The mammalian clock regulates major aspects of energy metabolism, including glucose and lipid homeostasis and mitochondrial oxidative metabolism. The biochemical basis for coordinated control of the circadian clock and diverse metabolic pathways is not well understood. Here we show that PGC-1alpha (Ppargc1a), a transcriptional coactivator that regulates energy metabolism, is rhythmically expressed in the liver and skeletal muscle of mice. PGC-1alpha stimulates the expression of clock genes, notably Bmal1 (Arntl) and Rev-erbalpha (Nr1d1), through coactivation of the ROR family of orphan nuclear receptors. Mice lacking PGC-1alpha show abnormal diurnal rhythms of activity, body temperature and metabolic rate. The disruption of physiological rhythms in these animals is correlated with aberrant expression of clock genes and those involved in energy metabolism. Analyses of PGC-1alpha-deficient fibroblasts and mice with liver-specific knockdown of PGC-1alpha indicate that it is required for cell-autonomous clock function. We have thus identified PGC-1alpha as a key component of the circadian oscillator that integrates the mammalian clock and energy metabolism.

The brain is assumed to be hypoactive during cardiac arrest. However, the neurophysiological state of the brain immediately following cardiac arrest has not been systematically investigated. In this study, we performed continuous electroencephalography in rats undergoing experimental cardiac arrest and analyzed changes in power density, coherence, directed connectivity, and cross-frequency coupling. We identified a transient surge of synchronous gamma oscillations that occurred within the first 30 s after cardiac arrest and preceded isoelectric electroencephalogram. Gamma oscillations during cardiac arrest were global and highly coherent; moreover, this frequency band exhibited a striking increase in anterior-posterior-directed connectivity and tight phase-coupling to both theta and alpha waves. High-frequency neurophysiological activity in the near-death state exceeded levels found during the conscious waking state. These data demonstrate that the mammalian brain can, albeit paradoxically, generate neural correlates of heightened conscious processing at near-death.global ischemia | global hypoxia | near-death experience | consciousness

Consciousness is determined both by level (e.g., being awake versus being anesthetized) and content (i.e., the qualitative aspects of experience). Subcortical areas are known to play a causal role in regulating the level of consciousness [1-9], but the role of the cortex is less well understood. Clinical and correlative data have been used both to support and refute a role for prefrontal and posterior cortices in the level of consciousness [10-22]. The prefrontal cortex has extensive reciprocal connections to wake-promoting centers in the brainstem and diencephalon [23, 24], and hence is in a unique position to modulate level of consciousness. Furthermore, a recent study suggested that the prefrontal cortex might be important in regulating level of consciousness [25] but causal evidence, and a comparison with more posterior cortical sites, is lacking. Therefore, to test the hypothesis that prefrontal cortex plays a role in regulating level of consciousness, we attempted to reverse sevoflurane anesthesia by cholinergic or noradrenergic stimulation of the prefrontal prelimbic cortex and two areas of parietal cortex in rat. General anesthesia was defined by loss of the righting reflex, a widely used surrogate measure in rodents. We demonstrate that cholinergic stimulation of prefrontal cortex, but not parietal cortex, restored wake-like behavior, despite continuous exposure to clinically relevant concentrations of sevoflurane anesthesia. Noradrenergic stimulation of the prefrontal and parietal areas resulted in electroencephalographic activation but failed to produce any signs of wake-like behavior. We conclude that cholinergic mechanisms in prefrontal cortex can regulate the level of consciousness.

Circadian melatonin production in the pineal gland and retina is under the control of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase. Because NAT activity varies diurnally, it has been considered both the melatonin rhythm-generating enzyme and the rate-limiting enzyme of melatonin synthesis. In rats with dramatically reduced NAT activity due to a H28Y mutation in NAT, melatonin levels remained the same as in wildtype controls, suggesting that NAT does not determine the rate of melatonin production at night. Using a combination of molecular approaches with a sensitive in vivo measurement of pineal diurnal melatonin production, we demonstrate that (i) N-acetylserotonin (NAS), the enzymatic product of NAT, is present in vast excess in the night pineals compared with melatonin; (ii) the continuous increase in NAT protein levels at late night does not produce a proportional increase in melatonin; and (iii) an increase in NAS in the same animal over several circadian cycles do not result in corresponding increase in melatonin output. These results strongly suggest that NAT is not the rate-limiting enzyme of melatonin formation at night.

