Invited Symposium: Pineal and its Hormone Melatonin



Materials & Methods


Discussion & Conclusion



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Distribution and Function of Melatonin Receptors in Rat CNS

Contact Person: Gregory M. Brown (gregorym.brown@utoronto.ca)


The pineal hormone melatonin has been implicated in the regulation of circadian rhythmicity and of seasonal reproductive responses in mammals. The biological functions of melatonin are mediated by melatonin receptors. Although the majority of the previous research on melatonin receptors has focused on the suprachiasmatic nucleus (SCN) and pars tuberalis (PT) as the major brain targets of melatonin, accumulating evidence indicates a more widespread distribution of the receptors in the brain(1). The retina is another source of melatonin in the CNS, but the hormone acts locally, ctivating its receptors located within the tissue(2). The precise localization of melatonin receptors in the brain and retina is still poorly understood. Two melatonin receptor subtypes, Mel1a and Mel1b have been cloned in mammals(3,4). Although both subtypes are linked to the inhibition of adenylyl cyclase, these receptors exhibit distinct expression patterns in the mammalian CNS, suggesting that the different subtypes may mediate different melatonin effects in the CNS. Yet the role of each receptor subtype in the diverse biological activities of melatonin remains unclear. Several studies implicate SCN GABAergic transmission in the generation and/or modulation of circadian rhythmicity(5,6). Thus, we hypothesize that melatonin may regulate GABAA receptor function in the SCN and thereby exert its effects on circadian rhythmicity. The goals of the present study are 1) to localize the Mel1a receptor in rat brain and retina and 2) to reveal the modulatory role of melatonin on GABAA receptor function.

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Materials and Methods

Anti-Mel1a receptor antibody was raised against a synthetic peptide corresponding to the third intracellular loop of the human Mel1a receptor (7). Western blots of the Mel1a receptor were performed in rat brain regions and retina. Immunocytochemistry for Mel1a was carried out in rat retina. Localization of Mel1a mRNA in rat retina was examined by in situ hybridization using digoxigenin-labeled cRNA probes. 

Standard whole-cell recordings were performed in SCN and CA1 neurons of adult rat hypothalamic and hippocampal slices, as well as in transfected HEK 293 cells. GABAA receptor-mediated whole-cell currents were induced by pressure-ejection of GABA. RT-PCR analysis was performed to examine the mRNA expression of Mel1a or Mel1b in rat SCN and hippocampal tissues, and in transfected HEK 293 cells.

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Western blotting of Mel1a receptor in the rat brain revealed a single immunoreactive band at approximately 37kD in all regions examined, i.e., cerebellum, medulla, midbrain, neocortex and hypothalamus (Fig. 1). The control blots which were treated with the antibody preabsorbed with the immunogen peptide showed no immunoreactive band. 

Fig. 1: Western blots of the Mel1a receptor in discrete regions of rat brain.

Western blotting of the Mel1a receptor in the rat retina showed a 37kD band as well, which was blocked with immunogen peptide.

Immunocytochemistry revealed a specific immunoreaction in the inner plexiform layer and the outer plexiform layer of the rat retina. Mel1a mRNA was localized to ganglion cells, amacrine cells and horizontal cells by in situ hybridization using antisense RNA probes. No hybridization signals were obtained with sense RNA probes. To test the effects of melatonin on GABAA receptor function in SCN, we have used whole-cell patch-clamp techniques to record GABAA receptor-mediated currents in SCN neurons of rat hypothalamic slices. Neurons located in the ventrolateral portion of the nucleus were recorded under voltage-clamp mode at a holding potential of -60 mV (Fig. 2A). Pressure ejection of GABA (10 mM) toward the neuron induced inward currents. Bath application of melatonin (1 nM) increased current amplitudes in 19 out of 30 cells tested (2893±198 pA for melatonin treatment versus 2268±216 pA for control; Student's t test, p < 0.05) with no effects on the remaining cells ( Fig 2B), suggesting that activation of melatonin receptors in the majority of these neurons can up-regulate GABAA receptor function.

Fig. 2: (A) A high magnification infrared DIC video image of rat SCN neurons.(B) Melatonin (1 nM) potentiates GABAA receptor-mediated whole-cell currents in a SCN neuron. (C) RT-PCR analysis of Mel1a gene expression in rat SCN. (D) A high magnification infrared DIC video image of rat hippocampal CA1neurons. (E) Melatonin (1 nM) inhibits GABAA receptor-mediated whole-cell currents in a CA1 neuron. (F) RT-PCR analysis of Mel1b gene expression in rat hippocampus. (G) Melatonin (1 nM) has no effect on GABA current in cells transfected with a1b2g2 (p 0.05, n =6), but enhances GABA current in cells transfected with the a1b2g2/Mel1a combination (p < 0.05, n =9) and inhibits GABA currents in cells transfected with a1b2g2/Mel1b (p < 0.05, n =6).

