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1Neuroscience Program, 2Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York; 3Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and 4Department of Anatomy and Neurobiology, Boston University, Boston Massachusetts
Submitted 15 December 2004; accepted in final form 2 April 2005
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ABSTRACT |
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-aminobutyric acid-C (GABAC)–receptor activity, as demonstrated by the ability of the GABAC-receptor antagonists, picrotoxin and TPMPA, to abolish the effects of melatonin. In contrast, neither the GABAA-receptor antagonist bicuculline nor the GABAB-receptor antagonist CGP 35348 diminished the effects of melatonin. RT-PCR analyses revealed expression of the 3 known melatonin receptors, MT1 (Mel1a), MT2 (Mel1b), and Mel1c. Because the effect of melatonin on tectal calcium increases was antagonized by an MT2-selective antagonist, 4-P-PDOT, we performed Western blot analyses with an antibody to the MT2 receptor; the data indicate that the MT2 receptor is expressed primarily as a dimeric complex and is glycosylated. The receptor is present in higher amounts at the end of the light period than at the end of the dark period, in a pattern complementary to the changes in melatonin levels, which are higher during the night than during the day. These results imply that melatonin, acting by MT2 receptors, modulates GABAC receptor activity in the optic tectum and that this effect is influenced by the light–dark cycle. |
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INTRODUCTION |
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; Wiechmann and Wirsig-Wiechmann 1993). In the tectum, melatonin binding has been reported both presynaptically (Brooks and Cassone 1992; Wiechmann et al. 1999) and postsynaptically (Krause et al. 1994). In Xenopus laevis, 3 G-protein–coupled melatonin receptors, denoted MT1 (Mel1a), MT2 (Mel1b), and Mel1c, have been identified and cloned (Ebisawa et al. 1994; Reppert et al. 1995b). Binding and molecular studies indicate that melatonin receptors undergo a daily rhythm of expression, increasing late during the light period and decreasing late during the dark period (Brooks and Cassone 1992; Wiechmann et al. 1999). Melatonin is synthesized at night by retinal photoreceptors where it is involved in photoreceptor outer disc shedding and phagocytosis (White and Fisher 1989), photomechanical movements (Pierce and Besharse 1987), increases in sensitivity of horizontal cells (Wiechmann et al. 1988), and changes in photoreceptor conductance (Brzezinski 1997). Melatonin also is synthesized at night in the pineal gland (Borjigin et al. 1999; Reiter 1991), from which it is released into the cerebrospinal fluid and blood; it modulates functions such as circadian rhythms, sleep, seasonal reproduction, neuroimmunological activities, and avian seasonal neuroplasticity (Arendt 2000; Brzezinski 1997; Guerrero and Reiter 2002).
Melatonin receptors mediate inhibitory effects of melatonin on increases of intracellular calcium induced by depolarizing influences (Zisapel and Laudon 1983). Using electrophysiological and fluorescence calcium-imaging approaches, this effect has been demonstrated in rat pituitary cells (Vanecek and Klein 1992) and in rat brain synaptosomes (Vacas et al. 1984). Melatonin also decreases the spontaneous discharge rate in cells of the striate cortex of the cat (Reuss and Kiefer 1989). In addition, several studies indicate that melatonin exerts an enhancing influence on GABAergic activity, increasing -aminobutyric acid (GABA) binding, turnover, and GABA-induced chloride ion uptake (Boatright et al. 1994; Rosenstein and Cardinali 1990; Rosenstein et al. 1989; Wan et al. 1999b). These observations raised the possibility that melatonin might influence tectal GABA receptors, which include ionotropic GABAA (Xiao et al. 1999) and GABAC receptors (Sivilotti and Nistri 1989) and metabotropic GABAB receptors (Sivilotti and Nistri 1988).
The prominent expression of melatonin receptors in the optic tectum and the evidence of melatonin's modulatory effects in other parts of the nervous system thus led us to test melatonin's effects by measuring calcium levels in tectal cells. For this purpose, we administered melatonin to tectal slices and compared the depolarization-induced calcium increase in slices pretreated with melatonin to nonpretreated slices. We also evaluated whether the effect of melatonin in the tectum is influenced by the light–dark cycle by assessing the effect of melatonin on tectal slices from animals killed at 2 different times in a 12 h:12 h light–dark cycle. Finally, we tested the possible involvement of GABA receptors as mediators of melatonin's effects. Our results support a role of melatonin in the tectal physiology of Xenopus through modulation of GABAC receptors.
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METHODS |
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4 wk before experiments were performed. Slice preparation and incubation times were equal for both groups of animals. All procedures were conducted with the approval of the State University of New York at Buffalo Animal Care Committee and in accordance with their regulations. Solutions and drugs
The ionic composition of the sucrose solution used for dissection and first hour recovery was (in mM): 3.3 KCl2, 0.8 MgCl2, 0.1 NaH2PO4, 10 dextrose, 223 sucrose, 1.8 CaCl2, and 26 NaHCO3, 1 pyruvic acid, 0.05 glutathione, 1 kynurenic acid, pH 7.4, 295–300 mOsm equilibrated with a gas mixture of 95% O2-5% CO2. The composition of the amphibian Ringer solution used for overnight incubation of the slices was (in mM): 111 NaCl, 3.3 KCl, 0.8 MgCl2, 26 NaHCO3, 1.8 CaCl2, 10 dextrose, and 0.1 NaH2PO4 bubbled with a gas mixture of 95% O2 and 5% CO2. The ionic composition of the recording solution was (in mM): 134.5 NaCl, 3.3 KCl, 0.8 MgCl2, 1.8 CaCl2, 10 dextrose, 5 HEPES, bubbled with O2, pH 7.4, 295–300 mOsm. The recording solution containing 22 mM KCl was prepared by substituting equimolar amounts of NaCl and KCl. Except as noted below, bicuculline (40 µM), 1 mM kynurenic acid, and 0.5 µM tetrodotoxin (TTX; Calbiochem, La Jolla, CA) were added to the recording solutions to block GABAA receptors, ionotropic glutamate receptors, and voltage-sensitive sodium channels, respectively. Melatonin stocks were dissolved in ethanol and then further diluted in the perfusion solution. The maximum concentration of ethanol applied to the brain slice was 0.0002%; tests of ethanol vehicle showed that this level of ethanol did not influence the responses of the slices. Stock solutions (1 mM) of the AM-ester form of Fluo-4 (a fluorescent calcium indicator) were made in absolute dimethylsulfoxide (DMSO). Aliquots were diluted with Ringer solution to give a final concentration of 1 µM. The nonionic detergent Pluronic F-127 (final concentration of 0.01%) was added to increase solubility.
