Melatonin inhibits tetraethylammonium-sensitive potassium channels of rod ON type bipolar cells via MT2 receptors in rat retina

Neuroscience 173 (2011) 19 –29

MELATONIN INHIBITS TETRAETHYLAMMONIUM-SENSITIVE POTASSIUM CHANNELS OF ROD ON TYPE BIPOLAR CELLS VIA MT2 RECEPTORS IN RAT RETINA X.-F. YANG, Y. MIAO, Y. PING, H.-J. WU, X.-L. YANG* AND Z. WANG*

Melatonin, a primary neurohormone synthesized and released during the nighttime mainly by the pineal gland, is known to regulate a variety of physiological processes in mammals mostly by activating two G-protein-coupled melatonin MT1 and MT2 receptors (Brzezinski, 1997; Vanecek, 1998; Dubocovich et al., 2003; Barrenetxe et al., 2004). In the vertebrate retina, melatonin is found to be synthesized by photoreceptors under the direct control of a circadian clock (Cahill and Besharse, 1992; Fukuhara et al., 2004; Tosini et al., 2007) and all three subtypes of melatonin receptors, namely MT1, MT2 and MT3, exist (Fujieda et al., 1999; Scher et al., 2003; Wiechmann, 2003; Wiechmann et al., 2004; Huang et al., 2005; Ping et al., 2008; Baba et al., 2009; Zhao et al., 2010), suggesting that retinal cells may be targets for melatonin action. There is growing evidence demonstrating neuromodulatory roles of melatonin in retinal information processing (Cosci et al., 1997; Vanecek, 1998; Tosini and Fukuhara, 2003; Wiechmann et al., 2003; Ribelayga et al., 2004; Huang et al., 2005; Alarma-Estrany and Pintor, 2007; Ping et al., 2008; Baba et al., 2009; Zhao et al., 2010). For instance, in the outer retina, in dark adapted isolated frog photoreceptors melatonin influences the cell membrane conductance (Cosci et al., 1997), and melatonin increases dark adaptation by acting directly on Xenopus rod photoreceptors (Wiechmann et al., 2003). In addition, melatonin modulates the function of cone-driven horizontal cells in teleost fish directly by altering AMPA receptor-mediated responses of these cells through the activation of MT1 receptors (Huang et al., 2005) and/or by influencing dopamine release from interplexiform and amacrine cells (Ribelayga et al., 2004). In carp rod-dominant ON type bipolar cells (Rod-ON-BCs), melatonin potentiates rod signals to these cells by modulating the activity of the metabotropic glutamate receptor expressed on these cells through the activation of MT2 receptors (Ping et al., 2008). Most recently, Baba et al. (2009) studied electroretinograms and retinal morphology in wild type and MT1 receptor deficient mice and found that melatonin modulates visual function and cell viability via the MT1 receptor. In the inner retina, our recent work also showed that melatonin potentiates glycine receptor-mediated currents in rat ganglion cells (RGCs) through a Ca2⫹-independent phospholipase C (PLC)/protein kinase C (PKC) signaling pathway by activating MT2 receptors (Zhang et al., 2007; Zhao et al., 2010). Voltage-gated K⫹ channels are one of key factors in determining the membrane excitability of neurons, and almost all neurons possess multiple K⫹ channels, which

Institutes of Brain Science, Institute of Neurobiology and State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China

Abstract—By challenging specific receptors, melatonin synthesized and released by photoreceptors regulates various physiological functions in the vertebrate retina. Here, we studied modulatory effects of melatonin on Kⴙ currents of rod-dominant ON type bipolar cells (Rod-ON-BCs) in rat retinal slices by patch-clamp techniques. Double immunofluorescence experiments conducted in isolated cell and retinal section preparations showed that the melatonin MT2 receptor was expressed in somata, dendrites and axon terminals of rat Rod-ON-BCs. Electrophysiologically, application of melatonin selectively inhibited the tetraethylammonium (TEA)-sensitive Kⴙ current component, but did not show any effect on the 4-aminopyridine (4-AP)-sensitive component. Consistent with the immunocytochemical result, the melatonin effect was blocked by co-application of 4-phenyl-2-propionamidotetralin (4-P-PDOT), a specific MT2 receptor antagonist. Neither protein kinase A (PKA) nor protein kinase G (PKG) seemed to be involved because both the PKA inhibitor Rp-cAMP and the PKG inhibitor KT5823 did not block the melatonin-induced suppression of the Kⴙ currents. In contrast, application of the phospholipase C (PLC) inhibitor U73122 or the protein kinase C (PKC) inhibitor bisindolylmaleimide IV (Bis IV) eliminated the melatonin effect, and when the Ca2ⴙ chelator BAPTAcontaining pipette was used, melatonin failed to inhibit the Kⴙ currents. These results suggest that suppression of the TEA-sensitive Kⴙ current component via activation of MT2 receptors expressed on rat Rod-ON-BCs may be mediated by a Ca2ⴙ-dependent PLC/inositol 1,4,5-trisphosphate (IP3)/PKC signaling pathway. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: patch-clamp, phospholipase C, protein kinase C, IP3. *Corresponding author. Tel: ⫹86-21-5423-7810; fax: ⫹86-21-5423-7643. E-mail addresses: [email protected] (Z. Wang) or xlyang@fudan. edu.cn (X.-L. Yang). Abbreviations: ACSF, artificial cerebral spinal fluid; BC, bipolar cell; Bis IV, bisindolylmaleimide IV; BKCa, large-conductance Ca2⫹-activated K⫹ channels; cAMP, cyclic adenosine monophosphate; [Ca2⫹]i, intracellular calcium concentration; cGMP, cyclic guanosine monophosphate; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol-bis(␤aminoethyl ether) N, N, N=, N=-tetraacetic acid; GCL, ganglion cell layer; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; INL, inner nuclear layer; IPL, inner plexiform layer; IP3, inositol 1,4,5trisphosphate; OPL, outer plexiform layer; PB, phosphate buffer; PBS, phosphate-buffered saline; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C; PTX, pertussis toxin; RGC, retinal ganglion cell; Rod-ON-BCs, rod-dominant ON-type bipolar cells; TEA, tetraethylammonium; 4-AP, 4-aminopyridine; 4-PPDOT, 4-phenyl-2-propionamidotetralin.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.11.028

19

20

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

are involved in regulating the membrane potential and spike frequency (Hille, 2001). Melatonin has been shown to modulate K⫹ channels in various cells and the actions are complex (Jiang et al., 1995; Huan et al., 2001; Hou et al., 2004; Liu et al., 2007). For instance, melatonin inhibits outward delayed rectifier K⫹ currents in hippocampal CA1 pyramidal neurons by a receptor-independent mechanism (Hou et al., 2004), but increases these currents in rat cerebellar granule cells via MT2 receptors (Huan et al., 2001; Liu et al., 2007). Several types of K⫹ channels are expressed in rat Rod-ON-BCs (Karschin and Wässle, 1990; Klumpp et al., 1995; Yazulla and Studholme, 1998; Hu and Pan, 2002). In the present work we aimed to examine effects of melatonin on K⫹ channels of Rod-ONBCs by patch-clamp techniques. Our results showed that, by activating G-protein coupled MT2 receptors, melatonin selectively inhibited tetraethylammonium (TEA)-sensitive, but not 4-aminopyridine (4-AP)-sensitive K⫹ channels of these cells through a Ca2⫹-dependent PLC/inositol 1,4,5trisphosphate (IP3)/PKC signaling pathway.

