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Learning Retardation and Enhanced Ethanol Preference Produced by Postnatal Pretreatments with Ethanol in Adult Rats
Learning
Retardation
by Postnatal
and
Enhanced
Pretreatments Tetsu
Behavioral
HAYASHI
Research
Ethanol with
Ethanol
and Sakutaro
December
in Adult
Produced Rats
TADOKORO
Institute, Gunma University Maebashi 371, Japan Accepted
Preference
School
of Medicine,
5, 1984
Abstract-Male neonates of Wistar strain rats were given 0.63-2.50 g/kg/day of ethanol, i.p., for 7 successive days from day 6 to 12 after birth. The acquisition processes of the discriminated lever-press avoidance response (intertrial interval: 25 sec, warning stimuli presentation: 5 sec, foot shock intensity: 110V, 0.5 mA, 50 Hz, AC) were investigated for 20 separate sessions from day 60 after birth. The preference test for ethanol was done beginning at 120 days of age. No significant differences in body weights were detected between saline and ethanol-pretreated groups. However, learning retardation was observed in all groups pretreated with ethanol. In these groups, slow responses to warning stimuli followed by escape responses from shocks delivered were often observed in early training sessions. An enhanced preference for ethanol was observed in all groups pretreated with ethanol for 7 successive days at maturity. These results suggest the possibility that learning ability and preference for ethanol in adult rats are strongly influenced by pretreatments with ethanol during the early postnatal period. In recent years, particular attention has been focused on alcohol as a teratogen. In the animal experiments, maternal ethanol ingestion during pregnancy has been reported to decrease the litter size or weights of the fetus (1-3). Morphological abnormalities have also been found in several organs of the neonates exposed to ethanol in utero (4, 5). Furthermore, the maternal ethanol ingestion produces several behavioral abnormalities, in particular learning deficits in the offspring at maturity (2, 6-9). These abnormalities observed in the animal experiments have been employed as a useful model of fetal alcohol syndrome (FAS) (10). However, few reports have been published concerning the effects of postnatal pretreatments with ethanol on behaviors or preference for ethanol in adult animals. The present study was designed to assess the effects of pretreatments with ethanol during --)n early postnatal period on learning ability and preference for ethanol in adult rats. Materials Animals
and
drug
and
Methods
treatments:
Eighty-two
male neonates obtained by 2 matings of 8 females with 3 male Wistar strain rats, which were supplied by the Institute of Experimental Animal Research of Gunma University School of Medicine, were used. Thirty-three rats were given i.p. several doses of ethanol (7.5-30% (V/V) solution), and the other 33 rats were given i.p. saline solution while they were kept with their mothers. Doses were made up to a volume of 10 ml/kg of body weight by diluting absolute ethanol with saline solution. The experimental schedules are presented in Table 1. As shown in the table, Groups I, II and III were given 0.63, 1.25 and 2.50 g/kg/day of ethanol, respectively, for 7 successive days from 6 to 12 days of age. Group IV was repeatedly given 2.50 g/kg/ session of ethanol immediately after training of the discriminated lever-press avoidance response for 20 separate sessions from 60 days of age. According to our experiences, 2.50 g/kg/day of ethanol for 7 successive days was the maximum safety dose for survival of the neonates. Groups V-VIII were prepared as controls for Groups I-IV, respectively, and were given the same
Table
volume
of saline
1.
Pretreatment
schedules
of
solution.
