(A) Abundance of pre-miR-132 and pre-miR-219-1 transcripts in pooled RNA samples of SCN and piriform cortex (CTX) tissue as a function of circadian time (CT 2, 6, 11, 15, 19, 22) was determined by semi-quantitative RT-PCR. Expression of gapdh served as the loading control. (B) Ribonuclease protection assays (RPA) for miR-219 (top) and miR-132 (bottom). Densitometric analysis of miR-219 (top) and miR-132 (bottom) is shown to the left. miRNA levels were normalized to 5S rRNA and expressed as fold expression. Mean values were determined from three independent experiments. (C) Quantitative real-time PCR analysis of pre-miR-219-1 (left) and pre-miR-132 (right) in the SCN of wild-type and mcry1/cry2 double mutant mice at CT 6 and CT 19. Data were normalized to gapdh levels and presented as fold expression.

(A–C) Representative actograms of wheel-running activity of C57Bl/6J mice which had received an infusion of (A) a negative control antagomir (scrambled), (B) miR-132 antagomir (40 μM, 3 μl) or (C) miR-219 antagomir at CT 2. Periods of darkness are shaded in gray. Activity onsets are indicated by blue and red lines. Red asterisk denotes infusion. The x-axis (top) indicates the Zeitgeber (ZT) time over a 24-hr cycle. The y-axis (left) indicates the nth day of the experiment. (D–F) Graphical representation of the period length (tau) of individual animals that were infused with (D) the negative control antagomir (scrambled), (E) miR-132 antagomir, or (F) miR-219 antagomir before (PRE) and after (POST) the infusion. (G) Quantitation of the effects of miR-219 antagomir on period length under free-running conditions. Values are presented as the mean difference ± SEM between post- and pre-infusion tau values (in hr). n=8–10 per group. * p<0.01 (two-tailed Student’s t-test). (H) Knockdown of miR-219 expression. miR-219 antagomir (40 μM, 3 μl) or vehicle were infused into the lateral ventricle at CT 2, and SCN tissue was subsequently harvested at CT 10. Abundance of mature miR-219 was determined by RPA. Abundance of 5S rRNA was used as the loading control.

(A) Cultured cortical neurons were transfected with expression constructs for pre-miR-132, pre-miR-219 (not shown) or pre-miR-1 (not shown), along with a DsRed.T3-NLS-PEST expression plasmid as a transfection marker (transfection ratio 3:1 of pre-miR construct: DsRed). Negative controls were cultures transfected with empty vector (pcDNA3.1), and adjacent untransfected neurons (DsRed-negative); internal Ca²⁺ levels were monitored using time-lapse digital microscopy of Fluo-4. Representative data from Fluo-4 loaded (green) untransfected and transfected (red) neurons stimulated with K⁺ (20 mM), glutamate (20 μM) and NMDA (20 μM). (B) Peak ± SEM Ca²⁺ responses of untransfected and transfected neurons. Data for each experiment were normalized to the peak response of untransfected neurons, which was set equal to a value of 1. * p<0.01. (C) HEK 293 cells were transfected with expression constructs for SCOP or FLAG-RFX4, along with pre-miR-132, pre-miR-219 or pre-miR-1 and DsRed.T3-NLS-PEST (transfection ratio 2:6:1 of target: pre-miR construct: DsRed). Forty-eight hours after transfection, cells were fixed and immunolabelled for SCOP or FLAG-RFX4. Representative panels show target expression when transfected with each pre-miRNA. Note the reduced expression of SCOP when cotransfected with pre-miR-219, and the reduced expression of FLAG-RFX4 when cotransfected with pre-miR-132. (D) Mean immunolabeling intensity for SCOP and FLAG-RFX4 under the three cotransfection conditions. Y-axis denotes fluorescent intensity units (0-4096 scale). Values are presented as mean ± SEM. n=200–250 cells per group. *p<0.05 (two-tailed Student’s t-test).

