THE ROLE OF BRAIN SEROTONIN IN THE MECHANISM OF THE CENTRAL ACTION OF RESERPINE

Transcription

1 Tnz JOUENAL or PisAssMAcoLooY AND EXPERIMENTAL THERAPEUTICS Copyright 1966 by The Williams & Wilkins Co. Vol. 152, No. 2 PrinirA in U.S.A. THE ROLE OF BRAIN SEROTONIN IN THE MECHANISM OF THE CENTRAL ACTION OF RESERPINE B. B. BRODIE, M. S. COMER,2 E. COSTA AND A. DLABAC3 Laboratory of Chemical Pharmacology, National Heart Institute, Bethesda, Maryland Accepted for publication November 17, 1965 ABSTRAcT BRODIE, B. B., M. S. COMER, E. COSTA AND A. DLABAC : The role of brain serotonin in the mechanism of the central action of reserpine. J. Pharmacol. 152 : , Reserpine sedation is associated with changes in brain serotonin (5-HT) rather than with catecholamines. Thus, after blockade of catecholamine synthesis, catecholamines can be reduced by 8% without producing sedation ; in contrast, reserpine elicits sedation in doses that reduce both 5-HT and catecholamines by only 55%. In addition, there is a time correlation between the effects of reserpine on behavior and impairment of the process that accumulates exogenous 5-HT in brain tissue. This process is recovered in 48 hr when rabbits have recovered from sedation, even though 5-HT stores are still largely depleted. After reserpine, the rate constant of 5-HT efflux is dose-dependent, and is maximal after a dose (5 mg/kg iv.) which depletes 5-HT at a half-life of about 7 mm. Intensity and duration of sedation are correlated with initial rates of 5-HT release rather than the final extent of depletion. After doses of reserpine that deplete the monoamines at a maximal rate, the steadystate levels calculated from the rate of synthesis and the rate constant of efflux are 12% of normal for 5-HT and 3% of normal for norcpinephrine (NE). Studies showing that 5-hydroxytryptophan (5-HTP) in doses that elicit excitation also causes the release of brain NE may remove a key argument against the view that reserpine acts through free 5-HT. More than 1 yr have passed since the discovery that reserpme impairs the mechanism that stores serotonin (5-HT) and reduces the content of this active substance in brain cells (Brodie et al., 1955 : Shore et al., 1957). This finding led to the speculation that 5-HT functions as a neurohumoral agent in brain and that the action of reserpine is mediated through free 5-HT which persistently occupies receptor sites (Brodie and Shore, 1957). The above view seemed plausible since the pharmacologic effects of reserpine were related temporally not to the concentration of drug in brain, but to a persistent inhibition of the 5-HT storage mechanism (Hess et a!., 1956). However, when reserpine was found to deplete norepinephrine (NE) (Holzbauer and Received for publication May 4, The experiments included in the paper were presented in preliminary form at the meeting of the American Society for Pharmacology and Experimental Therapeutics in San Francisco (Pharmacologist 5: 245, 1963) and at the Federation Meeting in Atlantic City (Fed. Proc. 24: 194, 1965). Fellow, National Multiple Sclerosis Society. 3Fellow, Geigy Research Laboratories. 34 Vogt, 1956 ; Bertler et al., 1956) as well as 5-HT, a deficiency of the adrenergic transmitter in central pathways was offered as an alternative explanation of sedation by reserpine (Carlsson et a!., 1957a). Since reserpine was shown to decrease adrenergic effects through a deficiency of NE in peripheral sympathetic nerves (Carlsson et a!., 1957b ; Brodie et al., 1957), it seemed logical to assume that sedation was associated with the loss of catecholamine stores in brain. In addition, the results of a number of studies seemed to be in conflict with the view that the behavioral and other central effects of reserpine are associated with changes in brain 5-lIT. 1) Although 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, elicits sedation in small doses, it causes a toxic excitation in large doses (Erspamer, 1961). 2) The se(lative action of reserpine is closely associated with the degree to which the storage of 5-HT is inhibited, but the recovery of animals from sedation is not closely related to the return of the brain amine level to the normal value (Brodie and Shore, 1957).

