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1521-009X/44/3/445–452$25.00 http://dx.doi.org/10.1124/dmd.115.067413 AND DISPOSITION Drug Metab Dispos 44:445–452, March 2016 Copyright ª 2016 by The American Society for and Experimental Therapeutics Supports CYP2D-Mediated Serotonin Synthesis in the

Anna Haduch, Ewa Bromek, Jacek Wójcikowski, Krystyna Gołembiowska, and Władysława A. Daniel

Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland

Received September 25, 2015; accepted January 7, 2016

ABSTRACT Melatonin is used in the therapy of and disorders and as (a monoaminoxidase inhibitor), the effect of melatonin was not a neuroprotective agent. The aim of our study was to demonstrate visible in the majority of the brain structures studied but could be that melatonin supported (via its deacetylation to 5-methoxytrypt- seen in all of them in 5,7-dihydroxytryptamine–lesioned animals Downloaded from ) CYP2D-mediated synthesis of serotonin from 5-methoxy- when serotonin storage and synthesis via a classic . We measured serotonin content in some brain pathway was diminished. Melatonin alone did not significantly regions (the cortex, , , striatum, increase extracellular serotonin concentration in the striatum of thalamus, , brain stem, , and cere- naïve rats but raised its content in -pretreated animals bellum) (model A), as well as its extracellular concentration in the (model B). The CYP2D inhibitor propafenone given intrastructurally striatum using an in vivo (model B) after melatonin prevented the melatonin-induced increase in striatal serotonin in dmd.aspetjournals.org (100 mg/kg i.p.) to male Wistar rats. Melatonin increased those animals. The obtained results indicate that melatonin supports the tissue concentration of serotonin in the brain structures stud- CYP2D-catalyzed serotonin synthesis from 5-methoxytryptamine in ied of naïve, sham-operated, or neurotoxin the brain in vivo, which closes the serotonin-melatonin-serotonin (5,7-dihydroxytryptamine)–lesioned rats (model A). Intracerebroven- biochemical cycle. The metabolism of exogenous melatonin to the tricular quinine (a CYP2D inhibitor) prevented the melatonin-induced serotonin may be regarded as a newly recognized increase in serotonin concentration. In the presence of pargyline additional component of its pharmacological action. at ASPET Journals on September 28, 2021

Introduction melatonin synthesis in them. Extrapineal melatonin seems not to be Melatonin is used in the treatment of some sleep disorders, including engaged in the regulation of the photoperiod (Acuña-Castroviejo et al., concurrent sleep disturbances in the course of different psychiatric 2014); however, together with pineal melatonin, extrapineal melatonin diseases such as and major depressive and seasonal protects cells against damage from oxidative due to its antioxidant affective disorders (Dolberg et al., 1998; Dalton et al., 2000; Shamir and anti-inflammatory activity. Moreover, melatonin exerts an anti- et al., 2000; Singh and Jadhav, 2014). Moreover, high doses of excitotoxic effect in the brain by reducing glutamate activity and melatonin are recommended for neuroprotection (Venegas et al., increasing that of GABA and it stimulates neurogenesis (Pandi-Perumal 2012; Acuña-Castroviejo et al., 2014). et al., 2008; Acuña-Castroviejo et al., 2014; Singh and Jadhav, 2014). The synthesis of endogenous melatonin in the is Being an amphiphilic substance, peripheral melatonin easily crosses the – governed by the light/dark cycle, which controls circadian and blood brain barrier (Green et al., 1975). Circulating melatonin is circannual rhythms. However, melatonin can also be produced in a metabolized mainly in the by isoforms of the substantial amount in extrapineal organs, including the gastrointestinal CYP1A subfamily (CYP1A1/A2/B1) to form 6-hydroxymelatonin and tract (in enterochromaffin cells), where it is not controlled by the then 6-sulfatoxymelatonin (Ma et al., 2005; Hardeland, 2010), but it can photoperiod (Pandi-Perumal et al., 2008; Venegas et al., 2012; Acuña- also be deacetylated to 5-methoxytryptamine (5-MT) (Rogawski et al., Castroviejo et al., 2014). The synthesis of melatonin from tryptophan 1979; Beck and Jonsson, 1981). via serotonin is catalyzed by N-acetyltransferase and hydroxyindole-O- In the brain, melatonin deacetylation to 5-MT seems to be of minor methyltransferase, these two being present in a variety of importance; both melatonin and 5-MT are formed mainly in the pineal organs/tissues such as the heart, liver, leukocytes, or the brain (e.g., the gland from which they are released to the circulation or to the third cerebral cortex and striatum), which also suggests the occurrence of ventricle. However, 5-MT formed from gut-derived melatonin in the liver crosses the blood–brain barrier (Beck and Jonsson, 1981; Acuña- Castroviejo et al., 2014) and, together with the brain-derived 5-MT, This research was supported by the European Union and the Polish Ministry of provides a direct substrate for CYP2D to form serotonin. Thus, the gut- Science and Higher Education [Grant DeMeTer POIG.01.01.02-12-004/09] and by derived or the exogenously supplied melatonin that provides most of the Polish Academy of Sciences Institute of Pharmacology [Statutory Funds]. the 5-MT for the organism may support serotonin formation by a dx.doi.org/10.1124/dmd.115.067413. CYP2D-mediated alternative pathway in vivo.