Groups of 5 or 6 animals were housed in stainless steel wire mesh cages of 38(D) x 25(VV)x20(H)cm after weaning at 21 days of age, and animals had free access to a solid diet of MF (Oriental Yeast Co., Tokyo) and tap water. The animals were kept in a room at 24±2'C with a 1 2 hr light-dark cycle (fluorescent illumination on 6:00-18:00 hr). However, the humidity was not controlled. Ethanol administration was conducted at 18:00 hr in all the groups except for Groups IV and VIII. In the latter groups, ethanol was given between 10:00-16:00 hr. The body weights were recorded every day during the ethanol treatment and one time per 10 days from 13 to 60 days of age. Discriminated lever-press avoidance schedule: The acquisition of discriminated avoidance response was investigated begin ning at 60 days of age. The animals were trained to avoid an electric shock by pressing a lever according to warning stimuli presented in an operant chamber (27(D)x20(1/\/)x18 (H)cm) (O'hara and Co., Ltd., Tokyo). The chamber was enclosed in a ventilated sound attenuating box. The avoidance schedule consisted of a 25 sec intertrial interval and a 5 sec presentation of the warning stimuli (buzzer and small lamp) (11 ). The electric shock of 110V, 0.5 mA, 50 Hz, AC was delivered through the stainless steel floor grids. The maximum duration of shock presentation was 5 sec, because an escape contingency was considered in the schedule. Each session consisted of 1 hr training per day, and the training was held every other day for 20 sessions. The indices for the evaluation of the acquisition processes were the response rate (no. of lever-pressings/
ethanol
and
experimental
conditions
min) and the avoidance rate (percent of correct response). The warm-up period data for the first 20 min of each session were excluded from the calculation of the mean avoidance and response rates in order to discard unstable behavioral responses (12). Behavior controlling and recording ap paratuses (GT 7705 and GT 7715, O'hara Co., Ltd., Tokyo) were placed in a separate room. Gross behaviors in the operant chamber were observed through a small window on the sound-attenuating box. Ethanol preference test: Continuous and simultaneous measurements of drinking and ambulatory activities were recorded auto matically beginning at 120 days of age with an ambulo-drinkometer (O'hara and Co., Ltd., Tokyo). A water tank was connected with a drinking spout through a cartridge, which accurately released a water drop of 0.05 ml. The short circuit generated by each drop was amplified to activate an electro magnetic counter. Numbers of counts were automatically recorded. Thus, the apparatus permitted an exact determination of the amount of water consumed. A tilting floor grid, which was set in a stainless steel cage (40(D)x25(W)x25(H)cm) was used for measurment of ambulatory activity. Any slight tilt of the floor caused by ambulatory movement of a rat was detected by a micro switch which activates an electromagnetic counter. The principles of the measurement have already been reported in detail by us (13, 14). Preference for drinking solutions was measured using a free-choice procedure in which tap water and 5% or 10% ethanol solutions were simultaneously presented to the rats by use of 2 separate drinking spouts
for 6 successive days. During the preference test period, positions of the 2 spouts were switched randomly (15). The rats were allowed access to tap water for 10 days between the tests. The preference rate, which indicates the amount of ethanol solution consumed as a percent of the total solution consumed during a 24 hr period, was used for the evaluation of the test results. Statistical analysis: Differences between groups were assessed statistically by one way ANOVA followed by Student's t-test. They were considered significant when P was equal to or less than 0.05. Results Body weight No significant differences in body weight between ethanol and saline-pretreated groups were detected during the ethanol treatment and thereafter (Fig. 1 ). The mean body weights of ethanol-pretreated groups (Groups I-III) at the beginning of the training were 225.3±7.2 g, 242.6±1 2.4 g and 222.1 ±1 5.6 g, respectively, and those of the saline-pretreated groups (average of Groups V-VII) at the same time were 252.7±6.5 g. Discriminated lever-press avoidance response Saline-pretreated groups: Unstable and ineffective lever-pressing was often observed
Fig.
1.
Changes
pretreatments pretreated changes
in body with
rats. in body
ethanol
weights in
saline
The pretreatments weights.
during and
ar.d
after
ethanol
produced
no
in the 1st-3rd sessions of the training, but the lever-pressing was stabilized by the 7th 8th sessions, and a stable behavioral baseline in response rate was established. These rats seldom left the lever even when foot shock was delivered and immediately pressed the lever to escape it. The avoidance rates reached 95% in the 7th session, and this level was maintained thereafter. The acquisition processes of avoidance conditioning in saline-pretreated rats are represented by dotted lines in Figs. 2-5. Ethanol-pretreated groups: Learning re tardation was produced in Groups I-III (Figs. 2-4). No significant differences in the mean response rate were detected in Groups -III when compared with that of the cor responding saline-pretreated groups. How ever, slow responses to the warning stimuli and subsequently escape responses were often seen in the cases where learning retardation was observed. These rats usually remained near the lever and pressed it when
Fig. 2. Effect of postnatal pretreatments with ethanol (0.63 g/kg/dayx7) on acquisition of con ditioned avoidance response (CAR) in adult rats. The pretreatments produced learning retardation dose-dependently. A------!\ Saline-pretreated (N=10), Group I (ethanol-pretreated, 0.63 g/kg/dayx7) (N=12), Upper panel: Changes in avoidance rates, Lower panel: Changes in response rates, • Significantly different from each corresponding 0 (at least P<0.01)
foot shock was delivered. Significantly lower levels avoidance rate were detected
of the mean in the 4th-7th
sessions in Group I, in the 2nd-7th sessions except for the 6th session in Group II and in the 3rd-9th sessions in Group III, when compared with those of the corresponding saline-pretreated groups. However, the avoidance rate was progressively improved in all ethanol-pretreated groups after the 10th session, and it reached 80-90% by the 20th session. Thus, no significant differences in the mean avoidance rate were detected between ethanol and saline-pretreated groups in the 10th session and thereafter. On the other hand, no differences in the response and avoidance rates were detected between Groups IV and VIII (Fig. 5). Ethanol preference test
Fig. 3. Effect of postnatal pretreatments with ethanol (1.25 g/kg/dayx7) on acquisition of CAR in adult rats. A------A Saline-pretreated (N=12), 0 Group II (ethanol-pretreated, 1.25 g/kg/ dayx7) (N=11). The data are shown in the same way as in Fig. 2.
