147
Behavioural Brain Research, 36 (1990) 147-154 Elsevier BBR00987
Long-term effects of developmental halothane exposure on radial arm maze performance in rats E d w a r d D. L e v i n ~, R a u l D e L u n a 2, E t s u r o U e m u r a 3 a n d R o b e r t E. B o w m a n 2 1Department of Psychiatry, Duke University, Durham, NC27710 (U.S.A.), 2Harlow Primate Lab, Department of Psychology, University of Wisconsin, Madison, W153715, (U.S.A.) and 3Department of Veterinary Anatomy, Iowa State University, Ames, IA 50011 (U.S.A.) (Received 23 June 1987) (Revised version received 19 May 1989) (Accepted 22 May 1989)
Key words: Halothane; Radial-arm maze; Behavioral teratology; Memory; Rat
Chronic exposure of rats to low levels of halothane during development, a treatment which retards synaptogenesis, was found to cause a long-term impairment of choice accuracy in the radial-arm maze. In Expt. 1, the relative importance of dose level and dosing regimen was examined. Dose level seemed the more critical variable for causing impaired choice accuracy. Exposure to 100 parts per million (ppm) of halothane in the air either on an intermittent or continuous schedule from day two of conception until 60 days after birth significantly impaired choice accuracy, whereas exposure to 25 ppm on a continuous schedule did not cause a deficit, even though with this condition the total amount of halothane exposure was about the same as with 100 ppm given intermittently. In Expt. 2, the 100 ppm intermittent exposure regimen was used to examine the relative importance of exposure during early and late developmental periods for producing the cognitive effects ofhalothane. Groups were divided into those exposed to halothane during gestation and until 30 days after birth (early exposure), those exposed from day 31 until day 90 (late exposure) and those exposed during both early and late periods (combined exposure). Adverse effects on choice accuracy were seen with all 3 types of exposure, but surprisingly, it was the late exposure that caused the most severe effects. These results show that developmental exposure to halothane which impairs synaptogenesis also causes long-lasting cognitive impairment. Halothane exposure can be a useful experimental tool for examining the relationship between synaptic and behavioral development.
INTRODUCTION
Halothane, a commonly used surgical anesthetic, has been found to cause adverse behavioral effects in operating room personnel chronically exposed to low concentrations in the air 5. Tests of cognitive function have been found to be particularly sensitive to low level effects of halothane 2~. The same is true in rodent models of toxicity, in which exposure during development is quite effective in causing both n e u r a l 7'8"13'18"21-23"25 and behavioral 6"12'13"18"19'23 impairment. The halo-
thane-induced cognitive impairment in rodents has been seen in tests of light-dark discrimination 1a'18'19, spontaneous alternation 13,23 and exploratory behavior in the radial-arm maze 12. The present study further examined halothane effects in the radial-arm maze, using choice accuracy as an index of cognitive performance. Radial-arm maze choice accuracy has been found to be sensitive to developmental exposure to other toxicants and drugs, including lead L4, trimentyltin z4 and diazepam 3. Regarding possible neurological sites for these effects, the radial-arm
Correspondence: E.D. Levin, Nicotine Research Lab (151), VA Medical Center, 508 Fulton St., Durham, NC 27705, U.S.A. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
148 maze has proven to be quite sensitive to certain types of brain lesions and pharmacological manipulations. The hippocampus and related areas are especially crucial for accurate radialarm maze performance2,1°,16,17, and this has been shown in particular for pharmacological manipulations of the cholinergic innervation of the hippocampus 11. These findings are particularly relevant to the present investigation because we have previously found that the hippocampus and related structures are very sensitive to the effects of developmental halothane exposure in retarding synaptogenesis 13"23.Halothane is quite effective in retarding synaptogenesis as was seen in the siblings of the rats tested in the present study 13'23. The retarded synaptogenesis has been particularly apparent in the hippocampus. This suggests that halothane exposure may be useful as an experimental tool for examining the critical phases of development for a variety of behavioral functions. Given its effects on hippocampal development, halothane exposure may be particularly useful as a tool in studying the neurological substrates of cognitive development. The first experiment in the present study was designed to further develop the use of halothane as an investigatory tool by determining the dosing parameters critical for producing cognitive effects. The second experiment was designed to use halothane as a tool in the investigation of critical periods of development of cognitive skills needed to solve the radial-arm maze. The relative effects of halothane exposure during early development was compared to exposure during juvenile development. In one of our previous studies lz halothane exposure during the juvenile period was crucial for producing a cognitive deficit in a light-dark discrimination shock escape task. The effect of halothane in suppressing synaptogenesis in the hippocampus 13may be particularly relevant to the effects of juvenile exposure, since this is a very late developing structure 14. Choice accuracy in the radial-arm maze should be quite good in detecting the functional effects of halothaneinduced hippocampal damage since it is very sensitive to the effects of hippoc ampal damage 1o,l 6,17.