ABBREVIATIONS: 5,7-DHT, 5,7-dihydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; 5-MT, 5-methoxytryptamine; aCSF, artificial ; DA, ; DMSO, dimethylsulfoxide; DRN, dorsal ; HPLC, high-performance liquid chromatography; MAO, monoaminoxidase; MRN, median raphe nuclei; NA, noradrenaline.

445 446 Haduch et al.

Recent studies have shown that, apart from the classic pathway of Materials and Methods serotonin formation from L-5-hydroxytryptophan, serotonin may also Chemicals be synthesized via CYP2D-catalyzed O-demethylation of 5-MT. Melatonin (hydrochloride), pargyline (hydrochloride), quinine (hydrochlo- Such a way of serotonin synthesis has been demonstrated for ride), propafenone (hydrochloride), serotonin (5-hydroxytryptamine or 5-HT; and rat cDNA-expressed CYP2D isoforms, as well as for brain hydrochloride) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopa- microsomes (Yu et al., 2003; Haduch et al., 2013). Moreover, using a mine (DA), noradrenaline (NA), 5,7-DHT (a creatinine sulfate salt), and ascorbic microdialysis model, the formation of serotonin via this alternative acid were purchased from Sigma-Aldrich (St. Louis, MO). Ketamine hydro- pathway has recently been shown to function in the brain in vivo chloride (Ketamine) and xylazine hydrochloride (Sedazin) came from Biowet (Haduch et al., 2015). (Puławy, Poland). All organic reagents were of high-performance liquid The aim of this study was to demonstrate that exogenous melatonin chromatography (HPLC) grade and were supplied by Merck (Darmstadt, supported (via deacetylation to 5-MT) CYP2D-mediated serotonin Germany). synthesis from 5-MT in the rat brain in vivo (Fig. 1) by measuring tissue or extracellular serotonin concentration after intraperitoneal Animals melatonin injection. Serotonin tissue content was measured in brain All experimental procedures were carried out in accordance with the National regions containing CYP2D and serotonergic innervation (model A). Institutes of Health Guide for the Care and Use of Laboratory Animals and were The experiment was carried out with naïve and 5,7-dihydroxytryptamine approved by the Bioethics Commission at the Polish Academy of Sciences (5,7-DHT)–pretreated animals in the absence/presence of the mono- Institute of Pharmacology, as compliant with the Polish law. The study was Downloaded from amine oxidase (MAO) inhibitor pargyline. Intraperitoneal pargyline conducted on male Wistar-Han rats (Charles River Laboratories, Sulzfeld, – was applied to prevent 5-MT (successively formed from melatonin Germany) weighing 300 325 g. The animals were housed in rooms with a controlled temperature and humidity on a 12-hour light/dark cycle and had free in the liver) and serotonin (formed from 5-MT in the brain) from access to tap water and standard laboratory food during the study. theoxidationbyMAOandthustopotentiate the effect of melatonin on the serotonin level in the brain (Prozialek and Vogel, 1978; The Tissue Levels of Serotonin in the Brain after Intraperitoneal Suzuki et al., 1981; Galzin and Langer, 1986; Raynaud and Pévet,

Melatonin Administration (Ex Vivo Measurement, Model A) dmd.aspetjournals.org 1991). Intracerebral 5,7-DHT (a neurotoxin specific to serotonergic ) was used to decrease the production of serotonin via a To study the supportive effect of exogenous melatonin on the alternative classic pathway from tryptophan and serotonin storage in neuronal pathway of serotonin synthesis from 5-MT in a brain tissue, melatonin was injected intraperitoneally (100 mg/kg i.p.) to naïve and 5,7-DHT–lesioned rats. terminals of the brain (Rogawski et al., 1979; Beck and Jonsson, The rats were anesthetized with ketamine HCl (75 mg/kg i.p.) and xylazine 1981) and, consequently, to enhance serotonin derived from the HCl (10 mg/kg i.p.) and were then placed in stereotaxic apparatus (David Kopf peripherally formed 5-MT (from melatonin), as well as to demon- Instruments, Tujunga, CA). All solutions were freshly prepared on the day of strate the role of cytochrome P450 present in neurons/terminals in experimentation. 5,7-DHT was dissolved in 0.9% NaCl with 0.05% ascorbic serotonin formation from 5-MT via an alternative pathway. The acid and was injected into the dorsal raphe nuclei (DRN) and median raphe at ASPET Journals on September 28, 2021 extracellular, functional concentration of serotonin in the striatum nuclei (MRN) at a concentration of 10 mg/ml(1ml, infused at a rate of 1 ml/min) (involved in motor functions) was measured in the absence/ into both raphe nuclei. The following coordinates were used (Paxinos and presence of pargyline using an in vivo microdialysis (model B). Watson, 2007): AP (anterior-posterior), –7.9; L (lateral), 0.0 from the bregma; The animals also received a CYP2D inhibitor to ascertain whether and V (ventral), –7.9 (MRN), –5.9 (DRN) from the surface of the dura. The CYP2D was engaged in the synthesis of serotonin from melatonin needle stayed in place for 5 minutes after injection before it was slowly withdrawn. Sham-operated animals were subjected to the same procedure as 5,7- via 5-MT O-demethylation. DHT–treated animals, but they received a vehicle (a 0.9% NaCl plus 0.05% ascorbic acid) instead of 5,7-DHT. Ten days after injection of 5,7-DHT (or vehicle), the rats received melatonin (100 mg/kg i.p.), an indirect exogenous substrate for serotonin synthesis and/or pargyline (75 mg/kg i.p., 30 minutes before melatonin) to prevent the melatonin-produced 5-MT and serotonin (formed from 5-MT) from MAO oxidation. Because of its insolubility in water, melatonin was dissolved in a 30% dimethylsulfoxide (DMSO). All were given according to the following schedule: 5,7-DHT or vehicle (raphe nuclei) → after 10 days, pargyline i.p. → after 30 minutes, melatonin i.p. or 30% DMSO → after 1 hour, decapitation → tissue serotonin (DA, NA). The rats were divided into 12 experimental groups (Table 1). Groups of nonoperated rats were as follows: control, 30% DMSO (2 ml/kg i.p.); melatonin (100 mg/kg i.p.); pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); and pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.). Groups of sham- operated rats were as follows: vehicle (1 ml, into the raphe nuclei) plus 30% DMSO (2 ml/kg i.p.); vehicle (1 ml, into the raphe nuclei) plus melatonin (100 mg/kg i.p.); vehicle (1 ml, into the raphe nuclei) plus pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); and vehicle (1 ml, into the raphe nuclei) plus pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.). Groups of operated (5,7-DHT–lesioned) rats were as follows: 5,7-DHT (1 ml, into the raphe nuclei) plus 30% DMSO (2 ml/kg i.p.); 5,7-DHT (1 ml, into the raphe nuclei) plus melatonin (100 mg/kg i.p.); 5,7-DHT (1 ml, into the raphe nuclei) plus pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); and 5,7-DHT (1 ml, into the raphe nuclei) plus pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.). Moreover, an additional group of naïve rats received bilateral intracerebro- m m Fig. 1. Metabolism of exogenous melatonin via deacetylation to 5-MT in the ventricular injections of the CYP2D inhibitor quinine (150 g/5 l i.c.v.), liver and subsequent O-demethylation to serotonin in the brain (the investigated melatonin (100 mg/kg i.p.), or quinine (30 minutes before melatonin) plus pathway). melatonin. The CYP2D competitive inhibitor quinine (Kobayashi et al., 1989) Melatonin Supports Serotonin Formation by Brain CYP2D 447