EXPERIMENTAL PROCEDURES All experimental procedures described here were in accordance with the National Institute of Health (NIH) guidelines for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996 and the guidelines of Fudan University on the ethical use of animals. During this study all efforts were made to minimize the number of animals used and their suffering. Male Sprague– Dawley rats weighing 100⬃150 g were obtained from SLAC Laboratory Animal Co. Ltd (Shanghai, China) and maintained under a 12 h/12 h light/dark cycle for at least 1 week before used.

Preparation of isolated rat Rod-ON-BC Retinal neurons were acutely dissociated by enzymatic and mechanical methods as previously described (Chen et al., 2004; Yu et al., 2006; Zhao et al., 2010) with minor modifications. In brief, animals were deeply anaesthetized with 25% urethane and killed by decapitation. Retinas were removed quickly and incubated in oxygenated Hank’s solution containing the following (in mM): NaCl 137, NaHCO3 0.5, NaH2PO4 1, KCl 3, CaCl2 2, MgSO4 1, sodium pyruvate 1, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 20 and glucose 16 adjusted to pH 7.4 with NaOH. The retinas were then digested in 5 mg/ml papain (Calbiochemical, San Diego, CA, USA) containing Hank’s solution, supplemented with 0.75 mg/ml L-cysteine for 33 min at 33.5–34.5 °C. After several rinses in Hank’s solution, the retinal neurons were mechanically dissociated with fire-polished Pasteur pipettes. Isolated retinal BCs were characterized by short bush-like dendrites emerging at one end of the soma and a long axon at the other end. These cells usually were PKC positive (Yu et al., 2006).

Immunohistochemistry and immunocytochemistry Immunofluorescence experiments were performed following the procedure described in detail previously (Chen et al., 2004), with minor modifications. Briefly, rats were anesthetized by 25% urethane and killed by decapitation. The eyecups were removed quickly and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 1 h at 4 °C, and then dehydrated with graded sucrose solutions at 4 °C. The retinas were vertically sectioned at 14 ␮m thickness on a freezing microtome (Leica, Nussloch, Germany). After washing with 0.01 M phosphate-buffered saline (PBS) (pH 7.4), the retinal sections were blocked for 1 h in 6% normal donkey serum (v/v) (Sigma, St. Louis, MO, USA),

1% normal bovine serum albumin and 0.2% Triton X-100 in PBS at room temperature, and then incubated with the primary antibodies overnight at 4 °C: goat polyclonal antibody against MT2 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal antibody against PKC alpha (1:1000 dilution; Abcam, Cambridge, MA, USA). Binding sites of the primary antibodies were visualized by incubating with DyLight™-488-conjugated donkey anti-goat IgG or Cy3-conjugated donkey antirabbit IgG 1:200 dilution (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The sections were counterstained by DAPI solution. Immunocytochemistry of isolated Rod-ON-BCs refers to Yu et al. (2006) for detailed description. Isolated cells were placed on a slide in PBS for 30 – 60 min at room temperature and fixed with 4% paraformaldehyde in 0.1 M fresh PB for 30 min, rinsed with PBS three times. They were then blocked for 1 h in PBS with 6% donkey serum plus 0.2% Triton X-100, followed by incubating with the anti-MT2 and anti-PKC alpha primary antibodies at working dilution of 1:200 and 1:1000 respectively for 2 h and further incubated with the secondary antibodies DyLight™-488-conjugated-conjugated donkey anti-goat IgG and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min at room temperature. The cells were visualized with a Leica SP2 confocal laser scanning microscope (Nussloch, Germany) using a 40⫻ oil-immersion objective lens. To avoid any possible reconstruction stacking artifact, double labeling was precisely evaluated by sequential scanning on single-layer optical sections at intervals of 1.0 ␮m.

Retinal slice preparation Retinal slices were prepared following a procedure previously described (Zhao et al., 2010). Following deep anesthesia and decapitation, rat eyes were removed quickly and immersed in an artificial cerebral spinal fluid (ACSF) solution (in mM: NaCl 126, KCl 2.5, CaCl2 2, MgCl2 1, NaH2PO4 1.25, NaHCO3 28, glucose 10, pH 7.4) bubbled with 95% O2 and 5% CO2. Retinae were then isolated and sliced vertically in a thickness of 200 –250 ␮m on a Narishige ST-20 slicer (Tokyo, Japan). Slices were transferred to a recording chamber and superfused continuously with oxygenated ACSF solution and viewed through a fixed-stage upright microscope (Axioskop 2 FS Mot, Zeiss, Germany) equipped with a 60⫻ water-immersion ceramic objective and DIC optics.

Identification of Rod-ON-BC Rat Rod-ON-BCs were identified according to the well-established criteria (Saito et al., 1985; Wong et al., 2005; Yu et al., 2006). ON BCs exhibit a soma in the distal part of the inner nuclear layer (INL), closely apposed to the outer plexiform layer (OPL), and an axon heading down to sublamina b of the inner plexiform layer (IPL) (Saito et al., 1985; Wong et al., 2005). Morphologically, the Rod-ON-BC has a relatively larger soma (⬎10 ␮m), compared to the Cone-ON-BC, and possesses a single characteristic enlarged axon terminal and flourishing and arborous dendrites. Electrophysiologically, due to its relatively larger soma, the Rod-ON-BC commonly has a larger capacitance than that of the Cone-ON-BC.

Whole-cell recording Whole-cell responses of BCs were conventionally recorded with pipettes of 6 –10 M⍀ resistance both in voltage-clamp and currentclamp modes, when filled with a solution containing (in mM): potassium gluconate 125, NaCl 5, CaCl2 1, MgCl2 1, ethylene glycol-bis(␤-aminoethyl ether) N, N, N=, N=-tetraacetic acid (EGTA) 4, HEPES 10, ATP-Mg 4, GTP-Na 0.5, phosphocreatine 10, pH 7.2 adjusted with KOH. Patch pipettes were made by pulling BF150-86-10 glass (Sutter Instrument Co., Novato, CA, USA) on a P-97 Flaming/Brown micropipette puller (Sutter Instrument Co.,

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

21

Novato, CA, USA) and fire polished (Model MF-830, Narishige, Tokyo, Japan) before recording. The pipettes were mounted on a motor-driven micromanipulator (MP-285, Sutter, Novato, CA, USA), and connected to an EPC10 patch-clamp amplifier (Heka, Germany). The series resistances were under 50 M⍀ and compensated for 80%. Cells with series resistances exceeding 50 M⍀ or showing large fluctuations during the recording were discarded. The holding current was often checked and only the data obtained in cells with the holding current of less than 80 pA during the first application of drugs were included in the present work. Data were filtered at 2 kHz, sampled at 5 kHz, and then stored for further analysis. Drug-containing ACSF were administrated in the bath medium through another inlet by a peristaltic pump (Rainin Instrument Co. Inc., Oakland, CA, USA). KT5823, Rp-cAMP, bisindolylmaleimide IV (Bis IV) and BAPTA were dialyzed into neurons after membrane rupture by including them in the pipette. All chemicals were obtained from Sigma Chemical Co. (Sigma-Aldrich, Inc., St. Louis, MO, USA), unless otherwise specified. Melatonin, 4phenyl-2-propionamidotetralin (4-P-PDOT; Tocris Bioscience, Ellisville, MO, USA), and KT5823 were first dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was less than 0.1% that had no effects on current and voltage responses of Rod-ON-BCs. All recordings were made at room temperature (20 –25 °C). Outward K⫹ currents of the Rod-ON-BCs were evoked by a serial voltage pulses with duration of 400 ms from a holding potential of ⫺60 mV to ⫹30 mV in an increment of 10 mV. The current amplitudes were detected at 350 ms of the voltage pulses. The data analysis was performed by using Clampfit 8.0 (Molecular Devices, Foster City, CA, USA) and Igor 4.0 (WaveMetrics, Lake Oswego, OR, USA).

