Reversal of fenfluramine and fluoxetine anorexia by 8-OH-DPAT is attenuated following raphe injection of 5,7-dihydroxytryptamine

Drugs that enhance serotonergic neurotransmission reduce food intake by directly or indirectly activating serotonergic receptors. In contrast drugs that inhibit serotonergic neurotransmission such as the 5-HT_1A agonist 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) stimulate food intake. The present study examined the effects of 8-OH-DPAT on the feeding suppressant action of the indirect 5-HT agonists fenfluramine (FEN; 0.63–2.5 mg/kg) and fluoxetine (FLU; 2.5–10 mg/kg), as well as the 5-HT_1B/2C agonist 1-(3-trifluoromethylphenyl)piperazine (TFMPP; 0.5–2 mg/kg). 8-OH-DPAT (62.5–250 μg/kg) was administered 5 min prior to FEN, FLU or TFMPP, injected 30 min before food access. While FEN, FLU and TFMPP dose-dependently reduced 2 h food intake, 8-OH-DPAT stimulated eating behavior. 8-OH-DPAT (62.5–250 μg/kg) pretreatment reversed the anorectic action of FEN (1.25 mg/kg) and FLU (5 mg/kg) but not TFMPP (1 mg/kg). Separate groups of rats were injected with 5,7-dihydroxytryptamine (5,7-DHT; 3 μg free base) into both the dorsal and median raphe, which resulted in extensive 5-HT depletion in hypothalamus (80%), striatum and hippocampus (90%). In both 5,7-DHT and vehicle-injected rats, FEN (1.25 mg/kg) and FLU (5 mg/kg) suppressed feeding. In 5,7-DHT treated rats, however, the ability of 8-OH-DPAT (125 μg/kg) to block FEN and FLU induced anorexia was attenuated. That is, 8-OH-DPAT pretreatment did not reverse the feeding inhibitory effects of either FEN or FLU. Further, the ability of FEN and FLU to suppress food intake was not altered by the 5,7-DHT lesion. These findings suggest that the reversal of FEN and FLU anorexia by 8-OH-DPAT is partially dependent on the integrity of brain 5-HT systems since their disruption compromises the ability of this 5-HT_1A agonist to antagonize the feeding suppressant action of either FEN or FLU. However, the ability of treatments which impair 5-HT neurotransmission to reverse FEN and FLU induced suppression of food intake may depend upon whether this impairment is acute and reversible (8-OH-DPAT), or chronic and irreversible (5,7-DHT).     

MDMA and fenfluramine reduce L-DOPA-induced dyskinesia via indirect 5-HT1A receptor stimulation

Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) pharmacotherapy in Parkinson's disease is often accompanied by the development of abnormal and excessive movements known as dyskinesia. Clinical and experimental studies indicate that indirect serotonin agonists can suppress dyskinesia without affecting the efficacy of L-DOPA. While the mechanism by which these effects occur is not clear, recent research suggests that serotonin 5-HT1A receptors may play a pivotal role. To test this, male Sprague-Dawley rats with unilateral 6-hydroxydopamine medial forebrain bundle lesions received 1 week of daily treatment with L-DOPA (12 mg/kg, i.p.) plus benserazide (15 mg/kg, i.p.). Beginning on the 8th day of treatment and every 3rd or 4th day thereafter, rats were pretreated with vehicle (0.9% NaCl), the serotonin and dopamine releaser 3,4-methylenedioxymethamphetamine (MDMA; 0.25 or 2.5 mg/kg, i.p.) or the serotonin releaser fenfluramine (FEN; 0.25 or 2.5 mg/kg, i.p.) 5 min prior to L-DOPA, after which abnormal involuntary movements (AIMs) and rotations were quantified every 20th minute for 2 h. Pretreatment with 2.5 mg/kg of either MDMA or FEN reduced AIMs. To determine the contribution of the 5-HT1A receptor to these effects, another group of L-DOPA-primed 6-hydroxydopamine-lesioned rats were pretreated with the 5-HT1A antagonist WAY100635 (0.5 mg/kg, i.p.), MDMA + WAY100635 (2.5 + 0.5 mg/kg, i.p.) or FEN + WAY100635 (2.5 + 0.5 mg/kg, i.p.) 5 min prior to L-DOPA and subsequent AIMs and rotation tests. The antidyskinetic effects of MDMA and FEN were reversed by cotreatment with WAY100635. These results suggest that 5-HT-augmenting compounds such as MDMA and FEN probably convey antidyskinetic properties in part via stimulation of 5-HT1A receptors.     

