1521-009X/44/3/445–452$25.00 http://dx.doi.org/10.1124/dmd.115.067413DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:445–452, March 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Melatonin Supports CYP2D-Mediated Serotonin Synthesis inthe Brain

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 sleep andmood disorders and asa neuroprotective agent. The aim of our study was to demonstratethat melatonin supported (via its deacetylation to 5-methoxytrypt-amine) CYP2D-mediated synthesis of serotonin from 5-methoxy-tryptamine. We measured serotonin tissue content in some brainregions (the cortex, hippocampus, nucleus accumbens, striatum,thalamus, hypothalamus, brain stem, medulla oblongata, and cere-bellum) (model A), as well as its extracellular concentration in thestriatum using an in vivo microdialysis (model B) after melatonininjection (100 mg/kg i.p.) to male Wistar rats. Melatonin increasedthe tissue concentration of serotonin in the brain structures stud-ied of naïve, sham-operated, or serotonergic neurotoxin(5,7-dihydroxytryptamine)–lesioned rats (model A). Intracerebroven-tricular quinine (a CYP2D inhibitor) prevented themelatonin-inducedincrease in serotonin concentration. In the presence of pargyline

(a monoaminoxidase inhibitor), the effect of melatonin was notvisible in the majority of the brain structures studied but could beseen in all of them in 5,7-dihydroxytryptamine–lesioned animalswhen serotonin storage and synthesis via a classic tryptophanpathway was diminished. Melatonin alone did not significantlyincrease extracellular serotonin concentration in the striatum ofnaïve rats but raised its content in pargyline-pretreated animals(model B). The CYP2D inhibitor propafenone given intrastructurallyprevented the melatonin-induced increase in striatal serotonin inthose animals. The obtained results indicate thatmelatonin supportsCYP2D-catalyzed serotonin synthesis from 5-methoxytryptamine inthe brain in vivo, which closes the serotonin-melatonin-serotoninbiochemical cycle. The metabolism of exogenous melatonin to theneurotransmitter serotonin may be regarded as a newly recognizedadditional component of its pharmacological action.

Introduction

Melatonin is used in the treatment of some sleep disorders, includingconcurrent sleep disturbances in the course of different psychiatricdiseases such as schizophrenia and major depressive and seasonalaffective disorders (Dolberg et al., 1998; Dalton et al., 2000; Shamiret al., 2000; Singh and Jadhav, 2014). Moreover, high doses ofmelatonin are recommended for neuroprotection (Venegas et al.,2012; Acuña-Castroviejo et al., 2014).The synthesis of endogenous melatonin in the pineal gland is

governed by the light/dark cycle, which controls circadian andcircannual rhythms. However, melatonin can also be produced in asubstantial amount in extrapineal organs, including the gastrointestinaltract (in enterochromaffin cells), where it is not controlled by thephotoperiod (Pandi-Perumal et al., 2008; Venegas et al., 2012; Acuña-Castroviejo et al., 2014). The synthesis of melatonin from tryptophanvia serotonin is catalyzed by N-acetyltransferase and hydroxyindole-O-methyltransferase, these two enzymes being present in a variety oforgans/tissues such as the heart, liver, leukocytes, or the brain (e.g., thecerebral cortex and striatum), which also suggests the occurrence of

melatonin synthesis in them. Extrapineal melatonin seems not to beengaged in the regulation of the photoperiod (Acuña-Castroviejo et al.,2014); however, together with pineal melatonin, extrapineal melatoninprotects cells against damage from oxidative stress due to its antioxidantand anti-inflammatory activity. Moreover, melatonin exerts an anti-excitotoxic effect in the brain by reducing glutamate activity andincreasing that of GABA and it stimulates neurogenesis (Pandi-Perumalet al., 2008; Acuña-Castroviejo et al., 2014; Singh and Jadhav, 2014).Being an amphiphilic substance, peripheral melatonin easily crosses theblood–brain barrier (Green et al., 1975). Circulating melatonin ismetabolized mainly in the liver by cytochrome P450 isoforms of theCYP1A subfamily (CYP1A1/A2/B1) to form 6-hydroxymelatonin andthen 6-sulfatoxymelatonin (Ma et al., 2005; Hardeland, 2010), but it canalso be deacetylated to 5-methoxytryptamine (5-MT) (Rogawski et al.,1979; Beck and Jonsson, 1981).In the brain, melatonin deacetylation to 5-MT seems to be of minor

importance; both melatonin and 5-MT are formed mainly in the pinealgland from which they are released to the circulation or to the thirdventricle. However, 5-MT formed from gut-derived melatonin in theliver crosses the blood–brain barrier (Beck and Jonsson, 1981; Acuña-Castroviejo et al., 2014) and, together with the brain-derived 5-MT,provides a direct substrate for CYP2D to form serotonin. Thus, the gut-derived or the exogenously supplied melatonin that provides most ofthe 5-MT for the organism may support serotonin formation by aCYP2D-mediated alternative pathway in vivo.

This research was supported by the European Union and the Polish Ministry ofScience and Higher Education [Grant DeMeTer POIG.01.01.02-12-004/09] and bythe Polish Academy of Sciences Institute of Pharmacology [Statutory Funds].

dx.doi.org/10.1124/dmd.115.067413.

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

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Recent studies have shown that, apart from the classic pathway ofserotonin formation from L-5-hydroxytryptophan, serotonin may alsobe synthesized via CYP2D-catalyzed O-demethylation of 5-MT.Such a way of serotonin synthesis has been demonstrated for humanand rat cDNA-expressed CYP2D isoforms, as well as for brainmicrosomes (Yu et al., 2003; Haduch et al., 2013). Moreover, using amicrodialysis model, the formation of serotonin via this alternativepathway has recently been shown to function in the brain in vivo(Haduch et al., 2015).The aim of this study was to demonstrate that exogenous melatonin

