Abstract
Degeneration of the cholinergic basal forebrain is implicated in the development of cognitive deficits and sleep/wake architecture disturbances in mild cognitive impairment (MCI) and Alzheimer’s disease (AD). Indirect-acting muscarinic cholinergic receptor agonists, such as acetylcholinesterase inhibitors (AChEIs), remain the only FDA-approved treatments for the cognitive impairments observed in AD that target the cholinergic system. Novel direct-acting muscarinic cholinergic receptor agonists also improve cognitive performance in young and aged preclinical species and are currently under clinical development for AD. However, little is known about the effects of direct-acting muscarinic cholinergic receptor agonists on disruptions of sleep/wake architecture and arousal observed in nonpathologically aged rodents, nonhuman primates, and clinical populations. The purpose of the present study was to provide the first assessment of the effects of the direct-acting M1/M4-preferring muscarinic cholinergic receptor agonist xanomeline on sleep/wake architecture and arousal in young and nonpathologically aged mice, in comparison with the AChEI donepezil, when dosed in either the active or inactive phase of the circadian cycle. Xanomeline produced a robust reversal of both wake fragmentation and disruptions in arousal when dosed in the active phase of nonpathologically aged mice. In contrast, donepezil had no effect on either age-related wake fragmentation or arousal deficits when dosed during the active phase. When dosed in the inactive phase, both xanomeline and donepezil produced increases in wake and arousal and decreases in nonrapid eye movement sleep quality and quantity in nonpathologically aged mice. Collectively, these novel findings suggest that direct-acting muscarinic cholinergic agonists such as xanomeline may provide enhanced wakefulness and arousal in nonpathological aging, MCI, and AD patient populations.
Keywords: cholinergic, Alzheimer’s disease, EEG, arousal, xanomeline, donepezil, nonpathological aging, sleep
Introduction
Basal forebrain cholinergic degeneration has been identified as an important factor in the clinical symptomatology of mild cognitive impairment (MCI) and Alzheimer’s disease (AD).1−4 Previous studies have demonstrated decreases in cortical and hippocampal cholinergic markers in aging and AD patient populations.5−9 In addition, preclinical and clinical studies have reported that reductions in cholinergic markers correlate with deficits in cognitive performance.10−12 Moreover, the cholinergic system has been shown to be crucial in modulating normal sleep/wake architecture and arousal with basal forebrain and brainstem cholinergic projections regulating wakefulness and rapid eye movement (REM) sleep.13−16 Given the importance of the cholinergic system in sleep/wake architecture control, age- and AD-related cholinergic degeneration have also been implicated in sleep/wake architecture and arousal deficits.17−19
Boosting cortical cholinergic signaling through indirect-acting muscarinic cholinergic receptor agonists such as acetylcholinesterase inhibitors (AChEIs) is the primary treatment for cognitive impairments in AD.20 The mechanism of action for AChEIs is through the prevention of the breakdown of synaptic acetylcholine (ACh).21 However, AChEIs produce only modest clinical efficacy due to progressive AD-related reductions in basal forebrain cholinergic synaptic signaling.22,23 AChEIs are also associated with numerous dose-limiting side effects, including nausea and diarrhea resulting from nonselective activation of peripheral muscarinic ACh receptors (mAChRs).24,25 Of the five different mAChR subtypes that are activated by ACh (M1–M5), M1 and M4 are highly expressed in cortical and limbic regions thought to be associated with arousal and cognition,26−28 whereas M2 and M3 display central and peripheral expression and are linked to the peripherally mediated adverse effects of AChEIs.29
As an alternative to AChEIs, multiple studies have investigated the effects of direct-acting muscarinic cholinergic receptor agonists that target the M1 and/or M4 mAChR subtypes for the treatment of impaired arousal and cognition in MCI and AD.30−32 For example, in one large multicenter trial, the M1/M4-preferring muscarinic cholinergic receptor agonist xanomeline produced significant effects on the behavioral disturbances in AD with a trend toward improvement in cognition.31,32 However, xanomeline, similar to other direct-acting muscarinic cholinergic receptor agonists, failed during clinical development due to dose-limiting adverse effects from the activation of peripheral mAChR subtypes.33 Recent clinical studies indicate that formulation of xanomeline with the peripherally restricted nonselective muscarinic receptor antagonist trospium, known as KarXT, may provide a broader therapeutic index for the use of direct-acting muscarinic cholinergic agonists34 (ClinicalTrials.gov: NCT03697252, NCT04659161, and NCT05511363).
While accumulating evidence supports the further clinical development of direct-acting muscarinic cholinergic receptor agonists, there have been limited studies to evaluate the effects of this mechanism on disruptions in sleep–wake architecture and/or arousal in nonpathological aging, MCI, and AD patient populations. To date, previous electroencephalography (EEG) studies by our group and others have investigated the effects of indirect- and direct-acting muscarinic cholinergic receptor agonists on the promotion of cholinergic signaling and subsequent changes in sleep/wake architecture and arousal during the inactive phase of young rodents35−38 when basal ACh levels are low.39 When dosed in young rats during the inactive phase, both the AChEI donepezil and xanomeline promoted wakefulness and/or increased gamma power during wake,35−38 a well-characterized correlate of arousal.40 Consistent with these findings, AChEIs have been reported to boost arousal in AD patients, as shown by a significant shift from low to high spectral power.41 However, AChEIs have also been shown to produce robust sleep disruptions in individuals with AD.24,42 In a recent meta-analysis of clinical studies in both healthy and AD patient populations, donepezil reduced stage 2 non-REM (NREM) sleep, sleep efficiency, and total sleep time.43 We have also demonstrated in young rats that donepezil dose-dependently decreases delta power (SWA; slow wave activity) during NREM sleep, a recognized measure of NREM sleep quality.35 Studies with the direct-acting muscarinic cholinergic agonist RS-86 revealed reductions in NREM sleep duration in healthy participants,44 while xanomeline decreased NREM sleep duration and delta power (SWA) during NREM sleep in young rats when dosed in the inactive phase.37 Yet, despite these studies in young preclinical species and healthy volunteers, little is known about the effects of direct-acting muscarinic cholinergic agonists on arousal and sleep–wake architecture in nonpathologically aged rodents or clinical populations across the circadian cycle.
Given the changes in ACh signaling across the circadian cycle (i.e., high in the active phase and low in the inactive phase39) and with aging (i.e., age-related reductions39), the current studies provide the first systematic assessment of the effects of xanomeline, in comparison with donepezil, on sleep/wake architecture, arousal, and sleep quality in aged and young mice across the circadian rhythm. We observed that aged mice displayed pronounced wake fragmentation and reductions in arousal during the active phase that could be reversed by xanomeline but not donepezil when dosed in the active phase. These studies provide the foundation for future sleep/wake architecture and spectral power assessments with xanomeline and other direct-acting muscarinic cholinergic receptor agonists in disease models of AD.
Results and Discussion
Nonpathologically Aged Mice Displayed Fragmentation of Wakefulness and Decreased REM Sleep during the Active Phase and Decreased NREM Sleep in the Inactive Phase
To understand the changes in sleep–wake architecture associated with nonpathological aging, we compared the baseline sleep–wake architecture and spectral power characteristics during saline vehicle treatment in young (3–4 months old) and aged (19–20 months old) C57B6/J mice from both the xanomeline and donepezil dosing studies. During the active (lights off) phase, we observed no change in wake duration on posthoc analyses [2 h bins: age, p = 0.0571; time, p < 0.0001; age × time, p = 0.0008 (Figure 1A) and 12 h bins: age, p = 0.0571; time, p < 0.0001; age × time, p = 0.0771 (Figure 1D)], an age-related increase in NREM sleep at ZT 16 (Zeitgeber time, where ZT 0 is the transition from lights off to on) (age, p = 0.1100; time, p < 0.0001; age × time, p = 0.0001) (Figure 1B) with no change in total NREM (age, p = 0.1100; time, p < 0.0001; age × time, p = 0.0107) (Figure 1E), and an age-related decrease in REM sleep at ZT 18 and 20 (age, p = 0.0260; time and age × time, p < 0.0001) (Figure 1C) with an overall decrease in REM sleep observed (age, p = 0.0260; time p < 0.0001; age × time, p = 0.1397) (Figure 1F). When assessing fragmentation of wakefulness during the active phase, we observed that nonpathologically aged mice displayed increased wake bout number (p < 0.0001) (Figure 2A) and decreased wake bout duration (p < 0.0017) (Figure 2B) and consequently displayed increased NREM sleep bout number (p < 0.0001) (Figure 2C) and decreased NREM sleep bout duration (p = 0.0001) (Figure 2D). During the inactive (lights on) phase, we observed increased wake at ZT 0 (age, p = 0.0571; time, p < 0.0001; age × time, p = 0.0008) (Figure 1A) with no overall change in wake (age, p = 0.0571; time, p < 0.0001; age × time, p = 0.0771) (Figure 1D), a decrease in NREM sleep at ZT 0 (age, p = 0.1100; time and age × time p = 0.0001) (Figure 1B) with an overall decrease in NREM sleep (age, p = 0.1100; time, p < 0.0001; age × time, p = 0.0107) (Figure 1E), and no change REM sleep (age, p = 0.0260; time p < 0.0001; age × time, p = 0.1397) (Figure 1F). During the inactive phase, no change in wake or NREM bout number or duration was observed (Supporting Information Table S1) (see Supporting Information Table S2 for full statistical analysis).
Figure 1.
Nonpathologically aged mice displayed reduced NREM sleep during the inactive phase and reduced REM sleep during the active phase. Shown is the duration of time in wake (A,D), NREM sleep (B,E), and REM sleep (C,F) in young (3–4 month-old) and nonpathologically aged (19–20 month-old) mice. Compared to young mice, nonpathologically aged mice displayed significantly increased wake at ZT 0, with no overall change in either phase (A,D). Nonpathologically aged mice displayed significantly increased NREM sleep at ZT 16 and significantly decreased NREM sleep at ZT 0, with a significantly decreased total NREM sleep during the inactive phase (B,E). Nonpathologically aged mice displayed a significantly decreased REM sleep at ZT 18 and 20 (C) and a significantly decreased total REM sleep between ZT 12 and ZT 24 (F). Data are expressed as means ± S.E.M. of 2 h bins (A–C); total duration of time in minutes in wake, NREM sleep, and REM sleep, respectively, ±S.E.M in 12 h bins (D–F); n = 28/group; open circles indicate p < 0.05 (C), * indicates p < 0.05, **p < 0.01, compared to young [repeated measures (RM) 2-way ANOVA matching by time followed by Sidak’s test]. See Supporting Information Table S2 for full statistical analysis.
Figure 2.
Nonpathologically aged mice displayed fragmented wakefulness during the active phase. Shown are the wake bout number (A), wake bout duration (B), NREM sleep bout number (C), and NREM sleep bout duration (D) in young and nonpathologically aged mice. Nonpathologically aged mice displayed increased wake bout number (A), reduced wake bout duration (B), increased NREM sleep bout number (C), and reduced NREM bout duration (D). Data are expressed as overall means ± S.E.M., n = 28/group. ** indicates p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to young (unpaired t-test). See Supporting Information Table S2 for full statistical analysis.