Data analysis

Fig. 1. Expression of MT2 receptors in rat Rod-ON-BCs. (A1–A4) Microphotographs showing double immunofluorescence labeling of an isolated Rod-ON-BC with the antibodies against PKC and MT2. The cell, which is PKC positive (A1), is characterized by a long axon with an enlarged terminal bulb. The dendrites, axon terminal and soma of the cell are all strongly stained by MT2 (A2). (A3) is the DAPI image of the cell. (A4) is the merged image of (A1), (A2) and (A3). Note that the labeling is concentrated on the membrane. Scale bar, 10 ␮m. (B1) and (B2) Microphotographs showing double immunofluorescence labeling of isolated BCs with the antibodies against MT2 and PKC. (B1) shows the labeling for MT2. (B2) shows double labeling for MT2 and PKC. The PKC-positive cell is a Rod-ON-BC (arrow), whereas the PKC-negative one may be an OFF type BC (arrowhead). Scale bar, 10 ␮m. (C1–C4) Confocal laser microphotographs of the vertical section of the rat retina, double stained with the antibodies against PKC and MT2. (C1) and (C2) show the staining for PKC (C1) and MT2 (C2), respectively. (C3) is the DAPI image of the section, and (C4) is the merged image of (C1), (C2) and (C3). PKC-positive ON type BCs are characterized by a long axon terminating in sublamina b and an enlarged terminal bulb (arrows in C4). Note that MT2 is extensively expressed on the dendrites, somata, axons, and axon terminals of almost all of the PKC-positive cells. (D1–D4) Confocal laser microphotographs of the vertical section of the rat retina, double stained with the antibodies against PKC and MT2. (D1) shows the staining for PKC. (D2) shows that no

The steady-state activation curve was fitted by the Boltzmann equation of the form G/Gmax⫽1/{1⫹exp[(Vh⫺Vm)/k]}, where Gmax is the maximum inward conductance, Vh is the half activation potential of the K⫹ currents, Vm is the test membrane potential, and k is the slope factor. The membrane conductance (G) of K⫹ current was calculated using the equation G⫽I/(Vm⫺Vrev), where Vrev is the reversal potential. The steady-state inactivation curve was fitted by the Boltzmann equation of the form I/Imax.⫽1/ {1⫹exp[(Vm⫺VH)/k]}, where I is the peak current measured from each prepulse, Imax. is the maximum peak current, VH is the prepulse voltage at which the current amplitude is half maximum, Vm is the prepulse, and k is the slope factor at VH. All data are presented as mean⫾SEM and statistical analysis was made using paired Student’s t-test.

RESULTS Expression of MT2 receptors on Rod-ON-BCs Expression of MT2 receptors on Rod-ON-BCs was first examined by double immunofluorescence labeling with the antibodies against MT2 receptors and PKC in both acutely isolated cells and retinal sections. The specificity of the MT2 antibody was tested in our previous study by Western

immunoflorescence labelling for MT2 receptors were found when the MT2 antibody was pre-absorbed with the immunizing antigen (blocking peptide, P). (D3) is the DAPI image of the section, and (D4) is the merged image of (D1), (D2) and (D3). Scale bar, 20 ␮m. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

22

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

Fig. 2. K⫹ currents of rat Rod-ON-BC consist of 4-AP- and TEAsensitive components. (A) Representative recordings from a RodON-BC showing outward K⫹ currents induced by a series of 400 ms depolarizing voltage pulses from a holding potential of ⫺60 mV to ⫹30 mV in an increment of 10 mV. (B, C) Current traces from the same cell as shown in (A), showing that extracellular application of 4 mM 4-AP (B) or 10 mM TEA (C) significantly suppressed the current amplitudes. (D) Bar chart summarizing the changes of K⫹ current amplitudes at ⫹30 mV after 4-AP and TEA application. n⫽4.

blot analysis and immunocytochemistry in isolated RGCs (Zhao et al., 2010). Fig. 1A1– 4 show a typical isolated RodON-BC, which was characterized by short dendrites emerging at one end of the soma and a long axon with an enlarged terminal bulb. This cell was labeled by the antibody against PKC (Fig. 1A1), a marker of ON type BCs. As shown in Fig. 1A2, 4, the dendrites, axon terminal and soma of the cell were strongly co-labeled by the MT2 antibody. All 20 RodON-BCs tested were all doubled labeled without exception. It should be pointed out that some PKC-negative BCs were also MT2-positive (arrowhead) (Fig. 1B1, 2), and MT2-positive signals were also found in almost all parts of these cells including dendrites, axon terminals and somata. Expression of MT2 receptors on Rod-ON-BCs was further confirmed by double immunofluorescence labeling conducted in retinal vertical sections. Fig. 1C shows the confocal laser microphotographs of a double-labeled section. The PKC-positive BCs (Fig. 1C1) were characterized with axons terminating in sublamina b at the vitreal border of the IPL, which is close to the ganglion cell layer (GCL). Labeling for MT2 was found extensively in the section (Fig. 1C2). From the merged image (Fig. 1C4), it was clear that almost all of the PKC-positive ON BCs (arrows), including the somata, dendrites and axon terminals, were double-labeled, demonstrating the expression of MT2 receptors on these cells. No immunoflorescence labeling for MT2 receptors was found (Fig. 1D2) when the antibody was pre-absorbed with the corresponding immunizing antigen (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Fig. 1D1– 4), further demonstrating that the labeling was specific. Modulation of Kⴙ channels by melatonin We first characterized outward K⫹ currents of Rod-ONBCs. A series of depolarizing voltage pulses from a holding potential of ⫺60 mV to 30 mV in an increment of 10 mV

induced outward K⫹ currents of increasing amplitudes (Fig. 2A). Perfusion of either 4-AP or TEA suppressed the currents (Fig. 2B, C). At ⫹30 mV test potential, the currents were inhibited by 4 mM 4-AP to 78.8⫾7.6% of control and by 10 mM TEA to 44.6⫾7.8% of control, respectively (n⫽4) (Fig. 2D), indicating that the outward K⫹ currents consisted of at least two components: 4-AP- and TEAsensitive ones. External application of 100 nM melatonin significantly and reversibly reduced the K⫹ current amplitudes (Fig. 3A–C). At ⫹30 mV test potential, melatonin reduced the currents to 69.2⫾9.0% of control (n⫽4, P⬍0.05), and after 5 min washout the currents recovered to 82.4⫾4.2% of control (n⫽4) (Fig. 3D). Melatonin-induced suppression of K⫹ currents was blocked by coapplication of 4-P-PDOT (100 nM), a specific MT2 receptor antagonist. Fig. 3E, F show that no suppression of the K⫹ currents was observed at all test potentials, when 100 nM 4-P-PDOT was co-applied with 100 nM melatonin. The

