Diurnal variation of brain activity in the human suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is the central clock for circadian rhythms. Animal studies have revealed daily rhythms in the neuronal activity in the SCN. However, the circadian activity of the human SCN has remained elusive. In this study, to reveal the diurnal variation of the SCN activity in humans, the SCN was localized, and its activity was investigated using perfusion imaging. We scanned each participant four times a day, every six hours, and higher activity was observed at noon while lower activity was recorded in the early morning. The SCN activity was then measured every thirty minutes for six hours from midnight to dawn and showed a decreasing trend and was comparable with the rodent SCN activity after switching off the lights. These results suggest that the diurnal variation of the human SCN follows the zeitgeber cycles of mammals and is modulated by physical lights rather than the local time.

Before the scan, we localized the SCN by using the resting state images collected in our 73 previous study 30 . Brain activity in the SCN over 24 hours was then measured. The 74 participants were scanned four times a day, every six hours, with a within-participant 75 design. Brain activity was further scanned every thirty minutes for six hours from 76 midnight to dawn to reveal more detailed characteristics of the diurnal variation 77 compared to previous animal data. 78 79

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Two experiments were conducted in this study. In the first experiment, the whole cycle 81 of the diurnal activity of the SCN was investigated by two perfusion images measuring 82 the CBF, which were acquired four times (18:00, 24:00, 6:00, 12:00 on local time) 83 within 24 hours (Experiment 1) (Fig. 1a, Supplementary Fig. 1). The lights in the MRI 84 room were on in all the scans. On the other hand, the second experiment investigated 85 the temporal trend of human SCN activity at night (Experiment 2). The perfusion 86 images were scanned every 30 min from 24:00 to 6:00 on local time (Fig. 1b) represented how likely the voxel is a boundary of a functional area, a "parcel" that 98 corresponds to a hypothalamic nucleus ( Fig. 2a/b). The low probability value in a voxel 99 of the map, therefore, indicates that the voxel is likely the center of a parcel. In the 100 probability map, the SCN was identified bilaterally above the optic chiasm and lateral to 101 the third ventricle ( Supplementary Fig. 2a/b). We also localized the surrounding nuclei 102 and listed the coordinates in Table 1. The coordinates of the voxel of the SCN ROI in 2-103 mm resolution for perfusion imaging were x = -2, y = 2, z = -16 for the left ROI and x = 104 2, y = 2, z = -16 for the right ROI ( Fig. 2a/b). 107 Experiment 1 investigated whether the SCN activity had increased or decreased signal 108 changes within 24 hours. We performed a one-way repeated-measures analysis of 109 variance (ANOVA) over the time (6:00, 12:00, 18:00, and 24:00 on local time). The 110 activity in the SCN was highest at 12:00 in the daytime and lowest at 6:00 in the 111 morning (Fig. 3a). The SCN activity was significantly modulated among the four scans 112 (F(3,78) = 3.38, P = 0.022). The activity at 12:00 was significantly higher than that at 113 6:00 (Tukey-Kramer test, P < 0.05). A similar trend was seen when the left and right 114 SCN were analyzed separately (Fig. 3a) and when the first and second scans were 115 analyzed separately (Fig. 3b).  124 In Experiment 2, the SCN activity was measured in detail every 30 min from 24:00 to 125 6:00. A regression analysis indicates that the SCN activity gradually decreased from 126 midnight to dawn (beta estimate = -1.40, t(215) = -2.41, P = 0.017) (Fig. 4a). 127 We then compared the activity of the human SCN with the rodent SCN activity 128 published previously 21 . The previous study is one of very few that collected data, still 129 available to date, from more mature mice that would match in age with the present 130 human study. In the previous study, the multiunit neural activity of the SCN was 131 reported every 30 minutes. The current study divided the rodent SCN data exposed to 132 darkness into two periods: one from zeitgeber time (ZT) 12 to ZT 18 and the other from 133 ZT 18 to ZT 24, where the time of lights on is defined as ZT 0. We examined whether 134 the human SCN activity during the night (24:00 to 6:00) was more compatible with the 135 activity during ZT 12-18 or with the activity during ZT 18-24 in rodents (Fig. 4b). In 136 other words, we examined whether the human SCN activity during the night is more  Amylase activity that monitors the stress level measured before and after the MRI 155 session was higher before the experiment (i.e., night) than after the experiment (i.e., 156 morning) (one-tailed t-test, t(19) = 1.75, P = 0.048), which is consistent with the 157 previous studies 34 , suggesting that the overnight scans were not so stressful to the 158 participants.

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On the other hand, the size of the SCN seems to be less than one voxel of the  Fig. 2b). It is also to be noted that the areas 208 surrounding the SCN are not classified as nuclei 40 . The estimated distance was 4.5 mm 209 between the SCN and supraoptic nucleus, and 5.5 mm between the SCN and 210 paraventricular nucleus, which is discriminable in the 2 mm voxel/smoothing size. As 211 demonstrated in Supplementary Fig. 2c, it is possible that the adjacent nucleus is 212 included in the 2 mm voxel, but the proportion seems limited.  The current study observed a decreasing trend in the SCN activity while the scan 238 room was dark, while increased activity was recorded just after the lights were turned

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Experimental designs 250 In this study, two experiments were conducted, wherein the first investigated the whole 251 cycle of the diurnal activity of the SCN in humans (Experiment 1). Two perfusion 252 images of pseudo-continuous arterial spin labeling (pCASL) in each participant were 253 acquired four times within 24 hours (18:00, 24:00, 6:00, 12:00 on local time) and were 254 used to calculate the CBF at a specific time (Fig. 1a). The lights in the MRI room were 255 turned on in all the scans, and participants were instructed to have meals 4.5 hours 256 before each scan and to rest at a hotel at night (Supplementary Fig. 1). It is a bit unusual 257 to have four meals a day, especially just before sleep at night. Therefore, we instructed 258 the participants to take at least small meals if they do not want to take ordinary meals.

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To administer scans for multiple (up to four) participants in one 24-h session, the exact  In the second experiment, we investigated the human SCN activity in more 263 detail during the night (Experiment 2). Participants stayed in the scanner throughout the 264 night, except for brief unavoidable interruptions, and were scanned every 30 min from 265 24:00 to 6:00 (Fig. 1b). The lights in the MRI room were switched off at 0:15 and on at 266 5:45 am. In each scan, one single perfusion image was acquired. EEG was recorded 267 during scanning using an MRI-compatible 32-ch international 10/20 EEG system. The     (Fig. 1c, 1d), where the probability value represents how likely the voxel 316 is a boundary of the functional area (parcel), or in other words, how unlikely the voxel 317 is a center of the parcel.

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The bilateral parcels of the SCN were identified above the optic chiasm, and the    The data and code that support the findings of this study are available at Dryad, except 365 for raw image data. The raw image data cannot be deposited in a public repository 366 because sharing raw image data was not included in the informed consent. Any 367 additional information required to reanalyze the data reported in this paper is available 368 from the corresponding author upon reasonable request.