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Genotype-dependent electroencephalographic, behavioral and analgesic correlates of morphine: An analysis in normal mice and in mice with septal lesions
Brain Research, 83 (1975) 135-141 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
135
GENOTYPE-DEPENDENT ELECTROENCEPHALOGRAPHIC, BEHAVIORAL AND ANALGESIC CORRELATES OF MORPHINE: AN ANALYSIS IN NORMAL MICE AND IN MICE WITH SEPTAL LESIONS
ALBERTO OLIVERIO Laboratorio di Psicobiologia e Psicofarmacologia C NR, 1, via Reno, 00198 Rome (Italy) (Accepted September 2nd, 1974)
SUMMARY
Electroencephalographic, locomotor, and analgesic responses were studied in two strains of inbred mice (C57BL/6J and DBA/2J) implanted with epidural cortical electrodes. For the C57 strain the injection of morphine produced a sharp increase of locomotor activity and the EEG showed high amplitude slow waves similar to sleep, while the same strain was less sensitive to the analgesic effects of the opiate. For the DBA strain the .locomotor activity was not enhanced following morphine and there was no alteration of the EEG pattern, while a pronounced analgesic response was evident in this strain. Septal lesions antagonized morphine analgesia in both strains while locomotor and electroencephalographic responses were unaffected in C57 mice. The findings suggest the following. (1) The electroencephalographic responses to morphine.are genetically determined. (2) The septal area is implicated in morphine-induced analgesia but not in the locomotor responses or in the brain electrical responses to the opiate. (3) The electroencephalographic alterations induced by morphine do not seem to be directly connected with the analgesic properties of the drug.
INTRODUCTION
A number of findings indicate that the effects of morphine on different behavioral patterns are modulated by the genetic make-up of the individual. For example, clear individual 4 or strain differences2,11,12 have been reported for the effects of opiates on the locomotor activity ('running fit'). Similarly, different studies show that both sensitivity and tolerance to opiate-induced analgesia and locomotor activity are genetically determined. A negative correlation between the degree of running and analgesic
136
A. OLIVERIO
responses has also been observed by using different strains of inbred mice. For example, when the strains C57BL/6J and DBA/2J are considered, C57 mice show the highest locomotor response, while the same strain was less sensitive than DBA mice to the analgesic effects of morphine or heroin 11. More recently, it was demonstrated that lesions of the limbic system, an area which is critical for eliciting analgesia:'. and which has been associated with opiate receptors s, antagonize morphine analgesia in both C57 and DBA strains, while the running fit syndrome was unaffected 1. Changes in cortical electrical activity in response to morphine administration have been studied 7,9,15, and clear interspecific differences have been reported'% The aim of the present research was to assess: (l) if the electroencephalographic responses to morphine are also modulated by genetic factors; (2) if morphine-induced changes in brain electrical activity are correlated to the differences in locomotor and analgesic responses evident between the strains of mice reported above; and (3) if lesion of the septum, which results in a reduction of the analgesic effects of morphine, also affects the EEG responses to the opiate. METHODS
The experiment was conducted on a total of 160 male mice belonging to the strains C57BL/6J (C57) and DBA/2J (DBA) procured from the Jackson Laboratory, Bar Harbor, Maine. The animals were 50-60 days old at the beginning of the experiment. Each group consisted of l0 animals. Surgery. The mice were anesthetized with sodium pentobarbital (60 mg/kg body weight) and stereotaxically-placed lesions were produced using a Kriegmodel Stoelting stereotaxic apparatus with a special mouse-head holderlL Lesions were made by passing 10-sec pulses of 2.0 mA anodal current from a Stoelting 58040 Lesions-Producing Device through a 0.04 mm diameter enamel-insulated stainlesssteel electrode. Septal lesions were produced by placing electrocoagulations :~ 0.04 mm lateral, 0.8 mm anterior, and 3.4 mm ventral to bregma. Forty C57 and 40 DBA mice were given septal lesions whilst 40 C57 and 40 DBA mice served as operated controls. After a one week recovery period 40 operated controls (20 C57 4- 20 DBA) and 40 mice with septal lesions (20 C57 4- 20 DBA) were implanted with 4 cortical electrodes as described by Valatx et al. 16,17. A one week recovery period elapsed between electrode implantation and the behavioral tests. Thus, the mice were 50-60 days old when they were subjected to the first surgical procedure for septal lesions or control operation; they were 57-67 days old when implanted with cortical electrodes, and 64-74 days old when subjected to behavioral tests. Behavioral tests. Two different groups of mice implanted with cortical electrodes (operated controls = 20 C57 4- 20 DBA; septal lesions = 20 C57 4- 20 DBA) were subjected to simultaneous measures of locomotor activity and EEG recording. In another experiment, 40 operated controls (20 C57 4- 20 DBA) and 40 mice with septal lesions (20 C57 4- 20 DBA) were tested for their hot-plate analgesic responses to morphine.
