Altered sleep latency and arousal regulation in mice lacking norepinephrine

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Abstract

Latency to sleep and the amount of sensory stimulation required to awaken an animal are measures of arousal threshold, which are ultimately modulated by an arousal regulation system involving many brain areas. Among these brain areas and network connections are wake-promoting nuclei of the brainstem and their corresponding neurotransmitters, including norepinephrine (NE). In this study, we used mice that are unable to produce NE to study its role in regulating sleep latency after a variety of interventions, and to study arousal from sleep after sleep deprivation (SD). Sleep latency was measured after gentle awakening or after injections of saline, caffeine or modafinil. Sleep latency was also measured before and after partial restoration of NE pharmacologically. Arousal threshold was measured by recording the number of decibels of white noise required to wake each mouse from NREM sleep after 0, 3 and 3+3 h SD (3 h SD followed by <2 min sleep, followed by an additional 3 h SD). Results showed that when mice were awakened without being touched, there were no differences in sleep latency between the genotypes. However, after an injection of saline, the control mice increased their sleep latency, whereas the NE-deficient mice did not. There were no group differences in sleep latency after treatment with either stimulant. The sleep latency difference between the genotypes was ameliorated by partial restoration of NE. The arousal threshold experiments revealed that significantly more noise was required to wake the NE-deficient mice after 3 and 3+3 h of SD. These findings show that mice lacking NE fall asleep more rapidly only after a mild stressor, such as an intraperitoneal injection. NE-deficient mice are also more difficult to wake up using audio stimulation after SD. The results presented here suggest that NE promotes wakefulness during transitions between sleep and wake under conditions involving mild stress and SD, but not under baseline circumstances.

Introduction

Throughout their daily lives, mammals experience three states of consciousness: wake, NREM sleep and REM sleep. The transitions between these states are physiologically regulated, and have evolutionary significance. For instance, studies have shown that autonomic responses to spontaneous and elicited awakenings from sleep are greatly and transiently elevated, which probably serve to heighten arousal in preparation for danger Horner et al., 1997, Trinder et al., 2003. In addition, the noradrenergic neurons of the locus coeruleus (LC) spontaneously fire not only during heightened periods of arousal, but they increase their firing just prior to a change in state between NREM sleep and wake, suggesting that the neurotransmitter norepinephrine (NE) participates in this alerting behavior upon waking (Aston-Jones and Bloom, 1981). In the present study, we investigate if NE plays a critical role in the arousal regulation of mice genetically deficient in NE. Because NE is traditionally thought of as a “wake-promoting” neurotransmitter, we hypothesized that the NE-deficient mice would have a shorter sleep latency after being awakened gently or with an injection of saline, and that they would require a higher level of noise to awaken them from sleep. We also tested their sleep latency after injections of common stimulants, to determine if the lack of NE altered the “normal” behavioral reaction to these two drugs.

The noradrenergic and serotonergic neurons of the reticular activating system of the brainstem, along with histaminergic neurons from the hypothalamic tuberomammilary nucleus, have been shown by electrophysiology to have high activity during the waking state, low activity during NREM sleep and to be nearly quiescent during REM sleep Aston-Jones and Bloom, 1981, McGinty and Harper, 1976, Steininger et al., 1999. Among these “wake active” nuclei is the noradrenergic LC. Increased NE levels and/or LC firing is highly associated with periods of increased arousal or attention, and cortical activation Aston-Jones and Bloom, 1981, Aston-Jones et al., 1994, Berridge and Foote, 1991, Berridge et al., 1993, Foote et al., 1991, Kodama et al., 2002, Rajkowski et al., 1994, Smith and Nutt, 1996. In addition, these “wake active” nuclei have anatomical and functional connections with the NREM sleep-generating ventral lateral preoptic area (VLPO), which uses the inhibitory neurotransmitter GABA to inhibit these nuclei during sleep Gallopin et al., 2000, Nitz and Siegel, 1997, Sherin et al., 1996, Sherin et al., 1998, Szymusiak et al., 1998. There is also strong evidence showing a causal reciprocal relationship between the activity of the reticular activating system (including NE in particular) and the cholinergic pedunculopontine tegmentum in the generation of REM sleep Crochet and Sakai, 1999, Hobson et al., 1975, Jones, 1991, Monti et al., 1988, Sakai, 1988, Singh and Mallick, 1996.

Given the participation of NE in the wake- and alertness-promoting systems in the brain, it follows that animals deficient in NE might have altered wake states or altered transitions to and from the waking state. Thomas et al. (1995) created mice that are chronically deficient in NE and epinephrine, by genetically targeting the dopamine β-hydroxylase gene; these mice are referred to here as either NE-deficient or “knockout” mice. The mice require pharmacological restoration of NE prenatally for survival, but after birth they develop normally without intervention. The mice are deficient in epinephrine also, but because there are very few adrenergic nuclei in the central nervous system, and sleep is a centrally generated state, we are primarily interested in how NE acts in the brain to modulate the sleep and wake states.

