Dale M. Edgar

Serotonin and the Mammalian Circadian System: I. In Vitro Phase Shifts by Serotonergic Agonists and Antagonists

The primary mammalian circadian clock, located in the suprachiasmatic nuclei (SCN), receives a major input from the raphe nuclei. The role of this input is largely unknown, and is the focus of this research. The SCN clock survives in vitro, where it produces a 24-hr rhythm in spontaneous neuronal activity that is sustained for at least three cycles. The sensitivity of the SCN clock to drugs can therefore be tested in vitro by determining whether various compounds alter the phase of this rhythm. We have previously shown that the nonspecific serotonin (5-HT) agonist quipazine resets the SCN clock in vitro, inducing phase advances in the daytime and phase delays at night. These results suggest that the 5-HT-ergic input from the raphe nuclei can modulate the phase of the SCN circadian clock. In this study we began by using autoradiography to determine that the SCN contain abundant 5-HT 1A and 5-HT1B receptors, very few 5-HT1C and 5-HT 2 receptors, and no 5-HT3 receptors. Next we investigated the ability of 5-HT-ergic agonists and antagonists to reset the clock in vitro, in order to determine what type or types of 5-HT receptor(s) are functionally linked to the SCN clock. We began by providing further evidence of 5-HT-ergic effects in the SCN. We found that 5-HT mimicked the effects of quipazine, whereas the nonspecific 5-HT antagonist metergoline blocked these effects, in both the day and night. Next we found that the 5-HT1A agonist 8-OH-DPAT, and to a lesser extent the 5-HT1A-1B agonist RU 24969, mimicked the effects of quipazine during the subjective daytime, whereas the 5-HT 1A antagonist NAN-190 blocked quipazines effects. None of the other specific agonists or antagonists we tried induced similar effects. This suggests that quipazine acts on 5-HT1A receptors in the daytime to advance the SCN clock. None of the specific agents we tried were able either to mimic or to block the actions of 5-HT or quipazine at circadian time 15. Thus, we were unable to determine the type of 5-HT receptor involved in nighttime phase delays by quipazine or 5-HT. However, since the dose-response curves for quipazine during the day and night are virtually identical, we hypothesize that the nighttime 5-HT receptor is a 5-HT1-like receptor.

A role for cryptochromes in sleep regulation

BackgroundThe cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes.ResultsUnder all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1ε did not change with sleep deprivation.ConclusionsThese results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

Serotonin and the Mammalian Circadian System: II. Phase-Shifting Rat Behavioral Rhythms with Serotonergic Agonists:

The suprachiasmatic nuclei (SCN) receive primary afferents from the median and dorsal raphe, but the role of these projections in circadian timekeeping is poorly understood. Studies of the SCN in vitro suggest that quipazine, a general serotonin (5-HT) receptor agonist, can produce circadian time-dependent phase advances and phase delays in circadian rhythms of neuronal activity. The present study addresses whether quipazine and the selective 5-HT 1A receptor agonist 8-OH-DPAT are similarly effective in vivo. Drinking and wheel-running patterns of male Wistar rats individually housed in constant darkness were monitored before and after subcutaneous administration of quipazine (5-10 mg/kg) at either circadian time (CT) 6 or CT 18, with and without running wheels available. Dose-dependent phase advances (20-180 min) were produced at CT 6. Significant phase shifts were not observed at CT 18. CT 6 quipazine-treated animals also showed a sustained and significant shortening of rhythm period (τ) following treatment (-0.28 hr; p < 0.002). τ shortening was inconsistently observed in CT 18 quipazine-treated rats. Neither quipazine-induced phase shifts nor τ effects were dependent on wheel-running activity per se. 8-OH-DPAT delivered via intracerebral ventricular treatment into the third ventricle (5 μl at 100 μM in saline) produced slightly smaller phase advances (20-90 min) at CT 6, but did not produce phase delays at CT 18 or changes in τ. These findings support in vitro evidence that 5-HT-ergic agonists can phase-shift the circadian pacemaker.