supported (via deacetylation to 5-MT) CYP2D-mediated serotoninsynthesis from 5-MT in the rat brain in vivo (Fig. 1) by measuringtissue or extracellular serotonin concentration after intraperitonealmelatonin injection. Serotonin tissue content was measured in brainregions containing CYP2D and serotonergic innervation (model A).The experiment was carried out with naïve and 5,7-dihydroxytryptamine(5,7-DHT)–pretreated animals in the absence/presence of the mono-amine oxidase (MAO) inhibitor pargyline. Intraperitoneal pargylinewas applied to prevent 5-MT (successively formed from melatoninin the liver) and serotonin (formed from 5-MT in the brain) fromthe oxidation by MAO and thus to potentiate the effect of melatoninon the serotonin level in the brain (Prozialek and Vogel, 1978;Suzuki et al., 1981; Galzin and Langer, 1986; Raynaud and Pévet,1991). Intracerebral 5,7-DHT (a neurotoxin specific to serotonergicneurons) was used to decrease the production of serotonin via aclassic pathway from tryptophan and serotonin storage in neuronalterminals of the brain (Rogawski et al., 1979; Beck and Jonsson,1981) and, consequently, to enhance serotonin derived from theperipherally formed 5-MT (from melatonin), as well as to demon-strate the role of cytochrome P450 present in neurons/terminals inserotonin formation from 5-MT via an alternative pathway. Theextracellular, functional concentration of serotonin in the striatum(involved in motor functions) was measured in the absence/presence of pargyline using an in vivo microdialysis (model B).The animals also received a CYP2D inhibitor to ascertain whetherCYP2D was engaged in the synthesis of serotonin from melatoninvia 5-MT O-demethylation.

Materials and Methods

Chemicals

Melatonin (hydrochloride), pargyline (hydrochloride), quinine (hydrochlo-ride), propafenone (hydrochloride), serotonin (5-hydroxytryptamine or 5-HT;hydrochloride) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopa-mine (DA), noradrenaline (NA), 5,7-DHT (a creatinine sulfate salt), and ascorbicacid were purchased from Sigma-Aldrich (St. Louis, MO). Ketamine hydro-chloride (Ketamine) and xylazine hydrochloride (Sedazin) came from Biowet(Puławy, Poland). All organic reagents were of high-performance liquidchromatography (HPLC) grade and were supplied by Merck (Darmstadt,Germany).

Animals

All experimental procedures were carried out in accordance with the NationalInstitutes of Health Guide for the Care and Use of Laboratory Animals and wereapproved by the Bioethics Commission at the Polish Academy of SciencesInstitute of Pharmacology, as compliant with the Polish law. The study wasconducted on male Wistar-Han rats (Charles River Laboratories, Sulzfeld,Germany) weighing 300–325 g. The animals were housed in rooms with acontrolled temperature and humidity on a 12-hour light/dark cycle and had freeaccess to tap water and standard laboratory food during the study.

The Tissue Levels of Serotonin in the Brain after IntraperitonealMelatonin Administration (Ex Vivo Measurement, Model A)

To study the supportive effect of exogenous melatonin on the alternativepathway of serotonin synthesis from 5-MT in a brain tissue, melatonin wasinjected intraperitoneally (100 mg/kg i.p.) to naïve and 5,7-DHT–lesioned rats.

The rats were anesthetized with ketamine HCl (75 mg/kg i.p.) and xylazineHCl (10 mg/kg i.p.) and were then placed in stereotaxic apparatus (David KopfInstruments, Tujunga, CA). All solutions were freshly prepared on the day ofexperimentation. 5,7-DHT was dissolved in 0.9% NaCl with 0.05% ascorbicacid and was injected into the dorsal raphe nuclei (DRN) and median raphenuclei (MRN) at a concentration of 10 mg/ml (1 ml, infused at a rate of 1 ml/min)into both raphe nuclei. The following coordinates were used (Paxinos andWatson, 2007): AP (anterior-posterior), –7.9; L (lateral), 0.0 from the bregma;and V (ventral), –7.9 (MRN), –5.9 (DRN) from the surface of the dura. Theneedle stayed in place for 5 minutes after injection before it was slowlywithdrawn. Sham-operated animals were subjected to the same procedure as 5,7-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 (orvehicle), the rats received melatonin (100 mg/kg i.p.), an indirect exogenoussubstrate for serotonin synthesis and/or pargyline (75 mg/kg i.p., 30 minutesbefore 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 drugs 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 ofnonoperated 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.); andpargyline (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 (100mg/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) pluspargyline (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) plusmelatonin (100 mg/kg i.p.); 5,7-DHT (1 ml, into the raphe nuclei) plus pargyline(75mg/kg i.p.) plus 30%DMSO (2ml/kg i.p.); and 5,7-DHT (1ml, into the raphenuclei) plus pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.).

Moreover, an additional group of naïve rats received bilateral intracerebro-ventricular injections of the CYP2D inhibitor quinine (150 mg/5 ml i.c.v.),melatonin (100 mg/kg i.p.), or quinine (30 minutes before melatonin) plusmelatonin. The CYP2D competitive inhibitor quinine (Kobayashi et al., 1989)

Fig. 1. Metabolism of exogenous melatonin via deacetylation to 5-MT in theliver and subsequent O-demethylation to serotonin in the brain (the investigatedpathway).

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was used in an appropriate intracerebral dose to produce an enzyme-specificmicromolar concentration of the inhibitor in the brain (Bromek et al., 2010;Haduch et al., 2015). Such an application of quinine allowed us to ascertainwhether CYP2D was engaged in the indirect synthesis of serotonin frommelatonin (via O-demethylation of the melatonin-formed 5-MT). The ratswere anesthetized with ketamine HCl (75 mg/kg i.p.) and xylazine HCl(10 mg/kg i.p.) and were then placed in the Kopf stereotaxic apparatus. Thecoordinates based on the Paxinos and Watson (2007) atlas were as follows:AP, –0.8; L, 61.5 from the bregma; and V, –3.5 from the dura. The freshlyprepared quinine at a concentration of 30 mg/ml was administered (5 ml,infused at a rate of 1 ml/min) into both lateral ventricles (150 mg perventricle). The needle was left in place for 5 minutes after injection before itsslow withdrawal. Control rats (sham-operated animals) were subjected to thesame procedure as the quinine-treated group, except that they received avehicle (0.9% NaCl) instead of quinine. The placement of the needle washistologically verified in 10 rats in a preliminary experiment and was thenchecked in two animals from each experimental group.

All drugs were given according to the following schedule: quinine i.c.v. orvehicle → after 30 minutes, melatonin i.p. or 30% DMSO → after 1 hour,decapitation → tissue serotonin.

