BRIEF COMMUNICATION
Suprachiasmatic Neuron Numbers and Rest–Activity Circadian Rhythms in Older Humans Joshua L. Wang, AB,1 Andrew S. Lim, MD,1,2 Wei-Yin Chiang, PhD,3,4 Wan-Hsin Hsieh, PhD,3,4 Men-Tzung Lo, PhD,3,4 Julie A. Schneider, MD,5 Aron S. Buchman, MD,5 David A. Bennett, MD,5 Kun Hu, PhD,1,3,4 and Clifford B. Saper, MD, PhD1 The suprachiasmatic nucleus (SCN) of the hypothalamus, the master mammalian circadian pacemaker, synchronizes endogenous rhythms with the external day– night cycle. Older humans, particularly those with Alzheimer disease (AD), often have difficulty maintaining normal circadian rhythms compared to younger adults, but the basis of this change is unknown. We report that the circadian rhythm amplitude of motor activity in both AD subjects and age-matched controls is correlated with the number of vasoactive intestinal peptide– expressing SCN neurons. AD was additionally associated with delayed circadian phase compared to cognitively healthy subjects, suggesting distinct pathologies and strategies for treating aging- and AD-related circadian disturbances. ANN NEUROL 2015;78:317–322
I
n mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus acts as the master circadian pacemaker to drive and synchronize endogenous rhythms with the external day–night cycle. The SCN must provide a stable, strong circadian output while remaining adaptable to differences between endogenous and exogenous cycles that arise from seasonal changes in the time of sunrise and endogenous circadian periods not exactly equal to 24 hours in length. The SCN contains a core region composed mainly of neurons expressing vasoactive intestinal peptide (VIP) and a shell region made up largely of arginine vasopressin (AVP)-expressing neurons,1 which work in a complementary manner to impart stability and flexibility to the circadian timing system. The VIP neurons of the SCN core receive direct light input from the ret-
ina and provide intranuclear projections to the rest of the SCN. These neurons use the VPAC2 receptor2 to promote synchronicity of SCN neurons, which is important for maintaining a high-amplitude circadian output, and to allow photic resetting. The AVP neurons of the SCN are relatively resistant to photic phase shifting,3 but provide extensive projections to SCN targets.4 In aged individuals, functional weakening of the circadian timing system and concomitant sleep impairments are associated with neurodegeneration and cognitive decline.5 Compared to young adults, older humans have been reported to have fewer VIP-immunoreactive (-ir) and AVP-ir neurons in the SCN.6,7 However, the relationship between the number of these SCN neurons and circadian activity rhythms in individual older community-dwelling adults is not known. To address this issue, we compared the numbers of surviving AVP-ir and VIP-ir neurons in the SCN with the actual circadian behavior of individual older adults, both with and without Alzheimer disease (AD).
Materials and Methods Human Subjects We studied 17 individuals (mean age 5 90.4 years at death; 4 males) from the Rush Memory and Aging Project (MAP)8 who had at least 1 week of actigraphy within the 18 months prior to death. The MAP is a longitudinal, community-based study of the chronic conditions of aging, in which motor activity of subjects is monitored biennially for up to 10 days using actigraphs (Actical; Philips Respironics, Best, the Netherlands) placed on subjects’ nondominant wrists (technical details of the actigraph recordings have