SYNAPSE 70:181–186 (2016)

Melatonin Receptor Activation Increases Glutamatergic Synaptic Transmission in the Rat Medial Lateral Habenula KATHERINE M. EVELY,1,2 RANDALL L. HUDSON,3 MARGARITA L. DUBOCOVICH,1 AND SAMIR HAJ-DAHMANE1,2* 1 Department of Pharmacology and Toxicology, University at Buffalo, State University of New York, Buffalo, New York 2 Research Institute on Addictions, University at Buffalo, State University of New York, Buffalo, New York 3 Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York

KEY WORDS

melatonin; habenula; EPSC; melatonin receptor; glutamate synapse

ABSTRACT Melatonin (MLT) is secreted from the pineal gland and mediates its physiological effects through activation of two G protein-coupled receptors, MT1 and MT2. These receptors are expressed in several brain areas, including the habenular complex, a pair of nuclei that relay information from forebrain to midbrain and modulate a plethora of behaviors, including sleep, mood, and pain. However, so far, the precise mechanisms by which MLT control the function of habenula neurons remain unknown. Using whole cell recordings from male rat brain slices, we examined the effects of MLT on the excitability of medial lateral habenula (MLHb) neurons. We found that MLT had no significant effects on the intrinsic excitability of MLHb neurons, but profoundly increased the amplitude of glutamate–mediated evoked excitatory post-synaptic currents (EPSC). The increase in strength of glutamate synapses onto MLHb neurons was mediated by an increase in glutamate release. The MLT-induced increase in glutamatergic synaptic transmission was blocked by the competitive MT1/ MT2 receptor antagonist luzindole (LUZ). These results unravel a potential cellular mechanism by which MLT receptor activation enhances the excitability of MLHb neurons. The MLT-mediated control of glutamatergic inputs to the MLHb may play a key role in the modulation of various behaviors controlled by the habenular complex. Synapse 70:181–186, 2016. VC 2016 Wiley Periodicals, Inc. INTRODUCTION Melatonin (MLT) or 5-methoxy-N-acetyltryptamine, a naturally occurring circadian hormone is primarily synthesised and released by the pineal gland and retina (Cardinali, 1981; Tosini, 2000). Levels of melatonin synthesis and secretion peak at night during the hours of darkness and are inhibited by light during the day (Reiter, 1991). The physiological effects of this circadian hormone are largely mediated by two G-protein coupled receptors, MT1 and MT2 (Dubocovich et al., 2010), which are distributed throughout the central nervous system (CNS). As such, in addition to the regulation of the circadian rhythm (Dubocovich, 2007; Zhu and Zee, 2012) melatonin is involved in the regulation of an array of physiological functions, such as seasonal reproduction (Reiter, 1980), neurogenesis (Ramırez-Rodrıguez et al., 2009), cancer (Mediavilla et al., 2010), pain (Wilhelmsen et al., 2011), sleep (Pandi-Perumal et al., 2008), Ó 2016 WILEY PERIODICALS, INC.

depression (Srinivasan et al., 2010, 2012) and anxiety (Lanfumey et al., 2013). Results from previous studies have shown that the habenular complex, a well-conserved structure thought to have evolved in close relation to the pineal gland (Guglielmotti and Cristino, 2006), expresses melatonin receptors (Adamah-Biassi et al., 2014; Weaver et al., 1989). Importantly, functional studies have indicated that some of the physiological and *Correspondence to: Samir Haj-Dahmane, Research Institute on Addictions, 1021 Main Street, State University of New York at Buffalo, Buffalo, NY 14302. E-mail: [email protected] Author Contributions: K.M.E conducted all electrophysiological experiments under the guidance of S.H-D. S.H-D. designed electrophysiological experiments. M.L.D. contributed to the pharmacological aspects of experimental design including reagent preparation. All authors contributed to the preparation of the manuscript and read the final version. Received 3 December 2015; Revised 13 January 2016; Accepted 20 January 2016 DOI: 10.1002/syn.21892 Published online 22 (wileyonlinelibrary.com).