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/5ml i.c.v.) plus 30% DMSO (2 ml/kg i.p.); vehicle (5 ml i.c.v.) plus melatonin(100mg/kg i.p.); and quinine (150mg/5ml i.c.v.) plusmelatonin (100mg/kg i.p.).

One hour after melatonin injection, the rats were decapitated and theirbrains were removed. The brains were cut into the cerebellum, hypothalamus,thalamus, nucleus accumbens, striatum, hippocampus, frontal cortex, the restof cortex, brain stem, andmedulla oblongata. Brain tissues were frozen on dryice and stored at –80�C until they were further analyzed.

Melatonin and the other pharmacological substances applied wereadministered at the same time of a day to rats coming from all of theexperimental groups. Consequently, all experimental procedures involvingcontrol groups and groups treated with melatonin were carried out at the sametime of a day (between 10:00 AM and 4:00 PM).

Extracellular Concentrations of Serotonin in the Striatum afterIntraperitoneal Administration of Melatonin: An In Vivo Microdialysis(Model B)

Microdialysis guides were implanted 7 days before melatonin (100 mg/kgi.p.) administration. The rats were anesthetized with ketamine (75mg/kg i.m.)and xylazine (10 mg/kg i.m.) and were then placed in stereotaxic apparatus(David Kopf Instruments). Vertical microdialysis guides (BioanalyticalSystems Inc., West Lafayette, IN) were implanted in the striatum using thefollowing coordinates (Paxinos and Watson, 2007): AP, +1.2; L, +3.0 fromthe bregma; and V, –3.2 from the surface of the dura.

On day 7 after the surgery, brain microdialysis probes (membranepolyacrylonitrile, 320 mm OD, 4 mm long; Bioanalytical Systems Inc.) wereplaced inside the guides implanted in the striatum and were connected to theUniventor 802 syringe pump (Agn Tho’s AB, Lidingö, Sweden), whichdelivered an artificial cerebrospinal fluid (aCSF) consisting of 145 mMNaCl,2.7 mM KCl, 1.0 mM MgCl2, and 1.2 mM CaCl2, pH 7.4, at a flow rate of2.0 ml/min.

Four baseline samples were collected from freely moving rats at 20-minuteintervals after a 165-minute washout period. Then, the appropriate drugs,propafenone (50 mM given using microdialysis probes) and/or melatonin(100 mg/kg i.p.), were administered to naïve or pargyline-treated rats(pargyline 75 mg/kg i.p., 60 minutes before melatonin). The applied effectivedoses of melatonin and the other pharmacological substances were chosen onthe basis of the results of our preliminary experiments (data not shown). Inthat experimental set, intracerebral propafenone (added to the aCSF) wasused instead of quinine as a specific and competitive CYP2D inhibitor (Xuet al., 1995; Zhou et al., 2013) to show the efficacy of another enzymeinhibitor in preventing melatonin effect. Moreover, in our preliminaryexperiment, propafenone was effective at a concentration 10 times lowerthan quinine (50mMversus 500mM), being thus also easier to dissolve in theaCSF. Dialysate fractions after melatonin injection were collected throughout180 minutes. All drugs were given according to the following schedule:microdialysis guide (in the striatum)→ after 7 days, pargyline i.p.→ after 60

TABLE1

The

effect

ofmelatonin

(100

mg/kg

i.p.)on

serotonintissuelevelin

thebrainstructures

ofnaïve,

sham

-operated,

and5,7-DHT–lesioned

rats(m

odel

A)

The

data

areexpressedas

themean6

S.E.M

.(n

=5–8).Statistical

significance

was

assessed

byatwo-way

(nonoperated

rats)or

three-way

(operatedrats)analysisof

variance

andFisher’stest.

Brain

Structure

Serotonin

TissueLevel

Nonoperated

Rats

OperatedRats

Sham-O

peratedRats

5,7-DHT–LesionedRats

Control

MEL

PARG

PARG

+MEL

Sham

MEL

PARG

PARG

+MEL

Lesion

MEL

PARG

PARG

+MEL

pg/mgof

tissue

Frontal

cortex

563.86

34.4

637.26

30.7

1127

.06

82.7***

1257

.06

44.8

506.66

20.1

685.16

31.4

+91

5.36

47.3+++

1035

.06

25.9

381.96

38.2+

542.06

45.0$

715.06

45.0

$$$

946.96

56.6

!!!

Restof

cortex

259.06

25.0

338.16

24.8*

523.96

21.5***

570.56

18.0

305.36

22.7

400.66

20.6

++

523.36

17.2+++

526.16

15.3

185.96

21.7+++

242.66

21.7

280.96

12.5

$$

403.76

38.7

!!!

Hippo

campu

s19

1.86

7.3

295.96

18.2**

555.76

32.4***

536.26

40.6

252.66

20.7

467.46

42.6

+++

413.86

19.8+++

428.16

22.0

33.5

610

.0+++

58.4

626

.577

.46

20.5

176.46

25.2

!!

Nucleus

accumbens

394.26

15.0

753.76

54.6***

794.06

35.5***

1136

.06

50.1###

368.86

43.3

1045

.86

134.8+

++

1266

.46

168.1+

++

1111

.86

46.4

261.06

28.7

612.06

103.8$

505.86

21.4

854.16

61.0

!!

Striatum

370.86

19.6

417.16

21.0

581.76

55.93**

759.36

48.6##

303.56

23.9

821.56

67.1

+++

623.06

64.7+++

834.56

30.4^

161.76

15.6

550.46

24.1$$$

574.36

27.9

$$$

896.96

113.2!!!

Thalamus

353.66

13.7

516.46

28.5*

922.66

66.16***

938.96

61.4

417.86

19.9

739.76

31.2

+++

773.36

23.6+++

908.46

27.5^

305.46

25.5+

678.36

19.6$$$

643.46

31.5

$$$

722.66

32.4

Hyp

othalamus

339.76

33.9

545.66

64.8*

673.86

55.7***

646.26

47.8

564.26

56.8

918.06

46.1

+++

962.06

55.9+++

897.06

41.1

236.36

38.9+++

320.56

52.8

374.76

53.3

$540.86

35.3

!