was used in an appropriate intracerebral dose to produce an -specific !!! !! !!! !!! !! !! ! !

! micromolar concentration of the inhibitor in the brain (Bromek et al., 2010; 3.3 56.6 38.7 113.2 32.4 35.3 20.9 61.0 133.0 25.2 Haduch et al., 2015). Such an application of quinine allowed us to ascertain 6 6 6 6 6 6 6 6 6 6 whether CYP2D was engaged in the indirect synthesis of serotonin from PARG + MEL 43.7 melatonin (via O-demethylation of the melatonin-formed 5-MT). The rats 946.9 403.7 896.9 722.6 540.8 474.0 1525.0 were anesthetized with ketamine HCl (75 mg/kg i.p.) and xylazine HCl 0.05 (versus lesion plus

$$$ $$ $$$ $$$ $ $$$ $$$ (10 mg/kg i.p.) and were then placed in the Kopf stereotaxic apparatus. The , $ 0.01 (versus sham-operated P

! coordinates based on the Paxinos and Watson (2007) atlas were as follows: 2.8 45.0 21.431.5 854.1 15.8 27.9 69.1 20.5 176.4 12.5 53.3 , P 6 6 6 6 6 6 6 6 6 6 AP, –0.8; L, 61.5 from the bregma; and V, –3.5 from the dura. The freshly PARG ++ prepared quinine at a concentration of 30 mg/ml was administered (5 ml, 30.0 574.3 715.0 505.8 643.4 406.0 1063.0 infused at a rate of 1 ml/min) into both lateral ventricles (150 mg per Lesioned Rats $

$ $$$ $$$ $$$ $$ ventricle). The needle was left in place for 5 minutes after injection before its $$$ slow withdrawal. Control rats (sham-operated animals) were subjected to the 26.5 77.4 45.0 21.7 280.9 19.6 52.854.2 374.7 11.7 24.1 5.1 103.8 6 6 6 6 6 6 6 6 6 6 MEL same procedure as the quinine-treated group, except that they received a vehicle (0.9% NaCl) instead of quinine. The placement of the needle was 58.4 542.0 242.6 678.3 320.5 952.7 234.4 histologically verified in 10 rats in a preliminary experiment and was then 0.001 (versus lesioned animals);

+ +++ +++ + +++ +++ checked in two animals from each experimental group. , +++ s test. ’ P All drugs were given according to the following schedule: quinine i.c.v. or 15.625.5 550.4 25.3 1.1 52.5 21.7 10.0 38.9 7.9 38.2 28.7 612.0 0.05 (versus sham-operated rats); lesioned rats (model A) – $$$ , 6 6 6 6 6 6 6 6 6 6 → → vehicle after 30 minutes, melatonin i.p. or 30% DMSO after 1 hour, Downloaded from P + decapitation → tissue serotonin. 161.7 305.4 411.2 The rats were divided into four experimental groups (Fig. 2) as follows: ^ ^ ^ control, vehicle (5 ml i.c.v.) plus 30% DMSO (2 ml/kg i.p.); quinine (150 mg/5 Operated Rats 8.2 14.0 15.322.0 185.9 33.5 30.4 27.5 41.1 236.3 53.2 129.6 25.9 381.9 46.4 261.0 163.2 ml i.c.v.) plus 30% DMSO (2 ml/kg i.p.); vehicle (5 ml i.c.v.) plus melatonin 6 6 6 6 6 6 6 6 6 6 (100 mg/kg i.p.); and quinine (150 mg/5 ml i.c.v.) plus melatonin (100 mg/kg i.p.). 58.8 PARG + MEL Lesion One hour after melatonin injection, the rats were decapitated and their 526.1 428.1 834.5 908.4 897.0 624.7 1035.0 1111.8 2026.0 were removed. The brains were cut into the , hypothalamus, dmd.aspetjournals.org 0.01 (versus lesioned animals);