2 1966 BRAIN 5-HT AND RESERPINE ACTION 341 The present paper shows that the central effects produced by reserpine are not attributable to the decline in brain NE level. However, the intensity of the central effects elicited by reserpine is closely associated with the rate of release of 5-HT, but not with the endogenous kvel of the brain amine. Moreover, the animals recover from sedation at a time when the process that accumulates exogenous 5-HT has recovered even though the endogenous stores are still largely depleted. Finally, the results show that the excitation produced by 5-HTP is associated with the release of brain NE. METHODS AND MATERIALS. Materials. Experiments were carried out in adult male rabbits weighing 2 to 3 kg (New Zealand white) and adult male rats weighing about 2 g (Sprague- Dawley). The reserpine alkaloid was prepared for intravenous injection in rabbits as described by Pletscher et al. (1956), and in rats by dissolving in acetic acid (.5 N). The levo form of ce-methyltyrosine, kindly donated by Merck Sharp and Dohme Research Laboratories, was prepared as a 2% solution as described by Spector et at. (1965) and injected intravenously in a volume of 2 ml/rat. 5-Hydroxy-im-tryptophan (California Corporation for Biochemical Research) was dissolved in isotonic saline. Chemical methods. Animals were killed and tissues removed as rapidly as possible and stored in a freezer. Brain 5-HT was determined by the fluorometric procedure of Bogdanski et al. (1956). For the assay of NE and dopamine, brain tissue was homogenized in 4 volumes of.4 N perchioric acid and centrifuged at about 1 X g for 1 mm. Two milliliters of supernatant fluid were transferred to a 15-ml test tube containing 5 mg of Al23, treated according to Crout (1961). The ph was adjusted to about 82 by addition of 4 ml of.5 M Tris buffer, ph 9. The catecholamines were adsorbed on the AhO5 by shaking, eluted with.2 N acetic acid and assayed fluorometrically according to the method of Chang (1964). When 5-HT was assayed after the administration of 5-HTP, the butanol extract was washed twice with borate buffer, ph 1, to remove traces of the amino acid. After 5-HTP admmistration, the apparent NE, dopamine and 5-UT in brain appeared to be identical with the authentic amines as demonstrated by fluorescent and activation spectra. Pharmacologic methods. The ability of brain to take up 5-UT was determined in rabbits from the increase in brain stem level 1 hr after injection of 5-HTP (5 mg/kg iv.). Only traces of the amino acid remained in brain ; thus, the rate of disappearance of 5-HT was not obscured by the continued formation of the amine. In rabbits, the central activity induced by reserpine was determined by the following indices: blepharospasm, lack of responsiveness to painful stimulus (applied by manual displacement of tarsometatarsal joint) and delay (3 sec or longer) in recovery of righting reflex after placing animals on their sides. The latter effect was usually the first to disappear, then the response to Pain and finally blepharospasm. Each sign was counted as 1, and in each animal the pharma- (ologic action of reserpine was scored from to 3. In rats, the central activity of reserpine or a- iiietlivl-tvrosine was determined by the following indices : 1 ) decrease in a ) exploratory activity and 1)) huddling behavior, 2) increase in muscle tone and 3) presence of blepharospasm. The intensity of each sign was given a score of 1 to 3, and in each animal the pharmacologic action of reserpine was given a score of to 12. la) Exploratory activity. Activity of rats was observed after placing them on a cage top in groups of four. Control animals generally explored their environment for a period of 4 to 6 mm, the duration being shorter in the morning than in late afternoon. A rating of 1 was given for cxploratory activity lasting 1 to 2 mm ; of 2, for 1 mm or less ; of 3 for complete lack of activity. ib) Huddling. One to two minutes after cessation of exploratory activity, control rats usually huddled. A rating of 1 was given for a delay greater than 2 mm ; of 2, for a delay greater than 4 mm ; of 3, if the rats did not huddle at all. 2) Increased muscle tone. A rating of 1 was given for a spastic gait ; of 2 for a hunchbacked posture ; of 3 for hyperextended paws. 3) Blepharospasm. A rating of 1 was given if closure of eyelids was intermittent ; of 2 if closure was continuous except when animals were disturbed ; of 3 if closure was continuous even when the animals were handled. After reserpine, the lack of exploratory activity was the first sign to appear, followed by increase in muscle tone, failure to huddle and blepharospasm. The first sign to disappear was increase in muscle tone, followed by failure to huddle, lack of exploratory activity and blepharospasm. The central activity of reserpine was also determined from the degree to which ethanol and hexobarbital were potentiated. In this test, the time for recovery of righting reflex after giving hexobarbital (1 mg/kg i.p.) or ethanol (3 g/kg i.p. in 5% solution) was determined in normal rats and in rats treated with various doses of reserpine.

