Ping Taishi

The Role of Cytokines in Physiological Sleep Regulation

Abstract: Several growth factors (GFs) are implicated in sleep regulation. It is posited that these GFs are produced in response to neural activity and affect input‐output relationships within the neural circuits where they are produced, thereby inducing a local state shift. These GFs also influence synaptic efficacy. All the GFs currently identified as sleep regulatory substances are also implicated in synaptic plasticity. Among these substances, the most extensively studied for their role in sleep regulation are interleukin‐1β (IL‐1) and tumor necrosis factor a (TNF). Injection of IL‐1 or TNF enhances non‐rapid eye movement sleep (NREMS). Inhibition of either IL‐1 or TNF inhibits spontaneous sleep and the sleep rebound that occurs after sleep deprivation. Stimulation of the endogenous production of IL‐1 and TNF enhances NREMS. Brain levels of IL‐1 and TNF correlate with sleep propensity; for example, after sleep deprivation, their levels increase. IL‐1 and TNF are part of a complex biochemical cascade regulating sleep. Downstream events include nitric oxide, growth hormone releasing hormone, nerve growth factor, nuclear factor kappa B, and possibly adenosine and prostaglandins. Endogenous substances moderating the effects of IL‐1 and TNF include anti‐inflammatory cytokines such as IL‐4, IL‐10, and IL‐13. Clinical conditions altering IL‐1 or TNF activity are associated with changes in sleep, for example, infectious disease and sleep apnea. As our knowledge of the biochemical regulation of sleep progresses, our understanding of sleep function and of many clinical conditions will improve.

Involvement of cytokines in slow wave sleep

Cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) play a role in sleep regulation in health and disease. TNFα or IL1β injection enhances non-rapid eye movement sleep. Inhibition of TNFα or IL1β reduces spontaneous sleep. Mice lacking TNFα or IL1β receptors sleep less. In normal humans and in multiple disease states, plasma levels of TNFα covary with EEG slow wave activity (SWA) and sleep propensity. Many of the symptoms induced by sleep loss, for example, sleepiness, fatigue, poor cognition, enhanced sensitivity to pain, are elicited by injection of exogenous TNFα or IL1β. IL1β or TNFα applied unilaterally to the surface of the cortex induces state-dependent enhancement of EEG SWA ipsilaterally, suggesting greater regional sleep intensity. Interventions such as unilateral somatosensory stimulation enhance localized sleep EEG SWA, blood flow, and somatosensory cortical expression of IL1β and TNFα. State oscillations occur within cortical columns. One such state shares properties with whole animal sleep in that it is dependent on prior cellular activity, shows homeostasis, and is induced by TNFα. Extracellular ATP released during neuro- and gliotransmission enhances cytokine release via purine type 2 receptors. An ATP agonist enhances sleep, while ATP antagonists inhibit sleep. Mice lacking the P2X7 receptor have attenuated sleep rebound responses after sleep loss. TNFα and IL1β alter neuron sensitivity by changing neuromodulator/neurotransmitter receptor expression, allowing the neuron to scale its activity to the presynaptic neurons. TNFαs role in synaptic scaling is well characterized. Because the sensitivity of the postsynaptic neuron is changed, the same input will result in a different network output signal and this is a state change. The top-down paradigm of sleep regulation requires intentional action from sleep/wake regulatory brain circuits to initiate whole-organism sleep. This raises unresolved questions as to how such purposeful action might itself be initiated. In the new paradigm, sleep is initiated within networks and local sleep is a direct consequence of prior local cell activity. Whole-organism sleep is a bottom-up, self-organizing, and emergent property of the collective states of networks throughout the brain.

Spontaneous and influenza virus-induced sleep are altered in TNF-α double-receptor deficient mice

Tumor necrosis factor-alpha (TNF-alpha) is associated with sleep regulation in health and disease. Previous studies assessed sleep in mice genetically deficient in the TNF-alpha 55-kDa receptor. In this study, spontaneous and influenza virus-induced sleep profiles were assessed in mice deficient in both the 55-kDa and 75-kDa TNF-alpha receptors [TNF-2R knockouts (KO)] and wild-type (WT) strain controls. Under baseline conditions the TNF-2R KO mice had less non-rapid eye movement sleep (NREMS) than WTs during the nighttime and more rapid eye movement sleep (REMS) than controls during the daytime. The differences between nighttime maximum and daytime minimum values of electroencephalogram (EEG) delta power during NREMS were greater in the TNF-2R KO mice than in WTs. Viral challenge (mouse-adapted influenza X-31) enhanced NREMS and decreased REMS in both strains roughly to the same extent. EEG delta power responses to viral challenge differed substantially between strains; the WT animals increased, whereas the TNF-2R KO mice decreased their EEG delta wave power during NREMS. There were no differences between strains in body temperatures or locomotor activity in uninfected mice or after viral challenge. Analyses of cortical mRNAs confirmed that the TNF-2R KO mice lacked both TNF-alpha receptors; these mice also had higher levels of orexin mRNA and reduced levels of the purine P2X7 receptor compared with WTs. Results reinforce the hypothesis that TNF-alpha is involved in physiological sleep regulation but plays a limited role in the acute-phase response induced by influenza virus.