+++ thalamus, nucleus accumbens, striatum, hippocampus, frontal cortex, the rest +++ +++ +++ +++ +++ +++ +++ +++ , +++ of cortex, brain stem, and medulla oblongata. Brain tissues were frozen on dry P 2.8 47.3 17.2 19.8 64.7 23.6 55.9 17.1 168.1 78.9 $$ ice and stored at –80C until they were further analyzed. 6 6 6 6 6 6 6 6 6 6 PARG 0.001 (versus PARG). Operated rats: Melatonin and the other pharmacological substances applied were , 58.9 P 915.3 523.3 413.8 623.0 773.3 962.0 546.6 administered at the same time of a day to rats coming from all of the 1266.4 1640.0 ### experimental groups. Consequently, all experimental procedures involving +++

+ ++ +++ +++ +++ +++ + +++ control groups and groups treated with melatonin were carried out at the same +++ pg/mg of tissue Sham-Operated Rats 5,7-DHT – at ASPET Journals on September 28, 2021 Serotonin Tissue Level time of a day (between 10:00 AM and 4:00 PM). 46.1 31.4 134.8 67.1 53.3 20.6 42.6 31.2 51.1 6.7 6 6 6 6 6 6 6 6 6 6 MEL

TABLE 1 Extracellular Concentrations of Serotonin in the Striatum after

0.05 (versus lesioned animals); Intraperitoneal Administration of Melatonin: An In Vivo Microdialysis 0.01 (versus PARG);

, (Model B) , P $ P 56.8 918.0 20.1 685.1 43.3 1045.8 23.9 821.5 26.7 397.7 22.720.7 400.6 467.4 19.9 739.7 69.5 1306.1 2.3 72.1

## Microdialysis guides were implanted 7 days before melatonin (100 mg/kg 6 6 6 6 6 6 6 6 6 6 i.p.) administration. The rats were anesthetized with ketamine (75 mg/kg i.m.)

368.8 303.5 256.5 and xylazine (10 mg/kg i.m.) and were then placed in stereotaxic apparatus (David Kopf Instruments). Vertical microdialysis guides (Bioanalytical ### ## ### Systems Inc., West Lafayette, IN) were implanted in the striatum using the 47.8 564.2 44.8 506.6 18.040.6 305.3 50.1 252.6 48.6 61.4 417.8 151.520.7 990.7 4.6 21.8 following coordinates (Paxinos and Watson, 2007): AP, +1.2; L, +3.0 from 6 6 6 6 6 6 6 6 6 6

0.001 (versus control); the bregma; and V, –3.2 from the surface of the dura. PARG + MEL Sham , On day 7 after the surgery, brain microdialysis probes (membrane P

0.05 (versus sham plus PARG); polyacrylonitrile, 320 mm OD, 4 mm long; Bioanalytical Systems Inc.) were ,

P placed inside the guides implanted in the striatum and were connected to the Univentor 802 syringe pump (Agn Tho’s AB, Lidingö, Sweden), which 55.7*** 646.2 82.7*** 1257.0 21.5***32.4*** 570.5 35.5*** 536.2 55.93** 1136.0 66.16*** 759.3 938.9 100.3*** 2091.0 27.2***4.6*** 683.7 81.7 0.001 (versus lesion plus PARG group). 6 6 6 6 6 6 6 6 6 6

PARG delivered an artificial cerebrospinal fluid (aCSF) consisting of 145 mM NaCl, , 2.7 mM KCl, 1.0 mM MgCl2, and 1.2 mM CaCl2, pH 7.4, at a flow rate of P

!!! 2.0 ml/min. 8). Statistical significance was assessed by a two-way (nonoperated rats) or three-way (operated rats) analysis of variance and Fisher 0.01 (versus control); *** – Four baseline samples were collected from freely moving rats at 20-minute Nonoperated Rats , intervals after a 165-minute washout period. Then, the appropriate drugs, P 64.8* 673.8 30.7 1127.0 24.8*18.2**54.6*** 523.9 555.7 21.0 794.0 28.5* 581.7 28.5 922.6 25.8**6.6 1901.0 438.8 80.6 propafenone (50 mM given using microdialysis probes) and/or melatonin 6 6 6 6 6 6 6 6 6 6 MEL (100 mg/kg i.p.), were administered to naïve or pargyline-treated rats

S.E.M. (n = 5 (pargyline 75 mg/kg i.p., 60 minutes before melatonin). The applied effective The effect of melatonin (100 mg/kg i.p.) on serotonin tissue level in the brain structures of naïve, sham-operated, and 5,7-DHT 6 doses of melatonin and the other pharmacological substances were chosen on the basis of the results of our preliminary experiments (data not shown). In 33.9 545.6 34.4 637.2 25.07.3 338.1 15.0 295.9 19.6 753.7 13.7 417.1 516.4 42.6 1124.0 20.21.8 378.8 50.1 6 6 6 6 6 6 6 6 6 6 that experimental set, intracerebral propafenone (added to the aCSF) was Control 0.05 (versus control); ** used instead of quinine as a specific and competitive CYP2D inhibitor (Xu , 370.8

P et al., 1995; Zhou et al., 2013) to show the efficacy of another enzyme 0.01 (versus lesion plus PARG group);

