Abhishek Singh

REM sleep loss increases brain excitability: role of noradrenalin and its mechanism of action

Ever since the discovery of rapid eye movement sleep (REMS), studies have been undertaken to understand its necessity, function and mechanism of action on normal physiological processes as well as in pathological conditions. In this review, first, we briefly surveyed the literature which led us to hypothesise REMS maintains brain excitability. Thereafter, we present evidence from in vivo and in vitro studies tracing behavioural to cellular to molecular pathways showing REMS deprivation (REMSD) increases noradrenaline level in the brain, which stimulates neuronal Na-K ATPase, the key factor for maintaining neuronal excitability, the fundamental property of a neuron for executing brain functions; we also show for the first time the role of glia in maintaining ionic homeostasis in the brain. As REMSD exerts a global effect on most of the physiological processes regulated by the brain, we propose that REMS possibly serves a housekeeping function in the brain. Finally, subject to confirmation from clinical studies, based on the results reviewed here, it is being proposed that the subjects suffering from REMS loss may be effectively treated by reducing either noradrenaline level or Na-K ATPase activity in the brain.

Activation of inactivation process initiates rapid eye movement sleep.

Interactions among REM-ON and REM-OFF neurons form the basic scaffold for rapid eye movement sleep (REMS) regulation; however, precise mechanism of their activation and cessation, respectively, was unclear. Locus coeruleus (LC) noradrenalin (NA)-ergic neurons are REM-OFF type and receive GABA-ergic inputs among others. GABA acts postsynaptically on the NA-ergic REM-OFF neurons in the LC and presynaptically on the latters projection terminals and modulates NA-release on the REM-ON neurons. Normally during wakefulness and non-REMS continuous release of NA from the REM-OFF neurons, which however, is reduced during the latter phase, inhibits the REM-ON neurons and prevents REMS. At this stage GABA from substantia nigra pars reticulate acting presynaptically on NA-ergic terminals on REM-ON neurons withdraws NA-release causing the REM-ON neurons to escape inhibition and being active, may be even momentarily. A working-model showing neurochemical-map explaining activation of inactivation process, showing contribution of GABA-ergic presynaptic inhibition in withdrawing NA-release and dis-inhibition induced activation of REM-ON neurons, which in turn activates other GABA-ergic neurons and shutting-off REM-OFF neurons for the initiation of REMS-generation has been explained. Our model satisfactorily explains yet unexplained puzzles (i) why normally REMS does not appear during waking, rather, appears following non-REMS; (ii) why cessation of LC-NA-ergic-REM-OFF neurons is essential for REMS-generation; (iii) factor(s) which does not allow cessation of REM-OFF neurons causes REMS-loss; (iv) the association of changes in levels of GABA and NA in the brain during REMS and its deprivation and associated symptoms; v) why often dreams are associated with REMS.

Involvement of the α1-adrenoceptor in sleep–waking and sleep loss-induced anxiety behavior in zebrafish

Sleep is a universal phenomenon in vertebrates, and its loss affects various behaviors. Independent studies have reported that sleep loss increases anxiety; however, the detailed mechanism is unknown. Because sleep deprivation increases noradrenalin (NA), which modulates many behaviors and induces patho-physiological changes, this study utilized zebrafish as a model to investigate whether sleep loss-induced increased anxiety is modulated by NA. Continuous behavioral quiescence for at least 6s was considered to represent sleep in zebrafish; although some authors termed it as a sleep-like state, in this study we have termed it as sleep. The activity of fish that signified sleep-waking was recorded in light-dark, during continuous dark and light; the latter induced sleep loss in fish. The latency, number of entries, time spent and distance travelled in the light chamber were assessed in a light-dark box test to estimate the anxiety behavior of normal, sleep-deprived and prazosin (PRZ)-treated fish. Zebrafish showed increased waking during light and complete loss of sleep upon continuous exposure to light for 24h. PRZ significantly increased sleep in normal fish. Sleep-deprived fish showed an increased preference for dark (expression of increased anxiety), and this effect was prevented by PRZ, which increased sleep as well. Our findings suggest that sleep loss-induced anxiety-like behavior in zebrafish is likely to be mediated by NAs action on the α1-adrenoceptor.

Protective role of noradrenaline in benzo[a]pyrene-induced learning impairment in developing rat.

