Neurochemical Research, VoL 17, No. 5, 1992, pp. 449--456

Effects ofp-Chloroamphetamine on Brain Serotonin Neurons* Ray W . Fuller 1

(Accepted November 11, 1991)

p-Chloroamphetamine (PCA) is a useful pharmacologic tool for selectively increasing brain serotonin function acutely by release of serotonin into the synaptic cleft. PCA produces behavioral, neurochemical and neuroendocrine effects believed due to serotonin release after doses in the range of 0.5-5 mg/kg. At higher doses and at longer times, PCA causes depletion of brain serotonin. The mechanisms of this depletion are not well understood but require the serotonin uptake carrier. Antagonism of PCA-induced depletion of brain serotonin is a useful means of assessing the ability of a compound to block the serotonin uptake carrier on brain serotonin neurons. PCA can also be used as a neurotoxic agent to deplete brain serotonin in functional studies, apparently by destroying some serotonergic nerve terminals. Used in this way, PCA has an advantage over 5,6- and 5,7dihydroxytryptamines in being effective by systemic injection, and it affects brain serotonergic projections with a different neuroanatomic specificity than the dihydroxytryptamines. KEY WORDS: p-Chloroamphetamine; serotonin; neurotoxicity; dihydroxytryptamines.

INTRODUCTION

for a minireview in the volume honoring Professor Aprison.

When I joined the Lilly Research Laboratories in 1963, two of my colleagues there--Drs. Jack Mills and Irwin Slater--introduced me to Professor Morris H. Aprison at Indiana University Medical Center as a local scientist who could help me learn about neurochemistry and about brain serotonin as a neurotransmitter. That same year, after I had begun experiments on brain serotonin, Dr. Mills suggested I investigate some halogenated amphetamines that he had synthesized, after Pletscher et al. (1) reported that p-chloromethamphetamine (Figure 1) depleted serotonin in rat brain. Because my acquaintance with halogenated amphetamines began the same year as my acquaintance with Professor Aprison, and I have enjoyed both acquaintances, I chose this topic

HISTORY OF p-CHLOROAMPHETAMINE When Pletscher and colleagues (1) first reported that p-chloromethamphetamine selectively depleted serotonin but not norepinephrine in rat brain, the compound was of interest because it did not directly inhibit either of the two enzymes in serotonin biosynthesis. Numerous chlorinated analogs of amphetamine had been synthesized and examined in the Lilly Research Laboratories as anorectic agents (2,3). When we studied those compounds, p-chloroamphetamine (PCA) emerged as the most potent serotonin depletor in the series (4,5). Following the important contributions of Sanders-Bush, Sulser and their co-workers (6-8), PCA became a much-used research tool for selective modification of serotonergic function in brain. Acutely, PCA releases serotonin and

1 Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 * Special issue dedicated to Dr. Morris H. Aprison.

449 0364-3190/92/0500-0449506.50/09 1992PlenumPublishingCorporation

450

Fuller m

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Fig. 1. Structuresofp-chloroamphetamineand someanalogs.

enhances serotonergic function, but depletes serotonin stores. The longer-term effect of high doses of PCA is depletion of serotonin and apparent diminution of serotonergic function.

Structure-Activity Relationships Several of the structural analogs of PCA that we have studied are shown in Figure 1. Our early studies (4,5) revealed that PCA was slightly more potent as a serotonin depletor than was p-chloromethamphetamine, the compound studied by Pletscher et al. (1). In fact, pchloromethamphetamine is rapidly and extensively metabolized to PCA in rats (9). Other N-alkyl analogs of PCA deplete serotonin, at least in part because they are metabolized to PCA (9,10). Huang et al. (11) reported that the c~-ethyl homolog of PCA was less potent than PCA in depleting brain serotonin in rats. Longer and shorter side chain homologs were inactive or less potent than PCA in depleting brain serotonin, as were analogs with the chloro substituent in other positions on the phenyl ring (12i. As we began our studies, we were surprised to see Pletscher et al. (13) report thatp-chloro-N-methylphenylethylamine depleted brain serotonin, for we found that the a-methyl branch was essential. I wrote Professor