Fig. 4. Effect of postnatal pretreatments with ethanol (2.50 g/kg/dayx7) on acquisition of CAR in adult rats. A------A Saline-pretreated (N=11), 0-0 Group III (ethanol-pretreated, 2.50 g/kg/ dayx7) (N=10). The data are shown in the same way as in Fig. 2.
Fig. 5. Effect of treatments with ethanol (2.50 g/ kg/session x20) during acquisition of CAR in 60 days-old rats. The treatments after maturation produced no learning retardation. A------A Treated with saline (N=8), J Group IV (treated with ethanol during the acquisition, 2.50 g/kg/session x20) (N=8) . The data are shown in the same way as in Fig. 2.
ambulation in 24 hr were recorded in the dark period, and no significant differences in the activities were detected between the groups. Saline-pretreated group: The saline pretreated group tended to consume more tap water than ethanol solution. In a preference test for 5% ethanol solution, ethanol solution consumed accounted for about 50% of the total solution consumed in the 1st and 2nd test days. However, the preference rate decreased thereafter, and it was 30% in an average over a period of 4 test days. The preference rate for 10% ethanol was 37% in an average throughout the total test periods (Fig. 6). The ambulatory activity observed in the saline-pretreated group did not differ sig nificantly from that obtained in the period preceding the preference test (pre-test period) throughout the total test periods. Ethanol-pretreated groups: Groups I-III consumed more 5% ethanol solution than tap water. Preference rates for 5% ethanol in these groups were 90% in an average throughout the total test periods, and they were significantly higher than that in the saline-pretreated group. However, there was no specific relationship between the
Fig. 6. Preference test for 5 or 10% ethanol solution in saline and ethanol-pretreated rats. Pretreatments with ethanol in the early postnatal period produced an enhanced preference for ethanol after maturation. C------C Saline-pretreated (N=10). Ethanol-pretreated, --A Group I (0.63 g/kg/ day x7) (N=10), El Group II (1.25 g/kg/ day x7) (N=10), 0-0 Group III (2.50 g/kg/ day x7) (N=10), A, /, • Significantly different from each corresponding 0 (at least P<0.05).
preference rates and the pretreated doses of ethanol. In the preference test for 10% ethanol, all ethanol-pretreated groups also tended to consume more ethanol than tap water. The preference rates for 10% ethanol in these groups averaged 70% throughout the total test periods, independently of the pretreated significantly
doses of ethanol. The rates were higher than that in the saline
pretreated group in the 1st and 4th test days in Group I, in the 1st-4th test days in Group II and in the 1st-5th test days in Group 111, respectively, as shown in Fig. 6. No significant differences in the ambulatory activity were obtained during the test periods in all the groups when compared with that obtained in the pre-test period.
Discussion The learning retardation observed in the present experiment resulted from the postnatal pretreatments with ethanol. The intensity of the learning retardation is con sidered to depend on the ethanol dosage pretreated. On the other hand, no significant difference in learning ability was detected between groups repeatedly given saline and ethanol solution immediately after the training sessions. These results suggest that there may be a critical period for producing the learning retardation following the postnatal ethanol -pretreatments.
may be due to hypoirritability. The offspring of mothers pretreated with 2-12.6 g/kg/day or 3.57-14.3 g/kg/day of ethanol throughout pregnancy were found to exhibit impaired two-way shuttle box avoidance (6) or passive avoidance learning (7), respectively. Similar learning deficits were also observed in the offspring of mothers pretreated with barbiturates. Ac cording to Middaugh et al. (17, 18), in the offspring of mice pretreated with 40 mg/kg of phenobarbital during the last 7 days of pregnancy, deficits in passive avoidance learning were found. Furthermore, a lack of responsiveness to environmental stimuli was pointed out for the learning deficit. It is, therefore, supposed that these results show a nature common in many respects with the effects of ethanol-pretreatment in the present experiment, although the method of the pretreatment is different.