, inhibitor in preventing melatonin effect. Moreover, in our preliminary P

!! experiment, propafenone was effective at a concentration 10 times lower 0.001 versus sham-operated rats (versus sham-operated rats); ^ m m

, than quinine (50 M versus 500 M), being thus also easier to dissolve in the

P aCSF. Dialysate fractions after melatonin injection were collected throughout +++

The data are expressed as the mean MEL, melatonin; PARG, pargyline. Nonoperated rats: * 180 minutes. All drugs were given according to the following schedule: Hypothalamus 339.7 Frontal cortex 563.8 Brain Structure Rest of cortexHippocampusNucleus accumbensStriatum 394.2 259.0 191.8 ThalamusBrain stemMedulla oblongataCerebellum 353.6 280.9 950.9 39.4 PARG group); rats); microdialysis guide (in the striatum) → after 7 days, pargyline i.p. → after 60 448 Haduch et al. Downloaded from

Fig. 2. Model A. The effect of intracerebral quinine administration on the melatonin-induced (100 mg/kg i.p.) increase in 5-HT tissue content in the following brain structures: frontal cortex (A), rest of the cortex (B), hippocampus (C), nucleus accumbens (D),

striatum (E), thalamus (F), hypothalamus (G), brain dmd.aspetjournals.org stem (H), medulla oblongata (I), and cerebellum (J). The data are expressed as the mean 6 S.E.M. (n =5–8). Statistical significance was assessed by a two-way analysis of variance and Fisher’stest.*P , 0.05 (versus control); **P , 0.01 (versus control); ***P , 0.001 (versus control); #P , 0.05 (versus melatonin); ##P , 0.01(versus melatonin); ###P , 0.001 (versus melatonin). The control values (in picograms per milligram of tissue) were as follows: 465.50 6 20.39 (frontal cortex), 352.08 6 17.42 (rest of the cortex), at ASPET Journals on September 28, 2021 270.26 6 7.94 (hippocampus), 424.01 6 24.26 (nucleus accumbens), 247.19 6 23.29 (striatum), 577.54 6 30.39 (thalamus), 459.33 6 34.76 (hypo- thalamus), 704.81 6 25.53 (brain stem), 560.21 6 18.68 (medulla oblongata), and 45.83 6 3.59 (cerebel- lum). MEL, melatonin; QUIN, quinine.

minutes, propafenone into the striatum and/or melatonin i.p. → in vivo plus melatonin (100 mg/kg i.p.); and pargyline (75 mg/kg i.p.) plus propafenone extracellular serotonin (dopamine). (50 mM) plus melatonin (100 mg/kg i.p.) The rats were divided into six experimental groups (Figs. 3 and 4): control, 30% At the end of the experiment, the rats were euthanized and their brains were DMSO (2 ml/kg i.p.); pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); histologically examined to validate probe placement. saline (2 mg/kg i.p.) plus melatonin (100 mg/kg i.p.); pargyline (75 mg/kg i.p.) plus Brain microdialysis procedures were carried out in rats from all of the propafenone (50 mM) plus 30% DMSO (2 ml/kg i.p.); pargyline (75 mg/kg i.p.) experimental groups at the same time of a day (between 10:00 AM and 6:00 PM). Melatonin Supports Serotonin Formation by Brain CYP2D 449

column (3 mm, 100 Â 3 mm; Thermo Scientific, Waltham, MA). The mobile

phase contained 0.1 M KH2PO4, 0.5 mM Na2EDTA, 80 mg/l sodium 1-octanesulfonate, and 4% methanol and was adjusted to pH 3.7 with 85%

H3PO4. The flow rate of the eluent was 0.6 ml/min. The potential of a 3-mm glassy carbon electrode was set at 0.7 V and a sensitivity of 5 nA/V. The column temperature was maintained at 30C. The Chromax 2007 program (Pol- Laboratory, Warsaw, Poland) was applied for the collection and analysis of data. Neurotransmitter concentrations in the dialysate were measured using a

composition of the mobile phase: 0.1 M KH2PO4, 0.5 mM Na2EDTA, 16 mg/l sodium 1-octanesulfonate, and 2% methanol, adjusted to pH 3.6 with 85%

H3PO4. The flow rate of the eluent was 0.7 ml/min.

