Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Schematic representation of the Mtnr1a ( MT 1 ) gene, indicating the location and size of the deletion. (B) Schematic diagram of PCR primers used for genotyping, with the expected PCR product sizes for MT +/+ , MT +/− and MT −/− provided in the table. (C) Gel image showing PCR genotyping results for MT +/+ , MT +/− and MT −/− mice. (D) Schematic representation of the Kcnma1 ( Slo1 ) gene, indicating the location and size of the deletion, and an α-helical representation of Slo1 highlighting the deleted amino acids within the core structure of the channel. (E) Schematic diagram of PCR primers used for genotyping, with the expected PCR product sizes for Slo1 +/+ , Slo1 +/− and Slo1 −/− provided in the table. (F) Gel image showing PCR genotyping results for Slo1 +/+ , Slo1 +/− and Slo1 −/− mice.

Article Snippet: The Mtnr1a cDNA (NM_008639, MR224599, OriGene, Rockville, MD, USA) and Slo1 cDNA (MBr5/3, provided by Dr. Lawrence Salkoff’s lab) were amplified using primer pairs 4378/4379 and 4376/4377, respectively ( ).

Techniques:

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Chromatogram showing the location (indicated by the arrowhead) of a 500-bp deletion in the Mtnr1a gene. (B) Nucleotide sequence encompassing the 500-bp deletion (underlined in red) and the flanking nucleotides matching the chromatogram. Amino acids encoded by exon 1 are underlined in green. (C) Chromatogram showing the location (indicated by the arrowhead) of an 866-bp deletion and a 3-bp insertion in the Kcnma1 gene. (D) Nucleotide sequence encompassing the 866-bp deletion (underlined in red), the 3-bp insertion (underlined in blue), and the flanking nucleotides matching the chromatogram. Amino acids encoded by exon 8 are underlined in green.

Article Snippet: The Mtnr1a cDNA (NM_008639, MR224599, OriGene, Rockville, MD, USA) and Slo1 cDNA (MBr5/3, provided by Dr. Lawrence Salkoff’s lab) were amplified using primer pairs 4378/4379 and 4376/4377, respectively ( ).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Schematic representation of the Mtnr1a ( MT 1 ) gene, indicating the location and size of the deletion. (B) Schematic diagram of PCR primers used for genotyping, with the expected PCR product sizes for MT +/+ , MT +/− and MT −/− provided in the table. (C) Gel image showing PCR genotyping results for MT +/+ , MT +/− and MT −/− mice. (D) Schematic representation of the Kcnma1 ( Slo1 ) gene, indicating the location and size of the deletion, and an α-helical representation of Slo1 highlighting the deleted amino acids within the core structure of the channel. (E) Schematic diagram of PCR primers used for genotyping, with the expected PCR product sizes for Slo1 +/+ , Slo1 +/− and Slo1 −/− provided in the table. (F) Gel image showing PCR genotyping results for Slo1 +/+ , Slo1 +/− and Slo1 −/− mice.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques:

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Chromatogram showing the location (indicated by the arrowhead) of a 500-bp deletion in the Mtnr1a gene. (B) Nucleotide sequence encompassing the 500-bp deletion (underlined in red) and the flanking nucleotides matching the chromatogram. Amino acids encoded by exon 1 are underlined in green. (C) Chromatogram showing the location (indicated by the arrowhead) of an 866-bp deletion and a 3-bp insertion in the Kcnma1 gene. (D) Nucleotide sequence encompassing the 866-bp deletion (underlined in red), the 3-bp insertion (underlined in blue), and the flanking nucleotides matching the chromatogram. Amino acids encoded by exon 8 are underlined in green.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Representative traces of electroencephalogram (EEG) and electromyogram (EMG) showing distinguishing features of REM sleep, NREM sleep, and wakefulness. (B) Representative hypnograms derived from EEG and EMG recordings for Slo1 +/+ , Slo1 −/− , MT 1 +/+ , and MT 1 −/− mice. (C) Quantification of sleep and wake durations during the subjective daytime.(D) Quantification of sleep and wake durations during the subjective nighttime. In panels C and D, the sample size ( n ) was 6 per group, including three males (blue data points) and three females (red data points). Asterisks indicate statistically significant differences between knockout mice and their corresponding littermate controls (* p < 0.05; ** p < 0.01; *** p < 0.001), while pound symbols indicate significant differences between Slo1 +/+ and MT 1 +/+ or between Slo1 −/− and MT 1 −/− (# p < 0.05; ## p < 0.01). Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Derivative Assay, Knock-Out

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Representative electroencephalogram (EEG) traces from Slo1 +/+ and Slo1 −/− mice. (B) Comparison of cumulative EEG power between Slo1 +/+ and Slo1 ⁻/⁻ mice. For Slo1 −/− mice, cumulative EEG power was calculated as the sum of the total power of all seizure events. For Slo1 +/+ mice, which exhibited no seizure activity, cumulative EEG power was calculated as the sum of the total power of randomly selected EEG segments. These segments matched the mean number (18) and mean duration (17.6 seconds) of seizure events observed in the Slo1 ⁻/⁻ group. (C) Comparison of cumulative EEG power between Slo1+/+ and Slo1⁻/⁻ mice, analyzed based on Zeitgeber time (ZT) and grouped into 4-hour intervals. In (B) and (C), the sample size (n) was 6 per genotype, including three males (blue data points) and three females (red data points). Statistical significance (***, p < 0.001) between Slo1⁻/⁻ mice and their littermate controls was determined using an unpaired t-test in (B) and a two-way mixed-design ANOVA followed by Tukey’s post hoc test in (C).

