Transcranial Direct Current Stimulation (tDCS) Ameliorates Stress-Induced Sleep Disruption via Activating Infralimbic-Ventrolateral Preoptic Projections.

Transcranial direct current stimulation (tDCS) is acknowledged for its non-invasive modulation of neuronal activity in psychiatric disorders. However, its application in insomnia research yields varied outcomes depending on different tDCS types and patient conditions. Our primary objective is to elucidate its efficiency and uncover the underlying mechanisms in insomnia treatment. We hypothesized that anodal prefrontal cortex stimulation activates glutamatergic projections from the infralimbic cortex (IL) to the ventrolateral preoptic area (VLPO) to promote sleep. After administering 0.06 mA of electrical currents for 8 min, our results indicate significant non-rapid eye movement (NREM) enhancement in naïve mice within the initial 3 h post-stimulation, persisting up to 16-24 h. In the insomnia group, tDCS enhanced NREM sleep bout numbers during acute stress response and improved NREM and REM sleep duration in subsequent acute insomnia. Sleep quality, assessed through NREM delta powers, remains unaffected. Interference of the IL-VLPO pathway, utilizing designer receptors exclusively activated by designer drugs (DREADDs) with the cre-DIO system, partially blocked tDCS's sleep improvement in stress-induced insomnia. This study elucidated that the activation of the IL-VLPO pathway mediates tDCS's effect on stress-induced insomnia. These findings support the understanding of tDCS effects on sleep disturbances, providing valuable insights for future research and clinical applications in sleep therapy.

Hypocretin role in posttraumatic stress disorder-like behaviors induced by a novel stress protocol in mice.

Introduction: Posttraumatic stress disorder (PTSD) is a psychiatric disorder developed in individuals who expose to traumatic events. These patients may experience symptoms, such as recurrent unwanted memory of the traumatic event, avoidance of reminders of the trauma, increased arousal, and cognitive difficulty. The hypocretinergic system originates from the lateral hypothalamic area (LHA) and projects diffusely to the whole brain, and hypocretin may be involved in the features of stress-related disorder, PTSD. Methods: Our study aimed to investigate the role of basolateral amygdala (BLA) hypocretin signals in the pathophysiology of PTSD-like symptoms induced by the modified multiple-prolonged stress (MPS) protocol. The BLA, a brain region involved in fear-related behaviors, receives the hypocretin projections. In this study, TCS1102, a dual hypocretin receptor antagonist, was used to block the hypocretin signal in BLA. Results: Our data indicated that the MPS protocol is a potential PTSD-like paradigm in mice. Meanwhile, the blockade of hypocretin signaling in the BLA relieved the MPS-induced fear response, and partially reduced PTSD-like anxiety behaviors performed by the open field test (OFT) and elevated plus maze (EPM) task. Discussion: Our findings suggest that the hypocretinergic system is a potential therapeutic approach for PTSD treatment. With further research, the hypocretin-based medication can be a candidate for human PTSD treatment.

A prolonged stress rat model recapitulates some PTSD-like changes in sleep and neuronal connectivity.

Chronic post-traumatic stress disorder (PTSD) exhibits psychological abnormalities during fear memory processing in rodent models. To simulate long-term impaired fear extinction in PTSD patients, we constructed a seven-day model with multiple prolonged stress (MPS) by modifying manipulation repetitions, intensity, and unpredictability of stressors. Behavioral and neural changes following MPS conveyed longitudinal PTSD-like effects in rats for 6 weeks. Extended fear memory was estimated through fear retrieval induced-freezing behavior and increased long-term serum corticosterone concentrations after MPS manipulation. Additionally, memory retrieval and behavioral anxiety tasks continued enhancing theta oscillation activity in the prefrontal cortex-basal lateral amygdala-ventral hippocampus pathway for an extended period. Moreover, MPS and remote fear retrieval stimuli disrupted sleep-wake activities to consolidate fear memory. Our prolonged fear memory, neuronal connectivity, anxiety, and sleep alteration results demonstrated integrated chronic PTSD symptoms in an MPS-induced rodent model.

Hypocretin in locus coeruleus and dorsal raphe nucleus mediates inescapable footshock stimulation (IFS)-induced REM sleep alteration.

