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The use of the club drugs 3,4-methylenedioxymethamphetamine (MDMA) and 5-methoxy-n,n-diisopropyltryptamine (Foxy) is of growing concern, especially as many of the effects, particularly during development, are unknown. The effects of these drugs upon homeostasis may be important since both are known to stimulate the hypothalamic-pituitary-adrenal axis. The purpose of this experiment was to examine alterations in rats in corticosterone and glucose following an acute exposure to these drugs at different stages of development: preweaning, adolescence, and adulthood. Both MDMA and Foxy increased corticosterone levels significantly at all ages examined, while glucose was elevated at all stages except at the adolescent time point (postnatal day 28). For both measures, there were no differences between the sexes with either drug. The data indicate that an acute exposure to these drugs alters CORT and glucose levels at all ages tested, raising the possibility that these changes may have effects on behavioral and cognitive function, as we and others have previously demonstrated.

The possible effect of melatonin, 5-methoxytryptamine, 5-methoxytryptophol, 6-chloromelatonin and 2-iodomelatonin on testosterone production by Leydig cells in vitro was investigated. The ability of individual indoles to inhibit testosterone production was found to depend on the concentration used. The relative inhibitory potency of the compounds tested was: 6-chloromelatonin greater than 2-iodomelatonin greater than melatonin greater than 5-methoxytryptamine greater than 5-methoxytryptophol. The results revealed that natural indoles which are synthesized in the pineal gland and their halogenized derivatives are capable of influencing directly testosterone production by Leydig cells. Also, these results demonstrated that melatonin exerts its remarkable antigonadotrophic effects, at least in part, through the direct decrease of testosterone production. Moreover, 6-chloromelatonin and 2-iodomelatonin, which are reported to inhibit melatonin binding to target tissues, possess properties of biological melatonin analogues under the conditions of the model system used.

The possibility that the terminal serotonin (5-HT) autoreceptor in the rat hippocampus is coupled to Gi, Go or Gs regulatory proteins was investigated using the electrically evoked overflow of [3H]5-HT from preloaded slices. Pertussis toxin, which inactivates Gi/o or cholera toxin, which stimulates GS, was injected directly in the hippocampus 3 to 11 days prior to the experiments. Hippocampus slices were prepared, loaded with [3H]5-HT, superfused continuously, and stimulated electrically 72 min (S1) and 116 min (S2) after the beginning of superfusion. In the absence of any drug, the evoked overflow of [3H]5-HT in S1 was not altered by either toxin. The enhancing effect of the 5-HT reuptake blocker paroxetine (1 μmol/I) on the evoked [3H]5-HT overflow was also unaltered by these toxins. 5-Carboxyamidotryptamine, a 5-HT autoreceptor agonist, inhibited in a concentration-dependent manner the stimulation-evoked release of [3H]5-HT The concentration-effect curve (0.001–0.1 μmol/I) for this drug was not altered by pretreatment with either pertussis or cholera toxin. Similarly, the effect of another 5-HT autoreceptor agonist, 5-methoxytryptamine (0.1 and I μmol/I), was not altered in the pretreated rats. In addition, the reduction of [3H]5-HT overflow obtained by increasing the stimulation frequency from 1 Hz to 5 Hz, which is due to an increase in terminal 5-HT autoreceptor activation at the higher frequency, was not altered by either toxin. The enhancing effect of the 5-HT autoreceptor antagonist methiothepin (1 μmol/I) on stimulation-evoked [3H]5-HT overflow was not changed by either pretreatment. N-Ethylmaleimide inactivates Gi/o proteins by alkylation. Preincubation with 30 μmol/I N-ethylmaleimide for 30 min did not alter the efficacy of the 5-HT reuptake blocker paroxetine to enhance [3H]5-HT overflow nor did it alter the attenuating effect of 5-methoxytryptamine and the differential effectiveness of 1 and 5 Hz stimulations on the overflow of [3H]5-HT In conclusion, the results suggest that the terminal 5-HT autoreceptor in the rat hippocampus may not be coupled to Gi, Go or Gs proteins.

A liquid chromatographic analysis with electrochemical detection of 5-methoxytryptamine, 5-methoxytryptophol, 5-methoxyindoleacetic acid and melatonin is described. Optimal elution conditions were determined by studying several variables: pH, buffer salt, counter ion and organic modifier. Measurement of 5-methoxyindoles in the pineal gland and plasma of hamsters has been performed after extraction. This method is specific and sensitive, and enables detection of 5-methoxyindoles in a pool of two hamster pineal glands. This is also the first time that these three 5-methoxyindoles have been measured simultaneously in plasma.

Our previous studies with a tissue culture model of neuronal development have shown that the development of serotonin neurons is dependent, at least in part, on the stimulation of high affinity serotonin receptors. One receptor inhibits the outgrowth of neurons, while the other promotes it. The present study was therefore undertaken to replicate these findings in a whole animal model system. Pregnant Sprague-Dawley rats were treated from gestational day 12 until birth with 0.1, 1.0 or 3.0 mg/kg 5-methoxytryptamine (5-MT). The pups were assessed for serotonin outgrowth by the selective synaptosomal uptake of [ 3 H]serotonin at postnatal days 1, 15 and 30 (D1, D15, D30). In addition, the pups were tested behaviorally for the neonatal serotonin syndrome at D5 (induced by quipazine), spontaneous alternation and open field activity at day 15 and lick suppression at day 30. At 1.0 mg/kg, the terminal outgrowth of serotonin neurons was inhibited, while the highest dose, 3.0 mg/kg, showed stimulation of outgrowth. The highest dose caused behavioral alterations which had abated by 30 days, while the intermediate dose (1.0 mg/kg) showed behavioral changes throughout. Interestingly, the lowest dose, 0.1 mg/kg, showed changes in uptake only at D1 and behavioral changes only at later timepoints, principally at D30. This suggests that serotonin not only plays a role in regulating the development of the neurons which produce it, but that it may also play a role in neurochemical imprinting—that is, changes in behavior in the adult may be due to changes in neurochemistry during development, even though that neurochemistry may have been corrected by the time the animal becomes an adult.

Experiments with male rats were staged to study effectiveness of radioprotectors of two classes of chemical compounds (aminothiols--cystamine and indolyl alkylamines--mexamine and indralin) against high-energy protons (120 MeV) at a minimal absolutely lethal dose (10 Gy) and more than lethal doses (11.0-14.0 Gy). The best protective effect was provided by intraabdominal indralin at a dose of 75 mg/kg. However, this protective effect of indralin weakens with a dose rise and fades away with the dose rising to the absolutely lethal or more than lethal level (14.0 Gy). Investigations of the effectiveness of shielding different segments of the rat's body from high-energy protons (120 MeV) at more than lethal doses showed a substantial reduction and then full loss of the shielding protective action. Evaluation of the effectiveness of combined protection (radioprotectors plus shielding) against high-energy protons at more than lethal doses led to the conclusion about an additive (at 1.0-13.0 Gy) or potentiative (at 14.0 Gy) effect. For instance, indralin (75 mg/kg) and shielding of the abdomen increased rat's survivability to 89.7% after exposure at 11.0-13.0 Gy and to 87.5% after exposure at 14.0 Gy. It should be kept in mind that the radioprotective action of this combination was also observed during exposure to more than lethal doses when the protective effect of shielding and chemical compounds is minimal or lost altogether.

