J. Pineal Res. 2001; 30:87–96

The modulation of neuronal activity bymelatonin: In vitro studies on mousehippocampal slices

Hogan MV, El-Sherif Y, Wieraszko A. The modulation of neuronalactivity by melatonin: in vitro studies on mouse hippocampal slices. J.Pineal Res. 2001; 30:87–96. © Munksgaard, Copenhagen

Abstract: The influence of melatonin on evoked potentials recordedfrom the CA1 field of mouse hippocampal slices was investigated.Melatonin (0.1–2.0 mM) and its analog, 6-chloromelatonin (0.1–0.5mM) depressed evoked potentials (EPSP and the population spike) in aconcentration-dependent manner. The melatonin-induced depressionwas followed by a slow recovery phase. Since the fiber potential wasnot affected, it was concluded that melatonin influenced synapticefficiency and/or cell excitability. Luzindole, an antagonist of MT2melatonin receptors, although slightly depressing evoked potentialswhen applied by itself (100 mM), blocked any further inhibition bymelatonin when added afterwards. We concluded that melatoninreduced synaptic efficiency and/or excitability of hippocampal neuronsmost likely through interaction with MT2 melatonin receptors, butother possible mechanisms of melatonin action are also considered.

M.V. Hogan1, Y. El-Sherif1,2 andA. Wieraszko1

1Department of Biology/CSI/IBR Center forDevelopmental Neuroscience, College ofStaten Island/CUNY, 2800 Victory Boulevard,Staten Island, New York 10314, USA; 2TheGraduate School & University Center, theCity University of NY, Ph.D. Program inBiology, Suite 4315, 365 Fifth Avenue, NewYork, New York 10016-4309

Key words: evoked potentials –hippocampal slices – luzindole – melatonin

Address reprint requests to Dr. AndrzejWieraszko, College of Staten Island/CUNY,2800 Victory Boulevard, Staten Island, NewYork 10314, USA.E-mail: Wieraszko@postbox.csi.cuny.edu

Received December 13, 1999;accepted March 8, 2000.

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) inmammals is produced mainly by the pineal gland[Borjigin et al., 1999] and to some extent by otherextrapineal tissues such as the retina and gas-trointestinal tract [Ralph, 1981]. Biosynthesis andsecretion of melatonin from the pineal gland oc-curs exclusively during the dark phase of the dailylight/dark cycle and is positively correlated with itsduration [Zawilska, 1996; Brzezinski, 1997]. Thisrhythmic light-dependent fluctuation in the mela-tonin level is regulated by a circadian clock local-ized in the suprachiasmatic nuclei (SCN) of thehypothalamus, whose activity is regulated bymelatonin itself through a feedback loop [Stein-lechner, 1996; Vanecek, 1998]. Melatonin has beenimplicated in the regulation of several periodicfunctions of mammalian physiology in a light-de-pendent fashion [Zawilska, 1996; Brzezinski,1997]. Since the melatonin produced in the pinealgland is not stored, its blood level varies accordingto the level of its production [Pang, 1985], ex-change with the circulatory system [Ferreira et al.,1996], and the efficiency of uptake mechanisms[Gitler et al., 1990].

Melatonin exerts its action mainly through G-protein-coupled receptors, which according to cur-rent nomenclature [Alexander and Peters, 1998]have been divided into three groups: mt1 (formerlyMel1a), MT2 (formerly Mel1b), and MT3 (for-merly ML2). So far only mt1 and MT2 have beencloned [Jockers et al., 1998] and found to befunctional in several mammalian tissues [Vanecek,1998] including brain [Nonno et al., 1995; Mazzuc-chelli et al., 1996; Cardinali et al., 1997; Al-Ghoulet al., 1998].

