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Is there a catecholamine-serotonin interaction in the control of locomotor activity?
Neuropharmacolo#y. 1975, 14. 501 506. Pergamon Press. Printed in Gt. Britain.
IS T H E R E A C A T E C H O L A M I N E - S E R O T O N I N INTERACTION IN THE CONTROL OF LOCOMOTOR ACTIVITY?* B. L. JACOBSand W. D. WISE Department of Psychology, Princeton University, Princeton, New Jersey 08540
and K. M . TAYLOR Department of Pharmacology, The Squibb Institute of Medical Research, Princeton, New Jersey 08540
(Accepted 25 November 1974) Summary--Previous studies have reported that rats given either midbrain raphe lesions or the serotonin synthesis inhibitor, p-chlorophenylalanine, show an increase above controls in their locomotor response to various doses of (+)-amphetamine. These data have been taken to be indicative of a catecholamine-serotonin interaction. The present data demonstrate that this increased response to amphetamine was obtained following lesions specific to the median raphe nucleus, whereas lesions of the dorsal raphe nucleus were without effect. These results also question the validity and generality of the catecholamine serotonin interaction hypothesis by demonstrating: (1) that when the data are plotted as a per cent of baseline activity, the interaction disappears; and (2) that a very similar effect is observed when amphetamine injections are replaced by scopolamine injections.
Decreases in functional brain serotonin, produced either through raphe lesions (KosTOWSKI, GIACALONE, GARATTINI a n d VALZELLI, 1 9 6 8 ; NEILL, GRANT a n d
GROSSMAN,
1972; JACOBS, WISE and TAYLOR, 1974a) or synthesis inhibition with p-chlorophenylalanine (CHRuSCIEL and HERMAN, 1969; FIBIGER and CAMPBELL, 1971: JACOBS, EUBANKS and WISE, 1974b) result in marked increases in locomotor activity in the rat. In addition, the locomotor increasing effects of amphetamine are potentiated either by raphe lesions (NEILL et al., 1972) or serotonin synthesis inhibition (MABRY and CAMPBELL, 1973). On the basis of these latter two findings with amphetamine, it has been argued that the catecholamines and serotonin interact in the control of locomotor activity in the rat. We have previously shown that selective lesions of the median raphe nucleus resulted in long-lasting increases in locomotor activity, whereas lesions of the dorsal nucleus were without effect (JACOBS et al., 1974a). The purpose of the present study was to examine whether the amphetamine-serotonin interaction hypothesis could be extended to animals with selective lesions of the dorsal or median raphe nucleus. A further objective was to test the validity and generality of this hypothesis by: (1) utilizing an anticholinergic drug which increases locomotor activity, and (2) by measuring the increases in activity as a percent of baseline rather than in terms of absolute levels of activity. METHODS
Activity Male Sprague-Dawley rats weighing 300-400 g were used in all experiments. Locomotor activity was measured by means of centre-balanced wire mesh stabilmeter cages (17.5 x 20.0 × 37-5cm) which tilted back and forth in the longitudinal direction, as the rat crossed the fulcrum. These cages served as the animal's home cage for the duration of the study. Crossings were recorded by means of a microswitch at one * Supported by NIMH Grants MH 23433 and MH 24711. 501
502
B.L. JACOBS,W. D. WISEand K. M. TAYLOR
end of the cage which was connected to an electronic counter in an adjacent room. The cages were housed in a well-ventilated temperature controlled room (22°C) with a 70dB masking noise. Animals were on a 12-hr light-dark schedule with "lights off" at 1000 hr. The room was lit by a 200W incandescent bulb.
