A Very Large Number of GABAergic Neurons Are Activated in the Tuberal Hypothalamus during Paradoxical (REM) Sleep Hypersomnia

Abstract

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD67 mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD67 in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD+, Fos-ir/MCH+, and GAD+/MCH+ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD+ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.

Immunohistochemical evidence for synaptic release of GABA from melanin-concentrating hormone containing varicosities in the locus coeruleus

“…MCH would thus have the capacity to modulate synaptic inputs to the LC and other target neurons, but not to inhibit them directly. Yet, previous in situ hybridization experiments found that many MCH neurons contained mRNA for GAD (Elias et al, 2001;Sapin et al, 2010) and thus suggested that they could utilize GABA as an inhibitory neurotransmitter. Our study shows for the first time that the MCH varicosities contain VGAT and thus would have the capacity to release GABA at synapses on target neurons (McIntire et al, 1997;Chaudhry et al, 1998).…”

“…Moreover, as we previously showed, Orx varicosities in the LC can also release glutamate onto glutamatergic synapses, since they contain the vesicular transporter for glutamate (VGluT2) and contact postsynaptic density-95 (PSD-95) puncta, the postsynaptic marker for glutamatergic synapses (Henny et al, 2010). Conversely, there is evidence to suggest that MCH neurons might utilize GABA, since their somata contain mRNA for glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA (Elias et al, 2001;Sapin et al, 2010), although their terminals have not yet been shown to contain or utilize GABA. It would thus appear possible that whereas Orx neurons could excite LC neurons in part through release of glutamate, MCH neurons could inhibit LC neurons through release of GABA.…”

Melanin-concentrating hormone neurons specifically promote rapid eye movement sleep in mice

“…MCH neurons contain the vesicular glutamate transporter 2, (Chee et al, 2015) and glutamic acid decarboxylase-67 (Sapin et al, 2010, Jego et al, 2013), but not the vesicular GABA transporter (Chee et al, 2015). Consistent with these observations, MCH terminals in the lateral septum release glutamate, but not GABA (Chee et al, 2015).…”

“…MCH, when injected intracerebroventricularly, produces a robust (~200%) increase in REM sleep (Verret et al, 2003) while infusion of an MCH receptor antagonist causes a significant reduction in REM sleep in rats (Ahnaou et al, 2008). MCH neurons heavily innervate the dorsolateral brainstem regions implicated in REM generation including the sublaterodorsal nucleus (SLD) and ventrolateral periaqueductal gray matter (vlPAG), (Hanriot et al, 2007, Peyron et al, 2009, Sapin et al, 2010, Clement et al, 2012) and local injection of MCH into the nucleus pontis oralis (in the subcoeruleus region which is considered to be an SLD-equivalent in cats) produces a significant increase (~70%) in REM sleep and reduces the latency to REM sleep (Torterolo et al, 2009). Juxtacellular recording studies show that MCH neurons are maximally active during REM sleep, silent during wake, and occasionally active in NREM sleep (Hassani et al, 2009), suggesting that they promote REM sleep.…”

The neuronal network responsible for paradoxical sleep and its dysfunctions causing narcolepsy and rapid eye movement (REM) behavior disorder

“…Our results also indicate that approximately 20% of these neurons co-contain the neuropeptide MCH. 102 In contrast, almost none of the neighboring hypocretin (Hcrt or orexin) neurons were c-Fosþ. Around 60% of all the MCH-immunoreactive neurons counted in the PeF, ZI and LHA were c-Fosþ 98,103 Further, it has been shown in head-restrained rats by means of juxtacellular labeling that the MCH and non-MCH GABAergic neurons fire quite exclusively during PS.…”