3,4-methylenedioxymethamphetamine (MDMA) administration to rats decreases brain tissue serotonin but not serotonin transporter protein and glial fibrillary acidic protein

Previous experiments conducted in this laboratory showed that administration of high-dose D-fenfluramine (D-FEN) and p-chloroamphetamine (PCA) decreased 5-HT transporter (SERT) binding and tissue 5-HT by 30-60% in caudate and whole brain tissue 2 days and 2 weeks after drug administration. However, protein expression as determined by Western blot analysis did not change in either tissue or time point, except for a 30% decrease in the caudate 2 days after PCA administration. In the present study, we studied the effect of MDMA and 5,7-dihydroxytryptamine (5,7-DHT) on tissue 5-HT levels and the protein expression level of SERT and glial fibrillary acidic protein (GFAP), a validated neurotoxicity marker. HYPOTHESIS: MDMA administration decreases SERT expression. METHODS: Two weeks after MDMA administration (7.5 mg/kg i.p., q 2 h x 3 doses) or 2 weeks after i.c.v. administration of 5,7,-DHT (150 microg/rat), male Sprague-Dawley rats were sacrificed and the caudate, cortex, and hippocampal tissue collected. Western blots for SERT and GFAP were generated using published methods. Tissue 5-HT levels were determined by HPLC coupled to electrochemical detection. RESULTS: MDMA treatment decreased tissue 5-HT in cortex, hippocampus, and caudate by about 50%. However, MDMA treatment had no significant effect on expression level of SERT and GFAP in any brain region. In contrast, 5,7-DHT reduced tissue 5-HT by more than 90%, decreased SERT protein expression by 20-35%, and increased GFAP by 30-39%. CONCLUSION: These data suggest the MDMA treatment regimen used here does not cause degeneration of 5-HT nerve terminals. Viewed collectively with our previous results and other published data, these data indicate that MDMA-induced persistent 5-HT depletion may occur in the absence of axotomy.     

Brainstem serotonergic hyperinnervation modifies behavioral supersensitivity to 5-hydroxytryptophan in the rat

Rat pups were injected intracisternally (i.c.) or intraperitoneally (i.p.) with 5,7-dihydroxytryptamine (5,7-DHT) or saline and challenged 2 and 14 weeks later with the 5-HT precursor 5-hydroxytryptophan (5-HTP), which evokes behavioral supersensitivity in adult rats, 5,7-DHT induced transient postinjection convulsions in rats injected i.c. but not i.p. Rats with either type of 5,7-DHT lesions displayed supersensitive behavioral responses to 5-HTP. However, rats lesioned by i.p. injections exhibited significantly greater shaking behavior (+1445%) in response to 5-HTP than their i.c. counterparts, who instead showed more forepaw myoclonus (+250%) and head weaving (+270%), the core features of the 5-HT syndrome. Differences in 5-HT syndrome behaviors were already present 2 weeks after lesioning, whereas the difference in shaking behavior was not. After 14 weeks, 5-HT was selectively depleted (-43 to -92%) in hippocampus, spinal cord, and frontal cortex, and differences between i.c. and i.p. 5,7-DHT routes were insignificant except in frontal cortex. Brainstem 5-HT concentrations were significantly increased (+35%) after i.p. 5,7-DHT injections in contrast to reduction (-89%) after i.c. 5,7-DHT; 5-hydroxyindole acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) ratios were decreased (-20%) with either route. These data suggest that brainstem 5-HT hyperinnervation following i.p. 5,7-DHT injection modifies the functional consequences of injury in abating the 5-HT syndrome, but does not result in complete recovery since shaking behavior is enhanced. Loss of presynaptically mediated autoregulation or receptor dysregulation may play a major role in behavioral supersensitivity induced by 5-HTP in rats with 5,7-DHT lesions. To the extent that the 5-HT syndrome is mediated by 5-HT1A receptors and shaking behavior by 5-HT2 sites, differential responses to injury of 5-HT1A and 5-HT2 receptors may contribute to these behavioral differences.     