MORPHINE AND E E G IN MICE
137
EEG recording and locomotor activity. Each mouse was placed in a 16 cm x 16 cm activity cage, 20 cm high. The walls consisted of gray plexiglass. The floor consisted of sixteen 4 cm x 4 cm metal plates, 1 m m apart. Circuitry was arranged so that when the mouse bridged two or more plates a cumulative counter was advanced and recorded one movement. The latency of the counter was 0.5 sec. The activity cage was electrically shielded and the cortical electrodes were connected to a Grass Polygraph by means of a connector which permitted the mouse to move freely in the cage. The locomotor activity and E E G were first recorded for 30 min in the absence of drug (saline). Then, the mice were intraperitoneally injected with 10.0 or 20.0 mg/kg of morphine hydrochloride and immediately subjected to a second 30 min session. Analgesia. The degree of tolerance to analgesia was determined by the hotplate method 4. The endpoint used was the licking of the forepaws or hindpaws. In controls this occurred with a mean latency of about 12 sec; a mouse was removed as soon as it reacted or if it failed to react within 30 sec. Anatomical control. Mice with brain lesions were anesthetized with ether and perfused pericardially with 5 ml of 0.9 ~ saline followed by 10 ml of 10 ~ formalinsaline. Histological verification of the lesion placement was based on microscopic examination of 50 # m frozen sections stained with cresylecht violet. All lesions varied from large, with complete destruction of the septal area, to smaller lesions that spared the lateral portions of the septum. On the basis of histological evaluation of the subject's brains at the end of the experiment, all the operated control animals and all the mice with septal lesions were assigned to the control or septal group respectively. RESULTS Locomotor activity and EEG Table I shows that operated control mice belonging to the C57 strain were more active than DBA mice under control (saline) conditions. When injected with morphine a clear-cut running fit was evident in the C57 strain while morphine did not exert TABLE I EFFECTS OF MORPHINE ON THE LOCOMOTOR ACTIVITY (MEAN ACTIVITY COUNTS ±
S.E.)
OF OPERATED
CONTROLS AND OF MICE WITH SEPTAL LESIONS
Strain Operated controls Saline
C57 DBA
225.7±26.1 98.6±11.2
Septal lesions
Morphine
Saline
Morphine
10.0
20.0 (mg/kg)
10.0
20.0 (mg/~g)
671.9£=51.3 81.7± 9.5
1110.8±118.6 271.6±30.1 60.5± 6.7 121.8i19.1
691.7±49.5 107.6±15.9
1050.1±41.6 90.5±18.6
When the morphine-induced running fit is considered, analysis of variance indicates significant differences between strains (F = 18.6, df = 2/56, P < 0.01). Differences between treatments (operated controls vs. septal) were not significant (F = 0.81, dr= 1/21, P > 0.05).
