A special thanks to Paul for his mentorship in worm-related matters and for the spontaneous conversations in Kerckhoff's and later Chen's halls. Previous work in the Prober lab has shown that the nocturnal hormone melatonin acts downstream of the circadian rhythm to promote sleep in zebrafish.
A review of melatonin’s sleep-promoting role across animal models
Introduction
Lerner had hoped that melatonin was "the long-sought factor" of vitiligo, a skin disorder he was studying, but injections of melatonin into adult males did not result in depigmentation, as it did in frogs. This basic light-pineal-melatonin pathway has been exhaustively studied and is well accepted among scientists who study melatonin and pineal-related processes, but the certain aspects of animal physiology - the output of melatonin -.
Synthesis and regulation of melatonin
The SCN is connected to the pineal gland via a multisynaptic pathway that begins with a single inhibitory connection from the SCN to the paraventricular nucleus (PVN). PVN neurons innervate the intermediolateral nucleus of the spinal cord (IML), which in turn innervates the superior cervical ganglion (SCG).
Functions of melatonin
This function of melatonin appears to be conserved in humans, as common variants of the MT2 melatonin receptor are associated with fasting glucose levels and risk of type 2 diabetes (Prokopenko et al., 2009; Lyssenko et al., 2009). However, melatonin treatment is generally not sedating in nocturnal species (Murakami et al., 2001; Huber et al., 1998; Langebartels et al., 2001), which is inconsistent with a general role for melatonin in sleep.
Melatonin studies using invertebrate model systems
These findings are also inconsistent with the drug experiments reported in Tanaka et al. 2020) conclude that PCDR-1 has pharmacological properties distinct from mammalian MT1 and MT2. Furthermore, Drosophila aanat1 mutants have normal sleep levels at baseline, although sleep recovery is enhanced (Shaw et al., 2000).
Melatonin studies using zebrafish
At night, aanat2 mutant animals slept about half as much as their wild-type control siblings, indicating that endogenous melatonin plays an important role in promoting sleep at night (Gandhi et al., 2015). Following the findings of Gandhi et al. 2015) under free-running conditions, ∆CLK animals maintained a wild-type circadian period of locomotor activity, and molecular circadian rhythms were normal in peripheral tissues (Livne et al., 2016).
Melatonin studies using avian species
These studies attempted to define the role of the pineal gland in the regulation of circadian rhythms by using locomotor behavior and body temperature as a readout of the circadian clock. The conventional interpretation of these studies is that Px (when coincident with loss of light signals) abolishes the circadian rhythm, and as the major hormonal output of the pineal gland, it is reasonable to assume that melatonin therefore orchestrates the circadian rhythm.
Melatonin studies using laboratory rodents
The ability of melatonin to play a role in circadian transmission is thought to be mediated by its action on the SCN (Cassone et al., 1986). To test the role of melatonin receptors in behavior, the laboratories of Steven Reppert and David Weaver generated MT1 and MT2 KO mice (Liu et al., 1997; Jin et al., 2003).
Human melatonin studies
A report by Ziegler et al. 2023) identify an association between heart disease and loss of melatonin in both humans with heart disease and in a mouse model of heart disease. Meanwhile, loss-of-function studies testing the role of melatonin in human sleep are scarce and difficult to interpret.
Mechanisms by which melatonin promotes sleep
In these early reports, strong melatonin binding was observed in the SCN and the pars tuberalis of the pituitary gland (Reppert et al., 1994). In the zebrafish, the optic tectum has been shown to process visual, auditory and water flow sensory information (Thompson et al., 2016). MT2 receptors are highly expressed in the reticular thalamic nucleus (Rt), whereas MT1 receptors are absent in this region (Ochoa-Sanchez et al., 2011; Lacoste et al., 2015).
In addition to the SCN, melatonin receptors (mainly MT2) were also detected in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus ( Lacoste et al., 2015 ).
Conclusions and future directions
The role of melatonin in sleep appears to have become less obvious (and/or more nuanced) in "higher" organisms. Understanding the role of melatonin in sleep is important to evaluate and optimize its use as a therapeutic agent. Finally, detailed investigation of sleep (ie, using EEG/EMG recordings) in diurnal rodents in the context of manipulations of melatonin gain and loss of function is required to determine the role of melatonin in sleep in these species.
Studies of melatonin's function in diurnal rodents will also aid in the development of melatonin-based therapies for human sleep disorders.
The genetic and neuronal substrates of melatonin signaling in
Loss of any single melatonin receptor-encoding gene has no overt effects on
Data from the video trackers report the locomotor activity of each larva over a time window defined by the experimenter. We observed a small but non-significant decrease in locomotor activity during the day in mtnr1aa-/- mutants (Fig. There was a significant decrease in daytime locomotor activity of mtnr1bb-/- mutants compared to their wild-type siblings (Figs 2,5 C).
Box plots use the Tukey method to determine whiskers and outliers; deviations are not shown. mtnr1bb mutants show reduced daytime locomotor activity but have wild-type sleep.
Sleep is defective in zebrafish lacking all three MT1 receptor paralogs
This was accompanied by a significant increase in locomotor activity at night (Fig. 2.7 D), a significant decrease in the average length of sleep (but also, interestingly, a small but significant increase in the number of sleeps) (Fig. 2.7 D). 2.7 G-H) and significantly longer latency to first sleep at night (Fig. 2.7 I) in triple mutants. We also observed a strong decrease in locomotor activity during the day (Figure 2.7 A, C) and a small but significant increase in sleep during the day (Figure 2.7 E). Triple -/- animals are significantly less active during the day and more active at night compared to triple +/+ controls.