Lesions Animals were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic instrument with the bite bar oriented 5ram above the ear bars. A bone flap over the midsaggital sinus was removed at an anterior-posterior coordinate above the midbrain raphe nuclei• The electrodes used for lesioning were thin tungsten wires (0'13 mm diameter) that were sharpened by etching and then insulated. Since the dorsal and median raphe nuclei are most extensive in the anterior posterior and dorso-ventral dimensions, and are quite narrow in the medial-lateral dimension, the lesioning of a particular nucleus was accomplished by making several small midline lesions with varying anterior-posterior and dorso-ventral coordinates. The coordinates at which a 160/~A d.c. current was passed for 20sec were: dorsal groul>-A-P (+) 0.5, D-V ( - ) 0.7, ( - ) 0.4, and ( - ) 0.1, A P (+) 0.2, D-V ( - ) 1.2, ( - ) 0.9, and ( - ) 0"6; median groul~-A-P (+) 0.5 and (+) 0'2, D-V ( - ) 3.0, ( - ) 2"6, ( - ) 2.2 (PELLEGRINO and CUSHMAN, 1967). Pharmacology Three groups, dorsal lesioned, median lesioned and control, consisting of 10 animals each, were used in all experiments. Pharmacological studies were not begun until one month after lesioning in order for the animals to completely recover from surgery and for their baseline activity to stabilize. On drug injection days, hourly activity measures were taken for the 3-hr period following "lights off" (1000hr). Activity during this period served as the baseline. All injections were administered intraperitoneally at 1300 hr. Hourly activity measures were then taken for the 2-hr period beginning 30 min post-injection (1330hr). In the first experiment, (+)-amphetamine sulphate was administered in doses of 0.0 (saline control), 0"5, 1.0 and 2.0mg/kg (expressed as the salt). Every animal received each drug dose in a semi-randomized order, and injections were spaced 5 days apart• The second experiment was identical in design except that the amphetamine injections were replaced by scopolamine hydrobromide injections of 0.0 (saline control), 0-125 and 0.250 mg/kg (expressed as the salt)•
Serotonin assay One month following the termination of all experiments, control and experimental rats were decapitated, the brain was quickly removed, and the forebrain was dissected away from the brainstem just anterior to the superior colliculus. The forebrain tissue was frozen over dry ice, weighed and stored at -14°C for the serotonin estimation, which took place within 48hr. The brainstem was placed in 10~o formalin for serial sectioning (40#m thick coronal sections stained with cresyl violet) in order to locate the site of the lesion. Serotonin was estimated by a modification of the procedure of BOGDANSKI,PLETSCHER, BRODIEand UDENFRIEND(1956). The frozen tissue was homogenized in 3ml of ice cold 0-4M perchloric acid containing 0.1°~ ascorbic acid and 0 1/o ethylenediaminetetraeetic acid. The homogenate was centrifuged at 10,0009 for 10 rain and the supernatant fluid was removed and placed in a stoppered test tube containing 0.5ml of 0"5M borate buffer pH 10. The mixture was adjusted to pH 10 with solid sodium carbonate and saturated with sodium chloride. Serotonin was extracted into 6ml of n-butanol. After centrifugation, the butanol phase was removed and placed in a stoppered test tube containing 1.0ml of 0.1M HCI and 7"5ml of n-heptane. After the tube had been shaken for 5 min and centrifuged at 2500 9 for 5 min, the aqueous phase was removed for serotonin estimation. Native fluorescence of serotonin was read at 295nm excitation, 540nm emission wavelengths, after the addition of 0"5ml 9M HC1. External and internal standards of serotonin (50ng) as well as reagent blanks were also determined. Five ng of serotonin produced a reading •
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Fig. 1. A: Dose-response curves for (+)-amphetamine sulphate (i.p.) in median lesioned, dorsal lesioned, and control animals. Data plotted are the mean hourly crossings for the 2-hr post-drug period. Baseline data represent the activity of uninjected animals. B: The data in A are replotted as a percentage of the pre-drug baseline activity. Each group represents 10 animals.
o f fluorescence twice that of the blank. The recovery o f serotonin added to tissue s a m p l e s w a s 82~o. Tissue c o n c e n t r a t i o n s were not corrected for recovery of serotonin. RESULTS As s h o w n in Figures 1A and 2A, the m e d i a n raphe lesion p r o d u c e d a long-lasting elevation in s p o n t a n e o u s activity of a p p r o x i m a t e l y 1 0 0 ~ a b o v e the activity o f the dorsal lesioned and control groups. Statistical analyses of the data plotted in Figure IA, i.e. m e a n hourly post-drug activity, s h o w that the d o s e of a m p h e t a m i n e significantly affected l o c o m o t o r activity (F = 21.38; 3,81 d.f.; P < 0"001), that the group in which the animals were placed had a significant effect (F = 7"20; 2,27 d.f.; P < 0.001), and x Median o Dorsal • Control
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Fig. 2. A: Dose-response curves for scopolamine hydrobromide (i.p.) in median lesioned, dorsal lesioned, and control animals. Da~a plotted are the mean hourly crossings for the 2-hr post-drug period. Baseline data represent the activity of uninjected animals. B: The data in A are replotted as a percentage of the pre-drug baseline activity. Each group represents 10 animals.