MATERIALS AND METHODS
Overview There were two experiments in this study. Expt. 1 examined the effect of 3 different dosing regimens of halothane exposure (25 parts per million [ppm] continuous, 100 ppm intermittent and 100 ppm continuous) from day two of conception until 60 days after birth. Expt. 2 compared the effects of 100 ppm of intermittent halothane exposure during early (day 2 of conception through day 30 after birth) and late (day 31 after birth through day 90 after birth) periods of development. In both experiments, rats were tested for spatial memory function in an 8-arm radial maze on 5 consecutive daily sessions.
Subjects Albino, Norwegian rats were obtained when 1-day pregnant (Holtzman Co., Madison, WI, U.S.A.). They were randomly sorted into treatment groups on the day of arrival and housed in litter cages with pine wood shavings. Rat chow (Wayne Lab Blox) and tap water were available ad libitum. A 12 h/12 h light-dark schedule was maintained throughout the study with behavioral testing conducted during the light period. The rats were housed in gas exposure chambers. ~ e n the rat pups were born, the litters were pared to e ~ t , as nearly sex-balanced as possible. The mothers were separated from the litters 25 days after birth. On day 30 the rats were separated into samesexed groups of four. On day 90 they were housed in same-sexed pairs. Halothane exposure
Halothane was added to the airstream by a Drager halothane vaporizer. The flow of air and halothane passed through flowmeters to regulate exposure levels. The system w a s calibrated and verified at weekly or shorter intervals by gas chromatography to deliver 25 or 100 ppm of halothane. The rats were h o u s e d in wire mesh litter cages placed inside air-tight chambers (Fisher Scientific Co., Pittsburgh, PA, U.S.A. and Boeker Scientific Co., P h i l a d e l p ~ PA, U.S.A.). These exposure chambers had internal dimen-
149 sions of 40 cm wide x 51 cm deep x 55 cm high. The airflow through the chambers was 10,000cc/min which provided approximately 5.35 air changes per hour. The rats in the intermittent halothane group were exposed to halothane in the air on a 'working week' schedule used in several previous studies in our laboratory 12,13,18"19,21-23, that is, halothane was added to the airstream for 8 h/day (approximately 09.00 to 17.00 h) for 5 days/week (Monday-Friday). The rats in the continuous halothane groups were exposed to halothane at all times except for onehour periods for animal care approximately 3 times/week.
Apparatus and procedure Beginning at approximately 150 days of age, the rats were tested in an 8-arm radial maze patterned after the design developed by Olton and Samuelson 15. The maze had a central platform 34 cm in diameter and arms 10.5 cm wide and 42 cm long. The floor of the central platform and the walls of the arms were made of wood painted fiat black. The floor of the arms was made of wire mesh. The top of the maze, 10 cm high, was made of clear Plexiglas so that the rats could make use of the many extramaze visual cues available in the test room. A video camera mounted above the central platform transmitted a picture of the rats' movements in the maze to a television monitor in a nearby room. During the course of pretraining and radial-arm maze testing, the rats were deprived of food for 23 h prior to testing and allowed free access to food for one hour after testing. Before maze testing each rat was confined to one arm and was trained for 4 days to find and eat Noyes food pellets (45 mg) dispensed at the end of the arm at a variable rate averaging one every 30 s. All of the rats ate at least 25 pellets in 15 min by the fourth day of training. After the preliminary training to familiarize the rats with the food rewards, the rats were tested on 5 consecutive days for their choice accuracy in retrieving rewards from the 8 arms of the maze. Before each session the entire maze was wiped out with a 1 ~o acetic acid solution to help avoid odor cues left by other rats. Arm entries were
scored when the rat first put its nose further than half-way down the choice arm. The first entry the rat made into each arm was rewarded by the dispensing of a 45-mg Noyes food pellet. Subsequent re-entries were not rewarded. The dependent measures consisted of arm entries until a choice was repeated (entries to repeat), number of entries in the first arm entries and response latency.