3 342 BRODIE ET AL. Vol. 15k RESULTS. Effect of.5-htp on level of brain catechokimines. Although small doses of 5-HTP are reported to elicit signs of sedation, large doses elicit excitation and sympathetic signs (Bogdanski et al., 1958). The following experiments were carried out to see whether doses of 5-HTP that produced excitation would also lead to the release of brain NE. In rats, the intravenous injection of 5-HTP (5 mg/kg) lowered the NE levels by about 5% (table 1) and elicited piloerection, panting and exopthalmos together with increased motor activity, circling movements, tremors and intermittent clonus of the paws. Injected intravenously into rabbits, 5-IITP (2 mg/kg) also decreased the stores of brain NE by about 5%. The animals displayed panting, exophthalmos and mydriasis, together with tremors, increased motor activity, ataxia, intermittent spasm of the paws, champing movements of the mouth and a tendency to walk backwards. Relationship of endogenous levels of brain 5-HT to recovery of rabbits from sedation. Within 6 hr after injection of reserpine (2 mg/kg iv.), the concentration of 5-HT in rabbit brain stem declined to about 1% of normal and remained at this level for more than 16 hr (fig. 1). In 36 hr, the level had risen perceptibly to about 2% of normal, and in 4 days to 5% of the normal value. Only after 7 to S days had elapsed was the normal value reached. Concurrent with the slight rise in 5-HT level, the gross behavior of the animals was almost normal in 36 hr, and in 48 hr the animals were completely recovered though the amine level was still barely 3% of normal. Treatment None 5-HTP TABLE 1 Effect of 5-HTP on brain NE level NE Content Rabbit brain stem Rat brain jsg/g ± S.D..57 ±.7 (4) I.51 ±.9 (9).29 ±.7 (4).27 ±.7 (4) Rats were given DL-5-HTP (5 mg/kg i.v.) and killed 9 mm later. Rabbits were given DL-5- HTP (2 mg/kg iv.) and killed 45 mm later. Figures in parentheses represent number of animals studied. HOURS AFTER RESERPINE FIG HT levels in rabbit brain stem at vanous times after reserpine (2 mg/kg i.v.). Control level is.88 ±.1 eg/g (mean ± S.D., n 12). Each bar represents the mean value of at least four animals. The difference between 5-UT levels in 16 and 36 hr is significant (P <.1), but the level in 16 hr is not significantly different from that in 6 hr (P >.5). Changes in brain stem NE (not shown) closely paralleled those of 5-UT. These results are in agreement with those of Shore et al. (1957), who reported that rabbits recovered from sedation at a time when brain 5-UT stores were still largely depleted, but recovery was concurrent with a small rise of endogenous 5-UT level. The rise in 5-UT level which accompanied the termination of the reserpine-induced sedation suggested that the capacity of brain tissue to take up 5-HT had been partly restored. A second dose of reserpine administered in 36 or 48 hr restored the animals to a somnolent state and the level of brain 5-HT to less than 1% of normal. Possible effect of reserpine on decarboxylation of 5-HTP. The possibility was studied that termination of the action of reserpine was associated with an increased capacity of neurons to accumulate 5-UT formed from administered 5-HTP. Previous studies have shown that the 5-UT so formed is readily held by the brain of normal animals (Bogdanski et al., 1958). Before comparing results from normal and reserpine-treated animals, it must be demonstrated that reserpine does not interfere with the functional activity of brain 5-UTP decarboxylase in vivo. This was tested by comparing the rise in brain 5-UT of normal and

4 1966 BRAIN 5-nT AND RESERPINE ACTION 34 reserpine-treated animals, 5 min after giving 5-UTP (1 mg/kg iv.) to rabbits pretreated with pargyline, an effective monoamine oxidase (MAO) inhibitor (2 mg/kg i.v.). Since the 5-UT level increased to the same extent in normal and reserpine-treated animals, it may be inferred that reserpine did not affect the formation of 5-HT from 5-UTP (table 2). Capacity of brain to take up 5-HT at vanou,s times after reserpine administration. Table 3 shows that 1 hr after administration of 5-UTP considerable amounts of 5-HT were retained by TABLE 2 Rate of 5-HT formation in brain stern of rabbits given 5-HTP 5- Treatment 5-HT Increase Pargyline (4) Pargyline + reserpine (4) a Mean score as described in METHODS. ± S.D..68 ± ±.21 Control animals and animals 4 hr after pretreatment with reserpine (2 mg/kg i.v.) were given