In the global cerebral ischemia, 5-hydroxytryptamine (serotonin, 5-HT) and 5-methoxytryptamine (5-MT) produce a significant neuroprotector effect closely correlated with their hypothermic action. This expands the spectrum of cerebral functions controlled by 5-HT. At the same time, melatonin and 2-iodmelatonin, which are chemically (but not pharmacologically) close to 5-HT and 5-MT, do not exhibit such protective effects. Probably, an important role in the interaction with 5-HT receptors belongs to the NH2 group of the indole ring.

Psychostimulants including amphetamines and cocaine, opioids including morphine, and some recreational drugs share the ability to cause drug dependence and addiction. Although these drugs of abuse primarily act on distinct molecular targets, such as monoamine transporters or receptors, they finally converge to common neural pathways. Several lines of evidence suggest that their chronic treatment leads to the enhancement of the mesocorticolimbic dopaminergic neurons from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and the medial prefrontal cortex (mPFC) and leads to abnormal glutamatergic function from the mPFC to the NAc and VTA. The neural adaptation of dopaminergic-glutamatergic system is considered to be critically implicated in neuropsychotoxic effects of these drugs of abuse. In addition, recent studies suggest that the serotonergic neurons from the raphe nuclei to limbic areas modulate the mesocorticolimbic dopaminergic-glutamatergic system and participate in the neuropsychotoxicity. In this review, our recent in vitro studies on the molecular targets and neural adaptation of methamphetamine, 3,4-methylenedioxymethanphetamine (MDMA, “ecstasy”), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT, “foxy”) using Xenopus oocytes, organotypic slice cultures of the mesocorticolimbic dopaminergic-glutamatergic system, and the raphe serotonergic system are introduced. Keywords:: drugs of abuse, methamphetamine, 3,4-methylenedioxymethanphetamine (MDMA, “ecstasy”), 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT, “foxy”), dopaminergic-glutamatergic system, serotonin transporter

The anxiolytic effect of diazepam (0.5, 1.0 and 2.0 mg/kg), buspirone (2.5 and 5.0 mg/kg), indorenate (2.5 and 5.0 mg/kg) and ipsapirone (5.0 and 10.0 mg/kg) was evaluated in male and female rats during the proestrus and metestrus phases. The burying behavior test was used to measure the anxiety levels. In this test, increases in the behavior latency are interpreted as prolonged reactivity, while reductions in the burying behavior are considered to reflect anxiolytic states. Diazepam increases in burying behavior latency were consistently higher than those observed after serotonergic anxiolytics. Buspirone, at no dose tested, affected the burying behavior latency, while indorenate and ipsapirone had only minor effects. Male individuals were more sensitive than females to the actions of diazepam on burying behavior. The serotonergic anxiolytics produce similar responses in both sexes. Metestrus females were much less sensitive to the action of all anxiolytics on burying behavior latency than proestrus females. Proestrus females were highly sensitive to the actions of diazepam on burying latency as compared both with males and metestrus females. Data show that a larger gender and within females variation occurs after treatment with diazepam as compared with the serotonergic anxiolytics. The results are discussed considering the relationships between ovarian hormones and the GABA-benzodiazepinic and serotonergic systems.

The systemic injection of diazepam (0.5 and 1.0 mg/kg), indorenate (5 and 10 mg/kg) and ipsapirone (2.5 and 5.0 mg/kg) reduced anxiety as tested in an exploratory avoidance model in mice. The injection of pindolol (2.0 mg/kg), alprenolol (5.0 mg/kg) or methiotepin (0.25 mg/kg) effectively prevented the anxiolytic action of indorenate and ipsapirone. The combined treatment of the antagonists with indorenate or ipsapirone did not reduce the motor activity, therefore suggesting that the inhibition of exploratory behaviour, after such combinations, was not mediated through a general motor impairment. Neither diazepam nor indorenate alone modified the motor activity; ipsapirone (5 and 2.5 mg/kg), however, reduced ambulation. This reduction was also prevented by administering pindolol, alprenolol or methiotepin. These observations suggest that the anxiolytic actions of indorenate and ipsapirone are mediated via stimulation of the 5-HT1A like receptor subtype.

The Harderian glands of Syrian hamsters contain melatonin and the enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) which synthesize melatonin from serotonin. Because the Harderian glands share this metabolic pathway with the pineal gland, we examined the effects of short-day photoperiods, which stimulate pineal-mediated gonadal regression, and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23), which has been described as a melatonin antagonist, on melatonin synthesis in the Harderian glands of the hamster. Harderian glands of male hamsters kept in short days had reduced NAT activity and melatonin concentration, but HIOMT activity was unchanged from that of longday controls. In males kept in short days, ML-23 restored melatonin concentrations to levels seen in long days but did not affect the short-day induced reduction in NAT activity. ML-23 had no effect upon NAT or HIOMT activity or melatonin concentration in male hamsters kept on long days. Harderian glands of female hamsters kept on short days had reduced melatonin concentrations, but NAT and HIOMT activities similar to those of long-day controls. ML-23 had no effect on Harderian NAT or HIOMT activities or melatonin concentration in females kept in short days. However, in females kept in long days, ML-23 treatment led to increased NAT activity and decreased melatonin concentrations. We conclude from these results that short-day photoperiods alter some aspects of melatonin synthesis in hamster Harderian glands and that these effects differ in males and females. ML-23 does not usually prevent the effects of short days on Harderian melatonin synthesis, suggesting that it is not a melatonin antagonist in the Syrian hamster.

Serotonin (5-HT) agonists, selective for 5-HT1, 5-HT2 or 5-HT3 receptor subtypes, were tested for their ability to mimic 5-HT in narrowing the tetraethylammonium-induced calcium-dependent plateau of action potentials recorded from frog sensory neurons. 5-Carboxamidotryptamine, 5-HT, alpha-methyl 5-HT and 5-methoxy-tryptamine possessed full agonist activity, with EC50S of 19 nM, 210 nM, 3.7 microM and 1.7 microM, respectively. 2-Methyl 5-HT was inactive. This agonist profile indicates that the calcium-dependent plateau in these sensory somata is modulated by a 5-HT1-like receptor.

The effect of late afternoon injections of melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-methoxyindole-3-acetic acid on testicular histology in mice were examined. Melatonin and 5-methoxytryptophol injections caused a reduction in the diameters of seminiferous tubules. The tests of melatonin-treated animals underwent some detectable regressive changes in the seminiferous tubules, whereas administration of 5-methoxytryptamine or 5-methoxytryptophol appeared to cause atrophy in some tubules. The percentage of aspermic tubules in melatonin-treated and methoxytryptamine-treated mice was significantly higher than that of the control. In involuted testes, some seminiferous tubules contained only Sertoli cells together with spermatogonia and spermatocytes, but no discernible spermatids and spermatozoa. Regressing spermatids and cell debris were frequently observed in the tubules. The tested of mice that received daily injections of 5-hydroxytryptophol and 5-methoxyindole-3-acetic acid were indistinguishable from those of the controls.

"Synaptic" ribbons and spherules are typical organelles of the mammalian pinealocytes, that present quantitative day-night variations parallel to pineal melatonin levels. The European hamster is a wild photoperiodic mammal whose pineal melatonin concentrations do not vary during the 24 hr light-dark cycle. The aim of this study was thus to see if a rhythmic variation in synaptic structures was nevertheless present in this species. It was found that, in contrast to all of the photoperiodic species studied to date, the European hamster pinealocyte contains no ribbons, and virtually no spherules. Moreover, constant administration of melatonin does not result in the occurrence of these organelles; nevertheless, the pinealocytes have the potentiality to produce spherules and, to a lesser extent, ribbons, as they are observed in ganglionectomized animals and in the deep pineal of some hamsters after ablation of the superficial part of the gland. The formation of pineal synaptic structures, normally lacking in the European hamster, thus seems directly related to the noradrenergic innervation of the gland.