The mechanism by which the healthy heart and brain die rapidly in the absence of oxygen is not well understood. We performed continuous electrocardiography and electroencephalography in rats undergoing experimental asphyxia and analyzed cortical release of core neurotransmitters, changes in brain and heart electrical activity, and brain-heart connectivity. Asphyxia stimulates a robust and sustained increase of functional and effective cortical connectivity, an immediate increase in cortical release of a large set of neurotransmitters, and a delayed activation of corticocardiac functional and effective connectivity that persists until the onset of ventricular fibrillation. Blocking the brain's autonomic outflow significantly delayed terminal ventricular fibrillation and lengthened the duration of detectable cortical activities despite the continued absence of oxygen. These results demonstrate that asphyxia activates a brainstorm, which accelerates premature death of the heart and the brain.asphyxic cardiac arrest | autonomic nervous system | coherence | directed connectivity | near-death experience

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCNs subregional organization to both photic input processing and rhythmic output control.circadian desynchronization ͉ dual oscillators ͉ suprachiasmatic I n mammals, circadian rhythms are governed by a master pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN) (1, 2). The SCN is a heterogeneous nucleus with major subregional differences in neurochemical phenotype, connectivity and patterns of gene expression (3-6). Light information is transmitted directly to the SCN via the retinohypothalamic tract (RHT) (7,8). In rats, RHT input is dense in the ventrolateral SCN (vlSCN), and relatively sparse in the dorsomedial SCN (dmSCN) (3). In this species, segregation of SCN afferents is paralleled by a segregation of efferent projections emerging from each subregion, and some SCN targets receive input from only the vl-or the dmSCN (9). This topographic organization of afferent and efferent projections suggests different roles for these subregions regarding processing of photic information and control of circadian outputs. Indeed, photic stimulation by light pulses applied during the subjective night or by abruptly shifting the light-dark (LD) cycle up-regulates expression of the clock gene Per1 in the vlSCN, inducing a transient desynchronization in gene expression between the two subregions (10-13). These data strongly suggest that the SCNЈs subregional organization is key to the processing of light information. Its role in the control of circadian outputs, however, is more difficult to assess and has been limited t...

Leading neuroscientific theories posit a central role for the functional integration of cortical areas in conscious states. Considerable evidence supporting this hypothesis is based on network changes during anesthesia, but it is unclear whether these changes represent state-related (conscious vs unconscious) or drug-related (anesthetic vs no anesthetic) effects. We recently demonstrated that carbachol delivery to prefrontal cortex (PFC) restored wakefulness despite continuous administration of the general anesthetic sevoflurane. By contrast, carbachol delivery to parietal cortex, or noradrenaline delivery to either prefrontal or parietal cortices, failed to restore wakefulness. Thus, carbachol-induced reversal of sevoflurane anesthesia represents a unique state that combines wakefulness with clinically relevant anesthetic concentrations in the brain. To differentiate the state-related and drug-related associations of cortical connectivity and dynamics, we analyzed the electroencephalographic data gathered from adult male Sprague Dawley rats during the aforementioned experiments for changes in functional cortical gamma connectivity (25-155 Hz), slow oscillations (0.5-1 Hz), and complexity (Ͻ175 Hz). We show that higher gamma (85-155 Hz) connectivity is decreased (p Յ 0.02) during sevoflurane anesthesia, an expected finding, but was not restored during wakefulness induced by carbachol delivery to PFC. Conversely, for rats in which wakefulness was not restored, the functional gamma connectivity remained reduced, but there was a significant decrease (p Ͻ 0.001) in the power of slow oscillations and increase (p Ͻ 0.001) in cortical complexity, which was similar to that observed during wakefulness induced after carbachol delivery to PFC. We conclude that the level of consciousness can be dissociated from cortical connectivity, oscillations, and dynamics.

Melatonin is a hormone secreted from the pineal gland specifically at night and contributes to a wide array of physiological functions in mammals. Melatonin is one of the most well understood output of the circadian clock located in the suprachiasmatic nucleus. Melatonin synthesis is controlled distally via the circadian clock located in the suprachiasmatic nucleus and proximally regulated by norepinephrine released in response to the circadian clock signals. To understand melatonin synthesis in vivo, we have performed microdialysis analysis of the pineal gland, which monitors melatonin as well as the precursor (serotonin) and intermediate (N-acetylserotonin) of melatonin synthesis in freely moving animals in realtime at high resolution. Our data revealed a number of novel features of melatonin production undetected using conventional techniques, which include (1) large interindividual variations of melatonin onset timing; (2) circadian regulation of serotonin synthesis and secretion in the pineal gland; and (3) a revised view on the rate-limiting step of melatonin formation in vivo. This article will summarize the main findings from our laboratory regarding melatonin formation in mammals.