Reduced efficacy of fluoxetine following MDMA ("Ecstasy")-induced serotonin loss in rats

Long-term serotonin (5-HT) neuronal loss is currently a major cause of concern associated with recreational use of the substituted amphetamine 3,4 methylenedioxymethamphetamine (MDMA; "Ecstasy"). Such loss may be problematic considering that psychiatric disorders such as depression and anxiety and responses to first line treatments for these disorders are associated with 5-HT. In this study the effects of prior exposure to MDMA on behavioural and central neurochemical changes induced by the serotonin (5-HT) re-uptake inhibitor and antidepressant fluoxetine were examined in rats. Animals were administered MDMA (10 mg/kg. i.p.) four times daily for two consecutive days. One week later the animals were subjected to treatment with fluoxetine (10 mg/kg, i.p.). Fluoxetine treatment groups received either acute (saline injections for 20 days followed by 3 fluoxetine treatments over 24 h) or chronic (once daily fluoxetine for 21 days) drug administration. Prior exposure to MDMA resulted in an attenuation of fluoxetine-induced swimming behaviour in the modified forced swimming test (FST); a behavioural test of antidepressant action. In parallel MDMA treatment resulted in significant regional depletions of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) accompanied by a reduction in cortical [3H] paroxetine binding to nerve terminal 5-HT transporters. MDMA-induced 5-HT loss was enhanced in animals following chronic fluoxetine administration. Elimination of fluoxetine and its metabolite norfluoxetine from the brain abolished this interaction between MDMA and fluoxetine treatment. Fluoxetine administration reduced both 5-HIAA and the 5-HIAA:5-HT metabolism ratio, which was attenuated in animals pre-treated with MDMA. Overall the results show that MDMA induces long-term 5-HT loss in the rodent brain and consequently diminishes behaviour and reductions in 5-HT metabolism induced by the antidepressant fluoxetine. These results have potential clinical relevance, suggesting that 5-HT re-uptake inhibitors such as fluoxetine may be less effective at treating depression in chronic abusers of MDMA.     

Effects of indolic neurotoxins on enteric serotonergic neurons

The effects of the indolic neurotoxins 5,6- and 5,7-dihydroxytryptamine (5,6-DHT; 5,7-DHT) on the enteric nervous system were examined. 5,6-DHT, in moderate dosage, 40 mg/kg, decreased uptake of tritiated 5-hydroxytryptamine (³H-5-HT) but not that of tritiated norepinephrine (³H-NE). However, selectivity of the neurotoxins for enteric serotonergic rather than adrenergic axons was enhanced by pretreating animals with desmethylimipramine to inhibit the catecholamine uptake mechanism. When this was done, 5,7-DHT was found to prevent the development of 5-HT histofluorescence (following injection of L-tryptophan) without affecting histofluorescence of NE. In contrast, 6-hydroxydopamine virtually abolished NE histofluorescence and uptake of ³H-NE without affecting development of 5-HT histofluorescence or uptake of ³H-5-HT. Electron microscopy revealed that 5,7-DHT induced dose-dependent lesions of axonal varicosities in the enteric nervous system. Early lesions, 1–4 hours following injection, resembled cytolysosomes and consisted of membrane enclosed regions of opaque cytoplasm containing synaptic vesicles. Affected varicosities contained a mixed population of large dense cored (～120nm) and small lucent vesicles (～70nm) but none exhibited pre- or postsynaptic membrane specializations. After 24 hours terminals degenerated and were engulfed by surrounding supporting cells. It is concluded that peripheral serotonergic neurons resemble central serotonergic neurons in susceptibility to the toxic effects of indolic neurotoxins. These neurotoxins are useful anatomical markers of serotonergic terminal varicosities.     

Endogenous levels of serotonin and 5-hydroxyindoleacetic acid in specific areas of the pigeon CNS: effects of serotonin neurotoxins

Endogenous levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were determined by high-performance liquid chromatography (HPLC) in specific regions of the pigeon central nervous system (CNS). High endogenous 5-HT levels in the visual wulst and brainstem and medium 5-HT content in the optic lobes were found. The cerebellum and retina showed low endogenous 5-HT levels. Similar endogenous 5-HIAA levels were measured in the visual wulst, optic lobes and brainstem, whereas the 5-HIAA content of the cerebellum and retina was significantly lower. The effects of para-chloroamphetamine (p-CA) and 5,7-dihydroxytryptamine (5,7-DHT) on the 5-HT and 5-HIAA content of the same regions were studied. Six days after p-CA treatment, the 5-HT content of the visual wulst, optic lobes, brainstem and the 5-HIAA content of the optic lobes and cerebellum markedly decreased. Nine days after 5,7-DHT administration, the 5-HT and 5-HIAA content of the visual wulst and optic lobes was significantly reduced. At longer survival times, serotonergic systems were differentially affected depending on both the neurotoxin treatment and the specific brain regions examined. The 5-HT content of the pigeon retina was not modified by p-CA treatment, whereas 5,7-DHT intravitreally injected caused a pronounced 5-HT depletion. Our results demonstrate that selective neurotoxins for serotonergic systems can provide a useful denervation tool for the study of serotonergic function in the pigeon CNS.     