138
A. OLIVERIO OPERATED CONTROLS I NORMAL BASELINE
2. BASELINE NORMAL SLEEP
3. IO MIN. AFTER MORPHINE
A
El
C
D
E
F
C57
SEPTAL LESIONS
G
H
I
K
L
C57
J DBA
i
Fig. 1. Effects of a single injection of morphine on the EEG tracing recorded from the cortex in normal mice (operated controls) and in mice with lesions of the septum. Samples of polygraphic recordings during waking (1, normal baseline), slow wave sleep (2, baseline normal sleep) and after the injection of morphine (20 mg]kg) in C57 and DBA mice. The normal sleep EEG (B) and EEG after morphine injection (C) are similar in the C57 strain. On the contrary, the waking EEG (D), and that after morphine injection (F) are similar in DBA mice. Septal lesions do not produce any major modification of the effects of morphine on the EEG patterns. any significant effect in DBA mice. Septal lesions resulted in increased locomotor activity in both strains but did not modify the morphine-induced running fit in C57 mice (Table I). Fig. 1 illustrates the characteristic wave patterns obtained from the recording sites in 2 mice. Recordings during waking were substantially similar to both strains. The normal baseline sleep E E G generally showed larger spindles in the DBA strain than in C57 mice. After a dose of morphine the E E G patterns were drastically altered in the C57 strain. This change occurred 3-10 min after the injection and lasted as long as 45-60 min. Fig. 1 shows samples of the E E G activity following morphine; tracings from normally sleeping C57 mice looked almost identical to recordings from the same subject after morphine administration, with a clear increase in voltage and the appearance of slow waves and spindles. On the contrary, morphine did not affect the E E G pattern of DBA mice at doses of 10 and 20 mg/kg. Fig. 1 also shows that septal lesions did not significantly affect the normal E E G baseline of either strains or the response to morphine evident in the operated C57 control mice.
Analgesia Untreated C57 and DBA (operated controls or mice with septal lesions) licked their paws within a mean latency of about 10-12 sec when placed on the hot-plate. Differences between the experimental groups (strain or lesion effects)were not signif-
MORPHINE AND E E G IN MICE
139
T A B L E II MORPHINE-INDUCED ANALGESIAIN OPERATED CONTROLS AND IN MICE WITH SEPTAL LESIONS
The numbers indicate the mean hot-plate latencies 4- S.E. (in sec) measured 30 min after the injection of morphine hydrochloride. Strain
C57 DBA
Operated controls
Septal lesions
Morphine
Morphine
10.0
20.0 (rng/kg)
10.0
20.0 (mg/kg)
16.7 ± 3.2 28.1 4- 1.0
23.6 ± 1.8 30.0 4- 0
12.0 4- 0.6 13.8 4- 1.5
15.9 ! 0.8 24.6 4- 1.2
Analysis of variance indicates significant differences between strains (F = 9.1, df = 1 / 2 1 , P < 0.01), between t r e a t m e n t s (operated controls vs. septal, F = 11.2, d f = 1/21, P < 0.01), a n d between doses (F = 8.7, df = 1/28, P < 0.01).
icant (P > 0.05). As previously shown~,11, ~z the analgesic effect of morphine was less evident in C57 than in DBA operated control mice. Septal lesions resulted in a clear reduction of the analgesic effects of morphine in both strains. Table II shows that at the dose of 20 mg/kg, morphine barely exerted an analgesic effect in C57 mice with lesions of the septum. DISCUSSION
As previously observed the effects of morphine on the running fit and analgesia are strain-dependent2,11,12,14 and a negative strain correlation between the behavioral measures was observed in C57 and DBA operated control mice. The results reported here demonstrate that the electroencephalographic response at cortical sites is modulated by genetic factors and that interspecific differences9 as well as intraspecific differences are dearly evident. Morphine exerts a clear-cut stimulation of the locomotor activity of the C57 strain, a change which is accompanied by a clear E E G alteration resulting in an electrocortical tracing similar to that evident in normally sleeping mice. On the contrary, the analgesic effects of the opiate are less evident in this strain than in DBA mice. In the latter strain, the injection of morphine was not followed by the appearance of higher levels of locomotor behavior or by any observable E E G modification. Thus, it would seem that in the mouse, or at least in these strains of mice, morphine-induced behavioral activation is correlated to a sleep-like E E G pattern, a dissociation between EEG and behavior similar to that described for some anticholinergic agents. It cannot be ruled out that a different electroencephalographic response to morphine might result from subcortical (rather than cortical) recording sites in the DBA strain. However, E E G responses recorded at cortical sites were found to be a reliable index of the bioelectric responses to morphine 15. The fact that morphine does not increase the locomotor activity of DBA mice further supports the possi-
140
A. OLIVERIO
bility that the lack of effect on EEG patterns may be ascribed to common neurophysiological mechanisms. Changes in electrocortical activity in response to morphine have been studied v, 9,aa but it is not known whether EEG responses to the drug are correlated to the analgesic effects of morphine. The results of the present study point out that locomotor or other excitatory patterns, rather than the analgesic effects of morphine, are possibly correlated to the electroencephalographic responses to the opiate. The fact that septal lesions antagonize morphine-induced analgesia but not the running fit or the sleeplike EEG activity seem to confirm this interpretation. A number of neurochemical and neurophysiological findings demonstrate that the strains of mice used in this experiment are also different for regional levels and turnover of brain noradrenaline and dopamine, levels of cholinergic enzymes in the cortex 3,6,1°, type of waking-sleep cycle and type of EEG activity 16,17. Thus, the different patterns of behavioral and EEG responses to morphine evident in these strains may be explained in terms of differences in the regional distribution of opiate receptors 8 as well as the reported neurochemical and neurophysiological strain variations. Experiments on the brain biochemical modifications induced by morphine in these strains will help elucidate this question.