been previously described9). A subset of subjects (n 5 7) had been diagnosed with AD, based on From the 1Program in Neuroscience and Division of Sleep Medicine, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada; 3Medical Biodynamics Program, Division of Sleep Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 4Center for Dynamical Biomarkers and Translational Medicine, National Central University, Chungli, Taiwan; and 5Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL Address correspondence to Dr Saper, 330 Brookline Avenue, Boston, MA 02215. E-mail: [email protected] Additional Supporting Information may be found in the online version of this article. Received Feb 4, 2015, and in revised form Apr 21, 2015. Accepted for publication Apr 22, 2015. View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana. 24432
C 2015 American Neurological Association V 317
ANNALS
of Neurology
FIGURE 1: Representative anatomic and actigraphic data. (A–D) Immunohistochemical staining of human suprachiasmatic nucleus for vasoactive intestinal peptide (A, C) and arginine vasopressin (B, D). Low-power (A, B, scale bar 5 1mm) and highpower views (C, D, scale bar 5 100lm) are shown from a single subject in consecutive sections. (E) Visualization of actigraphy data from an illustrative participant. Inset shows detail view of 48 hours of this recording. 3V 5 third ventricle; OC 5 optic chiasm; PVN 5 paraventricular nucleus; SCN 5 suprachiasmatic nucleus; SON 5 supraoptic nucleus. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]
318
Volume 78, No. 2
Wang et al: SCN and Rest–Activity Rhythms
TABLE 1. Demographic and Clinical Metrics of Study Participants
Characteristic
Suprachiasmatic Nucleus Available and 18 Months between Actigraphy and Death, n 5 24
Suprachiasmatic Nucleus Not Available, n 5 194
p
At time of actigraphy
89.6 (5.5) [83.1–101.3]
85.8 (7.5) [62.0–95.7]
88.0 (6.2) [71.6–100.8]
0.43
At time of death
90.4 (5.5) [83.9–101.6]
88.5 (7.2) [65.9–97.8]
89.5 (6.1) [72.7–102.7]
0.97
13 f76.5g/4 f23.5g
16 f66.7g/8 f33.3g
133 f68.6g/61 f31.4g
0.79
White Black
16 f94.1g 1 f5.8g
24 f100g 0 f0g
188 f96.9g 5 f2.6g
Other
0 f0g
0 f0g
1 f0.5g
Education, yr
14.1 (2.3) [10–18]
14.3 (1.9) [12–18]
14.7 (2.8) [8–25]
0.98
Body mass index, kg/m2
25.0 (4.3) [17.9–33.7]
28.5 (5.2) [20.9–40.8]
26.0 (5.0) [17.0–41.2]
0.09
Alzheimer disease
7 f41.2g
10 f41.7g
70 f36.1g
0.60
Depressive symptoms, No.
1.8 (1.9) [0–6]
2.2 (2.6) [0–8]
1.5 (2.0) [0–9]
0.49
Vascular risk factors, No.
1.4 (0.7) [0–2]
1.1 (0.83) [0–3]
1.3 (0.8) [0–3]
0.62
Vascular diseases, No.
0.53 (0.52) [0–1]
0.61 (1.0) [0–4]
0.77 (0.93) [0–4]
0.39
Self-reported sleep, h
7.5 (1.3) [5–10]
7.2 (1.6) [5–11]
7.4 (1.6) [4–12]
0.99
Age, yr
Sex, F/M Race
0.74
Values are expressed as mean (standard deviation) [range] or No. f%g. Continuous measures were tested across groups using the Kruskal–Wallis test; discrete measures were tested using the chi-square test; and race and sex were tested using Fisher exact test. F 5 female; M 5 male. cognitive impairment and pathological confirmation, using the National Institute of Neurological and Communicative Diseases and Stroke–Alzheimer’s Disease and Related Disorders Association and National Institute on Aging–Reagan Institute criteria, as described previously.10 The study was conducted in accordance with the latest version of the Declaration of Helsinki and was approved by the institutional review board of Rush University Medical Center. Written informed consent was obtained from all subjects.