Electrophysiological experiments have revealedthat the effect of exogenous melatonin is predomi-nantly inhibitory. Melatonin depressed neuronalactivity in the SCN [Mason and Brooks, 1988;Stehle et al., 1989], reduced GABAA receptor-me-diated current in hippocampal neurons [Wan etal., 1999], and attenuated neuronal excitability inthe guinea pig hippocampus [Zeise and Semm,1985], cerebellum [Semm and Vollrath, 1984], andrat striatum [Leon et al., 1998]. Although theaction of melatonin is mostly inhibitory, excitationof Purkinje cells in the pigeon cerebellum [Semmand Vollrath, 1984], enhancement of GABAA-me-diated current in rat SCN [Wan et al., 1999], chick

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spinal cord neurons [Wu et al., 1999], and activationof rat striatal neurons [Leon et al., 1998] have alsobeen reported. These diverse actions of melatoninalso correlate with biochemical data which show theinfluence of melatonin on the release/uptake ofneurotransmitters [Dubocovich, 1983; Faillace etal., 1996] and hormones [Watanabe et al., 1998;Yasin and Forsling 1998].

Besides interacting with its surface receptors,melatonin as a small lipophilic molecule can easilycross cellular membranes [Costa et al., 1995] andaccess nuclear binding sites described in severaltissues including the brain [Cohen et al., 1978;Acuna-Castroviejo et al., 1994; Carlberg andWiesenberg, 1995], where it can regulate gene ex-pression and protect DNA from free-radical in-duced damage [Reiter, 1998; Mor et al., 1999].Intracellular melatonin can also regulate the activ-ity of phosphorylating enzymes through the interac-tion with calmodulin [Benitez-King et al., 1996;Anton-Tay et al., 1998] and modify structuralproteins [Dubocovich, 1995; Benitez-King et al.,1996]. Thus, the various actions of melatonin in thebrain are exerted at multiple cellular levels and indifferent brain regions [Morgan et al., 1994; Nonnoet al., 1995; Mazzucchelli et al., 1996; Cardinali etal., 1997; Al-Ghoul et al., 1998].

We focused our attention on the influence ofmelatonin on the mammalian hippocampus, a brainstructure essential for memory formation [Squireand Backercave, 1991; Eichenbaum, 1992]. Sincethe presence of melatonin receptors in thehippocampus of several mammalian species hasbeen demonstrated [Weaver et al., 1989; Siuciak etal., 1990; DeReviers et al., 1991; Oaknin-Bendahanet al., 1992; Nonno et al., 1995; Wan et al., 1999],we postulate that a variable level of melatonin in thecirculatory system and in the CSF [Skinner andMalpaux, 1999] could significantly alter hippocam-pal functions and potentially influence the physiol-ogy of memory formation. Although previousstudies have demonstrated the influence of mela-tonin on hippocampal neurons [Zeise and Semm,1985], a detailed pharmacological characterizationof this influence could not be done, because goodagonists/antagonists of melatonin receptors wereunavailable at that time. Therefore, the main goalof the current research was to further characterizethe influence of melatonin on mouse hippocampalneurons.

Materials and methods

Hippocampal slice preparation

One-hundred and sixty-seven slices from 65Compton white mice of both sexes were used for

these experiments. The experiments were con-ducted in accordance with the guidelines of theCUNY Animal Care and Use Committee.Hippocampal slices were prepared as describedpreviously [Wieraszko and Seyfried, 1983]. Briefly,following decapitation, the brain was removed andboth hippocampi were dissected and placed intoice-cold Ringer solution consisting of (in mM):NaCl, 124; KCl, 3.1; KH2PO4, 1.3; MgSO4, 1.3;CaCl2, 3.1; NaHCO3, 25.5; and glucose, 10.0.Hippocampi were cut into slices (350 mm) with amanual tissue chopper and placed in an incubationchamber (33°C) constantly oxygenated with aCO2/O2 (5/95%) mixture.

Electrophysiological measurements

Following a 1 hr preincubation period, hippocam-pal slices were transferred to the interface record-ing chamber which was maintained at 33°C. Abipolar, stimulating electrode was placed on theSchaffer collateral-commissural fibers (Sch. coll.)and the recording electrodes were guided into thepyramidal or radiatum layer of CA1 to record thepopulation spike or EPSP, respectively. In orderto observe antidromically-evoked potentials, thestimulating and recording electrodes were placedin the alveus and pyramidal cell layer, respectively.The magnitude of the population spike and EPSPwas measured by a computer program as the aver-age distance between the highest negativity andpreceding and following positivities (see Fig. 1A,vertical dashed line). The strength of the stimula-tion in all tested slices was adjusted at the begin-ning of each experiment to approximately 75% ofthe maximal response. The potential was moni-tored at this level for the next 15–20 min, andexperiments were performed only on slices demon-strating stable responses to low frequency (0.03Hz) stimulation. Each experiment was performedon a separate slice. The working stock solution (10or 20 mL) was added directly to the 1 mL record-ing chamber with a Hamilton syringe to obtain thedesired final concentration. The change in the sizeof the potential was expressed as a percentage ofchange over the average control value observedbefore the treatment.