Nonpathologically Aged Mice Display Reduced Arousal during the Active Phase
An assessment of qEEG (quantitative EEG) spectral changes during each sleep state (wake, NREM sleep, and REM sleep) was performed to understand the deficits observed with nonpathological aging. During the active and inactive phases, a shift to lower powers was observed during wake (active: age, p = 0.0006; frequency and age × frequency, p < 0.0001; inactive: age, p = 0.1383; frequency and age × frequency, p < 0.0001) (Figure 3A,E). During the active phase, a consistent reduction in gamma power, a correlate of arousal was also observed (age, p < 0.0001; time and age × time interaction, both p = 0.0070) (Figure 3C); however, no change was seen during the inactive phase (age, p = 0.4365; time and time × age interaction, both p = 0.7044) (Figure 3H). During NREM sleep in the active phase, there was an increase in gamma power (age, p = 0.1320; frequency and age × frequency, p < 0.0001) (Figure 3B), with increased delta power (SWA) during NREM sleep in aged mice at the start of the active phase (age, p = 0.5943; time and age × time, p < 0.0001) (Figure 3D). During the inactive phase, there were no changes during NREM sleep (age, p = 0.2606; frequency and age × frequency, p < 0.0001) (Figure 3D) with no changes in delta power (SWA) during NREM sleep (age, p = 0.09673; frequency and age × frequency, p < 0.0001) (Figure 3I). During REM sleep, changes were observed in gamma power (age, p = 0.7072; frequency and age × frequency, p < 0.0001) (Figure 3G).
Figure 3.
Nonpathologically aged mice displayed reduced arousal in the active phase compared to young mice. Shown is the relative spectral power in nonpathologically aged (19–20 month) mice normalized to young (3–4 month) mice from 0.5 to 80 Hz during wake (A,E), NREM sleep (B,F), and REM sleep (G) during ZT 1–2 (drug baseline) in the active phase (A,B) and the inactive phase (E–G). Also shown are relative gamma power during wake normalized to young mice in 1 h bins (C,H) and relative delta power (SWA activity, 0.5–4 Hz) during NREM sleep normalized to young mice in 1 h bins (D,I) in young and nonpathologically aged mice across the active phase (C,D) and the inactive phase (H,I). During the active phase, nonpathologically aged mice displayed a main effect of age and an age × frequency interaction on relative spectral power during wake with a reduction in relative power in alpha and gamma frequencies (A). During NREM sleep, nonpathologically aged mice displayed an age × frequency interaction with an increase in relative gamma power (B). Nonpathologically aged mice showed an age-related reduction in gamma power across the active phase (C) and no change in delta power (SWA) during NREM sleep (D). In the inactive phase, nonpathologically aged mice displayed an age × frequency interaction with increased relative delta and reduced relative alpha power during wake (E), while during NREM sleep, no age-related changes were observed (F). During REM sleep, an age × frequency interaction was observed and nonpathologically aged mice displayed increased relative power at 34 and 76–79 Hz and reduced relative power at 54 and 56–59 Hz (G). During wake, no change in gamma power was seen (H), and no change in delta power (SWA) during NREM sleep was observed (I). Gray/tan shading represents frequency bands (Δ, delta 0.5–4 Hz; θ theta 4–8 Hz; α alpha, 8–13 Hz; β beta, 13–30 Hz; γ gamma 30–80 Hz). Data are expressed as means ± S.E.M. in 1 Hz bins (A,B,E–G) and means ± S.E.M. in 1 h bins (C,D,H,I), n = 27–28/group. Solid bars at the bottom of the graph indicate p < 0.05 compared to young (A,B,E,G). Open circles indicate p < 0.05 (C,D) (all RM 2-way ANOVA matching by time followed by Sidak’s test). See Supporting Information Table S2 for full statistical analysis.
Xanomeline Promoted Wake and Reversed Wake Fragmentation in Nonpathologically Aged Mice in the Active Phase
To examine the effects of the direct-acting M1/M4-preferring orthosteric mAChR agonist xanomeline on sleep/wake architecture in young and nonpathologically aged mice, we dosed mice with the vehicle or xanomeline 2–3 h into the active phase. Young mice displayed increased wake at ZT 18 following administration with the 10 mg/kg dose of xanomeline, while the 30 mg/kg dose of xanomeline increased wake at ZT 14 and 0 with a rebound decreased wake at ZT 20 (dose, time, and dose × time interaction, all p < 0.0001) (Figure 4A). Both the 3 and 10 mg/kg doses of xanomeline produced increased total wake from ZT 12–24, and 30 mg/kg produced increased total wake from ZT 0–12 (dose and time, both p < 0.0001; dose × time interaction, p = 0.0002) in the young mice (Figure 4D). Due to the increased wake, subsequent decreased NREM sleep was seen at ZT 18 with the 10 mg/kg dose of xanomeline and at ZT 14 and 0 with the 30 mg/kg dose of xanomeline in the young mice. A rebound increased NREM sleep was observed at ZT 20 with the 30 mg/kg dose of xanomeline (dose, time, and dose × time interaction; all p < 0.0001) (Figure 4B). There was decreased total NREM sleep at all doses of xanomeline from ZT 12–24 and decreased total NREM sleep at the 30 mg/kg dose of xanomeline from ZT 0–12 (dose and time, both p < 0.0001; dose × time interaction, p = 0.0012) in the young mice (Figure 4E). The 3 mg/kg dose of xanomeline increased REM sleep at ZT 0, and the 30 mg/kg dose of xanomeline increased REM sleep at ZT 20 (dose, p = 0.1296; time and dose × time interaction, both p < 0.0001) in the young mice (Figure 4C). Increased total REM sleep was observed from ZT 12–24 following dosing with the 30 mg/kg dose of xanomeline and from ZT 0–12 at the 3 mg/kg dose of xanomeline (dose, p = 0.1296; time and dose × time interaction, both p < 0.0001) in the young mice (Figure 4F).
Figure 4.
Xanomeline displayed wake promotion in the active phase in young and nonpathologically aged mice. Shown is the duration of time spent in wake (A,D,G,J), NREM sleep (B,E,H,K), and REM sleep (C,F,I,L) in young (A–F) and nonpathologically aged (G–L) mice following xanomeline administration 2 h into the active phase (see arrowhead). In young mice, 30 mg/kg xanomeline produced an initial increase in wake, followed by a rebound decrease in wake, while 10 mg/kg xanomeline produced an increased wake (A). Increased total wake over the 12 h of the active phase was observed at 3 and 10 mg/kg, while 30 mg/kg produced increased total wake in the subsequent inactive phase (D). 30 mg/kg xanomeline produced decreased NREM sleep following dosing with a rebound increased NREM sleep, and 10 mg/kg produced decreased NREM sleep (B); 3–30 mg/kg produced reduced NREM sleep during the active phase with the effects at 30 mg/kg, extending into the subsequent inactive phase (E). 30 mg/kg xanomeline increased REM sleep following dosing (C), with an overall increase in REM sleep observed during the active phase following dosing with 30 mg/kg xanomeline, while 3 mg/kg xanomeline produced increased REM during the subsequent inactive phase (F). In nonpathologically aged mice, 30 mg/kg xanomeline produced increased wake and 3 mg/kg produced reduced wake following dosing (G); 3 mg/kg produced reduced total wake in the active and subsequent inactive phases, and 10 mg/kg produced decreased wake in the inactive phase (J). 30 mg/kg reduced NREM sleep following dosing, and 3 mg/kg increased NREM sleep following dosing, with 3 and 10 mg/kg producing increased NREM sleep across the inactive phase (H). This resulted in an increased total NREM sleep at 3 and 10 mg/kg in the inactive phase following active phase dosing (K). Xanomeline had no effect on REM sleep immediately following active-phase dosing, but all doses reduced REM sleep in the inactive phase (I,L). Data are expressed as means ± S.E.M. of 2 h bins (A–C,G–I); open symbols indicate p < 0.05 compared to vehicle (2-way ANOVA matching by both factors followed by Dunnett’s test), or 12 h bins (D–F,J–L) * indicates p < 0.05, **p < 0.01, and ****p < 0.0001 compared to the vehicle (RM 1-way ANOVA followed by Dunnett’s test), n = 14/group; see Supporting Information Table S2 for full statistical analysis.
In nonpathologically aged mice, the 3 mg/kg dose of xanomeline decreased wake at ZT 14, with decreased wake also observed prior to dosing at ZT 12; while the 10 mg/kg dose of xanomeline decreased wake at ZT 10. In nonpathologically aged mice, the 30 mg/kg dose of xanomeline produced a more extended increase in wake at ZT 14 and 16 than observed in young mice. In addition, rebound decreased wake was observed at ZT 20 when xanomeline was dosed at 30 mg/kg (dose, time, and dose × time interaction, all p < 0.0001) in the nonpathologically aged mice (Figure 4G). Total wake from ZT 12–24 was reduced following administration of a 3 mg/kg dose of xanomeline, and total wake from ZT 0–12 was reduced following dosing with 3 and 10 mg/kg doses of xanomeline (dose and time, both p < 0.0001; dose × time interaction, p = 0.6602) in the nonpathologically aged mice (Figure 4J). The 3 mg/kg dose of xanomeline produced increased NREM sleep at ZT 14, 0, and 2 following dosing, with decreased NREM sleep at ZT 20. Xanomeline dosed at 10 mg/kg increased NREM sleep at ZT 0, 2, 6, and 10, with increased NREM also observed prior to dosing at ZT 12 in the nonpathologically aged mice. The 30 mg/kg dose of xanomeline produced decreased NREM sleep following dosing at ZT 14 and 16 (dose, time, and dose × time interaction, all p < 0.0001) (Figure 4H). The 3 and 10 mg/kg doses of xanomeline increased total NREM sleep from ZT 0–12 (dose and time, both p < 0.0001; dose × time interaction, p = 0.1754) in the nonpathologically aged mice (Figure 4K). The dose of 10 mg/kg of xanomeline reduced REM sleep at ZT 4, while 30 mg/kg dose of xanomeline reduced REM sleep at ZT 2 (dose, p = 0.3523; time, p < 0.0001 and dose × time interaction, p = 0.0035) (Figure 4I). All doses of xanomeline (3, 10, and 30 mg/kg) reduced total REM sleep from ZT 0–12 (dose, p = 0.3523; time, p < 0.0001; dose × time interaction, p = 0.0033) in the nonpathologically aged mice (Figure 4L).
Given the wake-promoting effects of xanomeline and age-related increase in wake fragmentation, we investigated the effects of xanomeline on wake fragmentation during the active phase in young and nonpathologically aged mice. Xanomeline had no effect on the wake bout number (no main effect of dose, p = 0.6576) or wake bout duration (no main effect of dose, p = 0.1084) from ZT 14–22 in young mice (Figure 5A,B). However, in nonpathologically aged mice, all doses of xanomeline reduced the wake bout number (main effect of dose, p < 0.0001), and the 30 mg/kg dose of xanomeline increased wake bout duration (main effect of dose, p < 0.0001) from ZT 14–22 (Figure 5C,D). Similar effects were observed on NREM sleep bout duration and number: xanomeline had no effect in young mice (NREM sleep bout duration, p = 0.3031; NREM sleep bout number, p = 0.3956) (Figure 6A,B), while in nonpathologically aged mice, all doses of xanomeline produced a decreased NREM bout number (main effect of dose, p < 0.0001) and increased NREM bout duration (main effect of dose, p < 0.0001) (Figure 6C,D).