Benzo[a]pyrene (B[a]P), a carcinogen, affects brain development, learning, and memory. Isolated studies have reported that B[a]P elevates noradrenaline (NA) level that may modulate neuronal growth, learning, and memory. Therefore, we investigated in vivo and in vitro the effects of B[a]P on learning and memory and its possible mechanism of action. Intracisternal administration of B[a]P on postnatal day 5 significantly reduced learning and memory in adolescent rats as observed by probe test using the Morris water maze. The density of both the subunits of the N‐methyl‐D‐aspartate (NMDA) receptor, NMDAR1 and NMDAR2B, significantly increased in the hippocampus. In vitro, B[a]P significantly increased NMDAR1 in both C6 and Neuro2a cell lines, whereas NMDAR2B was significantly increased in C6 but was significantly decreased in Neuro2a. Pretreatment with NA prevented the B[a]P‐induced effect on NMDAR1 expression in both cell lines. However, although NA prevented the B[a]P‐mediated increase in NMDAR2B expression in C6, it further potentiated the decrease of NMDAR2B in Neuro2a cells. Also, NA prevented the B[a]P‐induced increase in intracellular Ca2+ both in C6 and in Neuro2a. Our findings show that postnatal exposure of developing rats to B[a]P impairs learning and memory even when the rats became adolescent. We also observed that the effects were mediated by elevated intracellular Ca2+ levels and increased expression of NMDAR; furthermore, NA exerted a protective effect by modulating those factors. NA differentially affects neurons and glia, which may have a compensatory role during toxic insults, especially from B[a]P.

REM sleep deprivation-induced noradrenaline stimulates neuronal and inhibits glial Na-K ATPase in rat brain : In vivo and in vitro studies

Increased noradrenaline, induced by rapid eye movement (REM) sleep deprivation, stimulates Na-K ATPase activity in the rat brain. The brain contains neurons as well as glia and both possess Na-K ATPase, however, it was not known if REM sleep deprivation affects the enzyme in both types of cells identically. Rats were REM sleep deprived by the flowerpot method and free moving, large platform and recovery controls were carried out. Na-K ATPase activity was measured in membranes prepared from whole brain as well as from neuronal and glial fractions separated from REM sleep-deprived and control rats. The effects of noradrenaline (NA) in different fractions were studied with or without in vivo i.p. treatment of prazosin, an alpha1-adrenpceptor antagonist, as well as in vitro membranes prepared from neurons and glia separated from normal rat brain. Further, to confirm the findings, membranes were prepared from neuro2a and C6 cell lines treated with NA in the presence and absence of prazosin and Na-K ATPase activity was estimated. The results showed that neuron and neuro2a as well as glia and C6 possess comparable Na-K ATPase activity. After REM sleep deprivation the neuronal Na-K ATPase activity increased, while the glial enzyme activity decreased and these changes were mediated by NA acting on alpha1-adrenoceptor; comparable results were obtained by treating the neuro2a and C6 cell lines with NA. The opposite actions of NA on neuronal and glial Na-K ATPase activity probably help maintain neuronal homeostasis.

Mechanism of noradrenaline-induced stimulation of Na–K ATPase activity in the rat brain: implications on REM sleep deprivation-induced increase in brain excitability

Rapid eye movement (REM) sleep is a unique phenomenon expressed in all higher forms of animals. Its quantity varies in different species and with ageing; it is also affected in several psycho-somatic disorders. Several lines of studies showed that after REM sleep loss, the levels of noradrenaline (NA) increase in the brain. The NA in the brain modulates neuronal Na–K ATPase activity, which helps maintaining the brain excitability status. The detailed mechanism of increase in NA level after REM sleep loss and the effect of NA on stimulation of Na–K ATPase in the neurons have been discussed. The findings have been reviewed and discussed with an aim to understand the role of REM sleep in maintaining brain excitability status.