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Comparison, Activity Assay

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Membrane resistance ( R m ) and resting membrane potential (RMP) are unaffected by Slo1 −/− or MT 1 −/− . (B) Slo1 −/− or MT 1 −/− does not alter the frequency of APs induced by current injections. Left : Diagram of the current injection protocol. Middle : Representative current-clamp traces during 15-pA current injection. Right : AP frequency as a function of current injection. Filled and open triangles indicate statistically significant differences compared to the AP frequency at 0-pA current injection for the same group (Δ p < 0.05; ΔΔ p < 0.01; ΔΔΔ p < 0.001). Statistical analyses were performed using two-way mixed-design ANOVA followed by Tukey’s post hoc test. (C) Representative AP trace illustrating the quantification of AP threshold (TH), AP amplitude, APD 50 (AP duration at 50% repolarization), and AHP. (D) Voltage phase plots of group averaged APs induced by 15-pA current injection. Blue arrows indicate TH, AP peak, and maximum and minimum slopes. (E) Group-averaged APs induced by 15-pA current injection, displayed as actual (left) and normalized (right) waveforms. (F-K). Quantitative comparisons of AP-related parameters. Asterisks indicate statistically significant differences between groups (* p < 0.05; ** p < 0.01; *** p < 0.001), while “ns” indicates no significant difference. Statistical analyses were performed using two-way ANOVA (factors: genotype and time of day) followed by Tukey’s post hoc test. The number of neurons recorded ( n ) were: Slo1 +/+ 16, Slo1 −/− 12, MT 1 +/+ 11, MT 1 −/− 21, and MT 1 −/− ;Slo1 −/− double knockout (DKO) 19.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Membrane, Injection, Double Knockout

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Membrane resistance ( R m ) and resting membrane potential (RMP) are unaffected by Slo1 −/− or MT 1 −/− . (B) Slo1 −/− or MT 1 −/− does not alter the frequency of APs induced by current injections. Left : Diagram of the current injection protocol. Middle : Representative current-clamp traces during 15-pA current injection. Right : AP frequency as a function of current injection. Filled and open triangles indicate statistically significant differences compared to the AP frequency at 0-pA current injection for the same group (Δ p < 0.05; ΔΔ p < 0.01; ΔΔΔ p < 0.001). Statistical analyses were performed using two-way mixed-design ANOVA followed by Tukey’s post hoc test. (C) Voltage phase plots of group averaged APs induced by 15-pA current injection. (D) Group-averaged APs induced by 15-pA current injection, displayed as actual (left) and normalized (right) waveforms. (E-J). Quantitative comparisons of AP-related parameters. Asterisks indicate statistically significant differences between groups (* p < 0.05; *** p < 0.001), while “ns” indicates no significant difference. Statistical analyses were performed using two-way ANOVA (factors: genotype and time of day) followed by Tukey’s post hoc test. The number of neurons recorded ( n ) were: Slo1 +/+ 15, Slo1 −/− 19, MT 1 +/+ 16, and MT 1 −/− 18.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Membrane, Injection

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: (A) Representative immunohistochemistry images showing Slo1 immunoreactivity, DAPI staining, and merged images of the two in the bilateral SCN at ZT7 and ZT9 for Slo1+/+ and at ZT7 for Slo1−/−. (B) Quantification of Slo1 immunoreactivity across groups. Asterisks indicate statistically significant differences between groups (** p < 0.01; *** p < 0.001). Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. The sample size ( n ) was 3 animals per group, with the Slo1 immunoreactive signal (normalized to DAPI signal) for each animal derived from the average of five sections.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Immunohistochemistry, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Melatonin Enhances Sleep via MT 1 -Driven Activation of Slo1 in Suprachiasmatic Nucleus Neurons

doi: 10.1101/2025.03.12.642893

Figure Lengend Snippet: Potential physical interactions between MT 1 and Slo1 were explored using coimmunoprecipitation assays with HEK293T cells transfected HA-tagged MT 1 (MT 1 ::HA) and Flag-tagged Slo1 (Slo1::Flag) or their variants. (A) Full-length Slo1 and Slo1(1-388), but not Slo1(389-1164), coimmunoprecipitated with MT 1 . (B) Slo1(Δ44-113), which lacks a large portion of the S0-S1 loop, exhibited a much weaker interaction MT 1 compared to full-length Slo1. (C) The Slo1(44-113) peptide coimmunoprecipitated with MT 1 . (D) Both MT 1 (Δ1-46) and MT 1 (Δ313-353), which lack the N- and C-termini, respectively, coimmunoprecipitated with Slo1. (E) MT 1 (47-312), which lacks both the N- and C-termini, as well as MT 2 , coimmunoprecipitated with Slo1. (F) An unrelated transmembrane protein, CUB3, did not coimmunoprecipitate with MT 1 . Each gel image is representative of two independent experiments.