504
B. L. JAcoas, W. D. W~SE and K. M. TAYLOR Table 1. Forebrain serotonin (Izg/g) Group
Control Median lesioned Dorsal lesioned
n
Serotonin (gg/g)
"..~, Reduction
6 8 8
0.490 +/).023 0.325 + 0.010" 0.237 + 0.008*
33.7 51.7
Each value is the mean + S.E.M. Significance of difference bctween means of the lesioned groups from the control group: * P < 0.001 (two-tailed t-tests).
finally that there was a significant interaction between drug and group (F--5.91; 6,81 d.f.; P < 0"001). A further analysis indicated that the activity of the median group was significantly greater than that of either the dorsal or control groups (P < 0.01; 2,27 and 3,27 d.f. Newman-Keuls). These data imply that there is an amphetamine-serotonin interaction, and that it is specific to the median raphe nucleus. However, if the post-drug data are plotted (Figure I B) and statistically analyzed as a percent of the pre-drug baseline rather than as absolute post-drug activity counts (as above), the group curves completely overlap and the only variable producing a statistically significant effect is the dose of amphetamine (F = 28.01; 3,81 d.f.; P < 0.001). The lack of a statistically significant effect of group or an interaction between drug and group (analyses of variance, P > 010) using these measures, implies that there is no amphetamine-serotonin interaction. Much the same picture is seen when the results of the scopolamine experiment are examined. Statistical analyses of the data plotted in Figure 2A, indicate that the dose of scopolamine had a significant effect on activity (F = 9-75; 2,54 d.f.; P < 0.001), that the group that the animals were in had a significant effect (F = 5"80; 2,27 d.f.; P < 0.01), and that there was a significant interaction between drug and group (F = 4.01 ; 4,54 d.f.; P < 0-01). A further analysis indicated that the activity of the median group was significantly higher than that of either of the other two groups (P < 0.01; 2,27 and 3,27 d.f. Newman-Keuls). These data imply that there is a scopolamine-serotonin interaction, and that it is specific to the median nucleus. However, as with amphetamine, if the post-drug scopolamine data are plotted (Figure 2B) and statistically analyzed as a percent of the pre-drug baseline, the group curves completely overlap and the only variable producing a statistically significant effect is the dose of scopolamine (F = 13'09; 2,54 d.f.; P < 0.001). The lack of a statistically significant effect of group or of an interaction between drug and group (analyses of variance, P > 0.10) using these measures, implies that there is no scopolamine serotonin interaction. As shown in Table 1, both median and dorsal lesioned groups had significant reductions in forebrain serotonin content as compared with controls, 33-7 and 51"7~o respectively. Histological examination of the brainstem tissue indicated that at least 80~o of the target nucleus was destroyed in all cases, and in no case was there any damage to the other nucleus. These data are summarized in Figure 3 which displays the histological reconstruction of the smallest and largest lesions of both the dorsal and median nuclei at various levels. DISCUSSION
These results corroborate the previous findings of NEILL et al. 11972) and MABRV and CA~mBELL (1973) insofar as they show that a reduction in brain serotonin can potentiate the behavioural response to amphetamine. The present findings also extend the conclusions of these studies by demonstrating: (1) that the effect is seen following lesions specific to the median nucleus, and is not seen following lesioning of the dorsal nucleus; and (2) that a reduction in brain serotonin can also potentiate the behavioural response to an anticholinergic drug. On the other hand, the present data question the validity of the notion that an amphetamine-serotonin interaction really exists. There are two reasons for this. First, if one takes into account that the median lesioned animals have a higher baseline activity, then the percent increases produced by the various doses of amphetamine
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J Fig. 3. Coronal sections displaying the minimal damage (darkened area) and maximal damage (outlined in black) in the two lesioned groups. Top two lesions in all sections are of the dorsal nucleus and the bottom two lesions in all sections are of the median nucleus. Sections are taken every 0.4mm, with the top section at A-P (+) 0.2 and the bottom section at A-P ( - ) 1.0 (see PELLEGRINO and CUSHMAN, 1967, for abbreviations).
