Article Figures & Data
Figures
- Figure 1.
The axonal branching of cultured serotonin neurons is regulated by the neuritin protein level. A, Images of serotonin neurons from rat embryos. Immunodetection of serotonin-positive neurons treated with purified Fc or Neuritin-Fc protein (1 µg/ml) is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. B, Quantification of axon length (left) and the number of primary branches per 100 µm of axon (right) with various concentrations of Neuritin-Fc protein [n = 30/3 cells/independent experiments, one-way ANOVA followed by Dunnett's posttest; F(5,12) = 0.1109; p = 0.9963 (0.1 μg/ml Neuritin-Fc); p = 0.8566 (0.3 μg/ml); p = 0.9988 (1 μg/ml); p = 0.8480 (3 μg/ml); p = 0.6494 (10 μg/ml; axon length); F(5,12) = 0.3111; p = 0.9977 (0.1 μg/ml Neuritin-Fc); p = 0.0575 (0.3 μg/ml); ***p = 0.0006 (1 μg/ml); ***p = 0.0003 (3 μg/ml); and ***p < 0.0001 (10 μg/ml; number of branches) compared with neurons with Fc protein. The data were averaged and are presented by experimental conditions]. C, Images of serotonin neurons from neuritin+/+ (WT) or neuritin−/− (KO) mouse embryos with purified Fc or Neuritin-Fc protein (1 µg/ml). Immunodetection of serotonin-positive neurons is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. D, Quantification of axon length (top) and the number of primary branches per 100 µm of axon [bottom; n = 30/3 cells/independent experiments, two-way ANOVA followed by Sidak's posttest, treatment × genotype interaction F(1,8) = 0.0236; p = 0.882; genotype, F(1,8) = 2.151; p = 0.181; treatment, F(1,8) = 0.495; p = 0.502; p = 0.915 (WT) and p = 0.808 (KO; Fc vs Neuritin-Fc, respectively); with Fc, p = 0.616, and with Neuritin-Fc, p = 0.489 (WT vs KO; axon length); treatment × genotype interaction, F(1,8) = 10.11; p = 0.013; genotype, F(1,8) = 1.745; p = 0.223; treatment, F(1,8) = 57.13; p < 0.001; *p = 0.029 (WT); and ***p = 0.001 (KO; Fc vs Neuritin-Fc, respectively); with Fc, #p = 0.026, and with Neuritin-Fc, p = 0.400 (WT vs KO; number of branches). The data were averaged and are presented by experimental conditions]. Error bars, SD.
- Figure 2.
The axonal density and branching of serotonin neurons in brain regions are regulated by the neuritin protein. A, Schematic illustration of regions of interest for immunofluorescence observation. Regions of interest are indicated as red squares and are located in the mPFC (prelimbic, PL), hippocampus (CA3), and BLA. Line drawings are adapted from the Allen Mouse Brain Atlas. B, Reconstructed flattened images of the PL, CA3, and BLA from WT and KO mice. Immunodetection of serotonin transporter-positive neurons is shown. Scale bar, 100 µm. C, Quantification of the number of branch points along the serotonin transporter-positive axons and axon volume in the images [n = 4/6 region images/mouse, two-tailed t test with Welch's correction, t(8.7992) = 5.7429 and ***p < 0.001 in the mPFC; t(9.7826) = 0.7969 and p = 0.4444 in the hippocampus; and t(7.1474) = 2.9325 and *p = 0.021 in the amygdala (number of axonal branches); t(9.8606) = 2.8624 and *p = 0.017 in the mPFC; t(9.9553) = 0.3769 and p = 0.7141 in the hippocampus; and t(10.0000) = 3.0379 and *p = 0.012 in the amygdala (axonal volume). The data are averaged and presented by mouse experimental group]. D, Schematic illustration of the region of interest for immunofluorescence observation. The region of interest, located in the dorsal raphe nucleus (DR), is indicated by a red rectangle. The line drawing is adapted from the Allen Mouse Brain Atlas. E, Left, Images of DR from WT and KO mice. Immunodetection of serotonin-positive neurons is shown. Scale bar, 200 µm. Right, Quantification of the number of serotonin-positive cells in the DR (n = 3/3 sections/mouse; two-tailed t test with Welch's correction; t(3.9938) = 0.2195; p = 0.837. The data are averaged and presented by mouse experimental group). F, Left, Immunoblot images of lysates from the indicated brain regions from WT mice. Right, The quantification of the neuritin levels in each homogenate shown in the left panel [n = 6 animals; one-way ANOVA followed by Dunnett's post hoc test; F(3,20) = 5.9603; p = 0.9534 (mPFC); p = 0.1285 (hippocampus); ***p < 0.0001 (dorsal raphe), compared with the amygdala]. Error bars, S.D.