Brain

stem

950.96

42.6

1124

.06

28.5

1901

.06

100.3***

2091

.06

151.5

990.76

69.5

1306

.16

51.1

+16

40.0

678

.9+++

2026

.06

163.2^

411.26

25.3+++

952.76

54.2$$$

1063

.06

69.1

$$$

1525

.06

133.0!!

Medulla

oblongata

280.96

20.2

378.86

25.8**

438.86

27.2***

683.76

20.7###

256.56

26.7

397.76

53.3

+++

546.66

17.1+++

624.76

53.2

129.66

7.9+

++

234.46

11.7$$

406.06

15.8

$$$

474.06

20.9

!

Cerebellum

39.4

61.8

50.1

66.6

80.6

64.6*

**81

.76

4.6

21.8

62.3

72.1

66.7+

++

58.9

62.8+

++

58.8

68.2

14.0

61.1

52.5

65.1$

$$

30.0

62.8$

43.7

63.3!

MEL,melaton

in;PA

RG,pargyline.

Nonoperated

rats:*P

,0.05

(versuscontrol);**P,

0.01

(versuscontrol);***P

,0.001(versuscontrol);##P,

0.01

(versusPARG);

###P,

0.00

1(versusPARG).Operatedrats:+P,

0.05

(versussham

-operatedrats);

++P,

0.01

(versussham

-operated

rats);

+++P,

0.001versus

sham

-operatedrats(versussham

-operatedrats);^P

,0.05

(versussham

plus

PARG);

$P,

0.05

(versuslesioned

anim

als);$$P,

0.01

(versuslesion

edanim

als);$$$P,

0.001(versuslesioned

anim

als);! P

,0.05

(versuslesion

plus

PARG

grou

p);!!P,

0.01

(versuslesion

plus

PARG

grou

p);!!! P

,0.00

1(versuslesion

plus

PARG

grou

p).

Melatonin Supports Serotonin Formation by Brain CYP2D 447

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minutes, propafenone into the striatum and/or melatonin i.p. → in vivoextracellular serotonin (dopamine).

The rats were divided into six experimental groups (Figs. 3 and 4): control, 30%DMSO (2 ml/kg i.p.); pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.);saline (2 mg/kg i.p.) plus melatonin (100mg/kg i.p.); pargyline (75mg/kg i.p.) pluspropafenone (50 mM) plus 30% DMSO (2 ml/kg i.p.); pargyline (75 mg/kg i.p.)

plus melatonin (100 mg/kg i.p.); and pargyline (75 mg/kg i.p.) plus propafenone(50 mM) plus melatonin (100 mg/kg i.p.)

At the end of the experiment, the rats were euthanized and their brains werehistologically examined to validate probe placement.

Brain microdialysis procedures were carried out in rats from all of theexperimental groups at the same time of a day (between 10:00AM and 6:00 PM).

Fig. 2. Model A. The effect of intracerebral quinineadministration on the melatonin-induced (100 mg/kgi.p.) increase in 5-HT tissue content in the followingbrain structures: frontal cortex (A), rest of the cortex(B), hippocampus (C), nucleus accumbens (D),striatum (E), thalamus (F), hypothalamus (G), brainstem (H), medulla oblongata (I), and cerebellum (J). Thedata are expressed as the mean 6 S.E.M. (n = 5–8).Statistical significance was assessed by a two-wayanalysis of variance and Fisher’s test. *P, 0.05 (versuscontrol); **P , 0.01 (versus control); ***P , 0.001(versus control); #P , 0.05 (versus melatonin);##P , 0.01(versus melatonin); ###P , 0.001 (versusmelatonin). The control values (in picograms permilligram of tissue) were as follows: 465.50 6 20.39(frontal cortex), 352.08 6 17.42 (rest of the cortex),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), 560.21 618.68 (medulla oblongata), and 45.83 6 3.59 (cerebel-lum). MEL, melatonin; QUIN, quinine.

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Determination of Brain Neurotransmitters

The tissue concentrations of 5-HT, DA, NA, and 5-HIAA were measuredusing HPLC with electrochemical detection, according to the method of Haduchet al. (2015). Briefly, brain structures were homogenized in 20 volumes (v/w) ofice-cold 0.1 MHClO2 and were centrifuged at 15,000� g for 15 minutes at 4�C.The obtained supernatant (5 ml) was injected into the HPLC system. An externalstandard containing NA, DA, 5-HT, and 5-HIAA at concentrations of 50 ng/ml(or 2.5 ng/ml for the dialysate) was used. The chromatography system comprisedan LC-4C amperometric detector with a cross-flow detector cell (BioanalyticalSystems Inc.) as well as a 626 Alltech pump and a Hypersil Gold analytical

column (3 mm, 100 � 3 mm; Thermo Scientific, Waltham, MA). The mobilephase contained 0.1 M KH2PO4, 0.5 mM Na2EDTA, 80 mg/l sodium1-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-mmglassy carbon electrode was set at 0.7 V and a sensitivity of 5 nA/V. The columntemperature was maintained at 30�C. The Chromax 2007 program (Pol-Laboratory, Warsaw, Poland) was applied for the collection and analysis ofdata. Neurotransmitter concentrations in the dialysate were measured using acomposition of the mobile phase: 0.1 M KH2PO4, 0.5 mM Na2EDTA, 16 mg/lsodium 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 thedialysate fraction prior to drug administration was regarded as a basal value(100%). The data were statistically analyzed using two-way (nonoperated rats) orthree-way (operated rats) analysis of variance (model A), followed by Fisher’sleast significant differences post hoc test, or by a repeated-measures analysis ofvariance (model B), followed by Tukey’s post hoc test (Program Origin 7.5 orStatistica 9, respectively; StatSoft Inc., Tulsa, OK). The results were consideredto be statistically significant when P , 0.05.