Central administration of the somatostatin analog octreotide induces captopril-insensitive sleep responses

The effects of intracerebroventricular injections of the long-lasting somatostatin analog octreotide (Oct) were studied on sleep and behavior in rats. Pyrogen-free physiological saline and Oct (0.001, 0.01, 0.1 microgram) or vehicle were administered at light onset, and the electroencephalogram (EEG), motor activity, and cortical brain temperature were recorded during the 12-h light period. Plasma growth hormone (GH) concentrations were measured in samples taken at 30-min intervals after Oct. Oct (0.01 and 0.1 microgram) suppressed non-rapid eye movement sleep (NREMS) for 1-2 h. NREMS intensity (delta EEG activity during NREMS) dose dependently increased in hour 3 postinjection and thereafter (0.1 microgram). Plasma GH concentrations were suppressed after Oct (0.01 and 0.1 microgram), but pulses of GH secretions occurred 90-120 min postinjection in each rat. Oct (0.1 microgram) enhanced behavioral activity, including prompt drinking followed by grooming, scratching, and feeding. Intracerebroventricular injection of the angiotensin-converting enzyme inhibitor captopril (30 microgram, 10 min before Oct), blocked these behavioral responses but not the Oct-induced sleep alterations. The changes in sleep after intracerebroventricular Oct suggest an intracerebral action site and might result from Oct-induced variations in the sleep-promoting activity of GH-releasing hormone.The effects of intracerebroventricular injections of the long-lasting somatostatin analog octreotide (Oct) were studied on sleep and behavior in rats. Pyrogen-free physiological saline and Oct (0.001, 0.01, 0.1 μg) or vehicle were administered at light onset, and the electroencephalogram (EEG), motor activity, and cortical brain temperature were recorded during the 12-h light period. Plasma growth hormone (GH) concentrations were measured in samples taken at 30-min intervals after Oct. Oct (0.01 and 0.1 μg) suppressed non-rapid eye movement sleep (NREMS) for 1-2 h. NREMS intensity (delta EEG activity during NREMS) dose dependently increased in hour 3 postinjection and thereafter (0.1 μg). Plasma GH concentrations were suppressed after Oct (0.01 and 0.1 μg), but pulses of GH secretions occurred 90-120 min postinjection in each rat. Oct (0.1 μg) enhanced behavioral activity, including prompt drinking followed by grooming, scratching, and feeding. Intracerebroventricular injection of the angiotensin-converting enzyme inhibitor captopril (30 μg, 10 min before Oct), blocked these behavioral responses but not the Oct-induced sleep alterations. The changes in sleep after intracerebroventricular Oct suggest an intracerebral action site and might result from Oct-induced variations in the sleep-promoting activity of GH-releasing hormone.

Changes in rat sleep after single and repeated injections of the long-acting somatostatin analog octreotide.

Somnogenic activity is attributed to both growth hormone (GH) and GH-releasing hormone (GHRH). The aim of our experiments was to study sleep after suppression of the somatotropic axis by means of administration of a long-lasting somatostatin analog, octreotide. Rats received subcutaneous injections of physiological saline (baseline), octreotide (1, 10, and 200 μg/kg), or a control solution just before light onset, and sleep-wake activity and cortical brain temperature were recorded for 23 h. Each dose of octreotide slightly promoted rapid eye movement sleep (REMS) during the 12-h light period. Non-REM sleep (NREMS) was strongly suppressed for 1 h in response to 10 and 200 μg/kg octreotide. This was followed by slight increases in NREMS time and significant enhancements in electroencephalogram slow-wave activity during NREMS after 200 μg/kg octreotide. The octreotide-induced inhibition of the somatotropic axis, as determined by plasma GH levels, vanished by the time sleep increased. Another group of rats received 10 μg/kg octreotide twice a day for 5 days. This treatment elicited persistent decreases in both NREMS time and NREMS intensity. The results support the previously reported REMS-promoting activity of somatostatin in rats. The decreases in sleep after repeated injections of octreotide are attributed to a withdrawal of the normal sleep-promoting activity of GH. The role of GHRH-GH in octreotide-induced acute suppression of NREMS is currently not clear. Other mechanisms, such as mimicking central transmitter functions of somatostatin by octreotide, should also be considered.

TNFα siRNA reduces brain TNF and EEG delta wave activity in rats

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFα were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFα siRNA treatment specifically reduced TNFα mRNA by 45% in vitro without affecting interleukin-6 or gluR1–4 mRNA levels. In vivo the TNFα siRNAα reduced TNFα mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFα-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFα siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFα siRNA attenuates TNFα mRNA and TNFα protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFα is involved in CNS physiology including sleep regulation.