Plasticity and ontogeny of the central 5-HT transporter: effect of neonatal 5,7-dihydroxytryptamine lesions in the rat.

5,7-Dihydroxytryptamine (5,7-DHT) is unique as a serotonin (5-HT) neurotoxin in that i.p. injection of neonatal rats increases concentrations of 5-HT in brainstem while depleting 5-HT in cortex, hippocampus and spinal cord. To study the mechanism of this effect we measured the 5-HT transporter or uptake site, a presynaptic marker, using [3H]paroxetine binding. There were significant regional differences in Bmax of vehicle-injected rats: brainstem, diencephalon > striatum, cortex, spinal cord > hippocampus, cerebellum. There were also regional differences in the ontogeny of bindings sites: at postnatal day 7, [3H]paroxetine sites were 39% of adult levels in cortex compared to 63% in brainstem. Thirty days after 100 mg/kg 5,7-DHT i.p., Bmax of [3H]paroxetine binding was significantly increased in brainstem (+67%) and diencephalon (+136%), whereas it decreased in cortex (-59%), hippocampus (-94%) and spinal cord (-99%), striatum (-41%) and cerebellum (-37%). KD remained unaltered. In dose-response studies (0-200 mg/kg), 50 mg/kg was the threshold dose for Bmax effects and 200 mg/kg was lethal. In weekly time-course studies, changes were apparent 1 week after 5,7-DHT lesions. Binding site increases in diencephalon and brainstem were not maximal until 3 weeks after injection, whereas percent decreases in cortical sites remained unchanged at each week studied. Lesion effects on the ontogeny of [3H]paroxetine binding sites were region-dependent: cortical sites continued to increase with age but spinal sites did not. There was no significant recovery in spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotoxic effects of (±)fenfluramine and phentermine,alone and in combination,on monoamine neurons in the mouse brain

Until recently, (±)fenfluramine (FEN) was widely prescribed as an appetite suppressant. In animals, FEN is a potent and selective brain serotonin neurotoxin. The present studies assessed the effects of phentermine (PHEN), an appetite suppressant frequently used clinically in combination with FEN, on FEN-induced serotonin neurotoxicity. Groups (n = 6/group) of mice were treated with FEN (10 mg/kg), PHEN (20 mg/kg or 40 mg/kg), FEN (10 mg/kg) plus PHEN (20 mg/kg or 40 mg/kg), or vehicle twice daily for four days. Food intake and body weight were measured during and after drug treatment. Brains were evaluated for regional brain serotonin and dopamine axonal markers two weeks after drug treatment. PHEN enhanced the anorectic and weight-reducing effects of FEN. PHEN also significantly enhanced FEN's long-term toxic effects on 5-HT axons. This effect was evident in some (hypothalamus, striatum) but not all (hippocampus, cortex) brain regions examined. PHEN alone produced no long-term effects on 5-HT axonal markers. However, whether given alone or in combination with FEN, PHEN produced significant, dose-related decreases in striatal DA axonal markers. These results, coupled with those from previous studies, suggest that PHEN has the potential to exacerbate FEN-induced serotonin neurotoxicity, if utilized in certain doses. Further, the present results indicate that PHEN possesses dopamine (DA) neurotoxic potential. The relevance of these data to humans previously treated with FEN/PHEN is discussed. Synapse 30:239–246, 1998. © 1998 Wiley-Liss, Inc.     