Preparation of melatonin, 6-chloromelatonin, and luzindole

Melatonin, its agonist and antagonists are hydro-phobic molecules and they were all dissolved inethanol to obtain stock solutions which were thendiluted with Ringer’s to make a working stock.The working stock was added (10–20 mL) directlyto the recording chamber to give the desired final

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concentration. Since solubility of each of thesecompounds in Ringer’s was different, we estab-lished the following procedures to prepare solu-tions of each of these molecules. Melatonin(N-acetyl-5-methoxy-tryptamine-Sigma, St. Louis,MO) was prepared as a 250 mM stock solution in100% ethanol and diluted with Ringer’s to a work-ing stock of 62.5 mM in 25% ethanol. Sixteenmicroliters of the working solution was added tothe chamber to give a final concentration of 1 mM(0.4% ethanol). 6-Chloromelatonin (Sigma) wasprepared as a 100 mM stock solution in 100%ethanol and was diluted with Ringer’s to obtain a10 mM solution in 10% ethanol. Ten microliters ofworking solution was then added to obtain a finalconcentration of 100 mM 6-chloromelatonin (0.1%ethanol) in the recording chamber.

Luzindole (Tocris) was prepared as a 100 mMstock solution in 100% ethanol and diluted to 10mM working solution in 40% ethanol. It was

added to the recording chamber in the appropriatevolume to achieve a 100 mM final concentration(0.4% ethanol) of luzindole. Statistical analysiswas performed using the unpaired, two-tailed t-test.

Results

The main goal of our research was to determinethe influence of melatonin on hippocampal evokedpotentials. We evaluated changes in the popula-tion spike in order to determine if the number ofneurons activated by a single electrical pulse canbe modified by exogenous melatonin. On severaloccasions we also measured the effect of melatoninon the EPSP in order to investigate changes insynaptic efficacy and to determine if melatonin canaffect both parameters in a similar way.

Melatonin is a hydrophobic molecule and is nothighly water soluble. Therefore, we used ethanol

Fig. 1. The influence of ethanol andmelatonin on hippocampal evokedpotentials. In this and Figs. 3–5 theline in the graph shows the magnitudeof the evoked potential (populationspike or EPSP) recorded during thewhole experiment and the inserts de-pict the average of a few potentialsrecorded during the time period indi-cated by the horizontal bar at the endof arrows which mark the time ofrecording. The dashed and dottedlines shown on the potential Aa areimaginary lines drawn by the com-puter to calculate the value of thepotential (vertical dashed line). Cali-bration marks are shown in the lowerright corner of the figure. A, The influ-ence of ethanol (0.4%) on the popula-tion spike. Addition of ethanol isindicated by control vehicle ‘‘cv’’ ar-rows. Note that ethanol added threeconsecutive times at the concentrationof 0.4% had no significant effect onthe population spike. B, The influenceof 1 mM melatonin on the populationspike; addition of melatonin is markedby an arrow; potentials a, b, and cdepict the shape of the populationspike 10 min before addition of mela-tonin, at its maximal effect and at theend of recovery phase, respectively. C,The influence of 1 mM melatonin onEPSP. The rest of explanation as in B.

