Hypocretin1/OrexinA‐containing axons innervate locus coeruleus neurons that project to the Rat medial prefrontal cortex. Implication in the sleep‐wakefulness cycle and cortical activation

Abstract

The Hypocretin1/OrexinA (Hcrt1/OxA) neuropeptides are found in a group of posterolateral hypothalamus neurons and are involved in sleep-wakefulness cycle regulation. Hcrt1/OxA neurons project widely to brainstem aminergic structures, such as the locus coeruleus (LC), which are involved in maintenance of wakefulness and EEG activation through intense projections to the medial prefrontal cortex (mPFC). Moreover, defects of the Hcrt1/OxA system are linked to narcolepsy, a disorder characterized by excessive diurnal hypersomnia and REM state disturbance. We aimed to determine whether Hcrt1/OxA neurons innervate LC neurons (noradrenergic and nonnoradrenergic) that project to the mPFC, thereby sustaining behavioral wakefulness. To assess this, we used retrograde tracing from mPFC injections and either Hcrt1/OxA or tyrosine hydroxylase (TH) immunohistochemical labeling in single sections of rat LC. The retrograde tracer Fluorogold (FG) was microinjected into mPFC and, at optimal survival periods, sections through the LC were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Many LC neurons projecting to mPFC were nonnoradrenergic. Electron microscopy revealed a prominent localization of Hcrt1/OxA in unmyelinated axons and axon boutons (varicosities and axon terminals) within the LC. Hcrt1/OxA-immunoreactive axon boutons frequently apposed (104/1907) or made asymmetric excitatory-type synapses (60/1907) with FG-immunolabeled dendrites, indicating that Hcrt1/OxA can modulate the activity of LC neurons with cortical projections. Our results show that Hcrt1/OxA hypothalamic neurons likely excite LC neurons that project to the mPFC, and thus activate EEG and facilitate wakefulness. In narcoleptics, who are deficient in Hcrt1/OxA, impairment of this Hcrt1/OxA hypothalamic input to LC might contribute to the appearance of excessive daytime sleepiness.

Hypocretin1/orexinA-immunoreactive axons form few synaptic contacts on rat ventral tegmental area neurons that project to the medial prefrontal cortex

“…However, it is unknown, so far, which of these pathways is more relevant for arousal and wakefulness. Our results complement previous studies on Hcrt1/OxA innervation of brainstem wakefulness-related areas, [49–51], and allow us to establish cellular and subcellular bases by which the Hypocretinergic/orexinergic system excites VTA neurons that project to PL, activating the cerebral cortex. The VTA is not the only gateway for Hcrt1/OxA neurons to gain acces to the mPFC.…”

“…The common observation of Hcrt1/OxA axons close to blood vessels, similar to what has been reported in other brain areas [49–51], suggests an association between the Hcrt/Ox system and vasomotor control. In fact, Hcrt/Ox-R1 activation attenuates neurogenic vasodilation of dural vessels [52].…”

“…The VTA is not the only gateway for Hcrt1/OxA neurons to gain acces to the mPFC. Significant Hcrt1/OxA targeting has also been described in some other brainstem nuclei projecting to mPFC, such as the locus coeruleus [49], dorsal raphe nucleus [50] and laterodorsal tegmental nucleus [51]. Conceivably, all these structures contribute to cortical-activating actions of Hcrt1/OxA.…”

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

“…The LC also receives a dense innervation by Orx varicosities and has a high density of Orx1 receptors (Peyron et al, 1998;Hervieu et al, 2001;Cluderay et al, 2002). Whereas a proportion of the MCH varicosities contained VGAT and abutted gephyrin puncta in the LC here, a proportion of the Orx varicosities contained VGluT2 and contacted PSD-95, the postsynaptic density protein at glutamatergic synapses, in a previous study (Henny et al, 2010) and were also shown to make asymmetric synapses with the noradrenergic neurons (Horvath et al, 1999;Del Cid-Pellitero and Garzon, 2011). Thus the Orx neurons can excite noradrenergic neurons in the LC through excitatory action of their peptide together with that of glutamate to elicit wakefulness (Bourgin et al, 2000;Carter et al, 2010), when both the Orx neurons (Lee et al, 2005;Mileykovskiy et al, 2005) and LC neurons selectively discharge (Aston- Jones and Bloom, 1981).…”

Forebrain GABAergic projections to locus coeruleus in mouse

“…Early anatomical studies in rat suggested that some brainstem afferents terminate within the LC proper, while most other projections target the Peri-LC (Aston-Jones et al, 1986; Van Bockstaele et al, 2001). In addition it has been demonstrated that some projections from the preoptic and lateral hypothalamic areas, periaqueductal gray, and CAmy target the LC core (Luppi et al, 1995; Lee et al, 2005; Reyes et al, 2011), although some of the lateral hypothalamic hypocretin neurons may target non-noradrenergic LC projection neurons (Del Cid-Pellitero and Garzon, 2011). However, for many afferents it is still not clear whether they target interneurons or dendrites of noradrenergic neurons within the Peri-LC zone.…”