Quantification of the Circadian Activity Rhythm To estimate the circadian activity rhythm, we extracted the oscillatory component of 24 hours in motor activity using the empirical mode decomposition algorithm with a masking procedure11 and normalized its amplitude to the standard deviation of the fluctuations at smaller time scales. This method measures 24-hour rhythmicity in locomotor activity, which is somewhat irregular, August 2015
without making assumptions about the shape of the underlying waveform (eg, a sine wave with the cosinor method or a square wave with the circadian index; see Chou et al12). The software for the extraction of circadian activity rhythm is installed on the server of the Medical Biodynamics Program at Brigham and Women’s Hospital and will be available upon request. Activity acrophases and nadirs were measured from the raw actigraphy records as the 8 consecutive hours with the most and least total activity each day, respectively. These times were then expressed in relation to sunrise time during the actigraphic recording to minimize the effect of seasonal changes in light–dark cycles on activity behavior between subjects. We also calculated a number of previously reported circadian measures, including circadian index (the peak-to-trough amplitude measured between the 8 hours of greatest and least activity, normalized to the total activity in the day),12 interdaily stability (a measure of how similar the actigraphic records are from day to day),13 and intradaily variability (a measure of the fragmentation of the activity rhythm).13 319
ANNALS
of Neurology
Brain Tissue Processing and Immunohistochemistry Upon death, we manually dissected blocks of tissue from these subjects containing the hypothalamus from the chiasmatic region to the mammillary bodies. The blocks were coronally sectioned into four 1:4, 50lm series with 1 series immunohistochemically stained for VIP (anti-VIP; Immunostar, Hudson, WI; catalog # 20077, lot 1129001) and another stained for AVP (anti-AVP; Millipore, Billerica, MA; catalog # AB1565, lot LV1587742; Fig 1A–D). Preadsorption of either antibody with its target antigen prior to incubation of the tissue with the primary antibody completely abolished staining. We used methods identical to those recently described.9 As the VIP-ir and AVP-ir neurons are not always located at precisely overlapping rostral–caudal levels (ie, one population may begin or end a section or two rostral to the other), we only quantified AVP-ir or VIP-ir neurons in brains where there was at least 1 section rostral and 1 section caudal to the stained neurons. Visual inspection of the stained sections determined that the blocks from 14 subjects contained the entire VIP-ergic SCN and the blocks from 14 subjects contained the entire AVP-ergic SCN (17 total subjects).
Stereological Cell Counts Neuron counts of the SCN for each series were obtained using the optical fractionator stereological method, as previously described.9 To avoid bias in assigning borders to the cell group, we counted all immunoreactive neurons in the region bounded ventrally by the optic chiasm, medially by the third ventricle, dorsally by the paraventricular nucleus, and laterally by the supraoptic nucleus. Photomicrographs presented in Figure 1 were prepared using Photoshop CS6 (Adobe Systems, San Jose, CA). To show differences in distribution of the VIP-ir and AVP-ir neurons, minor adjustments to brightness and contrast were implemented to match the backgrounds of both photomicrographs, as the different antibodies used did not stain the tissue to the same darkness.
Results Clinical Characteristics The 17 subjects with actigraphic recordings
Suprachiasmatic Neuron Numbers and Rest–Activity Circadian Rhythms in Older Humans Joshua L. Wang, AB,1 Andrew S. Lim, MD,1,2 Wei-Yin Chiang, PhD,3,4 Wan-Hsin Hsieh, PhD,3,4 Men-Tzung Lo, PhD,3,4 Julie A. Schneider, MD,5 Aron S. Buchman, MD,5 David A. Bennett, MD,5 Kun Hu, PhD,1,3,4 and Clifford B. Saper, MD, PhD1 The suprachiasmatic nucleus (SCN) of the hypothalamus, the master mammalian circadian pacemaker, synchronizes endogenous rhythms with the external day– night cycle. Older humans, particularly those with Alzheimer disease (AD), often have difficulty maintaining normal circadian rhythms compared to younger adults, but the basis of this change is unknown. We report that the circadian rhythm amplitude of motor activity in both AD subjects and age-matched controls is correlated with the number of vasoactive intestinal peptide– expressing SCN neurons. AD was additionally associated with delayed circadian phase compared to cognitively healthy subjects, suggesting distinct pathologies and strategies for treating aging- and AD-related circadian disturbances. ANN NEUROL 2015;78:317–322
I
n mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus acts as the master circadian pacemaker to drive and synchronize endogenous rhythms with the external day–night cycle. The SCN must provide a stable, strong circadian output while remaining adaptable to differences between endogenous and exogenous cycles that arise from seasonal changes in the time of sunrise and endogenous circadian periods not exactly equal to 24 hours in length. The SCN contains a core region composed mainly of neurons expressing vasoactive intestinal peptide (VIP) and a shell region made up largely of arginine vasopressin (AVP)-expressing neurons,1 which work in a complementary manner to impart stability and flexibility to the circadian timing system. The VIP neurons of the SCN core receive direct light input from the ret-
ina and provide intranuclear projections to the rest of the SCN. These neurons use the VPAC2 receptor2 to promote synchronicity of SCN neurons, which is important for maintaining a high-amplitude circadian output, and to allow photic resetting. The AVP neurons of the SCN are relatively resistant to photic phase shifting,3 but provide extensive projections to SCN targets.4 In aged individuals, functional weakening of the circadian timing system and concomitant sleep impairments are associated with neurodegeneration and cognitive decline.5 Compared to young adults, older humans have been reported to have fewer VIP-immunoreactive (-ir) and AVP-ir neurons in the SCN.6,7 However, the relationship between the number of these SCN neurons and circadian activity rhythms in individual older community-dwelling adults is not known. To address this issue, we compared the numbers of surviving AVP-ir and VIP-ir neurons in the SCN with the actual circadian behavior of individual older adults, both with and without Alzheimer disease (AD).