Fig. 3. MT2 receptor-mediated suppression of outward K⫹ currents by melatonin in rat Rod-ON-BCs. (A) Representative outward K⫹ current recordings from a Rod-ON-BC in normal ACSF. (B, C) Current traces from the same cell as shown in (A), showing that extracellular application of melatonin (100 nM) reversibly inhibited the currents. (D) Bar chart summarizing the changes in K⫹ current amplitudes at ⫹30 mV after melatonin application. n⫽4, * P⬍0.05 vs control. (E) Representative outward K⫹ current recordings from another Rod-ON-BC. (F) Current traces from the same cell as shown in (E), showing that the K⫹ current amplitudes kept unchanged when extracellular co-application of melatonin (100 nM) and the MT2 receptor antagonist 4-P-PDOT (100 nM). (G) Bar chart summarizing the changes of K⫹ current amplitudes at ⫹30 mV after co-application of melatonin and 4-PPDOT. n⫽7. All data are presented as mean⫾SEM.

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

Fig. 4. Melatonin selectively suppresses TEA-sensitive K⫹ currents. (A) K⫹ currents of a Rod-ON-BC recorded in the presence of 4 mM 4-AP (control). (B) Current traces from the same cell as shown in (A), showing that extracellular application of melatonin (100 nM) inhibited the K⫹ currents in the presence of 4 mM 4-AP. (C) Bar chart summarizing the changes in TEA-sensitive current amplitudes at ⫹30 mV after melatonin application. n⫽6, *** P⬍0.001. (D) K⫹ currents of another Rod-ON-BC recorded in the presence of 10 mM TEA. (E) Current traces from the same cell as shown in (D), showing that extracellular application of melatonin (100 nM) did not change the K⫹ currents. (F) Bar chart summarizing the changes in 4-AP-sensitive current amplitudes at ⫹30 mV after melatonin application. n⫽5. All data are presented as mean⫾SEM.

average current obtained at ⫹30 mV test potential after 5 min perfusion of melatonin along with 4-P-PDOT was 95.6⫾2.6% of control (n⫽7, P⬎0.05) (Fig. 3G), suggesting that the inhibition of K⫹ currents by melatonin was mediated by MT2 receptors. We then examined effects of melatonin on 4-AP- and TEA-sensitive components respectively. Fig. 4A, B show the effect of 100 nM melatonin on the TEA-sensitive component of a Rod-ON-BC recorded at different test potentials. For this experiment, the cell was perfused with 4-AP (4 mM)-containing ACSF for 5 min, which reduced the K⫹ current amplitudes (Fig. 4A, control), prior to melatonin application. Addition of 100 nM melatonin significantly and reversibly suppressed the currents (Fig. 4B). Similar results were observed in all six cells tested. The bar chart shown in Fig. 4C shows the average data obtained in these six cells at ⫹30 mV test potential. Melatonin of 100 nM reduced the amplitude of the TEA-sensitive component to 68.5⫾7.1% of control (n⫽6, P⬍0.001). The effect of 100 nM melatonin on the 4-AP-sensitive component of another Rod-ON-BC is shown in Fig. 4D, E. The 4-AP-sensitive component of the K⫹ current (control) was obtained by perfusion of 10 mM TEA for 5 min (Fig. 4D). In the presence

23

of TEA, application of 100 nM melatonin did not change the current amplitudes (Fig. 4E). Similar results were obtained in other four cells. At ⫹30 mV the average current amplitude after melatonin application was 97.4⫾4.2% of control (n⫽5, P⬎0.05) (Fig. 4F). These results indicated that melatonin selectively inhibits the TEA-sensitive K⫹ current component in the rat Rod-ON-BC. Effects of melatonin on activation and inactivation of outward K⫹ currents were also examined in Rod-ON-BCs. For the activation protocol, outward K⫹ currents were evoked by 400 ms depolarizing pulses from ⫺60 mV to ⫹30 mV in an increment of 10 mV at intervals of 10 s. Just like shown in Fig. 3A, B, extracellular application of 100 nM melatonin reduced the outward K⫹ current amplitudes (Fig. 5A). Fig. 5B shows a plot of the normalized conductance as a function of membrane potential in normal ACSF (control) and in the presence of 100 nM melatonin. The Vh values of the K⫹ currents obtained before and after application of melatonin were ⫺0.34⫾2.08 mV and ⫺2.23⫾ 2.21 mV, respectively (n⫽7, P⬎0.05), suggesting no significant change in the voltage dependent steady-state activation of the outward K⫹ channels. For the inactivation protocol, outward K⫹ currents were evoked by conditioning prepulses from ⫺60 mV to different steps of membrane potentials (from ⫺80 mV to ⫹30 mV) prior to a 400 ms test pulse of 30 mV. As shown in Fig. 5C, melatonin decreased the current amplitudes, but appeared not to shift the steady-state inactivation curve of the K⫹ currents (Fig. 5D). The VH values obtained before and after melatonin application were ⫺40.0⫾2.1 mV and ⫺38.8⫾3.0 mV, respectively (n⫽4, P⬎0.05).

Signaling pathway of melatonin-induced suppression of Kⴙ currents As a G-protein coupled receptor, the activation of the MT2 receptor may regulate several intracellular signaling pathways, such as cyclic adenosine monophosphate (cAMP)protein kinase A (PKA) pathway (Vanecek, 1998; Von Gall et al., 2002; Markowska et al., 2002, 2004; Alarma-Estrany and Pintor, 2007), cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway (Faillace et al., 1996; Petit et al., 1999; Saenz et al., 2002), and PLCdependent pathway (McArthur et al., 1997; Lai et al., 2002; MacKenzie et al., 2002; Zhao et al., 2010). Possible involvement of the PKG signaling pathway was first tested. Fig. 6A–C show that extracellular application of 100 nM melatonin still reversibly reduced the K⫹ current amplitudes of a Rod-ON-BC that had been dialyzed intracellularly with 20 ␮M the PKG inhibitor KT5823 for 10 min (control). For this cell, the current amplitude at ⫹30 mV test potential was reduced to 210 pA from a control value of 319 pA after 100 nM melatonin application. On average, 100 nM melatonin reduced the current amplitudes (at ⫹30 mV) to 69.6⫾6.4% of control (n⫽6, P⬍0.01) (Fig. 6D). Similarly, melatonin-induced inhibition of K⫹ currents persisted when PKA was inhibited. The result obtained in a Rod-ON-BC, which was intracellularly dialyzed with the PKA inhibitor Rp-cAMP (10 ␮M) for 10 min before control