Chloromelatonin, 4-phenyl-2-propionamidotetralin (4-P-PDOT), and picrotoxin were purchased from Tocris (Ellisville, MO). Fluo-4 AM and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR). All the other chemicals were purchased from Sigma (St. Louis, MO).
Slice preparation
Animals were anesthetized by subcutaneous injection of 3-aminobenzoic acid ethyl ester (MS-222) and were then decapitated. Animals were killed at Zeitgeber time (ZT) 10 h except as noted. The brains were rapidly removed and placed in a chamber filled with chilled sucrose solution. Brains were then embedded in 4% low-melting-point agarose for slicing. The tissue was cut at 300 µM with a Vibratome (Vibratome, St. Louis, MO) while bathed in the same sucrose solution. Slices were then incubated in this solution for 1 h, after which they were transferred to chilled amphibian Ringer solution. Slices were incubated in this solution overnight. We compared slices prepared the same day with slices incubated overnight and found that overnight incubation produces a far higher yield of responsive tissue.
Labeling of tectal cells
Brain slices were labeled by soaking in a solution of Fluo-4 AM as described by Feller et al. (1996) (modifying the incubation time from 1 h to 7 min). After staining, slices were washed for 30 min with perfusion saline before use.
Calcium imaging
All experiments were performed at room temperature (20–24°C). Imaging experiments were performed with an Olympus BX51WI microscope equipped for epifluorescence. An Olympus 40 x (N.A. 0.8) water-immersion lens was used. Slices were placed in a perfusion chamber (Warner Instruments, Hamden, CT) with a 230-µL working bath volume and perfused at a rate of about 2 ml/min. Excitation light was provided by a 103 W/2 mercury short arc lamp (Olympus) and was attenuated by using neutral density filters to avoid photobleaching. Excitation and emission wavelengths were obtained by using a fluorescein-isothiocyanate (FITC) filter (excitation wavelength 490, emission 520). Images were collected with a Cooke Sensicam QE (Cooke, Auburn Hills, MI). Exposure times were between 100 and 300 ms, and frames were taken every 10 s. Acquisitions and analyses were performed with SLIDEBOOK software (Intelligent Imaging Innovations, Denver, CO).
Fluorescence was measured from regions of interest containing multiple cell bodies in layer 6, where cells that receive inputs from the retina and nucleus isthmi reside. Only slices that showed a similar increase in fluorescence to 3 initial depolarization stimuli were studied; about 60% of the slices met this criterion. Responses are presented as normalized 
F/F, where F is the resting fluorescence (before stimulation) and F is the peak change in fluorescence from resting levels. Normalization was accomplished by computing the ratio of the F/F response to melatonin to the F/F of a prior response to KCl. This normalization allowed us to standardize values across slices. Only responses to melatonin in slices that showed recovery, evidenced by subsequent response to KCl of nearly 95% of the initial response, were included in computations.
Pertussis toxin (PTX) treatment
Slices were incubated for 4 h in a 5% CO2, 95% O2 bubbled, static bath with 2 µg/ml PTX in Ringer solution. Control slices were incubated for equal amounts of time in Ringer solution.
Antagonist pretreatment
Slices were incubated for 1 h in a static bath, bubbled with 5% CO2, 95% O2, with 1 µM 4-P-PDOT in Ringer solution before perfusion was resumed and either melatonin or vehicle was applied. 4-P-PDOT was dissolved to 10 mM in 95% ethanol and diluted to 1 mM in 50% ethanol. Further serial dilutions were performed in Ringer solution.
RT/PCR
Total RNA was isolated from pooled samples of optic tectum, using an RNeasy total RNA isolation kit (Qiagen, Santa Clarita, CA), according to the manufacturer's instructions. Total RNA (500 ng/sample) was reverse-transcribed (RT) using components from an RNA PCR Kit (Perkin–Elmer, Norwalk, CT) in a volume of 10 µl. The cDNA was then amplified by the polymerase chain reaction (PCR), using oligonucleotide primers specific for the melatonin MT1, MT2, or Mel1c receptors, in a final volume of 50 µl. PCR primers were based on the Xenopus laevis melatonin receptor cDNA sequences (Ebisawa et al. 1994; Reppert et al. 1995b). For the MT1 receptor, the 5' primer sequence was [5'AAT CGC CTA TTC TCT AAT TCC GGC3'] and the 3' primer sequence was [5'AGA ATC TCC AAG GGG TGG ATA GAT3'], which corresponds to positions 16–39 and 426–403, respectively (GenBank accession number U31826), to generate a PCR product of 459 bp. For the MT2 receptor, the 5' primer sequence was [5'GAA AAG CTG TTC AGC CTG TGG3'] and the 3' primer sequence was [5'TTT GGG TGC TAC TTC TGT AGG3'], which corresponds to positions 16–36 and 423–403, respectively (GenBank accession number U31827), to generate a PCR product of 388 bp. For the Mel1c receptor, the 5' primer sequence was [5'ATG ATG GAG GTG AAT AGC AC3'] and the 3' primer sequence was [5'ACA CAG CAA CAA CCA GAT CG3'], which corresponds to positions 1–20 and 250–231 of the coding region, respectively (GenBank accession number U09561), to generate a PCR product of 251 bp. PCR reactions were performed with 1 cycle of 95°C for 5 min, then 40 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min, followed by 1 cycle of 72°C for 7 min; then 15 µl of the amplified cDNA was electrophoresed on an agarose gel and stained with ethidium bromide.