and even the forced administration of ethanol not always produces an enhanced preference for ethanol over a long period (25, 26). On the other hand, the offspring of mothers which received ethanol during pregnancy are reported to show an enhanced preference for ethanol at maturity (27, 28). These results show a similarity to those obtained in the present experiment, although the methods are different. The mechanism of the enhanced preference for ethanol observed in the present experiment is not yet elucidated. It is thought, however, that the phenomenon is not caused by a preference for taste or smell of ethanol or by position habit (15), but rather by a functional disorder of the central nervous system. According to Riley et al. (7), rats whose mothers consumed liquid diets containing 35% ethanol during 6-20 days of pregnancy showed a deficit in the passive avoidance test and consumed more lithium chloride solution (aversive taste) than did controls. As a possible cause of these phenomena, deficit in a central inhibitory system was emphasized. Krsiak et al. (29) reported that male offspring of mice pretreated with ethanol (1 g/kg/day) throughout the whole pregnancy period had lower brain serotonin concentrations than did controls. Furthermore, administration of p-chlorophenylalanine, the tryptophan hy droxylase inhibitor, increased ethanol intake in rats (30). On the contrary, 5-hydroxy tryptophan, the serotonin precursor, reduced ethanol intake (30). These findings suggest that there may be an important relationship between serotonergic function in the brain and ethanol intake. The results obtained in the present experiment suggest the possibility that learning ability or preference for ethanol in adult rats are strongly influenced by pre treatments with ethanol during the early postnatal period, even though the mechanism still remains to be elucidated. References
1 Lee, M. and Leichter, J.: Effect of litter size on the physical growth and maturation of the offspring of rats given alcohol during gestation. Growth 44, 327-335 (1980) 2
Abel,
E.L.:
Prenatal
effects
of
alcohol
on
adult
learning in rats. Pharmacol. Biochem. Behav. 10, 239-243 (1979) 3 Franzeska, G.E. and Margaret, G.C.: Effects of ethyl alcohol on development and social behavior in the offspring of laboratory mice. Psychopharmacology (Berlin) 62, 247-251 (1979) 4 West, J.R., Hodges, C.A. and Black, A.C.: Prenatal exposure to ethanol alters the organi zation of hippccampal mossy fibers in rats. Science 211, 957-959 (1981) 5 Barnes, D.E. and Walker, D.W.: Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus. Dev. Brain Res. 1, 333-340 (1981) 6 Bond, N.W. and DiGiusto, E.L.: Avoidance conditioning and Hebb-Williams maze per formance in rats treated prenatally with alcohol. Psychopharmacology (Berlin) 58, 69-71 (1978) 7 Riley, E.P., Lochry, E.A. and Shapiro, N.R.: Lack of response inhibition in rats prenatally exposed to alcohol. Psychopharmacology (Berlin) 62, 47-52 (1979) 8 Riley, E.P., Lochry, E.A., Shapiro, N.R. and Boldwin, J.: Response preservation in rats exposed to alcohol prenatally. Pharmacol. Biochem. Behav. 10, 255-259 (1979) 9 Bond, N.W. and DiGiusto, E.L.: Prenatal alcohol consumption and open-field behavior in rats: Effects of age at time testing. Psychopharma cology (Berlin) 52, 311-312 (1977) 10 Jones, K.L., Smith, D.W., Ulleland, C.N. and Streissguth, A.P.: Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1, 1267-1271 (1973) 11 Hayashi, T. and Tadokoro, S.: Learning deficits produced by postnatal pretreatment with chlorpromazine in adult rats. Neurobehav. Toxicol. 3, 27-35 (1981) 12 Hoffman, H.S., Fleshier, M. and Chorny, H.: Discriminated bar-press avoidance. J. Exp. Anal. Behav. 4, 309-316 (1961 ) 13 Kuribara, H., Hayashi, T., Alam, M.R. and Tadokoro, S.: Automatic measurement of drinking in rats: Effects of hypophysectomy. Pharmacol. Biochem. Behav. 9, 697-702 (1978) 14 Tadokoro, S., Kuribara, H., Shirasaka, K., Alam, M.R. and Fujimoto, K.: Fully automatic measure ment of behavioral rhythms in rats and its applications. Japan. J. Neuropsychopharmacol. 3, 785-803 (1981 ) (Abs. in English) 15 Myers, R.D. and Holman, R.B.: A procedure for eliminating position habit in preference aversion test for ethanol and other fluids. Psychol. Sci. 6, 235-236 (1966)
