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Effect of selective inhibitors of tyrosine and tryptophan hydroxylases on self-stimulation in the rat
EXPERIMENTAL
Effect Tryptophan
NEUROLOGY
of
27, 283-290
Selective
Hydroxylases
(1970)
Inhibitors on
of
Tyrosine
Self-Stimulation
and in the
Rat
S. GIBSON, E. G. MCGEER, AND P. L. MCGEER 1 Kirtsnten Laboratory of Ncrwological Research, Departrrlent of Psychiatry, University of British Cohnbia, Vancouver, Canada Received
December
31, 1969
The effect of various drugs on self-stimulation of rats having electrodes implanted in the medial forebrain bundle was measured. An increase in threshold for self-stimulation was observed in most rats following administration of: reserpine, 0.3 and 0.5 mg/kg ; L-a-methyl-P-tyrosine, 100 mgjkg ; DL-a-methyl-ptyrosine, 200 mg/kg ; DL-5-bromotryptophan, 290 mg/kg ; DL-P-chlorophenylalanine, 150 and 200 mg/kg; and DL-6-fluorotryptophan, 150 mg/kg. Reserpine, a depletor of both catecholamines and serotonin, was the most effective agent at increasing the threshold. This was followed by the tyrosine hydroxylase inhibitors a-methyl-P-tyrasine and DL-5-bromotryptophan. At the doses used, the tryptophan hydroxylase inhibitors nL+chlorophenylalanine and nt-6-fluorotryptophan were less effective. In general, the maximum effects of the drugs corresponded in time with the maximum depletion of the various amines. Measurement of amine levels suggested that catecholamine depletion was more influential at increasing the self&imulation threshold than serotonin depletion. Reserpine, which depletes all amines, seemed to have a larger effect on self-stimulation than would be indicated by the drop in amine levels. Over-all, the data suggest that both catecholamines and serotonin are involved in pathways for self-stimulating drive-reward with the catecholamines being more influential. Introduction
One of the most powerful self-stimulation centers in the rat is the medial forebrain bundle region of the posterolateral hypothalamus (7). This locus has dopaminergic, noradrenergic, and serotonergic fibers coursing rostrally through it (1, 2). It also has cholinergic structures in the vicinity (11). Self-stimulation of the region might therefore be expected to excite not only local structures but also fibers of all these aminergic systems. Such stimulation could have widespread effects on the midbrain, the septal region, the structures of the limbic lobe, and the hypothalamus. It is conceivable, however, that self-stimulation is not associated with activation of all these structures but is restricted to only 1 This work was supported by Grant No. M.4-3633 Council of Canada. 283
from the Medical
Research
284
GIBSON,
MCGEER,
AND
McGEER
some of them. Wise and Stein (14) have brought forward considerable evidence suggesting that self -stimulation is directed only at exciting noradrenergic neurons. The availability of selective inhibitors of the key enzymes for catecholamine and serotonin synthesis allows further exploration, and, in this communication, we present evidence that serotonergic neurons may also be involved in self-stimulation. Materials
and
Methods
Standard rat self-stimulating equipment (Neuropsych Co.) was used. The stimulator supplied a 60-cycle a-c voltage which could be varied linearly from O-17 v (O-6 root mean square volts). The impulse time was kept constant at 150 msec, so that for each bar press a train of approximately 15 pulses of the 6O-cycle sign wave variety was delivered. Male Hooded and Wistar rats, 200-300 g, were implanted with bipolar Teflon-coated electrodes in the medial forebrain bundle region of the posterior hypothalamus. Following the operation, the animals were trained to press the bar to receive electrical stimulation. The threshold voltage at which each animal would maintain a constant rate of stimulation and the rate itself were determined as follows. The voltage was set so as to produce a typical rate of stimulation which was determined over a 2-min period. The voltage was then manually decreased on the stimulator unit in steps of 1.1 v at 2-min intervals. The rate of stimulation was determined at each voltage level until a voltage was reached where the animal stopped stimulating. The voltage was then increased again until two or three consecutive settings produced the same rate of stimulation. The voltage setting where the maximum rate of stimulation was first reached was taken as the threshold voltage. Only animals that showed a stable threshold and rate for one week in daily tests