- Figure 3.
Neuritin KO mice exhibit depressive and anxiety behaviors. A, Time spent in the open arms of the EMPT by neuritin+/+ (WT) and neuritin−/− (KO) mice (n = 15 animals; two-tailed t test with Welch's correction; t(25.6710) = 3.3654; ***p = 0.0024). B, Latency time to feed in the NSFT (left; n = 15 animals; two-tailed t test with Welch's correction; t(20.1369) = 2.5142; *p = 0.0205) and food consumption of home cage feeding, following the NSFT (right; n = 15 animals; two-tailed t test with Welch's correction; t(27.9999) = 1.0963; p = 0.2823). C, Sucrose intake in the SPT of WT and KO mice (n = 15 animals; two-tailed t test with Welch's correction; t(27.9434) = 2.8203; **p = 0.0087). D, Immobility time in the TST of WT and KO mice (n = 15 animals; two-tailed t test with Welch's correction; t(25.5015) = 2.2941; *p = 0.0302). Error bars, SD.
- Figure 4.
UCS induces a reduction in neuritin expression, causing depressive and anxiety behaviors and a decrease in axon density and axonal branches of serotonin neurons. A, Schematic illustration of the experimental design for the UCS, followed by the EPMT, SPT, NSFT, and TST. B, Top, Immunoblot images of lysates from the indicated brain regions from control and UCS mice. Bottom, Quantification of neuritin levels in each homogenate shown in the top panel (n = 6 animals; two-tailed t test with Welch's correction; t(8.9269) = 2.5036 and *p = 0.034 in the mPFC; t(9.8671) = 2.7715 and *p = 0.020 in the hippocampus; t(7.6485) = 3.0701 and *p = 0.016 in the amygdala; and t(8.2625) = 0.4106 and p = 0.692 in the dorsal raphe). C, Time spent in the open arms of the EMPT in control and UCS mice (n = 14 animals; two-tailed t test with Welch's correction; t(23.8605) = 3.3409; ***p = 0.0027). D, Latency time to feed in the NSFT (left; n = 14 animals; two-tailed t test with Welch's correction; t(25.5676) = 3.0824; ***p = 0.0049) and food consumption of home cage feeding, following the NSFT (right; n = 14 animals; two-tailed t test with Welch's correction; t(25.5443) = 1.1029; p = 0.2804). E, Sucrose intake index of the SPT in control and UCS mice (n = 14 animals; two-tailed t test with Welch's correction; t(25.7981) = 2.6447; *p = 0.0137). F, Immobility time in the TST of control and UCS mice (n = 14 animals; two-tailed t test with Welch's correction; t(21.1291) = 2.0905; *p = 0.0488). G, Reconstructed flattened images of the PL, CA3, and BLA from control and UCS mice. Immunodetection of serotonin transporter-positive neurons is shown. Scale bar, 100 µm. H, Quantification of the number of branch points along the serotonin transporter-positive axons and the volume of axons in the images [n = 4/6 region images/mice; two-tailed t test with Welch's correction; t(7.3741) = 3.8808 and ***p = 0.005 in the mPFC; t(9.0694) = 2.3795 and *p = 0.0411 in the hippocampus; and t(8.9506) = 2.3972 and *p = 0.040 in the amygdala (number of axonal branches); t(8.9912) = 2.3857 and *p = 0.041 in the mPFC; t(9.9988) = 0.7659 and p = 0.4615 in the hippocampus; and t(9.5170) = 5.2341 and ***p < 0.001 in the amygdala (axonal volume). The data are averaged and presented by mouse experimental group]. I, Top, Images of dorsal raphe nuclei from control and UCS mice. Immunodetection of serotonin-positive neurons is shown. Scale bar, 200 µm. Bottom, Quantification of the number of serotonin-positive cells in the dorsal raphe nucleus (n = 3/3 sections/mouse; two-tailed t test with Welch's correction; t(3.2184) = 0.3816; p = 0.727). The data are averaged and presented by mouse experimental group. Error bars, SD.