Hypocretin (hcrt) is a stress-reacting neuropeptide mediating arousal and energy homeostasis. An inescapable footshock stimulation (IFS) could initiate the hcrt release from the lateral hypothalamus (LHA) and suppresses rapid eye movement (REM) sleep in rodents. However, the effects of the IFS-induced hcrts on REM-off nuclei, the locus coeruleus (LC) and dorsal raphe nucleus (DRN), remained unclear. We hypothesized that the hcrt projections from the LHA to LC or DRN mediate IFS-induced sleep disruption. Our results demonstrated that the IFS increased hcrt expression and the neuronal activities in the LHA, hypothalamus, brainstem, thalamus, and amygdala. Suppressions of REM sleep and slow wave activity during non-REM (NREM) sleep caused by the high expression of hcrts were blocked when a nonspecific and dual hcrt receptor antagonist was administered into the LC or DRN. Furthermore, the IFS also caused an elevated innate anxiety, but was limitedly influenced by the hcrt antagonist. This result suggests that the increased hcrt concentrations in the LC and DRN mediate stress-induced sleep disruptions and might partially involve IFS-induced anxiety.

Effects of N6 -(4-hydroxybenzyl) adenine riboside in stress-induced insomnia in rodents.

Adenosine exhibits a somnogenic effect; however, there is no adenosinergic hypnotic because of cardiovascular effects. This study investigated whether N6-(4-hydroxybenzyl) adenine riboside (T1-11), extracted from Gastrodia elata, produces somnogenic effects in rodents. We determined the involvement of adenosine 2A receptors (A2ARs) in GABAergic neurons of the ventrolateral preoptic area (VLPO) and the cardiovascular effects. Change of cage bedding is employed as a stressor to induce insomnia in rodents, and electroencephalograms and electromyograms were used to acquire and analyse sleep-wake activity. We found that intracerebroventricular administration of T1-11 before a dark period increased non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during a dark period, and T1-11-induced sleep increases were blocked by the A2AR antagonist, SCH58261, in naïve rats. Oral administration of T1-11 increased NREM sleep during both dark and light periods. Microinjection of the A2AR antagonist, SCH58261, into the VLPO blocked sleep effects of T1-11. In addition to the somnogenic effect in naïve mice, T1-11 suppressed the stress-induced insomnia and this suppressive effect was blocked by SCH58261. C-fos expression in GABAergic neurons of VLPO was increased after administration of T1-11 in Gad2-Cre::Ai14 mice, suggesting the activation of GABAergic neurons in the VLPO. T1-11 exhibited no effects on heart rate and the low frequency/high frequency ratio of heart rate variability. We concluded that T1-11 elicited somnogenic effects and effectively ameliorated acute stress-induced insomnia. The somnogenic effect is mediated by A2ARs to activate GABAergic neurons in the VLPO. This adenosine analogue could be a potential hypnotic because of no sympathetic and parasympathetic effects on the cardiovascular system.

Deep Brain Stimulation Increases Seizure Threshold by Altering REM Sleep and Delta Powers During NREM Sleep.

We previously demonstrated that seizure occurrences at different zeitgeber times alter sleep and circadian rhythm differently. On the other hand, the synchronized delta wave of electroencephalogram (EEG) during non-rapid eye movement (NREM) sleep facilitates seizure, while the desynchronized EEG of rapid eye movement (REM) sleep suppresses it. We also elucidated that unilateral deep brain stimulation (DBS) of the anterior nucleus of thalamus (ANT) suppresses seizure recurrence. In the present study, we intraperitoneally injected pentylenetetrazol (PTZ, 40 mg/kg) for 14 consecutive days (PTZ kindling) to induce spontaneous seizure in rats, and a 30-min (delivered 10 min before each PTZ injection) or a 3-h DBS of unilateral ANT (delivered 1 h before each PTZ injection) was applied to suppress seizure. The frequency of DBS stimulation was 200 Hz and the electrical current consisted of biphasic square pulses with 50-µA intensity, 100-µs pulse width, and 4.1-ms stimulation interval. Our results found that PTZ-induced spontaneous seizure did not cause a significant change in the quantity of NREM sleep but suppressed the amount of REM sleep. Unilateral ANT DBS prolonged the onset latency of ictal seizure, decreased the spontaneous seizure duration, and increased the survival rate but did not change the amplitude of epileptiform EEGs during ictal period. Unilateral ANT DBS did not significantly alter NREM sleep but increased the amount of REM sleep. An analysis of the spectrograms of fast Fourier transform indicated that the intensities of all frequencies were enhanced during the PTZ-induced ictal period and the subsequent spontaneous seizure. Thirty minutes of unilateral ANT DBS suppressed the augmentation of low-frequency (<10 Hz) intensities during the spontaneous seizure induced by PTZ kindling. We further found that consecutive injections of PTZ progressively increased the enhancement of the delta powers during NREM sleep, whereas unilateral ANT DBS inhibited this progressive enhancement. It was also noticed that 30 min of ANT DBS exhibited a better efficacy in epilepsy suppression than 3 h of ANT DBS. These results elucidated that unilateral ANT DBS enhanced the seizure threshold by increasing the amount of REM sleep and decreasing the progressive enhancement of delta power during NREM sleep to suppress spontaneous seizure recurrences in PTZ kindling-induced epileptic rats.

