emotional reactivity of animals (open-field) and on locomotor activity. Effects on emotional reactivity in an open-field test. The treated animals (T) did not differ from the control animals (C) either for the number of areas crossed [T: ~V7 = 63.35 _+ 5.38; C: M = 68.05 _+ 7.04; F(1,38) = 0.09; P > 0.05] or for boluses emitted during the test IT: M = 1.70 _+ 0.44; C: M = 2.40 + 0.41; F(1.38) = 1.32; P > 0.05]. Therefore, it appears that the Datura extract, 48 hr following treatment, produced no significant modification in emotional reactivity or in exploratory activity of animals placed in a novel environment. Effects on locomotor activity. The Datura extract produced no decrease in l o c o m o t o r activity, except during the first 30 rain of the test (Fig. 3). During that time, the treated animals (N = 17) crossed the light beams less frequently as compared to controls (N = 17) [F(1,32) = 10.14; P < 0.01]. There was, however, no difference between the treated and the control groups when the total duration of the test was taken into account. These results, in contrast to data of the previous experiment, seem to point to an action of the Datura extract on exploratory activity. The reason for this divergence is not clear and has to be analyzed in other situations involving exploratory activity. Thus the present experiment
358
UNGERER, SCHUBER, AND CHAUVIN
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120
150
180
210
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clearly demonstrates that the Datura extract, 48 hr following treatment, does not affect the general activity level of the animals. According to the results of these two behavioral tests, it appears that the effects of the Datura extract on the retention of the various learning tasks used do not seem to be due to an action on the level of emotionality or on general locomotor activity of the animals.
CHEMICAL ANALYSIS The activity of fractions that we obtained during the successive steps of chemical analysis were tested by comparing their effects on the avoidance component retention in Y-maze learning to those of the aqueous extract. The Y-maze learning and the retention test were carried out as previously described. Treatment was given 24 hr after learning. For each fraction, the dose injected was referred to the start weight of aqueous extract; the aqueous extract was injected at dose of 0.8 g/kg. The retention of the animals was tested 48 hr after treatment. The number of avoidance errors made by the animals during the retention test was recorded (Table 2).
Hydrolysis In order to determine if the activity of the aqueous extract could be suppressed by eliminating peptide bonds, acidic and basic hydrolysis of the aqueous extract was carried out. The aqueous extract (640 mg) was dissolved either in 10 ml of 0.1 N HC1 (acidic hydrolysis) or in 10 ml of 1 N NH4OH (basic hydrolysis) and heated to 100°C for 15 hr. The hydrolysates
a M ea n + SE.
E. D E A E and S ep had ex G-10
A 2.4 _+ 0.49
II 1.95 + 0.31 B 0.66 _+ 0.23
III 1.3 -+ 0.33
B 2.45 _+ 0.43
A 1.54 _+ 0.43
C. D E A E
I 2.31 _+ 0.30
Reliquate 1.3 -+ 0.26
Dialysate 3.2 _+ 0.62
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D. D E A E and S e p h a d e x G-25
0.1 N HC1 1.33
1 N NH4OH 1.6 -+ 0.45 a
A. H y d r o l y s i s
Avoidance errors NaC1 0.9 _+ 0.23 NaC1 0.9 +- 0.15 NaCI 1.09 _+ 0.34 NaC1 l . l _+ 0.31 NaCI 0.60 + 0.15
Datura 2.55 -+ 0.58 Datura 2.45 +- 0.51 Datura 3.18 -+_+0.68 Datura 3.1 -+ 0.50 Datura 2.11 +_ 0.38
F (3,34) = 7.38
F (4,67) = 3.77
F (3,39) = 4.772
F (3,42) = 6.11
F (3,34) - 2.87
F
TABLE 2 Effect of the F r a c t i o n s O b t a i n e d duri ng the S u c c e s s i v e Steps of C h e m i c a l A n a l y s i s on the R e t e n t i o n of Y - M a z e L e a r n i n g
< 0.001
< 0.01
< 0.006
< 0.01
= 0.05
P
360
UNGERER, SCHUBER, AND CHAUVIN