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Fig. 2. The influence of different concentrations of melatonin on hippocampal potentials. Solid and crossed bars representaverages during maximal effect of melatonin and at the recovery phase, respectively. A, The concentration-dependent depressionof the population spike. B, Attenuation of EPSP by 1 mM melatonin. The number of slices tested is indicated by the letter ‘‘n’’at the top of each bar. The delay (in min) between addition of melatonin and its maximal effect is indicated at the bottom of solidbars; the delay between melatonin addition and maximum recovery is shown at the bottom of crossed bars; * PB0.02,** PB0.0004, *** PB0.0001.

as a vehicle to dissolve and effectively distributemelatonin throughout the tissue. The first part ofour research was to determine if ethanol itself hadany influence on hippocampal, evoked potentials.As shown in Fig. 1A, ethanol added three consec-utive times (3×0.4%) had no significant effect onthe population spike (Fig. 1A; representative of 12experiments which all gave similar results) or onthe EPSP (n=3, data not shown). In the nextphase of our research we evaluated the influence ofmelatonin on the population spike and EPSP. Fig.1B,C illustrate the influence of 1 mM melatoninon the population spike and EPSP, respectively,and Fig. 2A,B (solid bars) show averaged resultsfor different melatonin concentrations for the pop-ulation spike and EPSP, respectively. Melatoninapplied at 0.1 mM had no apparent effect for theduration of experiments (approximately 60 min)and the magnitude of the population spikerecorded in the presence of 0.1 mM melatonin wasused as a control for all statistical comparisons(Fig. 2).

Melatonin applied at higher concentrations de-pressed the population spike in a dose-dependentfashion (Fig. 2). The population spike was reducedto 61.899.1% (n=12; PB0.02), 32.295.9%

(n=27; PB0.0004), and 14.395.0% (n=21;PB0.0001) following the application of 0.5, 1.0and 2.0 mM melatonin, respectively (Fig. 2). Notethat the EPSP seems to be less affected. Whilemelatonin at 1 mM reduced the population spikeby over 60%, EPSP was reduced by only 40% inthe same experimental conditions (compare Fig.2A,B). The delay between the time of melatoninapplication and its maximal depressive effect (indi-cated at the lower part of each solid bar in Fig. 2)was inversely related to the melatonin concentra-tion and lasted 16.193, 13.791 and 8.592 minfor 0.5, 1.0 and 2.0 mM melatonin, respectively.The melatonin-induced depression of the popula-tion spike and EPSP was not permanent and thepotentials recovered either fully or partially de-pending on the melatonin concentration (Fig.1B,C and Fig. 2A,B, crossed bars for the popula-tion spike and EPSP, respectively). While full re-covery was observed for 0.5 mM melatonin after62.392.6 min (n=9), only partial recovery wasrecorded following addition of 1.0 mM melatonin(64.997.5 min; n=25) or 2.0 mM melatonin(78.094.3 min; n=21). The potentials which re-covered after application of 1.0 and 2.0 mM mela-tonin were still significantly lower than controls

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(PB0.002 and PB0.01 for 1.0 and 2.0 mM mela-tonin, respectively).

A melatonin analog, 6-chloromelatonin (Cl-melatonin), was more potent in attenuation of thepopulation spike than melatonin itself. At 0.1 mMCl-melatonin depressed the population spike to84910% (n=7; n.s.) and 0.5 mM Cl-melatoninreduced the population spike to 23.597.1% (n=10; PB0.0007) (Fig. 3B). The time period betweenapplication of Cl-melatonin and its maximal effectwas also concentration-dependent and equalled19.794.0 and 13.993.1 min for 0.1 and 0.5 mMCl-melatonin, respectively. Recovery was also ob-served in these experiments. The population spikerecovered completely (10293%; n=4; with a de-lay of 68.0912.5 min) and up to 50% (51915%;n=10; with a delay of 73.0915 min) followingapplication of 0.1 and 0.5 mM Cl-melatonin, re-spectively (Fig. 3B).

When the population spike (Fig. 4, potential a2)was blocked with 2 mM kynurenic acid (a broadantagonist of glutamergic receptors) the remainingshort-latency potential (Fig. 4, potential a1), rep-resenting the number of activated Schaffer collat-eral-commissural fibers, which preceded the

population spike on our recordings, was not influ-enced by melatonin (compare potentials a1, b1,and c1 in Fig. 4).