To determine whether the enhancement of GABAA currents by melatonin is unique to neurons in the SCN or generalized to neurons in the central nervous system, we next investigated effects of melatonin on GABAA receptor-mediated currents in CA1 neurons in hippocampal slices (Fig. 2D). To our surprise, we did not observe melatonin-induced enhancement of the GABAA receptor-mediated currents in any neurons tested in this region. Melatonin treatment was instead found to decrease current amplitudes in 17 out of 25 CA1 neurons tested ( 1206±96 pA in melatonin treatment versus 1591±153 pA in control recording; p < 0.05; Fig. 2E). Thus, the effect of melatonin on GABAA receptor function in the responsive hippocampal neurons is opposite to that in responsive SCN neurons. Among many possibilities, the simplest explanation is that the SCN and the hippocampus may express different melatonin receptor subtypes which may mediate the opposite effects of melatonin on GABAA receptor function. Thus, we performed RT-PCR using primers specific for rat Mel1a and Mel1b to analyze the expression of Mel1a or Mel1b mRNA in rat SCN and hippocampus. As shown in Figure 2C, 2F, PCR products from the SCN specifically hybridized with the rat Mel1a oligonucleotide probes and in contrast, the hippocampal products were only recognized by the Mel1b probes. Subsequent cDNA subcloning and sequencing confirmed the identity of the products amplified from SCN tissue as rat Mel1a fragment and that from the hippocampus as rat Mel1b receptor fragment.

The opposite modulation of GABAA receptor function by melatonin in conjunction with the differential expression of Mel1a and Mel1b receptors in the SCN and hippocampus strongly suggests that Mel1a and Mel1b receptors may have distinct roles in modulating GABAA receptor function.

To test this hypothesis directly, we transiently co-transfected GABAA receptor a1b2g2 subunits with either Mel1a or Mel1b receptors into HEK293 cells. Overexpression of the Mel1a or Mel1b receptor genes in these cells was confirmed by RT-PCR analysis. We found that melatonin (1 nM) had no detectable effect on GABAA receptor-mediated whole-cell currents in cells expressing recombinant GABAA receptors only (1521±159 pA in control recording, 1510±154 pA in melatonin treatment; p 0.05, n = 6; Fig. 2G). However, melatonin (1 nM) increased the GABAA currents in cells expressing Mel1a receptors (1502±86 pA in control recording, 1818±112 pA in melatonin treatment; p < 0.05, n = 9; Fig. 2G), whereas it reduced the currents in cells expressing Mel1b receptors (1602±75 pA in control recording, 1265±68 pA in melatonin treatment; p < 0.05, n = 6; Fig. 2G). Thus these data confirm that the two melatonin receptor subtypes can mediate opposite effects on GABAA receptor function.

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Discussion and Conclusion

We have demonstrated expression of the Mel1a receptor protein in a variety of regions in rat brain. The physiological significance of melatonin receptors in those several brain regions is still not known. The widespread distribution of melatonin receptors shown in this study provides evidence for the diverse physiological functions of the hormone in the central nervous system. 

Mel1a receptor was immunocytochemically localized to the inner and outer plexiform layers and Mel1a receptor transcripts were localized to ganglion, amacrine and horizontal cells.  These results suggest that melatonin influences retinal physiology by acting on these retinal cell types via the Mel1a receptor expressed in their processes, located in the inner and outer plexiform layers. 

The lack of subtype-specific agonists and antagonists of melatonin receptors has made it impossible to determine the contributions of these two receptor subtypes to melatonin actions in the mammalian CNS. In the present work, we have provided strong evidence that these two receptors can mediate opposite effects of melatonin on a given biological function, suggesting that the receptor subtypes are critical determinants of the diversity of melatonin actions in the mammalian brain. GABA is the major inhibitory neurotransmitter in the mammalian SCN. In light of recent evidence indicating that GABAA receptor-mediated neurotransmission plays a pivotal role in the generation of cyclic firing activity of the SCN neurons, and since melatonin can act directly at the SCN to inhibit neuronal firing, the enhancement of GABAA receptor function by melatonin, through Mel1a receptors, may be responsible for the regulatory effects of melatonin on mammalian circadian time-keeping and melatonin's sleep-inducing effects. In addition, our results demonstrate that melatonin, through its Mel1b receptors, may directly affect mammalian hippocampal function.

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  1. Morgan, PJ, Barrett, P, Howell, HE, Helliwell, R (1994) Melatonin receptors: localization, molecular pharmacology and physiological significance. Neurochem. Int. 24:101-146. 
  2. Dubocovich, ML, Takahashi, JS (1987) Use of 2-[125I]Iodomelatonin to characterize melatonin binding sites in chicken retina. Proc. natn. Acad. Sci. U.S.A. 84:3916-3920 . 
  3. Reppert, SM, Weaver, DR, Ebisawa, T (1994) Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 13:1177-1185. 
  4. Reppert, SM, Godson, C, Mahle, CD, Weaver, DR, Slaugenhaupt, SA, Gusella, JF (1995) Molecular characterization of a second melatonin receptor expressed in human retina and brain: The Mel1b melatonin receptor. Proc. Natl. Acad. Sci. 12:8734-8738. 
  5. Ralph, M. R., Menaker, M. (1989) GABA regulation of circadian responses to light. I. Involvement of GABAA-benzodiazepine and GABAB receptors. J. Neurosci. 9:2858-2865. 
  6. Wagner, S., Castel, M., Gainer, H, Yarom, Y. (1997) GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity. Nature 387:598-603 
  7. Song, Y, Chan, CWY, Brown, GM, Pang, SF, Silverman, M (1997) Studies of the renal action of melatonin: evidence that the effects are mediated by 37kDa receptors of the Mel1a subtype localized primarily to the basolateral membrane of the proximal tubule. FASEB J. 11:93-100.. 
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Brown, GM; Fujieda, H; Qi, W; Pang, SF; (1998). Distribution and Function of Melatonin Receptors in Rat CNS. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/brown/brown0866/index.html
© 1998 Author(s) Hold Copyright