Neuropharmacology of Sleep and Wakefulness

γ-AMINOBUTYRIC ACID (GABA)

GABA is the major inhibitory neurotransmitter in the brain, and activation of GABA_A receptors causes neuronal inhibition by increasing chloride ion conductance. Because of their powerful inhibitory effects, GABA_A receptors are the targets of most sedative/hypnotic and general anesthetic drugs. GABA_A receptors exist as multiple subtypes (reviewed in⁹) and these subtypes are differentially located throughout the brain (reviewed in¹⁰). The differences in clinical effects caused by various benzodiazepine (e.g., diazepam) and non-benzodiazepine (e.g., eszopiclone) sedative/hypnotics are attributed to the relative selectivity of these drugs for different GABA_A receptor subtypes.¹⁰ The complexity imparted by the numerous GABA_A receptor subtypes is humbling. Although there is detailed knowledge about the many subunit isoforms that comprise GABA_A receptor subtypes,⁹ information is lacking about which of the many possible subtypes actually are expressed in specific brain regions,^11–13 and which subtypes are localized synaptically verses extrasynaptically.¹⁴ Extrasynaptically localized GABA_A receptors possess a delta subunit and have particular relevance for sleep medicine.^{15, 16}

A better understanding of the in vivo characteristics and anatomical localization of GABA_A receptor subtypes will contribute to rationale drug development. The preclinical studies described in this section illustrate the complexity of the problem and provide examples of how the effects of GABAergic drugs on behavior vary as a function of brain region. For example, although systemic administration of GABAmimetic drugs promotes sleep, sedation, or general anesthesia, enhancing GABAergic transmission within the pontine reticular formation actually increases wakefulness and decreases sleep. The pontine reticular formation is part of the ascending reticular activating system and contributes to the generation of REM sleep. Direct administration into the pontine reticular formation of drugs that increase GABAergic transmission increases wakefulness and inhibits sleep.^17–20 Similarly, pharmacologically increasing the concentration of endogenous GABA within the pontine reticular formation increases the time required for isoflurane to induce general anesthesia.²¹ Consistent with this finding are data showing that endogenous GABA levels in the pontine reticular formation are greater during wakefulness than during REM sleep²² or during the loss of wakefulness caused by isoflurane.²¹ Inhibiting GABAergic signaling at GABA_A receptors within the pontine reticular formation causes an increase in REM sleep and a decrease in wakefulness ^{18, 19, 23, 24}. Likewise, decreasing extracellular GABA levels in the pontine reticular formation of rat decreases wakefulness and increases sleep,²⁰ and shortens the time required for isoflurane to induce loss of consciousness.²¹ Furthermore, blocking GABA_A receptors in the pontine reticular formation increases time needed to regain wakefulness after isoflurane anesthesia.¹⁸ Considered together, these data demonstrate a wakefulness-promoting role for GABA in the pontine reticular formation.

In brain regions containing neurons that promote wakefulness, GABAergic inhibition has been shown to cause an increase in sleep. These brain regions include the dorsal raphé nucleus (Fig. 1; DRN), tuberomamillary nucleus of the posterior hypothalamus (Fig. 1; TMN), medial preoptic area (Fig. 1; MPO), and ventrolateral periaqueductal gray²⁵ (for reviews see7, 26, 27).

ACETYLCHOLINE

Acetylcholine is distinguished as being the first identified neurotransmitter. Although the first neurochemical theory of sleep²⁸ correctly posited that acetylcholine plays a primary role in generating the brain-activated states of wakefulness and REM sleep, cholinergic drugs are not part of the standard pharmacological armamentarium of sleep disorders medicine. Nonetheless, understanding the mechanisms by which cholinergic neurotransmission generates and maintains REM sleep is crucial, because acetylcholine interacts with other transmitter systems that are targets of sleep pharmacotherapy (e.g., GABAergic and monoaminergic). Much of the research on the regulation of sleep by acetylcholine has focused on transmission mediated by muscarinic cholinergic receptors. Five subtypes (M₁–M₅) of the muscarinic receptor have been identified,²⁹ and the M2 subtype plays a key role in the generation of REM sleep.³⁰