Hibernation in Black Bears: Independence of Metabolic Suppression from Body Temperature

Hibernating black bears suppress their metabolic rate to 25% of normal, but only slightly reduce their body temperature. Black bears hibernate for 5 to 7 months a year and, during this time, do not eat, drink, urinate, or defecate. We measured metabolic rate and body temperature in hibernating black bears and found that they suppress metabolism to 25% of basal rates while regulating body temperature from 30° to 36°C, in multiday cycles. Heart rates were reduced from 55 to as few as 9 beats per minute, with profound sinus arrhythmia. After returning to normal body temperature and emerging from dens, bears maintained a reduced metabolic rate for up to 3 weeks. The pronounced reduction and delayed recovery of metabolic rate in hibernating bears suggest that the majority of metabolic suppression during hibernation is independent of lowered body temperature.

Activity Feedback to the Mammalian Circadian Pacemaker: Influence on Observed Measures of Rhythm Period Length

In the mouse, activity is precisely timed by the circadian clock and is normally most intense in the early subjective night. Since vigorous activity (e.g., wheel running) is thought to induce phase shifts in rodents, the temporal placement of daily exercise/activity could be a determinant of observed circadian rhythm period. The relationship between spontaneous running-wheel activity and the circadian period of free-running rhythms was studied to assess this possibility. With ad libitum access to a running wheel, mice exhibited a free-running period (τ) of 23.43 ± 0.08 hr (mean ± SEM). When running wheels were locked, τ increased (23.88 ± 0.04 hr, p < 0.03), and restoration of ad libitum wheel running again produced a shorter period (τ = 23.56 ± 0.06 hr, p < 0.05). A survey of free- running activity patterns in a population of 100 mice revealed a significant correlation between the observed circadian period and the time of day in which spontaneous wheel running occurred (r = 0.7314, p < 0.0001). Significantly shorter periods were observed when running was concentrated at the beginning of the subjective night (τ = 23.23 ± 0.04), and longer periods were observed if mice ran late in the subjective night (τ = 23.89 ± 0.04), F (1, 99) = 34.96, p < 0.0001. It was previously believed that the period of the circadian clock was primarily responsive to externally imposed tonic or phasic events. Systematic influences of spontaneous exercise on τ demonstrate that physiological and/or behavioral determinants of circadian timekeeping exist as well.

Gene Expression in the Rat Brain during Sleep Deprivation and Recovery Sleep: An Affymetrix GeneChip® Study

Previous studies have demonstrated that macromolecular synthesis in the brain is modulated in association with the occurrence of sleep and wakefulness. Similarly, the spectral composition of electroencephalographic activity that occurs during sleep is dependent on the duration of prior wakefulness. Since this homeostatic relationship between wake and sleep is highly conserved across mammalian species, genes that are truly involved in the electroencephalographic response to sleep deprivation might be expected to be conserved across mammalian species. Therefore, in the rat cerebral cortex, we have studied the effects of sleep deprivation on the expression of immediate early gene and heat shock protein mRNAs previously shown to be upregulated in the mouse brain in sleep deprivation and in recovery sleep after sleep deprivation. We find that the molecular response to sleep deprivation and recovery sleep in the brain is highly conserved between these two mammalian species, at least in terms of expression of immediate early gene and heat shock protein family members. Using Affymetrix Neurobiology U34 GeneChips , we also screened the rat cerebral cortex, basal forebrain, and hypothalamus for other genes whose expression may be modulated by sleep deprivation or recovery sleep. We find that the response of the basal forebrain to sleep deprivation is more similar to that of the cerebral cortex than to the hypothalamus. Together, these results suggest that sleep-dependent changes in gene expression in the cerebral cortex are similar across rodent species and therefore may underlie sleep history-dependent changes in sleep electroencephalographic activity.