or scopolamine are no different across groups. This implies, at least, that the amphetamine/scopolamine-serotonin effect is not generalizable to a different measure. Furthermore, if one reanalyzes the data shown in Figure 2 of NEILL et al. (1972) and Figure 1 of MABRY and CAMPBELL (1973) in this way, it is clear that an interaction no longer appears (Table 2). The second reason for questioning the validity of the amphetamine-serotonin interaction is the existence of an apparently equally valid scopolamineserotonin interaction. The most parsimonious explanation o f both of these results is that serotonin depleted rats may be hyperreactive to a variety of agents which increase behavioural responsivity. This would make the amphetamine-serotonin interaction only a special case of a more general phenomenon. Finally, we would like to discuss a cautionary note. Since it is well known that no brain lesion produces destruction specific to the intended target, the data in the present study may be due, in part, to destruction of non-serotonergic neurones. PEPEU, GARAU and MULAS (1974) have shown, for example, that lesions of the median nucleus produce a 22~ reduction in forebrain acetylcholine. ",.l'. 14/7
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506
B. U JACOaS, W. D. WISE and K. M. TAYLOR
Table 2. Recalculation of previously published post amphetamine increases in activity as a percentage of baseline as opposed to absolute activity levels*
Amphetamine (mg/kg) 0'0 1"0 2"0 3"0
Amphetamine (mg/kg) 0.0 0.5 1.0 2-0 4"0
NEILL et al. (1972) Mean hourly crossings (Fig. 2) Saline Raphe lesion 48 130 220 350
190 515 810 875
MAaRY and CAMPBELL(1973) Mean hourly crossings (Fig. 1) Saline p-CPA 7 24 81 187 236
62 76 167 512 615
Per cent baseline activity (recalculated) Saline Raphe lesion 100 271 458 729
100 271 426 460
Per cent baseline activity (recalculated) Saline p-CPA 100 343 1157 2671 3371
100 122 269 825 992
* The data from NEILL et al. (1972) were derived from their Figure 2. The data from MABRY and CAMPBELL (1973) were generously given to us by the authors.
In conclusion, the existence of an amphetamine-serotonin interaction seems to depend on how we define "interaction". If it is defined as the absolute activity relative to control produced by a pharmacological treatment, then a case can be made for its validity, however, if we define it as percent increase above baseline, then the effect disappears. Even if we accept the former definition, its validity seems to be severely weakened by the existence of an apparently equally valid scopolamine-serotonin interaction. Basically, our data and those of NEILL et al. (1972) indicate that the magnitude of the response to a drug is largely determined by the baseline level from which the response begins. Thus, the behavioural hyperresponsivity of the serotonin depleted animal may be merely a manifestation of starting from an elevated baseline. Acknowledoements--The authors wish to thank Smith, Kline & French Laboratories for their generous gift of (+)-amphetamine sulphate. They also wish to thank DANIEL RUIMV for his contributions to various phases of these studies.