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behavioral effects of MLT may be mediated, at least in part, by melatonin receptor signaling in the habenula (deBorsetti et al. 2011; Zhao and Rusak, 2005). Structurally, the habenular complex is divided into medial and lateral nuclei, each with distinct connectivity (Aizawa et al., 2012; Herkenham and Nauta, 1979; Klemm, 2004). Functioning as a relay-station from forebrain to midbrain, the habenula is implicated in the regulation of numerous physiological functions; including sleep and stress homeostasis (Hikosaka, 2010). Despite accumulating evidence suggesting a functional link between the habenular complex and melatonergic system, very little is known about the cellular mechanisms by which MLT controls the function of habenula neurons. Here, we investigate the effect of MLT on the neuronal excitability and glutamatergic synaptic transmission in the rat medial lateral habenula (MLHb). Our results reveal that MLT increases the excitability of MLHb neurons by enhancing glutamatergic synaptic drive in the MLHb. MATERIALS AND METHODS Slice preparation Male Sprague–Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN) and group housed at the University at Buffalo Research Institute on Addictions Laboratory Animal Facility on a 12/12 light/dark cycle with free access to food and water. All experimental procedures were in accordance with the guideline of the National Institute of Health and were approved by the University at Buffalo Institutional Animal Care Use Committee. Rats (3- to 6-weeks old) were sacrificed by decapitation under isoflurane anesthesia between ZT 3 and ZT 5 (ZT 0: Lights ON). The brains were immediately dissected and placed in ice cold modified artificial cerebrospinal fluid (ACSF) of the following composition (mM); 110 choline chloride; 2.5 KCl; 0.5 CaCl2; 7 MgSO4; 1.25 NaH2PO4; 26.2 NaHCO3; 11.6 sodium Lascorbate; 3.1 sodium pyruvate and 25 glucose. Coronal slices (300 mm) containing the habenula was obtained using a vibrating blade microtome (Lancer series 1000; Leica Biosystems, St. Louis, MO). Slices were incubated for at least 45–60 min at 35–378C in regular ACSF of the following composition (mM), 119 NaCl; 2.5 CaCl2; 1.3 MgSO4; 1 NaH2PO4; 26.2 NaHCO3; and 11 glucose continuously gassed with 95% O2–5% CO2. After 1 h of recovery, slices were transferred to the recording chamber (Warner Instruments, Hamden, CT) fixed to an upright microscope (Olympus BX 51, Olympus, Tokyo, Japan). The slices were continuously perfused (2–3 mL min21) with ACSF saturated with 95% O2 to 5% CO2 maintained at 308C. Synapse

Electrophysiological recordings The lateral division of the habenula is further divided into a medial (MLHb) and lateral division. Neurons from the MLHb, adjacent to the border between the medial and lateral habenula were visualized with a BX51 Olympus microscope fitted with a 403 water-immersion lens, and equipped with differential interference contrast and infrared optical system. Somatic whole cell recordings were obtained with patch electrodes (3–5 MX) filled with internal solution containing (mM): 120 potassium gluconate; 10 KCl; 10 sodium phosphocreatine; 10 HEPES; 1 MgCl2; 1 EGTA; 2 Na2-ATP; 0.25 Na-GTP, at a pH of 7.3 and osmolarity of 280–290 mosmol1–1. For current clamp recordings, neurons were artificially maintained at 270 mV to avoid spontaneous activity while firing properties were assessed. Excitatory post-synaptic currents (EPSCs) were evoked using a monopolar stimulating electrode filled with ACSF and placed 50–100 mm from the recorded neuron. The duration (100–200 ms) and intensity (5–20 V) of the stimulus were adjusted to 75% of the maximum amplitude of EPSCs. EPSCs were evoked at a frequency of 0.1 Hz. In some experiments, a paired stimulus was delivered at inter-stimulus interval of 40 ms to determine the paired pulse ratio (PPR). Membrane currents were amplified with a Axoclamp 2B or Multiclamp 700B amplifier (Molecular Devices, Union City, CA), digitized with Digidata 1440 and collected using pCLAMP 10 software (Molecular Devices, Union City, CA). All chemicals used for ACSF preparation were purchased from Fisher Scientific (Pittsburgh, PA). Picrotoxin and GYKI 54,266 (4–(8-Methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin5-yl)-benzenamine hydrochloride) were purchased from Tocris Cookson (Ellisville, MO, USA). MLT (Nacetyl-5-methoxytryptamine) was purchased from Sigma Chemicals. Luzindole (N-acetyl-2-benzyltryptamine, LUZ) was synthesized by Dr. R. V. Rajnarayanan (Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14,214). For experiments testing the ability of LUZ to block the effect of MLT, slices were preincubated in ACSF containing LUZ (10 mM) for 30 min prior to recording. Data analysis Clampfit 10.2 software (Molecular Devices) was used to analyze EPSC recordings. EPSC amplitude was calculated as the difference between the average current during the 2 ms window at peak amplitude and the average baseline current taken 5 ms before the stimulus artifact. For each cell, recordings were normalized to a baseline of 10 min prior to drug application. PPR was calculated by dividing the amplitude of the second EPSC by the amplitude of