(A–B) Chromatin immunoprecipitation (ChIP) assay of PC12 cells using (A) an anti-CREB or (B) an anti-CLOCK antibody. RNA abundance was determined by real-time quantitative PCR. A nonspecific IgG was used as a negative control. In (A), the levels of immunoprecipitated (IP) DNA encompassing the promoter regions of miR-132, miR-219-1, c-Fos, and an r18S control were quantitated (right), and data for miR-132 (left) and c-Fos (middle) are presented as fluorescence intensity vs. number of PCR cycles. In (B), the levels of IP promoter DNA of miR-132, miR-219-1, mPeriod1 and an r18S control were determined (right), and data for miR-219-1 (left) and miR-132 (middle) are presented as fluorescence intensity vs. number of PCR cycles. (C) Position of murine (mmu-)miR-132 gene on chromosome 11 (top). Alignment scores are indicated by black bars in the conservation track of mouse, rat, human, dog and opossum (top). Two consensus CREs (italicized: yellow boxes) are located in the 5′ region of the miR-132 locus, and base mismatches from mouse in rat, human and dog are represented by lower case letters. Sequence alignments of mouse, rat, human and dog (bottom). (D) Position of murine (mmu-)miR-219-1 gene on chromosome 17 (top). Alignment scores are indicated in the conservation track (top). Two putative CREs (CRE1 and CRE2; italicized, yellow boxes) and an E-box motif (italicized, yellow box) are located in the 5′ region of the miR-219-1 locus (bottom). Mismatches from mouse are denoted by lower case letters. (E) Levels of pre-miR-219-1, but not of pre-miR-132 or gapdh, were elevated in PC12 cells co-transfected with expression constructs for CLOCK and BMAL1, as determined by semi-quantitative reverse transcription (RT)-PCR. (F) Quantitative analysis of CLOCK/BMAL1-dependent induction of pre-miR-219 in transfected PC12 cells as determined by real-time PCR. Data are presented as mean ± SEM nanograms of DNA. (G) Dominant-negative CREB (A-CREB) suppresses miR-132 induction. Primary cortical neurons were transfected with the expression constructs for A-CREB or GFP (negative control), were stimulated with forskolin (10 μM) or KCl (20 mM), and the abundance of pre-miR-132 transcript was determined by real-time PCR. Data are presented as mean ± SEM nanograms of cDNA.

(A) Mice received a single light pulse (LF; 15 min, 100 lux) at CT 6, 15, or 22, and were sacrificed 1 hr later. Abundance of pre-miR-132 and pre-miR-219-1 transcripts in pooled RNA samples of SCN and piriform cortex (CTX) tissue was determined by RT-PCR. Mice which had not received a light pulse (dark) but were sacrificed at the same circadian time served as negative controls. Expression of gapdh served as the loading control. (B) RPA analysis of mature miR-132 and miR-219 in the SCN 1 hr after light (+) treatment at CT 6, 15, or 22. Mice which had not received a light pulse (−) but were sacrificed at the same circadian time served as negative controls. Expression of 5S rRNA served as the loading control. Radiolabeled RNA size markers (M; in nucleotides) are shown to the right. P denotes undigested probe. (C) Mice were infused with the MEK inhibitor U0126 (10 mM, 3 μl) or vehicle 30 min prior to a single light flash (LF: 15 min, 100 lux). Mice were sacrificed 1 hr later and the abundance of pre-miR-132 and gapdh transcripts in the SCN were determined by RT-PCR. (D) Animals were infused with U0126 (10 mM), KN-62 (10 mM), or KT-5823 (1 mM), and the effects on basal (dark) and light- (LF: 15 min, 100 lux) induced pre-miR-132 expression were analyzed via real-time quantitative PCR. Data were normalized to gapdh levels and presented as fold expression. Mean values were determined from three independent experiments.