Experiment 1 There were 3 different halothane exposure groups and one control group. The experimental groups were all exposed to halothane from day 2 of gestation until 60 days after birth. One group was given 100 ppm of halothane on the intermittent 'working week' schedule and two groups were continuously exposed to halothane (one group to 25 ppm and one to 100 ppm) except for one-hour periods for animal care and testing approximately 3 times/week. The 100-ppm intermittent and 25-ppm continuous conditions resulted in approximately equal amounts of total exposure (dose x duration) to halothane. The controls were treated like the experimental groups except that no halothane was added to the alrstream of their chambers.
Experiment 2 There were 4 groups: controls, early exposure, late exposure and combined exposure. In this experiment, all of the exposure groups were given 100 ppm ofhalothane in the intermittent schedule. As in Expt. 1, the controls were treated in the same way as the other 3 groups except that no halothane was added to the airstream of their chambers. Rats in the early exposure group were the offspring of mothers given halothane from day 2 of gestation until parturition; the mothers and pups continued to be exposed until separation on day 25 and the pups alone were exposed until day 30. Rats in the late exposure group were offspring of mothers not exposed to halothane during pregnancy or nursing. The exposure of these offspring began on day 31 after birth and continued through day 90. The combined exposure group received both of these exposures. That is, they were exposed to halothane from day 2 of gestation until 90 days after birth.
150
Statistics The choice accuracy and latency data were evaluated by analysis of variance. Litter was the unit of variance in both experiments to guard against possible litter effects biasing the results of the statistical tests. In Expt. 1, the between-litters factor was the halothane dosing regimen, while in Expt. 2, the between-litters factors was the timing of exposure. In both experiments, the withinlitters factors consisted of the sex of rat and the 5 testing sessions. Orthogonal contrasts were made in both experiments to compare the different exposure groups. In Expt. 1, the first contrast compared the control group with the halothane-treated groups to determine the overall halothane effect. The second contrast compared the 25 ppm continuous with both of the 100 ppm halothane groups to determine the effect of dose level. The third contrast compared the 100 ppm intermittent group with the 100 ppm continuous group to determine the effect of dosing regimen. In Expt. 2, the first contrast compared the control group with all of the halothane exposure groups to evaluate the overall effects of halothane. The second contrast compared the late exposure group with the early and combined exposure groups to determine whether the early halothane had greater effects. The third contrast compared the early exposure group with the combined exposure group to determine whether the addition of the late exposure exacerbated any effects of the early exposure. Significant interactions were followed up by analyses of the simple main effects.