Melatonin assay
To extract melatonin, brain samples were sonicated in methanol and the supernatant was removed after centrifugation and diluted with water to contain 10% methanol. Melatonin was then extracted using C18 columns, and samples were dried (Savant Speed Vac) and reconstituted with assay buffer. The melatonin assay was conducted using a Buehlemann Lab radioimmunoassay kit (ALPCO Diagnostics, Windham, NH), according to protocol. This is a sensitive method with 0.33 pg/ml analytical and 0.62–0.80 pg/ml functional detectability. All samples were analyzed within the same extraction and assay procedure. Intraassay variability was 6.3%. Protein content in the pellet was measured using a BCA kit (Pierce Biotechnology, Rockford, IL).
Western blot
After incubation, slices were homogenized directly in 1 x Laemmli sample buffer. The lysates were denatured at 95°C for 10 min. Insoluble material was removed by centrifugation (14,000g for 10 min), and the protein concentration for each sample was measured by the method of Bradford (1976). Stringent denaturing conditions were achieved by separating denatured proteins by electrophoresis on a 6 M urea and 10% SDS–PAGE gel with prestained protein standards (Bio-Rad, Palo Alto, CA) and the proteins were transferred to nitrocellulose membranes. The membrane was blocked in tris-buffered saline (TBS) plus 5% nonfat dry milk (Carnation, Solon, OH) for 1 h at room temperature. Blots were incubated overnight at 4°C with antibodies generated against Xenopus MT2 receptors diluted 1:500 with blocking solution. After rinsing, blots were incubated with horseradish peroxidase–conjugated anti-rabbit antibodies (Pierce, Rockford, IL) diluted 1:20,000 for 1 h at room temperature. After washing, chemiluminescence detection was performed using the manufacturer's protocol (Supersignal West Pico Kit, Pierce). Immunoreactive bands were analyzed using a Bio-Rad GS-700 imaging densitometer and Multi-Analyst software (Version 1.1).
MT2 antibody
A polyclonal antibody directed against a 13 amino acid peptide (VKSEFKPRMQSDF), corresponding to a region of an intracellular loop of the Xenopus laevis MT2 receptor (Reppert et al. 1995b), was generated in rabbits (Invitrogen, Carlsbad, CA).
Statistical analysis
All data were analyzed by a 2-tailed, unpaired Student's t-test or by a one-way ANOVA followed by the Bonferroni post hoc test (SPSS Software, SPSS, Chicago, IL). Significance was defined as P < 0.05.
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RESULTS |
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To determine changes in tectal calcium levels, slices were incubated with the membrane-permeant form of Fluo-4, rinsed, and placed in a perfusion chamber (Fig. 1A). A rapid increase in Fluo-4 fluorescence was observed in response to depolarization (Fig. 1B). We tested increasing concentrations of KCl (15, 22, 26, and 30 mM) to establish a concentration of KCl that would permit us to observe either increases or decreases in calcium resulting from melatonin coapplication. Brain slices were stimulated at least 3 times with the same KCl concentration for 90 s. A 5-min interval was allowed between stimuli for washing. Each healthy slice was stimulated 8–10 times over a period of 1.5–2 h; responses to KCl were consistent over that period. The data were fitted to a Hill plot (not shown), and 22 mM KCl, which elicited a response of 80% of saturation levels, was chosen for subsequent experiments. In addition, for each graph presented in the RESULTS, every tectal slice contributing to the data was exposed to all the experimental conditions tested; the relatively high values of n for exposure to KCl reflect the repeated testing with KCl before and after each drug trial. Calcium transients were measured as F/F values.
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To investigate whether melatonin exerts an effect on depolarization-evoked calcium increases, tectal slices were pretreated for 4 min with melatonin followed by perfusion with 22 mM KCl plus melatonin. Pretreatment with melatonin inhibited the increase in calcium in tectal cells (Fig. 1C). This effect was concentration dependent, with a significant inhibition observed even at the lowest concentration tested, 50 nM (Fig. 2A). At the maximum melatonin concentration tested (5 µM), the peak Fluo-4 fluorescence averaged 58 ± 6% (SE) of control (n = 3, P < 0.05). The response to depolarization recovered to control levels after a 5-min washout period. In addition, we assessed whether melatonin had a similar inhibitory influence on increases of calcium elicited by a lower KCl concentration (15 mM); 0.05 µM melatonin elicited peak Fluo-4 fluorescence values that were 78% of control levels, essentially the same as those obtained with 22 mM KCl (data not shown).
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, we measured the tectal protein content to be about 630 µg and, based on these values, the amount of tectal melatonin was estimated as 0.57 ng. Because tectal volume is about 10 µl and the molecular weight of melatonin is 232.28 g/M, the concentration of melatonin in the tectum was estimated as 245 nM, close to the value used in most of our experiments. The specificity of the effect of melatonin was tested using chloromelatonin (0.5 µM), a melatonin analog. Pretreatment of tectal slices for 4 min with chloromelatonin followed by perfusion with 22 mM KCl plus chloromelatonin mimicked the effect of melatonin by decreasing the peak Fluo-4 fluorescence to 73% of control (n = 5, P < 0.05) (Fig. 2C). This effect is reversible.
