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Effect of Escherichia coli endotoxin on locomotor activity of mice
Tbxlcwr, 1969, Vol . 7. pp. 39-42
n Prag . Prlntad in Grast Br[t~in
EFFECT OF ESCHERICHIA COLT ENDOTOXIN ON LOCOMOTOR ACTIVITY OF MICE and RocER M. BROwxt
IIARBAN3 LAL*
Department of Pharmacology and Toxicology, University of Kansas, Lawronce, Kansas, U.S .A. (Acceptedfor publication 12 December 1968) Abstract-A single igjection of E. colt endotoxin (39-156 vg per kg, i.p .) decreased locomotor activity in mice. It also decreased the ability of amphetamine to stimulate spontaneous locomotion. The behavioral effects were not related to the effect on body temperature. INTRODUCTION
are heat-stable, high molecular complexes of phospholipids, polysaccharides, and proteins, contained within the cell wall ofboth pathogenic and non-pathogenic bacteria. Although it is a matter of common knowledge that systemic infections produce a variety of transient behavioral debilities, the precise relationship between the endotoxins and the disease-produced behavioral debilities is yet to be determined . With this purpose in mind, we began a series of studies on the effects of endotoxin on behavior. In our recent studies (LAL and BROwx, 1968) we observed that the lever-pressing activity of rats, trained to press a lever in order to obtain food or water, was depressed by small doses of the endotoxin. This depression of operant behavior may be due to a generalized debility produced by the endotoxin. It was therefore undertaken to measure directly the generalized debility produced by endotoxin injection. EIVDOTOXINS
METHOD
Swiss albino, random-bred, male mice, weighing 22-26 g were obtained from A . R. Schmidt and Company, and housed, 10 to a cage, 6 to 10 days before use. Photobeam activity cages described previously (W~IVZaL and BROnIe, 1966) were used to measure locomotor activity. Three light sources mounted on one side of the cage were focused on 3 respective photoelectric cells on the opposite side of the cage. The light beams passing perpendicularly to the long axis ofthe cage divided it into four sections of 4 x 8~5 in. each. Each photoelectric relay independently activated the `stepper' ofa Gerbrand Cumulative Recorder . The cages were housed in a sound-attenuated chamber whose temperature was regulated to 25 ~1 ° by a built-in compressor and cooling system . The mice received a single injection, 39-156 wg per kg, i.p. of E. coli endotoxin (Batch No. 0127 :B8 from Difco Laboratories, Detroit) dissolved in a vehicle of 005 M phosphatebuffered saline (pH 7~2), or they were injected with the vehicle alone. They were placed in the activity cages, 3 to a cage. The activity was recorded automatically as cumulative responses on Gerbrand Cumulative Recorders. Since the rate of locomotive activity is markedly 'Present address: Department of Pharmacology, University of Rhode Island, Kingston, Rhode Island 02881, U.S .A . tPresent address: Department of Pharmacology, University of Chicago, Chicago, Illinois 60637, U.S .A . 39
40
HARBANS LAL and ROGER M. BROWN
higher at the start, it was assumed to be due to exploratory motivation in the new environment . After the exploratory activity ceased the activity counts represent spontaneous locomotion of the animals. The first 60-min period was arbitrarily selected to measure exploratory activity while the second 60-min period was designated as spontaneous locomotion . As the variance of activity counts among activity cages was not significant (F> 005) the counts from 3 activity cages were pooled for statistical analysis . Since endotoxin has been reported to lower (HOLMES and MILLER, 1963) as well as raise (WINTER and Nuss, 1963) the body temperature, the rectal temperatures of the mice were recorded with a thermocouple device . RESULTS
In a preliminary experiment a single injection of E. coli endotoxin markedly depressed the exploratory and spontaneous locomotor activities of mice (Table 1). The depression was time-dependent . It began immediately after injection and the animals recovered within 24 hr after the single dose. In all subsequent experiments the activity was measured 2 hr after endotoxin administration . TABLE I .