- Figure 5.
Neuritin prevents UCS-induced abnormal behavior and decreased serotonin axon branching. A, Schematic illustration of the experimental design for virus injection followed by UCS and behavioral tests. B, Schematic showing the viral constructs used in this study. The CMV promoter sequence was followed by neuritin, the T2A cleavage peptide from Thosea asigna virus, and EGFP (top). Example coronal section prepared after the behavioral tests showing the expression of GFP in the BLA region indicated by the white dotted line (left side). Scale bar, 1 mm (bottom). C, Time spent in the open arms of EMPT in nonstressed control and UCS mice infected with control or neuritin virus [n = 15 animals; two-way ANOVA followed by Sidak's posttest; virus × stress interaction, F(1,56) = 2.702; p = 0.1058; virus, F(1,56) = 0.7215; p = 0.3993; stress, F(1,56) = 6.822; p = 0.0115; **p = 0.0078 (control) and p = 0.7464 (neuritin; nonstress vs UCS, respectively); nonstress, p = 0.8207; and stress, p = 0.1598 (control vs neuritin)]. D, Latency time to feed in the NSFT [left; n = 15 animals; two-way ANOVA followed by Sidak's posttest; virus × stress interaction, F(1,56) = 8.076; p = 0.0062; virus, F(1,56) = 9.603; p = 0.0030; stress, F(1,56) = 13.64; p = 0.0005; ***p < 0.0001 (control) and p = 0.7969 (neuritin; nonstress vs UCS, respectively); nonstress, p = 0.9794; and stress, ***p = 0.0002 (control vs neuritin)] and food consumption of home cage feeding, following the NSFT [right; n = 15 animals; two-way ANOVA followed by Sidak's posttest; virus × stress interaction, F(1,56) = 0.05134; p = 0.8216; virus, F(1,56) = 0.0008; p = 0.9782; stress, F(1,56) = 0.1056; p = 0.7466; p = 0.9967 (control) and p = 0.9162 (neuritin; nonstress vs UCS, respectively); nonstress, p = 0.9803; and stress, p = 0.9873 (control vs neuritin)]. E, Sucrose intake index of the SPT in nonstressed control and UCS mice infected with control or neuritin virus [n = 15 animals; two-way ANOVA followed by Sidak's posttest; virus × stress interaction, F(1,56) = 3.881; p = 0.0538; virus, F(1,56) = 0.4476; p = 0.0388; stress, F(1,56) = 8.248; p = 0.0057; **p = 0.0023 (control) and p = 0.7755 (neuritin; nonstress vs UCS, respectively); nonstress, p = 0.9933; and stress, *p = 0.0109 (control vs neuritin)]. F, Immobility time of the TST in nonstressed control and UCS mice infected with control or neuritin virus [n = 15 animals; two-way ANOVA followed by Sidak's posttest; virus × stress interaction, F(1,56) = 0.2348; p = 0.6299; virus, F(1,56) = 0.9395; p = 0.3366; stress, F(1,56) = 11.17; p = 0.0015; *p = 0.0180 (control) and p = 0.0940 (neuritin) (nonstress vs UCS, respectively); nonstress, p = 0.9288; and stress, p = 0.5216 (control vs neuritin)]. G, Reconstructed flattened images of the BLA from both virus-injected control and UCS mice. Immunodetection of serotonin transporter-positive neurons is shown. Scale bar, 100 µm. H, Quantification of the number of branch points along serotonin transporter-positive axon and the volume of axons in the images [n = 4/6 region images/mouse; two-way ANOVA followed by Sidak's posttest; virus × stress interaction, F(1,20) = 1.820; p = 0.1924; virus, F(1,20) = 3.118; p = 0.0927; stress, F(1,20) = 0.6279; p = 0.0210; p = 0.0259 (control) and p = 0.6671 (neuritin; nonstress vs UCS, respectively); nonstress, p = 0.9477; and stress, p = 0.0775 (control vs neuritin; number of axonal branches); virus × stress interaction, F(1,20) = 2.333; p = 0.1423; virus, F(1,20) = 3.891; p = 0.0625; stress, F(1,20) = 7.367; p = 0.0134; *p = 0.0141 (control) and p = 0.6534 (neuritin; nonstress vs UCS, respectively); nonstress, p = 0.9406; and stress, *p = 0.0443 (control vs neuritin; axonal volume). The data are averaged and presented by mouse experimental group]. Error bars, SD.