Materials and Methods Human Subjects We studied 17 individuals (mean age 5 90.4 years at death; 4 males) from the Rush Memory and Aging Project (MAP)8 who had at least 1 week of actigraphy within the 18 months prior to death. The MAP is a longitudinal, community-based study of the chronic conditions of aging, in which motor activity of subjects is monitored biennially for up to 10 days using actigraphs (Actical; Philips Respironics, Best, the Netherlands) placed on subjects’ nondominant wrists (technical details of the actigraph recordings have been previously described9). A subset of subjects (n 5 7) had been diagnosed with AD, based on From the 1Program in Neuroscience and Division of Sleep Medicine, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada; 3Medical Biodynamics Program, Division of Sleep Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 4Center for Dynamical Biomarkers and Translational Medicine, National Central University, Chungli, Taiwan; and 5Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL Address correspondence to Dr Saper, 330 Brookline Avenue, Boston, MA 02215. E-mail: [email protected] Additional Supporting Information may be found in the online version of this article. Received Feb 4, 2015, and in revised form Apr 21, 2015. Accepted for publication Apr 22, 2015. View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana. 24432
C 2015 American Neurological Association V 317
ANNALS
of Neurology
FIGURE 1: Representative anatomic and actigraphic data. (A–D) Immunohistochemical staining of human suprachiasmatic nucleus for vasoactive intestinal peptide (A, C) and arginine vasopressin (B, D). Low-power (A, B, scale bar 5 1mm) and highpower views (C, D, scale bar 5 100lm) are shown from a single subject in consecutive sections. (E) Visualization of actigraphy data from an illustrative participant. Inset shows detail view of 48 hours of this recording. 3V 5 third ventricle; OC 5 optic chiasm; PVN 5 paraventricular nucleus; SCN 5 suprachiasmatic nucleus; SON 5 supraoptic nucleus. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]
318
Volume 78, No. 2
Wang et al: SCN and Rest–Activity Rhythms
TABLE 1. Demographic and Clinical Metrics of Study Participants
Characteristic
Suprachiasmatic Nucleus Available and 18 Months between Actigraphy and Death, n 5 24
Suprachiasmatic Nucleus Not Available, n 5 194
p
At time of actigraphy
89.6 (5.5) [83.1–101.3]
85.8 (7.5) [62.0–95.7]
88.0 (6.2) [71.6–100.8]
0.43
At time of death
90.4 (5.5) [83.9–101.6]
88.5 (7.2) [65.9–97.8]
89.5 (6.1) [72.7–102.7]
0.97
13 f76.5g/4 f23.5g
16 f66.7g/8 f33.3g
133 f68.6g/61 f31.4g
0.79
White Black
16 f94.1g 1 f5.8g
24 f100g 0 f0g
188 f96.9g 5 f2.6g
Other
0 f0g
0 f0g
1 f0.5g
Education, yr
14.1 (2.3) [10–18]
14.3 (1.9) [12–18]
14.7 (2.8) [8–25]
0.98
Body mass index, kg/m2
25.0 (4.3) [17.9–33.7]
28.5 (5.2) [20.9–40.8]
26.0 (5.0) [17.0–41.2]
0.09
Alzheimer disease
7 f41.2g
10 f41.7g
70 f36.1g
0.60
Depressive symptoms, No.
1.8 (1.9) [0–6]
2.2 (2.6) [0–8]
1.5 (2.0) [0–9]
0.49
Vascular risk factors, No.
1.4 (0.7) [0–2]
1.1 (0.83) [0–3]
1.3 (0.8) [0–3]
0.62
Vascular diseases, No.