The level of cyclic AMP in NCB-20 cells was increased by serotonin (5-HT), 5-methoxytryptamine and 2-methyl-5-HT with EC 50 of 0.5 ± 0.1, 1.0 ± 0.1, 10 ± 0.1 μ M, respectively. The 5-HT-mediated increase of cyclic AMP content was completely blocked by metergoline but unaffected by 5-HT 3 antagonists, ICS 205–930, MDL 72222, quipazine and 5-HT 2 antagonist, ketanserin. Putative 5-HT 1A agonists (8-OH-DPAT, ipsapirone, and buspirone) and 5-HT 1B agonists (TFMPP and m -CPP) affected neither basal nor forskolin-dependent cyclic AMP accumulation. Receptor binding studies suggest that NCB-20 cells are devoid of 5-HT 1A and 5-HT 1B receptor sites. Application of 5-HT onto NCB-20 cells resulted in membrane depolarization by an evoked inward current which displayed rapid desensitization. 5-HT-mediated current had a reversal potential around 0 mV and was potently and reversibly inhibited by ICS 205–930. Our data suggest that in NCB-20 cells the 5-HT 3 receptor is involved in the generation of inward currents, while the 5-HT receptor coupled to adenylate cyclase does not seem to correspond to any of the known receptor subtypes.

Circadian involvement in Parkinson's disease (PD) and more specifically in nigro-striatal dopamine (NSD) function is of increasing interest to the neurosciences. Given that bright light therapy is of therapeutic value in PD, possible mechanisms underlying retinal involvement in this phenomenon was explored further by administering anti-Parkinsonian chemotherapies into the vitreus humour directly adjacent to the retina. 2 microl of a 100 mM solution of L-Dopa significantly improved motor function in the later stages of degeneration and during the day while the injection of 2 microl of a 10 mM solution of the melatonin receptor antagonist ML-23 improved motor function in the early stages of PD and during the dark phase of the light/dark cycle. The results suggest that the function of nigral cells is regulated by a more global system embracing circadian physiology that extends from the retina to the pineal. Furthermore, the induction of PD is characterised by an imbalance between melatonin and dopamine (DA) whereby this ratio is elevated at least 6 to 1 in favour of melatonin. The commonly observed treatment failures and side effects of DA replacement therapy probably result from increasing endogenous DA without taking parallel melatonin dysfunction into account. The proposed integrated function of the NSD and circadian systems may permit therapeutic targeting at a level which is safer, more effective and without the side effects of systemically administered regimens of DA replacement.

Experiments with male mice were performed to evaluate comparative effectiveness of radioprotectors cystamine, aminoethyl isothiuronium, mexamine and indralin against minimal absolutely lethal gamma-doses (9 Gy). The best protective effect was demonstrated by indralin at a dose of 75 mg/kg. Supportive data were received in experiments with rats. The radioprotective action of indralin consists mainly in quite successful preservation of the blood-forming components, i.e. the pool of stem cells in the marrow and spleen. Gamma-irradiation at superlethal doses (10 Gy and higher) weakens significantly or fully neutralizes these protectors in rodents. Shielding of radiosensitive organs with the help of lead and plastics proved to be a good protection of animals from minimal lethal gamma-doses. However, the superlethal doses of gamma-irradiation penetrated the shielding materials and disabled them to a large and full extent. Evaluation of effectiveness of the combined protection against superlethal gamma-doses by pharmaceutical agents and shielding revealed a potentiating effect. For instance, mexamine and shielding of the abdomen together increased survivability of rats to 76.7%. An even stronger effect was noted when shielding was combined with indraline which raised survivability to 100%. It should be emphasized that this combination is effective against superlethal gamma-doses that usually unassailable to radioprotectors and shielding.

The oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377–85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with β-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.

The effect of 5-methoxytryptamine (5-HT(4) receptor agonist) on the inotropic function of atrial myocardium was studied in children aged 2 months to 17 years, operated on for congenital heart disease. Functional activity of 5-HT(4) receptors was 8.4 times higher in dysfunction of the atrial septum in comparison with other congenital heart diseases. The positive inotropic effects of 5-HT(4) receptor agonist can promote compensation of myocardial work in children with pathological circulation.

Serotonin is involved in a wide range of physiological and patho-physiological mechanisms. In particular, 5-HT1A receptors are proposed to mediate stress-adaptation. The aim of this research was to investigate in adolescent rats: first, the consequences of perinatal exposure to 5-metoxytryptamine (5MT), a 5-HT1/5-HT2 serotonergic agonist, on behavioural-stress reactivity in elevated plus maze, open field and forced swim tests; secondly, whether the behavioural effects induced by perinatal exposure to 5MT on open field and forced swim tests were affected by the selective 5-HT1A receptor agonist LY 228729, a compound able to elicit a characteristic set of motor behaviours on these experimental models, and by the co-administration of the selective and silent 5-HT1A antagonist WAY 100635. Results indicate that a single daily injection of 5MT to, pregnant dams from gestational days 12 to 21 (1 mg/kg s.c.), and to the pups from postnatal days 2 to 18 (0.5 mg kg s.c.), induce in the adolescent rat offspring: an increase in the percentage of entries and time spent on the open arms in the elevated plus maze; a reduction in locomotor activity and rearing frequency, and an increase in the time spent on the central areas in the open field test; a decrease in immobility and an increase in swimming in the forced swim test. Acute administration of LY 228729 (1.5 mg/kg s.c.) strongly decreases rearing frequency and increases peripheral activity in the open field test, and decreases immobility and increases swimming in the forced swim test both in perinatally vehicle and 5MT-exposed offspring. Co-administration of WAY 100635 (0.25 mg/kg s.c.) abolishes the effects exerted by LY 228729. These results suggest that, in the adolescent rat, perinatal exposure to 5MT enhances the stress-related adaptive behavioural responses, presumably through a predominant action on presynaptic 5-HT1A receptors and does not deteriorate the functional response of 5-HT1A receptors to selective agonist and antagonist compounds.

A reduction in 5-HT1A receptor response enhances learning and memory performance in rats. Pre- and postnatal treatment with 5-methoxytryptamine (5MT), a non-selective serotonergic agonist, and early handling, reduce the number of 5-HT1A receptors in neonatal and pre-pubertal rat progeny. The aim of this study was to investigate in adolescent male rats the consequences of pre- and postnatal treatment with 5MT and its interaction with early handling on an object-place association learning task, the "Can test", a motivated, non-aversive, spatial/object discrimination task. Results show that a single daily injection of 5MT from gestational days 12 to 21 (1 mg/kg s.c.) and from postnatal days 2 to 18 to pups (0.5 mg/kg s.c.), increases the level of activity and the number of correct responses, and decreases the number of reference memory errors in the progeny as adolescent, compared to vehicle-treated rats. Similar effects are observed following a daily, brief, maternal separation of the pups from postnatal days 2 until 21. Furthermore, when 5MT-treated rats underwent to early handling procedure, the effects induced by 5MT increased handling-induced facilitation of the object-place association. These results suggest that pre- and postnatal treatment with 5MT enhances learning in the "Can test", probably due to a reduction in 5-HT1A receptors in the hippocampus. Whether the potentiation exerted by pre- and postnatal 5MT on early handling effects may be related to a further damping of 5-HT1A receptor response is not yet assessed; however, our data demonstrate that this association is able to induce long-term facilitative effects on spatial learning performance in a non-aversive spatial/object discrimination task in the adolescent rat offspring.