were used for drug studies. Control threshold voltages varied among the rats from 5-14 v (average 9) and stimulation rates from 50-250/min (average 117). The voltage difference between the threshold following administration of a drug and the control threshold was taken as the effect of the drug. Drug experiments were begun in the morning and run during the day. Whenever possible, the testing took place at the same time each day for a given animal. ii11 the drugs used were obtained from commercial sources and were administered intraperitoneally. L- And nr>-cr-methyl-p-tyrosine, DL-p-chlorophenylalanine, DL-5-bromotryptophan, and DL-6-fluorotryptophan were not soluble at neutral pH so they were injected as a suspension in 20% propylene glycol. The suspension was made by adding propylene glycol to a measured amount of drug, making the mixture basic with 3~ NaOH,
285
SELF-STIhiVLATIOiX
and shaking until the drug went into solution. The pH was brought back to 7 with 3~ HCl, and the volume adjusted with water so that each animal was injected with l-l.5 ml, Injection of drug-free suspensions made up by this procedure had no effect on self-stimulation. The animals were left at least 1 week between drug experiments and were not used for further studies if the control threshold showed changes. When the drug studies were completed, the animals were killed and the brains removed and fixed in formalin. Placement of the electrode in each rat was determined by histological esamination of brain slices stained with Luxol Fast Blue. In order to check that the drugs were producing the expected effects on amine levels the catecholamines and serotonin were determined by reported fluorometric methods (6) in the brains of control and druginjected male rats of species and size comparable to those used in the selfstimulation study. Results
A typical voltage-rate curve for self-stimulation is shown in Fig. 1. It illustrates the constant rate of self-stimulation which is achieved as the voltage is increased beyond the threshold. The changes are very sharp, indicating that this is a sensitive method of estimating drug effects. Figure 2 shows three typical examples of threshold changes versus time for rats injected with various drugs. The overall results are summarized
4)
,
10
12
14
16
18
20
VOLTAGE
1. Voltage vs. bar presses per minute for a typical Threshold equal to 14 v; rate equal to 171 * 20 press/min. FIG.
self-stimulating
rat,
286
GIBSON,
MCGEER,
4
0 HOURS
AND
12 AFTER
MCGEER
16
20
24
ADMINISTRATION
FIG. 2. Effect of drugs on threshold voltage of typical rats with time. V mA%fluorotryptophan, 100 mg/kg ; n n&bromotryptophan, 290 mg/kg ; 0 L-cr-methyltyrosine, 100 mg/kg.
in Table 1. As can be seen from the Table, drugs which cause a decrease in catecholamines, serotonin, or both were all effective in increasing the threshold for self-stimulation in the majority of rats tested. In some instances there was no response to the drugs, and on four occasions there were decreases, rather than increases, in threshold. These decreases OCcurred in three rats give p-chlorophenylalanine and in one of these same animals after DL-cu-methyl-p-tyrosine. Reserpine was tested for comparative purposes and proved to be the most effective drug used in changing the threshold for self-stimulation. At a dose of 0.3 mg/kg, an increase in threshold was observed in all animals, and at a dose of 0.5 mg/kg, self-stimulation was almost completely eliminated. Of the enzyme inhibitors used, Ba-methyl-fi-tyrosine was the most consistently effective. Out of 23 rats tested, 18 showed an increase in threshold, three showed no effect, and one showed a decrease. The maximum effect on self-stimulation was 5-9 hours after injection which is in agreement with the time of the maximum reported decrease in brain noradrenaline and dopamine content after the administration of this agent. Recovery of self-stimulation was complete within 24-48 hours, which is also in line with
287
SELF-STIMULATION
TABLE CHANGES
NO. rats 22 3 4 II 8 6 5 8 3 5
1~ SeL1~-5mrvL~~~o~
I>rllg ‘k dOW lieserpine, 0.3 nig,‘kg Iieserpine, 0.5 mg/kg r.-cr-Methyl-p-tyrosine, L-a-Methyl-p-tyrosine, DL-cy-Methq-I-P-tvrosille, DL-S-Bromotryptophau, DL-p-Chlorophen~lalanitle, DL-p-Chloropheuylalanine, m-6-Fluorotryptophau, m-6-Fluorotryptophan,
75 111gjkg 100 mg/kg 200 mg/kg” 290 nlg/kg 1.50 mg/kg 200 ~ng/kg 100 mgjkg 150 mg/kg
1
THHESHOLD
IIecr
None
INDC’CED
nY
VARIOUS
DRU<;~
Brain amine levels (70 of controls)” 5HT + 1.1 v + 2.2 v + 3.3 v NA
0
I)
12
8
2
0 0
0 2
0 1
0
3
95 70
1
0
-
_.