Cholinergic signaling originating from the laterodorsal tegmental and pedunculopontine tegmental nuclei (LDT/PPT) and the basal forebrain (see Fig. 1) promotes the cortically activated states of wakefulness and REM sleep (reviewed in³¹). LDT/PPT neurons can be divided into two populations based on discharge pattern. One population discharges maximally during wakefulness and REM sleep (referred to as Wake-On/REM-On) and another population fires only during wakefulness (Wake-On/REM-Off) (reviewed in²). This finding helps explain how acetylcholine can promote both wakefulness and REM sleep. LDT/PPT neurons project to numerous wakefulness-promoting brain regions.² Cholinergic terminals in the pontine reticular formation arise from the LDT/PPT,² and muscarinic receptors are present in the pontine reticular formation.^{30, 32, 33} Many studies have administered cholinomimetics to the pontine reticular formation and have demonstrated that cholinergic transmission in the pontine reticular formation induces REM sleep (reviewed in^{2, 31}). Electrically stimulating the LDT/PPT increases acetylcholine release in the pontine reticular formation³⁴ and increases REM sleep.³⁵ The release of endogenous acetylcholine in the pontine reticular formation is significantly greater during REM sleep than during wakefulness or NREM sleep.^36–38 Taken together, these data demonstrate that cholinergic projections from the LDT/PPT to the pontine reticular formation promote REM sleep.

Recent in vivo data obtained from normal rats demonstrate that the sedative/hypnotics zolpidem, diazepam, and eszopiclone differentially alter acetylcholine release in the pontine reticular formation.³⁹ Intravenous administration of eszopiclone prevented the REM phase of sleep, increased EEG delta power, and decreased acetylcholine release in rat pontine reticular formation.³⁹ These data provide the first functional evidence for a heterogeneous distribution of GABA_A receptor subtypes within the pontine reticular formation. The different effects of GABA_A receptor agonists on sleep have been attributed to brain region-specific distributions of GABA_A receptors and differences in sedative/hypnotic affinities for GABA_A receptor subtypes.⁴⁰ These preclinical data can be contrasted with human psychopharmacology where there has been no study convincingly demonstrating differential GABA_A subtype binding among benzodiazepine and non-benzodiazepine sleeping medications.⁴⁰ To date, the non-benzodiazepine, benzodiazepine-receptor agonist eszopiclone remains the only sleeping medication for which the long-term (6 months) effects have been characterized.^{41, 42}

Cholinergic neurons originating in the basal forebrain project throughout the entire cerebral cortex (reviewed in⁴³). Acetylcholine release in the basal forebrain is highest during REM sleep, lower during quiet wakefulness, and lowest during NREM sleep.⁴⁴ Cortical acetylcholine release is increased during wakefulness^{43, 45, 46} and REM sleep⁴⁵ as compared to NREM sleep. These support the interpretation that cholinergic transmission from the basal forebrain promotes cortical activation during wakefulness and REM sleep.

ADENOSINE

Adenosine is a breakdown product of adenosine triphosphate (ATP). Increases in endogenous adenosine levels in a specific brain region during a period of prolonged wakefulness indicate that the region has been metabolically active. Direct biochemical measures show that ATP levels increase during sleep in areas of the brain that are most active during wakefulness.⁴⁷ This finding provides direct support for the hypothesis that sleep serves a restorative function.⁴⁸

Four subtypes of adenosine receptors, A₁, A_2A, A_2B, and A₃, have been identified and are distributed widely throughout the brain. Adenosine A₁ and A_2A receptors are antagonized by caffeine and the idea that adenosine promotes sleep is supported by the ubiquitous consumption of caffeine to maintain wakefulness and enhance alertness. In humans, oral administration of caffeine prior to nocturnal sleep increases sleep latency and reduces sleep efficiency.⁴⁹ Furthermore, morning caffeine ingestion has been shown to decrease sleep efficiency and overall sleep during the subsequent night.⁵⁰ No adenosine agonists are presently available to promote sleep. Adenosine, however, is relevant for sleep medicine, as insomnia can be caused by consumption of caffeine or by the respiratory stimulant theophylline. Interestingly, adenosine can have analgesic effects and this action shows promise for clinical use.⁵¹

Adenosinergic transmission in brain regions that regulate sleep and wakefulness has been extensively investigated (reviewed in^{2, 52–54}). Activating adenosine A₁ receptors causes neuronal inhibition, and A₁ is the most abundant adenosine receptor subtype in brain. This section highlights selected studies supporting the interpretation that adenosine promotes sleep, at least in part, by inhibiting neurons in several key wakefulness-promoting brain areas.