24

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

Fig. 5. Melatonin does not change the voltage-dependent steady-state activation and inactivation of outward K⫹ currents of Rod-ON-BCs. (A) K⫹ currents of a Rod-ON-BC recorded before (left) and after application of 100 nM melatonin (right). Cell was held at ⫺60 mV and then stepped to 30 mV in 10 mV increments. (B) Plot of the normalized conductance as a function of membrane potential in normal ACSF (control) and in the presence of 100 nM melatonin. The data points are fitted with a Boltzman function. n⫽7. (C) K⫹ currents of another Rod-ON-BC recorded before (left) and after application of 100 nM melatonin (right). K⫹ currents were evoked by conditioning prepulses from a holding potential of ⫺60 mV to different steps of membrane potential (from ⫺80 mV to ⫹30 mV in 10 mV increments) prior to a 400 ms test pulse of 30 mV. (D) Steady-state inactivation curve in normal ACSF (control) and in the presence of 100 nM melatonin. Normalized points are fitted with a Boltzman function. n⫽4. All data are presented as mean⫾SEM.

recording, is shown in Fig. 6E–G. In the presence of RpcAMP, 100 nM melatonin reduced the K⫹ current amplitude from 172 to 113 pA at ⫹30 mV potential. On average, the K⫹ current amplitude by 100 nM melatonin (at ⫹30 mV) was reduced to 60.5⫾2.7% of control (n⫽5, P⬍0.01, Fig. 6H). It should be noted that the K⫹ currents were almost unchanged during intracellular dialysis of RpcAMP. After 10 min dialysis the current amplitudes were 93.3⫾3.5% of that at 0 min (n⫽5, P⬎0.05), suggesting that inhibition of adenylate cyclase did not reduce K⫹ currents by itself. These results suggest no involvement of PKA and PKG signaling pathways. Experiments involving with the PLC-PKC signaling pathway yielded different results. We dialyzed U73122 (5 ␮M), a PLC inhibitor (McArthur et al., 1997; Ross et al., 1998; Girón-Calle et al., 2002), into a Rod-ON-BC through the recording pipette for 10 min (control) and then observed effects of melatonin application on K⫹ currents of the cell (Fig. 7A, B). The K⫹ current amplitudes were almost unaffected by 100 nM melatonin. The average current amplitude at ⫹30 mV potential obtained from seven cells tested was 95.1⫾2.3% of control (n⫽7, P⬎0.05) (Fig. 7C). Activated PLC hydrolyzes membrane phosphatidylinositol into diacylglycerol (DAG) and IP3, which are known to regulate PKC directly or indirectly via Ca2⫹ (Way et al., 2000). Indeed, the melatonin effect on K⫹ currents of Rod-ON-BCs was PKC-dependent. When a cell was predialyzed with 5 ␮M Bis IV, a PKC inhibitor, for 10 min (control), extracellular application of 100 nM melatonin no

longer suppressed the K⫹ currents of the cell (Fig. 7D, E). Similar results were observed in all five cells tested and the data collected from these cells are summarized in Fig. 7F. The average K⫹ current amplitude (at ⫹30 mV) was 98.3⫾4.0% of control after melatonin application (P⬎ 0.05). The melatonin-induced inhibition of K⫹ currents is also dependent on intracellular Ca2⫹ ([Ca2⫹]i). When Ca2⫹ in the internal solution was removed and EGTA was replaced by 10 mM BAPTA, a fast Ca2⫹ chelator, a calculation using MaxChelator version 6.81 (Bers et al., 1994) indicated that [Ca2⫹]i may be reduced to 1 pM. Perfusion of 100 nM melatonin-containing ACSF did not change the K⫹ currents of the cell (Fig. 7G, H). The average current amplitude at ⫹30 mV test potential was 93.2⫾2.9% of control (n⫽7, P⬎0.05, Fig. 7I). TEA-sensitive component K⫹ current of neurons may be related to cell membrane potential (Fu et al., 1996; Sah, 1996; Hille, 2001). We finally tested how melatonin-induced inhibition of TEA-sensitive K⫹ currents may change the membrane potential of Rod-ON-BCs. As shown in Fig. 8, the membrane potential of the Rod-ON-BC, which was current-clamped, was around ⫺45 mV. Extracellular perfusion of 100 nM melatonin induced a membrane depolarization of about 3 mV with a long delay (⬃1 min), and the effect of melatonin was reversed by co-application of 100 nM 4-P-PDOT. Similar results were observed in seven cells tested, and the average depolarization was 2.26⫾ 0.36 mV (n⫽7).

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

25

observed in carp (Ping et al., 2008). It is of interest that MT2 receptors are also found in rat RGCs by immunocytochemistry, and these receptors are involved in modulation of glycine receptors (Zhang et al., 2007; Zhao et al., 2010). Although MT1 receptor mRNA was shown to be widely distributed in rat retina, the labeling was not detected in BCs and photoreceptors (Fujieda et al., 1999). In rat retinal section preparations, we did detect labeling for MT1 receptors in the INL, but the signal was very weak. Double immunofluorescence labeling with the antibodies against MT1 and PKC only revealed sparse and faint merged signals in Rod-ON-BCs. The distribution of MT1 receptors in rat retina seems somewhat different from that observed in mouse retina, in which MT1 receptor mRNA is abundant in both the ONL and the INL (Baba et al., 2009). Further investigation is required to clarify whether MT1 receptors are indeed expressed in Rod-ON-BCs. Melatonin selectively suppresses TEA-sensitive Kⴙ channels via MT2 receptors

Fig. 6. PKG and PKA signaling pathways are not involved in melatonin-induced inhibition of K⫹ channels. (A) Outward K⫹ currents from a Rod-ON-BC during internal infusion of 20 ␮M KT5823. (B, C) Current traces from the same cell as shown in (A), showing that extracellular application of melatonin (100 nM) persisted to inhibit the currents in a reversible manner. (D) Bar chart summarizing the changes in K⫹ current amplitudes at ⫹30 mV caused by 100 nM melatonin. n⫽6. ** P⬍0.01 vs. control. All the recordings were made in the presence of KT5823. (E) Outward K⫹ currents from another Rod-ON-BC during internal dialysis of 10 ␮M Rp-cAMP. (F, G) Current traces from the same cell as shown in (E), showing that extracellular application of melatonin (100 nM) reversibly inhibited the current amplitudes. (H) Bar chart summarizing the changes of K⫹ current amplitudes at ⫹30 mV caused by melatonin, n⫽5. ** P⬍0.01 vs. control. All the recordings were made in the presence of Rp-cAMP. All data are presented as mean⫾SEM.