Figure 5.
Xanomeline reduced the wake bout number and increased wake bout duration during the active phase in nonpathologically aged mice. Shown is the average wake bout number (A,C) and the average wake bout duration (B,D) in young (A,B) and nonpathologically aged (C,D) mice during the 8 h following dosing in the active phase. Xanomeline has no effect on the wake bout number (A) or duration (B) when dosed in the active phase in young mice. In nonpathologically aged mice, xanomeline dose-dependently reduced wake bout number (C) and increased wake bout duration (D). Data are expressed as overall means ± S.E.M., n = 14/group. ** indicates p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the vehicle (RM 1-way ANOVA followed by Dunnett’s test). See Supporting Information Table S2 for full statistical analysis.
Figure 6.
Xanomeline reduced the NREM bout number and increased NREM bout duration in the active phase in nonpathologically aged mice. Shown are the average NREM sleep bout number (A,C) and the average NREM sleep bout duration (B,D) in young (A,B) and nonpathologically aged (C,D) mice for 8 h following dosing in the active phase. Xanomeline had no effect on the NREM sleep bout number or duration when dosed in the active phase in young mice (A,B). In nonpathologically aged mice, xanomeline dose-dependently reduced the NREM sleep bout number (C) and increased NREM sleep bout duration (D). Data are expressed as overall means ± S.E.M., n = 14/group. ** indicates p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the vehicle (RM 1-way ANOVA followed by Dunnett’s test). See Supporting Information Table S2 for full statistical analysis.
Donepezil Had No Effect on Wake in Nonpathologically Aged Mice during the Active Phase
Next, we assessed the effects of donepezil, an AChEI approved for the treatment of cognitive impairments in AD, on sleep/wake architecture. In young mice, the 0.1 mg/kg dose of donepezil increased wake at ZT 18 and the 3 mg/kg dose of donepezil increased wake at ZT 14 with a reduction in wake at ZT 16 (dose, p = 0.3154; time, p < 0.0001; dose × time interaction, p = 0.0003) (Figure 7A) and had no effect on the overall wake from ZT 12–24 or ZT 0–12 (dose, p = 0.3154; time, p < 0.0001; dose × time interaction, p = 0.2740) (Figure 7D). In young mice, the 0.1 mg/kg dose of donepezil decreased NREM sleep at ZT 18, and the 3 mg/kg dose of donepezil decreased NREM sleep at ZT 14 with an increase in NREM sleep at ZT 16. Additionally, increased NREM sleep was seen at the 1 mg/kg dose of donepezil prior to dosing (dose, p = 0.4587; time and dose × time interaction, both p = 0.0001) (Figure 7B), with no effect on total NREM sleep observed at any dose from ZT 12–24 or 0–12 (dose, p = 0.4587; time, p < 0.0001; dose × time interaction, p = 0.1824) (Figure 7E). In young mice, the 0.1 and 0.3 mg/kg doses of donepezil decreased REM sleep at ZT 18 (dose, p = 0.2762; time, p < 0.0001; dose × time interaction, p = 0.0216) (Figure 7C), with no effect observed on overall REM sleep from ZT 12–24 and ZT 0–12 (dose, p = 0.2762; time, p < 0.0001; dose × time interaction, p = 0.4230) (Figure 7F).
Figure 7.
Donepezil had no effect on wake in the active phase in nonpathologically aged mice. Shown is the duration of time spent in wake (A,D,G,J), NREM sleep (B,E,H,K), and REM sleep (C,F,I,L) in young (A–F) and nonpathologically aged (G–L) mice following donepezil administration 2 h into the active phase (see the arrowhead). In young mice, 3 mg/kg of donepezil produced increased wake followed by a reduction in wake (A), with no effect on overall wake in the active phase (D). NREM sleep decreased following dosing with 3 mg/kg of donepezil before increasing (B) and no effect on overall NREM sleep during the active phase (E). 0.1 and 0.3 mg/kg donepezil reduced REM sleep following dosing (C), with no effect on overall REM sleep (F). In nonpathologically aged mice, there was no dose-related effect observed on wake (G,J) or NREM sleep (H,K). A dose-related increase in REM sleep was observed with 1 and 3 mg/kg donepezil (I), although no effect on total REM sleep was seen (L). Data are expressed as means ± S.E.M. of 2 h bins (A–C,G–I); open symbols indicate p < 0.05 compared to the vehicle (2-way ANOVA matching by both factors followed by Dunnett’s test) or 12 h bins (D–F,J–L), n = 14/group; see Supporting Information Table S2 for full statistical analysis.
In nonpathologically aged mice, donepezil had no effect on wake or NREM sleep when assessed as 2 h epochs (wake: dose, p = 0.5692; time, p < 0.0001; dose × time interaction, p = 0.0002; NREM: dose, p = 0.6304; time, p < 0.0001; dose × time interaction, p = 0.0002) (Figure 7G,H) or as the total amount of wake or NREM sleep, respectively, from ZT 12–24 or ZT 0–12 (wake: dose, p = 0.5692; time, p < 0.0001; dose × time interaction, p = 0.1623; NREM: dose, p = 0.6304; time, p < 0.0001; dose × time interaction, p = 0.2095) (Figure 7J,K). In nonpathologically aged mice, the 1 mg/kg dose of donepezil increased REM sleep at ZT 16 and 18, and the 3 mg/kg dose of donepezil increased REM sleep at ZT 20 (dose, p = 0.0033; time, p < 0.0001; dose × time interaction p = 0.0008) (Figure 7I), with a main effect of donepezil on total REM sleep between ZT 12–24 and ZT 0–12 but no effect at any specific dose on posthoc analysis (dose, p = 0.0033; time, p < 0.0001; dose × time interaction, p = 0.1763) (Figure 7L).
When assessing wake bout fragmentation following dosing in the active phase in young and nonpathologically aged mice, donepezil produced no effect on the wake bout number or average wake bout duration. However, dosing donepezil at 3 mg/kg in young mice during the active phase increased the NREM sleep bout number with no effect on NREM sleep bout duration. In nonpathologically aged mice, donepezil had no effect on the NREM sleep bout number, with a significant overall effect of dose on NREM sleep bout duration but no significant effect at any dose following posthoc analysis (Table 1).
Table 1. Donepezil Increased the NREM Bout Number in Young Mice in the Active Phase.
| young |
aged |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| wake |
NREM |
wake |
NREM |
||||||
| bout number (SEM), ZT 12–14 | average bout duration, ZT 12–14 (s) (SEM) | bout number (SEM), ZT 12–14 | average bout duration, ZT 12–14 (s) (SEM) | bout number (SEM), ZT 12–14 | average bout duration, ZT 12–14 (s) (SEM) | bout number (SEM), ZT 12–14 | average bout duration, ZT 12–14 (s) (SEM) | ||
| donepezil (mg/kg) | vehicle | 97.86 (5.384) | 194.0 (12.34) | 87.93 (4.686) | 117.0 (7.916) | 124.6 (12.32) | 166.1 (21.20) | 118.2 (12.47) | 100.4 (11.81) |
| 0.1 | 106.9 (3.741) | 172.1 (9.948) | 100.6 (4.046) | 102.7 (3.714) | 119.4 (13.12) | 203.1 (48.01) | 112.4 (12.56) | 105.5 (11.06) | |
| 0.3 | 97.93 (3.679) | 190.0 (8.692) | 89.14 (3.430) | 113.8 (6.711) | 136.5 (13.07) | 149.7 (17.10) | 130.3 (12.97) | 87.93 (9.329) | |
| 1 | 104.2 (5.233) | 183.8 (13.85) | 100.6 (5.366) | 101.1 (4.077) | 108.3 (11.22) | 174.1 (21.63) | 104.3 (10.63) | 123.0 (9.635) | |
| 3 | 107.6 (4.579) | 160.2 (8.768) | 105.1 (4.371)a | 109.3 (4.910) | 122.0 (8.971) | 147.1 (15.45) | 120.1 (9.290) | 101.2 (6.153) | |
| 1-way ANOVA | F4,52 = 1.263 | F4,52 = 2.244 | F4,52 = 3.765 | F4,52 = 2.241 | F4,52 = 9.779 | F4,52 = 0.8950 | F4,52 = 0.9223 | F4,52 = 2.805 | |
| P value | 0.2963 | 0.0768 | 0.0092 | 0.0772 | 0.4277 | 0.4737 | 0.4581 | 0.0349 | |
p < 0.05, Dunnett’s multiple comparisons compared to the vehicle condition.
Xanomeline and Donepezil Promoted Wake When Dosed in the Inactive Phase
Given the wake-promoting effects of xanomeline, we next assessed whether xanomeline and donepezil would be disruptive to sleep when dosed in the inactive phase. Xanomeline dose-dependently increased wake in young and nonpathologically aged mice when dosed in the inactive phase. In young mice, xanomeline produced the following dose-related changes in wake: 3 mg/kg dose of xanomeline increased wake at ZT 4, 10 mg/kg dose of xanomeline increased wake at ZT 2 and 4, with rebound reductions observed at ZT 18, and the 30 mg/kg dose of xanomeline increased wake at ZT 2 and 4 with rebound decreased wake seen at ZT 14, 16, and 18 (dose, p = 0.0006; time and dose × time interaction, both p < 0.0001) (Figure 8A). There was also an increase in total wake observed over 12 h from ZT 0–12 after the 10 and 30 mg/kg doses of xanomeline and reduced total wake observed from ZT 12–24 with the 30 mg/kg dose of xanomeline (dose, p = 0.0006; time, p < 0.0001 and dose × time interaction, both p < 0.0001) (Figure 8D). Consistent with wake promotion, NREM and REM sleep were reduced following dosing in the young mice. Xanomeline produced the following dose-related changes in NREM: the dose of 3 mg/kg of xanomeline reduced NREM sleep at ZT 4, the 10 mg/kg dose of xanomeline reduced NREM sleep at ZT 2 and 4 with rebound increased NREM sleep at ZT 18, and the 30 mg/kg dose of xanomeline decreased NREM sleep at ZT 2 and 4 with rebound increased NREM sleep observed at ZT 14, 16, and 18. Additionally, increased NREM sleep was observed at ZT 2 with 3 mg/kg xanomeline (dose, p = 0.0006; time and dose × time interaction, both p < 0.0001) in the young mice (Figure 8B). Total NREM sleep was decreased between ZT 0 and 12 following dosing with 10 and 30 mg/kg doses of xanomeline, and NREM sleep was increased between ZT 12 and 24 following dosing with the 30 mg/kg dose of xanomeline (dose, p = 0.0006; time and dose × time interaction, both p < 0.0001) (Figure 8E). Xanomeline produced the following dose-related changes in REM in young mice: the 10 mg/kg dose of xanomeline decreased REM sleep at ZT 2 and the 30 mg/kg dose of xanomeline decreased REM sleep at ZT 2 and 4 with a rebound increased REM sleep seen at ZT 8, 14, 18, and 20 (dose, p = 0.7367; time and dose × time interaction, both p < 0.0001) (Figure 8C). Reduced total REM sleep was observed with the 30 mg/kg dose of xanomeline between ZT 0 and 12 with a rebound increased total REM sleep between ZT 12 and 24 (p = 0.7367; time and dose × time interaction, both p < 0.0001) in the young mice (Figure 8F).