Prolonged wakefulness increases adenosine levels selectively in the basal forebrain (Fig. 1; BF) and cortex,⁵⁵ and increases adenosine A₁ receptor binding in human⁵⁶ and rat⁵⁷ brain. Pharmacologically increasing adenosine levels in the basal forebrain⁵⁸ or administering adenosine A₁ receptor agonists to the basal forebrain⁵⁴ causes an increase in sleep. Inactivating adenosine A₁ receptors in the basal forebrain decreases EEG delta power and NREM sleep time,⁵⁹ and immunohistochemical studies reveal that the basal forebrain contains A₁ receptors, but not A_2A receptors.⁶⁰ Cholinergic neurons in the basal forebrain project to the cortex and contribute to the EEG activation characteristic of wakefulness and REM sleep. Adenosine directly inhibits cholinergic neurons in the basal forebrain by activating A₁ receptors.⁶¹ Adenosine indirectly inhibits wakefulness-promoting hypocretin (orexin)-containing neurons in the lateral hypothalamus (Fig. 1; LH) by activating A₁ receptors.⁶² Blocking adenosine A₁ receptors in the lateral hypothalamus causes an increase in wakefulness and a decrease in sleep.⁶³ Histaminergic neurons in the tuberomammillary nucleus (Fig. 1; TMN) express adenosine A₁ receptors, and activating those receptors increases NREM sleep.⁶⁴ These complementary data suggest that adenosine promotes sleep by inhibiting wakefulness-promoting neurons localized to the basal forebrain, lateral hypothalamus, and tuberomammillary nucleus.

Adenosine also exerts sleep-promoting effects by actions at the level of the prefrontal cortex (Fig. 1; PFC) and the pontine reticular formation (Fig. 1; PnO, PnC). In vivo microdialysis experiments in mouse⁶⁵ have shown that adenosine acting at A₁ receptors in the prefrontal cortex inhibits traits that characterize wakefulness (including acetylcholine release in the prefrontal cortex and activation of the EEG), as well as the state of wakefulness. Activation of adenosine A₁ receptors in the prefrontal cortex also causes a decrease in the release of acetylcholine in the pontine reticular formation. These findings demonstrate that in the prefrontal cortex, adenosine A₁ receptors mediate a descending inhibition of wakefulness-promoting systems. Within the pontine reticular formation, activation of adenosine A_2A receptors increases time needed to recover from general anesthesia,⁶⁶ increases acetylcholine release,^{66, 67} and increases the amount of time spent in NREM sleep⁶⁷ and REM sleep.^{67, 68} The increase in REM sleep may be a result of the A_2A-mediated increase in acetylcholine release, because coadministration of a muscarinic receptor antagonist with the A_2A agonist blocks the REM sleep increase.⁶⁸ Studies examining the effects on sleep of adenosine receptor antagonists are required in order to conclude that endogenous adenosine within the pontine reticular formation modulates sleep. The finding that clinically used opioids, such as morphine and fentanyl, decrease adenosine levels in the pontine reticular formation⁶⁹ and disrupt REM sleep (reviewed in⁷⁰) suggests the possibility that adenosinergic transmission within the pontine reticular formation participates in REM sleep generation.

BIOGENIC AMINES

The monoamines have long been known to promote wakefulness. Serotonin (5-hydroxytryptamine; 5HT)-containing neurons of the dorsal raphé nucleus (Fig. 1; DRN), norepinephrine-containing neurons of the locus coeruleus (Fig. 1; LC), and histamine-containing neurons of the tuberomammillary nucleus (Fig. 1; TMN) discharge at their fastest rates during wakefulness, slow their firing in NREM sleep, cease discharging prior to and during REM sleep, and resume firing prior to the onset of wakefulness (reviewed in²). Dopaminergic neurons, by contrast, do not show major changes in firing rates across the sleep-wakefulness cycle.

GLUTAMATE

Glutamate is the main excitatory neurotransmitter in the brain and acts at α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) ionotropic receptors. Surprisingly, little is known about glutamatergic regulation of sleep and wakefulness. Sleep state-dependent changes in levels of endogenous glutamate change differentially across the brain (see Table 8 of¹¹⁶). For example, glutamate levels in some areas of rat cortex show increases in concentration during wakefulness and REM sleep, and decreases during NREM sleep.¹¹⁷ Microinjection and electrophysiological studies provide evidence that glutamate acts within the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus^118–120 (Fig. 1; PPT), the pontine reticular formation^121–123 (Fig. 1; PnO, PnC), and medial portions of the medullary reticular formation^{122, 124} to modulate traits and states of arousal. Glutamatergic neurons are present in rat pontine reticular formation¹²⁵ and neurons in the pontine reticular formation are capable of synthesizing glutamate for use as a neurotransmitter.¹²⁶ Glutamate elicits excitatory responses from pontine reticular formation neurons,^{127, 128} and glutamatergic and cholinergic transmission in the pontine reticular formation interact synergistically to potentiate catalepsy.¹²⁹ Given individually, agonists for AMPA, kainate, and NMDA receptors evoke excitatory responses from pontine reticular formation neurons.¹²¹ Dialysis delivery of the NMDA receptor antagonists ketamine or MK-801 to cat pontine reticular formation decreases acetylcholine release in the pontine reticular formation and disrupts breathing.³⁸