were then evaporated and dissolved in 20 ml of 0.9% NaCI. The animals treated with hydrolysates did not significantly differ from the NaC1 controls [F(1,34) = 1.05; P > 0.05] and made fewer errors compared to the animals treated with the aqueous extract [F(1,34) = 5.21; P < 0.05] (Table 2A). These results showed that the activity of the aqueous extract was suppressed by hydrolysis and that consequently, the active principle of this extract could be due to a peptide or a small protein. Dialysis The aqueous extract (480 mg dissolved in 10 ml of distilled water) was dialyzed against two changes of 10 vol of distilled water at 4°C for 24 hr each. The nondiffusible material (NDM) and the dialysate were then evaporated and dissolved in 0.9% NaC1. The dialysate impaired learning retention to the same extent as the aqueous extract [F(1,42) = 1.68; P > 0.05], whereas the nondiffusible material was without effect (NDM vs NaCI: F(1,42) = 0.06; P > 0.05) (Table 2B). These results indicate that the active principle has a molecular weight not greater than 20,000. Chromatography on DEAE-Cellulose and on Sephadex G-25 and G-IO The aqueous extract (480 mg) was dialyzed against distilled water; the dialysate, after freeze-drying, was dissolved in 0.05 M ammonium bicarbonate and layered onto a DEAE-cellulose column (1.6 x 35 cm). The column was eluted by a discontinuous gradient of 0.05 M (220 ml) and 0.3 M (270 ml) ammonium bicarbonate. Fractions collected were freeze-dried and then dissolved in 0.9% NaC1. Activity of the aqueous extract was concentrated in Fraction B (eluted at 0.3 M concentration), whereas Fraction A (eluted at 0.05 M concentration) was without effect on learning retention [Fraction A vs Datura:F(1,39) = 8.05; P < 0.01; Fraction B v s Datura: F(1,39) = 2.01; P > 0.05] (Table 2C). According to these results, the polarity of the active principle would be acidic. Fraction B, after evaporation and salt elimination, was dissolved in 0.025 M ammonium bicarbonate and layered onto a Sephadex G-25 Fine column (1.6 x 110 cm; void volume = 121 ml; flow rate = 12 ml/hr). Three fractions were collected, freeze-dried, and then dissolved in 0.9% NaCI: Fraction I (elution volume from 121 to 193 ml), Fraction II (elution volume from 193 to 229 ml), and Fraction III (elution volume from 229 to 380 ml). The extract activity was concentrated in Fraction I (Fraction I vs Datura: F(1,67) = 2.32; Fraction H vs Datura: F(1,67) = 4.87; Fraction III vs Datura: F(1,67) = 8.95; for o~-- .05 F(1,67) = 4.00] (Table 2D). According to these results, the molecular weight of the active principle is about 2000. Fraction I, after evaporation and salt elimination, was dissolved in 5 mM ammonium bicarbonate and layered onto a Sephadex G-10 column (1.6 x 50 cm; void volume = 43 ml; flow rate = 10 ml/hr). T w o fractions were collected, freeze-dried, and then dissolved in 0.9% NaCI: Fraction A
A FACTOR ISOLATED FROM Datura stramoniurn
361
corresponding to the void volume of the column and Fraction B corresponding to the bed volume (90 ml). The extract activity was concentrated in Fraction A (Fraction A vs Datura: [F(1,34) = 0.34; P > 0.05], whereas Fraction B was without effect (Fraction B vs NaCI: F(1,34) = 0.02; P > 0.05] (Table 2E). Consequently, it appears that the active principle is not retained by the Sephadex G-10 column. These results indicate that the active principle has a molecular weight greater than 700, which rules out the possibility that it may be an amino acid. Results of chemical analysis show that the active principle of the aqueous extract was hydrolyzable, dialyzable, and that its molecular weight is approximatively 2000. The Fraction A that we obtained after Sephadex G-10 chromatography was hydrolyzed by 6 N HC1 for 24 hr at 100°C. Chromatographic analysis revealed the presence of the following amino acids in the hydrolysate: Glu, Asp, Leu, Lys, Gly, and Ala.