Results

The Tissue Levels of Serotonin in the Brain after IntraperitonealAdministration of Melatonin (Ex Vivo Measurement, Model A)

Intact Rats. Melatonin and the applied pharmacological substances(the MAO inhibitor pargyline and the serotonergic neurotoxin 5,7-DHT) affected the tissue concentration of serotonin in the brain; thiseffect was structure dependent (Table 1). Melatonin (100 mg/kg i.p.)significantly increased serotonin content in the rest of the cortex(131%), hippocampus (154%), nucleus accumbens (191%), thalamus(146%), hypothalamus (161%), and medulla oblongata (135%) com-pared with the control (Table 1). A similar tendency was observed inother structures. The MAO inhibitor pargyline (75 mg/kg i.p.)potently increased serotonin concentration in all of the investigatedbrain structures, on average up to 207% of the control (frontal cortex,200%; rest of the cortex, 202%; hippocampus, 290%; nucleusaccumbens, 201%; striatum, 157%; thalamus, 261%; hypothalamus,198%; brain stem, 200%; medulla oblongata, 156%; and cerebellum,205%). However, melatonin administered to pargyline-pretreated ratsdid not elevate the serotonin concentration in most of the brainstructures studied, except for the nucleus accumbens, striatum, andmedulla oblongata (an increase up to 143%, 131%, and 156% of thecontrol, 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%), medullaoblongata (155%), and cerebellum (331%) (Table 1). Pargylinepotently increased tissue serotonin content in the brain structures ofsham-operated rats, on average up to 209% (frontal cortex, 181%; restof the cortex, 171%; hippocampus, 164%; nucleus accumbens, 344%;striatum, 205%; thalamus, 185%; hypothalamus, 171%; brain stem,166%; medulla oblongata, 213%; and cerebellum, 270%) (Table 1).Melatonin administered to pargyline-pretreated sham-operated ratssignificantly elevated serotonin concentration up to approximately134% in the striatum, 117% in the thalamus, and 124% in the brain stemcompared with pargyline-pretreated animals (Table 1).5,7-DHT–Lesioned Rats. The neurotoxin 5,7-DHT (10 mg/1 ml),

injected into the DRN and MRN, decreased tissue serotonin concen-tration in all of the brain structures studied, on average down to 54% of

Fig. 3. Model B. The melatonin-produced increase in extracellular serotoninconcentration in the striatum is prevented by the CYP2D inhibitor propafenone inpargyline-pretreated rats. Drugs were given as indicated with an arrow. The obtainedvalues are the mean 6 S.E.M. (n = 5–8). A repeated-measures analysis of varianceshowed effect of treatment (F = 5.25, P = 0), effect of time (F = 11.27, P = 0), andinteraction 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(P = 0.065; versus pargyline plus propafenone) (repeated-measures analysis ofvariance and Tukey’s post hoc test). MEL, melatonin; PARG, pargyline; PFN,propafenone.

Fig. 4. Model B. The melatonin-produced increase in extracellular dopamineconcentration in the striatum is prevented by the CYP2D inhibitor propafenone inpargyline-pretreated rats. Drugs were given as indicated with an arrow. The obtainedvalues are the mean 6 S.E.M. (n = 5–8). A repeated-measures analysis of varianceshowed effect of treatment (F = 5.25, P = 0), effect of time (F = 11.27, P = 0), andinteraction between both factors (F = 55.27, P = 0). #P, 0.001 (P = 0.00014; versuscontrol); *P , 0.001 (P = 0, 00014; versus pargyline); ^P , 0.001 (P = 0.00014;versus pargyline plus melatonin); !P , 0.1 (P = 0.053; versus pargyline pluspropafenone) (repeated-measures analysis of variance and Tukey’s post hoc test).MEL, melatonin; PARG, pargyline; PFN, propafenone.

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the sham-operated animals (frontal cortex, 75%; rest of the cortex, 61%;hippocampus, 13%; nucleus accumbens, 71%; striatum, 53%; thala-mus, 73%; hypothalamus, 42%; brain stem, 42%; medulla oblongata,51%; and cerebellum, 64%) (Table 1). The lesion was specific, since theconcentrations of the catecholaminergic neurotransmitters NA and DAwere not affected (data not shown). Melatonin (100 mg/kg i.p.)significantly enhanced the serotonin level in the brain structures of5,7-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 similartendency was observed in the hippocampus (174%), hypothalamus(136%), and rest of the cortex (130%). Pargyline markedly increasedserotonin level in the brain structures of 5,7-DHT–lesioned rats, onaverage up to 227% of the neurotoxin-lesioned animals (frontalcortex, 187%; rest of the cortex, 151%; hippocampus, 231%; nucleusaccumbens, 194%; striatum, 355%; thalamus, 211%; hypothalamus,159%; brain stem, 259%; medulla oblongata, 313%; and cerebellum,214%). Melatonin administered to the lesioned rats pretreated withpargyline elevated the serotonin concentration in all of the brainstructures tested, on average up to 150% of the pargyline-pretreatedlesioned rats (frontal cortex, 132%; rest of the cortex, 144%;hippocampus, 228%; nucleus accumbens, 169%; striatum, 156%;thalamus, 112%; hypothalamus, 144%; brain stem, 143%; medullaoblongata, 117%; and cerebellum, 146%).Effect of Quinine. In another experimental set, the specific CYP2D

inhibitor quinine, given to both lateral ventricles of the brain (150 mg/5ml i.c.v.) 30 minutes before melatonin (100 mg/kg i.p.), prevented themelatonin-evoked elevation in tissue serotonin content in the followingbrain structures of naïve rats: the rest of the cortex, hippocampus,striatum, thalamus, hypothalamus, brain stem, medulla oblongata, andcerebellum (Fig. 2). Such an effect of quinine was not observed in thefrontal cortex and nucleus accumbens. Quinine alone did not affect thetissue content of serotonin.

