Research articleREM sleep loss associated changes in orexin-A levels in discrete brain areas in rats
Introduction
Rapid eye movement sleep (REMS) is a unique reversible, physiological state expressed for varying duration; it repeats within the sleep state especially in the animals higher in evolutionary ladder. Normally it appears only after a period of non-REMS (NREMS) and barring in some diseased states (e.g. narcolepsy), REMS-like state does not appear during waking. Therefore, it was proposed that normally neurons responsible for REMS must be inhibited by the waking areas, while excited by the NREMS generating areas, which has been confirmed [1], [2] and subsequently the complex neural connections for the regulation of REMS has recently been constructed [3]. It essentially suggests that as long as noradrenergic (NA)-ergic REM-OFF neurons in the locus coeruleus (LC) remain active, which of course are under the influence of varieties of neurotransmitter inputs, REMS is not expressed and those neurons must cease activity for the expression of REMS [3], [4].
The levels of various neurotransmitters in different parts of the brain during REMS and REMS loss are likely to play significant role in REMS regulation during health and diseases. Orexin (Orx) is one of the many neurotransmitters involved in REMS regulation in health and diseases [5]. Orx synthesizing neurons are present in the perifornical (PeF) area, located at the lateral part of posterior hypothalamus (PH) and are generally active during waking, slow down and finally become quiescent during NREMS and REMS [6], [7], [8]. Activation of these neurons indeed increased waking and reduced REMS [9], [10], [11]. Recently, we have shown that Orx-ergic neurons indeed affect REMS by modulating the neurons in the LC [11]. Orx levels in the brain correspond to the activity of Orx-ergic neurons, i.e. higher during waking and REMS [12] and lower during NREMS. Interestingly, these Orx-ergic neurons project to many brain areas including the brainstem, LC, PH, which are classically known to be involved in the regulation of wakefulness-NREMS-REMS [11], [13], [14], [15] and to hippocampus and cortex, among many other areas which are apparently not involved in sleep-waking-REMS regulation [16]. Also, Orx-ergic neurons in PeF have local self-collateral inputs [17].
All the neurons even in any one functional area in the brain do not behave and perform identically. Thus, it is obvious that the levels of the neurotransmitters in different projected regions of the brain are likely to be different during alteration in one function. The levels of neurotransmitters would contribute to pre-disposition of expressions of behavior and variations in quality and quantity of expressions of behavior including diseases and symptoms thereof. Therefore, it is essential to estimate the levels of a neurotransmitter in relation to changes in a particular function simultaneously in multiple areas of the brain where the neurons from one area project to. Thus, to address this issue, in the present study, we estimated Orx-A levels in control, after REMS deprivation (REMSD) and after recovery from REMSD in different brain areas known to be involved in REMS regulation and in the areas although not involved in regulation of REMS as such, but are known to modulate function(s) affected by REMS loss. As Orx is of two types Orx-A and Orx-B, in this study, we estimated levels of Orx-A. To the best of our knowledge, this is the first such study especially after long-term REMSD.
Section snippets
Animals and REMSD
Experiments were conducted on inbred male Wistar rats (200–250 g; n = 20), maintained in 12:12 light/dark (L/D) cycle and were supplied with food and water ad libitum. All the efforts were made to minimize the number of rats and their suffering. Free moving control (FMC) rats (n = 5) were maintained in their normal dry home cages. Experimental rats (n = 5) were REMS deprived by the classical flowerpot method as reported earlier [18], [19]. In brief, for REMSD, the experimental rats were maintained for
Results
The standard plot of absorption at 450 nm by various concentration of Orx-A showed a linear relationship with R2 = 0.993. The wet tissue weight (mg ± SD) of each brain area across all rats taken for estimation of Orx-A was LC (24 ± 0.018), cortex (260 ± 0.113), PH (43 ± 0.022), PPT (25 ± .003) and hippocampus (126 ± 0.056). Under normal FMC condition detectable level (10–50 pg/g wet tissue) of Orx-A was estimated in samples from across the brain regions studied. The Orx-A levels were comparable in the FMC and
Discussion
The Orx-A level could be detected in different brain regions, which may be explained by the fact that orx-ergic PeF neurons are known to project to those brain areas [23]. The difference in relative levels of Orx-A could be due to the density of projections and intensity of firing rate of the Orx-ergic projecting neurons from the PeF. Orx-ergic neurons widely innervate different brain regions and send their densest projections to the LC [24], which possesses REM-OFF and wake-active neurons [25]
Conflict of interest
Authors declare no conflict of interest.
Author’s contribution
BNM designed the work; RM and MAK performed experiment and analyzed the data. All authors contributed in writing the final manuscript.
Acknowledgements
MAK received UGC fellowship. Research funding to BNM from Indian funding agencies from BUILDER program; Council of Scientific and Industrial Research; Department of Biotechnology and JC Bose fellowship (DST) is acknowledged.
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