Data Analysis An average neurotransmitter concentration of four stable samples of the dialysate fraction prior to drug administration was regarded as a basal value (100%). The data were statistically analyzed using two-way (nonoperated rats) or three-way (operated rats) analysis of variance (model A), followed by Fisher’s least significant differences post hoc test, or by a repeated-measures analysis of Fig. 3. Model B. The melatonin-produced increase in extracellular serotonin variance (model B), followed by Tukey’s post hoc test (Program Origin 7.5 or Downloaded from concentration in the striatum is prevented by the CYP2D inhibitor propafenone in pargyline-pretreated rats. Drugs were given as indicated with an arrow. The obtained Statistica 9, respectively; StatSoft Inc., Tulsa, OK). The results were considered , values are the mean 6 S.E.M. (n =5–8). A repeated-measures analysis of variance to be statistically significant when P 0.05. showed effect of treatment (F = 5.25, P = 0), effect of time (F = 11.27, P = 0), and interaction between both factors (F = 55.27, P = 0). *P , 0.001 (P = 0.000135; versus PARG); ^P , 0.001 (P = 0.00014; versus pargyline plus melatonin); !P , 0.1 Results (P = 0.065; versus pargyline plus propafenone) (repeated-measures analysis of variance and Tukey’s post hoc test). MEL, melatonin; PARG, pargyline; PFN, The Tissue Levels of Serotonin in the Brain after Intraperitoneal dmd.aspetjournals.org propafenone. Administration of Melatonin (Ex Vivo Measurement, Model A) Intact Rats. Melatonin and the applied pharmacological substances Determination of Brain (the MAO inhibitor pargyline and the serotonergic neurotoxin 5,7- The tissue concentrations of 5-HT, DA, NA, and 5-HIAA were measured DHT) affected the tissue concentration of serotonin in the brain; this using HPLC with electrochemical detection, according to the method of Haduch effect was structure dependent (Table 1). Melatonin (100 mg/kg i.p.) et al. (2015). Briefly, brain structures were homogenized in 20 volumes (v/w) of significantly increased serotonin content in the rest of the cortex ice-cold 0.1 M HClO2 and were centrifuged at 15,000 Â g for 15 minutes at 4C. (131%), hippocampus (154%), nucleus accumbens (191%), thalamus at ASPET Journals on September 28, 2021 The obtained supernatant (5 ml) was injected into the HPLC system. An external (146%), hypothalamus (161%), and medulla oblongata (135%) com- standard containing NA, DA, 5-HT, and 5-HIAA at concentrations of 50 ng/ml pared with the control (Table 1). A similar tendency was observed in (or 2.5 ng/ml for the dialysate) was used. The chromatography system comprised other structures. The MAO inhibitor pargyline (75 mg/kg i.p.) an LC-4C amperometric detector with a cross-flow detector cell (Bioanalytical potently increased serotonin concentration in all of the investigated Systems Inc.) as well as a 626 Alltech pump and a Hypersil Gold analytical brain structures, on average up to 207% of the control (frontal cortex, 200%; rest of the cortex, 202%; hippocampus, 290%; nucleus accumbens, 201%; striatum, 157%; thalamus, 261%; hypothalamus, 198%; brain stem, 200%; medulla oblongata, 156%; and cerebellum, 205%). However, melatonin administered to pargyline-pretreated rats did not elevate the serotonin concentration in most of the brain structures studied, except for the nucleus accumbens, striatum, and medulla oblongata (an increase up to 143%, 131%, and 156% of the control, respectively) (Table 1). Sham-Operated Rats. Melatonin (100 mg/kg i.p.) significantly elevated serotonin content in all of the brain structures studied in sham- operated rats: the frontal cortex (135%), the rest of the cortex (131%), hippocampus (185%), nucleus accumbens (284%), striatum (271%), thalamus (177%), hypothalamus (163%), brain stem (132%), medulla oblongata (155%), and cerebellum (331%) (Table 1). Pargyline potently increased tissue serotonin content in the brain structures of sham-operated rats, on average up to 209% (frontal cortex, 181%; rest of the cortex, 171%; hippocampus, 164%; nucleus accumbens, 344%; striatum, 205%; thalamus, 185%; hypothalamus, 171%; brain stem, Fig. 4. Model B. The melatonin-produced increase in extracellular dopamine 166%; medulla oblongata, 213%; and cerebellum, 270%) (Table 1). concentration in the striatum is prevented by the CYP2D inhibitor propafenone in Melatonin administered to pargyline-pretreated sham-operated rats pargyline-pretreated rats. Drugs were given as indicated with an arrow. The obtained values are the mean 6 S.E.M. (n =5–8). A repeated-measures analysis of variance significantly elevated serotonin concentration up to approximately showed effect of treatment (F = 5.25, P = 0), effect of time (F = 11.27, P = 0), and 134% in the striatum, 117% in the thalamus, and 124% in the brain stem interaction between both factors (F = 55.27, P = 0). #P , 0.001 (P = 0.00014; versus compared with pargyline-pretreated animals (Table 1). , ^ , control); *P 0.001 (P = 0, 00014; versus pargyline); P 0.001 (P = 0.00014; 5,7-DHT–Lesioned Rats. The neurotoxin 5,7-DHT (10 mg/1 ml), versus pargyline plus melatonin); !P , 0.1 (P = 0.053; versus pargyline plus propafenone) (repeated-measures analysis of variance and Tukey’s post hoc test). injected into the DRN and MRN, decreased tissue serotonin concen- MEL, melatonin; PARG, pargyline; PFN, propafenone. tration in all of the brain structures studied, on average down to 54% of 450 Haduch et al. the sham-operated animals (frontal cortex, 75%; rest of the cortex, 61%; concentration in pargyline-pretreated rats. Pargyline alone raised the hippocampus, 13%; nucleus accumbens, 71%; striatum, 53%; thala- extracellular dopamine concentration up to approximately 450% of the mus, 73%; hypothalamus, 42%; brain stem, 42%; medulla oblongata, basal level. Propafenone did not significantly affect the extracellular 51%; and cerebellum, 64%) (Table 1). The lesion was specific, since the concentration of dopamine in pargyline-pretreated animals (Fig. 4). concentrations of the catecholaminergic neurotransmitters NA and DA The basal extracellular level of serotonin in the striatum of naïve rats were not affected (data not shown). Melatonin (100 mg/kg i.p.) was 0.938 6 0.069 pg/10 ml, whereas that of dopamine equalled 7.5 6 significantly enhanced the serotonin level in the brain structures of 0.7 pg/10 ml and did not differ between the experimental groups. 