DISCUSSION Expression of MT2 receptors on rat Rod-ON-BCs MT2 receptors are widely expressed in both mammalian and non-mammalian retinas (Reppert et al., 1995; Wiechmann et al., 2004; Alarma-Estrany and Pintor, 2007; Savaskan et al., 2007; Huang et al., 2005; Ping et al., 2008; Zhao et al., 2010). Consistent with the observation that MT2 receptors are functionally expressed in Rod-ONBCs of carp (Ping et al., 2008), the present work, by double immunofluorescence labeling experiments in isolated cell and retinal section preparations, demonstrated for the first time that almost all parts, including somata, dendrites and axons, of rat Rod-ON-BCs abundantly expressed MT2 receptors. In addition, some PKC-negative BCs were also expressed MT2 receptors (arrowhead, see Fig. 1B). These results strongly suggest that melatonin synthesized in and released from photoreceptors (Fukuhara et al., 2004; Tosini et al., 2007) may modulate functions of BCs, just like

A major finding in this work was that melatonin suppressed the outward K⫹ currents of rat Rod-ON-BCs. Lots of previous studies reported modulatory effects of melatonin on various K⫹ channels in different regions of the central nervous system (Jiang et al., 1995; Huan et al., 2001; Hou et al., 2004; Liu et al., 2007). In rat hippocampal CA1 pyramidal neurons, melatonin at micromolar concentration inhibits outward delayed rectifier K⫹ currents by a direct interaction (Hou et al., 2004). Increase in these currents was, however, found in rat cerebellar granule cells, an effect that was mediated by MT2 receptors (Huan et al., 2001; Liu et al., 2007). Moreover, melatonin was shown to constrict rat cerebral arteries directly through inhibiting TEA-sensitive large-conductance Ca2⫹-activated K⫹ channels (BKCa), and the effect may be mediated by activating Gi or Go-coupled MT1 receptors (Geary et al., 1997). The melatonin receptor agonist 2-iodomelatonin-induced increase of open probability of BKCa was also reported in rat uterine myocytes, which was mediated by both pertussis toxin (PTX)-insensitive Gq/PLC and PTX-sensitive Gi/ cAMP/PKA signaling pathways (Steffens et al., 2003). Nevertheless, to our knowledge, this is the first work reporting that melatonin in the retina modulates voltagegated K⫹ channels by activating MT2 receptors, in addition to modulating ligand-gated channels (Huang et al., 2005; Ping et al., 2008; Zhao et al., 2010). As reported previously (Karschin and Wässle, 1990; Klumpp et al., 1995; Yazulla and Studholme, 1998; Hu and Pan, 2002), a variety of K⫹ channels are expressed on rat Rod-ON-BCs, including two outward K⫹ current components and an inward rectifying K⫹ current component. It should be mentioned that several G-protein coupled receptors, including the closely related MT1 and MT2 melatonin receptors, may form homo- and hetero-oligomeric complexes (Bouvier, 2001; Ayoub et al., 2002, 2004). In living HEK293 cells MT1 and MT2 receptors are preferentially to form heterodimer, and both MT2selective 4-P-PDOT and MT1-selective S26284 bind with high affinity to MT1/MT2 heterodimer. Occupation of either

26

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

Fig. 8. Melatonin induces membrane depolarization of Rod-ON-BCs. Representative recording from a Rod-ON-BC, which was currentclamped, showing that external application of 100 nM melatonin induced a membrane depolarization of about 3 mV. The effect was reversed by co-application of 4-P-PDOT (100 nM). Note that the depolarization emerged with a long delay (⬃1 min).

Fig. 7. Ca2⫹-dependent PLC-PKC signaling pathway is involved in melatonin-induced inhibition of K⫹ channels. (A) Outward K⫹ currents from a Rod-ON-BC during internal dialysis of 5 ␮M U73122. (B) Current traces from the same cell as shown in (A), showing that extracellular application of melatonin (100 nM) did not change the current amplitudes. (C) Bar chart summarizing the changes of K⫹ current amplitudes at ⫹30 mV due to melatonin application (n⫽7). (D) Outward K⫹ currents from another Rod-ON-BC during internal dialysis of 5 ␮M Bis IV. (E) Current traces from the same cell as shown in (D), showing that the current amplitudes kept unchanged after extracellular application of melatonin (100 nM). (F) Bar chart summarizing the changes of K⫹ current amplitudes at ⫹30 mV due to melatonin application (n⫽5). (G) Outward K⫹ currents from a still other Rod-ON-BC during internal dialysis of 10 mM BAPTA. (H) Current traces from the same cell as shown in (G), showing that the current amplitudes kept unchanged after extracellular application of melatonin (100 nM). (I) Bar

binding site is sufficient to induce a conformational change within the heterodimer (Ayoub et al., 2004). However, there are no data available concerning the formation of MT1/MT2 heterodimer in native mammalian retinas. Of course, we cannot exclude the possibility that melatonin inhibits K⫹ currents of Rod-ON-BCs by activating a possible MT1/MT2 heterodimer. Our experiments further demonstrated that melatonin selectively suppressed the TEA-, but not 4-AP-sensitive component. This suppression seemed to be due to an interference of the open state of the K⫹ channels, because melatonin did not affect activation and inactivation kinetics of these channels. In general, TEA-sensitive component K⫹ currents of neurons are considered to be related to cell membrane potential (Hille, 2001) and inhibition of these currents may result in membrane depolarization (Fu et al., 1996; Sah, 1996). In retinal BCs K⫹ currents have been characterized in several species (Kaneko and Tachibana, 1985; Tessier-Lavigne et al., 1988; Lasater, 1988; Karschin and Wässle, 1990; Gillette and Dacheux, 1995; Klumpp et al., 1995; Connaughton and Maguire, 1998; Han et al., 2000; Hu and Pan, 2002). While roles of K⫹ channels in BC processing are not fully clear and these currents seem not to confer ON or OFF properties of BCs (Lasater, 1988), they could affect the membrane depolarization level BCs could reach (Hu and Pan, 2002) and help sharp light responses of BCs (Lasater, 1988; Kaneko and Tachibana, 1985). Especially for ON BCs, which are at more hyperpolarized level in the dark than OFF BCs (Nelson and Kolb, 2004), inhibition of K⫹ currents would push the membrane potential to a more depolarized level. Indeed, as shown in Fig. 8, melatonin caused a membrane depolarization in Rod-ON-BCs, which may help increase visual sensitivity. It is of interest that in mouse retina melatonin increases the amplitude of the electroretinographic b-wave by activating MT1 receptors (Baba et al., 2009).

chart summarizing the changes of K⫹ current amplitudes at ⫹30 mV due to melatonin application (n⫽7). All the recordings were made in the presence of U73122 (for A and B), or Bis IV (for D and E), or BAPTA (for G and H). All data are presented as mean⫾SEM.