(A–C) Representative actograms of wheel-running activity of C57Bl/6J mice which received an infusion of (A) a negative control antagomir (scrambled), (B) miR-132 antagomir (40 μM, 3 μl), or (C) miR-219 antagomir at CT 14. All subjects received a single light pulse (15 min, 20 lux) at CT 15. Periods of darkness are shaded in gray. Activity onsets are indicated by blue lines. Red asterisk denotes light pulse. The x-axis (top) indicates the Zeitgeber (ZT) time over a 24-hr cycle. The y-axis (left) indicates the nth day of the experiment. (D) Quantitation of the effects of miR-132 antagomir on CT 15 light-induced phase shifts. Values are presented as mean ± SEM phase shift (in min). n=8–12 per group. * p<0.01 (two-tailed Student’s t-test). (E) Knockdown of miR-132 expression. miR-132 antagomir (40 μM, 3 μl) was infused into the lateral ventricle at CT 14, and a brief light pulse (100 lux, 15 min) was administered at CT 15. SCN tissue was subsequently harvested at CT 17. Control subjects were infused with vehicle and/or did not receive a light treatment. Abundance of mature miR-132 was determined by RPA. Abundance of 5S rRNA was used as the loading control.

(A) Effect of miR-132 and miR-219 on CLOCK/BMAL1-mediated Per1 transactivation. HEK293T cells were transfected with a Per1-luciferase reporter construct, in combination with the following: CLOCK and BMAL1 expression plasmids and constructs encoding pre-miR-132, pre-miR-219 or pre-miR-1. Cells were lysed 48 hrs post-transfection and assayed for luciferase activity. Y-axis denotes fold difference in luciferase activity relative to control samples transfected with the Per1-luciferase construct alone. Experiments were performed four times, and representative data were averaged from quadruplicate determinations. (B) Effect of miR-132 and miR-219 expression on Per1 transactivation in primary neurons under basal and stimulated conditions. Primary rat embryonic neurons were transfected with a Per1-luciferase reporter construct in combination with the expression vector for pre-miR-132, pre-miR-219 or pre-miR-1. Forty-eight hrs post-transfection, neurons were stimulated with 25 mM KCl and 5 μM forskolin and cell lysates were prepared 6 hrs later. Y-axis denotes fold difference in luciferase activity relative to control samples transfected with the Per1-luciferase construct alone. Experiments were performed four times, and representative data were averaged from quadruplicate determinations. (C) Generation of Per1-Venus BAC transgenic mice. The Venus-NLS-PEST-polyA cassette was inserted into the per1 locus on a 161-kb bacterial artificial chromosome (BAC) by homologous recombination. The BAC clone contains the full intergenic sequences of mPer1 and its flanking genes, including the 5′ promoter region. (D) The effect of miR-132 knockdown on light-induced Venus expression in the SCN of Per1-Venus BAC transgenic mice. miR-132 antagomir (40 μM, 3 μl) or drug vehicle (saline), were infused into the lateral ventricles of Per1-Venus BAC mice at CT 14, and a brief light pulse was administered at CT 15. Tissue was harvested at CT 19 for analysis of Venus expression by immunofluorescence. Dark control animals were not exposed to light but were sacrificed at the same circadian time. (E) Quantitation of light-induced Venus expression as a function of miR-132 abundance. Values are presented as mean ± SEM. n=3–8 mice per group. *p<0.05 (two-tailed Student’s t-test). (F) Immunohistochemical analysis of PER2 protein abundance in the SCN as a function of circadian time. (G) In a separate experiment, mice were infused with miR-132 antagomir (40 μM, 3 μl) or vehicle at CT 14 and exposed to light (100 lux, 15 min) at CT 15 (right). SCN tissue was harvested 29 hr later and immunostained for PER2. Quantitation of PER2 immunoreactivity in the SCN of miR-132 antagomir- or vehicle-infused mice (left). Values are presented as mean ± SEM. n=8–10 per group. *p<0.05 (two-tailed Student’s t-test).