Stress is a variable that can have large and wide-ranging effects on animal behavior. Acute stressors, such as restraint stress or footshock, can increase sleep latency after the stressor is discontinued Gonzolez et al., 1995, Koehl et al., 2002, Vazquez-Palacios and Velazquez-Moctezuma, 2000, although many studies do not report sleep latency Marinesco et al., 1999, Meerlo et al., 2001. Rodents experience increases in measures of physiological stress in response to intense stressors, such as restraint or cold stress Lenox et al., 1980, Meerlo et al., 2001; however, laboratory mice routinely experience less intense stressors, such as handling, cage changing and injections.

In the physiological response to stress, NE contributes to the stimulation of the hypothalamic–pituitary–adrenal axis Pacak et al., 1995, Tsigos and Chrousos, 2002. We have previously shown that NE-deficient mice have shorter sleep latencies after an injection of saline or a low dose of amphetamine; however, it is unclear if this phenotype is associated with the stress of handling (Hunsley and Palmiter, 2003). In the present study, sleep latency after a saline injection is compared with gentle awakening that does not include handling. In addition, the present study measures sleep latency both at the beginning of the light portion of the light/dark cycle and at the end, to determine if the sleep latency difference in the knockouts is influenced by a previous period of dark or light.

Another measure of arousal threshold is the amount of stimulation required to awaken an animal from sleep. In the current study, we recorded the amount of noise, in decibels, which was required to awaken mice from NREM sleep. In humans, investigators have studied arousal from all stages of sleep due to external noise generation Bonnet and Moore, 1982, Mullin and Kleitman, 1938. In general, arousal threshold from sleep varies according to time spent asleep and sleep stage (Bonnet and Moore, 1982), and is modifiable by sleep-promoting drugs Cluydts et al., 1995, Saletu and Grunberger, 1981, Saletu et al., 1980. Because NE-deficient mice are lacking a neurotransmitter that has traditionally been associated with wake-promotion, we predicted that the knockouts would be more difficult to wake up from NREM sleep than the controls. Several hours of sleep deprivation (SD) were included to potentially exacerbate this phenomenon.

Caffeine and modafinil, which are CNS stimulants, increase wake and locomotion in mice Duteil et al., 1990, Edgar and Seidel, 1997, Wisor et al., 2001. These stimulants utilize a variety of wake-promoting systems and associated neurotransmitters, including adenosine, dopamine, serotonin, hypocretin/orexin, acetylcholine and NE Chemelli et al., 1999, Duteil et al., 1990, Hadfield and Milio, 1989, Kirch et al., 1990, Porkka-Heiskanen et al., 1997, Strecker et al., 2000, Wisor et al., 2001. Because NE-deficient mice only lack NE, these stimulants should still increase waking behavior through the remaining, intact systems. Therefore, we sought to test the hypothesis that stimulation of nonnoradrenergic wake-promoting pathways would cause the knockout mice to exhibit a sleep latency duration that was similar to the control mice.

Section snippets

Animals

Mice lacking NE and Epi (knockouts) were created by inactivating the dopamine β-hydroxylase gene (Dbh; Thomas et al., 1995), and maintained on a mixed C57BL/6J and 129/SvCPJ hybrid background. Heterozygous mice were used as controls because they have normal NE levels (Thomas et al., 1998). Control and knockout mice were bred under specific pathogen-free conditions according to a protocol that allows production of the knockout animals (Thomas et al., 1995). Adult mice were housed in a modified

Sleep latency after gentle waking or saline injection

In this experiment, we investigated any potential differences in sleep latency between the genotypes according to method of waking, and between the morning/evening time points. When the mice were gently awakened (as described in Methods), there was no significant difference in latency to sleep between the genotypes either in the morning, or in the evening. However, when mice were handled and administered an intraperitoneal injection of saline (thereby introducing an element of stress), knockout

Discussion

We have shown previously that NE-deficient mice have a shorter sleep latency than controls after injection of low-dose amphetamine or saline (Hunsley and Palmiter, 2003). The results presented here suggest that this difference is mediated by some aspect of handling and/or injection, because when the mice were awakened using methods that were minimally stressful (no touching involved), there was no significant difference in latency to sleep between the genotypes. This was observed both at the

Acknowledgements

We acknowledge W. Curtis and M. Day for assistance with experimental procedures, and W. Curtis for animal care and data processing. We also thank C. Landis and the department of Biobehavioral Nursing at the University of Washington for the generous use of their animal sleep recording facilities, V. Denenberg for statistical consultation, D. Mills and E. Rubel for the use of their noise generating equipment, Sumitomo Pharmaceuticals for their donation of DOPS, and Cephalon for their donation of

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