Extracellular Concentrations of Serotonin and DA in the Striatumafter Intraperitoneal Administration of Melatonin: An In VivoMicrodialysis (Model B)

Melatonin (100 mg/kg i.p.) did not significantly affect the extracel-lular concentration of serotonin in the striatum of naïve rats (controlrats). For that reason, the animals were pretreated with pargyline toinhibit the oxidation of melatonin-produced 5-MT in the liver and ofserotonin in the brain and, consequently, to enhance the effect ofmelatonin on the intraneuronal and, possibly, also extraneuronal,serotonin level. As expected, when administered to animals pretreatedwith pargyline (75 mg/kg i.p.), melatonin increased serotonin concen-tration up to approximately 1000% of the pargyline-treated animals(Fig. 3). The CYP2D inhibitor propafenone (50 mM), given locallythrough a microdialysis probe, prevented the melatonin-induced in-crease in serotonin concentration, having decreased it to approximately25% of the pargyline- plus melatonin-treated animals. Pargyline aloneraised the extracellular serotonin concentration up to approximately325% of the basal level; when single points were compared, thepargyline-treated group was significantly different from the controlgroup (P , 0.0001, t test). Propafenone did not affect the extracellularconcentration of serotonin in pargyline-pretreated animals.Like in the case of serotonin, melatonin did not change the

extracellular concentration of dopamine in the striatum of naïve rats.However, when given to animals pretreated with pargyline, melatoninincreased dopamine concentration up to approximately 500% ofthe pargyline-pretreated animals. (Fig. 4). The CYP2D inhibitorpropafenone prevented the melatonin-evoked increase in the dopamine

concentration in pargyline-pretreated rats. Pargyline alone raised theextracellular dopamine concentration up to approximately 450% of thebasal level. Propafenone did not significantly affect the extracellularconcentration of dopamine in pargyline-pretreated animals (Fig. 4).The basal extracellular level of serotonin in the striatum of naïve rats

was 0.9386 0.069 pg/10 ml, whereas that of dopamine equalled 7.560.7 pg/10 ml and did not differ between the experimental groups.

Discussion

Serotonergic projections from the raphe nuclei of the brain steminnervate almost all of the brain structures that control importantphysiologic functions, including the hypothalamus (food intake,circadian rhythm, and thermoregulation), basal ganglia (motor func-tions), thalamus (sleep and epilepsy), hippocampus (stress, learning andmemory), and cortex (sleep and mood) (Törk, 1990; Di Giovanni et al.,2008). Recent studies revealed that, apart from the classic pathway ofserotonin formation from L-5-hydroxytryptophan, serotonin may alsobe synthesized in the brain via CYP2D-catalyzed O-demethylation of5-MT. Serotonin that was formed in that alternative way was shown invitro in microsomes derived from different brain structures (Haduchet al., 2013) and was found to function in vivo when we measured itstissue and extracellular concentrations in selected structures of the brainafter intracerebral administration of 5-MT (Haduch et al., 2015).Our study provides further evidence for the role of cytochrome P450

(CYP2D) in serotonin synthesis in the brain in vivo by showing thatexogenous melatonin administered intraperitoneally supports (viadeacetylation to 5-MT) serotonin formation by cytochrome P450. Toobserve serotonin formation from melatonin in vivo, in our experimentwe used its relatively high dose (100 mg/kg i.p.), which is pharmaco-logically acceptable. Such high doses of melatonin (up to 200 mg/kg aday) were shown to have low toxicity (Barchas et al., 1967; Jahnkeet al., 1999; Acuña-Castroviejo et al., 2014) and were used forpsychopharmacological behavioral tests in animals (Papp et al., 2003,2006). Furthermore, high doses of melatonin are recommended becauseof its protective properties as an antioxidant in saturating its in-tracellular therapeutic targets (Venegas et al., 2012; Acuña-Castroviejoet al., 2014). On the other hand, the rate of drug metabolism is muchfaster in rats (rodents) than in humans, so higher doses are necessary toreach similar plasma or tissue concentration in the two species. Thedose of melatonin used in our experiment was found to be effective inelevating tissue and extracellular concentrations of serotonin in the ratbrain.Melatonin increases the tissue content of serotonin in the brain

structures of naïve, sham-operated, or 5,7-DHT–lesioned rats (modelA). In the case of the brain stem (containing serotonergic neurons),melatonin is considerably more effective in 5,7-DHT–lesioned rats,which suggests the presence of a relatively larger pool of serotoninformed from tryptophan via a classic pathway than from 5-MT (formedfrom melatonin) via a CYP2D pathway, as well as its considerablereduction by the neurotoxins. As shown in naïve rats, the CYP2Dinhibitor quinine (Boobis et al., 1990; Bromek et al., 2010), given tolateral ventricles of the brain, prevents this effect in some brainstructures (e.g., cortex, hippocampus, striatum, thalamus, hypothala-mus, brain stem, medulla oblongata, and cerebellum), which indicatescontribution of CYP2D to the melatonin-produced elevation inserotonin concentration.As mentioned above, pargyline was applied to prevent 5-MT (formed

from melatonin in the liver) and serotonin (formed from 5-MT in thebrain) from the MAO oxidation to reinforce the effect of melatonin onthe serotonin level in the brain. In contrast with melatonin, both 5-MTand serotonin are rapidly metabolized MAO substrates, the enzyme

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being present in a large amount in the liver and brain (Suzuki et al.,1981; Galzin and Langer, 1986; Raynaud and Pévet, 1991; Hardeland,2010). However, the effect of melatonin on serotonin level is not visiblein the majority of structures of the pargyline-pretreated, nonlesionedanimals (both controls and sham-operated), since the amount of thetissue serotonin synthesized physiologically from endogenous sub-strates (mainly from tryptophan, but also from endogenous 5-MT) andprotected from MAO probably largely dominates the serotonin formedindirectly from exogenous melatonin (via melatonin deacetylation to5-MT and subsequent O-demethylation by CYP2D). However, themelatonin-induced increase in the tissue level of serotonin can be seenin all of the brain structures studied of pargyline-treated rats with apartial lesion of the serotonergic system. Under these experimentalconditions, the physiologic synthesis and storage of serotonin isdiminished by the neurotoxin 5,7-DHT specific to the serotonergicsystem, and the amount of serotonin formed by an alternative pathway(via 5-MTO-demethylation by CYP2D) is raised due to a supply of anexogenous substrate (i.e., 5-MT formed from exogenous melatonin inthe liver) (Rogawski et al., 1979; Beck and Jonsson, 1981), whichcrosses the blood–brain barrier (Acuña-Castroviejo et al., 2014).Moreover, both peripheral and brain 5-MT and serotonin formed fromit are protected by intraperitoneal pargyline against MAO oxidation.Melatonin also increases the functional, extracellular concentration

