Abstract
Orexin (ORX) neurons in the hypothalamus send their axons to arousal-promoting areas. We have previously shown that glutamatergic neurons in the lateral parabrachial nucleus (LPB) innervate ORX neurons. In this study, we examined potential pathways from the LPB to ORX neurons projecting to arousal-promoting areas in the brainstem by a combination of tract-tracing techniques in male Wistar rats. We injected the anterograde tracer biotinylated dextranamine (BDA) into the LPB and the retrograde tracer cholera toxin B subunit (CTb) into the ventral tegmental area, dorsal raphe nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental area, or locus coeruleus (LC). We then analyzed the BDA-labeled fibers and ORX-immunoreactive neurons in the hypothalamus. We found that double-labeled ORX and CTb neurons were the most abundant after CTb was injected into the LC. We also observed prominently overlapping distribution of BDA-labeled fibers, arising from neurons located in the lateral-most part of the dorsomedial nucleus and adjacent dorsal perifornical area. In these areas, we confirmed by confocal microscopy that BDA-labeled synaptophysin-immunoreactive axon terminals were in contiguity with cell bodies and dendrites of CTb-labeled ORX-immunoreactive neurons. These results suggest that the LPB innervates arousal-promoting areas via ORX neurons and is likely to promote arousal responses to stimuli.
Highlights
Orexin (ORX) is a small hypothalamic neuropeptide, synthesized by a cluster of neurons in the lateral hypothalamus
We injected biotinylated dextranamine (BDA) into the lateral parabrachial nuclei (LPB) to label neurons projecting to the ORX field in the hypothalamus anterogradely, and injected cholera toxin subunit B (CTb) into the ipsilateral ventral tegmental area (VTA), dorsal raphe nucleus (DR), pedunculopontine tegmental nucleus (PPT), laterodorsal tegmental area (LDT) or locus coeruleus (LC) to label neurons from the ORX field retrogradely (Fig. 1A,B)
We observed that ORX-IR neurons were distributed predominantly in the perifornix (PeF) and dorsal lateral hypothalamus (LH) in addition to the dorsomedial hypothalamic nucleus (DMH) across the tuberal hypothalamus (Supplementary Fig. 1 and Fig. 2B3–F5)
Summary
Orexin (ORX) is a small hypothalamic neuropeptide, synthesized by a cluster of neurons in the lateral hypothalamus. Within the ventral tegmental area (VTA), dorsal raphe nucleus (DR), pedunculopontine tegmental nucleus (PPT), laterodorsal tegmental area (LDT), locus coeruleus (LC)[5,6], monoaminergic neurons in the VTA, DR, and LC and cholinergic neurons in the PPT and LDT have been shown to receive projections from ORX neurons and these neurons are well known to promote arousal, projecting to widespread brain areas directly or indirectly[7]. Chemogenetic activation of the parabrachial nucleus induces activation of ORX neurons of the lateral hypothalamus and promotes wakefulness[19] These data allow us to hypothesize that LPB fibers could form synaptic contacts with ORX neurons projecting to brainstem arousal areas such as the VTA, DR, PPT, LDT and LC. We used a combination of tract-tracing techniques to provide novel evidence for the existence of pathways from the LPB to ORX hypothalamic neurons and to brainstem arousal areas in male Wistar rats
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