REFERENCES BOGO^NSKI, D. F., PLETSCnER, A., BRODtE, B. B. and UDENFRIEND, S. (1956). Identification and assay of serotonin in brain. J. Pharmac. exp. Ther. 117: 82-88. CHRUSlEL, T. L. and HERMAN, Z. S. (1969). Effect of dopamine on behavior in mice depleted of norepinephrine or serotonin. Psychopharmacologia 14: 124-134. FIBIGER, H. C. and CAMPBELL, B. A. (1971). The effect of parachorophenylalanine on spontaneous locomotor activity in the rat. Neuropharmacology 10: 25-32. JACOaS, B. L., WISE, W. D. and TAYLOR, K. M. (1974a). Differential behavioral and neurochemieal effects following lesions of the dorsal or median raphe nuclei in rats. Brain Res. 79: 353-361. JAcoas, B. L., EUaANKS, E. E. and WLSE,W. D. (1974b). Effect of indolealkylamine manipulations on locomotor activity in rats. Neuropharmacology 13: 575-583. KOSTOWSKX,W., GL~CALONE,W., GARATTINI, S. and VALZELLI, L. (1968). Studies on behavioral and biochemical changes in rats after lesions in midbrain raphe. Eur. J. Pharmac. 4: 371-376. MABRV, P. D. and CAMPBELL, B. A. (1973). Scrotonergic inhibition of catecholamine-induced behavioral arousal. Brain Res. 49:. 381-391. NEILL, D. B., GRANT, L. D. and GROSSMAN, S. P. (1972). Selective potentiation of locomotor effects of amphetamine by midbrain raphe lesions. Physiol. Behav. 9:. 655-657. PELLEGRINO, L. J. and CUSHMAN, A. J. (1967). A Stereotaxic Atlas of the Rat Brain. Appleton-Century-Crofts, New York. PEPEU, G., GARAU, L. and MULAS, M. L. (1974). Does 5-Hydroxytryptamine influence cholinergic mechanisms in the central nervous system? In: Advances in Biochemical Psychopharmacology (COSTA, E. and GREENGARO, P., Eds.), Vol. 10, pp. 247-252. Raven Press, New York.
IS T H E R E A C A T E C H O L A M I N E - S E R O T O N I N INTERACTION IN THE CONTROL OF LOCOMOTOR ACTIVITY?* B. L. JACOBSand W. D. WISE Department of Psychology, Princeton University, Princeton, New Jersey 08540
and K. M . TAYLOR Department of Pharmacology, The Squibb Institute of Medical Research, Princeton, New Jersey 08540
(Accepted 25 November 1974) Summary--Previous studies have reported that rats given either midbrain raphe lesions or the serotonin synthesis inhibitor, p-chlorophenylalanine, show an increase above controls in their locomotor response to various doses of (+)-amphetamine. These data have been taken to be indicative of a catecholamine-serotonin interaction. The present data demonstrate that this increased response to amphetamine was obtained following lesions specific to the median raphe nucleus, whereas lesions of the dorsal raphe nucleus were without effect. These results also question the validity and generality of the catecholamine serotonin interaction hypothesis by demonstrating: (1) that when the data are plotted as a per cent of baseline activity, the interaction disappears; and (2) that a very similar effect is observed when amphetamine injections are replaced by scopolamine injections.
Decreases in functional brain serotonin, produced either through raphe lesions (KosTOWSKI, GIACALONE, GARATTINI a n d VALZELLI, 1 9 6 8 ; NEILL, GRANT a n d
GROSSMAN,
1972; JACOBS, WISE and TAYLOR, 1974a) or synthesis inhibition with p-chlorophenylalanine (CHRuSCIEL and HERMAN, 1969; FIBIGER and CAMPBELL, 1971: JACOBS, EUBANKS and WISE, 1974b) result in marked increases in locomotor activity in the rat. In addition, the locomotor increasing effects of amphetamine are potentiated either by raphe lesions (NEILL et al., 1972) or serotonin synthesis inhibition (MABRY and CAMPBELL, 1973). On the basis of these latter two findings with amphetamine, it has been argued that the catecholamines and serotonin interact in the control of locomotor activity in the rat. We have previously shown that selective lesions of the median raphe nucleus resulted in long-lasting increases in locomotor activity, whereas lesions of the dorsal nucleus were without effect (JACOBS et al., 1974a). The purpose of the present study was to examine whether the amphetamine-serotonin interaction hypothesis could be extended to animals with selective lesions of the dorsal or median raphe nucleus. A further objective was to test the validity and generality of this hypothesis by: (1) utilizing an anticholinergic drug which increases locomotor activity, and (2) by measuring the increases in activity as a percent of baseline rather than in terms of absolute levels of activity. METHODS
Activity Male Sprague-Dawley rats weighing 300-400 g were used in all experiments. Locomotor activity was measured by means of centre-balanced wire mesh stabilmeter cages (17.5 x 20.0 × 37-5cm) which tilted back and forth in the longitudinal direction, as the rat crossed the fulcrum. These cages served as the animal's home cage for the duration of the study. Crossings were recorded by means of a microswitch at one * Supported by NIMH Grants MH 23433 and MH 24711. 501
502
B.L. JACOBS,W. D. WISEand K. M. TAYLOR
end of the cage which was connected to an electronic counter in an adjacent room. The cages were housed in a well-ventilated temperature controlled room (22°C) with a 70dB masking noise. Animals were on a 12-hr light-dark schedule with "lights off" at 1000 hr. The room was lit by a 200W incandescent bulb.