REFERENCES 1 CASTELLANO,C., ESPINET LLOVERA,B., AND OL1VERIO,A., Morphine induced running and analgesia in two strains of mice following septal lesions or modification of brain amines, Arch. Pharmacol., in press, 1974. 2 CASTELLANO,C., ANDOLIVERIO,A., A genetic analysis of morphine-induced running and analgesia in the mouse, submitted to Psychopharmacologia (Bed.), 1974, in press. 3 EBEL, A., HERMETET,J. D., AND MANDEL, P., Comparative study of acetylcholinesterase and choline-acetyltransferase enzyme activity in brain of DBA and C57 mice, Nature New Biol., 242 (1973) 56-57. 4 GOLDSTEIN,A., AND SHEEHAN, e., Tolerance to opiod narcotics. I. Tolerance to the 'running fit' caused by levorphanol in the mouse, d. Pharmacol. exp. Ther., 169 (1969) 175-184. 5 HERZ,!A., ALBUS,K., METYS,J., SCHUBERT,P., AND TESCHEMEKER,H., On the control sites for the antinociceptive action of morphine and Fentanyl, Neuropharmacol., 8 (1970) 539-551. 6 KEMPF, E., GREILSHAMER,J., MACK, G., AND MANDEL, P., Correlation of behavioral differences in three strains of mice with differences in brain amines, Nature (Lond.), 247 (1974) 483-485. 7 KHAZAN,N., WEEKS, J. R., ANDSCHROEDER,L. A., Electroencephalographic, electromicrographic and behavioral correlates during a cycle or self-maintained morphine addiction in the rat, J. Pharmacol. exp. Ther., 155 (1967) 521-531. 8 KUHAR, M. J., PERT, B. C., AND SNYDER, S. H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 447-450. 9 LONGO, V. G., Electroencephalographic Atlas for Pharmacological Research, Elsevier, Amsterdam, 1962, pp. 80-90. 10 MANDEL, P., EBEL, A., HERMETET, J. C., BOVET, D., ET OLIVERIO, A., Etudes des enzymes du syst6me cholinergique chez les hybrides F1 de souris se distinguant par leur aptitude au conditionnement, C. R. Acad. Sci. (Paris), 276 (1973) 395-398. 11 OLIVERIO,A., AND CASTELLANO,C., Genotype dependent sensitivity and tolerance to morphine and heroin: dissociation between opiate-induced running and analgesia in the mouse, Psychopharmacologia (Bed.), 1974, in press. 12 OL1VERIO, A., CASTELLANO,C., AND ELEFTrtERIOU, B. E., Morphine sensitivity and tolerance: a genetic investigation in the mouse, submitted to Psychopharmacologia (Bed.), (1974).
MORPHINE AND E E G IN MICE
141
13 OLIVERIO,A., CASTELLANO,C., AND MESSERI,P., Genotype dependent effects of septal lesions on different types of learning in the mouse, J. comp. physiol. Psychol., 82 (1973) 240-246. 14 SHUSTER,L., YU, G., AND ELEFTHERIOU,B. E., A genetic analysis of the response to morphine in mice. I. Analgesia, submitted to Psychopharmacologia (Berl.), (1974). 15 TEITELBAUM,H., CATRAVAS,G. N., AND McFARLAND, W. J., Reversal of morphine tolerance after medial thalamic lesions in the rat, Science, 185 (1974) 449-451. 16 VALATX,J. L., ET BUGAT,R., Facteurs g6n6tiques dans le d6terminisme du cycle veille-sommeil chez la souris, Brain Research, 69 (1974) 315-330. 17 VALATX,J. L., BUGAT, R., AND JOUVET, M., Genetic studies of sleep in mice, Nature (Lond.), 238 (1972) 226-227.
