Background: Melatonin (N-acetyl-5-methoxytryptamine) is the principal hormone produced by the pineal gland.  It is involved in regulating neuroendocrine function, and  it acts directly on the biological clock in the suprachiasmatic nucleus (SCN) to modulate the circadian rhythmicity of diverse biological functions such as sleep, hormonal and temperature cycles.

 The physiological effects of melatonin are mediated by high- affinity receptors which  have been cloned from several species including humans. These  receptors, which are encoded by distinct genes, have been divided into three separate subtypes:Mel1a, Mel1b and Mel1c.  These receptors share only ~60% homology, but they exhibit similar binding and pharmacological characteristics.  Moreover, all three subtypes are  linked to a pertussis toxin- sensitive inhibitory G- protein (Gi), which mediates the well-documented inhibitory effect of melatonin on the adenylyl cyclase (AC)-cAMP pathway.  The  Mel1a subtype was initially thought  to be the receptor responsible for the effects of melatonin on neuronal, circadian and reproductive function in mammals, in keeping with its presence in  the SCN and pars tuberalis. However, recent studies with Mel1a knockouts  indicate involvement of the  Mel1b subtype in mediating the circadian effects of melatonin in mice.   All three subtypes are expressed in lower vertebrates (eg Xenopus, chicken and zebra fish), whereas, to date,  only the Mel1a and Mel1b receptors  have been detected in mammals.  In contrast to the relative enrichment of the Mel1a   in the SCN, the Mel1b is primarily localized in  the retina, with lower expression in  the hippocampus and other CNS areas. It should be noted that the Mel1a and Mel1b subtypes are currently designated as melatonin  mt1 and MT2 receptors, respectively. However, the earlier nomenclature is used in the following comments, for consistency with that used  in the presented paper.

The Question: Several studies have shown that melatonin exerts a predominantly inhibitory effect on neuronal firing in the SCN. Since the principal inhibitory neurotransmitter , GABA, acts on GABAA
receptors in the  SCN to alter the activity of the circadian clock, the authors investigated whether the modulatory action of melatonin on clock function involves a GABAergic mechanism.

Evidence: 1)After confirming the presence of functional GABAA receptors in their SCN preparation, the authors demonstrated that melatonin (10nM) potentiates GABA-induced Cl- currents, as recorded in a whole-cell patch-clamp system. This effect was not due to a Cl- driving force as a Cl- - based intracellular solution was used.  Moreover, melatonin increased the slope of the current-voltage curve (I-V) without
altering the reversal potential, indicating that its enhancement of GABAergic function was due to increased GABAA receptor- activated whole-cell membrane conductance.

               2) The enhancing  effect of melatonin on GABAergic activity in SCN neurons appears to involve the Mel1a  subtype, since only this receptor was detected in the SCN by RT-PCR and southern blotting.

               3) In contrast to its effect in the SCN, melatonin inhibited GABA-induced Cl- currents in the hippocampus. However, only the Mel1b subtype was detected in the hippocampus by RT-PCR and southern blotting, suggesting that this receptor mediates the inhibitory action of melatonin  on GABAergic activity.

                4) In addition, the Mel1b antagonist, 4P-hippocampuPDOT, blocked the effect of melatonin in the hippocampus, but did not affect melatonin-induced  enhancement of GABAergic activity in the SCN.

                 5) This differential modulation of GABAergic activity, by the two melatonin receptor subtypes, was further confirmed by examining the effects of melatonin in HEK 293 cells co-transfected with either Mel1a or Mel1b receptors and GABAA receptors.  As observed in situ, the Mel1a or Mel1b receptors mediated enhancement or  inhibition of GABA -induced neuronal hyperpolarization, respectively.

Why is this paper important ? This is not the first paper to demonstrate a functional interaction between melatonin and GABA. However, this study has demonstrated for the first time that melatonin can either enhance or  suppress GABAergic function in the CNS, depending on the receptor subtype involved.

      In view of the widespread effects of GABA on neuronal function in the CNS, these findings set the stage for a better understanding of  the mechanisms underlying the diverse physiological effects of melatonin. As noted by the authors,  since the  two high-affinity melatonin receptors examined  are both coupled to similar signalling pathways, it is expected that other pathways underlie the differential effects of
melatonin on GABAergic activity.

      As noted earlier, melatonin inhibits  the AC pathway via activation of either Mel1a or Mel1b receptors, which are coupled to Gi.    In addition, there is evidence that melatonin can potentiate the stimulation of phospholipase C by other agents, probably via the beta/gamma subunits, which are released following activation of Gi. Since phosphorylation can either increase or decrease GABAergic activity, depending on the protein kinases and GABAA subunits involved,  it is possible that either suppression of the AC-cAMP-PKA pathway or activation of  the  PLC-PKC cascade by melatonin could alter GABAergic function. However, the precise mechanisms involved in the differential effects mediated by
Mel1a and Mel1b await clarification.
 

Abstract: Melatonin, a hormone principally produced and released by the pineal gland, has been shown to regulate a variety of biological functions including circadian rhythms, sleep-wake cycles and reproduction, presumably through activating  high-affinity G-protein- coupled receptors. We report here that these subtypes can differentially modulate the function of type-A  gamma-aminobutyric acid (GABAA) receptor, the principal neurotransmitter receptor mediating synaptic inhibition in the CNS. This work demonstrates that melatonin, through activation of different receptor subtypes, can exert opposite effects on the same substrate, suggesting that receptor subtype is the primary molecular basis for the diversity of melatonin’s effects.