In order to determine the mechanism of mela-tonin action we used luzindole, a specific MT2melatonin receptor antagonist [Dubocovich, 1998;Dubocovich et al., 1998]. Luzindole, at a concen-tration of 100 mM reduced the magnitude of thepopulation spike to 61.595.8% (n=13). The po-tential remained unchanged at this reduced leveland was only slightly reduced further to 52.396.4% (n=13, n.s.) by a subsequent addition of 1.0mM melatonin. The subsequent recovery of thepotential is very small (Fig. 5B, crossed bar). Fig.5A depicts a representative experiment and Fig.5B provides the averaged data. As indicated inFig. 5C the pattern of changes in EPSP followedthe changes in the population spike althoughEPSP reduced by luzindole was further attenuatedby the subsequent addition of melatonin. Thus, inthe presence of luzindole the influence of mela-tonin on EPSP seemed to be more pronouncedthan the effect on the population spike. The smallmelatonin-induced depression of EPSP was fol-lowed by a partial recovery period (Fig. 5C).

Fig. 3. The influence of 0.5mM 6-chloromelatonin on thepopulation spike. A, Exampleof an individual experiment.The addition of 6-chloromela-tonin is marked by an arrow.For further explanations seethe legend for Fig. 1. B, Theconcentration-dependent de-pression of the populationspike by 6-chloromelatonin-average results; *P B0.0007.For further explanation seethe legend for Fig. 2B.

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Fig. 4. The influence of mela-tonin on the population spikeand fiber potential recordedsimultaneously. Solid and dot-ted lines indicate the change inthe magnitude of the popula-tion spike and fiber potential,respectively. Potential ‘‘a’’shows the fiber potential (a1)and population spike (a2)recorded simultaneously fromthe same slice. Note that fol-lowing elimination of the popu-lation spike by addition of 2mM kynurenic acid (markedby an arrow) the fiber potentialremains unchanged (comparepotentials a and b). The subse-quent addition of 1mM mela-tonin had no influence on thefiber potential (compare poten-tials b and c). See legends toFigs. 1 and 2 for further de-tails. This figure represents oneof three experiments which allgave similar results.

However, our efforts to investigate this phe-nomenon further were hampered by the luzindole-induced variability in the size of EPSP.

Discussion

Melatonin and commercially available ligands ofits receptors are hydrophobic molecules with lim-ited solubility in water. Although according tosome reports (Shida et al., 1994) it is possible toincrease the solubility of melatonin in Ringer’s, wedecided to use ethanol as a vehicle to dissolve anddistribute melatonin and its analogs throughoutthe tissue. In the first part of our study we demon-strated that ethanol had no influence onhippocampal evoked potentials at the concentra-tions tested. Since in all experiments aimed to testthe influence of melatonin and its analogs the finalconcentration of ethanol never exceeded controlconcentrations, we attributed any observed effectsto the sole action of tested molecules.

Of the three types of potentials recorded in ourexperiments: fiber potential, excitatory postsynap-tic potential (EPSP), and population spike onlythe last two were depressed by melatonin. Conse-quently, we concluded that the properties of thepresynaptic axonal membrane (generation andpropagation of the action potential) are not influ-enced by melatonin. The most likely processes tobe affected are either synaptic transmission, post-synaptic membrane depolarization, and/or cell ex-

citability. Depression of the population spike maysuggest changes in the soma of the neurons, mod-ification of the synaptic efficiency, or a combina-tion of both. Attenuation of EPSP reflects changesthat occur exclusively at the synapse. Although thepopulation spike and EPSP were reduced, the for-mer was more affected. Therefore, one can specu-late that although the primary action of melatoninis concentrated at the stimulated synapse (paralleldepression of EPSP and population spike), otheractions of melatonin, expressed as a quantitativelydifferent effect on EPSP and population spike, arepossible. These could include changes in excitabil-ity of the postsynaptic cell, or modulation of theinhibition [Velazquez et al., 1999; Wan et al.,1999]. A decrease in cerebellar [Semm and Voll-rath, 1984] and hippocampal cell excitability [Zeiseand Semm, 1985] were indeed reported, althoughat much higher concentration range of melatonin(5–10 mM).