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the first (PPR 5 EPSC2/EPSC1). For each cell, the PPR was calculated during baseline and peak effect following drug application. The coefficient of variation (CV) was calculated by dividing the standard deviation by the EPSC mean amplitude. Graphs were plotted and statistics computed using Origin 8.0 software (Microcal Software, Northampton, MA). All comparisons were made using a student t test and P < 0.05 was considered significant. RESULTS Whole cell recordings were obtained from 60 neurons located in the dorsal region of the border between the medial and medial lateral habenula. On the basis of electrical properties, we found two distinct classes of neurons, previously described by Weiss and Veh (2011). Firing was evoked by a various hyperpolarizing and depolarizing current steps (i) or by a single hyperpolarizing pulse (ii–iii). First, “tonic firing neurons” can be described by regular firing of consecutive single action potentials (Fig. 1A). On the other hand, “burster neurons” fire in irregular bursts or groups of action potential (Fig. 1B). To test whether MLT alters the excitability of MLHb neurons, we first examined the effect of MLT on the holding current of MLHb neurons. Bath application of MLT (1 lM) did not significantly alter the holding current of all neurons sampled (Fig. 1C). Previous anatomical studies have shown that MLHb neurons receive glutamatergic input from various forebrain brain regions (Poller et al., 2013; Qin and Luo, 2009). Therefore, we next examined whether MLT can alters the strength of glutamatergic inputs impinging onto MLHb neurons. To that end, glutamatergic-mediated EPSCs were evoked in the presence of picrotoxin (100 lM) and recorded from neurons voltage clamped at 270 mV, a membrane potential where most, if not all, EPSCs are carried by AMPA receptors. Following stable baseline recording, bath application of MLT (1 lM) significantly increased the amplitude of EPSCs. A time course of a typical experiment depicting the effect of MLT on EPSC amplitude is shown in Figure 2A. On average, bath application of MLT (1 lM) increased the EPSC peak amplitude to 140% 6 11% of baseline, P < 0.05 (Fig. 2B, n 5 7), regardless of firing phenotype shown in Figure 1. Several mechanisms can underlie the MLTmediated increase in EPSC amplitude. These include an increase in the function and/or number of AMPA receptors, an increase in presynaptic glutamate release or a combination of the two. Therefore, in order to elucidate the mechanism by which MLT potentiates the amplitude of EPSCs, we examined its effect on the PPR and CV, parameters that reflect the probability of neurotransmitter release and how it changes throughout the time course of drug applica-