P < 0.10) decrement in the two 100-ppm groups when compared with the 25-ppm group. There was a significant main effect of sessions (F4,1~2 = 2.95, P < 0.025) with a significant linear trend (P < 0.05) reflecting a general improvement over the 5 sessions. Because there was a significant interaction of halothane x sex x session (F12 J~2 = 2.62, P < 0.01), the orthogonal contrasts were made within the analyses of the simple main effects. That is, the 3 contrasts (the controls vs all of the halothane groups, the 25-ppm group vs the two 100-ppm groups and the 100-ppm intermittent group vs the 100-ppm continuous group) were made for each sex on each session (Fig. 1). Significant halothane-related deficits were detected for the first two sessions. With the males during session two, the 25-ppm group has significantly better scores than the two 100-ppm groups (F1,28 = 7.03, P < 0.025), but the 100-ppm groups were not different from each other. With the females on session two, the controls were signifiExp I: Entries to Repeat: Males 75 70 65 60 55 50 C uJ
4.5
--..~---- 1O0C~ntt¢~
4.0 30
~ p ~ n ~s Hi)rbppm
25
~;eS310rls
RESULTS
Experiment 1 The two 100-ppm exposure groups had lower scores on the entries to repeat measure than the controls and the 25-ppm group. The controls had an overall average of 5.4 + 0.1 (mean + S.E.M.) entries to repeat and the 25-ppm continuous exposure group had nearly the same average (5.3 + 0.3), while the 100-ppm intermittent and 100-ppm continuous exposure groups had lower averages of 4.6 + 0.3 and 4.7 + 0.1 respectively. The orthogonal contrasts of the halothane groups showed a marginally significant (F1.28 = 3.86,
Expl: Entries to Repeat: Females 75 70 65 6.0 55 5.0 4.5
* • :
C~F~rr~e 256or~F~b I00 i'~ F ~
4.0 ~5 30 25
25pp~ ~ 103m~, *~p~E6
Fig. 1. Experiment 1: entries to repeat (mean + S.E:M:) over sessions.
151 Exp 2: Entries to Repeat
Exp I: Entries to Repeat 7,570-
6
6560-
5
C
C0~trol
55-
25P9mint
50-
4
• •
3
[] I00 ppm Cent
....~ ~
.-
.
--o--
/: jt
o~,
•
45. 4.035-
2
3.0" 25
,
Fema)e
hlal~
Sessions
Fig. 2. Experiment 1: entries to repeat (mean _+ S.E.M.) over sex.
cantly higher than all of the halothane groups (F1,28-----4.86, P < 0.05), but none of the halothane-treated groups were different from each other. As shown in Fig. 2, the impairment in the males given 100 ppm of halothane seemed to be somewhat more pronounced than in the females. Compared with their respective controls, the deficit in the males in the 100-ppm groups showed a deficit of approximately 18 ~o while the females showed a deficit of approximately 10~o. With the other choice accuracy measure, the number of different arms entered in the first 8 choices, there were no significant halothanerelated effects. There were also no halothanerelated effects in latency to finish the maze.
Experiment 2 As in Expt. 1, there was a halothane-related deficit with the entries to repeat measure. The controls averaged 5.1 + 0.2, the early exposure group averaged 4.6 + 0.4, the late exposure group averaged 4.1 + 0.3 and the combined exposure group averaged 4.9 + 0.2. There was a marginally significant main effect of halothane on this measure (F3.27 = 2.82, P < 0 . 1 0 ) . Also as in Expt. 1, there was a significant main effect of sessions (F4.~o8 = 8.01, P < 0.001) with a general improvement over successive sessions. Fig. 3 shows the performance of each group over the 5 sessions. Because there was a significant halothane × sex interaction (F3,27 = 4.62, P < 0.01), the 3 orthogonal contrasts (the controls vs all of the halothane groups, the late exposure group vs the early and combined exposure groups and the early exposure group vs the combined exposure
Fig. 3. Experiment 2: entries to repeat (mean + S.E.M.) over sessions.