Hypocretin in median raphe nucleus modulates footshock stimuli-induced REM sleep alteration.

Stress is one of major factors that cause sleep problems. Hypocretin represents a stress-related neuropeptide and is well known in maintaining physiological wakefulness. The hypocretinergic neurons originate in the lateral hypothalamic area (LHA) and transmit to several brain regions, including the median raphe nuclei (MRNs). The MRNs modulate both fear responses and sleep-wake activity; however, it remains unclear whether stress alters the levels of hypocretin to regulate MRNs and consequently disrupt sleep. In this paper, we employed the inescapable footshock stimuli (IFS) as a stressor and hypothesized that the IFS-induced sleep disruption is mediated by increased hypocretins in the MRNs. Our results demonstrate that the concentrations of hypocretin in the hypothalamus increased after IFS. Rapid eye movement (REM) sleep was reduced after footshock, and microinjection of non-selective hypocretin receptor antagonist TCS-1102 into the MRNs blocked the IFS-induced decrease of REM sleep. Furthermore, administration of hypocretins into the MRNs mimicked the IFS-induced REM sleep reduction. These results conclude that the increased levels of hypocretins in the MRNs mediate the IFS-induced REM sleep reduction.

Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

BACKGROUND: Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. RESULTS: Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. CONCLUSIONS: These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Low-frequency electroacupuncture suppresses focal epilepsy and improves epilepsy-induced sleep disruptions.

BACKGROUND: The positive effects of acupuncture at Feng-Chi acupoints on treating epilepsy and insomnia have been well-documented in ancient Chinese literature. However, there is a lack of scientific evidence to elucidate the underlying mechanisms behind these effects. Our previous study demonstrated that high-frequency (100 Hz) electroacupuncture (EA) at Feng-Chi acupoints deteriorates both pilocarpine-induced focal epilepsy and sleep disruptions. This study investigated the effects of low-frequency (10 Hz) EA on epileptic activities and epilepsy-induced sleep disruptions. RESULTS: In rats, the Feng-Chi acupoint is located 3 mm away from the center of a line between the two ears. Rats received 30 min of 10 Hz EA stimuli per day before each day's dark period for three consecutive days. Our results indicated that administration of pilocarpine into the left CeA at the beginning of the dark period induced focal epilepsy and decreased both rapid eye movement (REM) sleep and non-REM (NREM) sleep during the consequent light period. Low-frequency (10 Hz) EA at Feng-Chi acupoints suppressed pilocarpine-induced epileptiform EEGs, and this effect was in turn blocked by naloxone (a broad-spectrum opioid receptor antagonist), but not by naloxonazine (a µ-receptor antagonist), naltrindole (a δ-receptor antagonist) and nor-binaltorphimine (a κ-receptor antagonist). Ten Hz EA enhanced NREM sleep during the dark period, and this enhancement was blocked by all of the opioid receptor antagonists. On the other hand, 10 Hz EA reversed pilocarpine-induced NREM suppression during the light period, and the EA's effect on the sleep disruption was only blocked by naloxonazine. CONCLUSIONS: These results indicate that low-frequency EA stimulation of Feng-Chi acupoints is beneficial in improving epilepsy and epilepsy-induced sleep disruptions, and that opioid receptors in the CeA mediate EA's therapeutic effects.