- Figure 6.
FGF signaling is necessary for neuritin-mediated axonal branch formation in serotonin neurons. A, Images of serotonin neurons from rat embryos. PD173074 (100 nM) or AZD4547 (200 nM), FGFR inhibitors, and purified Fc or Neuritin-Fc protein (1 µg/ml) were applied at DIV 1. Immunodetection of serotonin-positive neurons is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. B, Quantification of the number of primary branches per 100 µm of axon (n = 30/3 neurons/independent experiments; two-way ANOVA followed by Sidak's post hoc test; neuritin treatment × inhibitor interaction, F(2,12) = 2.278; p = 0.1450; neuritin treatment, F(1,12) = 8.340; *p = 0.0136; inhibitor, F(2,12) = 24.37; ***p < 0.0001. *p = 0.0428 (Fc + DMSO vs Fc + PD173074); *p = 0.0142 (Fc + DMSO vs Fc + AZD4547); ***p = 0.0004 (neuritin-Fc + DMSO vs neuritin-Fc + PD173074); ***p = 0.0002 (neuritin-Fc + DMSO vs neuritin-Fc + AZD4547); *p = 0.0154 (Fc + DMSO vs neuritin-Fc + DMSO); p = 0.8321 (Fc + PD173074 vs neuritin-Fc + PD173074); p = 0.8194 (Fc + AZD4547 vs neuritin-Fc + AZD4547)]. The data were averaged and are presented as independent experiments. C, Images of serotonin neurons from rat embryos. siRNA against FGFR1 or control RNA was transfected before plating, and purified Fc or neuritin-Fc protein (1 µg/ml) was applied at DIV 1. Immunodetection of serotonin-positive neurons is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. D, Quantification of the number of primary branches per 100 µm of axon [n = 30/3 neurons/independent experiments; two-way ANOVA followed by Sidak's posttest; neuritin treatment × RNA interaction, F(1,8) = 7.527; p = 0.0253; neuritin treatment, F(1,8) = 1.378; p = 0.2741; RNA, F(1,8) = 27.79; ***p = 0.0008. p = 0.2107 (Fc + scRNA vs Fc + siRNA); ***p = 0.0009 (neuritin-Fc + scRNA vs neuritin-Fc + siRNA); *p = 0.0480 (Fc + scRNA vs Neuritin-Fc + scRNA); p = 0.5091 (Fc + siRNA vs neuritin-Fc + siRNA). The data are averaged and presented as independent experiments]. Error bars, SD.
- Figure 7.
FGF-2 is involved in neuritin-mediated axonal branching. A, Images of serotonin neurons from rat embryos. Each FGF (40 ng/ml) was applied at DIV 1. Immunodetection of serotonin-positive neurons is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. B, Quantification of the number of primary branches per 100 µm of axon [n = 30/3 cells/independent experiments; nested one-way ANOVA followed by Dunnett's posttest; F(7,711) = 1.720; comparing control, p = 0.9973 (to FGF-1); *p = 0.0489 (to FGF-2); p = 0.9789 (to FGF-4); p = 0.9999 (to FGF-5); p = 0.9995 (to FGF-7); p = 0.8013 (to FGF-8); and p = 0.9848 (to FGF-9). The data were averaged and are presented by experimental condition]. C, Images of serotonin neurons from WT and KO mouse embryos. FGF-2 (40 ng/ml) was applied at DIV 1. Immunodetection of serotonin-positive neurons is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. D, Quantification of the number of primary branches per 100 µm of axon [n = 22-26/4 neurons/independent experiments; two-way ANOVA followed by Sidak's posttest; genotype × FGF-2 interaction, F(1,12) = 8.802; p = 0.0118; genotype, F(1,12) = 244.1; ***p < 0.0001; FGF-2, F(1,12) = 14.11; ***p = 0.0027. ***p = 0.0009 (WT + control vs WT + FGF-2); p = 0.8296 (KO + control vs KO + FGF-2). The data were averaged and are presented by experimental condition]. E, Images of serotonin neurons from rat embryos. FGF-2 (40 ng/ml) and purified Fc or neuritin-Fc protein (1 µg/ml) were applied at DIV 1. Immunodetection of serotonin-positive neurons is shown. Arrows indicate axonal branch points. Scale bar, 50 µm. F, Quantification of the number of primary branches per 100 µm of axon (n = 30/3 neurons/independent experiments; two-way ANOVA followed by Sidak's posttest; neuritin × FGF-2 interaction, F(1,8) = 2.101; p = 0.1852; neuritin, F(1,8) = 6.341; p = 0.0359; FGF-2, F(1,8) = 9.574; ***p = 0.0148. p = 0.0246 (Fc + control vs Fc + FGF-2); ***p = 0.4792 (neuritin-Fc + control vs neuritin-Fc + FGF-2); *p = 0.0455 (Fc + control vs Neuritin-Fc + control); p = 0.7207 (Fc + FGF-2 vs neuritin-Fc + FGF = 2). The data were averaged and are presented as independent experiments. Error bars, SD.