16 Villescas, R., Zamenhof, S. and Guthrie, D.: The effects of early stress and undernutrition on the behavior of young adult rats and the cor relations between behavioral and brain parameters. Physiol. Behav. 23, 945-954 (1979) 17 Middaugh, L.D., Drake, S.T., Fraley, J.E. and Zemp, J.W.: Effects of subanesthetic doses of phenobarbital administered to pregnant mice on behavior of offspring. Fed. Proc. 31, 2147 (1972) 18 Middaugh, L.D., Santos, C.A., III and Zemp, J.W.: Effects of phenobarbital given to pregnant mice on behavior of mature offspring. Dev. Psychobiol. 18, 305-313 (1975) 19 Ho, B.T., Fritchie, G.E., Idanpaan-Heikkila, J.E. and Mclsaac, W.M.: Placental transfer and tissue distribution of ethanol-1 -14C: a radio autographic study in monkeys and hamsters. 0. J. Stud. Alcohol 33, 485-494 (1972) 20 Nomura, Y.: Functional development of the synaptic transmission in the rat CNS and its interaction with drugs. Folia Pharmacol. Japon. 76, 413-427 (1980) (Abs. in English) 21 Thadani, P.V., Christopher, L., Slotkin, T.A. and Schanberg, S.M.: Effects of maternal ethanol injection on amine uptake into synaptosomes of fetal and neonatal rat brain. J. Pharmacol. Exp. Ther. 200, 292-297 (1977) 22 Lucchi, L., Covelli, V., Spano, P.F. and Trabucchi, M.: Acute ethanol administration during pregnancy: Effects on central dopa minergic transmission in rat offspring. Neuro behav. Toxicol. Teratol. 6, 19-21 (1984) 23 Lucchi, L., Covelli, V., Petkov, V.V., Spano, P.F. and Trabucchi, M.: Effects of ethanol, given during pregnancy, on the offspring dopaminergic system. Pharmacol. Biochem. Behav. 19, 567 570 (1983) 24 Detering, N., Collins, R.M., Jr., Hawkins, R.L., Ozand, P.T. and Karahasan, A.: Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: A long-lasting effect in the hypothalamus. J. Neurochem. 36, 2094-2096 (1981) 25 Veale, W.L.: Ethanol selection in the rat following forced acclimation. Pharmacol. Biochem. Behav. 1,233-235(1973) 26 Myers, R.D. and Carey, R.: Preference factors in experimental alcoholism. Science 134, 469-470 (1961) 27 Bond, N.W. and DiGiusto, E.L.: Effects of prenatal alcohol consumption on open-field behavior and alcohol preference in rats. Psychopharmacologia (Berlin) 46, 163-165 (1976) 28
Phillips,
D.S.
and
Stainbrook,
G.L.:
Effects
of
early alcohol exposure upon adult learning ability and taste preferences. Physiol. Psychol. 4, 473-475 (1976) 29
Krsiak,
M.,
Elis,
J.,
Poschlova,
N. and
Masek,
K.:
during gestation. J. Stud. Alc. 38, 1696-1704 (1977) 30 Geller, I.: Effects of para-chlorophenylalanine and 5-hydroxytryptophan on alcohol intake in the rat. Pharmacol. Biochem. Behav. 1, 361 365 (1973)
Retardation
by Postnatal
and
Enhanced
Pretreatments Tetsu
Behavioral
HAYASHI
Research
Ethanol with
Ethanol
and Sakutaro
December
in Adult
Produced Rats
TADOKORO
Institute, Gunma University Maebashi 371, Japan Accepted
Preference
School
of Medicine,
5, 1984
Abstract-Male neonates of Wistar strain rats were given 0.63-2.50 g/kg/day of ethanol, i.p., for 7 successive days from day 6 to 12 after birth. The acquisition processes of the discriminated lever-press avoidance response (intertrial interval: 25 sec, warning stimuli presentation: 5 sec, foot shock intensity: 110V, 0.5 mA, 50 Hz, AC) were investigated for 20 separate sessions from day 60 after birth. The preference test for ethanol was done beginning at 120 days of age. No significant differences in body weights were detected between saline and ethanol-pretreated groups. However, learning retardation was observed in all groups pretreated with ethanol. In these groups, slow responses to warning stimuli followed by escape responses from shocks delivered were often observed in early training sessions. An enhanced preference for ethanol was observed in all groups pretreated with ethanol for 7 successive days at maturity. These results suggest the possibility that learning ability and preference for ethanol in adult rats are strongly influenced by pretreatments with ethanol during the early postnatal period. In recent years, particular attention has been focused on alcohol as a teratogen. In the animal experiments, maternal ethanol ingestion during pregnancy has been reported to decrease the litter size or weights of the fetus (1-3). Morphological abnormalities have also been found in several organs of the neonates exposed to ethanol in utero (4, 5). Furthermore, the maternal ethanol ingestion produces several behavioral abnormalities, in particular learning deficits in the offspring at maturity (2, 6-9). These abnormalities observed in the animal experiments have been employed as a useful model of fetal alcohol syndrome (FAS) (10). However, few reports have been published concerning the effects of postnatal pretreatments with ethanol on behaviors or preference for ethanol in adult animals. The present study was designed to assess the effects of pretreatments with ethanol during --)n early postnatal period on learning ability and preference for ethanol in adult rats. Materials Animals
and
drug
and
Methods
treatments:
Eighty-two
male neonates obtained by 2 matings of 8 females with 3 male Wistar strain rats, which were supplied by the Institute of Experimental Animal Research of Gunma University School of Medicine, were used. Thirty-three rats were given i.p. several doses of ethanol (7.5-30% (V/V) solution), and the other 33 rats were given i.p. saline solution while they were kept with their mothers. Doses were made up to a volume of 10 ml/kg of body weight by diluting absolute ethanol with saline solution. The experimental schedules are presented in Table 1. As shown in the table, Groups I, II and III were given 0.63, 1.25 and 2.50 g/kg/day of ethanol, respectively, for 7 successive days from 6 to 12 days of age. Group IV was repeatedly given 2.50 g/kg/ session of ethanol immediately after training of the discriminated lever-press avoidance response for 20 separate sessions from 60 days of age. According to our experiences, 2.50 g/kg/day of ethanol for 7 successive days was the maximum safety dose for survival of the neonates. Groups V-VIII were prepared as controls for Groups I-IV, respectively, and were given the same