135
GENOTYPE-DEPENDENT ELECTROENCEPHALOGRAPHIC, BEHAVIORAL AND ANALGESIC CORRELATES OF MORPHINE: AN ANALYSIS IN NORMAL MICE AND IN MICE WITH SEPTAL LESIONS
ALBERTO OLIVERIO Laboratorio di Psicobiologia e Psicofarmacologia C NR, 1, via Reno, 00198 Rome (Italy) (Accepted September 2nd, 1974)
SUMMARY
Electroencephalographic, locomotor, and analgesic responses were studied in two strains of inbred mice (C57BL/6J and DBA/2J) implanted with epidural cortical electrodes. For the C57 strain the injection of morphine produced a sharp increase of locomotor activity and the EEG showed high amplitude slow waves similar to sleep, while the same strain was less sensitive to the analgesic effects of the opiate. For the DBA strain the .locomotor activity was not enhanced following morphine and there was no alteration of the EEG pattern, while a pronounced analgesic response was evident in this strain. Septal lesions antagonized morphine analgesia in both strains while locomotor and electroencephalographic responses were unaffected in C57 mice. The findings suggest the following. (1) The electroencephalographic responses to morphine.are genetically determined. (2) The septal area is implicated in morphine-induced analgesia but not in the locomotor responses or in the brain electrical responses to the opiate. (3) The electroencephalographic alterations induced by morphine do not seem to be directly connected with the analgesic properties of the drug.
INTRODUCTION
A number of findings indicate that the effects of morphine on different behavioral patterns are modulated by the genetic make-up of the individual. For example, clear individual 4 or strain differences2,11,12 have been reported for the effects of opiates on the locomotor activity ('running fit'). Similarly, different studies show that both sensitivity and tolerance to opiate-induced analgesia and locomotor activity are genetically determined. A negative correlation between the degree of running and analgesic
136
A. OLIVERIO
responses has also been observed by using different strains of inbred mice. For example, when the strains C57BL/6J and DBA/2J are considered, C57 mice show the highest locomotor response, while the same strain was less sensitive than DBA mice to the analgesic effects of morphine or heroin 11. More recently, it was demonstrated that lesions of the limbic system, an area which is critical for eliciting analgesia:'. and which has been associated with opiate receptors s, antagonize morphine analgesia in both C57 and DBA strains, while the running fit syndrome was unaffected 1. Changes in cortical electrical activity in response to morphine administration have been studied 7,9,15, and clear interspecific differences have been reported'% The aim of the present research was to assess: (l) if the electroencephalographic responses to morphine are also modulated by genetic factors; (2) if morphine-induced changes in brain electrical activity are correlated to the differences in locomotor and analgesic responses evident between the strains of mice reported above; and (3) if lesion of the septum, which results in a reduction of the analgesic effects of morphine, also affects the EEG responses to the opiate. METHODS
The experiment was conducted on a total of 160 male mice belonging to the strains C57BL/6J (C57) and DBA/2J (DBA) procured from the Jackson Laboratory, Bar Harbor, Maine. The animals were 50-60 days old at the beginning of the experiment. Each group consisted of l0 animals. Surgery. The mice were anesthetized with sodium pentobarbital (60 mg/kg body weight) and stereotaxically-placed lesions were produced using a Kriegmodel Stoelting stereotaxic apparatus with a special mouse-head holderlL Lesions were made by passing 10-sec pulses of 2.0 mA anodal current from a Stoelting 58040 Lesions-Producing Device through a 0.04 mm diameter enamel-insulated stainlesssteel electrode. Septal lesions were produced by placing electrocoagulations :~ 0.04 mm lateral, 0.8 mm anterior, and 3.4 mm ventral to bregma. Forty C57 and 40 DBA mice were given septal lesions whilst 40 C57 and 40 DBA mice served as operated controls. After a one week recovery period 40 operated controls (20 C57 4- 20 DBA) and 40 mice with septal lesions (20 C57 4- 20 DBA) were implanted with 4 cortical electrodes as described by Valatx et al. 16,17. A one week recovery period elapsed between electrode implantation and the behavioral tests. Thus, the mice were 50-60 days old when they were subjected to the first surgical procedure for septal lesions or control operation; they were 57-67 days old when implanted with cortical electrodes, and 64-74 days old when subjected to behavioral tests. Behavioral tests. Two different groups of mice implanted with cortical electrodes (operated controls = 20 C57 4- 20 DBA; septal lesions = 20 C57 4- 20 DBA) were subjected to simultaneous measures of locomotor activity and EEG recording. In another experiment, 40 operated controls (20 C57 4- 20 DBA) and 40 mice with septal lesions (20 C57 4- 20 DBA) were tested for their hot-plate analgesic responses to morphine.