Figure 8.
Xanomeline increased wakefulness in the inactive phase in young and nonpathologically aged mice. Shown is the duration of time spent in wake (A,D,G,J), NREM sleep (B,E,H,K), and REM sleep (C,F,I,L) in young (A–F) and nonpathologically aged (G–L) mice following xanomeline administration 2 h into the inactive phase (see the arrowhead). In young mice, 10 and 30 mg/kg xanomeline produced increased wake with a subsequent rebound decreased wake in the active phase, and 3 mg/kg produced a transient wake increase (A), with increased total wake over the 12 h of the inactive phase observed at 10 and 30, and 30 mg/kg also produced decreased wake in the subsequent active phase (D). 10 and 30 mg/kg xanomeline produced decreased NREM sleep following dosing, with a rebound increase observed in the subsequent active phase, and 3 mg/kg produced increased NREM sleep at ZT 2 and decreased NREM sleep at ZT 4 (B), 10 and 30 mg/kg produced reduced NREM sleep during the inactive phase with increased NREM sleep observed at 30 mg/kg in the subsequent active phase (E). 10 and 30 mg/kg xanomeline decreased REM sleep following dosing, with rebound increased REM sleep seen in the 30 mg/kg group (C). An overall decrease in total REM sleep was seen during the inactive phase following dosing with 30 mg/kg xanomeline, and increased total REM sleep was observed in the subsequent active phase (F). In nonpathologically aged mice, 10 and 30 mg/kg xanomeline produced increased wake following dosing with a rebound decreased wake in the subsequent active phase (G); 10 and 30 mg/kg xanomeline increased total wake in the inactive phase and reduced total wake in the subsequent active phase (J). 10 and 30 mg/kg xanomeline reduced NREM sleep following dosing with a subsequent rebound increased NREM sleep in the active phase (H). This resulted in a decreased total NREM sleep at 10 and 30 mg/kg in the inactive phase and increased NREM sleep at all doses in the subsequent active phase (K). Xanomeline 10 and 30 mg/kg reduced REM sleep following inactive-phase dosing with subsequent rebound increased REM sleep (I). Total REM sleep was decreased in the inactive phase at 10 and 30 mg/kg xanomeline and increased in the subsequent active phase following 10 and 30 mg/kg xanomeline (L). Data are expressed as means ± S.E.M. of 2 h bins (A–C,G–I); open symbols indicate p < 0.05 compared to the vehicle (two-way ANOVA matching by both factors followed by Dunnett’s test) or 12 h bins (D–F,J–L); * indicates p < 0.05, **p < 0.01, and ****p < 0.0001 compared to the vehicle (RM 1-way ANOVA followed by Dunnett’s test), n = 13–14/group; see Supporting Information Table S2 for full statistical analysis.
In nonpathologically aged mice, the following pronounced wake-promoting effects of xanomeline were observed: the 10 mg/kg dose of xanomeline increased wake at ZT 2, with rebound decreased wake at ZT 12, 14, 18, and 20, while the dose of 30 mg/kg of xanomeline increased wake at ZT 2, 4, and 6 with rebound decreased wake seen at ZT 10, 12, 14, 16, 18, and 20 (dose, time, and dose × time interaction, all p < 0.0001) (Figure 8G). Total wake following dosing between ZT 0 and 12 was also increased with the 10 and 30 mg/kg doses of xanomeline, and a rebound decrease in the total wake was observed between ZT 12 and 24 (dose and dose × time interaction, both p < 0.0001; time, p = 0.0024) in the nonpathologically aged mice (Figure 8J). With the observed increased wake in the nonpathologically aged mice following dosing with xanomeline, the following decreases in NREM sleep were seen: the 10 mg/kg dose of xanomeline reduced NREM sleep at ZT 2 with rebound increased NREM sleep seen at ZT 12,14, and 20, while the 30 mg/kg dose of xanomeline decreased NREM sleep at ZT 2 and 4 with rebound increased NREM sleep seen at ZT 12, 14, 16, 18, and 20 (dose, p = 0.0011; time and dose × time interaction; both p < 0.0001) (Figure 8H). Total NREM sleep from ZT 0–12 was reduced following dosing with the 10 and 30 mg/kg doses of xanomeline, while increased NREM sleep from ZT 12–24 was observed at the 3, 10, and 30 mg/kg doses of xanomeline (dose, p = 0.0011; time and dose × time interaction, both p < 0.0001) (Figure 8K) in the nonpathologically aged mice. The following dose-related reductions in REM sleep were also observed with xanomeline in the nonpathologically aged mice: the 10 mg/kg dose of xanomeline reduced REM sleep at ZT 2 and 4, with rebound increased REM sleep observed at ZT 14, 16, 18, and 20, while the 30 mg/kg dose of xanomeline reduced REM sleep at ZT 2, 4, 6, and 8 with rebound increased REM sleep observed at ZT 10, 12, 14, 16, 18, and 20 (dose, p = 0.5442; time and dose × time interaction, both p < 0.0001) (Figure 8L). Total REM sleep from ZT 0–12 was reduced following dosing with the 10 and 30 mg/kg doses of xanomeline with rebound increased REM sleep seen at both doses from ZT 12–24 (dose, p = 0.5442; time and dose × time interaction, both p < 0.0001) (Figure 8L) in the nonpathologically aged mice.
Similar to xanomeline, donepezil increased wake following dosing in the inactive phase in young and nonpathologically aged mice. In the young mice, the 3 mg/kg dose of donepezil increased wake at ZT 2 with rebound decreases in wake observed at ZT 12, 14, and 20 (dose, p = 0.1761; time and dose × time interaction, both p < 0.0001) (Figure 9A). Total wake was increased following dosing with donepezil at 3 mg/kg between ZT 0 and 12, and reduced wake was observed between ZT 12 and 24 following dosing with the 0.1 and 3 mg/kg doses of donepezil (dose, p = 0.1761; time and dose × time interaction, both p < 0.0001) (Figure 9D) in the young mice. The following dose-related changes in NREM sleep were also observed with donepezil in the young mice: the 0.1 mg/kg dose of donepezil increased NREM sleep at ZT 8, while the 3 mg/kg dose of donepezil decreased NREM sleep at ZT 2 and 4 with a rebound increased NREM sleep at ZT 12 and 20 (dose, p = 0.2016; time and dose × time interaction, both p < 0.0001) (Figure 9B). The total NREM sleep between ZT 0 and 12 was reduced by the 3 mg/kg dose of donepezil, and between ZT 12 and 24 total NREM sleep was increased following dosing with the 0.1 and 3 mg/kg doses of donepezil (dose, p = 0.1761; time and dose × time interaction, both p < 0.0001) (Figure 9E). In the young mice, the following dose-related changes in REM sleep were also observed with donepezil: REM sleep was increased at ZT 4 following dosing with the 0.1 and 3 mg/kg doses of donepezil and reduced following the 3 mg/kg dose of donepezil at ZT 2 with a rebound increased REM sleep at ZT 20 (dose, p = 0.7558; time and dose × time interaction, both p < 0.0001) (Figure 9C). The total REM sleep was unchanged between ZT 0 and 12, and the total REM sleep was modestly increased with the 3 mg/kg dose of donepezil between ZT 12 and 24 (dose, p = 0.1761; time, p < 0.0001; dose × time interaction, p = 0.0417) (Figure 9F).
Figure 9.
Donepezil increased wakefulness in the inactive phase in young and nonpathologically aged mice. Shown is the duration of time spent in wake (A,D,G,J), NREM sleep (B,E,H,K), and REM sleep (C,F,I,L) in young (A–F) and nonpathologically aged (G–L) mice following donepezil administration 2 h into the inactive phase (see arrowhead). In young mice, 3 mg/kg donepezil increased wake following dosing, with decreased wake in the subsequent active phase (A). 3 mg/kg donepezil increased total wake in the inactive phase, and 0.1 and 3 mg/kg donepezil produced decreased total wake in the subsequent active phase (D). 3 mg/kg donepezil reduced NREM sleep following dosing, with subsequent rebound increased NREM sleep (B). 3 mg/kg donepezil produced decreased total NREM sleep in the inactive phase; 0.1 and 3 mg/kg increased NREM sleep in the subsequent active phase (E). 3 mg/kg donepezil reduced REM sleep following dosing, while 0.1 and 1 mg/kg increased REM sleep following dosing (C). There was no change in total REM sleep in the inactive phase following dosing; in the subsequent active phase, increased REM sleep was observed following 3 mg/kg donepezil (F). In nonpathologically aged mice, 3 mg/kg donepezil increased wake following dosing with rebound decreased wake in the subsequent active phase (G), this resulted in increased total wake during the inactive phase following 3 mg/kg donepezil dosing and reduced total wake in the subsequent active phase (J). 3 mg/kg donepezil produced reduced NREM sleep following dosing (H), with a reduction also seen in total NREM sleep during the inactive phase (K). 3 mg/kg donepezil produced an initial reduction in REM sleep, with subsequent rebound increased REM sleep (I); this resulted in increased total REM sleep in the active phase following inactive phase dosing (L). Data are expressed as means ± S.E.M. of 2 h bins (A–C,G–I); open symbols indicate p < 0.05 compared to vehicle (two-way ANOVA matching by both factors followed by Dunnett’s test) or 12 h bins (D–F,J–L) * indicates p < 0.05, **p < 0.01, and ****p < 0.0001 compared to the vehicle (RM one-way ANOVA followed by Dunnett’s test), n = 13–14/group; see Supporting Information Table S2 for full statistical analysis.
In the nonpathologically aged mice, the 3 mg/kg dose of donepezil increased wake at ZT 2 and 4 with a reduced wake at ZT 14 (dose, p = 0.0433; time and dose × time interaction, both p < 0.0001) (Figure 9G). As shown in Figure 9J, the overall wake was increased between ZT 0 and 12 and reduced between ZT 12 and 24 following administration of the 3 mg/kg dose of donepezil (dose, p = 0.0433; time and dose × time interaction, both p < 0.0001). The 3 mg/kg dose of donepezil also reduced NREM sleep at ZT 2 and 4 (dose, p = 0.0090; time and dose × time interaction, both p < 0.0001) (Figure 9H), with overall NREM sleep reductions between ZT 0 and 12 (dose, p = 0.0433; time and dose × time interaction, both p < 0.0001) in the nonpathologically aged mice (Figure 9K). The 3 mg/kg dose of donepezil decreased REM sleep at ZT 2 and 4, with rebound increased REM observed at ZT 10, 14, 16, and 18 in the nonpathologically aged mice. Additionally, an increase in REM sleep was observed at the 1 mg/kg dose of donepezil prior to dosing at ZT 0 (dose, p = 0.0214; time and dose × time interaction, both p < 0.0001) (Figure 9I). As shown in Figure 9L, there was no change in total REM sleep from ZT 0–12 following dosing with donepezil, while the 3 mg/kg dose of donepezil produced an increase in total REM sleep from ZT 12–24 (dose, p = 0.0214; time, p < 0.0001; dose × time interaction, p = 0.0004) in the nonpathologically aged mice (Figure 9L).