0 1 0 0
0 1 0 0 1 0 3
3 1 5 2 I
2
G
6.5
0 1
5 0
2.5 49
60
100 100
1
95 95 -
48 30 -._
110
4.5
3
0 0
2
0
2 2 1
1
0
1
I
80 70
0 Values are averages of those fouud in animals of conlparable size and species at the time of expected lasimum effect and are in general accord with the effects of these agents as reported in the literature. *Given in serial doses (10).
the time of recovery of amine levels. The fact that a-methyl-p-tyrosine in the higher dosesused reduced brain catecholamine levels by approximately 50% (10) was confirmed by measurementsin this laboratory. Dr&&omotryptophan, a selective tyrosine hydroxylase inhibitor (4)) produced an increase in threshold in all six animals where it was tried. For most of the rats the effect was mild, reaching its maximum 2-4 hours after injection which correlates with the time of maximum amine depression. Even at the high dose of 290 mg/kg, noradrenaline in these rats was only reduced by about 40%, so that the mildness of the effect as compared with that wrought by a-methyl-p-tyrosine would be in keeping with a less severe reduction in catecholamine levels. nL-5-Bromotryptophan had no effect on serotonin concentration. Dr.-p-Chlorophenylalanine, which is selective in its inhibition of tryptophan hydroxylase and therefore selective as a serotonin depletor, gave mixed results. Of 13 rats tested, nine showed an increase in threshold, one no effect, and three a decreasein threshold. The increase in threshold was observed 2-3 days after the injection which corresponds with the time of maximum decreasein serotonin levels (3) ; any decreasein threshold seemedto occur somewhat earlier (1-2 days). For those rats showing an increase in threshold, the effect was not as pronounced as with an equimolar dose of LYmethyl-p-tyrosine, even though at this dose of p-chlorophenylalanine brain serotonin levels are decreasedby about 7070, as evidenced both by literature reports and by confirmatory data obtained by us. DL-6-Fluorotryptophan, a selective inhibitor of tryptophan hydroxylase
288
GIBSON,
MCGEER,
AND
MCGEER
(5), was administered to eight rats. Five of these showed a mild increase in threshold and three no effect. The changes occurred l-3 hours after drug administration, the time of maximum effect on brain serotonin levels. The histological results showed that the electrodes were in the medial forebrain bundle or in tissue just dorsal to it in all the animals. The only two animals that showed several atypical responses had electrode placements in the same areas as did other rats that failed to show any unusual responses. All the animals were not given all the drugs, but each animal was given several drugs, and the histological results made it clear that the increases in threshold observed for catecholamine depletors versus serotonin depletors could not possibly be explained on the basis of different electrode placement. The only correlation between electrode placement and self-stimulation activity was that the more dorsal placements tended to produce higher rates and lower thresholds. Discussion
Our results on reduced self-stimulation following administration of LYmethyl-fi-tyrosine or reserpine are in agreement with other reports (8, 13). One hypothesis that has been proposed (14) is that reduction in noradrenaline caused by these drugs is responsible for the decreased selfstimulation activity, and indeed the time courses of the two effects correlate well both with these agents and with DL-5-bromotryptophan. However, with cu-methyl-p-tyrosine, noradrenaline had to be reduced much further than with reserpine to obtain equivalent changes in self-stimulation, and DL-5bromotryptophan produced only very mild changes in self-stimulation although it reduced the noradrenaline content as much as the dose (0.5 mg/ kg) of reserpine which eliminated self-stimulation in all three rats tested. This suggests that the relationship between self-stimulation and noradrenaline content must be subtle. Two possible explanations would be involvement of other amines or involvement of different pools of noradrenaline affected differently by reserpine, a-methyl-p-tyrosine, and m-5-bromotryptophan. Reserpine, of