The time necessary to observe melatonin andCl-melatonin actions was concentration-dependentand ranged from approximately 8 to 16 min, forhigher and lower concentrations of agonist, re-spectively. Since this time period included diffu-sion of the tested molecule and its action, weconcluded, accepting this short delay as an indica-tor, that both agonists interacting with melatoninreceptors influenced either fast synaptic processes,or the mechanisms directly involved in regulationof cell excitability. The inhibitory action of mela-

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tonin in our experiments was significantly reducedby luzindole, an antagonist of MT2 melatoninreceptors [Dubocovich et al., 1997]. It should bestressed, however, that luzindole itself exerted par-tial agonistic action and reduced the potential by40%. The subsequent addition of 1 mM melatoninhad no further significant effect, although 1 mMmelatonin reduced the potential by over 60%,when applied alone. This implies that there is asignificant overlap of luzindole and melatonin ac-tions. Therefore, we concluded that the action ofmelatonin was mediated mostly by MT2 receptors,which have already been described in the mousehippocampus [Oaknin-Bendahan et al., 1992; Wanet al., 1999]. This conclusion is further supported

by the ineffectiveness of prazosin in reducing ef-fects of melatonin (unpublished observation). Pra-zosin is an antagonist of MT3 melatonin receptors[Lucchelli et al., 1997; Alexander and Peters, 1998]which do not mediate any action of melatonin inmammalian tissues [Alexander and Peters, 1998].

The most appealing and straightforward expla-nation of the melatonin-induced attenuation of thepopulation spike and EPSP could be a MT2 recep-tor-mediated interference with the fast synapticmechanisms. MT2 receptors are localized in theretina presynaptically [Dubocovich, 1983; Dubo-covich et al., 1998] and if this is also the case in themouse hippocampus, they are in good position tomodulate neurotransmitter release. Indeed, mela-

Fig. 5. Inhibition of themelatonin-induced attenua-tion of evoked potentials byluzindole. A, Luzindole (100mM), although slightly attenu-ated the population spike (A)and EPSP (C) by itself, pre-vented the inhibitory actionof subsequently added mela-tonin (1 mM). B, The averageresults of luzindole action;solid bars represent the mag-nitude of the population spikeafter addition of luzindolealone (left) and after additionof melatonin in the presenceof luzindole (right); thecrossed bar depicts the recov-ery of the potential followingaddition of luzindole andmelatonin. For additional ex-planations see the legend toFig. 2. C, The influence ofmelatonin (1 mM) on EPSPin the presence of luzindole(100 mM). Note variations inthe size of EPSP following theaddition of luzindole. For therest of the explanation see toFig. 2 legend.

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tonin was shown to inhibit Ca2+ uptake in brainsynaptosomes [Zisapel and Laudon, 1983; Vacaset al., 1984], a process essential for the initiation ofneurotransmission. This action of melatonin mayreduce neurotransmitter release, and subsequentlyattenuate synaptic efficiency. Melatonin can alsoshift the balance between glutamate release anduptake in retina [Faillace et al., 1996] and it mayhave a similar effect in the hippocampus, whereglutamate is a major excitatory neurotransmitter[Wieraszko, 1983a]. We have demonstrated in thepast [Wieraszko, 1983b] and confirmed recently[El-Sherif et al., 1999] that any disturbance in thisequilibrium may have profound effects on theefficiency of hippocampal synapses. Melatonin canalso modulate glutamate-driven potentialsrecorded from hippocampal slices by direct inter-action with glutamergic receptors [Escames et al.,1996].

The recently observed activation of potassiumconductance and subsequent neuronal hyperpolar-ization [Jiang et al., 1995] induced by melatonincould provide an alternative explanation for itsinhibitory action. Potassium channels are acti-vated by subunits of pertussis toxin-sensitive Gproteins and it is well documented that most of themelatonin receptors are coupled to G proteins[Reppert et al., 1994; Fukawa et al., 1995;Vanecek, 1998] which regulate adenylate cyclaseactivity and the level of cAMP [Niles andHashemi, 1990; Niles et al., 1991]. However, notall melatonin effects have to be related to G-protein action, since melatonin receptors have theability to exist in high and low affinity states,which are G-protein coupled and uncoupled, re-spectively [Morgan et al., 1994]. Indeed, guaninenucleotide insensitive melatonin binding sites havebeen described in bovine hippocampus [Nonno etal., 1995]. Testing the influence of pertussis toxinmay help to distinguish between direct actions ofmelatonin and effects mediated by G-protein-linked melatonin receptors. The inhibitory actionof melatonin could also be mediated by an in-crease in inhibition [Zeise and Semm, 1985], butrecently it has been reported that melatonin medi-ated the attenuation of GABAA currents inhippocampal slices [Wan et al., 1999] which con-tradicts this suggestion. Also, the recently reportedanticonvulsant action of melatonin [Velazquez etal., 1999] appeared to be GABA-independent.