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tion. Bath application of MLT (1 lM) resulted in a significant decrease in the PPR (PPR control 5 1.04, PPR MLT 5 0.74, n 5 4, P < 0.05, Fig. 2C). Similarly, a decrease in the CV during application of MLT was observed (CV control 5 0.22, CV MLT 5 0.12, n 5 4, P < 0.05, Fig. 2D). Such findings indicate that the increase in EPSC amplitude is mediated by an increase in glutamate release. The physiological effects of melatonin are known to be mediated by receptor-dependent or receptorindependent mechanisms (Dubocovich et al., 2010). On the basis of the evidence that MLT receptors are present in the habenular complex of multiple species (Adamah-Biassi et al., 2014; Weaver et al., 1989), we hypothesized that the MLT-induced increase in glutamate release is mediated by activation of MLT receptors. We tested this notion by examining the impact of LUZ, a competitive non-selective MT1/MT2 receptor antagonist (Dubocovich, 1988), on the MLT-mediated potentiation of EPSC amplitude. We found that in slices treated with LUZ (10 lM) bath application of MLT (1 mM) had no significant effect on EPSC amplitude (n 5 6, Fig. 3). This pharmacological finding indicates that MLT enhances glutamatergic synaptic transmission in MLHb neurons via activation of MT1/ MT2 receptors. DISCUSSION Despite the close proximity and evolutionary relationship between the pineal gland and the habenular complex, very little is known about the functional role of MLT in the MLHb. In the present study, we report that in the MLHb MLT does not alter the resting membrane potential/holding current of either “tonic” or “bursting” neurons. In contrast, our results show that MLT profoundly increased the amplitude of glutamate-mediated EPSCs by enhancing excitatory drive in the habenular complex. The MLTmediated control of glutamatergic inputs is likely to play a critical role in numerous physiological functions controlled by the habenula. The MLT-mediated increase in the amplitude of glutamate-mediated EPSCs is consistently associated with a significant decrease in both the PPR and CV, two synaptic parameters that assess the probability of neurotransmitter release. Because a decrease of both parameters indicates an increase in glutamate release, we concluded that MLT enhances glutamatergic synaptic transmission to MLHb neurons by a presynaptic increase in glutamate release. Interestingly, previous studies have shown that MLT decreases glutamate release at Schaffer collateral CA1 hippocampal synapses (Choi et al., 2014). The discrepancy between this study and our finding could be attributed to the differing concentration of MLT, subtype/distribution of receptors, as well as, regional and cell specific effects. Importantly, the finding that Synapse

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Fig. 1. MLT has no effect on the holding current of two distinct classes of MLHb. neurons. A. Depicts typical tonic firing, (i) response to various hyperpolarizing and depolarizing current steps, (ii) response to a single hyperpolarizing pulse, *note: rebound firing, (iii)

enlarged timescale traces of typical tonic firing. B. Depicts typical burster firing, i–iii represent equivalent experimental parameters to A but illustrate a ‘bursting’ MLHb neuron. C. Average holding current is not significantly changed in response to MLT (1 lM).

the MLT-mediated increase in EPSC amplitude is blocked by a MT1/MT2 receptor antagonist LUZ indicates that this effect is signaled by presynaptic MLT receptors. However, future anatomical studies are required to determine the precise ultrastructural distribution of MT1 and MT2 receptors in the MLHb. Previous reports suggest that MLT receptor expression is confined to the medial habenula. However, the findings presented here may be attributed to the modulation of the unidirectional medial to lateral

intrinsic connectivity of the habenular nucleus (Bianco and Wilson, 2009). The most recognized signaling pathway associated with MT1 and MT2 receptors is through Gi/o inhibition of adenylyl cyclase resulting in decreased formation of cAMP (Drew et al., 2002), activation of inward rectifier potassium channels (GIRK) (Hablitz et al., 2015; van den Top et al., 2001) and inhibition of calcium currents (For review see, Dubocovich et al., 2010). Classically, activation of these signaling

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Fig. 2. MLT potentiates the amplitude of glutamate-mediated EPSCs. A. A representative experiment depicting the effect of MLT (1 lM) on the amplitude of EPSCs. Inset: EPSC traces before (black) and after (red) MLT, scale bar represents 50 pA and 10 ms. B. A summary graph representing the average effect of MLT (1 lM) on the amplitude of EPSCs (n 5 7). C. PPR before and after MLT (n 5 4). D. CV before and after MLT (n 5 4).

Fig. 3. LUZ blocks the MLT-mediated increase in amplitude of glutamate-mediated EPSCs. Average response to MLT (1 lM) in the presence LUZ (10 lM).

pathways decreases neuronal activity and inhibits neurotransmitter release (van den Top et al., 2001). Unexpectedly, here we find that activation of MT1/ MT2 increases glutamate release, suggesting that different signaling mechanisms mediate the effect of MT1/MT2 receptors in the MLHb. Interestingly, results from recent studies have shown that MT1/ MT2 heteromers can activate phospholipase C (PLC) (Baba et al., 2013), an enzyme known to metabolize phophatidyl-inositol biphosphate (PIP2), leading to the formation of 1,-2-dacylglycerol (1,2-DAG) and inositol triphosphate (IP3), which in turn, increases intracellular calcium and activates PKC. Usually,