Table
volume
of saline
1.
Pretreatment
schedules
of
solution.
Groups of 5 or 6 animals were housed in stainless steel wire mesh cages of 38(D) x 25(VV)x20(H)cm after weaning at 21 days of age, and animals had free access to a solid diet of MF (Oriental Yeast Co., Tokyo) and tap water. The animals were kept in a room at 24±2'C with a 1 2 hr light-dark cycle (fluorescent illumination on 6:00-18:00 hr). However, the humidity was not controlled. Ethanol administration was conducted at 18:00 hr in all the groups except for Groups IV and VIII. In the latter groups, ethanol was given between 10:00-16:00 hr. The body weights were recorded every day during the ethanol treatment and one time per 10 days from 13 to 60 days of age. Discriminated lever-press avoidance schedule: The acquisition of discriminated avoidance response was investigated begin ning at 60 days of age. The animals were trained to avoid an electric shock by pressing a lever according to warning stimuli presented in an operant chamber (27(D)x20(1/\/)x18 (H)cm) (O'hara and Co., Ltd., Tokyo). The chamber was enclosed in a ventilated sound attenuating box. The avoidance schedule consisted of a 25 sec intertrial interval and a 5 sec presentation of the warning stimuli (buzzer and small lamp) (11 ). The electric shock of 110V, 0.5 mA, 50 Hz, AC was delivered through the stainless steel floor grids. The maximum duration of shock presentation was 5 sec, because an escape contingency was considered in the schedule. Each session consisted of 1 hr training per day, and the training was held every other day for 20 sessions. The indices for the evaluation of the acquisition processes were the response rate (no. of lever-pressings/
ethanol
and
experimental
conditions
min) and the avoidance rate (percent of correct response). The warm-up period data for the first 20 min of each session were excluded from the calculation of the mean avoidance and response rates in order to discard unstable behavioral responses (12). Behavior controlling and recording ap paratuses (GT 7705 and GT 7715, O'hara Co., Ltd., Tokyo) were placed in a separate room. Gross behaviors in the operant chamber were observed through a small window on the sound-attenuating box. Ethanol preference test: Continuous and simultaneous measurements of drinking and ambulatory activities were recorded auto matically beginning at 120 days of age with an ambulo-drinkometer (O'hara and Co., Ltd., Tokyo). A water tank was connected with a drinking spout through a cartridge, which accurately released a water drop of 0.05 ml. The short circuit generated by each drop was amplified to activate an electro magnetic counter. Numbers of counts were automatically recorded. Thus, the apparatus permitted an exact determination of the amount of water consumed. A tilting floor grid, which was set in a stainless steel cage (40(D)x25(W)x25(H)cm) was used for measurment of ambulatory activity. Any slight tilt of the floor caused by ambulatory movement of a rat was detected by a micro switch which activates an electromagnetic counter. The principles of the measurement have already been reported in detail by us (13, 14). Preference for drinking solutions was measured using a free-choice procedure in which tap water and 5% or 10% ethanol solutions were simultaneously presented to the rats by use of 2 separate drinking spouts
for 6 successive days. During the preference test period, positions of the 2 spouts were switched randomly (15). The rats were allowed access to tap water for 10 days between the tests. The preference rate, which indicates the amount of ethanol solution consumed as a percent of the total solution consumed during a 24 hr period, was used for the evaluation of the test results. Statistical analysis: Differences between groups were assessed statistically by one way ANOVA followed by Student's t-test. They were considered significant when P was equal to or less than 0.05. Results Body weight No significant differences in body weight between ethanol and saline-pretreated groups were detected during the ethanol treatment and thereafter (Fig. 1 ). The mean body weights of ethanol-pretreated groups (Groups I-III) at the beginning of the training were 225.3±7.2 g, 242.6±1 2.4 g and 222.1 ±1 5.6 g, respectively, and those of the saline-pretreated groups (average of Groups V-VII) at the same time were 252.7±6.5 g. Discriminated lever-press avoidance response Saline-pretreated groups: Unstable and ineffective lever-pressing was often observed
Fig.