Role of GABA receptors in melatonin-induced inhibition of depolarization-mediated calcium increase
Several studies indicate that melatonin potentiates GABA-induced increases of chloride ion influx by regulating GABA-receptor activity (Rosenstein et al. 1989; Wan et al. 1999a; Wu et al. 1999). Because GABA-immunoreactive structures are found throughout the frog's optic tectum (Antal 1991; Li and Fite 2001; Rybicka and Udin 1994), we next studied whether the effect of melatonin on depolarization-evoked calcium increases in tectal slices is mediated by GABA-receptor activation. For this purpose, we examined the response of tectal cells to depolarization stimuli in conditions where GABAA-, GABAB-, or GABAC-receptor antagonists were added in combination with melatonin during depolarization (Fig. 3). Exogenous GABA was not added because depolarization was expected to cause release of GABA from tectal interneurons. In addition, to specifically test the effect of each GABA antagonist, bicuculline was omitted from the amphibian Ringer solution in these experiments. Bath application of the GABA-receptor antagonists alone had no significant effect on fluorescence increases elicited by 22 mM KCl compared with control levels (Fig. 3, black bars). Bicuculline (40 µM) and CPG 35348 (100 µM), GABAA- and GABAB-receptor antagonists, respectively, did not alter the effects of melatonin (Fig. 3, A and B). In contrast, picrotoxin (100 µM), a GABAA–C-receptor antagonist, counteracted the effect of melatonin (Fig. 3C). Furthermore, 2,5,6-tetrahydropyridine-4-yl methylphosphonic acid (TPMPA, 100 µM), a highly selective GABAC-receptor antagonist, significantly diminished the melatonin effect (Fig. 3D); changes in fluorescence resulting from 22 mM KCl/melatonin/TPMPA were not significantly different from the changes caused by 22 mM KCl alone (Fig. 3D). Because TPMPA, a purely GABAC-receptor antagonist, did not appear to be as effective an antagonist as picrotoxin, a GABAA–C-receptor antagonist, we also tested the 2 drugs in combination to evaluate further whether GABAA receptors might be playing a role. As Fig. 3D indicates, the effects of the antagonists were not additive. These results are consistent with the hypothesis that the effect of melatonin on tectal cells is mediated by GABAC receptors.
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We used RT-PCR to determine which of the 3 known Xenopus melatonin receptors are expressed in the tectum. PCR primers specific for all 3 melatonin receptors (MT1, MT2, and Mel1c) were reverse-transcribed and PCR-amplified (RT-PCR) from RNA isolated from Xenopus tectum. Agarose gel electrophoresis demonstrated PCR products of the expected size in tectum samples of late tadpoles, juveniles, and adult animals amplified with the MT2 and the Mel1c primers, but MT1 product was observed only in tadpoles and juveniles 
2 wk postmetamorphosis (Fig. 4). These results indicate that the Xenopus tectum expresses the 3 known melatonin receptors and suggest that their expression is influenced by age.
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EFFECT OF PERTUSSIS TOXIN (PTX).
To determine whether the effects of melatonin are mediated by melatonin receptors, which are G-protein coupled, we performed experiments in which tectal slices were preincubated with PTX, a blocker of inhibitory (Gi) and other (Go) G-proteins (Hildebrandt et al. 1983). Pretreatment of slices with PTX did not affect increases in Fluo-4 fluorescence resulting from KCl alone. Also, control slices preincubated in a static bath for 3–5 h in the absence of PTX showed the expected inhibitory effect of melatonin (66% of the response to KCl alone). In contrast, PTX pretreatment resulted in a significant inhibition of the melatonin effect on depolarization-induced calcium increases (Fig. 5); values in PTX-treated slices were 90% of control. These observations suggest that melatonin acts through a pertussis toxin–sensitive G-protein to decrease elevations of intracellular calcium induced by depolarization stimuli.
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), we sought to determine whether the MT2 receptor is also involved in the modulation of retinal input to the tectum by evaluating the effect of melatonin in tectal slices. For this purpose, we used 4-P-PDOT, a melatonin receptor antagonist with a 300-fold higher selectivity for the MT2 receptor over the MT1 receptor (Dubocovich et al. 1997). 4-P-PDOT (1 µM) counteracted the inhibitory effect of 50 nM melatonin in tectal cells (Fig. 6). Preincubation of slices with 4-P-PDOT had no effect on increases of fluorescence in response to 22 mM KCl alone. These results suggest that in tectal cells, the effect of melatonin on stimulated calcium increases is mediated by the MT2 receptor.
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Because the effect of melatonin on calcium increases in tectal slices was antagonized by 4-P-PDOT, an antagonist with a high selectivity for the MT2 receptor, we decided to test whether the protein for this receptor is present in the Xenopus tectum. Using sequence data for the MT2 receptor, we raised an antibody to a peptide corresponding to an intracellular loop of the receptor. With this antibody, we performed Western blot analysis using nonreducing SDS–PAGE, which revealed an immunoreactive band of about 85 kD (data not shown). Because molecular characterization of this receptor predicts a molecular mass of about 40 kD (Reppert et al. 1995a), we considered the possibility that the receptor is assembled as a dimer (Ayoub et al. 2002), and we therefore tested the effect of more stringent reducing conditions (as described in METHODS). This modification resulted in the detection of a lower band, with apparent molecular mass roughly corresponding to the expected monomeric species of the receptor (about 45 kD) (Fig. 7, left lane). In addition, isolated membranes of tectal tissue were treated with endoglycosidase H (Endo H), a recombinant glycosidase that cleaves oligosaccharides from N-linked glycoproteins (Maley et al. 1989). This treatment resulted in a decrease of the intensity of the higher and lower molecular weight immunoreactive bands and the subsequent detection of a new, nearly 38-kD band (Fig. 7, right lane). This effect is consistent with reports of sites for asparagine-linked glycosylation in the amino terminal of the MT2 receptor (Reppert et al. 1995a).