0.53 (0.52) [0–1]
0.61 (1.0) [0–4]
0.77 (0.93) [0–4]
0.39
Self-reported sleep, h
7.5 (1.3) [5–10]
7.2 (1.6) [5–11]
7.4 (1.6) [4–12]
0.99
Age, yr
Sex, F/M Race
0.74
Values are expressed as mean (standard deviation) [range] or No. f%g. Continuous measures were tested across groups using the Kruskal–Wallis test; discrete measures were tested using the chi-square test; and race and sex were tested using Fisher exact test. F 5 female; M 5 male. cognitive impairment and pathological confirmation, using the National Institute of Neurological and Communicative Diseases and Stroke–Alzheimer’s Disease and Related Disorders Association and National Institute on Aging–Reagan Institute criteria, as described previously.10 The study was conducted in accordance with the latest version of the Declaration of Helsinki and was approved by the institutional review board of Rush University Medical Center. Written informed consent was obtained from all subjects.
Quantification of the Circadian Activity Rhythm To estimate the circadian activity rhythm, we extracted the oscillatory component of 24 hours in motor activity using the empirical mode decomposition algorithm with a masking procedure11 and normalized its amplitude to the standard deviation of the fluctuations at smaller time scales. This method measures 24-hour rhythmicity in locomotor activity, which is somewhat irregular, August 2015
without making assumptions about the shape of the underlying waveform (eg, a sine wave with the cosinor method or a square wave with the circadian index; see Chou et al12). The software for the extraction of circadian activity rhythm is installed on the server of the Medical Biodynamics Program at Brigham and Women’s Hospital and will be available upon request. Activity acrophases and nadirs were measured from the raw actigraphy records as the 8 consecutive hours with the most and least total activity each day, respectively. These times were then expressed in relation to sunrise time during the actigraphic recording to minimize the effect of seasonal changes in light–dark cycles on activity behavior between subjects. We also calculated a number of previously reported circadian measures, including circadian index (the peak-to-trough amplitude measured between the 8 hours of greatest and least activity, normalized to the total activity in the day),12 interdaily stability (a measure of how similar the actigraphic records are from day to day),13 and intradaily variability (a measure of the fragmentation of the activity rhythm).13 319
ANNALS
of Neurology
Brain Tissue Processing and Immunohistochemistry Upon death, we manually dissected blocks of tissue from these subjects containing the hypothalamus from the chiasmatic region to the mammillary bodies. The blocks were coronally sectioned into four 1:4, 50lm series with 1 series immunohistochemically stained for VIP (anti-VIP; Immunostar, Hudson, WI; catalog # 20077, lot 1129001) and another stained for AVP (anti-AVP; Millipore, Billerica, MA; catalog # AB1565, lot LV1587742; Fig 1A–D). Preadsorption of either antibody with its target antigen prior to incubation of the tissue with the primary antibody completely abolished staining. We used methods identical to those recently described.9 As the VIP-ir and AVP-ir neurons are not always located at precisely overlapping rostral–caudal levels (ie, one population may begin or end a section or two rostral to the other), we only quantified AVP-ir or VIP-ir neurons in brains where there was at least 1 section rostral and 1 section caudal to the stained neurons. Visual inspection of the stained sections determined that the blocks from 14 subjects contained the entire VIP-ergic SCN and the blocks from 14 subjects contained the entire AVP-ergic SCN (17 total subjects).
Stereological Cell Counts Neuron counts of the SCN for each series were obtained using the optical fractionator stereological method, as previously described.9 To avoid bias in assigning borders to the cell group, we counted all immunoreactive neurons in the region bounded ventrally by the optic chiasm, medially by the third ventricle, dorsally by the paraventricular nucleus, and laterally by the supraoptic nucleus. Photomicrographs presented in Figure 1 were prepared using Photoshop CS6 (Adobe Systems, San Jose, CA). To show differences in distribution of the VIP-ir and AVP-ir neurons, minor adjustments to brightness and contrast were implemented to match the backgrounds of both photomicrographs, as the different antibodies used did not stain the tissue to the same darkness.
Results Clinical Characteristics The 17 subjects with actigraphic recordings