1.
Changes
pretreatments pretreated changes
in body with
rats. in body
ethanol
weights in
saline
The pretreatments weights.
during and
ar.d
after
ethanol
produced
no
in the 1st-3rd sessions of the training, but the lever-pressing was stabilized by the 7th 8th sessions, and a stable behavioral baseline in response rate was established. These rats seldom left the lever even when foot shock was delivered and immediately pressed the lever to escape it. The avoidance rates reached 95% in the 7th session, and this level was maintained thereafter. The acquisition processes of avoidance conditioning in saline-pretreated rats are represented by dotted lines in Figs. 2-5. Ethanol-pretreated groups: Learning re tardation was produced in Groups I-III (Figs. 2-4). No significant differences in the mean response rate were detected in Groups -III when compared with that of the cor responding saline-pretreated groups. How ever, slow responses to the warning stimuli and subsequently escape responses were often seen in the cases where learning retardation was observed. These rats usually remained near the lever and pressed it when
Fig. 2. Effect of postnatal pretreatments with ethanol (0.63 g/kg/dayx7) on acquisition of con ditioned avoidance response (CAR) in adult rats. The pretreatments produced learning retardation dose-dependently. A------!\ Saline-pretreated (N=10), Group I (ethanol-pretreated, 0.63 g/kg/dayx7) (N=12), Upper panel: Changes in avoidance rates, Lower panel: Changes in response rates, • Significantly different from each corresponding 0 (at least P<0.01)
foot shock was delivered. Significantly lower levels avoidance rate were detected
of the mean in the 4th-7th
sessions in Group I, in the 2nd-7th sessions except for the 6th session in Group II and in the 3rd-9th sessions in Group III, when compared with those of the corresponding saline-pretreated groups. However, the avoidance rate was progressively improved in all ethanol-pretreated groups after the 10th session, and it reached 80-90% by the 20th session. Thus, no significant differences in the mean avoidance rate were detected between ethanol and saline-pretreated groups in the 10th session and thereafter. On the other hand, no differences in the response and avoidance rates were detected between Groups IV and VIII (Fig. 5). Ethanol preference test
Fig. 3. Effect of postnatal pretreatments with ethanol (1.25 g/kg/dayx7) on acquisition of CAR in adult rats. A------A Saline-pretreated (N=12), 0 Group II (ethanol-pretreated, 1.25 g/kg/ dayx7) (N=11). The data are shown in the same way as in Fig. 2.
Fig. 4. Effect of postnatal pretreatments with ethanol (2.50 g/kg/dayx7) on acquisition of CAR in adult rats. A------A Saline-pretreated (N=11), 0-0 Group III (ethanol-pretreated, 2.50 g/kg/ dayx7) (N=10). The data are shown in the same way as in Fig. 2.