5-HT depletion with 5,7-DHT, PCA and PCPA in mice: differential effects on the sensitivity to 5-MeODMT, 8-OH-DPAT and 5-HTP as measured by two nociceptive tests

Depletion of 5-hydroxytryptamine (5-HT) in mice was produced by intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT, 80 micrograms) or by systemic injections of p-chloroamphetamine (PCA, 3 X 40 or 4 X 40 mg/kg), p-chlorophenylalanine (PCPA, 5 X 400 or 14 X 400 mg/kg) or combined PCA (3 X 40 mg/kg) + PCPA (11 X 400 mg/kg). Neither of the pretreatments altered nociception in the increasing temperature hot-plate test, whereas hyperalgesia was demonstrated in 5,7-DHT lesioned animals in the tail-flick test. 5,7-DHT-pretreatment enhanced the antinociceptive effect of the 5-HT agonists 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-hydroxytryptophan (5-HTP). This effect was observed after 2, 5 and 8 days in the tail-flick test and after 5 and 8 days in the hot-plate test. However, pretreatment with PCPA or PCA failed to alter the antinociception elicited by the 5-HT agonists, although a tendency towards enhancement of antinociception was found after combined treatment with PCA and PCPA. It is suggested that the injection of 5,7-DHT induces denervation supersensitivity of post-synaptic 5-HT receptors. The lack of such supersensitivity after PCPA-pretreatment which induces similar 5-HT depletion to 5,7-DHT, may suggest that other factors than the absence of 5-HT may contribute to the development of denervation supersensitivity. Alternatively, the three 5-HT depleting agents may produce a qualitatively different reduction of 5-HT.     

Ontogenetic Serotoninergic Lesioning Alters Histaminergic Activity in Rats in Adulthood

The aim of this study was to determine histamine content in the brain and the effect of histamine receptor antagonists on behavior of adult rats lesioned as neonates with the serotonin (5-HT) neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). At 3 days after birth Wistar rats were pretreated with desipramine (20 mg/kg ip) before bilateral icv administration of 5,7-DHT (37.5 μg base on each side) or saline—ascorbic (0.1%) vehicle (control). At 10 week levels of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were determined in frontal cortex, striatum, and hippocampus by an HPLC/ED technique. In the hypothalamus, frontal cortex, hippocampus and medulla oblongata, the level of histamine was analyzed by an immunoenzymatic method. Behavioral observations (locomotion, exploratory-, oral-, and stereotyped activity) were performed, and effects of DA receptor agonists (SKF 38393, apomorphine) and histamine receptor antagonists S(+)chlorpheniramine (H₁), cimetidine (H₂), and thioperamide (H₃) were determined. We confirmed that 5,7-DHT profoundly reduced contents of 5-HT and 5-HIAA in the brain in adulthood. Histamine content was also reduced in all examined brain regions. Moreover, in 5,7-DHT-lesioned rats the locomotor and oral activity responses to thioperamide were altered, and apomorphine-induced stereotype was intensified. From the above, we conclude that an intact central serotoninergic system modulates histamine H₃ receptor antagonist effects on the dopaminergic neurons in rats.     

Early post-natal administration of 5,7-dihydroxytryptamine destroys 5-HT neurons but does not affect spatial memory.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) may play an important role in learning and memory. It has also been suggested that 5-HT abnormalities may mediate some aspects of the cognitive disorders associated with Korsakoff syndrome and Alzheimer's Disease. The effect of intracisternally applied 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) on learning and memory in rodents was evaluated. Three-day-old rat pups were treated with pargyline (40 mg/kg, i.p.) followed by 5,7-DHT (50 micrograms/pup) and returned to the dam for a month. At 75 days of age, rats were tested on a learning set problem in the Morris water maze for 5 days followed by 30 days of testing in a 12-arm radial maze with 8 of the 12 arms baited. In the Morris water maze, the latency to locate the hidden platform did not differ significantly for 5,7-DHT treated and control rats (F less than 1.0). Similarly, 5,7-DHT treated rats performed comparably to controls on the 12-arm radial maze (F less than 1.0). At 106 days of age the assay of tryptophan hydroxylase activity in the dorsal raphe nuclei and hippocampus showed marked reduction (86%, 78%, respectively) in 5,7-DHT treated animals compared to vehicle injected controls. Immunocytochemical analysis was consistent with the biochemical results. In 5,7-DHT treated animals there was severe loss of neurons that bind 5-HT antibody in the dorsal and medial raphe nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proposed animal model of attention deficit hyperactivity disorder