EFFECTS OF E. CUI( ENDOTOXIN UN LOCOMOTOR ACTIVITY OF MICE
Activity counts+ Time after injection (hr)
Exploratory activity (0-60 min)
Spontaneous activity (60-120 min)
Vehicle 760 Endotoxin (156 wg/kg) 430 305 421 253 303 630
0 0 025 1 2 4 24
282 50 47 54 53 92 227
+Counts based on 3 mice in each activity cage at each time-interval . TABLE
2.
EFFECT OF E. CU%l ENDOTOXIN ON EXPLORATORY ACTIVITY OF MICE
Endotoxin G4g/kg) Vehicle 39 78 156 Source of variation Amongtl~eattnents Among cages within treatments Within tt~eatments
Activity counts (means fS. E.) (1`1)+ Experiment l Experiment 2± 1431 ~132 (3) 1625 f 103 (6) 342 f 69 (3) 244:~ 59 (6) 268 ~= 35 (3) 207f 25 (6) 253- 48 (3) 236- :. 58 (6) Analysis of variance (experiment 1) df Mean squares F' 3 1,638,243 115 8 12
18,392 142,017
013
+N is number of determinations, each determination consisted of activitycounts obtained from 3 mice in the activity cage. tExperiment 2 was a replicate of experiment 1 in order to determine the reproducibility of endotoxin effect at a different time.
Endotoxin and Locomotor Activity TABLE
3.
41
EFFECT OF E. COIi ENDOTOXIN ON LOCOMOTOR ACTIVITY OF MICE
Groups of mice 3 6 9 9
Exploratory activity Drug Mean1S .E .
_ S_po_ntane_ous activity Drug Mean~S .E,
None Saline Saline Endotoxin*
None Saline Amphetaminet Amphetaminet
1066T 96 1393 :133 1166 .!.144 318 48
233 f 48 276 25 1515313 590133
*156 Ecg per kg, 2 hr before testing, t5 mg per kg, immediately before testing. TABLE 4. FAILURE OF CORTL40NE PRETREATMENT TO PROTECT MICE AGAINST ENDOTOXIN INDUCED DEPRESSION OF EXPLORATORY AC11V17Y IN MICE
Drug
Dosc
ACtiVlty* (MeanfS. E.)
Vehicle Endotoxint Cortisone Endotoxin -ICortisone
1 ml/kg 156 F~g/kg 10 mg/kg 156 Wg/kg
1212 ~~ 173 238f 59 938 f101
10 mg/kg
397 :;. S'I
*Based upon activity of 9 groups of 3 mice each . t2 hr before testing. TABLE S .
EFFECT OF E. COI! ENDOTOXIN ON RECTAL TEMPERATURE OF MICE
Day of treatment Treatment 1 2 3 4
None None Vehicle Endotoxin (200 ~g/kg)
_.. __
1 hr
Rectal temperature (Mean *°C~ :S .E .) ., 2 hr 4 hr Shr
38~010~26 36~8~027 37~5~_0~28 37~9~-0~16
37~3f0~15 37~6f0~18 37~4~0~25 38~0~0~17
36~9f0~16 373-~0~18 37~8~025 37~7_~024
373±p~16 37~4f0~19 37~3 .~025 377-:~-0~?8
*Based upon 9 mia.
A dose as low as 39 Fig per kg depressed the exploratory activity in mice markedly (Table 2). From the data summarized in Table 3 it can be seen that the ability of amphetamine to enhance spontaneous locomotor activity was partially antagonized by pretreatment with endotoxin. In addition, cortisone failed to block the behavioral depression produced by endotoxin (Table 4). The dose of endotoxin slightly higher than that used in the behavioral study did not alter rectal temperature of the mice (Table S). DISCUSSION
E. coli endotoxin produced profound depression of exploratory and spontaneous locomotion . The magnitude of this depression was similar to that reported previously for food
42
HARBANS LAL and ROGER M. BROWN
or water-rewarded operant behavior (LAL and BROWN, 1968) . Further indication as to the extent ofdepression produced by endotoxin can be seen in the experiments with amphetamine pretreated mice. These data suggest that the depression by endotoxin and stimulation by amphetamine might be additive. The stimulant activity of amphetamine (5 mg per kg) is completely blocked in mice pretreated with a large dose (10 mg per kg) of endotoxin (LAL et al., 1965) . Further experiments using several doses of amphetamine are necessary to establish the exact relationship between the pharmacological properties of the toxin and those of amphetamine. Several toxic effects of endotoxin are prevented by pretreatment with cortisone (Knss, 1960) . The depression of locomotor activity by endotoxin was not antagonized by cortisone. Laboratory measurement of behavioral depression due to endotoxin is of considerable interest in view of theproposed role ofE. coli infections in causing mental retardation (Srn~t, 1954) and the behavioral debility generally associated with infectious diseases (IVIILLF1t, 1964) .
