Effects of Blindness on Sleep/Wakefulness States in Mice

Abstract

To examine the effects of blindness on sleep/wakefulness states, we compared locomotor activity and delayed recovery from isoflurane anesthesia induced by hypnotics during light and dark periods in sighted CBA/N and blind CBA/J mice. Locomotor activity around the switch from the dark to light period significantly differed in both mice. Delayed recovery induced by brotizolam was attenuated in both periods in CBA/J mice. In addition, the period specificity of delayed recovery caused by suvorexant or diphenhydramine in CBA/N mice was abolished in CBA/J mice. These results suggest that blindness impairs sleep quality.

INTRODUCTION

The body clock regulates sleep/wakefulness, as well as body temperature, blood pressure, hormone production, and neural and immune systems, to an approximately daily rhythm.^1–3) Since the endogenous body clock cycle is not a perfect 24-h period, organisms synchronize their biological processes with the 24-h rotation of the earth.^1,2,4) Several factors may cause entrainment, the most important of which is light stimulation.⁴⁾ Sleep disturbances are more common in visually impaired than in sighted individuals, and inadequate light stimulation may reduce the quantity (length) or quality of sleep.^5–8)

The retina of the mammalian eye contains three types of photoreceptor cells: rods, cones, and intrinsic photosensitive retinal ganglion cells (ipRGCs) that express melanopsin.⁹⁾ Rods and cones are mainly responsible for vision and ipRGCs for light responses other than vision. A previous study reported that mice lacking rods and cones showed functional circadian photoentrainment, pineal reactions to light, and the pupillary light reflex, whereas mice genetically engineered to lack melanopsin in addition to rods and cones lacked the majority of image- and non-image-forming responses.¹⁰⁾

Brain substances, such as gamma-aminobutyric acid (GABA), serotonin, noradrenaline, histamine, and orexin, regulate sleep and wakefulness.¹¹⁾ This regulation involves the sleep and arousal centers, which innervate and inhibit each other.¹²⁾ During sleep, the activity of the sleep center (GABAergic neurons) is increased, which suppresses the arousal center (orexin-producing neurons and monoaminergic neurons). On the other hand, during arousal, the activity of the arousal center increases and exceeds that of the sleep center.¹²⁾

We previously developed a method to evaluate the efficacy of hypnotics based on their ability to delay recovery from isoflurane anesthesia.¹³⁾ Using this method, we confirmed that delayed recovery induced by brotizolam was more pronounced in the light period (resting phase) than in the dark period (active phase) in C57BL/6J mice with normal retinas. In FVB/N and C3H/HeN mice, which lack rods and cones due to the rd1 mutation, delayed recovery induced by brotizolam was weaker, and to a similar extent in both light and dark periods.¹⁴⁾ These findings indicate that delayed recovery from isoflurane anesthesia induced by brotizolam reflects the depth of sleep and that blind mice with the rd1 mutation may not have deep sleep; however, it currently remains unclear whether this is due to differences in mouse strains.

The CBA strain includes CBA/J mice with the rd1 mutation and sub-strains without the rd1 modification (such as CBA/N and CBA/CaJ).^15–18) Previous studies reported that CBA/J mice were less sensitive to light and showed reduced circadian photosensitivity.^15,18) However, the sleep/wakefulness states in CBA/J mice remain unclear. Therefore, the effects of the rd1 mutation on the sleep/wake status were examined herein using mice with similar genetic backgrounds, namely, CBA/N and CBA/J mice.

MATERIALS AND METHODS

Animals

Six-week-old male CBA/N and CBA/J mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and Charles River Japan, Inc. (Yokohama, Japan), respectively. Mice were housed under a 12-h light/dark cycle, with lights on at 7 : 30 a.m. (Zeitgeber time = 0 [ZT0]) and lights off at 7 : 30 p.m. (Zeitgeber Time = 12 [ZT12]). Temperature was maintained at 23 ± 2 °C and relative humidity between 45 and 65%. Animal procedures were approved by the Animal Care and Use Committee of Setsunan University (k21-30 and k22-29) and were conducted according to the Japanese Pharmacological Society guidelines.

Materials

Diphenhydramine hydrochloride was purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and dissolved in water; brotizolam (Nichi-Iko Pharmaceutical Co., Ltd., Tokyo, Japan) and suvorexant (Belsomra^®, Merck & Co., Inc., Kenilworth, NJ, U.S.A.) were suspended in 0.5% CMC as previously described.^13,14)

Measurement of Spontaneous Locomotor Activity

Spontaneous locomotor activity was measured at 7–8 weeks of age using a previously reported method.¹⁴⁾

Drug Treatment

Brotizolam (0.75 mg/kg), suvorexant (40 mg/kg), or diphenhydramine (10 mg/kg) was orally administered one hour before the induction of isoflurane anesthesia, and the recovery time from the anesthetic was measured during the light or dark period at 9–17 weeks of age, as previously described.¹⁴⁾

Statistical Analysis

Experimental data were expressed as means ± standard error of the mean (S.E.M.). Statistical analyses were performed using the Student’s t-test for single comparisons. Differences were considered to be significant at p < 0.05.

RESULTS

In CBA/N and CBA/J mice, spontaneous locomotor activity was significantly higher between ZT12 and ZT24 than between ZT0 and ZT12, showing a nocturnal behavioral pattern (Fig. 1a). However, detailed comparisons every 3 h revealed that spontaneous locomotor activity at ZT0-3, ZT6-9, and ZT9-12 was significantly higher in CBA/J mice than in CBA/N mice. In contrast, spontaneous locomotor activity at ZT21-24 was markedly lower in CBA/J mice (Fig. 1b).