As shown in Table 2, when assessing wake and NREM sleep fragmentation following dosing in the inactive phase, xanomeline produced dose-dependent increases in the average wake bout duration in young animals and increased wake and decreased NREM sleep bout duration in nonpathologically aged mice. In comparison, donepezil at the 3 mg/kg dose reduced the wake and NREM sleep bout number and increased average wake and NREM sleep bout duration in young mice, while in the nonpathologically aged mice, this dose of donepezil decreased average NREM sleep bout duration (Table 2).
Table 2. Effects of Xanomeline and Donepezil on Wake and NREM Fragmentation during the Inactive Phase in Young and Nonpathologically Aged Micea .
| young |
aged |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| wake |
NREM |
wake |
NREM |
||||||
| bout number, ZT 2–10 (SEM) | average bout duration, ZT 2–10 (s), (SEM) | bout number, ZT 2–10 (SEM) | average bout duration, ZT 2–10 (s) (SEM) | bout number, ZT 2–10 (SEM) | average bout duration, ZT 2–10 (s), (SEM) | bout number, ZT 2–10 (SEM) | average bout duration, ZT 2–10 (s) (SEM) | ||
| xanomeline (mg/kg) | vehicle | 100.3 (3.552) | 77.83 (3.771) | 101.1 (3.478) | 189.9 (7.645) | 105.9 (3.996) | 79.65 (3.350) | 106.5 (3.517) | 174.4 (7.528) |
| 3 | 101.4 (4.343) | 90.89 (4.846) | 100.7 (4.489) | 179.9 (8.768) | 95.69 (3.834) | 87.63 (4.086) | 98.31 (3.795) | 189.2 (7.952) | |
| 10 | 100.4 (6.360) | 101.1 (7.655)* | 97.86 (6.557) | 180.7 (9.972) | 119.9 (10.75) | 88.35 (9.693) | 120.8 (10.78) | 156.2 (11.45) | |
| 30 | 93.79 (5.345) | 126.0 (8.73)**** | 91.07 (5.538) | 178.7 (9.147) | 96.77 (6.678) | 161.2 (14.95)**** | 95.69 (6.711) | 141.9 (8.663)** | |
| 1-way ANOVA | F3,39 = 0.5026 | F3,39 = 12.42 | F3,39 = 0.8155 | F3,39 = 0.4077 | F3,36 = 3.425 | F3,36 = 21.27 | F3,36 = 3.492 | F3,36 = 8.330 | |
| P value | 0.6827 | <0.0001 | 0.4931 | 0.7484 | 0.0272 | <0.0001 | 0.0253 | 0.0002 | |
| donepezil (mg/kg) | vehicle | 107.7 (3.864) | 65.90 (3.341) | 106.5 (4.281) | 183.3 (8.886) | 108.7 (5.486) | 73.65 (4.136) | 108.8 (5.634) | 181.2 (9.980) |
| 0.1 | 98.46 (5.297) | 69.40 (3.512) | 99.08 (5.317) | 204.0 (11.36) | 116.1 (8.822) | 72.66 (4.509) | 116.6 (8.723) | 172.1 (10.98) | |
| 0.3 | 106.4 (17.99) | 67.25 (3.278) | 106.5 (5.021) | 184.0 (9.716) | 119.9 (9.167) | 71.87 (4.036) | 121.5 (8.928) | 164.5 (11.68) | |
| 1 | 96.62 (5.424) | 72.83 (3.602) | 95.23 (5.021) | 209.1 (12.43) | 109.6 (8.478) | 75.43 (4.153) | 110.9 (8.528) | 182.8 (12.57) | |
| 3 | 86.23 (4.008)** | 91.93 (5.831)**** | 83.46 (3.640)*** | 226.4 (11.75)** | 123.2 (8.584) | 90.99 (8.022) | 122.9 (8.563) | 140.7 (8.501)* | |
| 1-way ANOVA | F4,48 = 4.998 | F4,48 = 7.566 | F4,48 = 6.272 | F4,48 = 4.481 | F4,52 = 0.6461 | F4,52 = 0.2230 | F4,52 = 0.6724 | F4,52 = 3.364 | |
| P value | 0.0019 | <0.0001 | 0.0004 | 0.0037 | 0.6322 | 0.0784 | 0.6141 | 0.0160 | |
*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, Dunnett’s multiple comparisons compared to the vehicle condition.
Xanomeline Increased Arousal in Nonpathologically Aged Mice during the Active Phase, While Donepezil Had No Effect
Next, we evaluated potential state-dependent changes in qEEG with xanomeline treatment in both young and nonpathologically aged mice. In the young mice, the 30 mg/kg dose of xanomeline produced increased theta and reduced alpha power (dose, p = 0.0725; time and dose × time interaction, both p < 0.0001) (Figure 10A). Modest reductions in gamma power during wake following dosing at the 10 and 30 mg/kg doses of xanomeline were also observed in the young mice (dose, p = 0.1824; time p < 0.0001 and dose × time interaction p = 0.0014) (Figure 10C), with general shifts to lower powers, including reductions in beta and alpha powers and increases in theta and delta powers across time (Supporting Information Figure S1). During NREM sleep following dosing with the 30 mg/kg dose of xanomeline, young mice displayed reductions in delta, alpha, and beta powers and increased theta and gamma powers (dose, time, and dose × time interaction, all p < 0.0001) (Figure 10B). Across time, delta power (SWA) during NREM sleep was decreased after administration with the 10 and 30 mg/kg doses of xanomeline, following a transient increase with the 30 mg/kg dose of xanomeline (dose, p = 0.4110; time and dose × time, p < 0.0001) (Figure 10D). Consistent with this reduction in delta power (SWA), increases in theta, gamma, and beta powers at the 10 mg/kg dose were observed in the young mice, with reductions in alpha and beta powers, and increased gamma power following the 30 mg/kg dose of xanomeline (Supporting Information Figure S2).
Figure 10.
During the active phase, xanomeline produced dose-dependent increases in arousal in nonpathologically aged mice and reduced delta power (SWA) during NREM sleep. Shown is the relative spectral power during wake (A,E) and NREM sleep (B,F) epochs only in 1–2 h following compound dosing relative to the 1 h predose baseline, gamma power during wake (C,G), and relative delta power (SWA) during NREM sleep (D,H) during the active phase in young (A–D) and nonpathologically aged (E–H) mice. In young animals, during wake 1–2 h postdose, 30 mg/kg xanomeline produced increased theta power and fluctuations across the gamma power range, and both 10 and 30 mg/kg produced reduced alpha power (A). During NREM sleep epochs 1–2 h postdose, 30 mg/kg reduced delta power and increased theta and gamma powers (B). 30 mg/kg xanomeline produced modest reductions in gamma power (C), and 10 and 30 mg/kg xanomeline reduced the delta power (SWA) during NREM sleep following a transient increase at 30 mg/kg (D). In nonpathologically aged mice, 10 mg/kg xanomeline decreased the delta power and increased the gamma power during wake epochs, and 30 mg/kg xanomeline increased delta, beta, and gamma power and reduced the theta and alpha power (E). Xanomeline had no effect on spectral power during NREM sleep; however, there were fewer than five mice that exhibited NREM sleep during the analysis window at the 30 mg/kg dose. Xanomeline dose-dependently increased the gamma power with increases seen at 3, 10, and 30 mg/kg during wake epochs (G) and produced reduced delta power (SWA) during NREM sleep at 10 mg/kg xanomeline, with no data available from 0–2 h postdose in the 30 mg/kg group due to insufficient mice displaying NREM sleep (H). Gray/tan shading represents frequency bands (Δ, delta 0.5–4 Hz; θ, theta 4–8 Hz; α, alpha, 8–13 Hz; β, beta, 13–30 Hz; γ, gamma 30–80 Hz). Data are expressed as means ± S.E.M. in 1 Hz bins (A,B,E,F) and means ± S.E.M. in 1 h bins. (C,D,G,H), n = 12–14/group; all time points in time courses contain n = 5–14 mice (C,D,G,H). Groups with fewer than 14 are due to not all mice displaying NREM sleep; # indicates fewer than five mice displayed NREM sleep in the 30 mg/kg dose group, so this was excluded. Solid bars indicate p < 0.05 compared to the vehicle (A,B,E,F); open symbols indicate p < 0.05 compared to the vehicle (C,D,G,H) (RM 2-way ANOVA) matching by both factors followed by Dunnett’s test for (A,C,E,G) and RM mixed effect model matching by both factors followed by Dunnett’s test for (B,D,F,H). See Supporting Information Table S2 for full statistical analysis.
In nonpathologically aged mice, xanomeline at the 30 mg/kg dose reduced the alpha power and increased the beta and gamma powers, consistent with increased arousal, with more modest increases in gamma power observed with the 10 mg/kg dose of xanomeline during wake (dose, p = 0.0004; frequency and dose × frequency, both p < 0.0001) (Figure 10E). As shown in Figure 10G, increases in gamma power were observed at all doses of xanomeline tested (dose, p = 0.0116; time and dose × time, p < 0.0001). In support of this, a shift to higher powers was observed at the 3 and 10 mg/kg doses of xanomeline with reductions in delta power at the 3 mg/kg dose and in delta and alpha power at the 10 mg/kg dose in the young mice. The 30 mg/kg dose of xanomeline shifted the frequencies from theta and alpha to beta and gamma, with a transient increase in delta power observed (Supporting Information Figure S1). Overall xanomeline had no dose-related effect on spectral power during NREM sleep in 1–2 h following dosing (dose, p = 0.8246; frequency p < 0.0001; dose × frequency, p = 0.4447) (Figure 10F). 10 mg/kg dose of xanomeline produced a transient decrease in delta power (SWA) during NREM sleep over time. The 30 mg/kg dose of xanomeline increased wakefulness in 2 h following dosing such that there were insufficient mice displaying NREM sleep to be analyzed (from −2 to 2 h postdose: dose, p = 0.031; time, p = 0.0448 and dose × time, p < 0.0001) (Figure 10H). In support of this decrease in delta power (SWA) during NREM, a shift to increased theta power was also observed with xanomeline in the nonpathologically aged mice, with additional shifts from alpha to beta and gamma powers (Supporting Information Figure S2).
In contrast, donepezil produced modest effects on gamma power, a correlate of arousal, during the active phase in young mice. The 3 mg/kg dose of donepezil increased the delta, beta, and lower gamma powers and reduced the alpha power (dose, p < 0.0001; frequency, p = 0.0483; dose × frequency, p < 0.0001) (Figure 11A), with no consistent dose-related effect on the total gamma power during wake (dose, p = 0.0744; time, p < 0.0001; dose × time, p = 0.0035) in the young mice (Figure 11C). In support of these observations, donepezil produced time-dependent increases in delta power and shifts from alpha to beta power were observed (Supporting Information Figure S3). During NREM sleep, the 3 mg/kg dose of donepezil produced a small increase in delta power in the young mice (dose, p = 0.7494; frequency, p < 0.0001; dose × frequency, p = 0.0260) (Figure 11B); however, no dose-related effect on delta power (SWA) during NREM sleep was observed with the posthoc tests (dose, p = 0.4884; time, p < 0.0001; dose × time interaction, p = 0.0035) (Figure 11D). Overall, the effects of donepezil on spectral power bands across time were modest and transient. Beta and gamma powers were reduced following administration with the 0.1 and 0.3 mg/kg doses of donepezil, while increased gamma power and reduced alpha power were seen with the 3 mg/kg dose of donepezil in the young mice (Supporting Information Figure S4).