course, reduces dopamine and serotonin as well as noradrenaline, and all three of these amines are involved in pathways running through the medial forebrain bundle region. The drugs used in this study were not suitable to differentiate between noradrenaline and dopamine, but the results obtained with m+chlorophenylalanine, as well as with r&d-fluorotryptophan, suggest that serotonin pathways are involved in self-stimulation. The time course of the effect of each of these drugs on self-stimulation is again in good correlation with their effect on central serotonin levels. The probability that serotonin is involved in self-stimulation is also suggested by the work of Poschel and Ninteman (9) who reported enhancement of
SELF-STIMULATION
289
self-stimulation upon administration of Shydroxytryptophan to rats pretreated with a monoamine oxidase (MrlO) inhibitor, and by the study of Stark, Boyd, and Fuller (12) who found that low doses of D-2-bromolysergic acid diethylamide (, BOL), a serotonin antagonist. decreased the threshold for hypothalamic self -stimulation in dogs while nL-pheniprazine hydrochloride (JB 516), an MAO inhibitor that causes an elevation of brain serotonin levels without a concomitant increase in noradrenaline, lowered the threshold for self-stimulation. The available data do suggest that a given selective percentage decrease in brain serotonin level generally has less effect on self-stimulation than a comparable selective decrease in brain catecholamine level. It may be that the catecholamines are involved in a more powerful reward system than is serotonin. Obviously, it would help greatly in our understanding of the roles of these amines in behavior and of the neuro-anatomical pathways associated with behavior if clear-cut definitions could be made of the neurotransmitters serving specific drive-reward systems. It would appear, however, that more sophisticated experimentation will be required before such distinctions are possible. References 1. ANDEN, N. E., A. CARLSSON, A. DAHLSTROM, K. FUXE, N. A. HILLARP, and P. R. LARSON. 1964. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sri. 3 : 523-530. 2. DAHLSTROM, A., and K. FUXE. 1964. Evidence for the existence of monoamine containing neurons in the central nervous system. .4&a Physiol. Stand. Supp. 232, 62: l-55. 3. ‘E;OE, K., and A. WEISSMAN. 1966. fi-Chlorophenylalanine: A specific depletor of brain serotonin. .I. Pharvlacol. Exp. Ther. 154 : 499-514. 4. MCGEER, E. G., P. L. MCGEER, and D. A. V. PETERS. 1967. Inhibition of brain tyrosine hydroxylase by Shalotryptophans. Lift Sci. 6 : 2221-2232. 5. MCGEER, E. G., D. A. V. PETERS, and P. L. MCGEER. 1968. Inhibition of rat brain tryptophan hydroxylase by 6-halotryptophans. Life Sci. 7 : 60-615. 6. MCGEER, P. L., E. G. MCGEER, and J. A. W’ADA. 1963. Serotonin and catecholamine levels in various cat brain areas following administration of psychoactive drugs or amine precursors. A.M.,4. Arch. Neural. 9 : 81-89. 7. OLDS, J. 1962. Hypothalamic substrates of reward. Physiol. Rev. 42: 55C604. 8. POSCHEL, B. P. H., and F. W. NINTEMAN. 1966. Hypothalamic self-stimulation. Its suppression by blockade of norepinephrine biosynthesis and reinstatement hy methamphetamine. Life Sci. 5 : 1 I-16. 9. POSCHEL, P., and F. NINTEMAN. 1968. Excitatory effects of 5 HTP on intracranial self-stimulation following MAO blockade, Life Sci. 7: 317-323. 10. RECH, R. H., H. K. BORYS, and K. E. MOORE. 1966. -4lterations in brain catecholamine levels in rats treated with a-methyl-tyrosine. J. Phamacol. Et-p. Thr-. 153 : 312-419. 11. SHUTE. C. C. D., and P. R. LEWIS. 1967. The ascending cholinergic reticular system : Neocortical, olfactory and suhcortical projections. Rrnirz SO : -197.522.
290
GIBSON,
MCGEER,
AND
MCGEER
P., E. S. BOYD, and R. W. FULLER. 1964. A possible role of serotonin in hypothalamic self-stimulation in dogs. J. Pharwzacol. Exp. Thu. 146 : 147-153. 13. STEIN, L. 1964. Self-stimulation of the brain and the central stimulant action of amphetamine. Fed. Proc. 23 : 836850. 14. WISE, C. D., and L. STEIN. 1969. Facilitation of brain self-stimulation by central administration of norepinephrine. Science 163 : 299-301. 12.