Amygdala opioid receptors mediate the electroacupuncture-induced deterioration of sleep disruptions in epilepsy rats.

BACKGROUND: Clinical and experimental evidence demonstrates that sleep and epilepsy reciprocally affect each other. Previous studies indicated that epilepsy alters sleep homeostasis; in contrast, sleep disturbance deteriorates epilepsy. If a therapy possesses both epilepsy suppression and sleep improvement, it would be the priority choice for seizure control. Effects of acupuncture of Feng-Chi (GB20) acupoints on epilepsy suppression and insomnia treatment have been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot). Therefore, this study was designed to investigate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi acupoints on sleep disruptions in rats with focal epilepsy. RESULTS: Our result indicates that administration of pilocarpine into the left central nucleus of amygdala (CeA) induced focal epilepsy and decreased both rapid eye movement (REM) sleep and non-REM (NREM) sleep. High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints, in which a 30-min EA stimulation was performed before the dark period of the light:dark cycle in three consecutive days, further deteriorated pilocarpine-induced sleep disruptions. The EA-induced exacerbation of sleep disruption was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting the involvement of amygdaloid opioid receptors. CONCLUSION: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints exhibits no benefit in improving pilocarpine-induced sleep disruptions; in contrast, EA further deteriorated sleep disturbances. Opioid receptors in the CeA mediated EA-induced exacerbation of sleep disruptions in epileptic rats.

Activation of amygdala opioid receptors by electroacupuncture of Feng-Chi (GB20) acupoints exacerbates focal epilepsy.

BACKGROUND: The effect of seizure suppression by acupuncture of Feng-Chi (GB20) acupoints has been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot), however, there is a lack of scientific evidence to prove it. This current study was designed to elucidate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi (GB20) acupoints on the epileptic activity by employing an animal model of focal epilepsy. METHODS: Administration of pilocarpine into the left central nucleus of amygdala (CeA) induced the focal epilepsy in rats. Rats received a 30-min 100 Hz EA stimulation of bilateral Feng-Chi acupoints per day, beginning at 30 minutes before the dark period and performing in three consecutive days. The broad-spectrum opioid receptor antagonist (naloxone), µ-receptor antagonist (naloxonazine), δ-receptor antagonist (naltrindole) and κ-receptor antagonist (nor-binaltorphimine) were administered directly into the CeA to elucidate the involvement of CeA opioid receptors in the EA effect. RESULTS: High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints did not suppress the pilocarpine-induced epileptiform electroencephalograms (EEGs), whereas it further increased the duration of epileptiform EEGs. We also observed that epilepsy occurred while 100 Hz EA stimulation of Feng-Chi acupoints was delivered into naïve rats. EA-induced augmentation of epileptic activity was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting that activation of opioid receptors in the CeA mediates EA-exacerbated epilepsy. CONCLUSIONS: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints has no effect to protect against pilocarpine-induced focal epilepsy; in contrast, EA further exacerbated focal epilepsy induced by pilocarpine. Opioid receptors in the CeA mediated EA-induced exacerbation of focal epilepsy.

Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats.

Theta rhythms generated in the hippocampus are controlled by the pacemaker in the medial septum-diagonal band of Broca (MS-DBB). The median raphe nucleus (MRN) transmits serotonergic signals to the MS-DBB, which suppresses the septo-hippocampus-produced theta waves, whereas GABAergic interneurons in the MRN facilitate the generation of theta oscillations. Animal studies have indicated that fear increases theta oscillations. Moreover, anxiolytics reduce reticular formation-elicited theta rhythms and theta blockade decreases anxiety. In this study, we hypothesized that the MRN mediates anxiety reduction caused by the theta blockade. Our results demonstrated that inescapable-footshock stimulation significantly increased the power of low-frequency theta oscillations (4-7 Hz) in rats. Both the electrical stimulation of MRN and administration of bicuculline into the MRN successfully desynchronized footshock-induced theta oscillations. Compared to the naïve rats, inescapable-footshock stimulation diminished the entry percentage and time spent in the open arms of the elevated plus maze (EPM), behavioral indicators of anxiety. Rats treated with either MRN stimulation or bicuculline administration to desynchronize theta oscillations reduced anxiety caused by the inescapable-footshock stimulation. Our results demonstrated that the electrical stimulation of MRN or blockade of the GABAergic pathways in the MRN interferes with theta oscillations and reduces anxiety, implicating the role of MRN.