In the intact organism exogenously appliedmelatonin is quickly cleared from the circulatorysystem due to accumulation by the tissue [Ferreiraet al., 1996] and metabolism in the liver [Borjiginet al., 1999]. In our experimental design the sliceswere not perfused and it could be assumed that

they were exposed to the steady concentration ofmelatonin and its analogs for the duration of theexperiments. However, the effect of melatonin inthe majority of experiments was biphasic: the de-pression was followed by a recovery phase. Asdiscussed above, there are several mechanismsthrough which melatonin by itself can depressevoked potentials. In addition to these melatonin-mediated effects, the kynurenines, bioactivemetabolites of melatonin [Pellicciari et al., 1994]which depress glutamergic neurotransmission inthe striatum [Leon et al., 1998] can, separately orjointly with melatonin be responsible for attenua-tion of hippocampal potentials. The observed de-lay for the maximal melatonin effect may be longenough to initiate melatonin metabolism. In mostof the experiments the attenuation was followedby a recovery. This recovery could be explained intwo ways. First, the melatonin level may decreasedue to its uptake and/or metabolism by the tissue.Second, assuming that the action of melatonin wasexerted through its receptors, one can speculatethat those receptors became desensitized and theeffect of melatonin and its analog diminished inspite of sustained agonists concentrations. Thisdesensitization would be inversely related to themelatonin concentration, since it was almost com-plete with lower concentrations of agonist. Al-though on the basis of our results we do not favorany of these two options, a melatonin-induceddown-regulation of its own receptors in vivo hasbeen observed [Gauer et al., 1993; Faillace et al.,1995] and experiments are currently under way toaddress these issues in our in vitro model.

Although the melatonin added to the slice-con-taining chamber was in the low millimolar concen-tration range, we do not know whatconcentrations of melatonin reached the neuronsinside of the hippocampal slice. The melatoninconcentration in the intact organism is not uni-form and varies depending on the structure andtime of the day [Pang and Brown, 1983; Skinnerand Malpaux, 1999]. While the concentration ofmelatonin in the plasma was reported to be in thenanomolar range [Cardinali et al., 1997], the braintissue has the ability to accumulate melatonin peri-odically to a level exceeding 50 times its plasmaconcentration [Pang et al., 1990; Cardinali et al.,1997]. Also, the distribution of melatonin withinthe brain is not uniform and several-fold differ-ences in the melatonin content were reported be-tween brain ventricles [Skinner and Malpaux,1999]. It is worth mentioning that melatonin isused in humans in concentrations ranging from 5mg/day in sleep disorders to 700 mg/day in cancerpatients [Brzezinski, 1997]. Therefore, melatonin

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concentrations interacting with neurons in thehippocampal slices may be physiologically andpharmacologically relevant.

In conclusion, melatonin, its analog and/ormetabolites attenuate hippocampal evoked poten-tials in a concentration-dependent, receptor-medi-ated manner. The influence of melatonin onhippocampal neurons may be of special impor-tance since the hippocampus is regarded as anintegral component of a number of memory sys-tems [Sherry and Schacter, 1987; Squire and Back-ercave, 1991]. Behavioral investigation ofhippocampal structure and cognitive function tendto emphasize either spatial memory acquisition[Nadel, 1991; Squire and Zola-Morgan, 1991], orthe role of non-spatial aspects of memory, such asdeclarative memory in humans [Squire and Zola-Morgan, 1991]. In the intact organism, hippocam-pal neurons will be exposed to melatoninsynthesized and secreted in a circadian rhythm.Considering our data, it reasonable to assume thatthe activity of hippocampal neurons would bedictated by the oscillating concentration of mela-tonin. Hence, melatonin can be considered as apowerful endogenous factor regulating the mecha-nisms of memory localized in the hippocampus.We are in the process of elucidating the exactmechanisms of melatonin action on hippocampalneurons.

Acknowledgments

Supported in part by an OMRD Fellowship to the CSI/IBR Center for Developmental Neuroscience to YasirEl-Sherif.

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