Triple -/- animals sleep more during the day and less at night than triple +/+ controls.
Sleep induced by exogenous melatonin is suppressed in MT1 receptor triple
Surprisingly, 1aa-/-; 1ab+/-; 1al+/- mutants were also strongly resistant to the activity decrease and sleep induced by melatonin. They did not suppress the effects of melatonin on locomotor activity quite as strongly as the triple null mutants (Fig. 2.9 A, C), but both genotypes were comparable in their suppression of melatonin-induced sleep (Fig. 2.9 D, F). . We observed that the effect of melatonin treatment on locomotor activity and sleep in mtnr1aa-/-; 1ab+/-; 1al+/- animals are comparable to those of mtnr1aa-/-; mtnr1ab-/-;.
Compared to triple heterozygous controls, which show an ∼4-fold increase in sleep after MT administration over the 3-h period, mtnr1aa-/-; 1ab+/-; 1al+/- and mtnr1aa-/-; 1ab-/-; 1al-/- larvae show strongly suppressed MT-induced sleep.
Loss of two copies of mtnr1aa and one copy of either mtnr1ab or mtnr1al is
Loss of both copies of mtnr1aa in an otherwise wild-type background does not produce any phenotype, as evidenced by the wild-type levels of locomotor activity and sleep and the wild-type responses to melatonin treatment in mtnr1aa-/- (1ab+/+;. Mutation of mtnr1aa in A 1ab+/- or 1ab-/- background is sufficient to significantly inhibit the effect of MT treatment on locomotor activity Mutation of mtnr1aa in a 1al-/- background is sufficient to significantly inhibit the effect of MT treatment on locomotor activity activity.
-D) Box plot of locomotor activity measurements during day 5 (C) and night 5 (D) for each genotype. mtnr1aa-/-; 1ab-/-; 1al-/- are significantly more active at night compared to triple heterozygotes. mtnr1aa-/-; 1ab-/-; 1al-/- animals sleep significantly less at night than triple heterozygotes.
A whole exon knockout allele of mtnr1aa phenocopies MT1 receptor triple
From CRISPR injections, we found two mtnr1aa (Ex1-KO) mutant lines in which the DNA sequence between the CRISPR guide targets (ie, the mtnr1aa exon 1 coding region) was apparently missing, as determined by PCR. We therefore predict that mtnr1aa mRNA is not transcribed in homozygous mtnr1aa(Ex1-KO)-/- animals and that genetic compensation of mtnr1aa by mtnr1ab and mtnr1al should be absent. Homozygous mtnr1aa (Ex1-KO) mutant larvae were significantly more active (Fig. 2.13 A, D) and slept less (Fig. 2.13 B, F) at night compared to their wild-type siblings.
The sleep defect was accompanied by a significant decrease in average sleep length (Fig. 2.13 G) and a longer latency to sleep (Fig. 2.13 I).
MT1 receptors are expressed broadly throughout the larval zebrafish brain
Expression pattern of mtnr1aa using fluorescence in situ hybridization (FISH). A) (Left) Illustration of the signal depicted in the right panels. There is prominent expression in the cell body layers (stratum periventriculare, SPV) of the optic tectum (TeO). Same as in (A), but a more ventral plane, showing mtnr1aa expression in the ventral layers of the TeO and in a bilateral group of cells (indicated by square) in the hindbrain.
The signal is evident in the optic tectum and in a bilateral cluster of cells in the hindbrain.
Behavioral responses to visual stimuli are suppressed in awake melatonin-
To control for sleep, we took two approaches in analyzing dim flash responses. First, we quantified the probability of responding to the 10% dark flash only among larvae that responded to the first 0% dark flash. It is still possible that larvae that responded to the 0% dark flash went into hibernation during the 20 s between the initial 0% dark flash and the 10% dark flash.
Furthermore, there was no significant difference between quipazine-treated larvae and vehicle-treated controls in the dim flash response (post-vehicle (0.46) v
Loss of melatonin signaling restores behavioral responses to certain visual
At the end of the 5-minute light period, the light-OFF transition elicits remarkably different behavioral responses during the subjective day versus during the night (Fig. 2.24 E). We found that, compared to aanat2+/- control siblings, who had a wild-type-like lack of response to light OFF at night, aanat2-/- mutant larvae showed a robust response to the transition (Fig. 2.24 B, G ). . During the 5-minute photoperiod preceding the light-OFF transition, aanat2-/- animals are noticeably more active than the aanat2+/- control siblings (Fig. 2.24 B, G).
Then, as before, we defined a response to light-OFF as a suprathreshold change in locomotor activity during the 1 second after the light-OFF transition.
Neuronal activity in the optic tectum is attenuated by melatonin
Effect of melatonin on sleep and brain temperature in the Djungarian hamster and the rat. Acute effects of melatonin on spontaneous and picrotoxin-evoked sleep-wake behavior in the rat. Role of the pineal gland in light-off triggering of paradoxical sleep in the rat (abstract).
Effects of melatonin and the melatonin receptor agonist S-20098 on the vigilance states, EEG spectra, and cortical temperature in the rat.
