Journal: Scientific Reports

Article Title: K ATP Channels Mediate Differential Metabolic Responses to Glucose Shortage of the Dorsomedial and Ventrolateral Oscillators in the Central Clock

doi: 10.1038/s41598-017-00699-3

Figure Lengend Snippet: Kir6.2/SUR1 combination of K ATP channels in the SCN neurones. ( A ) RT-PCR analysis indicating the expression of mRNA for pore-forming Kir6.2 and sulfonylurea subunits 1 in the SCN. Positive controls were performed using cDNA from rat brain. The expected PCR product sizes for Kir6.1, Kir6.2, SUR1, and SUR2 were 411, 385, 388, and 501 bp, respectively. Negative controls were performed using RT products with omission of reverse transcriptase (RT-) to examine the contamination of genomic DNA. ( B ) Cell-attached recordings showing the effects of the sulfonylurea on spontaneous firing of the SCN neurones (ZT 4–15). Firing responses of a representative cell to 200 μM tolbutamide (top left panel), 200 μM diazoxide (top middle panel), and 0.1 μM glibenclamide (top right panel). Note the lack of recovery of spontaneous firing after washout of glibenclamide. Daytime recordings (ZT 7). Bottom left panel: summary of experiments showing a moderate increase in firing rate by tolbutamide. Baseline spontaneous firing rate: 2.7 ± 0.3 Hz ( n = 50). Bottom right panel: summary of experiments showing a decrease in firing rate by diazoxide. Baseline spontaneous firing rate: 3.0 ± 0.3 Hz ( n = 17). * P < 0.05, *** P < 0.001.

Article Snippet: For immunohistochemical staining, sections (20 μm) were treated with 0.3% H 2 O 2 for 15 min to quench endogenous peroxidase, and then incubated overnight at 4 °C in PBS containing 2% serum, 0.3% Triton X-100, and primary antibodies against Kir6.2 (rabbit anti-Kir6.2; 1:5000; APC-020; Alomone Labs, Jerusalem, Israel) and AVP (rabbit anti-AVP; 1:3000; AB1565; Millipore, Temecula, CA, USA) .

Techniques: Reverse Transcription Polymerase Chain Reaction, Analysis, Expressing, Reverse Transcription, Polymerase Chain Reaction, Patch Clamp

Journal: Scientific Reports

Article Title: K ATP Channels Mediate Differential Metabolic Responses to Glucose Shortage of the Dorsomedial and Ventrolateral Oscillators in the Central Clock

doi: 10.1038/s41598-017-00699-3

Figure Lengend Snippet: Distribution and localization of Kir6.2 immunoreactivity (ir) in the SCN. ( A ) Distribution of the Kir6.2 (A1–3) and AVP (A4-6) immunoreactivity in the rostral (A1, A4), middle (A2, A5), and caudal (A3, A6) sections of the SCN. Scale bar: 200 µm. ( B ) Selective colocalization of Kir6.2-ir with AVP-ir (B1–3) but not with VIP-ir (B4–6). Note the colocalization of Kir6.2-ir and AVP-ir in and around the soma (B2) and in varicosities along the process (marked by arrowheads, B3). Note the reciprocal apposition of VIP-ir bouton-like swellings against a Kir6.2-ir soma (B5) and Kir6.2-ir bouton-like swellings against VIP-ir somata (B6). Note also bouton-like swellings (yellow) double-stained with Kir6.2 (green) and AVP (red) apposing Hoechst-stained cells (blue) in the ventrolateral region of the SCN (B7). Scale bar: 100 µm (B1, B4); 10 µm (B2, B3, B5–7). ( C ) Lack of colocalization of Kir6.2-ir with markers for three afferent inputs SERT-ir (C1, C2), vGluT2-ir (C3, C4), NPY-ir (C5, C6). Insets: co-distribution of SERT-ir, vGluT2-ir, and NPY-ir with Kir6.2-stained somata in the ventromedial region of the mid-SCN section. Scale bar: 100 µm (C1, C3, C5); 10 µm (C2, C4, C6, insets). OC: optic chiasm. 3 V: third ventricle. Asterisks mark Hoechst-stained nuclei.

Article Snippet: For immunohistochemical staining, sections (20 μm) were treated with 0.3% H 2 O 2 for 15 min to quench endogenous peroxidase, and then incubated overnight at 4 °C in PBS containing 2% serum, 0.3% Triton X-100, and primary antibodies against Kir6.2 (rabbit anti-Kir6.2; 1:5000; APC-020; Alomone Labs, Jerusalem, Israel) and AVP (rabbit anti-AVP; 1:3000; AB1565; Millipore, Temecula, CA, USA) .

Techniques: Staining, Immunofluorescence