Pletscher to point out the discrepancy and suggest we might exchange compound samples, but he replied that an error had been made in the paper and that the structure of the active compound should have had the methyl on the a-carbon not on the nitrogen; in other words, it was PCA not p-chloro-N-methyl-phenylethylamine. Thus our structure-activity studies agreed well. An especially interesting analog of PCA was the 13,13-difluoro compound. Fluorine is the smallest of the halogens. The two fluorine substituents on the [3-carbon change the shape of the PCA molecule very little, i.e., have minimum steric influence. But they greatly reduce the basicity of the nitrogen (its ability to accept a proton), reducing the pKa value (negative logarithm of the ionization constant) from above 9 to about 6.8 (14). At physiological pH, 13,13-difluoro-PCAexists mainly as an uncharged molecule, whereas PCA is almost entirely protonated (cationic). 13,13-Difluoro-PCAis very different from PCA in the rate of its metabolism and in its tissue distribution, illustrating the importance of ionization in drug metabolism and localization. PCA has a relatively long half-life in rats and localizes preferentially in tissues such as lung, kidney and brain. The [3,[3difluoro analog was rapidly metabolized (by oxidative deamination) and localized preferentially in fat, the tissue containing least amounts of PCA. The [3,[3-difluoro compound caused short-term depletion of brain serotonin when given at high doses in rats to produce equivalent brain levels as those obtained with PCA but did not cause long-term depletion of brain serotonin, apparently because the drug did not persist long enough in brain.

Characteristics of Serotonin Depletion Acute. When PCA is administered to rats, brain serotonin content is decreased within one or two hours and remains reduced as shown in Figure 2. Accompanying the acute decrease in serotonin content is a decrease in 5-hydroxyindoleacetic acid content (15) and a decrease in tryptophan hydroxylase activity measured in brain homogenates (7). Tryptophan hydroxylation in vivo measured by the accumulation of 5-hydroxytryptophan after decarboxylase inhibition is also decreased (16). Although total serotonin content is decreased, extracellular concentrations of serotonin measured by push-pull cannula techniques (17), by in vivo voltammetry (18,19) or by brain microdialysis (20) are increased. Adell et al. (21) reported that PCA still increased serotonin concentration in the microdialysis fluid even after reserpine treatment of the rats which markedly decreased basal concentrations of serotonin. They suggested that PCA

p-Chloroamphetamine and Brain Serotonin

451

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Fig. 2. Brain serotoninconcentrationsat various timesafter injection of a single dose ofp-chloroamphetamine(PCA) (0.1 mmole/kgi.p.) into rats. Mean values _+ standard errors for 5 rats per group are shown. Asterisksindicatesignificantdifferencefromcontrolgroup (P < .05).

releases serotonin from neuronal cytoplasm instead of from vesicular stores. The increase in extracellular concentrations of serotonin results in increased activation of synaptic receptors for serotonin, i.e., in enhanced serotonergic function, as shown by several changes. For instance, PCA injection causes myoclonus (22) and the serotonin behavioral syndrome--tremor, rigidity, Straub tail, hindlimb abduction, lateral head weaving and reciprocal forepaw treading (20,23). Additional acute behavioral effects of PCA thought to be due to serotonin release include inhibition of startle sensitization (24), suppression of sexual behavior in female rats (25) and the head twitch response in mice (26). Other acute effects of PCA mediated by release of serotonin into the synaptic cleft include activation of the pituitary-adrenocortieal axis (27), elevation of prolactin release (28) and increased plasma renin activity (29) in rats.

Long-Term. PCA is not only a serotonin-releasing drug whose acute effects are mediated by enhanced activation of postsynaptic serotonin receptors albeit accompanied by depletion of serotonin stores, but PCA at higher doses produces very long-term effects that are considered to represent neurotoxicity. The first clue to this came in 1970 when Frey (30) reported that brain serotonin in rats remained depleted for as long as 4 weeks after only 5 days of treatment with PCA. Sanders-Bush et al. (8) extended these findings and focused on the

long duration of PCA. Parameters specifically associated with serotonin neurons--content of serotonin and 5-HIAA, serotonin turnover, tryptophan hydroxylase, serotonin uptake capacity, serotonin transporters labeled with radioligands--are decreased for weeks or months by PCA (7,8,31-33). And many serotonin-containing nerve fibers detected by immunofluorescence are rendered undetectable by PCA (34). Further, evidence for nerve degeneration during the days immediately following PCA treatment has been provided (35). Not all serotonin neurons in the CNS are equally vulnerable to lesioning by PCA, but the full extent of the selectivity and its mechanistic basts are not understood. Mamounas and Molliver (34) have reported that PCA causes degeneration of fine serotonin axon terminals in rat cortex coming from dorsal raphe nuclei but spare coarse, beaded axons coming from median raphe nuclei. Fritschy et al. (36) reported that PCA selectively lesions serotonin axon terminals arising from the dorsal raphe nucleus while sparing projections from the raphe obscurus and raphe pallidus to the trigeminal motor nucleus. Blier et al. (19) reported that the dentate gyrus was resistant to the acute serotonin-releasing effect of PCA as well as to the prolonged serotonin-depleting effect. Possible Mechanisms