The effect of indorenate (5-methoxytryptamine, β -methyl carboxylate hydrochloride), a 5-HT 1A agonist, was investigated on the motor performance of rats with chronic spinal cord injury. Four months after a ninth thoracic vertebrae spinal cord contusion, 29 rats were randomly allocated into two groups: saline solution and indorenate-treated animals with daily doses incremented at weekly intervals. The locomotor performance of all rats was measured by the Basso, Beattie, and Bresnahan (BBB) rating scale. The results showed that at the end of the treatment, the motor activity of indorenate group was significantly better than that presented by saline solution group. The 80% of indorenate, (against 15% of saline solution) did not show detriment on motor activity. When we analysed the motor activity of rats with basal BBB lower than 10, a significant improvement of motor recovery in indorenate-treated animals was observed. The benefits observed in locomotor function at low doses followed by increasing doses could be associated with pharmacological treatment by indorenate, a well-known 5-HT 1A receptor agonist. Our results suggest a potential mechanism by which serotonergic agents may improve motor function in rats with chronic spinal cord injury. Most serotoninergic innervations of the spinal cord in mam- mals descend from the brainstem. Serotonin in spinal cord is closely related to motor activity (1). It is positively related to the level of tonic motor activity, especially as manifested in antigravity muscles and other muscle groups associated with gross motor activity. Serotonergic neurones also participate in the regulation of the central pattern generator (CPG) for locomotion (2). After spinal cord injury, the damage and loss of tracts related to motor activities, including serotoninergic innervations, cause loss of supraspinal control of motor functions and specifically loss of voluntary locomotor activity. Even though subjects with incomplete spinal cord injury can exhibit residual neurological capacities, they still suffer from locomotor and postural deficits such as difficulties in bearing weight, maintaining balance and adapting their walking speed (3). Nevertheless, in the spinal cord the necessary circuitry to generate basic locomotion through the so-called CPG a spinal cord circuitry exists that is capable of generating the locomotor command of each mus- cle of the limbs, setting the rhythm of locomotor cycles, and insuring intralimb and interlimb muscular coordinations. Activation of CPG can be attained by supplying the sub- lesional cord with the missing monoaminergic agents either by pharmacotherapy or by implanting serotonergic precur- sors. Pharmacological therapy with 5-HT agonists can induce significant motor function recovery of rats with acute spinal cord injury (4,5). However, this therapy has not been tested yet in individuals with chronic spinal cord injuries. More- over, until now, there is no effective therapy for these stages of injury, and thereby it is imperative to evaluate the effect of this strategy on the chronic phase of spinal cord injury. In this work, we investigated the effect of indorenate, a 5-HT 1A agonist with antihypertensive activity (6,7) and a neuropro- tection of the central nervous system (8), on the motor performance of rats with chronic spinal cord injury.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a synthetic orally active hallucinogenic tryptamine derivative, known also as Foxy or Foxy methoxy. However, few studies have examined its effects in vitro. In the present study, we investigated the actions of 5-MeO-DIPT against monoamine neurotransmitter transporters, including the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), using COS-7 cells heterologously expressing these transporters and rat brain synaptosomes. 5-MeO-DIPT specifically inhibited the uptake of [3H]serotonin (5-HT) by the SERT-expressing COS-7 cells and rat striatal synaptosomes in a high affinity manner at concentrations similar to those for cocaine. The effect was reversible and competitive. 5-MeO-DIPT failed to stimulate reverse transport of [3H]5-HT through SERT, while it prevented the releasing action of methamphetamine. 5-MeO-DIPT induced cell toxicity at high concentrations in COS-7 cells, and it was not influenced by the expression of SERT. These results demonstrated that 5-MeO-DIPT acts as a competitive SERT inhibitor and has an inability to cause reverse transport, underlying its serotonergic actions.

Integration of genomic data from pharmacokinetic pathways and drug targets is an emerging trend in bioinformatics, but is there a clear separation of pharmacokinetic pathways and drug targets? Should we also consider the potential interactions of endogenous substrates of drug-metabolizing enzymes with receptors and other molecular drug targets as we combine pharmacogenomic datasets? We discuss these overarching questions through a specific pharmacogenomic case study of the cytochrome P450 (CYP)2D6, serotonin and dopamine triad. Importantly, CYP2D6 may contribute to the regeneration of serotonin from 5-methoxytryptamine by virtue of its catalytic function as a 5-methoxyindolethylamine O-demethylase. Furthermore, serotonergic neurons provide a regulatory feedback on dopaminergic neurotransmission. Hence, we hypothesize that independent of its role as a pharmacokinetic gene, CYP2D6 may nuance the regulation of serotonergic and dopaminergic neurophysiology. Additionally, we reflect upon the contribution of hyperspecialization in biomedicine to the present disconnect between research on pharmacokinetics and drug targets, and the potential for remedying this important gap through informed dialogue among clinical pharmacologists, human geneticists, bioethicists and applied social scientists.

Many drugs are used or abused in social contexts without understanding the ramifications of their use. In this study, we examined the effects of a newly popular drug, 5-methoxy-diisopropyltryptamine (5-MEO-DIPT; 'foxy' or 'foxy-methoxy'). Two experiments were performed. In the first, 5-MEO-DIPT (0, 10, or 20 mg/kg) was administered to rats four times on a single day and animals were examined 3 days later. The animals that received 5-MEO-DIPT demonstrated hypothermia during the period of drug administration and delayed mild hyperthermic rebound for at least 48 h. Corticosterone levels in plasma were elevated in a dose-dependent manner compared to saline-treated animals with minor changes in 5-HT turnover and no changes in monoamine levels. In experiment 2, rats were examined in behavioral tasks following either 0 or 20 mg/kg of 5-MEO-DIPT. The animals treated with 5-MEO-DIPT showed hypoactivity and an attenuated response to (+)-methamphetamine-induced stimulation (1 mg/kg). In a test of path integration (Cincinnati water maze), 5-MEO-DIPT-treated animals displayed deficits in performance compared to the saline-treated animals. No differences were noted in the ability of the animals to perform in the Morris water maze or on tests of novel object or place recognition. The data demonstrate that 5-MEO-DIPT alters the ability of an animal to perform certain cognitive tasks, while leaving others intact and disrupts the endocrine system. 5-MEO-DIPT may have the potential to induce untoward effects in humans.

The absorption and fluorescence spectral characteristics of some biologically active indoles have been studied as a function of acidity and basicity (H_/pH/H(o)) in cationic (cetyltrimethylammonium bromide, CTAB), anionic (sodium dodecylsulphate, SDS) and aqueous phases at a given surfactant concentration. The prototropic equilibrium reactions of these probes have been studied in aqueous and micellar phases and apparent excited state acidity constant (pK(a)(*)) values are calculated. The probes show formation of different species on changing pH. Various species present in water, CTAB and SDS have been identified and the equilibrium constants have been determined by Fluorimetric Titration method. The fluorescence spectral data suggest the formation of oxonium ion through the excited state proton transfer reaction in highly acidic media and formation of photoproducts due to the base catalyzed auto-oxidative reaction in basic aqueous solutions. Variations in the apparent pK(a)(*) value have been observed in different media. The change in the apparent pK(a) values depends upon the solubilising power of the micelles, as well as on the location of the protonating site in the molecule. The observation about increase in pK(a)(*) values in SDS and decrease in CTAB compared to pure water for various equilibria is consistent with the pseudophase ion-exchange (PIE) model.