MORPHINE AND E E G IN MICE
137
EEG recording and locomotor activity. Each mouse was placed in a 16 cm x 16 cm activity cage, 20 cm high. The walls consisted of gray plexiglass. The floor consisted of sixteen 4 cm x 4 cm metal plates, 1 m m apart. Circuitry was arranged so that when the mouse bridged two or more plates a cumulative counter was advanced and recorded one movement. The latency of the counter was 0.5 sec. The activity cage was electrically shielded and the cortical electrodes were connected to a Grass Polygraph by means of a connector which permitted the mouse to move freely in the cage. The locomotor activity and E E G were first recorded for 30 min in the absence of drug (saline). Then, the mice were intraperitoneally injected with 10.0 or 20.0 mg/kg of morphine hydrochloride and immediately subjected to a second 30 min session. Analgesia. The degree of tolerance to analgesia was determined by the hotplate method 4. The endpoint used was the licking of the forepaws or hindpaws. In controls this occurred with a mean latency of about 12 sec; a mouse was removed as soon as it reacted or if it failed to react within 30 sec. Anatomical control. Mice with brain lesions were anesthetized with ether and perfused pericardially with 5 ml of 0.9 ~ saline followed by 10 ml of 10 ~ formalinsaline. Histological verification of the lesion placement was based on microscopic examination of 50 # m frozen sections stained with cresylecht violet. All lesions varied from large, with complete destruction of the septal area, to smaller lesions that spared the lateral portions of the septum. On the basis of histological evaluation of the subject's brains at the end of the experiment, all the operated control animals and all the mice with septal lesions were assigned to the control or septal group respectively. RESULTS Locomotor activity and EEG Table I shows that operated control mice belonging to the C57 strain were more active than DBA mice under control (saline) conditions. When injected with morphine a clear-cut running fit was evident in the C57 strain while morphine did not exert TABLE I EFFECTS OF MORPHINE ON THE LOCOMOTOR ACTIVITY (MEAN ACTIVITY COUNTS ±
S.E.)
OF OPERATED
CONTROLS AND OF MICE WITH SEPTAL LESIONS
Strain Operated controls Saline
C57 DBA
225.7±26.1 98.6±11.2
Septal lesions
Morphine
Saline
Morphine
10.0
20.0 (mg/kg)
10.0
20.0 (mg/~g)
671.9£=51.3 81.7± 9.5
1110.8±118.6 271.6±30.1 60.5± 6.7 121.8i19.1
691.7±49.5 107.6±15.9
1050.1±41.6 90.5±18.6
When the morphine-induced running fit is considered, analysis of variance indicates significant differences between strains (F = 18.6, df = 2/56, P < 0.01). Differences between treatments (operated controls vs. septal) were not significant (F = 0.81, dr= 1/21, P > 0.05).
138
A. OLIVERIO OPERATED CONTROLS I NORMAL BASELINE
2. BASELINE NORMAL SLEEP
3. IO MIN. AFTER MORPHINE
A
El
C
D
E
F
C57
SEPTAL LESIONS
G
H
I
K
L
C57
J DBA
i
Fig. 1. Effects of a single injection of morphine on the EEG tracing recorded from the cortex in normal mice (operated controls) and in mice with lesions of the septum. Samples of polygraphic recordings during waking (1, normal baseline), slow wave sleep (2, baseline normal sleep) and after the injection of morphine (20 mg]kg) in C57 and DBA mice. The normal sleep EEG (B) and EEG after morphine injection (C) are similar in the C57 strain. On the contrary, the waking EEG (D), and that after morphine injection (F) are similar in DBA mice. Septal lesions do not produce any major modification of the effects of morphine on the EEG patterns. any significant effect in DBA mice. Septal lesions resulted in increased locomotor activity in both strains but did not modify the morphine-induced running fit in C57 mice (Table I). Fig. 1 illustrates the characteristic wave patterns obtained from the recording sites in 2 mice. Recordings during waking were substantially similar to both strains. The normal baseline sleep E E G generally showed larger spindles in the DBA strain than in C57 mice. After a dose of morphine the E E G patterns were drastically altered in the C57 strain. This change occurred 3-10 min after the injection and lasted as long as 45-60 min. Fig. 1 shows samples of the E E G activity following morphine; tracings from normally sleeping C57 mice looked almost identical to recordings from the same subject after morphine administration, with a clear increase in voltage and the appearance of slow waves and spindles. On the contrary, morphine did not affect the E E G pattern of DBA mice at doses of 10 and 20 mg/kg. Fig. 1 also shows that septal lesions did not significantly affect the normal E E G baseline of either strains or the response to morphine evident in the operated C57 control mice.
