Perifornical orexinergic neurons modulate REM sleep by influencing locus coeruleus neurons in rats
Introduction
Rapid eye movement sleep (REMS) is a unique state when although one is behaviorally asleep, often one experiences dreams. This state may be objectively identified with reasonable success by the presence or absence of some electrophysiological signals; however, we are still unable to comment on the content, nature and quality of the dream(s). Typically REMS follows non-REMS (NREMS) and normally this state does not appear during or following the wake state, which however may happen in some diseased states e.g. narcolepsy. Although neural regulation of REMS is a complex process, it was proposed that the waking- and NREMS-inducing areas in the brain would have facilitatory and inhibitory influence on the REM-OFF and REM-ON neurons, respectively. It was subsequently confirmed in freely moving normally behaving cats that the brainstem and hypothalamic waking and NREMS-inducing areas, respectively, indeed exert opposite influence on the REM-OFF and REM-ON neurons (Thankachan et al., 2001, Mallick et al., 2004). Essentially, cessation of REM-OFF neurons in the Locus coeruleus (LC) is a pre-requisite for REMS generation, which as a corollary also meant that activation of LC-neurons prevents the appearance of REMS (Aston-Jones and Bloom, 1981a, Pal and Mallick, 2007). Integrating the findings from all such studies biological (Mallick et al., 2012) and mathematical (Kumar et al., 2012) models complimentary to each other for REMS regulation have been constructed recently.
The perifornical (PeF) area, an exclusive site for orexin (OX)-ergic neurons in the brain, influences REMS; however, its role, pathway, and relationship with the LC-REM-OFF neurons, particularly in relation to REMS regulation were unclear. Independent and isolated studies showed that PeF induces waking and reduces REMS (Koyama et al., 2003, Siegel, 2003, Alam and Mallick, 2008). Also, reduced level of OX and reduced number of OX-ergic neurons (Chemelli et al., 1999, Nishino et al., 2000, Hara et al., 2001, Gerashchenko et al., 2001a, Beuckmann et al., 2004) have been reported in animals showing increased REMS as well as in OXR-knockout animals (Chemelli et al., 1999, Willie et al., 2003). PeF neurons are active during waking and are quiescent during NREMS as well as tonic phase of REMS (Lee et al., 2005, Mileykovskiy et al., 2005). OX-ergic system has been reported to inhibit NREMS/REMS and increase waking (Koyama et al., 2003, Sakurai, 2005, Alam and Mallick, 2008, Sasaki et al., 2011). The neurons in the PeF project to most parts of the brain including the hypothalamus, the LC and the latero dorsal tegmentum/pedunculo pontine tegmentum (LDT/PPT) (Peyron et al., 1998, Greco and Shiromani, 2001, Marcus et al., 2001, Gerashchenko et al., 2001b, Gerashchenko et al., 2003) the site where REMS controlling neurons have been reported. Isolated and independent studies have shown that microinjection of OXR agonist/antagonist into the hypothalamus (Methippara et al., 2000), LC (Bourgin et al., 2000, Smith et al., 2003) and LDT/PPT (Bernard et al., 2003, Bernard et al., 2006, Nunez et al., 2006) could successfully modulate REMS. OX has been shown to exert excitatory effect on the LC noradrenalin (NA) neurons through orexin receptor1 (OX1R) (Hagan et al., 1999). However, direct evidence showing OX-ergic projections to LC modulating REMS was lacking. Therefore, in this study in freely moving chronically prepared rats REMS was quantified in control and experimental rats by either stimulating the PeF neurons alone or along with simultaneous micro-injection of OX1R antagonist into the LC. Also, to explore the effect of stimulation of the cell bodies in the PeF by ruling out the effect of stimulation of fibers of passage through the PeF, in separate experiments the PeF was stimulated electrically or by glutamate (chemically). The findings confirmed our recently proposed model (Mallick et al., 2012) that the PeF neurons prevent REMS by activating the LC-neurons.
Section snippets
Animals
Fifteen adult male Wistar rats weighing 300–350 g maintained on 12:12 light:dark cycle at 25 ± 1 °C ambient temperature having free access to food and water were randomly distributed in different groups. All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) of the Jawaharlal Nehru University. All efforts were made to minimize animal suffering and number of animals used in this study.
Drugs
The following chemicals from Sigma, USA were used in this study. l-Glutamic
Histology
The reconstructed representative histological sections through stimulation and microinjection sites in PeF and LC along with corresponding brain atlas sections (overlapped hemi-sections have been shown for convenience) have been shown in Fig. 2A, B. Both the PeF and LC sites were on target in (n = 6) rats, where as in (n = 4) rats one of the two sites was off target. Data from recordings of animals with correct stimulating electrodes and cannulae placements in the PeF (Fig. 2A) and cannulae in the
Discussion
The present study demonstrated that electrical and glutamatergic stimulation of the OX-ergic neurons in the PeF increased W and decreased NREMS as well as REMS. We also observed that electrical stimulation of the PeF neurons produced sustained (at least for 36 h) effects as compared to that of glutamatergic stimulation of the neurons in the PeF. Further, the latter effect was prevented by simultaneously blocking of OX1R in the LC suggesting that (i) the effects of activation of PeF were due to
Conclusion
Our findings suggest that the PeF OX-ergic neurons regulate REMS by modulating the LC REMS-related neurons and the effects are likely to be mediated by OX1R on the REM-OFF neurons. The stronger and sustained effect of mild electrical stimulation of PeF may be due to activation of reverberatory neuronal circuitry, although activation of nerve fibers passing through PeF cannot be ruled out. These in vivo findings in normal rats provide better understanding on the neural regulation of REMS,
Acknowledgments
Funding to BNM from Indian agencies-CSIR, DBT, JC Bose fellowship and UGC meritorious fellowship to MAK is acknowledged.
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Equal contribution.