STARK,
Effect Tryptophan
NEUROLOGY
of
27, 283-290
Selective
Hydroxylases
(1970)
Inhibitors on
of
Tyrosine
Self-Stimulation
and in the
Rat
S. GIBSON, E. G. MCGEER, AND P. L. MCGEER 1 Kirtsnten Laboratory of Ncrwological Research, Departrrlent of Psychiatry, University of British Cohnbia, Vancouver, Canada Received
December
31, 1969
The effect of various drugs on self-stimulation of rats having electrodes implanted in the medial forebrain bundle was measured. An increase in threshold for self-stimulation was observed in most rats following administration of: reserpine, 0.3 and 0.5 mg/kg ; L-a-methyl-P-tyrosine, 100 mgjkg ; DL-a-methyl-ptyrosine, 200 mg/kg ; DL-5-bromotryptophan, 290 mg/kg ; DL-P-chlorophenylalanine, 150 and 200 mg/kg; and DL-6-fluorotryptophan, 150 mg/kg. Reserpine, a depletor of both catecholamines and serotonin, was the most effective agent at increasing the threshold. This was followed by the tyrosine hydroxylase inhibitors a-methyl-P-tyrasine and DL-5-bromotryptophan. At the doses used, the tryptophan hydroxylase inhibitors nL+chlorophenylalanine and nt-6-fluorotryptophan were less effective. In general, the maximum effects of the drugs corresponded in time with the maximum depletion of the various amines. Measurement of amine levels suggested that catecholamine depletion was more influential at increasing the self&imulation threshold than serotonin depletion. Reserpine, which depletes all amines, seemed to have a larger effect on self-stimulation than would be indicated by the drop in amine levels. Over-all, the data suggest that both catecholamines and serotonin are involved in pathways for self-stimulating drive-reward with the catecholamines being more influential. Introduction
One of the most powerful self-stimulation centers in the rat is the medial forebrain bundle region of the posterolateral hypothalamus (7). This locus has dopaminergic, noradrenergic, and serotonergic fibers coursing rostrally through it (1, 2). It also has cholinergic structures in the vicinity (11). Self-stimulation of the region might therefore be expected to excite not only local structures but also fibers of all these aminergic systems. Such stimulation could have widespread effects on the midbrain, the septal region, the structures of the limbic lobe, and the hypothalamus. It is conceivable, however, that self-stimulation is not associated with activation of all these structures but is restricted to only 1 This work was supported by Grant No. M.4-3633 Council of Canada. 283
from the Medical
Research
284
GIBSON,
MCGEER,
AND
McGEER
some of them. Wise and Stein (14) have brought forward considerable evidence suggesting that self -stimulation is directed only at exciting noradrenergic neurons. The availability of selective inhibitors of the key enzymes for catecholamine and serotonin synthesis allows further exploration, and, in this communication, we present evidence that serotonergic neurons may also be involved in self-stimulation. Materials
and
Methods
Standard rat self-stimulating equipment (Neuropsych Co.) was used. The stimulator supplied a 60-cycle a-c voltage which could be varied linearly from O-17 v (O-6 root mean square volts). The impulse time was kept constant at 150 msec, so that for each bar press a train of approximately 15 pulses of the 6O-cycle sign wave variety was delivered. Male Hooded and Wistar rats, 200-300 g, were implanted with bipolar Teflon-coated electrodes in the medial forebrain bundle region of the posterior hypothalamus. Following the operation, the animals were trained to press the bar to receive electrical stimulation. The threshold voltage at which each animal would maintain a constant rate of stimulation and the rate itself were determined as follows. The voltage was set so as to produce a typical rate of stimulation which was determined over a 2-min period. The voltage was then manually decreased on the stimulator unit in steps of 1.1 v at 2-min intervals. The rate of stimulation was determined at each voltage level until a voltage was reached where the animal stopped stimulating. The voltage was then increased again until two or three consecutive settings produced the same rate of stimulation. The voltage setting where the maximum rate of stimulation was first reached was taken as the threshold voltage. Only animals that showed a stable threshold and rate for one week in daily tests were used for drug studies. Control threshold voltages varied among the rats from 