Sleep and circadian abnormalities in a transgenic mouse model of Alzheimer’s disease: A role for cholinergic transmission

The Tg2576 mouse model of Alzheimers disease (AD) exhibits age-dependent amyloid beta (Abeta) deposition in the brain. We studied electroencephalographically defined sleep and the circadian regulation of waking activities in Tg2576 mice to determine whether these animals exhibit sleep abnormalities akin to those in AD. In Tg2576 mice at all ages studied, the circadian period of wheel running rhythms in constant darkness was significantly longer than that of wild type mice. In addition, the increase in electroencephalographic delta (1-4 Hz) power that occurs during non-rapid eye movement sleep after sleep deprivation was blunted in Tg2576 mice relative to controls at all ages studied. Electroencephalographic power during non-rapid eye movement sleep was shifted to higher frequencies in plaque-bearing mice relative to controls. The wake-promoting efficacy of the acetylcholinesterase inhibitor donepezil was lower in plaque-bearing Tg2576 mice than in controls. Sleep abnormalities in Tg2576 mice may be due in part to a cholinergic deficit in these mice. At 22 months of age, two additional deficits emerged in female Tg2576 mice: time of day-dependent modulation of sleep was blunted relative to controls and rapid eye movement sleep as a percentage of time was lower in Tg2576 than in wild type controls. The rapid eye movement sleep deficit in 22 month-old female Tg2576 mice was abolished by brief passive immunization with an N-terminal antibody to Abeta. The Tg2576 model provides a uniquely powerful tool for studies on the pathophysiology of and treatments for sleep deficits and associated cholinergic abnormalities in AD.

Sleep homeostasis in suprachiasmatic nuclei-lesioned rats: effects of sleep deprivation and triazolam administration.

The electroencephalogram (EEG) and electromyogram of rats with lesions in the suprachiasmatic nuclei (SCNx) were recorded during two series of 24-h baseline, 6-h sleep deprivation (SD), and 24-h recovery. At recovery onset, rats were injected i.p. with vehicle (VEH) control solution or 0.4 mg/kg triazolam (TRZ) in a balanced crossover design. Consecutive 10-s epochs were scored for vigilance states and EEG power spectra were computed. Arousal states were uniformly distributed during 24-h baseline (wake 47% of recording time, non-rapid-eye movement sleep (nonREMS) 47%, REMS 7%), and EEG spectra (0-25 Hz) were devoid of significant trends. State-specific EEG power spectra profiles in SCNx rats were similar to those of intact animals reported previously. However, EEG delta power (0.5-3.5 Hz) of nonREMS was markedly lower in SCNx rats. Recovery from 6-h SD was characterised by a short-lasting reduction of REMS, and a long-lasting increase of nonREMS time at the cost of wakefulness. EEG delta power rebounded during the first 8 h in recovery, and fell below baseline level after 12 h in recovery. During 0-2 h TRZ recovery, rats spent more time in nonREMS with higher EEG slow wave activity as compared to the corresponding VEH recovery period. EEG slow wave activity fell below baseline levels 10 h after TRZ injection and termination of SD. We conclude that major features of homeostatic sleep EEG regulation are present in SCNx rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible glial role in the mammalian circadian clock.

The primary mammalian circadian clock is located within the suprachiasmatic nuclei (SCN), but the cellular organization of the clock is not yet known. We investigated the potential role of glial cells in the clock mechanism by determining whether disrupting glial activity affects the in vitro circadian rhythm of neuronal activity and the in vivo circadian activity rhythm in rats. We used two agents (octanol and halothane) that block gap junctions, and one (fluorocitrate) that inhibits glial metabolism. All three agents disrupted the circadian pattern of neuronal activity. Octanol flattened the rhythm at the highest concentration (200 microM) and induced a small phase delay at a lower concentration (66 microM). Halothane and fluorocitrate induced ultradian rhythmicity. Fluorocitrate injected into the SCN of an intact rat induced arrhythmicity for about 1 week, after which the rhythm reappeared with a 1.6 h delay. These results suggest that glia play an important role in the SCN circadian clock.

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a Novel Metabotropic Glutamate 2 Potentiator with Potential Anxiolytic/Antidepressant Properties: In Vivo Profiling Suggests a Link between Behavioral and Central Nervous System Neurochemical Changes

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC50 = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.