Lesions Animals were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic instrument with the bite bar oriented 5ram above the ear bars. A bone flap over the midsaggital sinus was removed at an anterior-posterior coordinate above the midbrain raphe nuclei• The electrodes used for lesioning were thin tungsten wires (0'13 mm diameter) that were sharpened by etching and then insulated. Since the dorsal and median raphe nuclei are most extensive in the anterior posterior and dorso-ventral dimensions, and are quite narrow in the medial-lateral dimension, the lesioning of a particular nucleus was accomplished by making several small midline lesions with varying anterior-posterior and dorso-ventral coordinates. The coordinates at which a 160/~A d.c. current was passed for 20sec were: dorsal groul>-A-P (+) 0.5, D-V ( - ) 0.7, ( - ) 0.4, and ( - ) 0.1, A P (+) 0.2, D-V ( - ) 1.2, ( - ) 0.9, and ( - ) 0"6; median groul~-A-P (+) 0.5 and (+) 0'2, D-V ( - ) 3.0, ( - ) 2"6, ( - ) 2.2 (PELLEGRINO and CUSHMAN, 1967). Pharmacology Three groups, dorsal lesioned, median lesioned and control, consisting of 10 animals each, were used in all experiments. Pharmacological studies were not begun until one month after lesioning in order for the animals to completely recover from surgery and for their baseline activity to stabilize. On drug injection days, hourly activity measures were taken for the 3-hr period following "lights off" (1000hr). Activity during this period served as the baseline. All injections were administered intraperitoneally at 1300 hr. Hourly activity measures were then taken for the 2-hr period beginning 30 min post-injection (1330hr). In the first experiment, (+)-amphetamine sulphate was administered in doses of 0.0 (saline control), 0"5, 1.0 and 2.0mg/kg (expressed as the salt). Every animal received each drug dose in a semi-randomized order, and injections were spaced 5 days apart• The second experiment was identical in design except that the amphetamine injections were replaced by scopolamine hydrobromide injections of 0.0 (saline control), 0-125 and 0.250 mg/kg (expressed as the salt)•
Serotonin assay One month following the termination of all experiments, control and experimental rats were decapitated, the brain was quickly removed, and the forebrain was dissected away from the brainstem just anterior to the superior colliculus. The forebrain tissue was frozen over dry ice, weighed and stored at -14°C for the serotonin estimation, which took place within 48hr. The brainstem was placed in 10~o formalin for serial sectioning (40#m thick coronal sections stained with cresyl violet) in order to locate the site of the lesion. Serotonin was estimated by a modification of the procedure of BOGDANSKI,PLETSCHER, BRODIEand UDENFRIEND(1956). The frozen tissue was homogenized in 3ml of ice cold 0-4M perchloric acid containing 0.1°~ ascorbic acid and 0 1/o ethylenediaminetetraeetic acid. The homogenate was centrifuged at 10,0009 for 10 rain and the supernatant fluid was removed and placed in a stoppered test tube containing 0.5ml of 0"5M borate buffer pH 10. The mixture was adjusted to pH 10 with solid sodium carbonate and saturated with sodium chloride. Serotonin was extracted into 6ml of n-butanol. After centrifugation, the butanol phase was removed and placed in a stoppered test tube containing 1.0ml of 0.1M HCI and 7"5ml of n-heptane. After the tube had been shaken for 5 min and centrifuged at 2500 9 for 5 min, the aqueous phase was removed for serotonin estimation. Native fluorescence of serotonin was read at 295nm excitation, 540nm emission wavelengths, after the addition of 0"5ml 9M HC1. External and internal standards of serotonin (50ng) as well as reagent blanks were also determined. Five ng of serotonin produced a reading •
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Fig. 1. A: Dose-response curves for (+)-amphetamine sulphate (i.p.) in median lesioned, dorsal lesioned, and control animals. Data plotted are the mean hourly crossings for the 2-hr post-drug period. Baseline data represent the activity of uninjected animals. B: The data in A are replotted as a percentage of the pre-drug baseline activity. Each group represents 10 animals.