PEPTIDES

Many peptides are known to modulate sleep (reviewed in¹³⁰). The present chapter focuses on hypocretin (orexin), leptin, and ghrelin because of their relevance for sleep disorders medicine.

OPIOIDS

Opioids are the major class of drugs used to treat acute and chronic pain, and one side effect of opioids is sleep disruption. Sleep disruption, in turn, exacerbates pain^{183, 184} and increases the dose of opioids required for successful pain management (reviewed in^{69, 70}). Clinically relevant doses of opioids given to otherwise healthy humans disrupt sleep (reviewed in¹⁸⁵). For example, a single intravenous infusion of morphine in healthy volunteers decreases stages 3 and 4 NREM sleep, decreases REM sleep, and increases stage 2 NREM sleep.¹⁸⁶ A nighttime dose of morphine or methadone also decreases stages 3 and 4 NREM sleep while increasing stage 2 NREM sleep.¹⁸⁷ Constant infusion of analgesic doses of remifentanil overnight decreases REM sleep in healthy volunteers.¹⁸⁸ The cycle of opioid-induced sleep disruption leading to increased pain and increased opioid requirement is recognized as a significant clinical problem that must be addressed at the mechanistic level.¹⁸⁹

Opioid-induced disruption of REM sleep is mediated, at least in part, by decreasing acetylcholine release in the pontine reticular formation.⁷⁰ Opioids also decrease adenosine levels in the basal forebrain and in the pontine reticular formation,⁶⁹ two brain regions where adenosine has sleep-promoting effects. Local administration of morphine into the pontine reticular formation of cat¹⁹⁰ or rat¹⁹¹ increases wakefulness and decreases REM sleep.

FUTURE DIRECTIONS

This selective overview was completed during the summer of 2010, a date also marking the 20^th anniversary of the human genome project. The stunning successes – and unmet hopes – of genomic approaches to medicine were highlighted in the June 12^th and 14^th issues of The New York Times.^{192, 193} These two articles offer a sobering reminder that taking a molecule from pre-clinical discovery to commercially available drug typically requires 15 or more years. This time interval is without any mandate to understand the mechanisms of drug action. As a former director of research and development at Wyeth noted¹⁹³ “Genomics did not speed up drug development. It gave us more rapid access to new molecular targets.” Potential molecular targets can be rapidly interrogated with high throughput screening programs that use a cell line transfected to contain a reporter construct. But identifying potential molecular targets leaves unanswered the question of whether the candidate targets will be druggable in vivo. This complexity is exemplified by sedative/hypnotic medications commonly used in sleep medicine. GABA_A receptors are drug targets that promotes a sleep-like state by unknown actions⁴⁰ when they are activated in some brain regions, yet GABA_A receptors enhance wakefulness when activated selectively in the posterior hypothalamus¹⁹⁴ or pontine reticular formation.^{18, 19, 21} As busy as Fig. 1 may seem, it barely hints at the complexity of data that must be logically integrated if we are to derive a coherent model of the endogenous neurochemical processes that regulate states of sleep and wakefulness.

Recent progress in understanding the basic neuropharmacology of sleep can be appreciated by comparing the 1990 and the 2005 editions of Brain Control of Wakefulness and Sleep.² The incorporation of basic neuropharmacology into sleep disorders medicine is readily apparent by comparing the first and most recent editions of Principles and Practice of Sleep Medicine.¹⁹⁵ Future progress is most likely to come from a systems biology approach that seeks to integrate genomic, cellular, network, and behavioral levels of analysis.¹⁹⁶ The focus on sleep medications in the Clinics of North America series demonstrates the cross-cutting relevance of sleep for the practice of medicine. The pressing clinical problem of sleep disorders medicine will continue to stimulate advances in understanding the neurochemical regulation of sleep.