group) were made for each sex (Fig. 4). For the males, the contrast of the controls vs all of the halothane groups was marginally significant (F1.27 -- 3.90, P < 0.10), with the control group ranking higher than each of the halothane-treated groups. The contrast of the late exposure group vs the early and combined exposure groups was significant (FI,27 = 11.95, P < 0 . 0 1 ) . Surprisingly, this effect was due to a pronounced deficit in the late exposure group. There was no difference between the early and combined exposure groups. For the females none of the orthogonal contrasts revealed significant effects. In this experiment averaged over all halothane groups, the males showed a deficit of 1 9 ~ while the females showed a deficit of only 8 ~o. This is very similar to the first experiment where the males exposed to 100 ppm of halothane showed a deficit of 18~ while the females showed a deficit of 10~o. With the measure of number of arms entered in the first 8 choices, there was also a significant halothane-related deficit. This was seen in the comparison of the controls with all of the haloExp2: Entries to Repeat 7 6
• Control • E~ m Late
4
[9 c o ~ *p< tO
w 2
Male
Female
**p
Behavioural Brain Research, 36 (1990) 147-154 Elsevier BBR00987
Long-term effects of developmental halothane exposure on radial arm maze performance in rats E d w a r d D. L e v i n ~, R a u l D e L u n a 2, E t s u r o U e m u r a 3 a n d R o b e r t E. B o w m a n 2 1Department of Psychiatry, Duke University, Durham, NC27710 (U.S.A.), 2Harlow Primate Lab, Department of Psychology, University of Wisconsin, Madison, W153715, (U.S.A.) and 3Department of Veterinary Anatomy, Iowa State University, Ames, IA 50011 (U.S.A.) (Received 23 June 1987) (Revised version received 19 May 1989) (Accepted 22 May 1989)
Key words: Halothane; Radial-arm maze; Behavioral teratology; Memory; Rat
Chronic exposure of rats to low levels of halothane during development, a treatment which retards synaptogenesis, was found to cause a long-term impairment of choice accuracy in the radial-arm maze. In Expt. 1, the relative importance of dose level and dosing regimen was examined. Dose level seemed the more critical variable for causing impaired choice accuracy. Exposure to 100 parts per million (ppm) of halothane in the air either on an intermittent or continuous schedule from day two of conception until 60 days after birth significantly impaired choice accuracy, whereas exposure to 25 ppm on a continuous schedule did not cause a deficit, even though with this condition the total amount of halothane exposure was about the same as with 100 ppm given intermittently. In Expt. 2, the 100 ppm intermittent exposure regimen was used to examine the relative importance of exposure during early and late developmental periods for producing the cognitive effects ofhalothane. Groups were divided into those exposed to halothane during gestation and until 30 days after birth (early exposure), those exposed from day 31 until day 90 (late exposure) and those exposed during both early and late periods (combined exposure). Adverse effects on choice accuracy were seen with all 3 types of exposure, but surprisingly, it was the late exposure that caused the most severe effects. These results show that developmental exposure to halothane which impairs synaptogenesis also causes long-lasting cognitive impairment. Halothane exposure can be a useful experimental tool for examining the relationship between synaptic and behavioral development.
INTRODUCTION
Halothane, a commonly used surgical anesthetic, has been found to cause adverse behavioral effects in operating room personnel chronically exposed to low concentrations in the air 5. Tests of cognitive function have been found to be particularly sensitive to low level effects of halothane 2~. The same is true in rodent models of toxicity, in which exposure during development is quite effective in causing both n e u r a l 7'8"13'18"21-23"25 and behavioral 6"12'13"18"19'23 impairment. The halo-
thane-induced cognitive impairment in rodents has been seen in tests of light-dark discrimination 1a'18'19, spontaneous alternation 13,23 and exploratory behavior in the radial-arm maze 12. The present study further examined halothane effects in the radial-arm maze, using choice accuracy as an index of cognitive performance. Radial-arm maze choice accuracy has been found to be sensitive to developmental exposure to other toxicants and drugs, including lead L4, trimentyltin z4 and diazepam 3. Regarding possible neurological sites for these effects, the radial-arm
Correspondence: E.D. Levin, Nicotine Research Lab (151), VA Medical Center, 508 Fulton St., Durham, NC 27705, U.S.A. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
148 maze has proven to be quite sensitive to certain types of brain lesions and pharmacological manipulations. The hippocampus and related areas are especially crucial for accurate radialarm maze performance2,1°,16,17, and this has been shown in particular for pharmacological manipulations of the cholinergic innervation of the hippocampus 11. These findings are particularly relevant to the present investigation because we have previously found that the hippocampus and related structures are very sensitive to the effects of developmental halothane exposure in retarding synaptogenesis 13"23.Halothane is quite effective in retarding synaptogenesis as was seen in the siblings of the rats tested in the present study 13'23. The retarded synaptogenesis has been particularly apparent in the hippocampus. This suggests that halothane exposure may be useful as an experimental tool for examining the critical phases of development for a variety of behavioral functions. Given its effects on hippocampal development, halothane exposure may be particularly useful as a tool in studying the neurological substrates of cognitive development. The first experiment in the present study was designed to further develop the use of halothane as an investigatory tool by determining the dosing parameters critical for producing cognitive effects. The second experiment was designed to use halothane as a tool in the investigation of critical periods of development of cognitive skills needed to solve the radial-arm maze. The relative effects of halothane exposure during early development was compared to exposure during juvenile development. In one of our previous studies lz halothane exposure during the juvenile period was crucial for producing a cognitive deficit in a light-dark discrimination shock escape task. The effect of halothane in suppressing synaptogenesis in the hippocampus 13may be particularly relevant to the effects of juvenile exposure, since this is a very late developing structure 14. Choice accuracy in the radial-arm maze should be quite good in detecting the functional effects of halothaneinduced hippocampal damage since it is very sensitive to the effects of hippoc ampal damage 1o,l 6,17.