Indorenate (5-methoxytryptamine-β-methylcarboxylate) is a 5-HT1A receptor agonist that produces antihypertensive, anxiolytic, antidepressant and anticonvulsant effects. However, there is evidence suggesting that these effects could involve the activation of benzodiazepine (BZD) receptors but not the activation of a1-adrenergic receptors. The goal of this study was to analyse the effect of indorenate on a1-adrenergic and BZD receptor binding in specific rat brain areas by using in-vitro autoradiography. Coronal brain sections from male Wistar rats were used for labelling 5-HT1A (3H-8-OH-DPAT, 2 nm), a1-adrenergic (3H-prazosin, 2 NM) and BZD (3H-flunitrazepam, 2 NM) receptor binding in the presence or absence of indorenate (1 μm). Indorenate totally displaced 3H-8-OH-DPAT binding in all the brain areas evaluated. It decreased 3H-prazosin binding just in the frontal (30%) and sensorimotor (32%) cortices and in the thalamus (21%). Additionally, indorenate diminished 3H-flunitrazepam binding only in the cingulate (10%) and piriform (18%) cortices as well as in the dorsal raphe nucleus (18%). These results confirm that indorenate is a 5-HT1A ligand and suggest the possible participation of a1-adrenergic and BZD receptors in its pharmacological properties.

Background Japanese men who have sex with men (MSM), especially those living in large metropolitan areas such as Tokyo and Osaka, are facing a growing HIV/AIDS epidemic. Although the Internet is used as a new venue for meeting sex partners, it can also serve as a useful research tool for investigating the risk behaviours of Japanese MSM. This Internet survey explored the extent of substance use and its association with sexual risk behaviours among Japanese MSM. Methods Between 28 February 2003 and 16 May 2003 MSM were recruited through 57 Japanese gay-oriented Web sites, gay magazines, and Internet mailing lists. Participants completed a structured questionnaire anonymously through the Internet. Results In total, 2,062 Japanese MSM completed the questionnaire. The average age of participants was 29.0 years and 70.5% identified as gay, 20.8% as bisexual, and 8.7% as other. Overall, 34.5% reported never using a substance, 45% reported ever using one type of substance (lifetime reported single substance users), and 19.6% had used more than 1 type of substance (lifetime reported multiple substance users) in their lifetimes. The substances most commonly used were amyl nitrite (63.2%), 5-methoxy-N, N-diisopropyltryptamine (5MEO-DIPT) (9.3%), and marijuana (5.7%). In the multivariate analysis, unprotected anal intercourse, having had 6 or more sexual partners, visiting a sex club/gay venue in the previous 6 months, a lower education level, and being 30 to 39 years of age were associated with both lifetime single and lifetime multiple substance use. Lifetime reported multiple substance use was also correlated with having a casual sex partner, having symptoms of depression, being diagnosed as HIV-positive, and greater HIV/AIDS-related knowledge. Conclusion This is the first Internet-based research focused on the sexual and substance use behaviours of MSM in Asia. Our findings suggest a compelling need for prevention interventions to reduce HIV risk-related substance use behaviours among Japanese MSM. The results also suggest that the Internet is potentially a useful tool for collecting behavioural data and promoting prevention interventions among this population.

Several experiments have demonstrated that pineal gland plays a physiological anticancer role. Melatonin (MLT), its most investigated hormone, is a natural anticancer agent. However, MLT would not be the only endocrine molecule responsible for the anticancer property of the pineal gland. In fact, another pineal indole hormone, the 5-methoxytryptamine (5-MTT), has appeared to exert in vitro an antitumour activity superior to that of MLT itself. Previous studies have already shown the therapeutic anticancer action of MLT in association with chemotherapy also in human neoplasms. This study was performed to evaluate the influence of 5-MTT at physiological doses (1 mg/day orally during light phase) on the efficacy of chemotherapy with cisplatin plus etoposide in advanced non-small cell lung cancer patients with respect to that obtained in patients treated by chemotherapy alone or chemotherapy plus pharmacological doses of MLT (20 mg/day orally during the dark phase of the day). The study included 100 patients, who were randomised to receive chemotherapy alone or in association with MLT or 5-MTT. The overall response rate achieved in both patients concomitantly treated with MLT or 5-MTT was significantly higher with respect to that obtained in patients treated with chemotherapy alone. Moreover, both MLT and 5-MTT significantly reduced some chemotherapy-related toxicities, namely thrombocytopenia and neurotoxicity. This preliminary study shows that less known pineal hormone 5-MTT may exert at low doses the same anticancer therapeutic effect in association with chemotherapy, which may be obtained by pharmacological doses of the most investigated pineal hormone MLT.

Lipids, including cholesterol, are critical components of the cell membrane where they are enriched in microdomains, lipid rafts, which organize and concentrate receptors and intracellular proteins involved in signal transduction. The present study examined the effects of cholesterol depletion on serotonin (5-HT) binding and signaling via 5-hydroxytryptamine(7) (5-HT(7)) receptors in stably transfected HeLa cells. Immunohistochemical, ligand-binding and biotinylation experiments demonstrated that the studied cells expressed high levels of 5-HT(7) receptors at their surface with a pharmacological profile resembling 5-HT(7) receptors in native tissue. Depletion of cholesterol, by combined treatment with mevastatin, fumonisin B(1) and mevalonate or methyl-beta-cyclodextrin (MbetaCD), caused highly significant reductions in the B(max) values of [(3)H]5-HT- and [(3)H]-(R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol ([(3)H]SB269970)-binding to 5-HT(7) receptors. Cholesterol depletion also reduced the total level of 5-HT(7) receptor protein determined by Western blot analysis. None of the examined treatments affected the affinity of [(3)H]5-HT- or [(3)H]SB269970-binding to 5-HT(7) receptors. Treatment with serotonin caused strong inductions in the phosphorylation states of Ser(63)-ATF-1 and Ser(133)-CREB. These effects of serotonin on signal transduction were significantly counteracted by pre-treatment with cholesterol synthesis inhibitors. Altogether, the present study demonstrates that cholesterol depletion decreases binding of both agonist and antagonist radioligands to 5-HT(7) receptors and counteract 5-HT(7) receptor-mediated intracellular signaling.

A new Schiff base of gossypol with 5-methoxytryptamine (GSTR) and a new hydrazone of gossypol with dansylhydrazine (GHDH) have been synthesized and studied by Fourier transform infrared (FTIR), 1H and 13C nuclear magnetic resonance (NMR), ultraviolet-visible (UV-VIS), electrospray ionization–mass spectroscopy (ESI-MS) as well as the parametric method PM5. The spectroscopic methods have provided clear evidence that GSTR exists in chloroform solution as an enamine–enamine tautomer, whereas GHDH is present in chloroform as a N-imine–N-imine tautomer. The fluorescence spectra of both compounds indicate that their quantum yield of fluorescence is increased by one or two orders of magnitude compared to that of pure gossypol. The ESI-MS spectra of the 1:1 mixtures of GSTR or GHDH with formic acid have demonstrated that both compounds exist as protonated monomers in the gas phase, whereas GHDH can also exist in a stable protonated dimeric structure. The structures of the stable tautomers are calculated and visualized using the PM5 semiempirical method. The intra- and intermolecular hydrogen bonds within these structures are discussed. © 2006 Wiley Periodicals, Inc. Biopolymers 82: 521–535, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Contemporary theory regarding the cause and treatment of neuropsychiatric disease strongly suggests that as the human body ages it gradually loses the intrinsic safeguards that protect it from oxidative damage. Melatonin is one hormone that serves this function in that it possesses antioxidative properties in the mammalian body and brain. Melatonin has been shown to prevent the progressive degeneration produced by neurotoxins employed in experimental models to mimic the degenerative events in various neuropsychiatric disease states. There are an abundance of models for numerous disease states demonstrating that melatonin can inhibit oxidative stress and by such a mechanism it is presumed to exert a therapeutic effect. While a similar scenario has been revealed with in vitro work relating specifically to Parkinson's disease, clinical work with melatonin in this disorder demonstrates that it is devoid of any remarkable therapeutic effects. More recent preclinical and clinical work has reliably demonstrated that melatonin in fact may be without therapeutic efficacy and may even worsen the condition. On this pretense, attempts to reduce the bioavailability of melatonin using a melatonin receptor antagonist have been found to completely restore behavioral and regulatory function in the presence of chronically reduced levels of dopamine, without producing side effects commonly seen with traditional dopamine replacement therapy. The unavoidable conclusion from this work suggests that within the dynamic framework of the mammalian brain, hormones may play a duel, and possibly ambivalent, role in homeostasis and in the etiology of disease. Such a position requires a reevaluation of the etiology, the role of dopamine, the neurochemical characteristics of Parkinson's disease and the validity of the models employed to study this and other neuropsychiatric disorders.

