Neuropeptidergic Input from the Lateral Hypothalamus to the Suprachiasmatic Nucleus Alters the Circadian Period in Mice

Abstract

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which transmits circadian information to other brain regions and regulates the timing of sleep and wakefulness. Neurons in the lateral hypothalamus (LH), particularly those producing melanin-concentrating hormone (MCH) and orexin, are key regulators of sleep and wakefulness. Although the SCN receives nonphotic input from other brain regions, the mechanisms of functional input from the LH to the SCN remain poorly understood. Here, we show that orexin and MCH peptides influence the circadian period within the SCN of both sexes. When these neurons are ablated, the circadian behavioral rhythms are lengthened under constant darkness. Using anterograde and retrograde tracing, we found that orexin and MCH neurons project to the SCN. Furthermore, the application of these peptides to cultured SCN slices shortened circadian rhythms and reduced intracellular cAMP levels. Additionally, pharmacological reduction of intracellular cAMP levels similarly shortened the circadian period in SCN slices. These findings suggest that orexin and MCH peptides from the LH contribute to the modulation of the circadian period in the SCN.

Keywords: cAMP; circadian rhythm; lateral hypothalamus; neuropeptides; suprachiasmatic nucleus.

Figure 1.

Ablation of orexin and MCH neurons shows a lengthening of circadian behavioral rhythms. A, Schematic drawing of the tTA-driven DTA expression system. B, C, Schematic drawing of the coronal brain slice and fluorescence images in the LH. Scale bar, 200 µm. D, Representative examples of locomotor activity rhythms for control and orexin neuron-ablated (Orexin Nx) mice (left), the circadian period under DD calculated by periodogram (middle; control, n = 10; male 4/female 6; orexin Nx, n = 7; male 3/female 4), and daily locomotor activity profiles of these mice under LD and DD (right). E, Representative examples of locomotor activity rhythms of control and MCH neurons ablated (MCH Nx) mice (left), the circadian period under DD calculated by periodogram (middle; control, n = 8; male 5/female 3; MCH Nx, n = 7; male 4/female 3), and daily locomotor activity profiles of these mice under LD and DD (right). F, Representative examples of locomotor activity rhythms of control and orexin and MCH neurons ablated (Orexin-MCH Nx) mice (left), the circadian period under DD calculated by periodogram (middle; control, n = 11; male 5/female 6; orexin and MCH Nx, n = 10; male 4/female 6; *p < 0.05; Student's t test), and daily locomotor activity profiles of these mice under LD and DD (right).

Figure 2.

Anterograde and retrograde identification of the projections of orexin and MCH neurons in the LH to the SCN and peri-SCN. A, Schematic drawing of anterograde tracing of orexin neurons in the LH. EGFP fused synaptophysin were expressed in orexin or MCH neurons in the LH (injection site). B, Representative example of fluorescent image of synaptophysin-EGFP in the LH. Scale bar, 1,000 µm. C, Representative example of fluorescent images of synaptophysin-EGFP expressed in orexin neurons in the anterior, middle, and posterior SCN (left). Magnified images in the dorsal and ventral areas of the SCN are described on the right. D, Representative example of fluorescent images of synaptophysin-EGFP expressed in MCH neurons in the anterior, middle, and posterior SCN (left). Magnified images in the dorsal and ventral areas of the SCN are described on the right. Scale bar, 100 µm (left) and 10 µm (right). E, Schematic drawing of anterograde tracing in WT mice. F, Representative example of fluorescent image of synaptophysin-EGFP in the LH of WT mice. There is no expression in the LH (injection site). Scale bar, 1,000 µm. G, Representative example of fluorescent images of synaptophysin-EGFP expressed in WT SCN. Magnified images in the dorsal and ventral areas of the SCN are described on the right. Scale bar, 100 µm (left) and 10 µm (middle and right). H, Schematic drawing of retrograde tracing from the SCN. Retrobeads were injected in the SCN. I, Fluorescent image of retrobeads injected in the SCN. Scale bar, 100 µm. J, Representative fluorescent image of retrobeads in the LH (magenta, retrobeads; yellow, orexin; light blue, MCH). Scale bar, 100 µm (left) and 10 µm (right). K, The percentage of retrobead-positive neurons in the orexin or MCH-positive neurons in the LH (n = 3).

Figure 3.