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To determine whether the inhibitory influence of melatonin on depolarization-induced calcium increase is influenced by the light–dark cycle, the effects of melatonin on tectal tissue obtained from animals killed at 2 different ZT were compared. We found that the effect of melatonin was more pronounced in animals killed at ZT 10 (ZT 0 = lights ON) than in animals killed at ZT 23 (Fig. 8A). The decrease of F/F at ZT 10 was significantly different from the values obtained at ZT 23 (n = 15, P < 0.05).
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DISCUSSION |
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Melatonin modulation of GABAC-receptor function and possible impact on tectal activity
A link between melatonin and GABAergic pathways was previously reviewed (Rosenstein and Cardinali 1990), and several groups provided evidence of such links in Xenopus retina, where melatonin receptor immunoreactivity is colocalized with GABAergic amacrine cells (Wiechmann and Wirsig-Wiechmann 2001b), and the GABA-receptor blockers, picrotoxin and bicuculline, block melatonin-induced suppression of dopamine release (Boatright et al. 1994).
We also demonstrated that suppression of depolarization-induced calcium accumulation by melatonin involves GABAC receptor activation. Picrotoxin and TPMPA, nonselective and selective GABAC antagonists, respectively, reversed the melatonin-induced inhibition of calcium increase (Fig. 3).
Pharmacological experiments have indicated that GABAC receptors are present in the frog tectum (Sivilotti and Nistri 1989). In addition, studies in the mammalian superior colliculus, the mammalian counterpart of the frog tectum, have suggested a postsynaptic role of GABAC receptors in GABAergic transmission (Kirischuk et al. 2003). These reports reveal that GABAC receptors mediate an excitatory action of GABA on the activity of projection neurons induced by optic nerve stimulation (Boller and Schmidt 2003; Pasternack et al. 1999; Platt and Withington 1998; Sivilotti and Nistri 1989). These results appear to contradict our observations that GABAC receptors mediate the inhibitory effect of melatonin. However, these authors attributed the excitatory action of GABAC receptors to disinhibition, a multisynaptic effect. Because we used TTX, bicuculline, and kynurenic acid, we observed only direct effects. In contrast, retinotectal activity initiates a chain of events, in which disinhibition may be crucial for triggering the firing of tectal projection neurons that mediate prey catching or predator avoidance. If so, the presence of melatonin would alter such cascades of activity.
Because GABAC receptors are down-modulated by a cAMP-dependent protein kinase (PKA) (Dong and Werblin 1994; Wellis and Werblin 1995), and melatonin decreases cAMP (Wiechmann and Wirsig-Wiechmann 1993), melatonin could initiate an intracellular signaling pathway involving inhibition of adenylyl cyclase, a decrease of cAMP, and relief of cAMP-dependent down-modulation of GABAC receptors (Fig. 9). The resulting increase in chloride influx by the GABAC receptor would then oppose the depolarizing effect of KCl and thereby reduce the opening of voltage-sensitive calcium channels. This model is supported by reports that melatonin-induced hyperpolarization of plasma membrane is an upstream event to its influence on calcium influx through voltage-dependent channels (Vanecek 1999; Vanecek and Klein 1992). The impact of melatonin on tectal functioning is thus mediated in part by GABAC receptors, and the circadian periods when melatonin levels are high are predicted to be periods of high GABAC-mediated disinhibition within the tectum. However, melatonin also affects other components of the visual system that influence tectal function; as noted above, melatonin receptors directly affect retinal functioning (Brzezinski 1997; Wiechmann et al. 1988), and our work in progress also shows that melatonin influences the responses of retinotectal axons (Prada and Udin, unpublished observations). Moreover, melatonin also affects calcium levels in the nucleus isthmi, a midbrain structure with reciprocal connections to the tectum (Gruberg and Udin 1978; R. Lima and S. Udin, unpublished observations). The net effect of these multiple sites of melatonin's influences has yet to be established.
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We propose that the effect of melatonin in tectal slices is mediated at least in part by the MT2 receptor, for several reasons: 1) PCR and Western blotting show expression of this receptor in the tectum; 2) diurnal variations in responses to melatonin correlate well with changes in MT2 receptor expression; and 3) the MT2 receptor antagonist 4-P-PDOT (Dubocovich et al. 1997) counteracts the effect of melatonin in tectal cells. In the case of interpretation of the 4-P-PDOT data, however, some caution is required because the selectivity of the drug has not been established in Xenopus.
In Xenopus, evidence that MT2 receptors are also expressed in the retina (Wiechmann and Wirsig-Wiechmann 2001a) also supports the idea that this receptor contributes to the effect of melatonin on visual function.
In the optic tectum of Xenopus, evidence of melatonin receptors has been indicated by autoradiographic receptor binding (Mazzucchelli et al. 1996). Our data imply that a substantial amount of binding is likely to involve the MT2 receptor. In addition, our Western blot analyses performed under different denaturing conditions suggest that MT2 is expressed primarily as a dimeric complex. This result is in line with a report that demonstrated ligand-independent dimerization of melatonin receptors (Ayoub et al. 2002). Our Western blot analyses revealed 2 immunoreactive bands of about 45 and 85 kDa in the membrane fraction of the optic tectum. The predicted molecular weight of the MT2 receptor is about 40 kDa, not including posttranslational modifications (Reppert et al. 1995a). We hypothesize that the higher band observed in our immunoblots represents the glycosylated dimeric form of the receptor and that the lower band represents the glycosylated monomer form, as indicated by the appearance of a new, nearly 38-kDa band after treatment of membrane fractions with Endo H. Glycosylation of this receptor is supported by analyses of its sequence, which contains a consensus N-terminal glycosylation site (Reppert et al. 1995a). It is interesting that Endo H treatment did not result in a lower band of the dimeric form. This observation suggests that glycosylation plays an important role in stabilization of the dimeric complexes.
