REM sleep modulation by perifornical orexinergic inputs to the pedunculo-pontine tegmental neurons in rats

Highlights

• Glutamatergic stimulation of PeF neurons increased waking and decreased NREMS and REMS.

• PeF stimulation-induced effects were prevented by simultaneous injection of OX1R antagonist into the PPT.

• Increased waking was possibly due to activation of wake-active neurons in PPT and other brain areas.

• Opposite effects on waking and NREMS could be due to reciprocal connection between wake- and NREMS-related neurons.

• Decreased REMS was possibly due to GABA-ergic inhibition of REM-ON neurons in the PPT.

Abstract

Rapid eye movement sleep (REMS) is regulated by the interaction of the REM-ON and REM-OFF neurons located in the pedunculo-pontine-tegmentum (PPT) and the locus coeruleus (LC), respectively. Many other brain areas, particularly those controlling non-REMS (NREMS) and waking, modulate REMS by modulating these REMS-related neurons. Perifornical (PeF) orexin (Ox)-ergic neurons are reported to increase waking and reduce NREMS as well as REMS; dysfunction of the PeF neurons are related to REMS loss-associated disorders. Hence, we were interested in understanding the neural mechanism of PeF-induced REMS modulation. As a first step we have recently reported that PeF Ox-ergic neurons modulate REMS by influencing the LC neurons (site for REM-OFF neurons). Thereafter, in this in vivo study we have explored the role of PeF inputs on the PPT neurons (site for REM-ON neurons) for the regulation of REMS. Chronic male rats were surgically prepared with implanted bilateral cannulae in PeF and PPT and electrodes for recording sleep-waking patterns. After post-surgical recovery sleep-waking-REMS were recorded when bilateral PeF neurons were stimulated by glutamate and simultaneously bilateral PPT neurons were infused with either saline or orexin receptor1 (OX1R) antagonist. It was observed that PeF stimulation increased waking and decreased NREMS as well as REMS, which were prevented by OX1R antagonist into the PPT. We conclude that the PeF stimulation-induced reduction in REMS was likely to be due to inhibition of REM-ON neurons in the PPT. As waking and NREMS are inversely related, subject to confirmation, the reduction in NREMS could be due to increased waking or vice versa. Based on our findings from this and earlier studies we have proposed a model showing connections between PeF- and PPT-neurons for REMS regulation.

Introduction

Rapid eye movement sleep (REMS) is an instinct behavior and does not have voluntary regulation. It has been identified by the presence of electrophysiological signals and has been detected in all mammals, including humans (Campbell and Tobler, 1984). Normally REMS is not expressed during waking and it appears only after the subject has spent some time in non-REMS (NREMS) (Jouvet, 1975, Gottesmann, 2001). The quantity of REMS gradually decreases through aging; however, it is never absent in life (Roffwarg et al., 1966, Campbell and Tobler, 1984). REMS is affected in most of the altered psycho-somato-patho-physiological states (Petit et al., 1993, Boeve et al., 2007) and many of the physiological processes are affected depending on the duration of experimental REMS deprivation (REMSD) (Kushida et al., 1989). Thus, quite understandably REMS regulation is extremely complex and it exerts global effect throughout the body.

Based on published data from this lab and that of others we have recently proposed a comprehensive model for REMS regulation (Mallick et al., 2012). Essentially the REM-OFF neurons in the locus coeruleus (LC) must cease firing and simultaneously the REM-ON neurons in the pedunculo-pontine area (PPT) must become active for the generation and continuation of REMS. These REM-OFF and REM-ON neurons are under the influence of many factors and brain areas, including those regulating NREMS and wakefulness (Thankachan et al., 2001, Mallick et al., 2004, Mallick et al., 2012). Normally REMS does not appear during waking; however, in REMS-associated disorder, narcolepsy, REMS like symptom (atonia) appears during waking (Chemelli et al., 1999, Zeitzer et al., 2006). The postero-lateral perifornical area (PeF), the exclusive site of orexin (Ox)-ergic neurons in the brain, is involved in waking as well as narcolepsy (Chemelli et al., 1999, Estabrooke et al., 2001, Alam et al., 2002) suggesting its role in REMS regulation. Direct modulation of PeF neurons (Alam and Mallick, 2008, Sasaki et al., 2011, Choudhary et al., 2014), presence of Ox-ergic receptors (OX1R) on LC and PPT neurons (Peyron et al., 1998, Nambu et al., 1999) and modulation of REMS by infusion of Ox or OX1R-agonist or antagonist into the LC (Hagan et al., 1999, Horvath et al., 1999, Choudhary et al., 2014) or PPT (Koyama et al., 2004, Takakusaki et al., 2005, Kim et al., 2009) supports the role of Ox on REMS. The Ox-ergic PeF neurons are likely to modulate the REMS and the effect may be mediated by influencing either or both the LC and PPT neurons. In support, recently we have shown that the PeF-induced modulation of REMS is mediated through the LC (Choudhary et al., 2014). However, Ox activates (Hagan et al., 1999, Kim et al., 2009) both the LC (site of REM-OFF) and the PPT (site of REM-ON) neurons, which behave in an opposite manner in relation to REMS and Ox-level has been found to significantly increase in the LC but not in the PPT after REMSD (Mehta et al., 2015). Thus, although Ox is likely to modulate PPT-REM-ON neurons for modulation of REMS, it was necessary to study if PeF-induced modulation of REMS is mediated by Ox acting on the PPT neurons. Therefore, in this study we have investigated if chemical stimulation of the PeF neurons modulated REMS and if the effect was prevented by simultaneous blocking of OX1R antagonist into the PPT. We have observed that stimulation of the PeF Ox-ergic neurons decreased REMS and indeed the effect was prevented by simultaneous infusion of OX1R antagonist into the PPT. To our knowledge this is the first direct evidence confirming that the PeF stimulation influences the PPT neurons for modulating REMS.