pargyline (2 mg/kg i.v.). One hour later, animals were given DL-5-HTP (1 mg/kg i.v.) and then killed after 5 mm. The increase in 5-UT is amine content of brain stem from animals given pargyline and 5-HTP minus that from animals given pargyline alone. Figures in parentheses represent number of experiments. TABLE 3 Central effects and retention of exogenous 5-HT by rabbit brain stem at various times after reserpine Rabbits were given DL-5-HTP (5 mg/kg i.v.) at various times after injection of reserpine (2 mg/kg iv.) and killed 1 hr later. Values for 6 and 16 hr are significantly different (P <.1) from those of other times. Figures in parentheses represent number of animals studied. Hours after Reserpine Increas e in Brain S-HT Central Effect? pg ± S.D. 1.1 ±.21 (4) 6.39 ±.6 (5) ±.8 (5) ±.7 (5) ±.7 (6) ±.11 (6) ±.13 (4) HOURS AFTER 5 HIP ADMINISTRATION FIG. 2. Time course of disappearance of 5-UT taken up by brain stem 1 hr after the administration of 5-HTP (5 mg/kg i.v.) to control rabbits (striped bars) and to rabbits treated with reserpine (2 mg/kg i.v.) 5 hr (solid bars) and 6 hr (crosshatched bars) previously. Increase in 5-HT is amine content of brain stem from animals given 5-HTP minus that from animals not given 5-HTP. Each bar represents mean value of at least four rabbits. the brain stem of normal rabbits. In contrast, 6 and 16 hr after pretreatment with reserpine, the brain stem held only small amounts of 5-HT. In 36 hr, however, the brain stem was able to accumulate considerable amounts of 5-UT. The ability of the brain to accumulate 5-UT became progressively greater with the time lapse between the administration of reserpine and 5-UTP, and in 6 hr was almost equal to that of the controls. These data suggest that the recovery of the brain cell mechanism which takes up 5-UT was far more complete than the ability to store the endogenous amine. Further evidence of this was obtained from the persistence of the amine formed from 5-UTP (fig. 2). In control rabbits, the amine disappeared slowly and was held for more than 6 hr after the injection of 5-HTP. In contrast, in animals that received 5-UTP 5 hr after treatment with reserpine, the 5-UT disappeared rapidly and was retained at most for 3 hr. When animals were given 5-UTP 6 hr after reserpine, the 5-UT again disappeared slowly and was retained for more than 6 hr.

5 344 BRODIE ET AL. Vol. 152 Relationship between the central effects of neserpine and #{244}-IIT uptake. Data in table 3 also show that the central effects of reserpine are temporally associated with the ability of rabbit brain stem to accumulate exogenous 5-UT. In 16 lir, the amount of 5-HT retained by brain after 5-I-fTP was no greater than in 6 hr and the animals were still profoundly sedated. By 36 hr, when the central effects of reserpine had largely disappeared, the brain stem accumulated exogenous 5-HT in considerable amounts. Relationship between central effects of reserpine and rate of release of #{244}-HT. After reserpine administration, 5-HT levels declined at initial rates that were proportional to the dose between.1 and.9 mg/kg (fig. 3). Expressing the initial rates of depletion in terms of kd, the proportion of 5-UT stores that disappear per minute, the maximal initial rate of depletion (kd max =.86 min ) was produced by 1 mg/kg, and this was only slightly higher than that produced by 5 mg/kg mg/kg A Reserprne.-..9mg/ kg of Reserpine -.3mg/kg of Reserpine A-A.1mg/kg Reserprne I I I I I TIME IN MINUTES FIG. 3. Exponential decline of brain 5-UT in rats after reserpine administration. The rate constants of depletion (k4) for various doses of reserpine were as follows:.77 min1 for 5 mg/kg;.33 min for.9 mg/kg;.11 min for.3 mg/kg;.3 min for.1 mg/kg. Each point represents the average value for at least four rats. Figure 4 shows that the intensity and duration of the pharmacologic responses to reserpine were functions of the 5-HT release rate. The minimal dose to produce overt sedation (.3 mg/kg) released 5-HT at about 13% of the maximal rate, and produced its peak effect after a latency period of about 4 hr. A dose of.9 mg/kg released 5-UT at about 38% of the maximal rate, and after a latency period of 2 to 4 hr, elicited a maximal degree of central activity, based on the indices described in METHODS. This activity was sustained at a peak for about 2 hr and then diminished. In contrast, 5 or 1 mg/kg of drug, which released 5-HT at the maximal rate, elicited