Electrical stimulation of left anterior thalamic nucleus with high-frequency and low-intensity currents reduces the rate of pilocarpine-induced epilepsy in rats.

PURPOSE: Bilateral electrical stimulation of anterior nuclei of thalamus (ANT) has shown promising effects on epileptic seizures. However, bilateral implantation increases the risk of surgical complications and side effects. This study was undertaken to access the effectiveness of a stimulation paradigm involving high frequency and low intensity currents to stimulate the left ANT in rats. METHODS: Male Sprague-Dawley rats were implanted with electroencephalogram (EEG) electrodes, and an additional concentric bipolar stimulation electrode into either the left or right ANT. The stimulus was a train of pulses (90 µs duration each) delivered with a frequency of 200 Hz and a current intensity of 50 µA. Thalamic stimuli were started 1 h before the first intraperitoneal pilocarpine injection (i.p., 300 mg/kg), and were applied for 5 h. RESULTS: EEG documented seizure activity and status epilepticus (SE) developed in 87.5% of rats treated with no ANT stimulation after a single dose of pilocarpine. Left ANT stimulation significantly increased the tolerance threshold for pilocarpine-induced EEG seizure activity; 20% of rats developed their EEG documented seizure activity after receiving the first dose, whereas 50%, 10% and 20% of rats did not develop seizure activity until they had received the 2nd, 3rd and 4th pilocarpine injection at 1-h intervals. Moreover, left thalamic stimulation reduced the occurrences of both EEG documented seizure activity and SE induced by single-dose pilocarpine to 25%. However, our result demonstrated that little effect on the occurrence rate of seizures and SE was found when rats received right ANT stimulation. CONCLUSIONS: These results suggest that continuously 5-h left ANT stimulation with high frequency and low intensity currents, beginning from 1h before the pilocarpine administration, may successfully reduce the occurrence rate of EEG documented seizure activity and SE development in rats.

Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats.

STUDY OBJECTIVES: Controversial sleep disruptions (e.g., poor nighttime sleep and daytime somnolence) are common in epilepsy patients. Sleep is known to be regulated by homeostatic factors, which mediate sleep propensity, and the circadian oscillator, a clocklike mechanism. However, it is unknown how epileptic episodes that occur at different zeitgeber times (ZTs) alter sleep regulation. This study was designed to elucidate the sleep disruptions associated with epilepsy and their underlying mechanisms by delivering kindled epilepsy at different ZTs: ZT0, ZT6, and ZT13. DESIGN: Kindled epilepsy was induced at 3 different ZTs, and sleep-wake activities were analyzed before and after full-blown seizure. Ribonuclease protection assay, radioimmunoassay, and immunohistochemistry were respectively employed to determine the levels of interleukin-1 mRNA, corticosterone, and PER1 protein. SETTING: The experiments were performed at Neurophysiology Laboratory at National Taiwan University. PARTICIPANT AND INTERVENTIONS: Male Sprague-Dawley rats were implanted with electroencephalograph (EEG) electrodes, a bipolar stimulating electrode, and a guide cannula. Kindling stimuli were delivered via a bipolar electrode placed in the right central nucleus of the amygdala. MEASUREMENT AND RESULTS: Kindled epilepsy occurring at ZT0 and ZT13 predominantly affected homeostatic factors, whereas ZT6-kindling stimuli altered the circadian oscillator. ZT0-kindling decreased rapid eye movement (REM) and non-REM (NREM) sleep, which was mediated by corticotrophin-releasing hormone, but did not alter the rhythm of sleep-wake fluctuation. On the other hand, ZT13-kindling enhanced interleukin-1 and consequently increased NREM sleep without altering the sleep-wake fluctuation. Nevertheless, the expression of PER1 protein in suprachiasmatic nucleus of the hypothalamus and the circadian rhythm of sleep fluctuation were respectively advanced 6 h and 2 h when kindling stimulation was delivered at ZT6. Shifts of sleep circadian rhythm and PER1 oscillation induced by ZT6-kindling were blocked by administration of hypocretin receptor antagonist SB334867 into the SCN, indicating the involvement of hypocretin. CONCLUSION: These observations suggest that the occurrence of epilepsy at different ZTs alters sleep processes differently. CITATION: Yi PL; Chen YJ; Lin CT; Chang FC. Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats. SLEEP 2012;35(12):1651-1665.

