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

Melanin-concentrating hormone (MCH) is synthesized by neurons located in the hypothalamus and projecting to widespread regions of the brain, including the locus coeruleus (LC), through which MCH could modulate sleep-wake states. Yet MCH does not appear to exert direct postsynaptic effects on target neurons, including the noradrenergic LC neurons. Previous studies using in situ hybridization showed that MCH neurons synthesize glutamic acid decarboxylase (GAD) and could thus utilize GABA as a neurotransmitter. To determine whether MCH varicosities can release GABA, we examined by fluorescent microscopy in the LC, whether their terminals also contain the vesicular transporter for GABA (VGAT). In dual-immunostained sections, we found that approximately 6% of MCH varicosities was immunopositive for VGAT and a similar proportion for synaptophysin, the presynaptic marker for small synaptic vesicles, whereas <1% was positive for the vesicular glutamate transporter (VGluT2). Moreover, of the MCH varicosities, ∼5% abutted puncta that were immunostained for gephyrin, the postsynaptic marker for GABAergic synapses. In triple-immunostained sections viewed with confocal laser scanning microscopy, we established that MCH varicosities that also contained VGAT or abutted upon gephyrin puncta contacted the tyrosine hydroxylase-immunostained neurons of the LC. Our results suggest that although MCH neurons can influence noradrenergic LC neurons through paracrine release and indirect effects of their peptide, they can also do so through synaptic release and direct postsynaptic effects of GABA and thus serve to inhibit the LC neurons during sleep, when they are silent, and the MCH neurons discharge.

Medial prefrontal cortex receives input from dorsal raphe nucleus neurons targeted by hypocretin1/orexinA-containing axons.

The medial prefrontal cortex (mPFC) is strongly involved in cognition and behavior. It receives input from brainstem nuclei implicated in behavioral wakefulness and electrographic cortical activation, such as the dorsal raphe nucleus (DRN). Moreover, the hypocretinergic/orexinergic (Hcrt/Ox) hypothalamic neurons innervate DRN, thus modulating its activity and presumably allowing transitions between sleep-wakefulness cycle states. Dysfunction in this system is associated with narcolepsy. In this study we aimed to determine the precise location of DRN neurons projecting to mPFC and the extent to which they contain serotonin (5-hydroxytryptamine); we have also assessed whether Hcrt1/OxA neurons innervate DRN neurons that could sustain behavioral wakefulness through their projections to mPFC. The retrograde tracer Fluorogold was injected into mPFC and DRN sections were processed for double immunolabeling of anti-Fluorogold and either anti-5-hydroxytryptamine or anti-Hcrt1/OxA antisera. Most DRN neurons projecting to mPFC were located in the ventral sector of the rostral and intermediate DRN, and around half of them were serotonergic. Hcrt1/OxA-immunoreactivity in DRN was observed in unmyelinated axons and axon boutons (varicosities or axon terminals). Hcrt1/OxA immunoreactivity was observed within the cytoplasm and in dense-cored vesicles of these axons. Hcrt1/OxA-labeled boutons established both asymmetric synapses (n=30) and appositional contacts (n=102) with Fluorogold-labeled dendrites belonging to DRN neurons projecting to mPFC. Our results show that Hcrt1/OxA neurons may exert a direct synaptic influence on DRN neurons that could facilitate wakefulness, although other non-synaptic actions through volume transmission are also suggested.

[Modulation by the hypocretinergic/orexinergic neurotransmission system in sleep-wakefulness cycle states]. / El sistema de neurotransmisión hipocretinérgico/orexinérgico en la regulación de los estados de vigilia y sueño.

INTRODUCTION: The hypocretins/orexins are neuropeptides synthesized by a small neuronal cell group located in the posterior and lateral hypothalamus. These peptides have been considered modulators of the sleep-wakefulness cycle since their discovery in 1998; the hypocretinergic/orexinergic system is very active during wakefulness. In addition, the absence of either these peptides or their receptors is associated to narcolepsy-cataplexy, a disease in which the sleep-wakefulness cycle is completely disorganized. DEVELOPMENT: Hypocretinergic/orexinergic neurons directly activate the cerebral cortex and neuronal cell groups of the reticular activating system containing noradrenaline, serotonin, dopamine, acetylcholine and histamine, through which they may also indirectly activate the cerebral cortex and enhance wakefulness; as well, these neurons inhibit REM sleep generation in the ventral pontine tegmentum. The decrease in the activity of hypocretinergic/orexinergic neurons during sleep inhibits the aminergic and cholinergic neurons of the reticular activating core, decreasing cortical activation and renewing REM sleep generation in the ventral pontine tegmentum. CONCLUSIONS: Hypocretins/orexins modulate wakefulness and EEG activation in part through their actions on reticular core neurons projecting to the cortex and suppress REM sleep generation through inhibition of ventral pontine tegmentum neurons within the ventral oral pontine tegmentum. The hypoactivity of this system supports the sleep cycle fragmentation and general disorganization appearing in narcolepsy, as well as momentary interruption of wakefulness by REM sleep episodes.