a maximum of central activity in 3 to 4 mm. This activity \5 sustained for 2 to 3 hr and then diminished. Since each of the indices of central action has a limited intensity, the activity of reserpine was also determined from the potentiation of ethanol and hexobarbital as measured by recovery of the righting reflex. The data in table 4 show that 5 mg/kg of reserpine was more effective than.9 mg/kg in potentiating the action of ethanol and hexobarbital. The effects of 5 and 1 mg/kg of reserpine were not significantly different. The data in table 5 show the central effects and the 5-UT levels in rats at various times after reserpine administration. At first, the levels declined exponentially (fig. 3) and tended to level off in about 1 hr. After the smaller reserpine doses, the levels of 5-UT declined to a lower value over the next 3 hr, but the central effects were related to the initial rate of release rather than to the final degree of depletion. For example, in 4 hr both.9 and 5 mg/kg of the drug had lowered brain 5-UT stores by about 88%, but the effects of the larger dose were considerably more intense and prolonged, as shown in figure 4. Central effects produced by blockade of catechokimine synthesis. The central effects produced by the blockade of catecholamine synthesis were compared with those elicited by reserpine. The synthesis of catecholamines was inhibited by a-methyl-tyrosine, a synthetic amino acid which blocks the biosynthesis of catecholamines in vivo and, given in repeated doses, decreases the motor activity in various animal species (Spector et a!., 1965). In our experiments, a-methyl-tyrosine (2

6 1966 BRAIN 5-nT AND RESERPINE ACTION I I I I I 1 - Reserp/ne./mg/kg - k#{246} rn/ri 5- - I Resrp#na.Smg/kg - C,) I- C., U. U. Ui Cl) C., U. 1 Li Cl) mg/kg I #{149} I HOURS AFTER RESERPINE - I FIG. 4. Relationship between the central effects of reserpine and the rate constants of 5-HT depletion in rats. Each point represents the mean score of responses obtained with eight animals. The rate constants after various doses of reserpine are shown on right side of figure. The peak score after.3 mg/kg of reserpine comprises rigidity, blepharospasm, decreased exploratory activity and decreased huddling activity. mg/kg iv.) was administered to rats, and the central effects were related to the levels of brain amine. In 12 hr, when the levels of NE and dopamine had declined by about 76 and 81%, the animals showed little or no decrease in exploratory activity. In 18 hr, however, there was a marked decline in motor activity when the brain levels of catecholamines were reduced by about 93%. At this time, the animals displayed ptosis but no tremors or muscle rigidity; indeed, the muscles were flaccid. After the injection of reserpine (.9 mg/kg iv.), the relationship between the levels of brain catecholamines and the central effects was quite different; the drug, in doses that lowered the levels of NE, dopamine and 5-UT by 7 to 8%, evoked marked central effects, and exploratory activity was almost completely absent (table 6). DIscussIoN. Considerable evidence opposes the view that the sedative effects of reserpine are caused by the lack of brain NE (Vogt, 1961 ; Brodie and Costa, 1962). In addition, our results show that a-methyl-tyrosine, which blocks catecholamine synthesis (Spector et at., 1965), does not elicit sedation until cerebral catecholamines are reduced by 9% or more. This is not surprising, since NE stores in peripheral neurons must be largely depleted before nerve function is lost (Gaffney et at., 1963). In contrast, reserpine produces overt sedation in doses that deplete stores of 5-UT and NE in brain only by about 55%. In correlating the central effects of reserpine with changes in brain 5-UT, one should consider the possible consequences of the continuous formation of amine in the absence of a storage mechanism. After 5-HT stores arc