MATERIALS AND METHODS
Overview There were two experiments in this study. Expt. 1 examined the effect of 3 different dosing regimens of halothane exposure (25 parts per million [ppm] continuous, 100 ppm intermittent and 100 ppm continuous) from day two of conception until 60 days after birth. Expt. 2 compared the effects of 100 ppm of intermittent halothane exposure during early (day 2 of conception through day 30 after birth) and late (day 31 after birth through day 90 after birth) periods of development. In both experiments, rats were tested for spatial memory function in an 8-arm radial maze on 5 consecutive daily sessions.
Subjects Albino, Norwegian rats were obtained when 1-day pregnant (Holtzman Co., Madison, WI, U.S.A.). They were randomly sorted into treatment groups on the day of arrival and housed in litter cages with pine wood shavings. Rat chow (Wayne Lab Blox) and tap water were available ad libitum. A 12 h/12 h light-dark schedule was maintained throughout the study with behavioral testing conducted during the light period. The rats were housed in gas exposure chambers. ~ e n the rat pups were born, the litters were pared to e ~ t , as nearly sex-balanced as possible. The mothers were separated from the litters 25 days after birth. On day 30 the rats were separated into samesexed groups of four. On day 90 they were housed in same-sexed pairs. Halothane exposure
Halothane was added to the airstream by a Drager halothane vaporizer. The flow of air and halothane passed through flowmeters to regulate exposure levels. The system w a s calibrated and verified at weekly or shorter intervals by gas chromatography to deliver 25 or 100 ppm of halothane. The rats were h o u s e d in wire mesh litter cages placed inside air-tight chambers (Fisher Scientific Co., Pittsburgh, PA, U.S.A. and Boeker Scientific Co., P h i l a d e l p ~ PA, U.S.A.). These exposure chambers had internal dimen-
149 sions of 40 cm wide x 51 cm deep x 55 cm high. The airflow through the chambers was 10,000cc/min which provided approximately 5.35 air changes per hour. The rats in the intermittent halothane group were exposed to halothane in the air on a 'working week' schedule used in several previous studies in our laboratory 12,13,18"19,21-23, that is, halothane was added to the airstream for 8 h/day (approximately 09.00 to 17.00 h) for 5 days/week (Monday-Friday). The rats in the continuous halothane groups were exposed to halothane at all times except for onehour periods for animal care approximately 3 times/week.
Apparatus and procedure Beginning at approximately 150 days of age, the rats were tested in an 8-arm radial maze patterned after the design developed by Olton and Samuelson 15. The maze had a central platform 34 cm in diameter and arms 10.5 cm wide and 42 cm long. The floor of the central platform and the walls of the arms were made of wood painted fiat black. The floor of the arms was made of wire mesh. The top of the maze, 10 cm high, was made of clear Plexiglas so that the rats could make use of the many extramaze visual cues available in the test room. A video camera mounted above the central platform transmitted a picture of the rats' movements in the maze to a television monitor in a nearby room. During the course of pretraining and radial-arm maze testing, the rats were deprived of food for 23 h prior to testing and allowed free access to food for one hour after testing. Before maze testing each rat was confined to one arm and was trained for 4 days to find and eat Noyes food pellets (45 mg) dispensed at the end of the arm at a variable rate averaging one every 30 s. All of the rats ate at least 25 pellets in 15 min by the fourth day of training. After the preliminary training to familiarize the rats with the food rewards, the rats were tested on 5 consecutive days for their choice accuracy in retrieving rewards from the 8 arms of the maze. Before each session the entire maze was wiped out with a 1 ~o acetic acid solution to help avoid odor cues left by other rats. Arm entries were
scored when the rat first put its nose further than half-way down the choice arm. The first entry the rat made into each arm was rewarded by the dispensing of a 45-mg Noyes food pellet. Subsequent re-entries were not rewarded. The dependent measures consisted of arm entries until a choice was repeated (entries to repeat), number of entries in the first arm entries and response latency.