Dopamine (DA) neurons are implicated in the hyperlocomotion of neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of attention deficit hyperactivity disorder (ADHD). Because serotonin (5-HT) neurons mediate some DA agonist effects, we investigated the possible role of 5-HT neurons on locomotor activity. Rats were treated at 3 days after birth with vehicle or 6-OHDA (134 μg ICV; desipramine pretreatment, 20 mg/kg IP, 1 h), and at 10 weeks with vehicle or 5,7-dihydroxytryptamine (5,7-DHT; 75 μg ICV; pretreatment with desipramine and pargyline, 75 mg/kg IP, 30 min), to destroy DA and/or 5-HT fibers. Intense spontaneous hyperlocomotor activity was produced in rats lesioned with both 6-OHDA and 5,7-DHT. Locomotor time in this group was 550 ± 17 s in a 600 s session, vs. 127 ± 13 s in the 6-OHDA group and <75 s in 5,7-DHT and intact control groups (p < 0.001). Oral activity dose-effect curves established that 5,7-DHT attenuated DA D₁ receptor supersensitivity and further sensitized 5-HT_2c receptors. Acute treatment with dextroamphetamine (0.25 mg/kg SC) reduced locomotor time in 6-OHDA+5,7-DHT-lesioned rats to 76 ± 37 s (p < 0.001). Striatal DA was reduced by 99% and 5-HT was reduced by 30% (vs. 6-OHDA group). Because combined 6-OHDA (to neonates) and 5,7-DHT (to adults) lesions produce intense hyperlocomotion that is attenuated by amphetamine, we propose this as a new animal model of ADHD. The findings suggest that hyperactivity in ADHD may be due to injury or impairment of both DA and 5-HT neurons.     

Plasma levels of parent compound and metabolites after doses of either d-fenfluramine or d-3,4-methylenedioxymethamphetamine (MDMA) that produce long-term serotonergic alterations

Plasma levels of parent compounds and metabolites were determined in adult rhesus monkeys after doses of either 5mg/kg d-fenfluramine (FEN) or 10mg/kg d-3, 4-methylenedioxymethamphetamine (MDMA) i.m. twice daily for four consecutive days. These treatment regimens have been previously shown to produce long-term serotonin (5-HT) depletions. Peak plasma levels of 2.0+/-0.4 microM FEN were reached within 40min after the first dose of FEN, and then declined rapidly, while peak plasma levels (0.4+/-0.1 microM) of the metabolite norfenfluramine (NFEN) were not reached until 6h after dosing. After the seventh (next to last) dose of FEN, peak plasma levels of FEN were 35% greater than after the first dose while peak NFEN-levels were 500% greater. The t(1/2) for FEN was 2.6+/-0.3h after the first dose and 3.2+/-0.2h after the seventh. The estimated t(1/2) for NFEN was more than 37.6+/-20.5h. Peak plasma levels of 9.5+/-2.5 microM MDMA were reached within 20min after the first dose of MDMA, and then declined rapidly, while peak plasma levels (0.9+/-0.2 microM) of the metabolite 3,4-methylenedioxyamphetamine (MDA) were not reached until 3-6h after dosing. After the seventh (next to last) dose of MDMA, peak plasma levels of MDMA were 30% greater than the first dose while peak MDA levels were elevated over 200%. The t(1/2) for MDMA was 2.8+/-0.4h after the first and 3.9+/-1.1h after the seventh dose. The estimated t(1/2) for MDA was about 8.3+/-1.0h. Variability in plasma levels of MDMA and MDA between subjects was much greater than that for FEN and NFEN. This variability in MDMA and MDA exposure levels may have lead to variability in the subsequent disruption of some behaviors seen in these same subjects. There were 80% reductions in the plasma membrane-associated 5-HT transporters 6 months after either the FEN or MDMA dosing regimen indicating that both treatments produced long-term serotonergic effects.     

N-tert-butyl-alpha-phenylnitrone protects against 3,4-methylenedioxymethamphetamine-induced depletion of serotonin in rats

The present study examined the effect of N-tert-butyl-alpha-phenylnitrone (PBN) on 3,4-methylenedioxmathamphetamine (MDMA)-induced depletion of serotonin in the CNS. Rats were treated with two concurrent injections of MDMA (20 mg/kg, s.c.), PBN (50-400 mg/kg dissolved in ethanol, 50 mg/ml of 25% ethanol, i.p.), saline or 25% ethanol, alone or in combination, 6 h apart, and sacrificed 5 days later. Rectal temperature was measured prior to and hourly following the drug injection for 5 h. Monoamine levels in the tissue were measured by HPLC. Density of the 5-HT transporters was assayed by [3H]paroxetine binding. Rectal temperature of rats increased after MDMA, decreased after PBN, ethanol, PBN plus ethanol, and MDMA plus ethanol, and was not significantly altered after MDMA plus PBN. Levels of 5-HT and 5-HIAA in the frontal cortex, hippocampus, striatum, and brain stem of rats decreased significantly after MDMA or MDMA plus ethanol, but not after MDMA plus PBN, PBN plus ethanol (PBN dissolved in ethanol), or ethanol as compared to the saline controls. Levels of 5-HT and 5-HIAA in the brain tissues of rats treated with MDMA plus PBN were elevated as compared to those treated with MDMA plus saline. Similar results were observed in the density of 5-HT transporters in the frontal cortex and hippocampus. These results indicate that scavenging of free radicals of MDMA metabolites or reactive oxygen species by PBN and with lowering of body temperature protected against MDMA-induced depletion of serotonin transmitter.     