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

Physiological significance of the melatonin effect is worthwhile to be further explored. Ca2ⴙ-dependent PLC/IP3/PKC signaling pathway mediates the melatonin effect on Kⴙ currents Activation of MT2 receptors may regulate the activity of the cAMP/PKA pathway (Vanecek, 1998; Masana and Dubocovich, 2001) or lead to changes in cGMP/PKG activity (Petit et al., 1999; Ping et al., 2008). In carp Rod-ON-BCs, activation of MT2 receptors increases cGMP levels by inhibiting phosphodiesterase (PDE), thus resulting in a potentiation of rod signals to these cells (Ping et al., 2008). In uterine myocytes, 2-iodomelatonin antagonizes the ␤-adrenoceptor agonist isoprenaline-induced inhibition of BKCa in pregnant cells and potentiation of BKCa in nonpregnant cells, which are mediated by PTX-sensitive Gi/ cAMP/PKA signaling pathway (Steffens et al., 2003). Moreover, nitric oxide-cGMP-PKG signaling pathway-mediated activation of ATP-sensitive K⫹ channels and BKCa was found to be responsible for melatonin-induced local peripheral antinociception (Hernández-Pacheco et al., 2008). However, the data provided in this work showed that melatonin persisted to suppress the K⫹ currents of Rod-ON-BCs when PKA and PKG were inhibited by RpcAMP and KT5823 respectively (Fig. 6), suggesting no involvement of these pathways. It is worth of noting that these two signaling pathways are also not involved in melatonin-induced potentiation of glycine currents of RGCs, an action that is mediated by MT2 receptors as well (Zhao et al., 2010). Accumulating evidence demonstrates that the PLCPKC signaling pathway may be a major one following activation of MT2 receptors (McArthur et al., 1997; Lai et al., 2002; MacKenzie et al., 2002; Alarma-Estrany and Pintor, 2007; Zhao et al., 2010). In rat suprachiasmatic nucleus (SCN) slices activation of MT2 receptors produces a phase shift through an increase in PLC-linked PKC activity (Gerdin et al., 2004; Dubocovich et al., 2005). Our recent work in rat RGCs also demonstrated that melatonininduced potentiation of glycine currents is mediated by increasing the PLC-PKC activity (Zhao et al., 2010). The present work provides new evidence to support the involvement of this signaling pathway in MT2 receptor-mediated effects. Acknowledgments—This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500805; 2007CB512205; 2011CB504602), the Natural Science Foundation of China (31070966; 30900427; 30870803; 30930034), Pujiang Talent Project of the Shanghai Science and Technology Committee (08PJ1401600), and the 211 Project sponsored by the Ministry of Education of China.

REFERENCES Alarma-Estrany P, Pintor J (2007) Melatonin receptors in the eye: location, second messengers and role in ocular physiology. Pharmacol Ther 113:507–522. Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M, Jockers R (2002) Monitoring of ligand-independent dimer-

27

ization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 277:21522–21528. Ayoub MA, Levoye A, Delagrange P, Jockers R (2004) Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers. Mol Pharmacol 66:312–321. Baba K, Pozdeyev N, Mazzoni F, Contreras-Alcantara S, Liu C, Kasamatsu M, Martinez-Merlos T, Strettoi E, Luvone PM, Tosini G (2009) Melatonin modulates visual function and cell viability in the mouse retina via the MT1 melatonin receptor. Proc Natl Acad Sci U S A 106:15043–15048. Barrenetxe J, Delagrange P, Martinez JA (2004) Physiological and metabolic functions of melatonin. J Physiol Biochem 60:61–72. Bers DM, Patton CW, Nuccitelli R (1994) A practical guide to the preparation of Ca2⫹ buffers. Methods Cell Biol 40:3–29. Bouvier M (2001) Oligomerization of G-protein-coupled transmitter receptors. Nat Rev Neurosci 2:274 –286. Brzezinski A (1997) Melatonin in humans. N Engl J Med 336:186 –195. Cahill GM, Besharse JC (1992) Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina. Vis Neurosci 8:487– 490. Chen L, Yu YC, Zhao JW, Yang XL (2004) Inwardly rectifying potassium channels in rat retinal ganglion cells. Eur J Neurosci 20: 956 –964. Connaughton VP, Maguire G (1998) Differential expression of voltagegated K⫹ and Ca2⫹ currents in bipolar cells in the zebrafish retinal slice. Eur J Neurosci 10:1350 –1362. Cosci B, Longoni B, Marchiafava PL (1997) Melatonin induces membrane conductance changes in isolated retinal rod receptor cells. Life Sci 60:1885–1889. Dubocovich ML, Hudson RL, Sumaya IC, Masana MI, Manna E (2005) Effect of MT1 melatonin receptor deletion on melatonin-mediated phase shift of circadian rhythms in the C57BL/6 mouse. J Pineal Res 39:113–120. Dubocovich ML, Rivera-Bermúdez MA, Gerdin MJ, Masana MI (2003) Molecular pharmacology, regulation and function of mammalian melatonin receptors. Front Biosci 8:d1093– d1108. Faillace MP, Keller Sarmiento MI, Rosenstein RE (1996) Melatonin effect on the cyclic GMP system in the golden hamster retina. Brain Res 711:112–117. Fu XW, Wu SH, Brezden BL, Kelly JB (1996) Potassium currents and membrane excitability of neurons in the rat’s dorsal nucleus of the lateral lemniscus. J Neurophysiol 76:1121–1132. Fujieda H, Hamadanizadeh SA, Wankiewicz E, Pang SF, Brown GM (1999) Expression of mt1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin. Neuroscience 93:793–799. Fukuhara C, Liu C, Ivanova TN, Chan GCK, Strom DR, Iuvone PM, Tosini G (2004) Gating of the cAMP signalling cascade and melatonin synthesis by the circadian clock in mammalian retina. J Neurosci 24:1803–1811. Geary GG, Krause DN, Duckles SP (1997) Melatonin directly constricts rat cerebral arteries through modulation of potassium channels. Am J Physiol 273:H1530 –H1536. Gerdin MJ, Masana MI, Rivera-Bermúdez MA, Hudson RL, Earnest DJ, Gillette MU, Dubocovich ML (2004) Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin. FASEB J 18: 1646 –1656. Gillette MA, Dacheux RF (1995) GABA- and glycine-activated currents in the rod bipolar cell of the rabbit retina. J Neurophysiol 74: 856 – 875. Girón-Calle J, Srivatsa K, Forman HJ (2002) Priming of alveolar macrophage respiratory burst by H2O2 is prevented by