REFERENCES J. E. and Mu.LErt, N. E. (1963) Effects of bacterial endotoxin on water intake, food intake, and body temperature in the albino rat. J. exp . Med.118, 649. Kris, E. H. (1960) Effect of corticosteroids and hormones of pregnancy on the lethal action of bacterial endotoxin. Ann. N. Y. Acad. Sct . 88, 107. LnL, H. and BxowN, R. (1968) Effect of bacterial endotoxin on operant behavior of the rat. Tozic. appl. Pharmac . 14, 41 . LPL, H ., BROWN, R., $ROOFS, D. and WENZEL, D. (1965) Effect of bacterial endotoxin on operant behavior and drug-induced CNS activity. Pharmacotogist 7, 101 . MILLER, N. E. (1964) Some psychophysiological studies of motivation and of the behavioral effects of illness. Butl. Br. psycho!. Soc. 17 (55), 1 . $àfITH, E. S. (1954) Purulent meningitis in infants and children.J. Pediat . 45, 425. WENZEL, D. G. and BRODIE, L. L. (1966) Effect of group size and cage design on voluntary running activity of amphetamine-treated mice . Archs int . Pharmacodyn. ThEr. 159, 154. WINTER, C. A. and Ntrs4, G. W. (1963) Pyretogenic effects of bacterial lipopolysaccharide and the assay of antipyretic drugs in rats . Toxtc. app!. Pharmac. 5, 247. Hot,ntFS,
n Prag . Prlntad in Grast Br[t~in
EFFECT OF ESCHERICHIA COLT ENDOTOXIN ON LOCOMOTOR ACTIVITY OF MICE and RocER M. BROwxt
IIARBAN3 LAL*
Department of Pharmacology and Toxicology, University of Kansas, Lawronce, Kansas, U.S .A. (Acceptedfor publication 12 December 1968) Abstract-A single igjection of E. colt endotoxin (39-156 vg per kg, i.p .) decreased locomotor activity in mice. It also decreased the ability of amphetamine to stimulate spontaneous locomotion. The behavioral effects were not related to the effect on body temperature. INTRODUCTION
are heat-stable, high molecular complexes of phospholipids, polysaccharides, and proteins, contained within the cell wall ofboth pathogenic and non-pathogenic bacteria. Although it is a matter of common knowledge that systemic infections produce a variety of transient behavioral debilities, the precise relationship between the endotoxins and the disease-produced behavioral debilities is yet to be determined . With this purpose in mind, we began a series of studies on the effects of endotoxin on behavior. In our recent studies (LAL and BROwx, 1968) we observed that the lever-pressing activity of rats, trained to press a lever in order to obtain food or water, was depressed by small doses of the endotoxin. This depression of operant behavior may be due to a generalized debility produced by the endotoxin. It was therefore undertaken to measure directly the generalized debility produced by endotoxin injection. EIVDOTOXINS
METHOD
Swiss albino, random-bred, male mice, weighing 22-26 g were obtained from A . R. Schmidt and Company, and housed, 10 to a cage, 6 to 10 days before use. Photobeam activity cages described previously (W~IVZaL and BROnIe, 1966) were used to measure locomotor activity. Three light sources mounted on one side of the cage were focused on 3 respective photoelectric cells on the opposite side of the cage. The light beams passing perpendicularly to the long axis ofthe cage divided it into four sections of 4 x 8~5 in. each. Each photoelectric relay independently activated the `stepper' ofa Gerbrand Cumulative Recorder . The cages were housed in a sound-attenuated chamber whose temperature was regulated to 25 ~1 ° by a built-in compressor and cooling system . The mice received