5,7-DHT–lesioned rats: frontal cortex (142%), nucleus accumbens (234%), striatum (340%), thalamus (222%), brain stem (232%), medulla oblongata (181%), and cerebellum (375%) (Table 1). A similar Discussion tendency was observed in the hippocampus (174%), hypothalamus Serotonergic projections from the raphe nuclei of the brain stem (136%), and rest of the cortex (130%). Pargyline markedly increased innervate almost all of the brain structures that control important serotonin level in the brain structures of 5,7-DHT–lesioned rats, on physiologic functions, including the hypothalamus (food intake, average up to 227% of the neurotoxin-lesioned animals (frontal , and ), basal ganglia (motor func- cortex, 187%; rest of the cortex, 151%; hippocampus, 231%; nucleus tions), thalamus (sleep and epilepsy), hippocampus (stress, and accumbens, 194%; striatum, 355%; thalamus, 211%; hypothalamus, memory), and cortex (sleep and mood) (Törk, 1990; Di Giovanni et al., 159%; brain stem, 259%; medulla oblongata, 313%; and cerebellum, 2008). Recent studies revealed that, apart from the classic pathway of Downloaded from 214%). Melatonin administered to the lesioned rats pretreated with serotonin formation from L-5-hydroxytryptophan, serotonin may also pargyline elevated the serotonin concentration in all of the brain be synthesized in the brain via CYP2D-catalyzed O-demethylation of structures tested, on average up to 150% of the pargyline-pretreated 5-MT. Serotonin that was formed in that alternative way was shown in lesioned rats (frontal cortex, 132%; rest of the cortex, 144%; vitro in microsomes derived from different brain structures (Haduch hippocampus, 228%; nucleus accumbens, 169%; striatum, 156%; et al., 2013) and was found to function in vivo when we measured its thalamus, 112%; hypothalamus, 144%; brain stem, 143%; medulla tissue and extracellular concentrations in selected structures of the brain oblongata, 117%; and cerebellum, 146%). after intracerebral administration of 5-MT (Haduch et al., 2015). dmd.aspetjournals.org Effect of Quinine. In another experimental set, the specific CYP2D Our study provides further evidence for the role of cytochrome P450 inhibitor quinine, given to both lateral ventricles of the brain (150 mg/5 (CYP2D) in serotonin synthesis in the brain in vivo by showing that ml i.c.v.) 30 minutes before melatonin (100 mg/kg i.p.), prevented the exogenous melatonin administered intraperitoneally supports (via melatonin-evoked elevation in tissue serotonin content in the following deacetylation to 5-MT) serotonin formation by cytochrome P450. To brain structures of naïve rats: the rest of the cortex, hippocampus, observe serotonin formation from melatonin in vivo, in our experiment striatum, thalamus, hypothalamus, brain stem, medulla oblongata, and we used its relatively high dose (100 mg/kg i.p.), which is pharmaco- cerebellum (Fig. 2). Such an effect of quinine was not observed in the logically acceptable. Such high doses of melatonin (up to 200 mg/kg a at ASPET Journals on September 28, 2021 frontal cortex and nucleus accumbens. Quinine alone did not affect the day) were shown to have low toxicity (Barchas et al., 1967; Jahnke tissue content of serotonin. et al., 1999; Acuña-Castroviejo et al., 2014) and were used for psychopharmacological behavioral tests in animals (Papp et al., 2003, 2006). Furthermore, high doses of melatonin are recommended because Extracellular Concentrations of Serotonin and DA in the Striatum of its protective properties as an antioxidant in saturating its in- after Intraperitoneal Administration of Melatonin: An In Vivo tracellular therapeutic targets (Venegas et al., 2012; Acuña-Castroviejo Microdialysis (Model B) et al., 2014). On the other hand, the rate of is much Melatonin (100 mg/kg i.p.) did not significantly affect the extracel- faster in rats (rodents) than in , so higher doses are necessary to lular concentration of serotonin in the striatum of naïve rats (control reach similar plasma or tissue concentration in the two species. The rats). For that reason, the animals were pretreated with pargyline to dose of melatonin used in our experiment was found to be effective in inhibit the oxidation of melatonin-produced 5-MT in the liver and of elevating tissue and extracellular concentrations of serotonin in the rat serotonin in the brain and, consequently, to enhance the effect of brain. melatonin on the intraneuronal and, possibly, also extraneuronal, Melatonin increases the tissue content of serotonin in the brain serotonin level. As expected, when administered to animals pretreated structures of naïve, sham-operated, or 5,7-DHT–lesioned rats (model with pargyline (75 mg/kg i.p.), melatonin increased serotonin concen- A). In the case of the brain stem (containing serotonergic neurons), tration up to approximately 1000% of the pargyline-treated animals melatonin is considerably more effective in 5,7-DHT–lesioned rats, (Fig. 3). The CYP2D inhibitor propafenone (50 mM), given locally which suggests the presence of a relatively larger pool of serotonin through a microdialysis probe, prevented the melatonin-induced in- formed from tryptophan via a classic pathway than from 5-MT (formed crease in serotonin concentration, having decreased it to approximately from melatonin) via a CYP2D pathway, as well as its considerable 25% of the pargyline- plus melatonin-treated animals. Pargyline alone reduction by the neurotoxins. As shown in naïve rats, the CYP2D raised the extracellular serotonin concentration up to approximately inhibitor quinine (Boobis et al., 1990; Bromek et al., 2010), given to 325% of the basal level; when single points were compared, the lateral ventricles of the brain, prevents this effect in some brain