7 346 BRODIE ET AL. Vol. 152 emptied, the release is no longer limited by the normal biologic controls, and the amine diffuses directly from sites of synthesis onto receptors. Concentration at receptors will now be constant and will be lower or higher than normal depending on relative rates of appearance and disappearance (Brodie and Shore, 1957). A major argument against the view that reserpine acts through free 5-UT is based on the premise that the excitation elicited by 5-UTP represents the action of 5-UT released TABLE 4 Influence of reserpine on duration of action of ethanol and hexobarbital ire rats Rats received ethanol (3 g/kg i.p.) in 5% solution or hexobarbital (1 mg/kg i.p.) 1 hr after various doses of reserpine. Rats given the hypnotics without reserpine served as controls. Figures in parentheses refer to number of animals in each series. Dose of Reserpine mg/hg None iv Ethanol Duration of Hypnosis miss * S.D. Hexobarbital 18 ± 1.6 (11) 2 ± 2.9 (6) bob ± 3.1 () 45b 9.9 (5) 144 ± 36.2 (9) fj4b 12.2 (5) 146 ± 35. (6) 65 ± 1.1 (5) a Time from loss to return of righting reflex. b Values are significantly different from the precedingvalue (P <.5). from normal sites of storage. Our studies, however, show that 5-HTP causes the release of NE. Moreover, 5-HTP does not elicit excitation in animals depleted of NE stores (Carisson et at., 1957a). Since 5-UT releases catecholamines from granules in vitro (Carisson et at., 1963) and from the isolated heart (this laboratory, unpublished data), it is probable that 5-HTP causes excitation through the release of catecholamines. Another difficulty in accepting the view that reserpine sedation is mediated through free 5-UT is the inferential nature of the evidence that the amount of 5-UT increases at the receptors. In approaching this problem, this laboratory has formulated a model, describing the storage of 5-UT and other monoamines, which has proved useful in interpreting the action of various drugs on nerve endings (Costa and Brodie, 1964). The theoretical model consists of two compartments : 1 ) a mobile pool containing free 5-HT in dynamic equilibrium with 2) a larger pool of amine complexed in granules. According to this model, the level of indole in the mobile pool is maintained by a carrier mechanism at the neuronal membrane that counterbalances the outward diffusion of 5-UT and takes up the amines from extracellular fluid in the vicinity of receptors (Brodie and Beaven, 1963). Reserpine is postulated to inhibit the carrier mechanism, thereby releasing the amine onto MAO, which is partly inside, but mostly outside, the nerve ending (Potter et at., 1965). TABLE 5 Central effect8 and brain 5-HT levels at various times after re8erpine Dose of Reserpine Hours after Reserpine.1 mg/kg iv..3 mg/kg i.v..9 mg/kg iv. 5. mg/kg Lv. Brain 5-HT Central effects Brain 5-HT Central effects Brain 5-HT Central effects Brain 5-HT Central effects % of co,trols ± S.D. 76±8 67±7 59±5 % of confrols * S.D. 8±8 61±9 51±18 38±6 35± % of controls ± S.D. 73±1 45±8 28±3 22±3 16±7 14± % of controls * S.D. 39±6 24±5 12±5 1±5 12± Brain 5-HTlevel of controls,.51 sg/g ± S.D..6 (it = 12). Each value is the average of eight animals.

8 1966 BRAIN 5-icr AND RESERPINE ACTION 347 The carrier mechanism in the membrane may be regarded as a barrier to the free diffusion of 5-HT. The efficacy of the mechanism may be expressed as the degree to which 5-UT efflux is increased when the process is blocked by reserpine. Normally, synthesis and efflux are equal, 6.3 mjg/g/min (Tozer and Neff, personal communication) ; hence, the rate constant for 5-UT efflux (k) will be 6.3 clivided by the brain 5-UT level (51 mj.tg/g), i.e.,.13 min. In analyzing the effects of reserpine, it is helpful to consider first its effects on a static system in which there is no synthesis of 5-UT. According to our model, the 5-HT level would decline exponentially with a rate constant of.13 min, since amine loss is no longer balanced by formation. In fact Dlabac (unpublished data) has demonstrated that after blockade of catecholamine synthesis by a-methyl-tyrosine, the brain NE level declines exponentially at a rate constant corresponding to the rate of synthesis at the normal steady state. Reserpine would merely increase the rate constant of efflux, thereby increasing the speed at which the level declines to zero. On plotting ln ([5-UT]/[5-UT]) vs. time, a series of lines would be obtained whose slopes describe the rate constants. The picture is more complicated since 5-UT is formed at a constant rate. Hence, the actual change of concentration with time will be K - k [5-UT], where K is the constant rate of synthesis and k is the first-order rate constant of efflux. The explicit expression describing the concentration of amine is now 5-UT = - ( - [5..HT]) er where [5-H! ] is the concentration of 5-UT in time t, [5-UT] is the initial concentration and k is rate constant of efflux after various doses of reserpine. The rate constants will now