Both the acute and the long-term depletion of brain serotonin by PCA are dependent upon the membrane serotonin transporter on serotonin neurons, and the depletion of serotonin is antagonized by inhibitors of the transporter (37,38). Probably PCA is accumulated into serotonin nerve terminals by the membrane transporter, and that accumulation is necessary for the depletion of serotonin. One injection of a long-acting serotonin uptake inhibitor such as fluoxetine, given before PCA injection, prevents both the acute and the long-term depletion of serotonin. However, the acute and long-term effects can be dissociated. For instance, fluoxetine can be given 4 hours after PCA injection, when acute depletion of serotonin has already occurred. In that case, serotonin levels return to normal rapidly and remain normal thereafter (39). A single injection of a short-acting uptake inhibitor such as chlorimipramine, when given before PCA, prevents serotonin depletion for a few hours, but PCA persists in the brain longer than does chlorimipramine, and at 24 hours serotonin is depleted as much by PCA as if chlorimipramine had not been given (40). Some analogs of PCA such as the 13,[3-difluoro compound cause the acute but not long-term depletion of serotonin (14). The

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N-cyclopropyl analog initially increases rather than decreases brain serotonin due to its inhibition of monoamine oxidase, but causes the long-lasting depletion of serotonin via metabolism of PCA (10). Early studies had not revealed a dependence of PCA neurotoxicity on availability of serotonin stores (39), but Berger et al. (41) have reported recently that severe depletion of serotonin by the combination of p-chlorophenylalanine and reserpine provides substantial protection against the serotonergic neurotoxicity of PCA. They suggested specifically that serotonin release from platelets resulted in the formation of a neurotoxic metabolite of serotonin. Commins et al. (35) have specifically suggested that endogenous serotonin may undergo conversion to 5,6dihydroxytryptamine as a consequence of PCA treatment and that the neurotoxicity toward serotonergic neurons may be mediated by this 5,6-dihydroxytryptamine, which they detected in rat hippocampus by high performance liquid chromatography after a single i.p. dose of PCA. Axt and Seiden (42) reported that pretreatment with c~-methyl-tyrosine, an inhibitor of tyrosine hydroxylation, attenuated the depletion of serotonin by PCA in rat striatum and hippocampus. They suggested that newly synthesized dopamine was involved in the neurotoxic effects of PCA toward serotonin neurons. PCA is known to release dopamine and increases its concentration in microdialysis fluid samples from rat striatum (43). Although i.p. administration of cysteine can attenuate the neurotoxic effect of PCA toward brain serotonin neurons in rats (44), it may do so by decreasing brain concentrations of PCA instead of by modifying the mechanism of neurotoxicity (45). Is a Metabolite of PCA Involved?

The possibility that a metabolite of PCA might be involved in its neurotoxicity toward brain serotonin neurons has long been considered (15,32,46), but no compelling evidence has yet been presented. In our early structure-activity studies of PCA, some analogs were studied because they were potential metabolites of PCA. Most of these did not deplete serotonin, e.g., 13-hydroxyPCA and 3-chloro-4-hydroxyamphetamine. One analog, N-hydroxy-PCA, depleted brain serotonin, but it turned out to be extensively metabolized to PCA rather than the converse (47). Miller et al. (48) found that PCA could be converted to chemically reactive intermediates by hepatic and brain microsomal preparations in vitro, but that does not prove that such conversion is involved in the effects of PCA on brain serotonin neurons in vivo. Recent evidence consistent with the possibility of produc-

tion of a toxic metabolite is the finding of Berger et al. (49) that direct intracerebral application of PCA, even continuous infusion for two days, failed to elicit axonal degeneration as seen with systemic administration of PCA. However, as yet, no specific metabolite of PCA has been implicated as being responsible for the short- or longterm depletion of brain serotonin, nor is it proven that any metabolite of PCA is involved. Functional Consequences of the Long-term Serotonin Depletion