5-14 v (average 9) and stimulation rates from 50-250/min (average 117). The voltage difference between the threshold following administration of a drug and the control threshold was taken as the effect of the drug. Drug experiments were begun in the morning and run during the day. Whenever possible, the testing took place at the same time each day for a given animal. ii11 the drugs used were obtained from commercial sources and were administered intraperitoneally. L- And nr>-cr-methyl-p-tyrosine, DL-p-chlorophenylalanine, DL-5-bromotryptophan, and DL-6-fluorotryptophan were not soluble at neutral pH so they were injected as a suspension in 20% propylene glycol. The suspension was made by adding propylene glycol to a measured amount of drug, making the mixture basic with 3~ NaOH,
285
SELF-STIhiVLATIOiX
and shaking until the drug went into solution. The pH was brought back to 7 with 3~ HCl, and the volume adjusted with water so that each animal was injected with l-l.5 ml, Injection of drug-free suspensions made up by this procedure had no effect on self-stimulation. The animals were left at least 1 week between drug experiments and were not used for further studies if the control threshold showed changes. When the drug studies were completed, the animals were killed and the brains removed and fixed in formalin. Placement of the electrode in each rat was determined by histological esamination of brain slices stained with Luxol Fast Blue. In order to check that the drugs were producing the expected effects on amine levels the catecholamines and serotonin were determined by reported fluorometric methods (6) in the brains of control and druginjected male rats of species and size comparable to those used in the selfstimulation study. Results
A typical voltage-rate curve for self-stimulation is shown in Fig. 1. It illustrates the constant rate of self-stimulation which is achieved as the voltage is increased beyond the threshold. The changes are very sharp, indicating that this is a sensitive method of estimating drug effects. Figure 2 shows three typical examples of threshold changes versus time for rats injected with various drugs. The overall results are summarized
4)
,
10
12
14
16
18
20
VOLTAGE
1. Voltage vs. bar presses per minute for a typical Threshold equal to 14 v; rate equal to 171 * 20 press/min. FIG.
self-stimulating
rat,
286
GIBSON,
MCGEER,
4
0 HOURS
AND
12 AFTER
MCGEER
16
20
24
ADMINISTRATION
FIG. 2. Effect of drugs on threshold voltage of typical rats with time. V mA%fluorotryptophan, 100 mg/kg ; n n&bromotryptophan, 290 mg/kg ; 0 L-cr-methyltyrosine, 100 mg/kg.
in Table 1. As can be seen from the Table, drugs which cause a decrease in catecholamines, serotonin, or both were all effective in increasing the threshold for self-stimulation in the majority of rats tested. In some instances there was no response to the drugs, and on four occasions there were decreases, rather than increases, in threshold. These decreases OCcurred in three rats give p-chlorophenylalanine and in one of these same animals after DL-cu-methyl-p-tyrosine. Reserpine was tested for comparative purposes and proved to be the most effective drug used in changing the threshold for self-stimulation. At a dose of 0.3 mg/kg, an increase in threshold was observed in all animals, and at a dose of 0.5 mg/kg, self-stimulation was almost completely eliminated. Of the enzyme inhibitors used, Ba-methyl-fi-tyrosine was the most consistently effective. Out of 23 rats tested, 18 showed an increase in threshold, three showed no effect, and one showed a decrease. The maximum effect on self-stimulation was 5-9 hours after injection which is in agreement with the time of the maximum reported decrease in brain noradrenaline and dopamine content after the administration of this agent. Recovery of self-stimulation was complete within 24-48 hours, which is also in line with
287
SELF-STIMULATION
TABLE CHANGES
NO. rats 22 3 4 II 8 6 5 8 3 5
1~ SeL1~-5mrvL~~~o~
I>rllg ‘k dOW lieserpine, 0.3 nig,‘kg Iieserpine, 0.5 mg/kg r.-cr-Methyl-p-tyrosine, L-a-Methyl-p-tyrosine, DL-cy-Methq-I-P-tvrosille, DL-S-Bromotryptophau, DL-p-Chlorophen~lalanitle, DL-p-Chloropheuylalanine, m-6-Fluorotryptophau, m-6-Fluorotryptophan,
75 111gjkg 100 mg/kg 200 mg/kg” 290 nlg/kg 1.50 mg/kg 200 ~ng/kg 100 mgjkg 150 mg/kg
1
THHESHOLD
IIecr
None
INDC’CED
nY
VARIOUS
DRU<;~
Brain amine levels (70 of controls)” 5HT + 1.1 v + 2.2 v + 3.3 v NA
0
I)
12
8
2
0 0
0 2
0 1
0
3
95 70
1
0
-
_.