Rapid Eye Movement Sleep Is Reduced in Prolactin-Deficient Mice

Prolactin (PRL) is implicated in the modulation of spontaneous rapid eye movement sleep (REMS). Previous models of hypoprolactinemic animals were characterized by changes in REMS, although associated deficits made it difficult to ascribe changes in REMS to reduced PRL. In the current studies, male PRL knock-out (KO) mice were used; these mice lack functional PRL but have no known additional deficits. Spontaneous REMS was reduced in the PRL KO mice compared with wild-type or heterozygous littermates. Infusion of PRL for 11-12 d into PRL KO mice restored their REMS to that occurring in wild-type or heterozygous controls. Six hours of sleep deprivation induced a non-REMS and a REMS rebound in both PRL KO mice and heterozygous littermates, although the REMS rebound in the KOs was substantially less. Vasoactive intestinal peptide (VIP) induced REMS responses in heterozygous mice but not in KO mice. Similarly, an ether stressor failed to enhance REMS in the PRL KOs but did in heterozygous littermates. Finally, hypothalamic mRNA levels for PRL, VIP, neural nitric oxide synthase (NOS), inducible NOS, and the interferon type I receptor were similar in KO and heterozygous mice. In contrast, tyrosine hydroxylase mRNA was lower in the PRL KO mice than in heterozygous controls and was restored to control values by infusion of PRL, suggesting a functioning short-loop negative feedback regulation in PRL KO mice. Data support the notion that PRL is involved in REMS regulation.

Vagotomy attenuates brain cytokines and sleep induced by peripherally administered tumor necrosis factor-α and lipopolysaccharide in mice.

STUDY OBJECTIVE Systemic tumor necrosis factor-α (TNF-α) is linked to sleep and sleep altering pathologies in humans. Evidence from animals indicates that systemic and brain TNF-α have a role in regulating sleep. In animals, TNF-α or lipopolysaccharide (LPS) enhance brain pro-inflammatory cytokine expression and sleep after central or peripheral administration. Vagotomy blocks enhanced sleep induced by systemic TNF-α and LPS in rats, suggesting that vagal afferent stimulation by TNF-α enhances pro-inflammatory cytokines in sleep-related brain areas. However, the effects of systemic TNF-α on brain cytokine expression and mouse sleep remain unknown. DESIGN We investigated the role of vagal afferents on brain cytokines and sleep after systemically applied TNF-α or LPS in mice. MEASUREMENTS AND RESULTS Spontaneous sleep was similar in vagotomized and sham-operated controls. Vagotomy attenuated TNF-α- and LPS-enhanced non-rapid eye movement sleep (NREMS); these effects were more evident after lower doses of these substances. Vagotomy did not affect rapid eye movement sleep responses to these substances. NREMS electroencephalogram delta power (0.5-4 Hz range) was suppressed after peripheral TNF-α or LPS injections, although vagotomy did not affect these responses. Compared to sham-operated controls, vagotomy did not affect liver cytokines. However, vagotomy attenuated interleukin-1 beta (IL-1β) and TNF-α mRNA brain levels after TNF-α, but not after LPS, compared to the sham-operated controls. CONCLUSIONS We conclude that vagal afferents mediate peripheral TNF-α-induced brain TNF-α and IL-1β mRNA expressions to affect sleep. We also conclude that vagal afferents alter sleep induced by peripheral pro-inflammatory stimuli in mice similar to those occurring in other species.

Whisker Stimulation increases expression of Nerve Growth Factor- and Interleukin-1β-immunoreactivity in the Rat Somatosensory Cortex

Activity-dependent changes in cortical protein expression may mediate long-term physiological processes such as sleep and neural connectivity. In this study we determined the number of nerve growth factor (NGF)- and interleukin-1beta (IL1beta)-immunoreactive (IR) cells in the somatosensory cortex (Sctx) in response to 2 h of mystacial whisker stimulation. Manual whisker stimulation for 2 h increased the number of NGF-IR cells within layers II-V in activated Sctx columns, identified by enhanced Fos-IR. IL1beta-IR neurons increased within layers II-III and V-VI in these activated columns and IL1beta-IR astrocytes increased in layers I, II-III and V as well as the external capsule beneath the activated columns. These whisker-stimulated increases in the Sctx did not occur in the auditory cortex. These data demonstrate that expression of NGF or IL1beta in Sctx neurons and IL1beta in Sctx astrocytes is, in part, afferent input-dependent.

Food Restriction Alters the Diurnal Distribution of Sleep in Rats

The purpose of the present study was to determine the effects of restricting food and water intake to the light period on sleep and brain temperature (Tbr). Sprague-Dawley male rats were anesthetized and provided with electrodes and thermistors for electroencephalographic (EEG) and Tbr recordings. Baseline recordings were performed after a 3-week recovery period. After baseline recordings, access to food and water was restricted (FWR) to the light period for 29 days. During FWR, the diurnal distribution of rapid-eye-movement sleep (REMS) and Tbr were reversed, while the distribution of non-REMS (NREMS) between the dark and light periods was attenuated. Daily food and water intake, body weight, and the diurnal distribution of EEG slow-wave activity within NREMS remained unchanged. In a separate study, sham-operated and pinealectomized rats were studied in a similar manner. The sleep responses of pinealectomized and sham-operated rats to FWR were similar. Further, FWR did not affect melatonin levels in the sham-operated rats, thereby suggesting that the pineal gland does not mediate the effects of FWR on sleep.