Activation of GABAergic pathway by hypocretin in the median raphe nucleus (MRN) mediates stress-induced theta rhythm in rats.

The frequency of electroencephalograms (EEGs) is predominant in theta rhythm during stress (e.g., footshock) in rats. Median raphe nucleus (MRN) desynchronizes hippocampal theta waves via activation of GABAergic neurons in the medial septum-diagonal band of Broca (MS-DBB), a theta rhythm pacemaker. Increased hypocretin mediates stress responses in addition to the maintenance of wakefulness. Hypocretin receptors are abundant in the MRN, suggesting a possible role of hypocretin in modulating stress-induced theta rhythm. Our results indicated that the intensity of theta waves was enhanced by footshock and that a hypocretin receptor antagonist (TCS1102) suppressed the footshock-induced theta waves. Administration of hypocretin-1 (1 and 10 µg) and hypocretin-2 (10 µg) directly into the MRN simulated the effect of footshock and significantly increased theta waves. Co-administration of GABA(A) receptor antagonist, bicuculline, into the MRN blocked the increase of theta waves induced by hypocretins or footshock. These results suggested that stress enhances the release of hypocretins, activates GABAergic neurons in the MRN, blocks the ability of MRN to desynchronize theta waves, and subsequently increases the intensity of theta rhythm.

Kappa-opioid receptors in the caudal nucleus tractus solitarius mediate 100 hz electroacupuncture-induced sleep activities in rats.

Previous results demonstrated that 10 Hz electroacupuncture (EA) of Anmian acupoints in rats during the dark period enhances slow wave sleep (SWS), which involves the induction of cholinergic activity in the caudal nucleus tractus solitarius (NTS) and subsequent activation of opioidergic neurons and µ-receptors. Studies have shown that different kinds of endogenous opiate peptides and receptors may mediate the consequences of EA with different frequencies. Herein, we further elucidated that high-frequency (100 Hz)-EA of Anmian enhanced SWS during the dark period but exhibited no direct effect on rapid eye movement (REM) sleep. High-frequency EA-induced SWS enhancement was dose-dependently blocked by microinjection of naloxone or κ-receptor antagonist (nor-binaltorphimine) into the caudal NTS, but was affected neither by µ- (naloxonazine) nor δ-receptor antagonists (natatrindole), suggesting the role of NTS κ-receptors in the high-frequency EA-induced SWS enhancement. Current and previous results depict the opioid mechanisms of EA-induced sleep.

Effect of cannabidiol on sleep disruption induced by the repeated combination tests consisting of open field and elevated plus-maze in rats.

Patients with post-traumatic stress disorder (PTSD) frequently complain of having sleep disturbances, such as insomnia and rapid eye movement (REM) sleep abnormality. Cannabidiol (CBD), a psycho-inactive constituent of marijuana, reduces physiological non-REM (NREM) sleep and REM sleep in normal rats, in addition to generating its anxiolytic effect. However, the effects of CBD on anxiety-induced sleep disturbances remain unclear. Because anxiety progression is caused by persistent stress for a period of time, we employed the repeated combination tests (RCT) consisting of a 50-min open field (OF) and a subsequent 10-min elevated plus-maze (EPM) for four consecutive days to simulate the development of anxiety. Time spent in the centre arena of OF and during open arms of the EPM was substantially decreased in latter days of RCT, suggesting the habituation, which potentially lessens anxiety-mediated behavioural responses, was not observed in current tests. CBD microinjected into the central nucleus of amygdala (CeA) significantly enhanced time spent in centre arena of OF, increased time during the open arms and decreased frequency of entry to the enclosed arms of EPM, further confirming its anxiolytic effect. The decrease of NREM sleep during the first hour and the suppression of REM sleep during hours 4-10 after the RCT represent the similar clinical observations (e.g. insomnia and REM sleep interruption) in PTSD patients. CBD efficiently blocked anxiety-induced REM sleep suppression, but had little effect on the alteration of NREM sleep. Conclusively, CBD may block anxiety-induced REM sleep alteration via its anxiolytic effect, rather than via sleep regulation per se. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Biphasic effects of baicalin, an active constituent of Scutellaria baicalensis Georgi, in the spontaneous sleep-wake regulation.