A number of effects in animals have suggested that the long-term depletion of brain serotonin by PCA influences brain function, although such animals are quite normal in gross appearance. For instance, Vorhees et al. (50) reported that rats treated with a 5 mg/kg i.p. dose of PCA showed hypoactivity and increased defecation in open-field testing for as long as 30 days. They showed facilitated acquisition in a shock avoidance Y-maze task for up to 15 days. Brain serotonin was still reduced by 41% after 38 days. Grabowska and Michaluk (51) reported that PCA injected at 2 mg/kg daily for 5 days did not affect locomotion measured 3 days after the last dose in rats but it intensified the stimulation caused by apomorphine. Those findings and other data in the paper were taken by the authors as confirming "'our hypothesis about the possible inhibitory role of serotonin in the apomorphineinduced locomotor stimulation in rats." Ogren et al. (52) reported evidence of impaired serotonergic function after depletion of brain serotonin by two doses of PCA, which resulted in 63% depletion of serotonin concentration in whole brain. Pretreatment with zimelidine completely prevented the depletion of serotonin. Eight days after PCA treatment, rats failed in the acquisition of a two-way conditioned avoidance response. Rats that had been pretreated with zimelidine and then given PCA acquired the avoidance response at least as well as control rats. The ability of zimelidine to reverse both the behavioral and the biochemical deficit is evidence that the reduction in serotonin content caused the reduced response acquisition. The long-term effects of PCA-induced depletion of brain serotonin on learning depends markedly on the learning task used. Altman et al. (53) studied two types of positively reinforced complex spatial discrimination tasks in rats. A week after two doses of PCA to deplete brain serotonin, performance in the eight-arm radial-arm maze was not affected. Learning was significantIy enhanced in the PCA-treated rats trained in the Stone 14unit T-maze. The improved acquisition was completely

p-Chloroamphetamine and Brain Serotonin

abolished when zimelidine was co-administered to antagonize the serotonin depletion by PCA. Gob et al. (54) gave a single dose of PCA to rats on postnatal day 8 and studied open-field behavior through postnatal day 30. Ambulatory activity and other measures of exploratory activity and grooming were increased during postnatal days 11-14, but exploratory activity was significantly diminished in the fourth week. Learning of avoidance behavior in rats has been reported to be decreased after injection of PCA on postnatal day 8 (55). Nabeshima et al. (56) gave one dose of PCA (10 mg/kg i.p.) 13 days earlier and observed that the intensity of head-weaving, turning, forepaw-treading, hindlimb abduction and Straub tail induced by 5-methoxyN,N-dimethyltryptamine and the intensity of head-twitch, turning and back-pedalling induced by phencyclidine were markedly increased. Similar increases in the responses to 5-methoxy-N,N-dimethyltryptamine and phencyclidine had been reported after lesioning of serotonin neurons with intracerebroventricular 5,7-dihydroxytryptamine (57). Serotonin Depletion by a High Dose of PCA Attenuates Subsequent Effects Mediated by Acute Serotonin Release by a L o w Dose of PCA

Table I shows that the acute, corticosterone-elevat~ng effec~ of PCA in rats, previously shown to be dependent on serotonin release (27), is attenuated by pretreatment with a high dose of PCA that causes prolonged depletion of brain serotonin stores. Such pretreatment with neurotoxic doses of PCA has been shown to antagonize or prevent many acute effects of PCA that are mediated by serotonin release, such as the antinociceptive effect in mice (58). Kutscher and Yamamoto

Table I. Serum CorticosteroneElevationbyp-Chloroamphetamine

in ControlRats and in Rats Treated One WeekEarIierwith p-Chloroamphetamine Dose of PCA, mg/kg given i.p. at -1 hr 0 1 3

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29 + 1b

43 • 2b

12• 12 -4- 2 21 • 5b

PCA given at 20 mg/kg 1 week earlier. In a separategroup of such treatedrats, brain serotoninconcentrationwas reducedto 0.24 -_ 0.02 ixg/g from 0.61 • 0.02 Ixg/gin control rats, a 61% reduction(P < _ool). P < .05, increasefrom correspondingzero dose group. Mean values • standzrderrors fc~r5 rats per group are shown.