o f fluorescence twice that of the blank. The recovery o f serotonin added to tissue s a m p l e s w a s 82~o. Tissue c o n c e n t r a t i o n s were not corrected for recovery of serotonin. RESULTS As s h o w n in Figures 1A and 2A, the m e d i a n raphe lesion p r o d u c e d a long-lasting elevation in s p o n t a n e o u s activity of a p p r o x i m a t e l y 1 0 0 ~ a b o v e the activity o f the dorsal lesioned and control groups. Statistical analyses of the data plotted in Figure IA, i.e. m e a n hourly post-drug activity, s h o w that the d o s e of a m p h e t a m i n e significantly affected l o c o m o t o r activity (F = 21.38; 3,81 d.f.; P < 0"001), that the group in which the animals were placed had a significant effect (F = 7"20; 2,27 d.f.; P < 0.001), and x Median o Dorsal • Control
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Fig. 2. A: Dose-response curves for scopolamine hydrobromide (i.p.) in median lesioned, dorsal lesioned, and control animals. Da~a plotted are the mean hourly crossings for the 2-hr post-drug period. Baseline data represent the activity of uninjected animals. B: The data in A are replotted as a percentage of the pre-drug baseline activity. Each group represents 10 animals.
504
B. L. JAcoas, W. D. W~SE and K. M. TAYLOR Table 1. Forebrain serotonin (Izg/g) Group
Control Median lesioned Dorsal lesioned
n
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"..~, Reduction
6 8 8
0.490 +/).023 0.325 + 0.010" 0.237 + 0.008*
33.7 51.7
Each value is the mean + S.E.M. Significance of difference bctween means of the lesioned groups from the control group: * P < 0.001 (two-tailed t-tests).
finally that there was a significant interaction between drug and group (F--5.91; 6,81 d.f.; P < 0"001). A further analysis indicated that the activity of the median group was significantly greater than that of either the dorsal or control groups (P < 0.01; 2,27 and 3,27 d.f. Newman-Keuls). These data imply that there is an amphetamine-serotonin interaction, and that it is specific to the median raphe nucleus. However, if the post-drug data are plotted (Figure I B) and statistically analyzed as a percent of the pre-drug baseline rather than as absolute post-drug activity counts (as above), the group curves completely overlap and the only variable producing a statistically significant effect is the dose of amphetamine (F = 28.01; 3,81 d.f.; P < 0.001). The lack of a statistically significant effect of group or an interaction between drug and group (analyses of variance, P > 010) using these measures, implies that there is no amphetamine-serotonin interaction. Much the same picture is seen when the results of the scopolamine experiment are examined. Statistical analyses of the data plotted in Figure 2A, indicate that the dose of scopolamine had a significant effect on activity (F = 9-75; 2,54 d.f.; P < 0.001), that the group that the animals were in had a significant effect (F = 5"80; 2,27 d.f.; P < 0.01), and that there was a significant interaction between drug and group (F = 4.01 ; 4,54 d.f.; P < 0-01). A further analysis indicated that the activity of the median group was significantly higher than that of either of the other two groups (P < 0.01; 2,27 and 3,27 d.f. Newman-Keuls). These data imply that there is a scopolamine-serotonin interaction, and that it is specific to the median nucleus. However, as with amphetamine, if the post-drug scopolamine data are plotted (Figure 2B) and statistically analyzed as a percent of the pre-drug baseline, the group curves completely overlap and the only variable producing a statistically significant effect is the dose of scopolamine (F = 13'09; 2,54 d.f.; P < 0.001). The lack of a statistically significant effect of group or of an interaction between drug and group (analyses of variance, P > 0.10) using these measures, implies that there is no scopolamine serotonin interaction. As shown in Table 1, both median and dorsal lesioned groups had significant reductions in forebrain serotonin content as compared with controls, 33-7 and 51"7~o respectively. Histological examination of the brainstem tissue indicated that at least 80~o of the target nucleus was destroyed in all cases, and in no case was there any damage to the other nucleus. These data are summarized in Figure 3 which displays the histological reconstruction of the smallest and largest lesions of both the dorsal and median nuclei at various levels. DISCUSSION
These results corroborate the previous findings of NEILL et al. 11972) and MABRV and CA~mBELL (1973) insofar as they show that a reduction in brain serotonin can potentiate the behavioural response to amphetamine. The present findings also extend the conclusions of these studies by demonstrating: (1) that the effect is seen following lesions specific to the median nucleus, and is not seen following lesioning of the dorsal nucleus; and (2) that a reduction in brain serotonin can also potentiate the behavioural response to an anticholinergic drug. On the other hand, the present data question the validity of the notion that an amphetamine-serotonin interaction really exists. There are two reasons for this. First, if one takes into account that the median lesioned animals have a higher baseline activity, then the percent increases produced by the various doses of amphetamine
Catecholamine-serotonin interaction?