Analgesia Untreated C57 and DBA (operated controls or mice with septal lesions) licked their paws within a mean latency of about 10-12 sec when placed on the hot-plate. Differences between the experimental groups (strain or lesion effects)were not signif-
MORPHINE AND E E G IN MICE
139
T A B L E II MORPHINE-INDUCED ANALGESIAIN OPERATED CONTROLS AND IN MICE WITH SEPTAL LESIONS
The numbers indicate the mean hot-plate latencies 4- S.E. (in sec) measured 30 min after the injection of morphine hydrochloride. Strain
C57 DBA
Operated controls
Septal lesions
Morphine
Morphine
10.0
20.0 (rng/kg)
10.0
20.0 (mg/kg)
16.7 ± 3.2 28.1 4- 1.0
23.6 ± 1.8 30.0 4- 0
12.0 4- 0.6 13.8 4- 1.5
15.9 ! 0.8 24.6 4- 1.2
Analysis of variance indicates significant differences between strains (F = 9.1, df = 1 / 2 1 , P < 0.01), between t r e a t m e n t s (operated controls vs. septal, F = 11.2, d f = 1/21, P < 0.01), a n d between doses (F = 8.7, df = 1/28, P < 0.01).
icant (P > 0.05). As previously shown~,11, ~z the analgesic effect of morphine was less evident in C57 than in DBA operated control mice. Septal lesions resulted in a clear reduction of the analgesic effects of morphine in both strains. Table II shows that at the dose of 20 mg/kg, morphine barely exerted an analgesic effect in C57 mice with lesions of the septum. DISCUSSION
As previously observed the effects of morphine on the running fit and analgesia are strain-dependent2,11,12,14 and a negative strain correlation between the behavioral measures was observed in C57 and DBA operated control mice. The results reported here demonstrate that the electroencephalographic response at cortical sites is modulated by genetic factors and that interspecific differences9 as well as intraspecific differences are dearly evident. Morphine exerts a clear-cut stimulation of the locomotor activity of the C57 strain, a change which is accompanied by a clear E E G alteration resulting in an electrocortical tracing similar to that evident in normally sleeping mice. On the contrary, the analgesic effects of the opiate are less evident in this strain than in DBA mice. In the latter strain, the injection of morphine was not followed by the appearance of higher levels of locomotor behavior or by any observable E E G modification. Thus, it would seem that in the mouse, or at least in these strains of mice, morphine-induced behavioral activation is correlated to a sleep-like E E G pattern, a dissociation between EEG and behavior similar to that described for some anticholinergic agents. It cannot be ruled out that a different electroencephalographic response to morphine might result from subcortical (rather than cortical) recording sites in the DBA strain. However, E E G responses recorded at cortical sites were found to be a reliable index of the bioelectric responses to morphine 15. The fact that morphine does not increase the locomotor activity of DBA mice further supports the possi-
140
A. OLIVERIO
bility that the lack of effect on EEG patterns may be ascribed to common neurophysiological mechanisms. Changes in electrocortical activity in response to morphine have been studied v, 9,aa but it is not known whether EEG responses to the drug are correlated to the analgesic effects of morphine. The results of the present study point out that locomotor or other excitatory patterns, rather than the analgesic effects of morphine, are possibly correlated to the electroencephalographic responses to the opiate. The fact that septal lesions antagonize morphine-induced analgesia but not the running fit or the sleeplike EEG activity seem to confirm this interpretation. A number of neurochemical and neurophysiological findings demonstrate that the strains of mice used in this experiment are also different for regional levels and turnover of brain noradrenaline and dopamine, levels of cholinergic enzymes in the cortex 3,6,1°, type of waking-sleep cycle and type of EEG activity 16,17. Thus, the different patterns of behavioral and EEG responses to morphine evident in these strains may be explained in terms of differences in the regional distribution of opiate receptors 8 as well as the reported neurochemical and neurophysiological strain variations. Experiments on the brain biochemical modifications induced by morphine in these strains will help elucidate this question.