In vitro quantitative studies of the oxidative metabolism of (5-methoxy-N,N-diisopropyltryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) metabolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics. Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-deisopropylase activities. The apparent Km value of CYP2D6 was close to that for 5-MeO-DIPT O-demethylation, and the Km values of other CYP enzymes were similar to those of the low-Km (CYP2C19), intermediate-Km (CYP1A2, CYP2C8 and CYP3A4) and high-Km phases (CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition studies, quinidine (1 microM), an inhibitor of CYP2D6, almost completely inhibited human liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10 microM. Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9 inhibitor and ketoconazole, a CYP3A4 inihibitor (5 microM each) suppressed about 60%, 45%, 15% and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50 microM substrate. In contrast, omeprazole (10 microM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by human liver microsomes, whereas at the same concentration the inhibitor suppressed the reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-MeO-DIPT N-deisopropylase enzymes in the human liver.

To clarify the role of diluents in the preparation of molecularly imprinted polymers utilizing only hydrogen bonding, we investigated the effects of diluents by using different solvents. Melatonin (N-acetyl-5-methoxytryptamine), an amide bond and indole ring-containing hormone was chosen as the target molecule. N-Propionyl-5-methoxytryptamine was used as the pseudo template, methacrylic acid as the functional monomer, and solvents were used as diluents. Interactions between the template, the functional monomer, melatonin, and the solvents, were observed by 1H NMR spectroscopy. The polymers were evaluated by high-performance liquid chromatography. The results suggest the hydrogen bonding-acceptor capacity of the solvent is the most important factor in the preparation of molecularly imprinted polymers for hydrogen bonding-donating molecules. Hydrogen bonding between the template, the functional monomer, and solvent can be estimated from the chemical shifts in 1H NMR spectra of those molecules in the solvent.

This study describes the diel rhythms in several indoleamines, melatonin, and related 5-methoxyindoles in the pineal organ of rainbow trout in vivo. In addition, the effect of different photoperiod conditions was evaluated. Melatonin levels displayed clear daily rhythms in the pineal organ of rainbow trout kept experimentally under long (LD 16:08), neutral (LD 12:12), and short (LD 08:16) photoperiods. Duration of melatonin signal was dependent on the night length of prevailing photoperiod, while peak amplitude was higher when lengthening the photoperiod. Significant daily rhythms in 5-HT content, the precursor of melatonin synthesis, were found in neutral and short photoperiod with increases of the amine content just after the light–dark interphase and decreases in the middle of the night, which were more important under short photoperiod. In contrast, no significant 24-h cyclic variation was found in pineal 5-HT content under long photoperiod. Daily profiles in the content of the main 5-HT oxidative metabolite, the 5-hydroxyindoleacetic acid (5-HIAA), outlined those of the amine precursor. The chronograms of both aminergic compounds contrast with those of 5-hydroxytryptophan content, which displayed a net tendency to increase at night. This study also provides evidence for the existence of daily cyclic changes in the content of 5-methoxytryptamine (5-MT), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptophol (5-MTOL) in trout pineal organ, which were also dependent on photoperiod. The 24-h profiles in 5-MT content correlated well with those of 5-HT, showing a peak at the first hour of darkness in all photoperiodic conditions, and a decay at midnight only in both neutral and long photoperiods. Similarly, the content of 5-MTOL also displayed high values during the day–night transition in trout kept under neutral and long photoperiods, followed by a slow decay all along the night. Finally, levels of 5-MIAA increased in all photoperiods when lights were turned off, being this nocturnal increase maximal in fish kept under LD 16:08. These results suggest that light–dark cycle modulates daily rhythms in pineal indoles and non-melatonin 5-methoxyindoles by acting mainly through the melatonin synthesis activity, which limits the availability of 5-HT for the oxidative and direct methylation pathways. In addition, it seems that a nocturnally increased synthesis of 5-HT might be a requirement for the optimal formation of melatonin and other 5-methoxyindoles in the pineal organ when trout remain under short photoperiods.

Brain serotonin 5-HT(7) receptors are known to be expressed in neurons and astrocytes. We now report the presence of these receptors in a third type of cell, microglial cells. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human microglial MC-3 cell line. The maximal effect of 5-HT was 3.4+/-0.3-fold stimulation (mean+/-S.E.M., n=5) above basal levels. The rank order of agonist potency (pEC50 values) was 5-CT (7.09)>5-HT (6.13)>or=5-MeOT (5.78)>>8-OH-DPAT (ca. 5). The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT7 receptor antagonist SB-269970 (pA2 value 9.03). Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT7 receptor in extracts of MC-3 cells. The presence of two splice variants of the 5-HT7 receptor (5-HT7(a/b)) was visualized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis with specific primers. In real-time PCR studies, the mRNA for interleukin-6 (IL-6) was found to be increased by 2.5-fold in MC-3 cells after 1 h incubation with 5-CT (1 microM) and this effect was fully blocked by the 5-HT7 receptor antagonist SB-269970 (1 microM). These data show that functional 5-HT7 receptors are present in human microglial MC-3 cells, suggesting that they are involved in neuroinflammatory processes.

The effects of two serotonergic (5-HT1A) receptor agonists (8-OH-DPAT; 0.01, 0.1, 0.3, 1 mg/kg, s.c., and Indorenate; 1, 3, 10 mg/kg, i.p.) were evaluated in three type of seizures in male Wistar rats: clonic-tonic convulsions induced by pentylenetetrazol (PTZ, 60 mg/kg, i.p.), status epilepticus (SE) of limbic seizures produced by kainic acid (KA, 10 mg/kg, i.p.) and tonic-clonic seizures by amygdala kindling. 8-OH-DPAT decreased the incidence of tonic seizures and the mortality rate induced by PTZ. Indorenate increased the latency to the PTZ-induced seizures and decreased the percentage of rats showing tonic extension and death. Concerning KA, 8-OH-DPAT augmented the latency and reduced the frequency of wet-dog shake (WDS) and generalized seizure (GS). At high doses it diminished the occurrence and delayed the establishment of SE. Indorenate augmented the latency to WDS, GS and SE, and diminished the number of GS. 8-OH-DPAT and Indorenate did not alter the expression of kindled seizures. However, Indorenate enhanced the refractoriness to subsequent seizures during the postictal depression. Some effects induced by 8-OH-DPAT and Indorenate on seizures evaluated and postictal depression were fully or partially blocked by WAY100635. These results suggest that 5-HT1A receptor agonists modify epileptic activity depending on the type of seizure.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a new psychoactive tryptamine derivative, has been synthesised by the Speeter and Anthony procedure. This synthetic route was characterised by ESI-MS-MS, ESI-TOF-MS and NMR. Side products have been identified as 3-(2-N,N-diisopropylamino-ethyl)-1H-indol-5-ol (5), 2-N,N-diisopropylamino-1-(5-methoxy-1H-indol-3-yl)-ethanol (6), 2-(5-methoxy-1H-indol-3-yl)-ethanol (7) and 2-N,N-diisopropylamino-1-(5-methoxy-1H-indol-3-yl)-ethanone (8).