Article Snippet: Mtnr1a (MT 1 ) and Kcnma1 (Slo1) knockout mice were generated by the Jackson Laboratory (JAX) using CRISPR-mediated gene editing of zygotes derived from CBA/CaJ mice (JAX Stock# 000654).

Techniques: Transfection

Journal: Animals : an Open Access Journal from MDPI

Article Title: Melatonin in Male Dromedary Camel ( Camelus dromedarius ) Seminal Plasma and Its Specific MT1 and MT2 Receptors on Sperm Membranes

doi: 10.3390/ani15010083

Figure Lengend Snippet: Distribution of MT1 melatonin receptors in camel spermatozoa, evaluated by the indirect immunofluorescence method. Immunostaining in the head (H), post-acrosome (PA), neck (N), tail (T) and cytoplasmic droplet (CD) was evidenced. Magnification 1000×. MT1 receptors ( A , D , G , J ), Hoechst staining ( B , E , H , K ), and bright field ( C , F , I , L ) are shown.

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Immunofluorescence, Immunostaining, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Melatonin in Male Dromedary Camel ( Camelus dromedarius ) Seminal Plasma and Its Specific MT1 and MT2 Receptors on Sperm Membranes

doi: 10.3390/ani15010083

Figure Lengend Snippet: Indirect immunofluorescence controls. Samples were incubated with only the MT1 ( A ) or MT2 ( C ) primary or secondary antibody ( E ) and ( G ) for Alexa Fluor 594 anti-mouse antibody and Alexa Fluor 488 anti-rabbit antibody, respectively). Magnification 1000×. Fluorescence after 30 s exposition ( A , C , E , G ) and bright field ( B , D , F , H ) are shown.

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Immunofluorescence, Incubation, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Melatonin in Male Dromedary Camel ( Camelus dromedarius ) Seminal Plasma and Its Specific MT1 and MT2 Receptors on Sperm Membranes

doi: 10.3390/ani15010083

Figure Lengend Snippet: Comparison among age classes of males at different melatonin receptors localizations (MT1 ( A ) and MT2 ( B )) in acrosome (A), post-acrosome (PA), head (H), neck (N), tail (T), cytoplasmic droplet (CD) and apical edge (AE). Values are shown as percentage of localization of n = 439 spermatozoa. * indicates p < 0.05.

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Comparison

Journal: Animals : an Open Access Journal from MDPI

Article Title: Melatonin in Male Dromedary Camel ( Camelus dromedarius ) Seminal Plasma and Its Specific MT1 and MT2 Receptors on Sperm Membranes

doi: 10.3390/ani15010083

Figure Lengend Snippet: Representative Western blot images showing the presence of MT1 ( A ) and MT2 ( B ) melatonin receptors in protein extracts from camel spermatozoa (M: Molecular weight marker (kDa), C: Camel sperm proteins, (+): Positive ram control).

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Western Blot, Molecular Weight, Marker, Control

Journal: Antioxidants

Article Title: Protective Effects of Exogenous Melatonin Administration on White Fat Metabolism Disruption Induced by Aging and a High-Fat Diet in Mice

doi: 10.3390/antiox13121500

Figure Lengend Snippet: Primer pairs for mRNAs were as follows.

Article Snippet: The following primary antibodies were employed: AANAT (ab3505, Abcam, Waltham, MA, USA), MTNR1A (PA5-75749, Thermofisher, Beijing, China), CEBPB (AF6202, Affinity Biosciences, Changzhou, China), MMP9 (AF5228, Affinity Biosciences, Changzhou, China), IL-1β (16806-1-AP, Proteintech, Wuhan, China), and NF-κB (8242, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Sequencing

Journal: Antioxidants

Article Title: Protective Effects of Exogenous Melatonin Administration on White Fat Metabolism Disruption Induced by Aging and a High-Fat Diet in Mice

doi: 10.3390/antiox13121500

Figure Lengend Snippet: Melatonin increases AANAT and MTNR1A expression in eWAT. ( A ) AANAT and MTNR1A in eWAT detected by immunofluorescence double staining (scale bar is 50 μm); ( B ) immunofluorescence analysis of CEBPB in eWAT (scale: 50 μm); ( C – E ) statistical graph of MTNR1A,AANAT and CEBPB positive expression rate; ( F ) Western blot analysis of MTNR1A protein expression in eWAT; ( G ) statistical graph of protein expression; ( H ) relative expression of MTNR1A, AANAT and CEBPB mRNA expression by qPCR analysis. All data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The following primary antibodies were employed: AANAT (ab3505, Abcam, Waltham, MA, USA), MTNR1A (PA5-75749, Thermofisher, Beijing, China), CEBPB (AF6202, Affinity Biosciences, Changzhou, China), MMP9 (AF5228, Affinity Biosciences, Changzhou, China), IL-1β (16806-1-AP, Proteintech, Wuhan, China), and NF-κB (8242, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Immunofluorescence, Double Staining, Western Blot