453

(59) studied the acute responses to PCA 2 to 4 days after a 10 mg/kg dose of PCA, a dose sufficient to produce long-term depletion of brain serotonin. The abnormal behaviors elicited by acute PCA (forepaw treading, circling, headweaving and inching) were attenuated, as was the inhibition of normal behaviors such as walking, inactivity, rearing and grooming by acute PCA, suggesting that the neurotoxic dose of PCA had limited the availability of serotonin stores for release by the acute injection. Similar results have been reported by Nabeshima et al. (56). Marsden (60) found that the behavioral effects and the serotonin-releasing effect of a PCA injection were attenuated in rats that had received a PCA injection the previous day. Archer et al. (61) reported that depletion of brain serotonin by long-term PCA treatment completely blocked the retention impairment resulting from acute PCA, showing the importance of serotonin stores to the acute effects of PCA. Properties of PCA Relative to Indoleamine Serotonin Neurotoxins

Unlike 5,6- and 5,7-dihydroxytryptamines, which do not cross the blood-brain barrier and have to be given intraventricularly or directly into brain tissue, PCA can be given systemically to deplete serotonin in the CNS (62). The regional pattern of serotonin depletion by PCA differs from that of the dihydroxytryptamines (63,64). Bjorkum and Berge (65) used PCA and 5,6-dihydroxytryptamine as tools for selective chemical Iesioning of ascending vs. descending serotonergic projections. PCA preferentially lesioned ascending projections as evident from a 76% loss of cortical synaptosomal capacity to take up serotonin but only a 35% loss in spinal cord. In contrast, 5,6-dihydroxytryptamine did not reduce cortical uptake but decreased spinal synaptosomal uptake by 83%. The magnitude of the antinociceptive effect due to serotonin release by an acute low dose of PCA in rats was decreased by pretreatment with a neurotoxic dose of PCA, and the duration of the antinociceptive effect was decreased by 5,6-dihydroxytryptamine. The authors concluded that both ascending and descending serotonergic pathways contribute to PCA-induced antinociception. The depletion of serotonin by PCA is more easily blocked by uptake inhibitors than is depletion by dihydroxytryptamines. The blockade of serotonin depletion by PCA provides an excellent means of determining if some consequence truly is due to serotonin depletion and also provides a useful way of evaluating in vivo effects of new inhibitors of the serotonin transporter found by in vitro techniques. Both the acute and the proIonged

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depletion of brain serotonin by PCA depends on the membrane transporter, so antagonism of PCA-induced depletion of brain serotonin is useful for evaluating potency and duration of action of serotonin uptake inhibitots (37,66,67). Use of PCA for Chemical Lesioning of Serotonergic Pathways

Although PCA has largely been used to lesion rat brain serotonin neurons, it can also be used to deplete serotonin in other species such as mice (58,68,69) and pigeons (64). PCA can be a useful tool for chemical lesioning of brain serotonin neurons, and it has been used for example in obtaining evidence for serotonergic pathway involvement in the antinociceptive effects of nefopam (70), evidence for a sex-specific role of serotonin in punishment-induced behavioral suppression in rats (71), evidence that serotonergic neuronal systems inhibitorily regulate dopamine-dependent behavior induced by phencyclidine in rats (72), evidence for involvement of serotonergic processes in the learning of two types of positively reinforced complex spatial discrimination tasks in rats (53), evidence for involvement of serotonin in the suckling-induced release of prolactin in lactating rats (73), and evidence for serotonergic mediation of the decrease in 3,4-methylenedioxymethamphetamine-induced inhibition of dopamine release as measured by in vivo voltammetry in rat striatum (74). Dilsaver et al. (75) used PCA lesions to test the possible involvement of serotonin in oxotremorine-induced hypothermia in rats. PCA-induced lesions did not attenuate the hypothermic response to oxotremorine in alaproclate-pretreated rats. The ability of alaproclate, an inhibitor of serotonin uptake, to potentiate the oxotremorine response may have occurred through non-serotonergic mechanisms. PCA lesioning of serotonin neurons abolished regional cerebral metabolic responses to mchlorophenylpiperazine, a serotonin agonist, but not responses to other non-serotonergic drugs in rats, allowing Freo et al. (76) to interpret that m-chlorophenylpiperazine reduces cerebral metabolic rates for glucose via a presynaptic action on serotonin neurons.

SUMMARY The continuing study of PCA suggests it can increase brain serotonin function acutely at low doses by release of serotonin and can cause prolonged depletion of brain serotonin after high doses. The prolonged depletion probably reflects a neurotoxic action analogous

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to that of 5,6- and 5,7-dihydroxytryptamines. Both the acute and the long-term effects of PCA are dependent upon the membrane uptake carrier on serotonin neurons, possibly because PCA is concentrated in serotonin neurons via that uptake carrier. The possibilities that a metabolite of PCA or an endogenous substance such as dopamine or a metabolite of serotonin is involved in the long-term effects of PCA remain under investigation.

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