DISCUSSION The Datura aqueous extract greatly impaired retention in the three learning situations used in this study. According to the results of control experiments, this impairment does not seem to be due to an action on the performance level of animals nor to an action on acquisition processes. Furthermore, the Datura extract had no effect on general locomotor activity nor on emotional reactivity of animals, as shown by actographic and open-field test results. In some experimental conditions, the Datura extract seemed to affect exploratory activity of animals. However, it appears difficult to ascribe the retention deficit produced by the Datura extract to such an action. Indeed, it is important to emphasize that the Datura extract injected 48 hr before maze learning did not disturb acquisition, though the animal was placed in a novel situation. However, the Datura extract greatly impaired performance in the retention tests, even though this situation then did not involve any novel component for the animal. Nevertheless, it should be emphasized that the Datura extract could affect some motivational processes and thus interfere with performance during retention testing. Further experiments are necessary to verify this hypothesis. However, if we consider all our results, these are in agreement with an interpretation of the Datura effects on learning retention in terms of an action on memory processes. Such an interpretation leads to several considerations. First, in none of the three learning tasks used, did the Datura extract erase all memory of the learned task. Second, the retention of a learned task is affected by the Datura extract for a relatively long period of time (24 hr), if we compare this time to the maximum effectiveness delays of other drugs or treatments that affect memory retention (Soumireu-Mourat, 1976). Nevertheless, 24-hr effectiveness delays have been observed with various treatments, especially with scopolamine (Wiener and Deutsch, 1968), physostigmine (Lewis and Bregman, 1972;
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Puerto et al., 1976), pentylenetetrazol (Pearlman et al., 1961), KC1 (Avis and Carlton, 1968; Buresova and Bures, 1971), and puromycin (Flexner et al., 1963; Ungerer, 1969). Treatments which impair memory with such long delays would not be likely to affect memory formation processes, but rather, would interfere either with memory recall or memory expression or would accelerate forgetting. The Datura extract, in view of its long gradient of effectiveness, could affect, as these other treatments, the same processes. However, according to the fact that the Datura effects are time dependent, we cannot rule out the possibility that the Datura extract could affect some slow memory formation processes (McGaugh, 1966). Finally, it should be emphasized that the Datura extract has long-lasting effects on memory retention since these effects were still observed 48 hr following treatment. The mechanisms by which the Datura extract impairs memory processes remains unknown. The presence of several amino acids in the active fraction obtained after DEAE-cellulose and gel-Sephadex filtration point to the possibility that the active principle of the Datura extract could be a small peptide. In agreement with this hypothesis, it should be noted that several studies in the last decade have shown the role of peptides in some behavioral processes. This role is not related to a hormonal action but seems to be due to either stimulating or inhibiting effects on cerebral activity (Iversen, 1974). It should likewise be recalled that peptide analogs of ACTH and vasopressin (ACTHI.~07-L-Phe, desglycinamide vasopressin) facilitated learning and delayed extinction of avoidance learning in rats independently of all endocrine effects (De Wied, 1973). These peptides appear to have a specific effect on cerebral activity. Indeed, when D-Phe was substituted to L-Phe in position 7 of ACTHI_10, the peptide so obtained delayed acquisition and increased the rate of extinction and, therefore, had behavioral effects opposite to those of ACTH~_~07-L-Phe. According to Schottman et al. (1972) the behavioral effects of these two peptides could be related to their action on cerebral protein synthesis. In fact, ACTH~_107-L-Phe had a stimulating effect on leucine incorporation in macromolecules of the brain stem, whereas ACTH~_~07-D-Phe caused a decrease in leucine incorporation. However, such an action cannot be ascribed in the case of the Datura extract, as shown by the lack of effects on valine incorporation in cerebral proteins (Ungerer, 1975). At present, analysis of the aqueous extract is being pursued to isolate and to specify the chemical nature of the active principle. This study will allow us to determine whether this active principle presents some analogies to the behaviorally active peptides discussed above. REFERENCES Archer, J. (1973). Tests for emotionality in rats and mice: A review. Anita. Behav. 21, 205-235. Arena, J. M. (1954). Poisoning in infants and children: I. Accidents and emergencies. Pediat. Clin. North Amer. 1:777.
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