The effect of melatonin was prevented by pretreatment of the slices with PTX (Fig. 5). Because PTX blocks the effects of agents that act through Gi/Go proteins (Katada and Ui 1982), we postulate that melatonin's effect involves a G-protein–mediated mechanism. Altogether, these data indicate that in tectal slices, melatonin decreases depolarization-induced calcium increase through a receptor-mediated mechanism that probably involves the MT2 receptor. The present data, however, do not preclude an involvement of the Mel1c receptor in the effect of melatonin on tectal cells.
Tectal melatonin content
We have calculated that the concentration of melatonin in the Xenopus tectum is about 250 nM. This concentration is much higher than the circulating concentration of the hormone reported in amphibians (0.5–1 nM) (d'Istria et al. 1994; Wright et al. 2003). However, several studies have reported that the brain concentration of melatonin greatly exceeds its plasma concentration (Cardinali et al. 1997; Hedlund et al. 1977), a result that probably stems from the ability of brain tissue to accumulate melatonin (Ferreira et al. 1996).
Most of our experiments were conducted using 500 nM melatonin, which is about twice the dark-period value that we have estimated for tectal melatonin. Because we used bath perfusion and slices of 300 µm thickness, the effective concentration of melatonin reaching the center of the slice, where the healthiest cells are most likely to be located, should be close to the physiological level.
Influence of the light–dark cycle on melatonin's effect and on receptor density
Not surprisingly, the effect of melatonin on tectal slices was influenced by the light–dark cycle. Melatonin's effect was less pronounced in tissue removed late during the dark period (ZT 23) than late in the light period (ZT 10). This finding is consistent with a decrease in MT2 receptor protein in tecta of animals killed at ZT 23 as compared with ZT 10 (Fig. 8). These results support previous studies indicating that melatonin receptors undergo a diurnal rhythm of expression (Guerrero et al. 2000; Masana et al. 2000; Wiechmann and Smith 2001). Furthermore, melatonin levels in the Xenopus brain were high during the dark period and low during the light period (Fig. 2B), consistent with the possibility that high levels of melatonin downregulate melatonin receptor expression. Our data in Xenopus are in general agreement with observations in other species (Aste et al. 2001; Brooks and Cassone 1992) and indicate that the light–dark cycle influences the levels of melatonin and of its receptors. The net effect of the changes in melatonin and melatonin receptors is likely to be dominated by the large magnitude of the circadian fluctuations in melatonin levels rather than by the relatively small changes in melatonin receptor numbers, although our data do not directly address this point.
Melatonin's effect on calcium levels
The present results are consistent with previous work indicating an inhibitory effect of melatonin on calcium accumulation resulting from excitatory inputs (Ayar et al. 2001; Dubocovich 1983; Vacas et al. 1984; Vanecek and Klein 1992; Zisapel and Laudon 1983). Several studies indicate that the effects of melatonin on calcium accumulation include a downstream influence on calcium influx through calcium channels (Ayar et al. 2001; Mei et al. 2001; Vanecek and Klein 1995). Our results suggest that in tectal cells, melatonin regulates calcium increases by an action involving an inhibitory G-protein and GABA receptors. We hypothesize that melatonin may in part regulate calcium levels by enhancing GABA-mediated hyperpolarization, which would then decrease calcium influx through voltage-sensitive calcium channels.
In conclusion, we have demonstrated that the optic tectum of Xenopus has functional melatonin receptors. In tectal slices, exogenous melatonin inhibits increases in cytosolic calcium evoked by depolarization stimuli with 22 mM KCl. Our data indicate that this effect is receptor mediated and involves activation of GABAC receptors. In addition, the light–dark cycle influences melatonin levels in the Xenopus brain, the effect of melatonin in tectal slices, and the density of expression of the MT2 receptor.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. B. Udin, Professor of Physiology and Biophysics, 553 Biomedical Research Building, 3435 Main Street, SUNY at Buffalo, Buffalo, NY 14214 (E-mail: sudin{at}buffalo.edu)
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REFERENCES |
|---|
|
Arendt J. Melatonin, circadian rhythms, and sleep. N Engl J Med 343: 1114–1116, 2000.
Aste N, Cozzi B, Stankov B, and Panzica G. Sexual differences and effect of photoperiod on melatonin receptor in avian brain. Microsc Res Tech 55: 37–47, 2001.[CrossRef][Web of Science][Medline]
Ayar A, Martin DJ, Ozcan M, and Kelestimur H. Melatonin inhibits high voltage activated calcium currents in cultured rat dorsal root ganglion neurones. Neurosci Lett 313: 73–77, 2001.[CrossRef][Web of Science][Medline]
Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M, and Jockers R. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 277: 21522–21528, 2002.
Besharse JC and Dunis DA. Methoxyindoles and photoreceptor metabolism: activation of rod shedding. Science 219: 1341–1343, 1983.
Boatright JH, Rubim NM, and Iuvone PM. Regulation of endogenous dopamine release in amphibian retina by melatonin: the role of GABA. Vis Neurosci 11: 1013–1018, 1994.[Web of Science][Medline]
Boller M and Schmidt M. GABAC receptors in the rat superior colliculus and pretectum participate in synaptic neurotransmission. J Neurophysiol 89: 2035–2045, 2003.