0 1 0 0
0 1 0 0 1 0 3
3 1 5 2 I
2
G
6.5
0 1
5 0
2.5 49
60
100 100
1
95 95 -
48 30 -._
110
4.5
3
0 0
2
0
2 2 1
1
0
1
I
80 70
0 Values are averages of those fouud in animals of conlparable size and species at the time of expected lasimum effect and are in general accord with the effects of these agents as reported in the literature. *Given in serial doses (10).
the time of recovery of amine levels. The fact that a-methyl-p-tyrosine in the higher dosesused reduced brain catecholamine levels by approximately 50% (10) was confirmed by measurementsin this laboratory. Dr&&omotryptophan, a selective tyrosine hydroxylase inhibitor (4)) produced an increase in threshold in all six animals where it was tried. For most of the rats the effect was mild, reaching its maximum 2-4 hours after injection which correlates with the time of maximum amine depression. Even at the high dose of 290 mg/kg, noradrenaline in these rats was only reduced by about 40%, so that the mildness of the effect as compared with that wrought by a-methyl-p-tyrosine would be in keeping with a less severe reduction in catecholamine levels. nL-5-Bromotryptophan had no effect on serotonin concentration. Dr.-p-Chlorophenylalanine, which is selective in its inhibition of tryptophan hydroxylase and therefore selective as a serotonin depletor, gave mixed results. Of 13 rats tested, nine showed an increase in threshold, one no effect, and three a decreasein threshold. The increase in threshold was observed 2-3 days after the injection which corresponds with the time of maximum decreasein serotonin levels (3) ; any decreasein threshold seemedto occur somewhat earlier (1-2 days). For those rats showing an increase in threshold, the effect was not as pronounced as with an equimolar dose of LYmethyl-p-tyrosine, even though at this dose of p-chlorophenylalanine brain serotonin levels are decreasedby about 7070, as evidenced both by literature reports and by confirmatory data obtained by us. DL-6-Fluorotryptophan, a selective inhibitor of tryptophan hydroxylase
288
GIBSON,
MCGEER,
AND
MCGEER
(5), was administered to eight rats. Five of these showed a mild increase in threshold and three no effect. The changes occurred l-3 hours after drug administration, the time of maximum effect on brain serotonin levels. The histological results showed that the electrodes were in the medial forebrain bundle or in tissue just dorsal to it in all the animals. The only two animals that showed several atypical responses had electrode placements in the same areas as did other rats that failed to show any unusual responses. All the animals were not given all the drugs, but each animal was given several drugs, and the histological results made it clear that the increases in threshold observed for catecholamine depletors versus serotonin depletors could not possibly be explained on the basis of different electrode placement. The only correlation between electrode placement and self-stimulation activity was that the more dorsal placements tended to produce higher rates and lower thresholds. Discussion
Our results on reduced self-stimulation following administration of LYmethyl-fi-tyrosine or reserpine are in agreement with other reports (8, 13). One hypothesis that has been proposed (14) is that reduction in noradrenaline caused by these drugs is responsible for the decreased selfstimulation activity, and indeed the time courses of the two effects correlate well both with these agents and with DL-5-bromotryptophan. However, with cu-methyl-p-tyrosine, noradrenaline had to be reduced much further than with reserpine to obtain equivalent changes in self-stimulation, and DL-5bromotryptophan produced only very mild changes in self-stimulation although it reduced the noradrenaline content as much as the dose (0.5 mg/ kg) of reserpine which eliminated self-stimulation in all three rats tested. This suggests that the relationship between self-stimulation and noradrenaline content must be subtle. Two possible explanations would be involvement of other amines or involvement of different pools of noradrenaline affected differently by reserpine, a-methyl-p-tyrosine, and m-5-bromotryptophan. Reserpine, of course, reduces