Hippocampal serotonin re-uptake and nocturnal locomotor activity after microinjections of 5,7-DHT in the fornix-fimbria

The role of the hippocampal 5-hydroxytryptamine (5-HT) terminals in the control of locomotor activity was investigated by lesioning 5-HT axons in the fimbria with 5,7-dihydroxytryptamine (5,7-DHT). Rats pretreated with desimipramine (10 mg/kg, i.p.) received microinjections of 5,7-DHT (0, 1, 3, 5 or 10 μg in 0.4 μl ascorbic Ringer's solution) into the fornix-fimbria. On the fourteenth to twenty-first nights after operation, nocturnal locomotor activity was measured in photocell cages. Twenty-eight to thirty days after operation degeneration of 5-HT terminals was assessed by measuring in vitro [³H]5-HT re-uptake in slices of dorsal hippocampus, ventral hippocampus and the septum.Groups injected with 5,7-DHT showed hyperactivity in the night period and increased decrements of activity between tests, both of which were related to the dose of neurotoxin. A reduction of [³H]5-HT re-uptake was found in dorsal hippocampus which was related to the dose of 5,7-DHT, but ventral hippocampal and septal [³H]5-HT re-uptake were not systematically reduced. For each rat, levels of dorsal and ventral hippocampal [³H]5-HT re-uptake were negatively correlated with the mean nocturnal activity from the 7 nights of testing. Levels of dorsal, but not ventral hippocampal [³H]5-HT re-uptake were negatively correlated with the mean nightly decrement of activity. No correlations were found between septal [³H]5-HT and these activity measures. These results, indicate that the increase in nocturnal locomotor activity caused by generalized depletion of 5-HT in the brain may be due to disruption of hippocampal 5-HT terminals supplied by the fornix-fimbria.     

Plastic responses of neonatal 5-hydroxytryptamine1B receptors to 5,7-dihydroxytryptamine lesions mapped by quantitative autoradiography

We previously found different effects on behavior, serotonin (5-HT) concentrations, 5-HT uptake sites, and 5-HT_1A binding sites of neonatal 5,7-dihydroxytryptamine (5,7-DHT) lesions depending on the route of 5,7-DHT injection. To study the impact of early lesions on 5-HT_1B sites as putative 5-HT terminal autoreceptors, we labelled them autoradiographically with [³H]5-HT 4 months after intraperitoneal (i.p.) or intracisternal (i.c.) 5,7-DHT injection during the first postnatal week and quantitated specific binding in 22 brain regions. Changes were confined to the subiculum and substantia nigra, regions with the most 5-HT_1B-specific binding and projection areas of structures with high mRNA expression. Both routes of 5,7-DHT injection were associated with increases in specific binding in subiculum (24% for i.p. and 47% for i.c. route). In contrast, there was a 32% increase in specific binding in the substantia nigra in rats with lesions made i.c. but not i.p. No significant differences were found in nucleus accumbens, caudate-putamen or other brain areas. In saturation homogenate binding studies of 5-HT_1B sites using [¹²⁵I]iodocyanopindolol 1 month after i.p. injections, neonatal 5,7-DHT lesions did not significantly alter B_max or K_d in the neocortex, striatum, diencephalon or brainstem. These data indicate the differential effects of the route of neonatal 5,7-DHT injections on plasticity of 5-HT_1B receptor recognition sites and suggest the presence of a subpopulation of post-synaptically located 5-HT_1B sites which increases in response to denervation. The data also suggest that sprouting of 5-HT neurons after neonatal 5,7-DHT lesions does not involve 5-HT_1B sites.     