Fig. 5. Effect of treatments with ethanol (2.50 g/ kg/session x20) during acquisition of CAR in 60 days-old rats. The treatments after maturation produced no learning retardation. A------A Treated with saline (N=8), J Group IV (treated with ethanol during the acquisition, 2.50 g/kg/session x20) (N=8) . The data are shown in the same way as in Fig. 2.
ambulation in 24 hr were recorded in the dark period, and no significant differences in the activities were detected between the groups. Saline-pretreated group: The saline pretreated group tended to consume more tap water than ethanol solution. In a preference test for 5% ethanol solution, ethanol solution consumed accounted for about 50% of the total solution consumed in the 1st and 2nd test days. However, the preference rate decreased thereafter, and it was 30% in an average over a period of 4 test days. The preference rate for 10% ethanol was 37% in an average throughout the total test periods (Fig. 6). The ambulatory activity observed in the saline-pretreated group did not differ sig nificantly from that obtained in the period preceding the preference test (pre-test period) throughout the total test periods. Ethanol-pretreated groups: Groups I-III consumed more 5% ethanol solution than tap water. Preference rates for 5% ethanol in these groups were 90% in an average throughout the total test periods, and they were significantly higher than that in the saline-pretreated group. However, there was no specific relationship between the
Fig. 6. Preference test for 5 or 10% ethanol solution in saline and ethanol-pretreated rats. Pretreatments with ethanol in the early postnatal period produced an enhanced preference for ethanol after maturation. C------C Saline-pretreated (N=10). Ethanol-pretreated, --A Group I (0.63 g/kg/ day x7) (N=10), El Group II (1.25 g/kg/ day x7) (N=10), 0-0 Group III (2.50 g/kg/ day x7) (N=10), A, /, • Significantly different from each corresponding 0 (at least P<0.05).
preference rates and the pretreated doses of ethanol. In the preference test for 10% ethanol, all ethanol-pretreated groups also tended to consume more ethanol than tap water. The preference rates for 10% ethanol in these groups averaged 70% throughout the total test periods, independently of the pretreated significantly
doses of ethanol. The rates were higher than that in the saline
pretreated group in the 1st and 4th test days in Group I, in the 1st-4th test days in Group II and in the 1st-5th test days in Group 111, respectively, as shown in Fig. 6. No significant differences in the ambulatory activity were obtained during the test periods in all the groups when compared with that obtained in the pre-test period.
Discussion The learning retardation observed in the present experiment resulted from the postnatal pretreatments with ethanol. The intensity of the learning retardation is con sidered to depend on the ethanol dosage pretreated. On the other hand, no significant difference in learning ability was detected between groups repeatedly given saline and ethanol solution immediately after the training sessions. These results suggest that there may be a critical period for producing the learning retardation following the postnatal ethanol -pretreatments.
may be due to hypoirritability. The offspring of mothers pretreated with 2-12.6 g/kg/day or 3.57-14.3 g/kg/day of ethanol throughout pregnancy were found to exhibit impaired two-way shuttle box avoidance (6) or passive avoidance learning (7), respectively. Similar learning deficits were also observed in the offspring of mothers pretreated with barbiturates. Ac cording to Middaugh et al. (17, 18), in the offspring of mice pretreated with 40 mg/kg of phenobarbital during the last 7 days of pregnancy, deficits in passive avoidance learning were found. Furthermore, a lack of responsiveness to environmental stimuli was pointed out for the learning deficit. It is, therefore, supposed that these results show a nature common in many respects with the effects of ethanol-pretreatment in the present experiment, although the method of the pretreatment is different.
and even the forced administration of ethanol not always produces an enhanced preference for ethanol over a long period (25, 26). On the other hand, the offspring of mothers which received ethanol during pregnancy are reported to show an enhanced preference for ethanol at maturity (27, 28). These results show a similarity to those obtained in the present experiment, although the methods are different. The mechanism of the enhanced preference for ethanol observed in the present experiment is not yet elucidated. It is thought, however, that the phenomenon is not caused by a preference for taste or smell of ethanol or by position habit (15), but rather by a functional disorder of the central nervous system. According to Riley et al. (7), rats whose mothers consumed liquid diets containing 35% ethanol during 6-20 days of pregnancy showed a deficit in the passive avoidance test and consumed more lithium chloride solution (aversive taste) than did controls. As a possible cause of these phenomena, deficit in a central inhibitory system was emphasized. Krsiak et al. (29) reported that male offspring of mice pretreated with ethanol (1 g/kg/day) throughout the whole pregnancy period had lower brain serotonin concentrations than did controls. Furthermore, administration of p-chlorophenylalanine, the tryptophan hy droxylase inhibitor, increased ethanol intake in rats (30). On the contrary, 5-hydroxy tryptophan, the serotonin precursor, reduced ethanol intake (30). These findings suggest that there may be an important relationship between serotonergic function in the brain and ethanol intake. The results obtained in the present experiment suggest the possibility that learning ability or preference for ethanol in adult rats are strongly influenced by pre treatments with ethanol during the early postnatal period, even though the mechanism still remains to be elucidated. References
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