REFERENCES 1 CASTELLANO,C., ESPINET LLOVERA,B., AND OL1VERIO,A., Morphine induced running and analgesia in two strains of mice following septal lesions or modification of brain amines, Arch. Pharmacol., in press, 1974. 2 CASTELLANO,C., ANDOLIVERIO,A., A genetic analysis of morphine-induced running and analgesia in the mouse, submitted to Psychopharmacologia (Bed.), 1974, in press. 3 EBEL, A., HERMETET,J. D., AND MANDEL, P., Comparative study of acetylcholinesterase and choline-acetyltransferase enzyme activity in brain of DBA and C57 mice, Nature New Biol., 242 (1973) 56-57. 4 GOLDSTEIN,A., AND SHEEHAN, e., Tolerance to opiod narcotics. I. Tolerance to the 'running fit' caused by levorphanol in the mouse, d. Pharmacol. exp. Ther., 169 (1969) 175-184. 5 HERZ,!A., ALBUS,K., METYS,J., SCHUBERT,P., AND TESCHEMEKER,H., On the control sites for the antinociceptive action of morphine and Fentanyl, Neuropharmacol., 8 (1970) 539-551. 6 KEMPF, E., GREILSHAMER,J., MACK, G., AND MANDEL, P., Correlation of behavioral differences in three strains of mice with differences in brain amines, Nature (Lond.), 247 (1974) 483-485. 7 KHAZAN,N., WEEKS, J. R., ANDSCHROEDER,L. A., Electroencephalographic, electromicrographic and behavioral correlates during a cycle or self-maintained morphine addiction in the rat, J. Pharmacol. exp. Ther., 155 (1967) 521-531. 8 KUHAR, M. J., PERT, B. C., AND SNYDER, S. H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 447-450. 9 LONGO, V. G., Electroencephalographic Atlas for Pharmacological Research, Elsevier, Amsterdam, 1962, pp. 80-90. 10 MANDEL, P., EBEL, A., HERMETET, J. C., BOVET, D., ET OLIVERIO, A., Etudes des enzymes du syst6me cholinergique chez les hybrides F1 de souris se distinguant par leur aptitude au conditionnement, C. R. Acad. Sci. (Paris), 276 (1973) 395-398. 11 OLIVERIO,A., AND CASTELLANO,C., Genotype dependent sensitivity and tolerance to morphine and heroin: dissociation between opiate-induced running and analgesia in the mouse, Psychopharmacologia (Bed.), 1974, in press. 12 OL1VERIO, A., CASTELLANO,C., AND ELEFTrtERIOU, B. E., Morphine sensitivity and tolerance: a genetic investigation in the mouse, submitted to Psychopharmacologia (Bed.), (1974).
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13 OLIVERIO,A., CASTELLANO,C., AND MESSERI,P., Genotype dependent effects of septal lesions on different types of learning in the mouse, J. comp. physiol. Psychol., 82 (1973) 240-246. 14 SHUSTER,L., YU, G., AND ELEFTHERIOU,B. E., A genetic analysis of the response to morphine in mice. I. Analgesia, submitted to Psychopharmacologia (Berl.), (1974). 15 TEITELBAUM,H., CATRAVAS,G. N., AND McFARLAND, W. J., Reversal of morphine tolerance after medial thalamic lesions in the rat, Science, 185 (1974) 449-451. 16 VALATX,J. L., ET BUGAT,R., Facteurs g6n6tiques dans le d6terminisme du cycle veille-sommeil chez la souris, Brain Research, 69 (1974) 315-330. 17 VALATX,J. L., BUGAT, R., AND JOUVET, M., Genetic studies of sleep in mice, Nature (Lond.), 238 (1972) 226-227.