AIM OF THE STUDY: Baicalin is an active compound originating from the root of Scutellaria baicalensis Georgi, which has been used for anti-inflammation, anti-bacteria, anti-hypertension, anti-allergy and sedation since ancient China, though the neuronal mechanisms involved in the sedative effect is still unclear. Baicalin possesses the ability to decrease the expression of pro-inflammatory cytokines and nuclear factor (NF)-κB activity. Furthermore, baicalin has demonstrated an anxiolytic-like effect via activation of γ-aminobutyric acid-A (GABA(A)) receptors. Pro-inflammatory cytokines (e.g. interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α) and the GABAergic system promote sleep. This study was designed to determine whether the GABA(A) receptor activation and/or the suppression of pro-inflammatory cytokines mediate(s) baicalin-induced sleep alterations. MATERIALS AND METHODS: Baicalin was intracerebroventricularly (ICV) administered 20 min either prior to the beginning of the light period or before the onset of the dark period. Electroencephalogram (EEG) and gross body movement were acquired for sleep analysis. Pharmacological blockade of IL-1 and GABA(A) receptors were employed to elucidate the involvements of IL-1 and GABA(A) receptors in baicalin-induced sleep alterations. IL-1ß concentrations obtained after baicalin administration in several distinct brain regions were determined by ELISA. RESULTS: ICV administration of baicalin decreased slow wave sleep (SWS) during the first 2h of the light period. Rapid eye movement sleep (REMS) was not altered. The blockade of IL-1ß-induced SWS enhancement by baicalin suggests that the antagonism of IL-1 receptors is involved in baicalin-induced SWS decrement during the light period. However, IL-1ß concentrations during the light period were not altered after baicalin administration. In contrast, baicalin increased both SWS and REMS during hours 8-10 of the dark (active) period when baicalin was administered at the beginning of the dark period, and its effects were blocked by the GABA(A) receptor antagonist bicuculline. CONCLUSION: Baicalin exhibits biphasic effects on sleep-wake regulation; the decrease of SWS during the light period and increases of SWS and REMS during the dark period. Inhibition of IL-1 action and enhancement of GABA(A) receptor activity may mediate baicalin's effects during the light and dark period, respectively.

Endogenous opiates in the nucleus tractus solitarius mediate electroacupuncture-induced sleep activities in rats.

Electroacupuncture (EA) possesses various therapeutic effects, including alleviation of pain, reduction of inflammation and improvement of sleep disturbance. The mechanisms of EA on sleep improvement, however, remain to be determined. It has been stated in ancient Chinese literature that the Anmian (EX17) acupoint is one of the trigger points that alleviates insomnia. We previously demonstrated that EA stimulation of Anmian acupoints in rats during the dark period enhances non-rapid eye movement (NREM) sleep, which involves the induction of cholinergic activity in the nucleus tractus solitarius (NTS). In addition to cholinergic activation of the NTS, activation of the endogenous opioidergic system may also be a mechanism by which acupuncture affects sleep. Therefore, this study was designed to investigate the involvement of the NTS opioidergic system in EA-induced alterations in sleep. Our present results indicate that EA of Anmian acupoints increased NREM sleep, but not rapid eye movement sleep, during the dark period in rats. This enhancement in NREM sleep was dose-dependently blocked by microinjection of opioid receptor antagonist, naloxone, and the µ-opioid receptor antagonist, naloxonazine, into the NTS; administrations of δ-receptor antagonist, natrindole, and the κ-receptor antagonist, nor-binaltrophimine, however, did not affect EA-induced alterations in sleep. Furthermore, ß-endorphin was significantly increased in both the brainstem and hippocampus after the EA stimuli, an effect blocked by administration of the muscarinic antagonist scopolamine into the NTS. Our findings suggest that mechanisms of EA-induced NREM sleep enhancement may be mediated, in part, by cholinergic activation, stimulation of the opiodergic neurons to increase the concentrations of ß-endorphin and the involvement of the µ-opioid receptors.