Serotonin 5-HT(7) receptors are present in astrocytes. Understanding their role in this type of cell would greatly benefit from the identification of astroglial cell lines expressing this receptor type. The aim of the present study was to assess the expression of native 5-HT(7) receptors and 5-HT(7) receptor mRNA in a number of human glioblastoma cell lines, by means of cAMP measurements, Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human glioblastoma cell lines, U-373 MG, U-138 MG, U-87 MG, DBTRG-05MG, T98G, H4, CCF-STTG1 and Hs 683. The rank order of potency was 5-CT5-HT=5-MeOT8-OH-DPAT. The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT(7) receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89-1.13) and pA(2) values of 8.69-9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT(7) receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT(7) receptor (5-HT(7(a/b/d))) was visualized by RT-PCR analysis with specific primers in all human glioblastoma cell lines. In conclusion, human glioblastoma cell lines express functional 5-HT(7) receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT(7) receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines.

The hypothesis explored in this review is that the high levels of serotonin in the blood seen in some autistic children (the so-called hyperserotonemia of autism) may lead to some of the behavioral and cellular changes also observed in the disorder. At early stages of development, when the blood-brain Barrier is not yet fully formed, the high levels of serotonin in the blood can enter the brain of a developing fetus and cause loss of serotonin terminals through a known negative feedback function of serotonin during development. The loss of serotonin innervation persists throughout subsequent development and the symptoms of autism appear. A review of the basic scientific literature on prenatal treatments affecting serotonin is given, in support of this hypothesis, with an emphasis on studies using the serotonin agonist, 5-methoxytryptamine (5-MT). In work using 5-MT to mimic hyperserotonemia, Sprague-Dawley rats are treated from gestational day 12 until postnatal 20. In published reports, these animals have been found to have a significant loss of serotonin terminals, decreased metabolic activity in cortex, changes in columnar development in cortex, changes in serotonin receptors, and "autistic-like" behaviors. In preliminary cellular findings given in this review, the animals have also been found to have cellular changes in two relevant brain regions: 1. Central nucleus of the amygdala, a brain region involved in fear-responding, where an increase in calcitonin gene related peptide (CGRP) was found 2. Paraventricular nucleus of the hypothalamus, a brain region involved in social memory and bonding, where a decrease in oxytocin was found. Both of these cellular changes could result from loss of serotonin innervation, possibly due to loss of terminal outgrowth from the same cells of the raphe nuclei. Thus, increased serotonergic activity during development could damage neurocircuitry involved in emotional responding to social stressors and may have relevance to the symptoms of autism.

A new mechanism has been recently proposed, whereby melatonin may participate in the ongoing process of neuronal degeneration in models of neurodegenerative disorders, such as Parkinson's disease (PD). Antagonism of the melatonin receptor in rats using constant light or pinealectomy induced recovery and reduced the mortality typically associated with dopamine (DA) degeneration. In additional studies, employing ML-23 in the 6-OHDA-treated rat, remission from experimental PD was achieved using this drug in a post 6-OHDA treatment regime. To permit the further assessment of ML-23 as a potential clinical candidate for the treatment of PD, the present study was undertaken to determine the efficacy of ML-23 in the 1-methyl-4-phenyl, 1,2,3,6 tetrahydropyridine (MPTP) model in the common marmoset. ML-23 was administered orally in a dose of 3 mg/kg twice daily to half of the animals, while the other half received vehicle only, in a blinded protocol, for 56 days. The effects of the treatment on positive and negative features of MPTP-induced PD were assessed, including horizontal and vertical movement, head checking, general behaviour and Parkinsonian condition, raisin board performance, the ability to remove a foot label, palatable and dry food intake, water consumption, bradykinaesia, and the positive symptoms of tremor, obstinate progression, and agitation. On all parameters, ML-23 produced a significant remission from MPTP-induced Parkinsonism, and this effect did not abate when ML-23 treatment was withdrawn. In a further pilot study involving a crossover of two animals, one animal treated previously with MPTP plus vehicle showed some remission of negative and positive features, although ML-23 treatment was not commenced until 8 weeks post-MPTP. Conversely, a recurrence of Parkinsonian signs was not observed when ML-23 treatment was withdrawn and substituted with oral vehicle. Dopamine transporter was severely impaired in all marmosets treated with ML-23 or vehicle for the duration of the study. These results suggest that a novel mechanism involving melatonin is involved in the primary aetiology of the chronic aspects of PD, and such a mechanism is not related to the antioxidative function of this hormone. From these preliminary results, it is concluded that ML-23 and other melatonin analogues have an important role to play in the treatment and clinical management of Parkinson's disease.

Animal models for neuropsychiatric disorders are implemented for the purpose of investigating a single or multiple aspects of a specific disease entity. In Parkinson's disease (PD) several models have been utilised to study the biochemical and behavioural consequences of dopamine (DA) neurone degeneration with the intent of further understanding the aetiology of this disease and improving its treatment. While the bilateral 6-hydroxydopamine (6-OHDA) model has been used to produce a broad spectrum of neurochemical and behavioural deficits characterising DA degeneration in humans, this model is traumatic, labour intensive and is associated with high mortality due to the acute effects of the neurotoxin. Consequently, the unilateral 6-OHDA model was developed and implemented. In this model damage to the ascending DA system is produced on one side of the brain thereby inducing postural rotation. This movement is exaggerated by activating the remaining DA systems with apomorphine or amphetamine thereby making it more quantifiable. In view of the less traumatic effects on homeostasis and relative ease with which this model can be implemented it has been used routinely for the purpose of screening potential anti-Parkinsonian drugs for clinical use. However, like any model, the use of the unilateral rotation model has its limitations. It is proposed that the process of exaggerating DA function using this paradigm limits the discovery of potential anti-PD drugs to those which are effective in counteracting an exaggerated DA response. This factor may account for the high incidence of unwanted side effects including involuntary movement, tardive dyskinaesia (TD) and psychosis which are commonly observed in DA replacement therapy. Secondly, this approach limits potential drug candidates to those acting exclusively on brain DA systems. This too is a problem in the sense that PD is known to be a disease involving numerous systems in the human brain and potential therapies acting via other neurochemical systems are being excluded when this model is used exclusively. The object of the present paper is to report the discovery of a non-DA drug possessing potent anti-Parkinsonian qualities which were revealed using the bilateral 6-OHDA model of PD as a screening tool. When the same drug was retested in the traditional unilateral screening model no effect was observed, while more advanced models confirmed its efficacy. These results illustrate that the implementation of appropriate models for revealing new treatment strategies for PD should be broadly based so that single treatment entities are not exclusively pursued for diseases whose aetiologies are multifaceted. Premature extrapolation of findings from a single, early stage model to its clinical counterpart can be detrimental to advancing new treatment strategies, induce false hope, and increase morbidity in PD patients.