Orexin and MCH receptors expressed in the SCN and peri-SCN. A, Representative FISH with fluorescent Nissl staining images in the SCN (gray, Nissl; light blue, orexin receptor 1; magenta, MCH receptor 1). B, Representative FISH with fluorescent Nissl staining images in the SCN (gray, Nissl; light blue, orexin receptor 2; magenta, MCH receptor 1). Scale bar, 100 µm (left) and 10 µm (right). C–E, Results of single-cell RNA sequencing using data from Morris et al. Avp, Vip, Grp, or Nms expression patterns in OX1R-, OX2R-, or MCHR1-positive (>0.2, green) or OX1R-, OX2R-, or MCHR1-negative (<0.2, red) cells in the SCN are shown in each panel.

Figure 4.

Orexin and MCH peptides shorten circadian PER2::LUC rhythms in the SCN slice. A, Representative double-plotted PER2::LUC rhythms in the SCN slice. Stars in each panel indicate the timing of peptide or vehicle application. B, The circadian period of PER2::LUC rhythms in the SCN before and after application of peptides (vehicle, n = 11; orexin-A, n = 11; MCH, n = 13; orexin-A and MCH, n = 12; **p < 0.01; mixed-effect model with post hoc Sidak's multiple-comparison test). C, The normalized amplitude of PER2::LUC rhythms in the SCN slice after peptide application. The amplitudes were normalized with the difference in peak–trough before the administration as 1.0.

Figure 5.

Orexin and MCH peptides modulate Ca²⁺ dynamics in the SCN neurons. A, F, Fluorescence images of GCaMP8s expressing Vgat neurons in the SCN. Scale bar, 20 µm. B, G, The intensity of fluorescence changes obtained from SCN neurons before, during, and after treatment with orexin-A or MCH peptides. Green, magenta, and gray lines indicate neurons with increased, decreased, and unchanged fluorescence intensity upon peptide administration, respectively. Fluorescence intensity is expressed as z-score. C, H, Pie chart showing the percentage of cells that exhibited increase, decrease, or no change in fluorescence intensity during peptide administration. The paired Wilcoxon signed-rank test (p < 0.05) was used to compare the average intensity 2 min before and during peptide application. D, I, Difference in fluorescence intensity between orexin-A and MCH administrations. Each symbol represents one neuron (n = 41 from 3 mice at ZT 4–10; n = 55 from 4 mice at ZT 14–20). E, J, Fluorescence intensity changes in SCN neurons before, during, and after treatment with aCSF, glutamate, and GABA during the subjective day and night. A–E for day and F–J for night.

Figure 6.

Orexin and MCH peptides modulate cAMP dynamics in the SCN neurons. A, F, Fluorescence images of cAMPinG1-expressing Vgat neurons in the SCN. Scale bar, 20 µm. B, G, Fluorescence intensity changes from SCN neurons before, during, and after treatment with orexin-A or MCH peptides. Green, magenta, and gray lines indicate neurons with decreased or unchanged fluorescence intensity upon peptide administration, respectively. Fluorescence intensity is expressed as z-score. C, H, Pie chart showing the percentage of cells that exhibited decrease or unchanged fluorescence intensity during peptide administration. Paired Wilcoxon signed-rank test (p < 0.05) was used to compare average intensity 2 min before and during peptide application. D, I, Difference in fluorescence intensity between orexin-A and MCH administrations. Each symbol represents one neuron (n = 51 from 3 mice at ZT 4–10; n = 53 from 4 mice at ZT 14–20). E, J, Fluorescence intensity changes in SCN neurons before, during, and after treatment with aCSF, glutamate, and GABA during the subjective day and night. A–E for day and F–J for night.

Figure 7.

Pharmacological reduction of cAMP levels shortens circadian PER2::LUC rhythms in the SCN slice. A, Representative double-plotted PER2::LUC rhythms in the SCN slice. Stars in each panel indicate the timing of vehicle or MDL-12,330A (5.0 µM) application. B, Circadian period of PER2::LUC rhythms in SCN slices before and after MDL application (vehicle, n = 5; MDL, n = 5; **p < 0.01; mixed-effect model with post hoc Sidak's multiple-comparison test).

Figure 8.

A hypothetical model of the regulation of the circadian period in the SCN by orexin and MCH neurons. Orexin and MCH neurons in the LH regulate wakefulness and sleep, respectively. Neuronal activity of orexin neurons is typically high during wakefulness and intermediate during NREM sleep, whereas the neuronal activity of MCH neurons is high during REM sleep and intermediate during wakefulness. Orexin and MCH peptides are released from neuronal terminals located in the SCN and peri-SCN, where they reduce cAMP levels and shorten circadian rhythms in the SCN.