Figure 11.
In the active phase, during wake, donepezil had no effect on arousal in young and nonpathologically aged mice and produced shifts to higher powers during NREM sleep in nonpathologically aged mice. Shown is the relative spectral power during wake (A,E) and NREM sleep (B,F) epochs only in 1–2 h following compound dosing relative to the 1 h predose baseline, gamma power during wake (C,G), and SWA (relative delta power) during NREM sleep (D,H) during the active phase in young (A–D) and nonpathologically aged (E–H) mice. In young mice, during wake, epochs 3 mg/kg donepezil increased delta, beta, and gamma power and reduced alpha power (A). Donepezil produced no dose-related effect on spectral power during NREM sleep (B), with inconsistent effects observed on gamma power during wake across the active phase (C) and no effect on delta power (SWA) during NREM sleep across the active phase (D). In nonpathologically aged mice, 3 mg/kg donepezil increased delta and beta power and reduced alpha and gamma power during wake epochs (E). During NREM sleep, donepezil increased gamma power (F). No significant change in gamma power during wake across the active phase (G) or delta power (SWA) during NREM sleep around dosing time was observed, with a modest increase in delta power (SWA) 5 and 8 h after dosing with 1 mg/kg donepezil (H). Gray/tan shading represents frequency bands (Δ, delta 0.5–4 Hz; θ, theta 4–8 Hz; α, alpha, 8–13 Hz; β, beta, 13–30 Hz; γ, gamma 30–80 Hz). Data are expressed as means ± S.E.M. in 1 Hz bins (A,B,E,F) and means ± S.E.M. in 1 h bins. (C,D,G,H), n = 12–14/group; all time points in time courses contain n = 10–14 mice (C,D,G,H). Groups with fewer than 14 are due to not all mice displaying NREM sleep. Solid bars indicate p < 0.05 compared to the vehicle (A,B,E,F); open symbols indicate p < 0.05 compared to the vehicle (C,D,G,H) (RM 2-way ANOVA) matching by both factors followed by Dunnett’s test for (A,C,E,G) and RM mixed-effect model matching by both factors followed by Dunnett’s test for (B,D,F,H). See Supporting Information Table S2 for full statistical analysis.
In the nonpathologically aged mice, during wake epochs, the 3 mg/kg dose of donepezil increased the delta and beta powers and reduced the alpha and gamma powers (dose, p < 0.4513; frequency and dose × frequency, p < 0.0001) (Figure 11E), with no effect on the total gamma power during wake over time (dose, p = 0.9580; time p < 0.0001; dose × time p = 0.6851) (Figure 11G). Similar to young mice, donepezil increased the delta power with a shift from the alpha to theta power and decreased the theta power in the nonpathologically aged mice (Supporting Information Figure S3). During NREM sleep, the 3 mg/kg dose of donepezil reduced the delta power and increased gamma power in the nonpathologically aged mice (dose, p = 0.0013, frequency, and dose × frequency, both p < 0.0001) (Figure 11F), with modest increases in delta power (SWA) during NREM sleep observed 5 and 8 h following administration with the 1 mg/kg dose of donepezil (dose, p = 0.0316; time and dose × time interaction, both p < 0.0001) (Figure 11H). Additionally, the alpha power increased with the 0.1, 0.3, and 1 mg/kg doses of donepezil in the nonpathologically aged mice, while gamma power was reduced with the 0.1 mg/kg dose of donepezil and increased with the 3 mg/kg dose of donepezil (Supporting Information Figure S4).
Xanomeline and Donepezil Reduced NREM Sleep Quality When Dosed in the Inactive Phase
Given the wake and arousal-promoting effects observed with xanomeline, we next assessed the effects of xanomeline and donepezil on the relative spectral power when dosed in the inactive phase in both the young and nonpathologically aged mice. In the young mice, xanomeline dose-dependently increased the gamma power and reduced the delta power during wake. Additionally, the 30 mg/kg dose of xanomeline increased the theta power (dose, p = 0.0211; frequency and dose × frequency, both p < 0.0001) (Figure 12A). An increased total gamma power during wake across time following the 30 mg/kg dose of xanomeline was observed, with transient reductions observed at the 3 and 10 mg/kg doses of xanomeline (dose, p < 0.0266; time and dose × time, both p < 0.0001) (Figure 12D). Consistent with this shift to higher frequencies, reductions in delta power were also observed with xanomeline in the young mice. Transient increases in theta power were noted at the 3 and 10 mg/kg doses of xanomeline with a decrease in theta power following the 30 mg/kg dose of xanomeline. The alpha and beta powers were reduced following dosing with the 10 and 30 mg/kg doses of xanomeline with increased alpha power observed with the 3 mg/kg dose of xanomeline in the young mice (Supporting Information Figure S5). During NREM sleep, all doses of xanomeline produced reductions in delta power; the 30 mg/kg dose of xanomeline also decreased the alpha power and increased theta and gamma powers (dose, frequency, and dose × frequency interaction, all p < 0.0001) (Figure 12B). The dose-dependent reductions in delta power (SWA) produced by xanomeline were observed across time during NREM sleep in the young mice (−2 to 1 h postdose: dose, p = 0.0163; time and dose × time interaction, both p < 0.0001; 2 to 8 h postdose: dose, p = 0.0293; time and dose × time interaction, both p < 0.0001) (Figure 12E). Consistent with the xanomeline-induced decreases in delta power (SWA), a shift to theta power was also seen with decreases in alpha and increases in beta and gamma powers in the young mice (Supporting Information Figure S6). During REM sleep, the 10 mg/kg dose of xanomeline reduced the delta power in the young mice (dose, p = 0.8089; frequency and dose × frequency interaction, both p < 0.0001) (Figure 12C).
Figure 12.
During the inactive phase, xanomeline increased arousal during wake and reduced the delta power (SWA) in NREM sleep in young and nonpathologically aged mice. Shown is the relative spectral power during wake (A,F), NREM sleep (B,G), and REM sleep (C,H) epochs only in 1–2 h following compound dosing relative to the 1 h predose baseline, gamma during wake (D,I), and relative delta power (SWA) during NREM sleep (E,J), following xanomeline dosing during the inactive phase in young (A–E) and nonpathologically aged (F–J) mice. In young mice, during wake epochs, 3, 10, and 30 mg/kg xanomeline decreased the delta power and increased the gamma power. 3 mg/kg increased the alpha power, and 30 mg/kg increased the theta power in 1–2 h following dosing (A). During NREM sleep, all doses decreased the delta power and 30 mg/kg additionally increased the theta power, reduced the alpha power, and increased the gamma power in 1–2 h following dosing (B). During REM sleep, 10 mg/kg xanomeline decreased the delta power and increased the theta power; insufficient mice displayed REM sleep in the 30 mg/kg xanomeline group in 1–2 h following dosing (C). Gamma power during wake was dose-dependently increased following 10 and 30 mg/kg xanomeline (D), and delta power (SWA) during NREM sleep displayed a dose-dependent decrease following all doses of xanomeline (E). In nonpathologically aged animals, during wake epochs, 30 mg/kg xanomeline produced decreased theta and alpha powers and increased beta and gamma powers in 1–2 h following dosing (F). During NREM sleep, 10 mg/kg xanomeline decreased delta, alpha, and beta powers and increased theta and gamma powers; insufficient mice displayed NREM sleep in the 30 mg/kg xanomeline group in 1–2 h following dosing (G). During REM sleep, 10 mg/kg xanomeline decreased the delta power and increased the alpha power; insufficient mice displayed REM sleep in the 30 mg/kg xanomeline group in 1–2 h following dosing (H). Gamma power during wake was increased following dosing with 10 and 30 mg/kg xanomeline (I), with a dose-dependent decrease in delta power (SWA) during NREM observed (J). Gray/tan shading represents frequency bands (Δ, delta 0.5–4 Hz; θ, theta 4–8 Hz; α, alpha, 8–13 Hz; β, beta, 13–30 Hz; γ, gamma 30–80 Hz). Data are expressed as means ± S.E.M. in 1 Hz bins (A–C,F–H) and means ± S.E.M. in 1 h bins (D,E,I,J), n = 7–14/group; all time points in time courses contain n = 7–14 mice (D,E,I,J). Groups with fewer than 13–14 are due to not all mice displaying NREM or REM sleep; # indicates fewer than five mice display NREM or REM sleep in the 30 mg/kg dose group, so this was excluded. Solid bars indicate p < 0.05 compared to the vehicle (A,B,C,F,G,H); open symbols indicate p < 0.05 compared to the vehicle (D,E,I,J); ** indicates the main effect of dose p < 0.01 (J) (RM 2-way ANOVA) matching by both factors followed by Dunnett’s test for (A,D,F,I) and RM mixed-effect model matching by both factors followed by Dunnett’s test for (B,C,E,G,H,J). See Supporting Information Table S2 for full statistical analysis.
In nonpathologically aged mice during wake, xanomeline reduced the alpha power and increased beta and gamma powers, consistent with increased arousal (dose, frequency, and dose × frequency interaction, all p < 0.0001) (Figure 12F). Xanomeline produced dose-dependent increases in the gamma power across time (dose, p = 0.0049; time and dose × time, both p < 0.0001) (Figure 12I). Consistent with this shift to higher powers, reduced delta frequency was seen following a transient increase after administration of the 30 mg/kg dose of xanomeline, along with reduced theta and alpha powers and increased beta power in the nonpathologically aged mice (Supporting Information Figure S5). During NREM sleep, the 3 and 10 mg/kg doses of xanomeline reduced the delta power, while the 10 mg/kg dose of xanomeline decreased the alpha power and increased the gamma power in the nonpathologically aged mice (dose, p = 0.2131; frequency and dose × frequency interaction, both p < 0.0001) (Figure 12G). Xanomeline produced a dose-dependent reduction in delta power (SWA) during NREM sleep across time in the nonpathologically aged mice with significance at all doses (−2 to 1 h postdose: dose, time, and dose × time interaction, all p < 0.0001; 2 to 8 h postdose: dose, p = 0.0080; time and dose × time, both p < 0.0001) (Figure 12J). Similar to young mice, increased theta power, decreased alpha, and increased gamma power were observed in the nonpathologically aged mice (Supporting Information Figure S6). During REM sleep, the 10 mg/kg dose of xanomeline decreased the delta power and increased the alpha power in the nonpathologically aged mice (dose, p = 0.9999; frequency, p < 0.0001; dose × frequency p = 0.0020) (Figure 12H).
Donepezil also produced disruptions during NREM sleep when dosed in the inactive phase. In the young mice during wake, the 1 and 3 mg/kg doses of donepezil decreased the delta power and increased the alpha and gamma powers, consistent with increased arousal (dose, p = 0.0002; frequency and dose × frequency, both p < 0.0001) (Figure 13A). Donepezil increased the gamma power across time during wake with the 1 and 3 mg/kg doses in the young mice (dose, p = 0.0739; time, p < 0.0001; dose × time, p = 0.0011) (Figure 13D). Consistent with this shift to higher powers, reductions in delta and theta powers were seen, with transient increases in theta power following administration of the 3 mg/kg dose of donepezil (Supporting Information Figure S7).