28

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29

phosphatidylcholine-specific phospholipase C inhibitor tricyclodecan-9-yl-xanthate (D609). J Pharmacol Exp Ther 301: 87–94. Han Y, Jacoby RA, Wu SM (2000) Morphological and electrophysiological properties of dissociated primate retinal cells. Brain Res 875:175–186. Hernández-Pacheco A, Araiza-Saldaña CI, Granados-Soto V, Mixcoatl-Zecuatl T (2008) Possible participation of the nitric oxidecyclic GMP-protein kinase G-K⫹ channels pathway in the peripheral antinociception of melatonin. Eur J Pharmacol 596: 70 –76. Hille B (2001) Potassium channels and chloride channels. In: Ion channels of excitable membrane, 3rd ed. (Hille B, ed), pp 131–167. Sunderland, UK: Sinauer Associates, Inc. Hou SW, Zheng P, Sun FY (2004) Melatonin inhibits outward delayed rectifier potassium currents in hippocampal CA1 pyramidal neuron via intracellular indole-related domains. J Pineal Res 36:242–249. Hu HJ, Pan ZH (2002) Differential expression of K⫹ currents in mammalian retinal bipolar cells. Vis Neurosci 19:163–173. Huan CL, Zhou MO, Wu MM, Zhang ZH, Mei YA (2001) Activation of melatonin receptor increases a delayed rectifier K⫹ current in rat cerebellar granule cells. Brain Res 917:182–190. Huang H, Lee SC, Yang XL (2005) Modulation by melatonin of glutamatergic synaptic transmission in the carp retina. J Physiol (Lond) 569:857– 871. Jiang ZG, Nelson CS, Allen CN (1995) Melatonin activates an outward current and inhibits Ih in rat suprachiasmatic nucleus neurons. Brain Res 687:125–132. Kaneko A, Tachibana M (1985) A voltage-clamp analysis of membrane currents in solitary bipolar cells dissociated from Carassius auratus. J Physiol (Lond) 358:131–152. Karschin A, Wässle H (1990) Voltage- and transmitter-gated currents in isolated rod bipolar cells of rat retina. J Neurophysiol 63: 860 – 876. Klumpp DJ, Song EJ, Pinto LH (1995) Identification and localization of K⫹ channels in the mouse retina. Vis Neurosci 12:1177–1190. Lai FP, Mody SM, Yung LY, Kam JY, Pang CS, Pang SF, Wong YH (2002) Molecular determinants for the differential coupling of G␣16 to the melatonin MT1, MT2 and Xenopus Mel1c receptors. J Neurochem 80:736 –745. Lasater EM (1988) Membrane currents of retinal bipolar cells in culture. J Neurophysiol 60:1460 –1480. Liu LY, Hoffman GE, Fei XW, Li Z, Zhang ZH, Mei YA (2007) Delayed rectifier outward K⫹ current mediates the migration of rat cerebellar granule cells stimulated by melatonin. J Neurochem 102:333–343. MacKenzie RS, Melan MA, Passey DK, Witt-Enderby PA (2002) Dual coupling of MT1 and MT2 melatonin receptors to cyclic AMP and phosphoinositide signal transduction cascades and their regulation following melatonin exposure. Biochem Pharmacol 63:587–595. Markowska M, Mrozkowiak A, Pawlak J, Skwarło-Son´ta K (2004) Intracellular second messengers involved in melatonin signal transduction in chicken splenocytes in vitro. J Pineal Res 37: 207–212. Markowska M, Mrozkowiak A, Skwarło-Son´ta K (2002) Influence of melatonin on chicken lymphocytes in vitro: involvement of membrane receptors. Neuro Endocrinol Lett 23:67–72. Masana MI, Dubocovich ML (2001) Melatonin receptors signalling: finding the path through the dark. Sci STKE 107:pe39. McArthur AJ, Hunt AE, Gillette MU (1997) Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn. Endocrinology 138: 627– 634. Nelson R, Kolb H (2004) ON and OFF pathways in the vertebrate retina and visual system. In: The visual neurosciences, vol. 1 (Chalupa LM, Werner JS, eds), pp 260 –278. London, UK: The MIT Press.

Petit L, Lacroix I, de Coppet P, Strosberg AD, Jockers R (1999) Differential signaling of human Mel1a and Mel1b melatonin receptors through the cyclic guanosine 3=-5=-monophosphate pathway. Biochem Pharmacol 58:633– 639. Ping Y, Huang H, Zhang XJ, Yang XL (2008) Melatonin potentiates rod signals to ON type bipolar cells in fish retina. J Physiol (Lond) 586:2683–2694. Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA, Gusella JF (1995) Molecular charaterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci U S A 92:8734 – 8738. Ribelayga C, Wang Y, Mangel SC (2004) A circadian clock in the fish retina regulates dopamine release via activation of melatonin receptors. J Physiol (Lond) 554:467– 482. Ross AW, Webster CA, Thompson M, Barrett P, Morgan PJ (1998) A novel interaction between inhibitory melatonin receptors and protein kinase C-dependent signal transduction in ovine pars tuberalis cells. Endocrinology 139:1723–1730. Saenz DA, Turjanski AG, Sacca GB, Marti M, Doctorovich F, Sarmiento MI, Estrin DA, Rosenstein RE (2002) Physiological concentrations of melatonin inhibits the nitridergic pathway in the Syrian hamster retina. J Pineal Res 33:31–36. Sah P (1996) Ca2⫹-activated K⫹ currents in neurones: types, physiological roles and modulation. Trends Neurosci 19:150 –154. Saito T, Kujiraoka T, Yonaha T, Chino Y (1985) Reexamination of photoreceptor-bipolar connectivity patterns in carp retina: HRP-EM and Golgi-EM studies. J Comp Neurol 236:141–160. Savaskan E, Jockers R, Ayoub M, Angeloni D, Fraschini F, Flammer J, Eckert A, Müller-Spahn F, Meyer P (2007) The MT2 melatonin receptor subtype is present in human retina and decreases in Alzheimer’s disease. Curr Alzheimer Res 4:47–51. Scher J, Wankiewicz E, Brown GM, Fujieda H (2003) AII amacrine cells express the MT1 melatonin receptor in human and macaque retina. Exp Eye Res 77:375–382. Steffens F, Zhou XB, Sausbier U, Sailer C, Motejlek K, Ruth P, Olcese J, Korth M, Wieland T (2003) Melatonin receptor signaling in pregnant and nonpregnant rat uterine mycytes as probed by large conductance Ca2⫹-activated K⫹ channel activity. Mol Endocrinol 17:2103–2115. Tessier-Lavigne M, Attwell D, Mobbs P, Wilson M (1988) Membrane currents in retinal bipolar cells of the axolotl. J Gen Physiol 91:49 –72. Tosini G, Davidson AJ, Fukuhara C, Kasamatsu M, Castanon-Cervantes O (2007) Localization of a circadian clock in mammalian photoreceptors. FASEB J 21:3866 –3871. Tosini G, Fukuhara C (2003) Photic and circadian regulation of retinal melatonin in mammals. J Neuroendocrinol 15:364 –369. Vanecek J (1998) Cellular mechanisms of melatonin action. Physiol Rev 78:687–721. Von Gall C, Stehle JH, Weaver DR (2002) Mammalian melatonin receptors: molecular biology and signal transduction. Cell Tissue Res 309:151–162. Way KJ, Chou E, King GL (2000) Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci 21:181–187. Wiechmann AF (2003) Differential distribution of Mel1a and Mel1c melatonin receptors in Xenopus laevis retina. Exp Eye Res 76:99 –106. Wiechmann AF, Udin SB, Summers Rada JA (2004) Localization of Mel1b melatonin receptor-like immunoreactivity in ocular tissues of Xenopus laevis. Exp Eye Res 79:585–594. Wiechmann AF, Vrieze MJ, Dighe R, Hu Y (2003) Direct modulation of rod photoreceptor responsiveness through a Mel1c melatonin receptor in transgenic Xenopus Laevis retina. Invest Ophthalmol Vis Sci 44:4522– 4531. Wong KY, Cohen ED, Dowling JE (2005) Retinal bipolar cell input mechanisms in giant danio. II. Patch-clamp analysis of on bipolar cells. J Neurophysiol 93:94 –107.

X.-F. Yang et al. / Neuroscience 173 (2011) 19 –29 Yazulla S, Studholme KM (1998) Differential distribution of Shaker-like and Shab-like K⫹-channel subunits in goldfish retina and retinal bipolar cells. J Comp Neurol 396:131–140. Yu YC, Cao LH, Yang XL (2006) Modulation by brain natriuretic peptide of GABA receptors on rat retinal ON-type bipolar cells. J Neurosci 26:696 –707.

29

Zhang M, Cao LH, Yang XL (2007) Melatonin modulates glycine currents of retinal ganglion cells in rat. Neuroreport 18:1675– 1678. Zhao WJ, Zhang M, Miao Y, Yang XL, Wang Z (2010) Melatonin potentiates glycine currents through a PLC/PKC signaling pathway in rat retinal ganglion cells. J Physiol (Lond) 588:2605–2619.

(Accepted 13 November 2010) (Available online 18 November 2010)