Experiment 1 There were 3 different halothane exposure groups and one control group. The experimental groups were all exposed to halothane from day 2 of gestation until 60 days after birth. One group was given 100 ppm of halothane on the intermittent 'working week' schedule and two groups were continuously exposed to halothane (one group to 25 ppm and one to 100 ppm) except for one-hour periods for animal care and testing approximately 3 times/week. The 100-ppm intermittent and 25-ppm continuous conditions resulted in approximately equal amounts of total exposure (dose x duration) to halothane. The controls were treated like the experimental groups except that no halothane was added to the alrstream of their chambers.
Experiment 2 There were 4 groups: controls, early exposure, late exposure and combined exposure. In this experiment, all of the exposure groups were given 100 ppm ofhalothane in the intermittent schedule. As in Expt. 1, the controls were treated in the same way as the other 3 groups except that no halothane was added to the airstream of their chambers. Rats in the early exposure group were the offspring of mothers given halothane from day 2 of gestation until parturition; the mothers and pups continued to be exposed until separation on day 25 and the pups alone were exposed until day 30. Rats in the late exposure group were offspring of mothers not exposed to halothane during pregnancy or nursing. The exposure of these offspring began on day 31 after birth and continued through day 90. The combined exposure group received both of these exposures. That is, they were exposed to halothane from day 2 of gestation until 90 days after birth.
150
Statistics The choice accuracy and latency data were evaluated by analysis of variance. Litter was the unit of variance in both experiments to guard against possible litter effects biasing the results of the statistical tests. In Expt. 1, the between-litters factor was the halothane dosing regimen, while in Expt. 2, the between-litters factors was the timing of exposure. In both experiments, the withinlitters factors consisted of the sex of rat and the 5 testing sessions. Orthogonal contrasts were made in both experiments to compare the different exposure groups. In Expt. 1, the first contrast compared the control group with the halothane-treated groups to determine the overall halothane effect. The second contrast compared the 25 ppm continuous with both of the 100 ppm halothane groups to determine the effect of dose level. The third contrast compared the 100 ppm intermittent group with the 100 ppm continuous group to determine the effect of dosing regimen. In Expt. 2, the first contrast compared the control group with all of the halothane exposure groups to evaluate the overall effects of halothane. The second contrast compared the late exposure group with the early and combined exposure groups to determine whether the early halothane had greater effects. The third contrast compared the early exposure group with the combined exposure group to determine whether the addition of the late exposure exacerbated any effects of the early exposure. Significant interactions were followed up by analyses of the simple main effects.