activation of this signaling pathway increases neuronal excitability and neurotransmitter release. Thus, it is possible that MT1/MT2 receptor heteromers mediate the increase in glutamatergic synaptic transmission to MLHb neurons via the PLC signaling cascade. Further, molecular studies are required to test this notion and identify the signaling cascade by which MLT receptors regulate the strength of glutamate synapses in the MLHb. The melatonin rich dorsal third ventricle is located directly adjacent to the habenula and is in close proximity to the MLT secreting pineal gland. Third ventricle levels of MLT are believed to come directly from the pineal gland and are reported to be up to twenty times greater than in peripheral circulation (Skinner and Malpaux, 1999). Hence, the habenula is ideally positioned to be the target of high concentration firstwave MLT secretions. It is not understood whether the sensitized state of habenular melatonin receptors has been altered due to consistent exposure to high levels of MLT. The affinity of MLT for its receptor is in the picomolar range (Dubocovich et al., 2010). However, it is conceivable that MLT receptors in this region are isoforms with altered affinity. Also, due to surrounding brain matter and active metabolism, a higher concentration of drug is often required to reach the target site when recording from brain slices of considerable thickness. For these reasons, we believe that the micromolar concentration of melatonin used in the present study is appropriate. Synapse

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However, further investigation characterizing the pharmacodynamics of habenular MLT receptors and the possibility of isoforms is warranted. This study demonstrates for the first time that the neurohormone MLT modulates the strength of glutamatergic synapses by activating presynaptic MLT receptors within the rat habenula. Whether MLT modulates GABAergic transmission in the region remains to be determined and will be the subject of future investigation. There is considerable overlap in behaviors implicated by both the habenula and the melatonergic system. The findings presented here provide evidence for functional habenular MLT receptors and provide a foundation for forming future hypotheses regarding the involvement of MLT in the modulation of signaling to midbrain regions responsible for various behavioral outputs. ACKNOWLEDGMENTS The authors thank Dr. R. V. Rajnarayanan (Department of Pharmacology and Toxicology, University at Buffalo, NY) for generously providing luzindole. They thank Marina Popovska-Gorevski for reviewing and providing comments on the manuscript. REFERENCES Adamah-Biassi EB, Zhang Y, Jung H, Visapragada S, Miller RJ, Dubocovich ML. 2014. Distribution of MT1 melatonin receptor promoter-driven RFP expression in the brains of BAC C3H/HeN transgenic mice. J Histochem Cytochem 62:70–84. Aizawa H, Kobayashi M, Tanaka S, Fukai T, Okamoto H. 2012. Molecular characterization of the subnuclei in the rat habenula. J Comp Neurol 520:4051–4066. Baba K, Benleulmi-Chaachoua A, Journ e AS, Kamal M, Guillaume JL, Dussaud S, Gbahou F, Yettou K, Liu C, Contreras-Alcantara S, Jockers R, Tosini G. 2013. Heteromeric MT1/MT2 melatonin receptors modulate photoreceptor function. Sci Signal 6:ra89. Bianco IH, Wilson SW. 2009. The habenular nuclei: A conserved asymmetric relay station in the vertebrate brain. Philos Trans R Soc Lond B Biol Sci 364:1005–1020. Cardinali DP. 1981. Melatonin.l A mammalian pineal hormone. Endocr Rev 2:327–346. Choi TY, Kwon JE, Durrance ES, Jo SH, Choi SY, Kim KT. 2014. Melatonin inhibits voltage sensitive Ca(21) channel-mediated neurotransmitter release. Brain Res 1557:34–42. de Borsetti NH, Dean BJ, Bain EJ, Clanton JA, Taylor RW, Gamse JT. 2011. Light and melatonin schedule neuronal differentiation in the habenular nuclei. Dev Biol 358:251–261. Drew JE, Barrett P, Conway S, Delagrange P, Morgan PJ. 2002. Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors. Biochim Biophys Acta 1592:185–192. Dubocovich ML. 1988. Luzindole (N-0774): A novel melatonin receptor antagonist. J Pharmacol Exp Ther 246:902–910. Dubocovich ML. 2007. Melatonin receptors: Role on sleep and circadian rhythm regulation. Sleep Med 8:34–42. Dubocovich ML, Delagrange P, Krause DN, Sugden D, Cardinali DP, Olcese J. 2010. International union of basic and clinical pharmacology. LXXV. Nomenclature, classification, and pharmacology

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