pargyline-treated group was significantly different from the control structures (e.g., cortex, hippocampus, striatum, thalamus, hypothala- group (P , 0.0001, t test). Propafenone did not affect the extracellular mus, brain stem, medulla oblongata, and cerebellum), which indicates concentration of serotonin in pargyline-pretreated animals. contribution of CYP2D to the melatonin-produced elevation in Like in the case of serotonin, melatonin did not change the serotonin concentration. extracellular concentration of dopamine in the striatum of naïve rats. As mentioned above, pargyline was applied to prevent 5-MT (formed However, when given to animals pretreated with pargyline, melatonin from melatonin in the liver) and serotonin (formed from 5-MT in the increased dopamine concentration up to approximately 500% of brain) from the MAO oxidation to reinforce the effect of melatonin on the pargyline-pretreated animals. (Fig. 4). The CYP2D inhibitor the serotonin level in the brain. In contrast with melatonin, both 5-MT propafenone prevented the melatonin-evoked increase in the dopamine and serotonin are rapidly metabolized MAO substrates, the enzyme Melatonin Supports Serotonin Formation by Brain CYP2D 451 being present in a large amount in the liver and brain (Suzuki et al., Miguez et al., 1995), since its low doses (#1 mg/kg i.p.) were found to 1981; Galzin and Langer, 1986; Raynaud and Pévet, 1991; Hardeland, increase serotonin concentration in some brain structures (Anton-Tay 2010). However, the effect of melatonin on serotonin level is not visible et al., 1968; Miguez et al., 1994). in the majority of structures of the pargyline-pretreated, nonlesioned The results presented herein are in agreement with some previous animals (both controls and sham-operated), since the amount of the findings suggesting that the CYP2D-mediated formation of serotonin tissue serotonin synthesized physiologically from endogenous sub- from 5-MT may take place in vivo. Thus humanized CYP2D6 strates (mainly from tryptophan, but also from endogenous 5-MT) and transgenic mice show a higher concentration of serotonin and its protected from MAO probably largely dominates the serotonin formed metabolite 5-HIAA in (Yu et al., 2003) and the brain indirectly from exogenous melatonin (via melatonin deacetylation to (Cheng et al., 2013), and intracerebral administration of 5-MT increases 5-MT and subsequent O-demethylation by CYP2D). However, the the extracellular concentration of serotonin in the brain, as has been melatonin-induced increase in the tissue level of serotonin can be seen shown using a brain microdialysis in living rats (Haduch et al., 2015). in all of the brain structures studied of pargyline-treated rats with a Moreover, individuals with no or a defective CYP2D6 gene (expressing partial lesion of the serotonergic system. Under these experimental a poor metabolizer phenotype) are more anxiety prone (Bertilsson et al., conditions, the physiologic synthesis and storage of serotonin is 1989; González et al., 2008; Cheng et al., 2013), such a behavior being diminished by the neurotoxin 5,7-DHT specific to the serotonergic provoked by a low serotonin level in the limbic system of the brain system, and the amount of serotonin formed by an alternative pathway (Hensler, 2006). (via 5-MT O-demethylation by CYP2D) is raised due to a supply of an In conclusion, our study indicates that exogenous melatonin supports Downloaded from exogenous substrate (i.e., 5-MT formed from exogenous melatonin in CYP2D-catalyzed serotonin synthesis from 5-MT in vivo. CYP2D- the liver) (Rogawski et al., 1979; Beck and Jonsson, 1981), which catalyzed serotonin synthesis from the melatonin-derived 5-MT closes crosses the blood–brain barrier (Acuña-Castroviejo et al., 2014). the serotonin-melatonin-serotonin biochemical cycle. Hence, the Moreover, both peripheral and brain 5-MT and serotonin formed from therapeutic effect of melatonin may stem not only from its action on it are protected by intraperitoneal pargyline against MAO oxidation. melatonin receptors and from its radical scavenger properties (Singh Melatonin also increases the functional, extracellular concentration and Jadhav, 2014) but also from its metabolism to the of serotonin under specific conditions (model B). In fact, the neurotransmitter serotonin. The latter mechanism may be regarded as a dmd.aspetjournals.org extracellular level of the neurotransmitter depends not only on its newly recognized additional component of the pharmacological action amount synthesized in the but also on its release into the of melatonin. These findings are of both physiologic and pharmaco- synaptic cleft and into the neuron. The effect of melatonin on logical importance, since melatonin can be formed endogenously and these presynaptic processes has not yet been well recognized, but the administered as a drug in the therapy of sleep and mood disorders, as available data suggest such a possibility (Pandi-Perumal et al., 2008). well as a neuroprotective agent. Thus, exogenous melatonin does not significantly increase extracellular serotonin in naïve rats but raises it in pargyline-pretreated animals. In at ASPET Journals on September 28, 2021 the striatum of pargyline-pretreated rats, melatonin potently elevates Authorship Contributions serotonin concentration, yet such a strong effect does not appear when Participated in research design: Haduch, Daniel. ł the tissue content of serotonin is measured (model A). However, in this Conducted experiments: Haduch, Bromek, Wójcikowski, Go embiowska. Performed data analysis: Haduch, Gołembiowska, Daniel. experimental model and in these conditions (model B), the vesicle- Wrote or contributed to the writing of the manuscript: Haduch, Daniel. stored neurotransmitter does not mask the amount of serotonin synthesized from exogenous melatonin. In addition, as mentioned above, the 5-MT freshly synthesized from melatonin in the liver and the References serotonin formed from it in the brain are protected from MAO. 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