be described by a series of lines obtained by plotting in ([5-UT] - [5T]n/[5T1 _ [5-HT]j vs. time, where 5-UT, 5-UT. and 5-UT are the concentrations initially, at time t and at infinite time (steady state) respectively. These values describe the rates at which 5-HT declines, not to zero, but to a new steady state. In practice, it is much simpler to calculate the rate constants by adding.13 min1, the normal rate and k = k +.13 min1. TABLE 6 Relationship between brain amine levels and central effects of a-methyl-tyrosine and reser pine in rats Treatment Central effects were evaluated 12 hr after administration of a-methyl-tyrosine (2 mg/kg i.v.) and 1 hr after reserpine (.9 mg/kg iv.). Figures in parentheses represent number of experiments. a-methyltyrosine (5) Reserpine (6) Depletion of Brain Amines Score of Central Effects NEpe5HT EAG H B M GEA = decreased exploratory activity; H = lack of huddling ; B = blepharospasm ; MT = increased muscle tone. constant of 5-HT efflux, to kd, the initial rate constant of depletion (table 7). The level of available 5-UT in brain after complete inhibition of the storage process may be calculated from the expression described above for the concentration of 5-HT. At infinite time, the exponential term is zero, and the equation becomes 5-UT = (K/kr) where K is rate of synthesis and hr is rate constant of effiux. It is obvious, therefore, that reserpine cannot reduce the 5-UT level to zero since (K/kr) >. Table 7 shows the steady state values of 5-HT after various doses of reserpine. When 5-HT is released at a maximal rate constant, the level declines to about 12% of normal, or 63 mtg/g. These levels represent 4 Rate of efflux - rate of synthesis net loss. At the steady state, the net loss is zero. Hence, rate of efflux (koco) rate of synthesis (K), where k is rate constant of efflux (min1) before reserpine administration, C is initial concentration and K is rate of synthesis. Immediately after reserpine administration krco K = kc where kr is new rate constant of efflux (misc ) and k4 is the initial rate constant of decline (min1). On rearrangement, this becomes k-k5= =.Ol3min

9 348 BRODIE ET AL. Vol. 152 TABLE 7 Effects of reserpine on blockade of 5-HT storage process and on relative rates of 5-HT efflux Reserpine Dose Blockade of Storage Processa Rate Constant of 5HT Effitix (kr) Steady State Level Calculated Experimental Difference Time to Reach Half Total Decline at Steady State mg/kg iv. % min % normal mis ± ± ± ± ± #{176}[kd/(kd max)] X 1. the 5-UT available to receptors after reserpine administration. Similar calculations for heart and brain NE levels indicate that these are only about 3% of normal. The time required for reserpine to reduce the brain 5-UT levels to one-half the total decline at the new steady state values is determined from the equation t11, = (.693/kr). The values (table 7) reveal that small doses of reserpine release small amounts of 5-HT over a relatively long time, while a dose of reserpine that elicits a maximal rate of release depletes 5% of the amine in about 7 mm. Our present results indicate that considerable amounts of free 5-HT are present in brain after complete inhibition of storage. This is consistent with the view that reserpine sedation is mediated through a constant occupancy of serotonergic receptors and with the finding that recovery of rabbits from sedation coincides with recovery of the process that takes up exogenous 5-UT. Restoration of the latter mechanism would terminate sedation by talcing up 5-HT from receptors into nerve endings. Our results are not a proof that the central effects of reserpine are causally related to changes in 5-UT. However, the results mdicate that the pharmacologic effects elicited by reserpine are related to the initial rate at which 5-UT is released. In addition, the disappearance of the sedative action of the drug is temporally correlated with the ability of brain tissue to accumulate exogenous 5-UT. However, it is not yet clear why the brain is able to accumulate exogenous 5-UT, long before the endogenous levels of amine reach the norma! value. The same discrepancy may also be inherent in our results which show that, 1 hr or later after reserpine injection, the 5-UT levels decline exponentially to the level calculated from the rate constant of efflux and then tend to decline to a much lower level (table 7). In 1 hr, for example,.9 mg/kg of reserpine depletes 5-UT by about 75%, but