- Figure 8.
Inhibition of FGF signaling causes depressive and anxiety behaviors and decreases the axon density and the axonal branch number of serotonin neurons in different brain regions. A, Top, Immunoblot images of lysates from the indicated brain regions of WT and neuritin KO mice. Bottom, Quantification of the phospho-FGFR1 levels in each homogenate shown in the top panel (n = 6 animals; two-tailed t test with Welch's correction; t(9.8348) = 2.4149 and *p = 0.036 in the mPFC; t(8.3236) = 3.6104 and **p = 0.006 in the hippocampus; and t(9.8689) = 3.2916 and **p = 0.008 in the amygdala). B, Top, Immunoblot images of brain lysates from CMC- and AZD4547-treated mice. Bottom, Quantification of the phospho-FGFR1 levels shown in the top panel (n = 4 animals; two-tailed t test with Welch's correction; t(5.9983) = 3.6516; *p = 0.011). C, Schematic illustration of the experimental design for the inhibition of FGF signaling. AZD4547 (50 mg/kg) or vehicle (1% CMC, 0.9% NaCl, 5% DMSO; 1 ml/kg) was orally administered for four sets of 5 consecutive days at 2 d intervals for a total of 20 d, followed by EPMT, SPT, NSFT, and TST. D, Time spent in the open arms by vehicle (CMC)-treated and AZD4547-treated EMPT mice (n = 14 animals; two-tailed t test with Welch's correction; t(23.2076) = 2.5752; *p = 0.0169). E, Latency time to feed in the NSFT (left; n = 14 animals; two-tailed t test with Welch's correction; t(22.2276) = 3.7419; ***p = 0.0011) and food consumption of home cage feeding, following the NSFT (right; n = 14 animals; two-tailed t test with Welch's correction; t(25.6728) = 1.3447; p = 0.1905). F, Sucrose intake in the SPT of CMC- and AZD4547-treated mice (n = 14 animals; two-tailed t test with Welch's correction; t(25.9522) = 2.6447; *p = 0.0116). G, Immobility time of the TST in CMC- and AZD4547-treated mice (n = 14 animals; two-tailed t test with Welch's correction; t(24.9604) = 2.5753; *p = 0.0163). H, Reconstructed flattened images of the PL, CA3, and BLA from CMC- and AZD4547-treated mice. Immunodetection of serotonin transporter-positive neurons is shown. Scale bar, 100 µm. I, Quantification of the number of branch points along the serotonin transporter-positive axons and axon volume in the images [n = 4/6 region images/mouse; two-tailed t test with Welch's correction; t(7.8441) = 2.8100 and *p = 0.023 in the mPFC; t(5.9624) = 0.5689 and p = 0.5902 in the hippocampus; and t(8.5825) = 3.4783 and **p = 0.007 in the amygdala (number of axonal branches); t(9.0980) = 2.3038 and *p = 0.046 in the mPFC; t(8.4435) = 2.7328 and *p = 0.025 in the hippocampus; and t(9.7989) = 5.1331 and ***p < 0.001 in the amygdala (axonal volume). The data are averaged and presented by mice]. J, Top, Images of dorsal raphe nuclei from CMC-treated and AZD4547-treated mice. Immunodetection of serotonin-positive neurons is shown. Scale bar, 200 µm. Bottom, Quantification of the number of serotonin-positive cells in the dorsal raphe nucleus [n = 3/3 sections/mouse; two-tailed t test with Welch's correction; t(3.2184) = 0.3816; p = 0.727. The data are averaged