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J Fig. 3. Coronal sections displaying the minimal damage (darkened area) and maximal damage (outlined in black) in the two lesioned groups. Top two lesions in all sections are of the dorsal nucleus and the bottom two lesions in all sections are of the median nucleus. Sections are taken every 0.4mm, with the top section at A-P (+) 0.2 and the bottom section at A-P ( - ) 1.0 (see PELLEGRINO and CUSHMAN, 1967, for abbreviations).
or scopolamine are no different across groups. This implies, at least, that the amphetamine/scopolamine-serotonin effect is not generalizable to a different measure. Furthermore, if one reanalyzes the data shown in Figure 2 of NEILL et al. (1972) and Figure 1 of MABRY and CAMPBELL (1973) in this way, it is clear that an interaction no longer appears (Table 2). The second reason for questioning the validity of the amphetamine-serotonin interaction is the existence of an apparently equally valid scopolamineserotonin interaction. The most parsimonious explanation o f both of these results is that serotonin depleted rats may be hyperreactive to a variety of agents which increase behavioural responsivity. This would make the amphetamine-serotonin interaction only a special case of a more general phenomenon. Finally, we would like to discuss a cautionary note. Since it is well known that no brain lesion produces destruction specific to the intended target, the data in the present study may be due, in part, to destruction of non-serotonergic neurones. PEPEU, GARAU and MULAS (1974) have shown, for example, that lesions of the median nucleus produce a 22~ reduction in forebrain acetylcholine. ",.l'. 14/7
D
506
B. U JACOaS, W. D. WISE and K. M. TAYLOR
Table 2. Recalculation of previously published post amphetamine increases in activity as a percentage of baseline as opposed to absolute activity levels*
Amphetamine (mg/kg) 0'0 1"0 2"0 3"0
Amphetamine (mg/kg) 0.0 0.5 1.0 2-0 4"0
NEILL et al. (1972) Mean hourly crossings (Fig. 2) Saline Raphe lesion 48 130 220 350
190 515 810 875
MAaRY and CAMPBELL(1973) Mean hourly crossings (Fig. 1) Saline p-CPA 7 24 81 187 236
62 76 167 512 615
Per cent baseline activity (recalculated) Saline Raphe lesion 100 271 458 729
100 271 426 460
Per cent baseline activity (recalculated) Saline p-CPA 100 343 1157 2671 3371
100 122 269 825 992
* The data from NEILL et al. (1972) were derived from their Figure 2. The data from MABRY and CAMPBELL (1973) were generously given to us by the authors.
In conclusion, the existence of an amphetamine-serotonin interaction seems to depend on how we define "interaction". If it is defined as the absolute activity relative to control produced by a pharmacological treatment, then a case can be made for its validity, however, if we define it as percent increase above baseline, then the effect disappears. Even if we accept the former definition, its validity seems to be severely weakened by the existence of an apparently equally valid scopolamine-serotonin interaction. Basically, our data and those of NEILL et al. (1972) indicate that the magnitude of the response to a drug is largely determined by the baseline level from which the response begins. Thus, the behavioural hyperresponsivity of the serotonin depleted animal may be merely a manifestation of starting from an elevated baseline. Acknowledoements--The authors wish to thank Smith, Kline & French Laboratories for their generous gift of (+)-amphetamine sulphate. They also wish to thank DANIEL RUIMV for his contributions to various phases of these studies.
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