Figure 13.
In the inactive phase, donepezil decreased the delta power (SWA) during NREM and increased arousal wake in young and nonpathologically aged mice. Shown is the relative spectral power during wake (A,F), NREM sleep (B,G), and REM sleep (C,H) epochs only in 1–2 h following compound dosing relative to the 1 h predose baseline, gamma during wake (D,I), and relative delta power (SWA) during NREM sleep (E,J), following xanomeline dosing during the inactive phase in young (A–E) and nonpathologically aged (F–J) mice. In young mice, during wake, 1 and 3 mg/kg donepezil decreased the delta power and increased the alpha power and gamma powers in 1–2 h following dosing (A). During NREM sleep, 1 mg/kg donepezil produced an increased gamma power, while 3 mg/kg produced a modest increase in the theta power and a decreased beta power in 1–2 h following dosing (B). No dose-related effects on the spectral power during REM sleep were observed in 1–2 h following dosing; in the 3 mg/kg donepezil dose, insufficient mice displayed REM sleep, so this was excluded (C). Donepezil produced increased gamma power with effects seen at 1 and 3 mg/kg (D) and produced a transient decrease in delta power (SWA) during NREM sleep followed by a rebound increase at 3 mg/kg (E). In nonpathologically aged mice, during wake epochs, 3 mg/kg donepezil reduced the theta power and increased beta and gamma powers in 1–2 h following dosing (F). During NREM sleep, 3 mg/kg donepezil reduced the delta power and increased beta and gamma powers in 1–2 h following dosing (G). No dose-related effect on the REM relative spectral power was observed in 1–2 h following dosing; the 3 mg/kg donepezil dose had insufficient mice displaying REM sleep, so it was excluded (H). Donepezil increased the gamma power following dosing in the 3 mg/kg group (I). 3 mg/kg donepezil produced reduced the delta power (SWA) during NREM sleep (L). Gray/tan shading represents frequency bands (Δ, delta 0.5–4 Hz; θ, theta 4–8 Hz; α, alpha, 8–13 Hz; β, beta, 13–30 Hz; γ, gamma 30–80 Hz). Data are expressed as means ± S.E.M. in 1 Hz bins (A–C,F–H) and means ± S.E.M. in 1 h bins (D,E,I,J), n = 9–14/group; all time points in time courses contain n = 10–14 mice (D,E,I,J). Groups with fewer than 13–14 are due to not all mice displaying NREM or REM sleep; # indicates that fewer than five mice display NREM or REM sleep in the 3 mg/kg dose group, so this was excluded. Solid bars indicate p < 0.05 compared to the vehicle (A–C,F–H); open symbols indicate p < 0.05 compared to the vehicle (D,E,I,J) (RM 2-way ANOVA) matching by both factors followed by Dunnett’s test for (A,D,F,I) and RM mixed-effect model matching by both factors followed by Dunnett’s test for (B,C,E,G,H,J). See Supporting Information Table S2 for full statistical analysis.
During NREM sleep, the 1 mg/kg dose of donepezil produced modestly increased gamma power and the 3 mg/kg dose of donepezil modestly decreased the delta power and increased the theta power in the young mice (dose, p = 0.0290, frequency and dose × frequency, both p < 0.0001) (Figure 13B). The decreased delta power (SWA) during NREM sleep observed with the 3 mg/kg dose of donepezil was followed by a small rebound (dose, p = 0.5619; time and dose × time, both p < 0.0001) (Figure 13E). Consistent with this shift away from the delta power, increased theta, beta, and gamma powers were observed with donepezil, with rebound reductions in theta, alpha, and beta powers in the young mice (Supporting Information Figure S8). During REM sleep, there were no dose-related effects produced by donepezil in the young mice (dose, p = 0.8469; frequency, p = 0.0082 and dose × frequency, p > 0.9999) (Figure 13C).
During wake, the nonpathologically aged mice displayed decreased theta power and increased beta and gamma powers following the 3 mg/kg dose of donepezil (dose, p = 0.0007; frequency and dose × frequency, both p < 0.0001) (Figure 13F). An increased gamma power across time during wake was also observed following the 3 mg/kg dose of donepezil (dose, p = 0.0917; time and dose × time, both p < 0.0001) (Figure 13I). In support of this shift to higher powers, the 3 mg/kg dose of donepezil produced decreased theta and alpha powers with increased beta power, as well as a transient modest increase in delta power in the nonpathologically aged mice (Supporting Information Figure S7). When assessing the relative spectral power during NREM sleep, the 3 mg/kg dose of donepezil reduced the delta power and increased beta and gamma powers in the nonpathologically aged mice (dose, frequency, and dose × frequency, all p < 0.0001) (Figure 13G). This resulted in a decreased delta power (SWA) during NREM sleep across time following the 3 mg/kg dose of donepezil (2 to 8 h postdose: dose, p = 0.0399; time and dose × time, both p < 0.0001) (Figure 13J). Similar to young mice, the nonpathologically aged mice displayed increased theta, beta, and gamma powers, with reduced alpha power after donepezil treatment (Supporting Information Figure S8). Donepezil produced no dose-related effect on the spectral power during REM sleep in the nonpathologically aged mice (dose, p = 0.9466; frequency, p < 0.0015; dose × frequency, p = 0.4598) (Figure 13H).
Xanomeline and Donepezil Produced Cholinergic Adverse Effects at Higher Doses in Nonpathologically Aged Mice
We assessed whether xanomeline and donepezil produced adverse side effects associated with the activation of peripheral M2 and M3 mAChRs in nonpathologically aged mice during the inactive and active phases at doses that produced increased wakefulness and enhanced qEEG correlates of arousal, using the modified Irwin neurological test battery (Supporting Information Tables S3 and S4). During the active phase, the 30 mg/kg dose of xanomeline and the 3 mg/kg dose of donepezil produced significant adverse effects, consistent with the activation of peripheral M2 and M3 mAChRs when compared to the vehicle-treated mice, and the 30 mg/kg dose of xanomeline induced greater adverse effects compared to the 3 mg/kg dose of donepezil (main effect of dose, p < 0.0001; and time, p = 0.0002; xanomeline vs the vehicle, p < 0.0001; donepezil vs the vehicle: dose, p = 0.0238; and xanomeline vs donepezil, p = 0.0003) (Supporting Information Table S3). Similarly, during the inactive phase, significant adverse effects consistent with the activation of peripheral M2 and M3 mAChRs were observed following administration of the 30 mg/kg dose of xanomeline and the 3 mg/kg dose of donepezil compared to vehicle conditions; however, no difference between the xanomeline- and donepezil-treated mice was observed (main effect of dose and time, p < 0.0001; xanomeline vs the vehicle, p < 0.0001; donepezil vs the vehicle: dose, p < 0.0001; and no effect of xanomeline vs donepezil) (Supporting Information Table S4).
In summary, nonpathologically aged mice displayed multiple disruptions in sleep/wake architecture and arousal across the circadian rhythm. During the active phase, aged mice showed increased fragmentation of wake, as denoted by increased numbers of wake bouts and reduced wake bout duration. This observed fragmentation in wake in nonpathological aging was consistent with previous rodent studies45 and analogous to increased daytime napping observed in aging and AD clinical populations.46,47 Furthermore, this is the first study demonstrating that nonpathological aging in mice produces significant deficits in arousal during the active but not the inactive phase, characterized by a decreased gamma power during wake epochs. Similar changes in arousal have been reported in clinical literature, where AD populations display shifts in the spectral power from high to low frequency during wake, and in normal aging, where decreased gamma power during wake is seen.48,49 In the inactive phase, aged mice showed only modest decreases in total NREM sleep duration, with no observed change in wake or arousal. Overall, the impact of aging in mice resulted in circadian-dependent changes in sleep/wake architecture and arousal, highlighting the importance in future studies of evaluating preclinical AD disease models alone or in combination with novel pharmacological challenges across the diurnal rhythm.
Numerous studies have explored the changes in cholinergic signaling associated with circadian rhythm and/or aging.1,5,39 Previous studies have demonstrated that central ACh levels are highest during the active phase and lowest in the inactive phase in rodents.39 With increasing age, cholinergic signaling in rodents stops displaying its normal circadian changes,39 which may explain the more profound wake fragmentation and arousal deficits observed in the present study in the active phase of nonpathologically aged mice. Changes in cholinergic function during nonpathological aging in mice may also explain the observed differences in the efficacy of indirect- and direct-acting muscarinic cholinergic agonists on normalizing wake fragmentation and arousal deficits across the circadian cycle. Specifically, during the inactive phase when cholinergic signaling is low, donepezil and xanomeline increased wakefulness and arousal in the young mice. In the nonpathologically aged mice, donepezil and xanomeline increased wakefulness, with donepezil modestly increasing arousal, while xanomeline robustly enhanced arousal. In contrast, during the active phase in the young mice when cholinergic signaling is high, donepezil produced no effect on wake and arousal, whereas xanomeline produced an increase in wake with no effect on arousal. During the active phase in the nonpathologically aged mice, donepezil again had no effect on wake or arousal, while xanomeline produced marked increases in both wakefulness and arousal. We previously demonstrated that young rodents display reduced arousal during wake in the inactive phase.37 In light of these and the present findings, we hypothesize that young rodents in the active phase exhibit optimal arousal associated with high levels of cholinergic signaling during wake such that there is an insufficient dynamic range in cholinergic tone to further boost arousal. However, the nonpathologically aged mice displayed a deficit in arousal during the active phase, which may be attributed to the previously described age-related reductions in cholinergic signaling. Such a deficit in cholinergic signaling during the active phase in nonpathologically aged mice suggests that boosting arousal may be possible through the direct activation of M1 and/or M4 mAChRs using the direct-acting muscarinic cholinergic agonist xanomeline. However, in contrast, boosting diminished cholinergic signaling with the AChEI donepezil may not provide sufficient enhancement of central cholinergic signaling at cortical M1 and/or M4 mAChR subtypes to observe improvements in arousal. Ongoing studies are evaluating the integrity of cholinergic basal forebrain projections, signaling, and muscarinic receptor density in nonpathologically aged mice to confirm this hypothesis.
The present findings support further development of pharmacological approaches, such as direct-acting muscarinic cholinergic agonists like xanomeline, to boost cholinergic signaling at M1 and M4 mAChRs in aging, MCI, and AD. Historical in vitro studies suggested that the direct-acting muscarinic cholinergic agonist xanomeline displays partial agonism preferentially at the M1 and M4 mAChRs,50 which was thought to contribute to its improved adverse effect profile and enhanced efficacy observed relative to AChEIs. However, the present in vivo data using the modified Irwin neurological test battery do not support the idea of an improved adverse effect profile of xanomeline; we actually observed that xanomeline causes more pronounced adverse effects relative to donepezil. To achieve a broader therapeutic index, a formulation of xanomeline, with the peripherally restricted nonselective mAChR antagonist trospium, known as KarXT, has been developed, which is currently undergoing clinical trials34 (ClinicalTrials.gov: NCT03697252, NCT04659161, and NCT05511363). With regards to the mechanism of action of xanomeline, recent in vitro pharmacology studies have demonstrated that xanomeline exhibits unique biased agonism activity, with significant bias away from ERK1/2 phosphorylation and Ca2+ mobilization signaling pathways compared to Gαi2 activation, at the recombinant human M4 mAChR subtype,51 which may also account for its unique efficacy profile in preclinical and clinical studies.