of serotonin under specific conditions (model B). In fact, theextracellular level of the neurotransmitter depends not only on itsamount synthesized in the neuron but also on its release into thesynaptic cleft and reuptake into the neuron. The effect of melatonin onthese presynaptic processes has not yet been well recognized, but theavailable data suggest such a possibility (Pandi-Perumal et al., 2008).Thus, exogenous melatonin does not significantly increase extracellularserotonin in naïve rats but raises it in pargyline-pretreated animals. Inthe striatum of pargyline-pretreated rats, melatonin potently elevatesserotonin concentration, yet such a strong effect does not appear whenthe tissue content of serotonin is measured (model A). However, in thisexperimental model and in these conditions (model B), the vesicle-stored neurotransmitter does not mask the amount of serotoninsynthesized from exogenous melatonin. In addition, as mentionedabove, the 5-MT freshly synthesized frommelatonin in the liver and theserotonin formed from it in the brain are protected from MAO. TheCYP2D inhibitor propafenone (Xu et al., 1995; Zhou et al., 2013)prevents the melatonin-induced increase in extracellular serotonin,which testifies to engagement of this cytochrome P450 isoform in thesynthesis of serotonin from the melatonin-derived 5-MT.Melatonin simultaneously increases extracellular dopamine con-

centration in the striatum, this effect being prevented by the CYP2Dinhibitor propafenone. The above results are in line with our previousfindings obtained after local injection of 5-MT to the striatum(Haduch et al., 2015); moreover, they also support our earlierhypothesis that increases in 5-MT and serotonin (evoked by theinjection of 5-MT or melatonin) stimulate 5-HT2 heteroreceptorslocated on dopaminergic terminals and indirectly enhance the releaseof dopamine in this structure (Di Giovanni et al., 2008; Navailles andDe Deurwaerdère, 2011).The obtained results indicate that exogenous melatonin administered

peripherally supports CYP2D-catalyzed serotonin synthesis from 5-MTin the brain in vivo, as shown by the elevation in tissue and extracellularserotonin concentrations in the brain after melatonin administration andits prevention by the two CYP2D inhibitors quinine and propafenone.However, it cannot be excluded that in spite of providing the substrate(5-MT) for an alternative pathway of serotonin synthesis, melatoninmay also stimulate the endogenous synthesis of serotonin or affect otherregulatory processes of the neurotransmitter (i.e., its release or uptake;

Miguez et al., 1995), since its low doses (#1 mg/kg i.p.) were found toincrease serotonin concentration in some brain structures (Anton-Tayet al., 1968; Miguez et al., 1994).The results presented herein are in agreement with some previous

findings suggesting that the CYP2D-mediated formation of serotoninfrom 5-MT may take place in vivo. Thus humanized CYP2D6transgenic mice show a higher concentration of serotonin and itsmetabolite 5-HIAA in blood plasma (Yu et al., 2003) and the brain(Cheng et al., 2013), and intracerebral administration of 5-MT increasesthe extracellular concentration of serotonin in the brain, as has beenshown using a brain microdialysis in living rats (Haduch et al., 2015).Moreover, individuals with no or a defective CYP2D6 gene (expressinga poor metabolizer phenotype) are more anxiety prone (Bertilsson et al.,1989; González et al., 2008; Cheng et al., 2013), such a behavior beingprovoked by a low serotonin level in the limbic system of the brain(Hensler, 2006).In conclusion, our study indicates that exogenous melatonin supports

CYP2D-catalyzed serotonin synthesis from 5-MT in vivo. CYP2D-catalyzed serotonin synthesis from the melatonin-derived 5-MT closesthe serotonin-melatonin-serotonin biochemical cycle. Hence, thetherapeutic effect of melatonin may stem not only from its action onmelatonin receptors and from its radical scavenger properties (Singhand Jadhav, 2014) but also from its metabolism to the monoaminergicneurotransmitter serotonin. The latter mechanism may be regarded as anewly recognized additional component of the pharmacological actionof melatonin. These findings are of both physiologic and pharmaco-logical importance, since melatonin can be formed endogenously andadministered as a drug in the therapy of sleep and mood disorders, aswell as a neuroprotective agent.

Authorship ContributionsParticipated in research design: Haduch, Daniel.Conducted experiments: Haduch, Bromek, Wójcikowski, Gołembiowska.Performed data analysis: Haduch, Gołembiowska, Daniel.Wrote or contributed to the writing of the manuscript: Haduch, Daniel.

References

Acuña-Castroviejo D, Escames G, Venegas C, Díaz-Casado ME, Lima-Cabello E, López LC,Rosales-Corral S, Tan DX, and Reiter RJ (2014) Extrapineal melatonin: sources, regulation,and potential functions. Cell Mol Life Sci 71:2997–3025.

Anton-Tay F, Chou C, Anton S, and Wurtman RJ (1968) Brain serotonin concentration: elevationfollowing intraperitoneal administration of melatonin. Science 162:277–278.

Barchas J, DaCosta F, and Spector S (1967) Acute pharmacology of melatonin. Nature 214:919–920.

Beck O and Jonsson G (1981) In vivo formation of 5-methoxytryptamine from melatonin in rat. JNeurochem 36:2013–2018.

Bertilsson L, Alm C, De Las Carreras C, Widen J, Edman G, and Schalling D (1989) Debriso-quine hydroxylation polymorphism and personality. Lancet 1:555.

Boobis AR, Sesardic D, Murray BP, Edwards RJ, Singleton AM, Rich KJ, Murray S, de la TorreR, Segura J, and Pelkonen O, et al. (1990) Species variation in the response of the cytochromeP-450-dependent monooxygenase system to inducers and inhibitors. Xenobiotica 20:1139–1161.

Bromek E, Haduch A, and Daniel WA (2010) The ability of cytochrome P450 2D isoforms tosynthesize dopamine in the brain: An in vitro study. Eur J Pharmacol 626:171–178.

Cheng J, Zhen Y, Miksys S, Beyo�glu D, Krausz KW, Tyndale RF, Yu A, Idle JR, and GonzalezFJ (2013) Potential role of CYP2D6 in the central nervous system. Xenobiotica 43:973–984.

Dalton EJ, Rotondi D, Levitan RD, Kennedy SH, and Brown GM (2000) Use of slow-releasemelatonin in treatment-resistant depression. J Psychiatry Neurosci 25:48–52.

Di Giovanni G, Di Matteo V, Pierucci M, and Esposito E (2008) Serotonin-dopamine interaction:electrophysiological evidence. Prog Brain Res 172:45–71.