Evidence for a postsynaptic action of the serotonergic anxiolytics: Ipsapirone, indorenate and buspirone

In the present experiment we analyzed whether the antianxiety action of the serotonergic 1A agonists buspirone (5 mg/kg), ipsapirone (5 mg/kg), indorenate (5 mg/kg), and 8-OH-DPAT (0.5 mg/kg) were mediated through the stimulation of pre- or postsynaptic serotonergic receptors. The experimental anxiety values were determined with the burying behavior test, where a reduction in the cumulative time of burying behavior was interpreted as a reduction in anxiety. To that purpose we analyzed the putative anxiolytic action of these drugs in animals with lesion of the serotonergic fibers after the intracerebroventricular (ICV) injection of 5,7-dihydroxytyptamine (5,7-DHT, 10 or 150 micrograms/10 microliters). The neurochemical analysis shows that these treatments produce a statistically significant reduction in 5-HT and 5-HIAA levels in various brain areas. The results of the behavioral experiments reveal that buspirone, ipsapirone, and indorenate produced exactly the same reduction in burying behavior in lesioned animals as compared with control rats. The reduction in burying behavior produced by 8-OH-DPAT was effectively prevented by the lesion with 5,7-DHT. These data suggest that the anxiolytic effect of buspirone, ipsapirone, and indorenate is mediated via the stimulation of postsynaptic receptors, while the somatodendritic receptors are involved in the antianxiety effect of 8-OH-DPAT.     

Monoaminergic denervation of the rat hippocampus: Microiontophoretic studies on pre- and postsynaptic supersensitivity to norepinephrine and serotonin

The responsiveness of hippocampal CA₃ pyramidal neurons to microiontophoretic applications of serotonin (5-HT), norepinephrine (NE), γ-aminobutyric acid (GABA) and isoproterenol (ISO) was assessed in rats following 5,7-dihydroxy-tryptamine (5,7-DHT) and 6-hydroxydopamine (6-OHDA) pretreatments and bilateral locus coeruleus lesions. The intraventricular administration of 200 μg (free base) of 5,7-DHT and of 6-OHDA produced 89% and 93% decreases of 5-HT and NE respectively. None of these pretreatments modified the initial responsiveness to, or recovery from iontophoretic application of 5-HT. In 6-OHDA pretreated and locus-lesioned rats, the initial effectiveness of NE was not altered but its effect was markedly prolonged. However, there was no such prolongation of the effect of ISO which is not a substrate for the high affinity NE reuptake. The effect of GABA was not affected by these pretreatments. Acute pharmacological blockade of the NE reuptake with desipramine (5 mg/kg, i.p.) similarly induced a prolongation of the effect of iontophoretically applied NE, while fluoxetine (10 mg/kg, i.p.) a 5-HT reuptake blocker, failed to alter the recovery of pyramidal cells from iontophoretic application of 5-HT.

It is concluded that 5-HT denervation induces neither pre- nor postsynaptic types of supersensitivity in hippocampal pyramidal cells, contrasting with the previously shown supersensitivity of ventral lateral geniculate and amygdaloid neurons following 5-HT denervation. NE denervation fails to induce a postsynaptic type of supersensitivity but leads to a marked prolongation of the response to NE indicative of a presynaptic mechanism. These results underscore the necessity for regional studies of neurotransmitters and drug action.     

Involvement of the serotonergic neuronal system in phencyclidine-induced place aversion in rats.

The possible involvement of the serotonergic neuronal system in aversive motivation produced by phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] was investigated using a place-conditioning paradigm in rats. PCP (4 mg/kg, i.p.) produced place aversion in this task as reported previously (Kitaichi K, Noda Y, Hasegawa T, Furukawa H, Nabeshima T. Acute phencyclidine induces aversion, but repeated phencyclidine induces preference in the place conditioning test in rats. Eur J Pharmacol 1996;318:7-9). The blockade of serotonin2A (5-HT2A) receptors using the antagonist ritanserin (3 and 10 mg/kg, p.o.) significantly attenuated this aversive property of PCP whereas lesions of serotonergic neurons using 5,7-dihydroxytryptamine (5,7-DHT, 100 microg/animal, i.c.v.) failed to affect it. Repeated PCP treatment (10 mg/kg, i.p. for 14 days), which is enough to diminish the stereotyped 5-HT2A receptor-mediated head-twitch behavior, also decreased the place aversion. These results suggest that the serotonergic neuronal system, specifically the 5-HT2A receptor, may play a critical role in producing PCP-induced place aversion.