in 4 hr the loss increases to about 9%. It is possible that reserpine first releases the amines by inhibiting the carrier process at nerve endings. In the absence of amines, adenosine triphosphate or other granular components needed to form the amine complex may gradually disappear, but the re-formation of these granular constituents lags behind recovery of the uptake process. It may be pertinent that after the administration of the benzoquino!izines, which are short acting, both behavior and brain amine levels quickly return to normal (Pletscher et a!, 1962). REFERENCES BERTLER, A., CARLSSON, A. AND ROSENGREN, E. : Release by reserpine of catecholamines from rabbits hearts. Naturwissenschaften 43 : 521, BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B. UDENFRIEND, S. : Identification and assay of serotonin in brain. J. Pharmacol. 117: 82-88, BOGDANSKI, D. F., WEISSBACH, H. AND UDEN- FRIEND, S. : Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J. Pharmacol. 122: , BRODIE, B. B. AND BEAVEN, M. A. : Neurochemical transducer systems. Med exp. 8: , BRODIE, B. B. AND COSTA, E. : Some current views on brain monoamines. in Monoamines et Syst#{232}me Nerveux Central, ed. by J. de Ajuriaguerra, pp , Georg et Cie, Gen#{232}ve, BRODIE, B. B., OLIN, J. S., KUNTZMAN, R. G. ud SHORE, P. A. : Possible interrelationship between release of brain norepinephrine and serotonin by reserpine. Science 125 : , BRODIE, B. B., PLETSCHER, A. AND SHORE, P. A.:

10 BRAIN 5-icr AND RESERPINE ACTION Evidence that serotonin has a role in brain function. Science 122: 968, BRODIE, B. B. AND SHORE, P. A. : A concept for the role of serotonin and norepinephrine as chemical mediators in the brain. Ann. N.Y. Acad. Sci. 66: , CutLssoN, A., HILLARP, N. A. n WALDECK, B.: Analysis of the Mg-ATP dependent mechanism in the amine granules of the adrenal medulla. Acta physiol. scand. 59: suppl. 215, 1-38, CARLSSON, A., LwixuIsT, M. AND MAGNUSSON, T.: 3,4-Dihydroxyphenylalanine and 5-hydroxytryptophan as reserpine antagonists. Nature, Lond. 18: 12, 1957a. CARLSSON, A., RO5ENGREN, E., BERTLER, A. AND NILSSON, J. : Effect of reserpine on the metabolism of catecholamines. In Psychotropic Drugs, ed. by S. Garattim and V. Ghetti, pp , Elsevier Publishing Co., Amsterdam, 1957b. CHANO, C. C. : A sensitive method for spectrophotofluorometric assay of catecholamines. Tnt. J. Neuropharmacol. 3: , COSTA, E. AND BRODIE, B. B. : Concept of the neurochemical transducer as an organized molecular unit at sympathetic nerve endings. in Progress in Brain Research, Biogenic Amines, ed. by H. E. Himwich and W. A. Himwich, pp , Elsevier Publishing Co., Amsterdam, CROUT, R. J.: Catecholamines in urine, in Standard Methods of Clinical Chemistry, vol. 3, pp. 62-8, Academic Press, New York, ERSPAMER, V.: Recent research in the field of 5-hydroxytryptamine and related indolealkylamines. in Fortschritte Arzneimittefforschung, ed. by E. Jucker, vol. 3, pp , Birkhauser Verlag, Basel, GAFFNEY, T. E., CHID5EY, C. A. AND BRATJNWALD, E. : Study of the relationship between the neurotransmitter store and adrenergic nerve block induced by reserpine and guanethidine. Circulation Res. 12: , HESS, S. M., SHORE, P. A. AND BRODIE, B. B. : Persistence of reserpine action after the disappearance of drug from brain ; effect on serotonin. J. Pharmaco!. 118: 84-89, HOLZBAUER, M. AND VOCT, M. : Depression by reserpine of the noradrena!ine concentration in the hypothalamus of the cat. J. Neurochem. 1 : 8-11, PLETSCHER, A., BRossI, A. AND Gzy, K. F. : Benzoquinolizine derivatives : A new class of monoamine decreasing drugs with psychotropic action. Tnt. Rev. Neurobiol. 4: , PLETScHER, A., SHORE, P. A. tm BRODIE, B. B.: Serotonin as a mediator of reserpine action in brain. J. Pharmacol. 116: 84-89, POTTER, L. T., COOPER, T., WILLIAM, V. AND WOLFE, D. E. : Synthesis, binding, release and metabolism of NE in normal and transplanted dog hearts. Circulation Res. 16: , SHORE, P. A., PLETSCHER, A., TOMICH, E. G., CARLSSON, A., KUNTZMAN, R. AND BRODIE, B. B.: Role of brain SilT in reserpine action. Ann. N.Y. Aead. Sci. 66: , SPECTOR, S., SJOERDSMA, A. AND UDENFRIEND, S.: Blockade of endogenous norepinephrine synthesis by a-methyl-tyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmacol. 147: 86-95, Voor, M.: Discussion. in Ciba Foundation Symposium, Adrenergic Mechanisms, ed. by J. R. Vane, G. E. W. Wolstenho!me and M. O Connor, rc , Little, Brown & Co., Boston, 1961.