Fig. 1. Locomotor Activity by CBA/N and CBA/J Mice

(a, b) Locomotor activity by CBA/N and CBA/J mice was plotted and compared every 12 h (a) or every 3 h (b). Blue (circle) represents data on CBA/N mice and red (square) represents data on CBA/J mice. Male mice were used at 7–8 weeks of age (n = 12 for each group). All displayed values are means ± S.E.M. * p < 0.05, ** p < 0.01.

Brotizolam significantly delayed recovery from isoflurane anesthesia in both the light and dark periods, and this effect was robust in the light period in both mice (Fig. 2a). However, the delayed impact in both periods was less in CBA/J mice than in CBA/N mice (Fig. 2a). Suvorexant significantly delayed recovery only in the light period in CBA/N mice, whereas it caused a significant delay in both the light and dark periods in CBA/J mice (Fig. 2b). Diphenhydramine significantly delayed recovery in the dark period only in CBA/N mice, and had no effect on recovery from anesthesia in either period in CBA/J mice (Fig. 2c).

Fig. 2. Effects of Hypnotics on Isoflurane Anesthesia in CBA/N and CBA/J Mice

(a–c) Delayed recovery induced by brotizolam (a), suvorexant (b), or diphenhydramine (c) was compared during the light and dark periods in CBA/N and CBA/J mice. Diurnal experiments were conducted between ZT6 and ZT9 under normal light and nocturnal experiments between ZT12 and ZT15 under dim red light. Male mice were used at 9–17 weeks of age (n = 10–12 for each group). All displayed values are means ± S.E.M. * p < 0.05, ** p < 0.01.

DISCUSSION

Spontaneous locomotor activity by CBA/N and CBA/J mice was significantly higher in the dark period than in the light period. In CBA/J mice, which have lost rods and cones due to the rd1 mutation, ipRGCs may have contributed to the entrainment of the body clock under the light/dark cycle.¹⁹⁾ However, detailed comparisons of spontaneous locomotor activity revealed significant differences around the switch from the dark to light period. Milosavljevic et al. previously demonstrated using chemical genetic techniques that the selective activation of ipRGCs in mice in the dark increased arousal and anxiety behaviors.²⁰⁾ Therefore, the significant increases observed in spontaneous locomotor activity during the light period in CBA/J mice may be due to arousal and anxiety behaviors.²¹⁾

In CBA/N mice, delayed recovery from isoflurane anesthesia induced by brotizolam was more pronounced in the light period than in the dark period. In contrast, delayed recovery caused by diphenhydramine was only observed in the dark period. These results were considered to be reasonable because the arousal center is activated during the dark period and the sleep center during the light period in mice.¹²⁾ Delayed recovery induced by brotizolam was weaker in both periods in CBA/J mice than in CBA/N mice. In addition, delayed recovery induced by diphenhydramine was not observed in CBA/J mice in the light or dark period. These results suggest that CBA/J mice do not achieve deep sleep during the light period and may not be sufficiently aroused during the dark period.

Delayed recovery induced by suvorexant was only observed in the light period in CBA/N mice, whereas it was similar in both periods in CBA/J mice. Orexin-producing neurons project to the nuclei of monoaminergic and cholinergic neurons, and function to maintain arousal through the control of these neurons.^12,22) On the other hand, orexin-producing neurons receive inhibitory inputs from serotonergic and noradrenergic nerves, suggesting a role in the maintenance of constant orexin neuron activity by inhibiting orexin neurons during arousal.²²⁾ Therefore, the weak activation of orexin neurons was detected in CBA/J mice in both the light and dark periods due to changes in inhibition by the arousal center. However, it is important to note that among the strains examined, besides CBA/N and CBA/J mice shown herein, only C57BL/6J mice exhibited delayed recovery from isoflurane anesthesia induced by suvorexant. Although C57BL/6J mice showed the same degree of delay in both the dark and light phases,¹⁴⁾ C57BL/6N mice, another substrain of C57BL/6, showed no delay in either the light or dark phase (unpublished data). Although the reason for this currently remains unclear, factors other than blindness may contribute to delayed recovery from isoflurane anesthesia caused by suvorexant. A previous study reported that rod-cone and melanopsin-based pathways were both necessary for modulating the effects of light and dark on sleep, in contrast to circadian photo-entrainment.²³⁾ In addition, Wang et al. showed that the awake and non-REM conditions frequently shifted between each other in the hours following the transition from the light to dark period in mice with ipRGCs, but no rods or cones, suggesting unstable sleep/wakefulness states.²⁴⁾ These findings are consistent with the present results, indicating that sleep quality is impaired in mice with the rd1 mutation.

Since CBA/N and CBA/J mice are different inbred mouse strains differentiated from CBA/H mice derived from the DBA species, mutations other than the rd1 mutation are also present.²⁵⁾ For example, a deficiency in Bruton’s tyrosine kinase (Btk), which is essential for B cell differentiation and maturation, has been reported in CBA/N.^25,26) However, since no mutations affecting sleep have been detected in either mouse type, the differences observed in the present study may be attributed to the rd1 mutation. Ruggiero et al. also suggested that the expression of neuropeptides and neuronal activity in the SCN differed between CBA/J mice and CBA/N mice.²⁷⁾ Therefore, the compensatory biological changes induced by early retinal degeneration in CBA/J mice may have affected the results of the present experiments.

In summary, locomotor activity by CBA/J mice around the switch from the dark to light period significantly differed from that in CBA/N mice. Delayed recovery induced by brotizolam was attenuated in both periods in CBA/J mice. The period specificity of delayed recovery caused by suvorexant or diphenhydramine in CBA/N mice was abolished in CBA/J mice. These results suggest that blindness impairs sleep quality. In addition, the methods used in this experiment may help screen the sleep/wake states in various knockout mice.

Conflict of Interest

The authors declare no conflict of interest.

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