and presented by mouse experimental group]. K, Top, Immunoblot images of lysates from the indicated brain regions of CMC-treated and AZD-treated mice. Bottom, Quantification of the neuritin levels in each homogenate shown in the top panel (n = 6 animals; two-tailed t test with Welch's correction; t(5.7366) = 2.6420 and *p = 0.040 in the mPFC; t(9.1375) = 2.3643 and *p = 0.042 in the hippocampus; t(9.4551) = 0.3952 and p = 0.701 in the amygdala; and t(8.8334) = 0.7257 and p = 0.487 in the dorsal raphe). L, Schematic illustration of the role of neuritin in FGF signaling. Top, With neuritin, FGFR1 is recruited and remains on the cell surface, which enables FGFR1 to interact with FGF-2 more easily, activate FGF signaling, and induce axonal branching. Bottom, Without neuritin, FGFR1 levels on the cell surface is insufficient. Therefore, the interaction between FGF-2 and FGFR1 is decreased, resulting in a decrease in axonal branch formation. Error bars, SD.
- Figure 9.
The expression of FGF-2 is not altered by neuritin KO, whereas UCS conditioning decreases FGF-2 levels in the brain. A, Immunoblot images of lysates from the indicated brain regions of WT and KO mice (left). Quantification of FGF-2 levels in each homogenate shown in the left panel [n = 6 animals; two-tailed t test with Welch's correction; t(9.5067) = 0.8029 and p = 0.442 (p18); t(9.9793) = 0.3943 and p = 0.702 (p22); and t(9.9935) = 0.3881 and p = 0.706 (p24) in the mPFC; t(9.0873) = 0.1160 and p = 0.910 (p18); t(8.1054) = 1.3213 and p = 0.222 (p22); and t(8.9200) = 1.0118 and p = 0.338 (p24) in the hippocampus; and t(7.3899) = 1.9547 and p = 0.089 (p18); t(9.8938) = 0.9786 and p = 0.351 (p22); and t(9.7849) = 0.2906 and p = 0.777 (p24) in the amygdala; right]. B, Immunoblot images of lysates from the indicated brain regions of control and UCS mice (left). The quantification of FGF-2 levels in each homogenate shown in the left panel [n = 6 animals; two-tailed t test with Welch's correction; t(8.4195) = 1.8086 and p = 0.106 (p18); t(5.7998) = 1.2097 and p = 0.273 (p22); and t(7.5382) = 2.7151 and *p = 0.028 (p24) in the mPFC; t(9.5575) = 0.7081 and p = 0.496 (p18); t(9.6633) = 2.9531 and *p = 0.015 (p22); and t(8.5514) = 3.2652 and *p = 0.010 (p24) in the hippocampus; and t(9.7837) = 3.5130 and **p = 0.006 (p18); t(9.2929) = 0.3411 and p = 0.741 (p22); and t(9.9027) = 2.4668 and *p = 0.034 (p24) in the amygdala; bottom]. Error bars, SD.
Tables
Stressor Duration Description # of TIMES used 1 Cold stress 15 min Put in 4°C conditions 5 2 Forced swimming 10 min Put in water at room temperature 4 3 Hot plate 5 min Put on hot plate (40°C) 5 4 Food deprivation 12 h No food pellets 4 5 Cage tilting 2 h Cages are elevated from one side and kept tilted at 45° 6 6 Wet bed 2 h A cage with damp bedding with water at room temperature 4 7 Empty bed 2 h Empty cage with no bedding. 4 8 Light 12 h Overnight illumination (light on overnight) 4 9 Dark 3 h Light off and put in complete dark during the daytime 6 10 Stroboscope 4 h Emits a stroboscope flashing light (20 rpm) during the night time 4