REM sleep and its Loss-Associated Epigenetic Regulation with Reference to Noradrenaline in Particular

Sleep is an essential physiological process, which has been divided into rapid eye movement sleep (REMS) and non-REMS (NREMS) in higher animals. REMS is a unique phenomenon that unlike other sleep-waking states is not under voluntary control. Directly or indirectly it influences or gets influenced by most of the physiological processes controlled by the brain. It has been proposed that REMS serves housekeeping function of the brain. Extensive research has shown that during REMS at least noradrenaline (NA) -ergic neurons must cease activity and upon REMS loss, there are increased levels of NA in the brain, which then induces many of the REMS loss associated acute and chronic effects. The NA level is controlled by many bio-molecules that are regulated at the molecular and transcriptional levels. Similarly, NA can also directly or indirectly modulate the synthesis and levels of many molecules, which in turn may affect physiological processes. The burgeoning field of behavioral neuroepigenetics has gained importance in recent years and explains the regulatory mechanisms underlying several behavioral phenomena. As REMS and its loss associated changes in NA modulate several pathophysiological processes, in this review we have attempted to explain on one hand how the epigenetic mechanisms regulating the gene expression of factors like tyrosine hydroxylase (TH), monoamine oxidase (MAO), noradrenaline transporter (NAT) control NA levels and on the other hand, how NA per se can affect other molecules in neural circuitry at the epigenetic level resulting in behavioral changes in health and diseases. An understanding of these events will expose the molecular basis of REMS and its loss-associated pathophysiological changes; which are presented as a testable hypothesis for confirmation.

Regulation of Brain Na-K ATPase Activity by Noradrenaline with Particular Reference to Normal and Altered Rapid Eye Movement Sleep

Rapid eye movement sleep (REMS) is naturally expressed at least in all the mammals, including humans, studied so far. It is regulated by interplay among complex neuronal circuitry in the brain involving various neurotransmitters. Although the precise function and role of REMS is yet to be deciphered, loss of REMS increases brain excitability; however, the mechanism of action was unknown. As Na-K ATPase is the key molecule that maintains ionic homeostasis across neuronal membrane and modulates the excitability status of neurons, we proposed that REMS deprivation (REMSD) could affect the neuronal Na-K ATPase activity. On the other hand, evidences suggest that REMSD would elevate noradrenaline (NA) level in the brain and it has been proposed that REMS maintains brain NA at an optimum level. Therefore, while attempting to understand and explain the mechanism of action we hypothesized that REMSD-induced elevated NA could modulate Na-K ATPase activity in the brain and thus modulates the neuronal and brain excitability. In this chapter first we discuss the mechanism of increase in NA level in the brain after REMSD. Then we discuss the effect of such elevated NA on neuronal and glial Na-K ATPase activity. We observed that REMSD-induced increase in NA affected neuronal and glial Na-K ATPase activities in opposite manner, while it increased neuronal Na-K ATPase, and it decreased the same in glia. An intricate regulation of Na-K ATPase activity in neurons and glia is likely to be responsible for maintenance of ionic homeostasis in the brain during normal situation, which when disturbed including upon REMS loss patho-physiological changes and symptoms are expressed.

Noradrenergic β-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na–K ATPase Subunit Expression in C6 Cells

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na–K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na–K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca++ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on β-adrenoceptor decreased Na–K ATPase activity and mRNA expression of the catalytic α2-Na–K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca++ played a critical role in such decreased α2-Na–K ATPase expression. In contrast, noradrenaline acting on β-adrenoceptor up-regulated the expression of regulatory β2-Na–K ATPase subunit, which although was cAMP and Ca++ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na–K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.

Targeting modulation of noradrenalin release in the brain for amelioration of REMS loss-associated effects

Abstract Rapid eye movement sleep (REMS) loss affects most of the physiological processes, and it has been proposed that REMS maintains normal physiological processes. Changes in cultural, social, personal traits and life-style severely affect the amount and pattern of sleep, including REMS, which then manifests symptoms in animals, including humans. The effects may vary from simple fatigue and irritability to severe patho-physiological and behavioral deficits such as cognitive and behavioral dysfunctions. It has been a challenge to identify a molecule(s) that may have a potential for treating REMS loss-associated symptoms, which are very diverse. For decades, the critical role of locus coeruleus neurons in regulating REMS has been known, which has further been supported by the fact that the noradrenalin (NA) level is elevated in the brain after REMS loss. In this review, we have collected evidence from the published literature, including those from this laboratory, and argue that factors that affect REMS and vice versa modulate the level of a common molecule, the NA. Further, NA is known to affect the physiological processes affected by REMS loss. Therefore, we propose that modulation of the level of NA in the brain may be targeted for treating REMS loss-related symptoms. Further, we also argue that among the various ways to affect the release of NAlevel, targeting α2 adrenoceptor autoreceptor on the pre-synaptic terminal may be the better option for ameliorating REMS loss-associated symptoms.