In previous studies, we have reported that selective M1 mAChR stimulation enhances wake and arousal,35 suggesting that the wake- and arousal-promoting effects of xanomeline observed in the present study may be primarily M1-mediated. Different approaches to provide greater M1 activation without producing dose-limiting adverse effects, seen both in clinical studies22,24,33 and in the present data, include compounding the direct-acting muscarinic cholinergic agonist xanomeline with the peripherally restricted muscarinic antagonist trospium34 (ClinicalTrials.gov: NCT03697252, NCT04659161, and NCT05511363). Alternatively, allosteric ligands have been shown to display improved muscarinic selectivity compared to direct-acting orthosteric agonists and may provide a different mechanism through which to achieve greater selectivity with improved M1 or M4 activation than seen with the indirect-acting AChEIs.35,52,53 Ongoing studies are investigating whether M1 and M4 mAChR-positive allosteric modulators display age- and circadian rhythm-dependent effects on sleep–wake architecture and arousal.
Sleep disruptions are a well-characterized symptom of AD, with recent work suggesting that sleep disruptions may also lead to increased AD pathology.54 Numerous studies have indicated that AChEIs, including donepezil, may lead to increased sleep disturbances.43 Our current data set supports this, with donepezil reducing NREM sleep quality in aged mice when dosed in the inactive phase. Xanomeline administered in the inactive phase produced a similar decrease in NREM sleep quality. This provides further evidence that the time in the circadian cycle when these compounds are administered is vitally important. One consideration of the present data is that the reported effects were observed following acute dosing. In clinical populations, the donepezil dose is escalated over several weeks until a stable chronic maintenance dose is achieved.23 The NREM sleep disruptions and peripheral side effects observed with donepezil and xanomeline may decrease with chronic dosing. Future studies will be needed to assess the effects of xanomeline on sleep/wake architecture following chronic dosing.
In conclusion, this study is the first to systematically assess circadian-dependent pharmacological effects of the direct-acting muscarinic cholinergic agonist xanomeline and indirect-acting muscarinic cholinergic agonist donepezil on sleep/wake architecture and spectral power in young and aged mice. The data presented here indicate that when considering treatment for a disease process that occurs during aging, it is of critical importance to understand the efficacy of the compound in the aging process across the circadian cycle. These findings support the future development of ligands such as xanomeline that directly target M1 and/or M4 mAChRs subtypes. Future studies in higher-order species will be essential to test the hypothesis that direct-acting muscarinic cholinergic agonists provide improved symptomatic benefits if dosed during the day in MCI and AD populations compared to indirect-acting muscarinic cholinergic agonists such as the AChEI donepezil.
Materials and Methods
Subjects
Young adult (3–4 month-old, n = 28) and aged (19–20 month-old, n = 40; 28 for EEG, 12 for assessment of cholinergic side effects) wildtype male C56BL/6J mice (The Jackson Laboratory) served as subjects. Prior to study initiation, all mice were socially housed. Following the surgical implantation of EEG telemetry devices, all animals were individually housed. Mice were housed in humidity-controlled rooms and maintained in a 12/12 h light–dark cycle with food and water available ad libitum. All studies were approved by the Vanderbilt University Animal Care and Use Committee, and experimental procedures conformed to guidelines established by the National Research Council Guide for the Care and Use of Laboratory Animals.
Compounds
Xanomeline (synthesized in-house, 3–30 mg/kg) and donepezil (AstaTech inc, Bristol, PA, 0.1–3 mg/kg) were dissolved in saline. All compounds were dosed at a volume of 10 mL/kg via intraperitoneal (I.P.) injection. The dose range for both has been shown to modulate sleep/wake architecture in rats,35,37 and the top dose of both was where dose-limiting adverse side effects were observed (Supporting Information Tables S3 and S4).
Electroencephalography
Surgery
A telemetric transmitter (HD-X02, Data Science International [DSI], Minneapolis, MN) was implanted in all mice using previously described methods.35,55 A 2–3 cm midline incision was made over the skull. A frontoparietal EEG lead was placed, with the frontal co-ordinate at +1.5 mm AP, −2 mm ML and the parietal co-ordinate at −3 mm AP, 2 mm ML, which was secured with screws and covered with dental cement (Patterson Dental, Saint Paul, MN). A second biopotential lead for recording the electromyogram (EMG) was placed in the nuchal muscle. Mice were recovered for a minimum of 10 days postsurgery prior to recording.
EEG Recording and Sleep Staging
EEG and EMG recordings were performed for 24 h starting at either lights on (inactive phase) or lights off (active phase) with the mice housed in their home cage. Ponemah software (v3.0, DSI) was used to capture EEG and EMG waveforms. A wireless receiver (RCP-1, DSI) below each home cage transmitted data which was continuously sampled at 500 Hz. Dosing with xanomeline (3–30 mg/kg or saline vehicle i.p.) or donepezil (0.1–3 mg/kg or saline vehicle i.p.) was performed 2–3 h into either the active or inactive phase. For all experiments, time is displayed in zeitgeber time, where ZT 0 indicates a transition from the active to the inactive phase (lights off to lights on).
Following the recording, all traces were manually scored by trained observers blinded to age and dose. The recordings were scored in 5 s epochs using Neuroscore 3.3.1 software (DSI) as wake, NREM sleep, or REM sleep based on previously published characteristic patterns by our group.35,37,55,56 The duration of time in each state (wake, NREM sleep, and REM sleep) separated into 2 or 24 h bins, served as the primary dependent measures to assess the age and pharmacological effects. Fragmentation of sleep and wake was assessed by calculating the average NREM sleep or wake bout length and number of NREM sleep or wake bouts for the 8 h following dosing, thus remaining within the phase of dosing.
qEEG Spectral Power Analysis
Once the data was divided by the sleep stage, the relative spectral power from the qEEG trace was calculated in 1 Hz bins between 0.5 and 80 Hz using a fast Fourier transform with a Hamming window overlap ratio of 0.5. Within each 1 Hz interval relative, the spectral power was binned by the sleep stage (wake, NREM sleep, or REM sleep). To understand pharmacological effects, this was averaged across a predose baseline, 1–2 h following light change, and a postdose period, 1–2 h following dosing. The postdosing period was then represented within wake, NREM sleep, and REM sleep, respectively, as a percent change to the predose period in the same state. Power band analysis across time within wake and NREM sleep was calculated by binning the spectral power from 0.5 to 4 Hz (delta), 4 to 8 Hz (theta), 8 to 13 Hz (alpha), 13 to 30 Hz (beta), and 30 to 80 Hz (gamma) during wake and NREM sleep, respectively. This was averaged each hour from 2 h predose to 8 h postdose and represented relative to 1–2 h following light change. When assessing age-dependent changes, the 1 h periods are normalized to the same 1 h periods in young mice.
Assessing Cholinergic Adverse Effects
Donepezil and xanomeline effects on autonomic and somatomotor functions were assessed in nonpathologically aged C57BL/6J mice in the active and inactive phases. Assessments were performed 30, 60, 120, and 240 min after I.P. administration of 30 mg/kg xanomeline, 3 mg/kg donepezil, or the saline vehicle. For assessment, a modified Irwin neurological test battery57 was used as described in our previous work.53 In brief, numerous autonomic and somatomotor behavioral end points were observed by blinded, trained observers (see Supporting Information Tables S3 and S4, for a full list of behaviors assessed), and each behavior was scored as 0 (normal), 1 (mild effect), or 2 (marked effect). For each behavioral end point, the score was averaged across subjects, and then the sum of the average scores for all the behavioral end points was used to calculate the total score.
Statistics
qEEG analysis and sleep/wake architecture are displayed as means ± S.E.M. Two-way repeated analysis of variance (ANOVA; repeating by both factors for pharmacological studies; repeated by one factor for age-related comparisons) was used when assessing the relative spectral power change from 0.5 to 80 Hz, individual power bands across time, and for all sleep/wake architecture assessments. When data was absent due to animals not entering into NREM or REM sleep during the analysis period, a RM mixed-effect model (REML) was applied. If data for an entire dose was absent at a given time point when assessing individual power bands across time, two-way repeated ANOVA (or REMLs) was applied; one from the start of recording until the highest dose group displayed NREM sleep (including vehicle and all doses except the top dose) and one from the initiation of NREM sleep in the highest dose group following dosing (including vehicle and all doses). When assessing wake and NREM sleep average bout length and bout number, an unpaired t-test was used to compare young and aged mice and a RM one-way ANOVA (or REML if missing data due to mice not entering NREM sleep) to compare dosing conditions. Sidak’s multiple-comparison test was performed to compare young and aged cohorts. Otherwise, Dunnett’s multiple comparison test was performed to compare dosing conditions to the vehicle. For the modified Irwin neurological test battery, a total adverse event score was compared using a two-way ANOVA with the vehicle condition grouped within the phase as a saline vehicle was used for both compounds, and no differences were seen between vehicle treatments. Tukey’s multiple-comparison test was used to compare the main effect of dose across the vehicle-, xanomeline-, and donepezil-treated conditions within the phase. All statistical analysis and graphing were performed using GraphPad Prism version 9.4.1 (see Supporting Information Table S2 for full statistical analysis).
Acknowledgments
We thank Dr. Michael Bubser for critical comments on the manuscript and Edith Duncan, Mary-Wilson Screws, Kate Friedman, Laura Wan, Arthur Yu, and Jackson Blough for their efforts with data processing. Studies were performed in part through the use of the Murine Neurobehavioral Core laboratory at the Vanderbilt University Medical Center. Graphical abstract was created with BioRender.com.
Glossary
Abbreviations
- Ach
acetylcholine
- AchEI
acetylcholinesterase inhibitors
- AD
Alzheimer’s disease
- EEG
electroencephalography
- EMG
electromyography
- MCI
mild cognitive impairment
- mAChR
muscarinic acetylcholine receptor
- NREM
nonrapid eye movement
- qEEG
quantitative electroencephalography
- REM
rapid eye movement
- SWA
slow wave activity
- ZT
zeitgeber time
Supporting Information Available
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.2c00592.
Author Contributions
J.K.R.: conceptualization, methodology, investigation, visualization, formal analysis, writing-original draft, and editing. S.M.I.: formal analysis, writing–review, and editing. L.B.T.: investigation, writing–review, and editing. C.W.L.: reagents. C.K.J.: conceptualization, methodology, resources, writing–review and editing, funding acquisition.
This work was supported by a National Institute of Aging grant to C.K.J. (grant AG054622)
The authors declare the following competing financial interest(s): Dr. Jones and Dr. Lindsley are inventors on patents protecting allosteric modulators and orthosteric antagonists of muscarinic acetylcholine receptors. Dr. Jones and Dr. Lindsley have received royalties from Acadia Pharmaceuticals and Neumora Therapeutics. Dr Jones owns personally-acquired stock in Karuna Therapeutics. The remaining authors have nothing to disclose.
Supplementary Material
References
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