Chapter 2: The genetic and neuronal substrates of melatonin signaling in
2.3 Sleep induced by exogenous melatonin is suppressed in MT1 receptor triple
2.3 Sleep induced by exogenous melatonin is suppressed in MT1 receptor triple
the MT1 family of melatonin receptors. 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 did 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 is comparable to that of mtnr1aa-/-; mtnr1ab-/-;
mtnr1al-/-. The simplest interpretation from these data is that melatonin induces sleep exclusively through mtnr1aa, and that the other MT1 receptors are largely dispensable.
However, we note that mtnr1aa-/- mutants are not defective for sleep (Fig. 2.1), and we only observe sleep defects in animals that have mutations in all three MT1 paralogs (Fig.
2.7). Thus, while mtnr1aa appears to play a primary role in mediating melatonin’s effects, there must be an interaction between mtnr1aa and the other MT paralogs. In the next section, we measure the responses of MT1 double mutants to melatonin treatment to examine this relationship.
We also tested the effect of melatonin treatment on MT2 family receptor mutants.
Among the four genotypes, we found no significant difference in their responses to melatonin (Fig. 2.10). We conclude that melatonin promotes sleep primarily, and perhaps exclusively, via MT1 receptors.
Figure 2.9. Induction of sleep by melatonin treatment requires MT1 paralogs. (A) Average locomotor activity of 6-dpf mtnr1aa+/-; 1ab+/-; 1al+/- (black), mtnr1aa+/-; 1ab-/-;
1al-/- (blue), mtnr1aa-/-; 1ab+/-; 1al+/- (pink), and mtnr1aa-/-; 1ab-/-; 1al-/- (red) larvae during 3 hours before and after administering 1 µM melatonin (MT) (at t = 0; indicated by the black arrow). (B) Locomotor activity for larvae of each genotype during 3 hrs before (‘Pre’) and after (‘Post’) MT administration, not including the 20 mins preceding or following MT administration. Lines represent mean +/- s.d. (C) Mean locomotor activity changes after MT administration normalized to pre-treatment baseline locomotor activity ((post-MT mean activity – pre-MT mean activity) / pre-MT mean activity). Error bars represent s.d.
mtnr1aa-/-; 1ab+/-; 1al+/- and mtnr1aa-/-; 1ab-/-; 1al-/- animals are significantly impaired for MT-induced locomotor activity decrease compared to the triple heterozygous sibling controls. (D) Fraction of larvae that are asleep per minute for 3 hrs before and after MT administration (black arrow). (E) Same as in (B) but fraction of animals asleep. (F) Fold change in sleep amount after MT administration (post-MT sleep amount of each larva / pre-MT mean sleep amount of genotype). Compared to triple heterozygous controls, which show a ~4-fold increase in sleep after MT administration over the 3-hr period, mtnr1aa-/-; 1ab+/-; 1al+/- and mtnr1aa-/-; 1ab-/-; 1al-/- larvae show strongly suppressed MT-induced sleep. Data are pooled from 8 video-tracker experiments. Number of animals represented in each genotype are indicated in parentheses in legend. For panels (B) and (E), ÜÜÜ = P < 0.001; Two-way ANOVA. For panels (C) and (F), ÜÜÜ = P < 0.001 vs.
mtnr1aa+/-; 1ab+/-; 1al+/-; Kruskal-Wallis test with Dunn’s multiple comparisons test. Non- significant comparisons to the control genotype (+/-; +/-; +/-) are not annotated.
-150 -100 -50 0 50 100 150 Time (mins) since +1µM MT 0
0.1 0.2 0.3 0.4 0.5 0.6
Fraction asleep
PrePost PrePost
PrePost PrePost 0
5 10 15
Activity (secs/min)
-150 -100 -50 0 50 100 150 Time (mins) since +1µM MT 0
1 2 3 4 5 6 7 8
Activity (secs/min)
A B
D
PrePost PrePost
PrePost PrePost 0.0
0.2 0.4 0.6 0.8 1.0
Fraction asleep
E +/
-; +/-; +/- +/-; -/-; -/-
-/-; +/-; +/- -/-; -/-; -/- -1.0
-0.8 -0.6 -0.4 -0.2 0.0
Norm. activity change after melatonin C
✱✱✱
✱✱✱
+/-; +/-; +/- (165) -/-; +/-; +/- (163)
+/-; -/-; -/- (143) -/-; -/-; -/- (144) mtnr1aa; mtnr1ab; mtnr1al
+/-; +/-; +/- +/-; -/-; -/-
-/-; +/-; +/- -/-; -/-; -/- 0
1 2 3 4 5 6 7
Fold change in sleep after melatonin F
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✱✱✱
✱✱✱
✱✱✱
Figure 2.10. MT2 paralogs are dispensable for sleep induced by exogenous melatonin. (A) Average locomotor activity of 6-dpf mtnr1ba+/-; 1bb+/- (black), mtnr1ba+/-
; 1bb-/- (dark blue), mtnr1ba-/-; 1bb+/- (light blue), and mtnr1ba-/-; 1bb-/- (orange) larvae during 3 hours before and after administering 1 µM melatonin (MT) (at t = 0; indicated by the black arrow). (B) Locomotor activity for larvae of each genotype over the 3 hrs before (‘Pre’) and after (‘Post’) MT administration, not including the 20 mins preceding or following MT administration. Lines represent mean +/- s.d. (C) Mean locomotor activity changes after MT administration normalized to pre-treatment baseline locomotor activity ((post-MT mean activity – pre-MT mean activity) / pre-MT mean activity). Error bars represent s.d.
(D) Fraction of larvae that are asleep per minute for 3 hrs before and after MT administration (black arrow). (E) Same as in (B) but fraction of animals asleep. (F) Fold change in sleep amount after MT administration (post-MT sleep amount of each larva / pre-MT mean sleep amount of genotype). Data are pooled from 2 video-tracker experiments. Number of animals represented in each genotype are indicated in parentheses in legend. For panels (B) and (E), ns = not significant (P > 0.05); Two-way ANOVA. For panels (C) and (F), no genotype was significantly different from the control genotype (+/-; +/-); Kruskal-Wallis test with Dunn’s multiple comparisons test.
-150 -100 -50 0 50 100 150 Time (mins) since +1µM MT 0
1 2 3 4 5 6 7 8
Activity (secs/min)
-150 -100 -50 0 50 100 150 Time (mins) since +1µM MT 0
0.1 0.2 0.3 0.4 0.5 0.6
Fraction asleep
PrePost PrePost
PrePost PrePost 0
2 4 6 8 10
Activity (secs/min)
A B
D E
PrePost PrePost
PrePost PrePost 0.0
0.2 0.4 0.6 0.8 1.0
Fraction asleep
+/-; +/- +/-; -/-
-/-; +/- -/-; -/- -1.0
-0.8 -0.6 -0.4 -0.2 0.0
Norm. activity change after melatonin +/-; +/- (38) C
-/-; +/- (60) +/-; -/- (43) -/-; -/- (39) mtnr1ba; mtnr1bb
+/-; +/- +/-; -/-
-/-; +/- -/-; -/- 0
5 10 15 20 25 30
Fold change in sleep after melatonin F ns
ns
2.4 Loss of two copies of mtnr1aa and one copy of either mtnr1ab or mtnr1al is