a single injection, 39-156 wg per kg, i.p. of E. coli endotoxin (Batch No. 0127 :B8 from Difco Laboratories, Detroit) dissolved in a vehicle of 005 M phosphatebuffered saline (pH 7~2), or they were injected with the vehicle alone. They were placed in the activity cages, 3 to a cage. The activity was recorded automatically as cumulative responses on Gerbrand Cumulative Recorders. Since the rate of locomotive activity is markedly 'Present address: Department of Pharmacology, University of Rhode Island, Kingston, Rhode Island 02881, U.S .A . tPresent address: Department of Pharmacology, University of Chicago, Chicago, Illinois 60637, U.S .A . 39
40
HARBANS LAL and ROGER M. BROWN
higher at the start, it was assumed to be due to exploratory motivation in the new environment . After the exploratory activity ceased the activity counts represent spontaneous locomotion of the animals. The first 60-min period was arbitrarily selected to measure exploratory activity while the second 60-min period was designated as spontaneous locomotion . As the variance of activity counts among activity cages was not significant (F> 005) the counts from 3 activity cages were pooled for statistical analysis . Since endotoxin has been reported to lower (HOLMES and MILLER, 1963) as well as raise (WINTER and Nuss, 1963) the body temperature, the rectal temperatures of the mice were recorded with a thermocouple device . RESULTS
In a preliminary experiment a single injection of E. coli endotoxin markedly depressed the exploratory and spontaneous locomotor activities of mice (Table 1). The depression was time-dependent . It began immediately after injection and the animals recovered within 24 hr after the single dose. In all subsequent experiments the activity was measured 2 hr after endotoxin administration . TABLE I .
EFFECTS OF E. CUI( ENDOTOXIN UN LOCOMOTOR ACTIVITY OF MICE
Activity counts+ Time after injection (hr)
Exploratory activity (0-60 min)
Spontaneous activity (60-120 min)
Vehicle 760 Endotoxin (156 wg/kg) 430 305 421 253 303 630
0 0 025 1 2 4 24
282 50 47 54 53 92 227
+Counts based on 3 mice in each activity cage at each time-interval . TABLE
2.
EFFECT OF E. CU%l ENDOTOXIN ON EXPLORATORY ACTIVITY OF MICE
Endotoxin G4g/kg) Vehicle 39 78 156 Source of variation Amongtl~eattnents Among cages within treatments Within tt~eatments
Activity counts (means fS. E.) (1`1)+ Experiment l Experiment 2± 1431 ~132 (3) 1625 f 103 (6) 342 f 69 (3) 244:~ 59 (6) 268 ~= 35 (3) 207f 25 (6) 253- 48 (3) 236- :. 58 (6) Analysis of variance (experiment 1) df Mean squares F' 3 1,638,243 115 8 12
18,392 142,017
013
+N is number of determinations, each determination consisted of activitycounts obtained from 3 mice in the activity cage. tExperiment 2 was a replicate of experiment 1 in order to determine the reproducibility of endotoxin effect at a different time.
Endotoxin and Locomotor Activity TABLE
3.
41
EFFECT OF E. COIi ENDOTOXIN ON LOCOMOTOR ACTIVITY OF MICE
Groups of mice 3 6 9 9
Exploratory activity Drug Mean1S .E .
_ S_po_ntane_ous activity Drug Mean~S .E,
None Saline Saline Endotoxin*
None Saline Amphetaminet Amphetaminet
1066T 96 1393 :133 1166 .!.144 318 48
233 f 48 276 25 1515313 590133
*156 Ecg per kg, 2 hr before testing, t5 mg per kg, immediately before testing. TABLE 4. FAILURE OF CORTL40NE PRETREATMENT TO PROTECT MICE AGAINST ENDOTOXIN INDUCED DEPRESSION OF EXPLORATORY AC11V17Y IN MICE
Drug
Dosc
ACtiVlty* (MeanfS. E.)