Dolberg OT, Hirschmann S, and Grunhaus L (1998) Melatonin for the treatment of sleep dis-turbances in major depressive disorder. Am J Psychiatry 155:1119–1121.

Galzin AM and Langer SZ (1986) Potentiation by deprenyl of the autoreceptor-mediated in-hibition of [3H]-5-hydroxytryptamine release by 5-methoxytryptamine. Naunyn SchmiedebergsArch Pharmacol 333:330–333.

González I, Peñas-Lledó EM, Pérez B, Dorado P, Alvarez M, and LLerena A (2008) Relationbetween CYP2D6 phenotype and genotype and personality in healthy volunteers. Pharma-cogenomics 9:833–840.

Green AR, Hughes JP, and Tordoff AF (1975) The concentration of 5-methoxytryptamine in ratbrain and its effects on behaviour following its peripheral injection. Neuropharmacology 14:601–606.

Melatonin Supports Serotonin Formation by Brain CYP2D 451

at ASPE

T Journals on M

arch 1, 2021dm

d.aspetjournals.orgD

ownloaded from

Haduch A, Bromek E, Kot M, Kami�nska K, Gołembiowska K, and Daniel WA (2015) Thecytochrome P450 2D-mediated formation of serotonin from 5-methoxytryptamine in the brainin vivo: a microdialysis study. J Neurochem 133:83–92.

Haduch A, Bromek E, Sadakierska-Chudy A, Wójcikowski J, and Daniel WA (2013) The cat-alytic competence of cytochrome P450 in the synthesis of serotonin from 5-methoxytryptaminein the brain: an in vitro study. Pharmacol Res 67:53–59.

Hardeland R (2010) Melatonin metabolism in the central nervous system. Curr Neuropharmacol8:168–181.

Hensler JG (2006) Serotonergic modulation of the limbic system. Neurosci Biobehav Rev 30:203–214.

Jahnke G, Marr M, Myers C, Wilson R, Travlos G, and Price C (1999) Maternal and de-velopmental toxicity evaluation of melatonin administered orally to pregnant Sprague-Dawleyrats. Toxicol Sci 50:271–279.

Kobayashi S, Murray S, Watson D, Sesardic D, Davies DS, and Boobis AR (1989) The speci-ficity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat isthe inverse of that in man. Biochem Pharmacol 38:2795–2799.

Ma X, Idle JR, Krausz KW, and Gonzalez FJ (2005) Metabolism of melatonin by human cy-tochromes p450. Drug Metab Dispos 33:489–494.

Miguez JM, Martin FJ, and Aldegunde M (1994) Effects of single doses and daily melatonintreatments on serotonin metabolism in rat brain regions. J Pineal Res 17:170–176.

Miguez JM, Martin FJ, and Aldegunde M (1995) Effects of pinealectomy and melatonin treat-ments on serotonin uptake and release from synaptosomes of rat hypothalamic regions. Neu-rochem Res 20:1127–1132.

Navailles S and De Deurwaerdère P (2011) Presynaptic control of serotonin on striatal dopaminefunction. Psychopharmacology (Berl) 213:213–242.

Pandi-Perumal SR, Trakht I, Srinivasan V, Spence DW, Maestroni GJ, Zisapel N, and CardinaliDP (2008) Physiological effects of melatonin: role of melatonin receptors and signal trans-duction pathways. Prog Neurobiol 85:335–353.

Papp M, Gruca P, Boyer PA, and Mocaër E (2003) Effect of agomelatine in the chronic mildstress model of depression in the rat. Neuropsychopharmacology 28:694–703.

Papp M, Litwa E, Gruca P, and Mocaër E (2006) Anxiolytic-like activity of agomelatine andmelatonin in three animal models of anxiety. Behav Pharmacol 17:9–18.

Paxinos G and Watson C (2007) The Rat Brain in Stereotaxic Coordinates, Academic Press,London.

Prozialek WC and Vogel WH (1978) Deamination of 5-methoxytryptamine, serotonin andphenylethylamine by rat MAO in vitro and in vivo. Life Sci 22:561–569.

Raynaud F and Pévet P (1991) 5-Methoxytryptamine is metabolized by monoamine oxidase A inthe pineal gland and plasma of golden hamsters. Neurosci Lett 123:172–174.

Rogawski MA, Roth RH, and Aghajanian GK (1979) Melatonin: deacetylation to 5-methoxy-tryptamine by liver but not brain aryl acylamidase. J Neurochem 32:1219–1226.

Shamir E, Laudon M, Barak Y, Anis Y, Rotenberg V, Elizur A, and Zisapel N (2000) Mel-atonin improves sleep quality of patients with chronic schizophrenia. J Clin Psychiatry 61:373–377.

Singh M and Jadhav HR (2014) Melatonin: functions and ligands. Drug Discov Today 19:1410–1418.

Suzuki O, Katsumata Y, and Oya M (1981) Characterization of eight biogenic indoleamines assubstrates for type A and type B monoamine oxidase. Biochem Pharmacol 30:1353–1358.

Törk I (1990) Anatomy of the serotonergic system. Ann N Y Acad Sci 600:9–34, discussion 34–35.

Venegas C, García JA, Escames G, Ortiz F, López A, Doerrier C, García-Corzo L, López LC,Reiter RJ, and Acuña-Castroviejo D (2012) Extrapineal melatonin: analysis of its subcellulardistribution and daily fluctuations. J Pineal Res 52:217–227.

Yu AM, Idle JR, Byrd LG, Krausz KW, Küpfer A, and Gonzalez FJ (2003) Regeneration ofserotonin from 5-methoxytryptamine by polymorphic human CYP2D6. Pharmacogenetics 13:173–181.

Xu BQ, Aasmundstad TA, Bjørneboe A, Christophersen AS, and Mørland J (1995) Ethyl-morphine O-deethylation in isolated rat hepatocytes. Involvement of codeine O-demethylationenzyme systems. Biochem Pharmacol 49:453–460.

Zhou K, Khokhar JY, Zhao B, and Tyndale RF (2013) First demonstration that brain CYP2D-mediated opiate metabolic activation alters analgesia in vivo. Biochem Pharmacol 85:1848–1855.

Address correspondence to: Prof. Władysława Anna Daniel, Institute ofPharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, Poland.E-mail: nfdaniel@cyf-kr.edu.pl

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