dopamine and serotonin as well as noradrenaline, and all three of these amines are involved in pathways running through the medial forebrain bundle region. The drugs used in this study were not suitable to differentiate between noradrenaline and dopamine, but the results obtained with m+chlorophenylalanine, as well as with r&d-fluorotryptophan, suggest that serotonin pathways are involved in self-stimulation. The time course of the effect of each of these drugs on self-stimulation is again in good correlation with their effect on central serotonin levels. The probability that serotonin is involved in self-stimulation is also suggested by the work of Poschel and Ninteman (9) who reported enhancement of
SELF-STIMULATION
289
self-stimulation upon administration of Shydroxytryptophan to rats pretreated with a monoamine oxidase (MrlO) inhibitor, and by the study of Stark, Boyd, and Fuller (12) who found that low doses of D-2-bromolysergic acid diethylamide (, BOL), a serotonin antagonist. decreased the threshold for hypothalamic self -stimulation in dogs while nL-pheniprazine hydrochloride (JB 516), an MAO inhibitor that causes an elevation of brain serotonin levels without a concomitant increase in noradrenaline, lowered the threshold for self-stimulation. The available data do suggest that a given selective percentage decrease in brain serotonin level generally has less effect on self-stimulation than a comparable selective decrease in brain catecholamine level. It may be that the catecholamines are involved in a more powerful reward system than is serotonin. Obviously, it would help greatly in our understanding of the roles of these amines in behavior and of the neuro-anatomical pathways associated with behavior if clear-cut definitions could be made of the neurotransmitters serving specific drive-reward systems. It would appear, however, that more sophisticated experimentation will be required before such distinctions are possible. References 1. ANDEN, N. E., A. CARLSSON, A. DAHLSTROM, K. FUXE, N. A. HILLARP, and P. R. LARSON. 1964. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sri. 3 : 523-530. 2. DAHLSTROM, A., and K. FUXE. 1964. Evidence for the existence of monoamine containing neurons in the central nervous system. .4&a Physiol. Stand. Supp. 232, 62: l-55. 3. ‘E;OE, K., and A. WEISSMAN. 1966. fi-Chlorophenylalanine: A specific depletor of brain serotonin. .I. Pharvlacol. Exp. Ther. 154 : 499-514. 4. MCGEER, E. G., P. L. MCGEER, and D. A. V. PETERS. 1967. Inhibition of brain tyrosine hydroxylase by Shalotryptophans. Lift Sci. 6 : 2221-2232. 5. MCGEER, E. G., D. A. V. PETERS, and P. L. MCGEER. 1968. Inhibition of rat brain tryptophan hydroxylase by 6-halotryptophans. Life Sci. 7 : 60-615. 6. MCGEER, P. L., E. G. MCGEER, and J. A. W’ADA. 1963. Serotonin and catecholamine levels in various cat brain areas following administration of psychoactive drugs or amine precursors. A.M.,4. Arch. Neural. 9 : 81-89. 7. OLDS, J. 1962. Hypothalamic substrates of reward. Physiol. Rev. 42: 55C604. 8. POSCHEL, B. P. H., and F. W. NINTEMAN. 1966. Hypothalamic self-stimulation. Its suppression by blockade of norepinephrine biosynthesis and reinstatement hy methamphetamine. Life Sci. 5 : 1 I-16. 9. POSCHEL, P., and F. NINTEMAN. 1968. Excitatory effects of 5 HTP on intracranial self-stimulation following MAO blockade, Life Sci. 7: 317-323. 10. RECH, R. H., H. K. BORYS, and K. E. MOORE. 1966. -4lterations in brain catecholamine levels in rats treated with a-methyl-tyrosine. J. Phamacol. Et-p. Thr-. 153 : 312-419. 11. SHUTE. C. C. D., and P. R. LEWIS. 1967. The ascending cholinergic reticular system : Neocortical, olfactory and suhcortical projections. Rrnirz SO : -197.522.
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P., E. S. BOYD, and R. W. FULLER. 1964. A possible role of serotonin in hypothalamic self-stimulation in dogs. J. Pharwzacol. Exp. Thu. 146 : 147-153. 13. STEIN, L. 1964. Self-stimulation of the brain and the central stimulant action of amphetamine. Fed. Proc. 23 : 836850. 14. WISE, C. D., and L. STEIN. 1969. Facilitation of brain self-stimulation by central administration of norepinephrine. Science 163 : 299-301. 12.
STARK,