1. The main aim of this investigation was to delineate the distribution of the 5-HT(7) receptor in human brain. Autoradiographic studies in guinea-pig and rat brain were also carried out in order to revisit and compare the anatomical distribution of 5-HT(7) receptors in different mammalian species. 2. Binding studies were performed in rat frontal cortex membranes using 10 nm [(3)H]mesulergine in the presence of raclopride (10 microm) and DOI (0.8 microm). Under these conditions, a binding site with pharmacological characteristics consistent with those of the 5-HT(7) receptors was identified (rank order of binding affinity values: 5-CT5-HT5-MeOTmesulergine approximately methiothepin8-OH-DPAT=spiperone approximately (+)-butaclamolimipramine approximately (+/-)-pindololondansetron approximately clonidine approximately prazosin). 3. The autoradiographic studies revealed that the anatomical distribution of 5-HT(7) receptors throughout the human brain was heterogenous. High densities were found over the caudate and putamen nuclei, the pyramidal layer of the CA2 field of the hippocampus, the centromedial thalamic nucleus, and the dorsal raphe nucleus. The inner layer of the frontal cortex, the dentate gyrus of the hippocampus, the subthalamic nucleus and superior colliculus, among others, presented intermediate concentrations of 5-HT(7) receptors. A similar brain anatomical distribution of 5-HT(7) receptors was observed in all three mammalian species studied. 4. By using [(3)H]mesulergine, we have mapped for the first time the anatomical distribution of 5-HT(7) receptors in the human brain, overcoming the limitations previously found in radiometric studies with other radioligands, and also revisiting the distribution in guinea-pig and rat brain.

We tested the expression of genes coding receptors of a cutaneous serotoninergic/melatoninergic system in whole human skin and in normal and pathologic cultured skin cells. Evaluation of serotonin (5HT), melatonin (MT), and melatonin-related receptors (MRR) showed expression of the isoforms 5HT2B, 5HT7, and MT1 genes in almost all the tested samples. Expression of other isoforms was less prevalent; 5HT2C, MRR, and MT2 were rarely detected. We also found novel isoforms for MT2, MRR, and 5HT2B and documented the process of RNA editing for 5HT2C. Testing for functional activity of these receptors with serotonin and melatonin (10(-14) to 10(-10) M) showed variable effects depending on cell type and culture conditions. Thus, serotonin stimulated proliferation of melanocytes in medium deprived of growth factors, while inhibiting cell growth in the presence of growth factors. Melatonin inhibited both apoptosis of HaCaT keratinocytes incubated in serum-free media, and proliferation of cells cultured in medium supplemented with serum. Melatonin also increased the numbers of viable fibroblasts incubated in serum free medium. N-acetylserotonin (NAS) and 5 methoxytryptamine (5MTT) were generally without effect on cell proliferation, with the exception of an inhibition of melanocyte proliferation at the higher 5MTT concentration of 10(-10) M. Thus, skin cells represent a true target for the products of the serotoninergic/melatoninergic cutaneous pathway with their actions modulating cell proliferation or viability.

5-Methoxytryptamine (5-MT) is a potent antioxidant and has radioprotective action. N-acetyl-5-methoxytryptamine (melatonin, NA-5-MT) is a free radical scavenger and antioxidant, which protects against oxidative damage due to a variety of toxicants. The infrared spectra of 5-MT, NA-5-MT and new synthesized N-phenylsulfonamide-5-methoxytryptamine (PS-5-MT) were investigated in the region between 4000 and 400 cm−1. Vibrational assignments of the molecules have been made for fundamental modes on the basis of the group vibrational concept, infrared intensity and comparison with the assignments for related molecules. X-ray powder diffraction patterns of molecules were also recorded. In order to optimize the geometries of the molecules, molecular mechanic calculations (MM3) were performed. Conformational analysis of 5-MT, NA-5-MT and PS-5-MT was also established by the using PM3 method.

Polymorphic cytochrome P450 2D6 (CYP2D6) is expressed in several types of central neurons but its function in human brain is currently unknown. Using recombinant enzymes and CYP2D6-transgenic mice, we established that 5-methoxytryptamine (5-MT), a metabolite and precursor of melatonin, is a specific and high-turnover endogenous substrate of CYP2D6. This potent serotonergic neuromodulator in numerous physiological systems binds tightly to recombinant CYP2D6 enzyme with an equilibrium dissociation constant (K(s)) of 23.4 micromol/l, and is O-demethylated to serotonin (5-hydroxytryptamine, 5-HT) with a high turnover of 51.7 min(-1) and low Michaelis-Menten constant of 19.5 micromol/l. The production of 5-HT from 5-MT catalyzed by CYP2D6 was inhibited by selective serotonin reuptake inhibitors, and their inhibition potency (K(i), micromol/l) decreased in the order of fluoxetine (0.411)norfluoxetine (1.38)fluvoxamine (10.1)citalopram (10.9). Liver microsomes prepared from CYP2D6-transgenic mice showed about 16-fold higher 5-MT O-demethylase activity than that from wild-type mice. After the intravenous co-administration of 5-MT (10 mg/kg) and pargyline (20 mg/kg), serum 5-HT level was about 3-fold higher in CYP2D6-transgenic mice than wild-type mice. When dosed with alpha,alpha,beta,beta-d -5-MT, alpha,alpha,beta,beta-d4-5-HT was detected in transgenic mouse serum, and its content was much higher than wild-type mouse. alpha,alpha,beta,beta-d4-5-HT was not produced in CYP2D6-transgenic mice pretreated with quinidine. The regeneration of 5-HT from 5-MT provides the missing link in the serotonin-melatonin cycle. Up to 10% of the population lacks this enzyme. It is proposed that this common inborn error in 5-MT O-demethylation to serotonin influences a range of neurophysiologic and pathophysiologic events.

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.

Indorenate (5-methoxytryptamine beta-methylcarboxylate, INDO) is a serotonin (5-hydroxytryptamine, 5-HT) agonist that has affinity for 5-HT(1A/1B/2C) receptors. Unlike other anxiolytics such as 5-HT receptor agonists, INDO may not share tolerance or dependency with the benzodiazepine anxiolytics. It has been reported that the discriminative stimulus properties of 5-HT(1A/1B/2C) agonists, but not those of 5-HT(3/4) agonists, generalize to INDO. Therefore, the aim of the present study was to obtain further evidence on the differential involvement of 5-HT(1A/1B/2C) receptors in the discriminative stimulus properties of INDO by evaluating its interactions with antagonists of the 5-HT(1A), 5-HT(1B), 5-HT(2C), and 5-HT(3/4) receptor subtypes. Rats were trained to discriminate INDO from saline in a conditioned taste aversion paradigm. For Group D(+)S(-), administration of INDO signalled that saccharin flavour was followed by LiCl, while injection of vehicle signalled safe consumption of saccharin solution. Group D(-)S(+) had the contingencies reversed. After this training, rats had generalization tests where INDO administration was preceded by different doses of the following antagonists: WAY100635 (5-HT(1A)), NAN190 (5-HT(1A)), methiothepin (5-HT(1A/1B/2C)), GR127935 (5-HT(1B/1D)), ketanserin (5-HT(2A/2C)), ritanserin (5-HT(2C/2A)), mesulergine (5-HT(2C/2A)), metergoline (5-HT(2C/2A)), SB206553 (5-HT(2B/2C)), and tropisetron (5-HT(3/4)). In Group D(+)S(-), the order of potency to block the discriminative stimulus properties of INDO was WAY100635>ketanserin>ritanserin>GR127935>mesulergine congruent with SB206553>metergoline>methiothepin>NAN190, while in Group D(-)S(+), the order was WAY100635>GR127935>ketanserin>ritanserin>mesulergine congruent with SB206553>metergoline>methiothepin>NAN190. Tropisetron did not produce any alteration of the discriminative control by INDO. These results suggest that the discriminative signal of INDO is mediated by 5-HT(1A/2C/1B) receptors and that blockade of any of its components produces a degradation of its discriminative effects.