Borjigin J, Li X, and Snyder SH. The pineal gland and melatonin: molecular and pharmacologic regulation. Annu Rev Pharmacol Toxicol 39: 53–65, 1999.[CrossRef][Web of Science][Medline]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
Brooks DS and Cassone VM. Daily and circadian regulation of 2-iodomelatonin binding in the chick brain. Endocrinology 131: 1297–1304, 1992.
Brzezinski A. Melatonin in humans. N Engl J Med 336: 186–195, 1997.
Cardinali DP, Golombek DA, Rosenstein RE, Cutrera RA, and Esquifino AI. Melatonin site and mechanism of action: single or multiple? J Pineal Res 23: 32–39, 1997.[Web of Science][Medline]
d'Istria M, Monteleone P, Serino I, and Chieffi G. Seasonal variations in the daily rhythm of melatonin and NAT activity in the Harderian gland, retina, pineal gland, and serum of the green frog, Rana esculenta. Gen Comp Endocrinol 96: 6–11, 1994.[CrossRef][Web of Science][Medline]
Dong CJ and Werblin FS. Dopamine modulation of GABAC receptor function in an isolated retinal neuron. J Neurophysiol 71: 1258–1260, 1994.
Dubocovich ML. Melatonin is a potent modulator of dopamine release in the retina. Nature 306: 782–784, 1983.[CrossRef][Medline]
Dubocovich ML, Masana MI, Iacob S, and Sauri DM. Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor. Naunyn Schmiedebergs Arch Pharmacol 355: 365–375, 1997.[CrossRef][Web of Science][Medline]
Ebisawa T, Karne S, Lerner MR, and Reppert SM. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores. Proc Natl Acad Sci USA 91: 6133–6137, 1994.
Faillace MP, Sarmiento MIK, and Rosenstein RE. Melatonin effect on [3H]glutamate uptake and release in the golden hamster retina. J Neurochem 67: 623–628, 1996.[Web of Science][Medline]
Feller MB, Delaney KR, and Tank DW. Presynaptic calcium dynamics at the frog retinotectal synapse. J Neurophysiol 76: 381–400, 1996.
Ferreira SA, Rollag MD, and Glass JD. Pharmacokinetics of extracellular melatonin in Siberian hamster forebrain. Brain Res 733: 318–320, 1996.[CrossRef][Web of Science][Medline]
Gruberg ER and Udin SB. Topographic connections between the nucleus isthmi and the tectum of the frog Rana pipiens. J Comp Neurol 179: 487–500, 1978.[CrossRef][Web of Science][Medline]
Guerrero HY, Gauer F, Schuster C, Pevet P, and Masson-Pevet M. Melatonin regulates the mRNA expression of the MT1 melatonin receptor in the rat pars tuberalis. Neuroendocrinology 71: 163–169, 2000.[CrossRef][Web of Science][Medline]
Guerrero JM and Reiter RJ. Melatonin-immune system relationships. Curr Top Med Chem 2: 167–179, 2002.[CrossRef][Medline]
Hedlund L, Lischko MM, Rollag MD, and Niswender GD. Melatonin: daily cycle in plasma and cerebrospinal fluid of calves. Science 195: 686–687, 1977.
Hildebrandt JD, Sekura RD, Codina J, Iyengar R, Manclark CR, and Birnbaumer L. Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature 302: 706–709, 1983.[CrossRef][Medline]
Katada T and Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci USA 79: 3129–3133, 1982.
Kirischuk S, Akyeli J, Iosub R, and Grantyn R. Pre- and postsynaptic contribution of GABAC receptors to GABAergic synaptic transmission in rat collicular slices and cultures. Eur J Neurosci 18: 752–758, 2003.[CrossRef][Web of Science][Medline]
Krause DN, Siuciak JA, and Dubocovich ML. Unilateral optic nerve transection decreases 2-[125I]-iodomelatonin binding in retinorecipient areas and visual pathways of chick brain. Brain Res 654: 63–74, 1994.[CrossRef][Web of Science][Medline]
Li Z and Fite KV. GABAergic visual pathways in the frog Rana pipiens. Vis Neurosci 18: 457–464, 2001.[CrossRef][Web of Science][Medline]
Maley F, Trimble RB, Tarentino AL, and Plummer TH Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180: 195–204, 1989.[CrossRef][Web of Science][Medline]
Masana MI, Benloucif S, and Dubocovich ML. Circadian rhythm of MT1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse. J Pineal Res 28: 185–192, 2000.[Web of Science][Medline]
Mazzucchelli C, Capsoni S, Angeloni D, Fraschini F, and Stankov B. Expression of the melatonin receptor in Xenopus laevis: a comparative study between protein and mRNA distribution. J Pineal Res 20: 57–64, 1996.[Web of Science][Medline]
Mei YA, Lee PP, Wei H, Zhang ZH, and Pang SF. Melatonin and its analogs potentiate the nifedipine-sensitive high-voltage-activated calcium current in the chick embryonic heart cells. J Pineal Res 30: 13–21, 2001.[CrossRef][Web of Science][Medline]
Pasternack M, Boller M, Pau B, and Schmidt M. GABAA and GABAC receptors have contrasting effects on excitability in superior colliculus. J Neurophysiol 82: 2020–2023, 1999.
Pierce ME and Besharse JC. Melatonin and rhythmic photoreceptor metabolism: melatonin-induced cone elongation is blocked at high light intensity. Brain Res 405: 400–404, 1987.[CrossRef][Web of Science][Medline]
Platt B and Withington DJ. GABA-induced long-term potentiation in the guinea-pig superior colliculus. Neuropharmacology 37: 1111–1122, 1998.[CrossRef][Web of Science][Medline]
Reiter RJ. Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12: 151–180, 1991.
Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA, and Gusella JF. Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci USA 92: 8734–8738, 1995a.
Reppert SM, Weaver DR, Cassone VM, Godson C, and Kolakowski L Jr. Melatonin receptors are for the birds: molecular analysis of two receptor subtypes differentially expressed in chick brain. Neuron 15: 1003–1015, 1995b.[CrossRef][Web of Science][Medline]
Reuss S and Kiefer W. Melatonin administered systemically alters the properties of visual cortex cells in cat: further evidence for a role in visual information processing. Vision Res 29: 1089–1093, 1989.[CrossRef][Web of Science][Medline]
Rosenstein R and Cardinali D. Central GABAergic mechanisms as targets for melatonin activity in brain. Neurochem Int 17: 373–379, 1990.
Rosenstein R, Esteves E, and Cardinal D. Time-dependent effect of glutamic acid decarboxylase and Cl2+ influx in rat hypothalamus. J Neuroendocr 1: 443–447, 1989.
Rybicka KK and Udin SB. Ultrastructure and GABA immunoreactivity in layers 8 and 9 of the optic tectum of Xenopus laevis. Eur J Neurosci 6: 1567–1582, 1994.[CrossRef][Web of Science][Medline]
Sivilotti L and Nistri A. Complex effects of baclofen on synaptic transmission of the frog optic tectum in vitro. Neurosci Lett 85: 249–254, 1988.[CrossRef][Web of Science][Medline]
Sivilotti L and Nistri A. Pharmacology of a novel effect of gamma-aminobutyric acid on the frog optic tectum in vitro. Eur J Pharmacol 164: 205–212, 1989.[CrossRef][Web of Science][Medline]
Stenkamp DL, Iuvone PM, and Adler R. Photomechanical movements of cultured embryonic photoreceptors: regulation by exogenous neuromodulators and by a regulable source of endogenous dopamine. J Neurosci 14: 3083–3096, 1994.[Abstract]
Vacas MI, Keller Sarmiento MI, and Cardinali DP. Pineal methoxyindoles depress calcium uptake by rat brain synaptosomes. Brain Res 294: 166–168, 1984.[CrossRef][Web of Science][Medline]
Vanecek J. Inhibitory effect of melatonin on GnRH-induced LH release. Rev Reprod 4: 67–72, 1999.[Abstract]
Vanecek J and Klein D. Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells. Endocrinology 130: 701–707, 1992.
Vanecek J and Klein DC. Melatonin inhibition of GnRH-induced LH release from neonatal rat gonadotroph: involvement of Ca2+ not cAMP. Am J Physiol Endocrinol Metab 269: E85–E90, 1995.
Wan Q, Wan H-Y, Liu F, Braunton J, Niznik HB, Pang SF, Brown GM, and Wang YT. Differential modulation of GABAA receptor function by Mel1a and Mel1b receptors. Nat Neurosci 2: 401–403, 1999b.[CrossRef][Web of Science][Medline]
Wellis DP and Werblin FS. Dopamine modulates GABAc receptors mediating inhibition of calcium entry into and transmitter release from bipolar cell terminals in tiger salamander retina. J Neurosci 15: 4748–4761, 1995.[Abstract]
White MP and Fisher LJ. Effects of exogenous melatonin on circadian disc shedding in the albino rat retina. Vision Res 29: 167–179, 1989.[CrossRef][Web of Science][Medline]
Wiechmann AF, Campbell LD, and Defoe DM. Melatonin receptor RNA expression in Xenopus retina. Mol Brain Res 63: 297–303, 1999.[Medline]
Wiechmann AF and Smith AR. Melatonin receptor RNA is expressed in photoreceptors and displays a diurnal rhythm in Xenopus retina. Brain Res Mol Brain Res 91: 104–111, 2001.[Medline]
Wiechmann AF and Wirsig-Wiechmann CR. Distribution of melatonin receptors in the brain of the frog Rana pipiens as revealed by in vitro autoradiography. Neuroscience 52: 469–480, 1993.[CrossRef][Web of Science][Medline]
Wiechmann AF and Wirsig-Wiechmann CR. Melatonin receptor mRNA and protein expression in Xenopus laevis nonpigmented ciliary epithelial cells. Exp Eye Res 73: 617–623, 2001a.[CrossRef][Web of Science][Medline]
Wiechmann AF and Wirsig-Wiechmann CR. Multiple cell targets for melatonin action in Xenopus laevis retina: distribution of melatonin receptor immunoreactivity. Vis Neurosci 18: 695–702, 2001b.[CrossRef][Web of Science][Medline]
Wiechmann AF, Yang X-L, Wu SM, and Hollyfield JG. Melatonin enhances horizontal cell sensitivity in salamander retina. Brain Res 453: 377–380, 1988.[CrossRef][Web of Science][Medline]
Wright ML, Duffy JL, Guertin CJ, Alves CD, Szatkowski MC, and Visconti RF. Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog, Rana catesbeiana. Gen Comp Endocrinol 130: 120–128, 2003.[CrossRef][Web of Science][Medline]
Wu FS, Yang YC, and Tsai JJ. Melatonin potentiates the GABAA receptor-mediated current in cultured chick spinal cord neurons. Neurosci Lett 260: 177–180, 1999.[CrossRef][Web of Science][Medline]
Xiao J, Wang Y, and Wang SR. Effects of glutamatergic, cholinergic and GABAergic antagonists on tectal cells in toads. Neuroscience 90: 1061–1067, 1999.[CrossRef][Web of Science][Medline]
Zisapel N and Laudon M. Inhibition by melatonin of dopamine release from rat hypothalamus: regulation of calcium entry. Brain Res 272: 378–381, 1983.[CrossRef][Web of Science][Medline]
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INTRODUCTION