P < 0.10) decrement in the two 100-ppm groups when compared with the 25-ppm group. There was a significant main effect of sessions (F4,1~2 = 2.95, P < 0.025) with a significant linear trend (P < 0.05) reflecting a general improvement over the 5 sessions. Because there was a significant interaction of halothane x sex x session (F12 J~2 = 2.62, P < 0.01), the orthogonal contrasts were made within the analyses of the simple main effects. That is, the 3 contrasts (the controls vs all of the halothane groups, the 25-ppm group vs the two 100-ppm groups and the 100-ppm intermittent group vs the 100-ppm continuous group) were made for each sex on each session (Fig. 1). Significant halothane-related deficits were detected for the first two sessions. With the males during session two, the 25-ppm group has significantly better scores than the two 100-ppm groups (F1,28 = 7.03, P < 0.025), but the 100-ppm groups were not different from each other. With the females on session two, the controls were signifiExp I: Entries to Repeat: Males 75 70 65 60 55 50 C uJ
4.5
--..~---- 1O0C~ntt¢~
4.0 30
~ p ~ n ~s Hi)rbppm
25
~;eS310rls
RESULTS
Experiment 1 The two 100-ppm exposure groups had lower scores on the entries to repeat measure than the controls and the 25-ppm group. The controls had an overall average of 5.4 + 0.1 (mean + S.E.M.) entries to repeat and the 25-ppm continuous exposure group had nearly the same average (5.3 + 0.3), while the 100-ppm intermittent and 100-ppm continuous exposure groups had lower averages of 4.6 + 0.3 and 4.7 + 0.1 respectively. The orthogonal contrasts of the halothane groups showed a marginally significant (F1.28 = 3.86,
Expl: Entries to Repeat: Females 75 70 65 6.0 55 5.0 4.5
* • :
C~F~rr~e 256or~F~b I00 i'~ F ~
4.0 ~5 30 25
25pp~ ~ 103m~, *~p~E6
Fig. 1. Experiment 1: entries to repeat (mean + S.E:M:) over sessions.
151 Exp 2: Entries to Repeat
Exp I: Entries to Repeat 7,570-
6
6560-
5
C
C0~trol
55-
25P9mint
50-
4
• •
3
[] I00 ppm Cent
....~ ~
.-
.
--o--
/: jt
o~,
•
45. 4.035-
2
3.0" 25
,
Fema)e
hlal~
Sessions
Fig. 2. Experiment 1: entries to repeat (mean _+ S.E.M.) over sex.
cantly higher than all of the halothane groups (F1,28-----4.86, P < 0.05), but none of the halothane-treated groups were different from each other. As shown in Fig. 2, the impairment in the males given 100 ppm of halothane seemed to be somewhat more pronounced than in the females. Compared with their respective controls, the deficit in the males in the 100-ppm groups showed a deficit of approximately 18 ~o while the females showed a deficit of approximately 10~o. With the other choice accuracy measure, the number of different arms entered in the first 8 choices, there were no significant halothanerelated effects. There were also no halothanerelated effects in latency to finish the maze.
Experiment 2 As in Expt. 1, there was a halothane-related deficit with the entries to repeat measure. The controls averaged 5.1 + 0.2, the early exposure group averaged 4.6 + 0.4, the late exposure group averaged 4.1 + 0.3 and the combined exposure group averaged 4.9 + 0.2. There was a marginally significant main effect of halothane on this measure (F3.27 = 2.82, P < 0 . 1 0 ) . Also as in Expt. 1, there was a significant main effect of sessions (F4.~o8 = 8.01, P < 0.001) with a general improvement over successive sessions. Fig. 3 shows the performance of each group over the 5 sessions. Because there was a significant halothane × sex interaction (F3,27 = 4.62, P < 0.01), the 3 orthogonal contrasts (the controls vs all of the halothane groups, the late exposure group vs the early and combined exposure groups and the early exposure group vs the combined exposure
Fig. 3. Experiment 2: entries to repeat (mean + S.E.M.) over sessions.
group) were made for each sex (Fig. 4). For the males, the contrast of the controls vs all of the halothane groups was marginally significant (F1.27 -- 3.90, P < 0.10), with the control group ranking higher than each of the halothane-treated groups. The contrast of the late exposure group vs the early and combined exposure groups was significant (FI,27 = 11.95, P < 0 . 0 1 ) . Surprisingly, this effect was due to a pronounced deficit in the late exposure group. There was no difference between the early and combined exposure groups. For the females none of the orthogonal contrasts revealed significant effects. In this experiment averaged over all halothane groups, the males showed a deficit of 1 9 ~ while the females showed a deficit of only 8 ~o. This is very similar to the first experiment where the males exposed to 100 ppm of halothane showed a deficit of 18~ while the females showed a deficit of 10~o. With the measure of number of arms entered in the first 8 choices, there was also a significant halothane-related deficit. This was seen in the comparison of the controls with all of the haloExp2: Entries to Repeat 7 6
• Control • E~ m Late
4
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Female
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