Vehicle Endotoxint Cortisone Endotoxin -ICortisone
1 ml/kg 156 F~g/kg 10 mg/kg 156 Wg/kg
1212 ~~ 173 238f 59 938 f101
10 mg/kg
397 :;. S'I
*Based upon activity of 9 groups of 3 mice each . t2 hr before testing. TABLE S .
EFFECT OF E. COI! ENDOTOXIN ON RECTAL TEMPERATURE OF MICE
Day of treatment Treatment 1 2 3 4
None None Vehicle Endotoxin (200 ~g/kg)
_.. __
1 hr
Rectal temperature (Mean *°C~ :S .E .) ., 2 hr 4 hr Shr
38~010~26 36~8~027 37~5~_0~28 37~9~-0~16
37~3f0~15 37~6f0~18 37~4~0~25 38~0~0~17
36~9f0~16 373-~0~18 37~8~025 37~7_~024
373±p~16 37~4f0~19 37~3 .~025 377-:~-0~?8
*Based upon 9 mia.
A dose as low as 39 Fig per kg depressed the exploratory activity in mice markedly (Table 2). From the data summarized in Table 3 it can be seen that the ability of amphetamine to enhance spontaneous locomotor activity was partially antagonized by pretreatment with endotoxin. In addition, cortisone failed to block the behavioral depression produced by endotoxin (Table 4). The dose of endotoxin slightly higher than that used in the behavioral study did not alter rectal temperature of the mice (Table S). DISCUSSION
E. coli endotoxin produced profound depression of exploratory and spontaneous locomotion . The magnitude of this depression was similar to that reported previously for food
42
HARBANS LAL and ROGER M. BROWN
or water-rewarded operant behavior (LAL and BROWN, 1968) . Further indication as to the extent ofdepression produced by endotoxin can be seen in the experiments with amphetamine pretreated mice. These data suggest that the depression by endotoxin and stimulation by amphetamine might be additive. The stimulant activity of amphetamine (5 mg per kg) is completely blocked in mice pretreated with a large dose (10 mg per kg) of endotoxin (LAL et al., 1965) . Further experiments using several doses of amphetamine are necessary to establish the exact relationship between the pharmacological properties of the toxin and those of amphetamine. Several toxic effects of endotoxin are prevented by pretreatment with cortisone (Knss, 1960) . The depression of locomotor activity by endotoxin was not antagonized by cortisone. Laboratory measurement of behavioral depression due to endotoxin is of considerable interest in view of theproposed role ofE. coli infections in causing mental retardation (Srn~t, 1954) and the behavioral debility generally associated with infectious diseases (IVIILLF1t, 1964) .
REFERENCES J. E. and Mu.LErt, N. E. (1963) Effects of bacterial endotoxin on water intake, food intake, and body temperature in the albino rat. J. exp . Med.118, 649. Kris, E. H. (1960) Effect of corticosteroids and hormones of pregnancy on the lethal action of bacterial endotoxin. Ann. N. Y. Acad. Sct . 88, 107. LnL, H. and BxowN, R. (1968) Effect of bacterial endotoxin on operant behavior of the rat. Tozic. appl. Pharmac . 14, 41 . LPL, H ., BROWN, R., $ROOFS, D. and WENZEL, D. (1965) Effect of bacterial endotoxin on operant behavior and drug-induced CNS activity. Pharmacotogist 7, 101 . MILLER, N. E. (1964) Some psychophysiological studies of motivation and of the behavioral effects of illness. Butl. Br. psycho!. Soc. 17 (55), 1 . $àfITH, E. S. (1954) Purulent meningitis in infants and children.J. Pediat . 45, 425. WENZEL, D. G. and BRODIE, L. L. (1966) Effect of group size and cage design on voluntary running activity of amphetamine-treated mice . Archs int . Pharmacodyn. ThEr. 159, 154. WINTER, C. A. and Ntrs4, G. W. (1963) Pyretogenic effects of bacterial lipopolysaccharide and the assay of antipyretic drugs in rats . Toxtc. app!. Pharmac. 5, 247. Hot,ntFS,