Orexin/hypocretin peptides play a central role in the integrated control of feeding/reward and behavioural activation, principally via interactions with other neural systems. A brainstem area involved in behavioural activation is the nucleus incertus (NI), located in the posterior ventromedial central grey. Several studies have implicated NI in control of arousal/stress and reward/feeding responses. Orexin receptor mRNA expression identifies NI as a putative target of orexin modulation. Therefore, in this study we performed neural tract-tracing and immunofluorescence staining to characterise the orexinergic innervation of NI. Our results indicate a convergent innervation of the NI area by different orexin neuron populations, with an abundance of orexin-A-containing axons making putative synaptic contacts with relaxin-3-positive NI neurons. The influence of orexin-A on NI neuron activity was investigated using patch-clamp recordings. Orexin-A depolarised the majority (64%) of recorded neurons and this effect was maintained in the presence of tetrodotoxin and glutamate and GABA receptor antagonists, indicating a likely postsynaptic action. Voltage-clamp experiments revealed that in 'type I' NI neurons comprising relaxin-3-positive cells, orexin-A acted via L-type calcium channels, whereas in 'type II' relaxin-3-negative neurons, activation of a sodium/calcium exchanger was involved. A majority of the orexin-A sensitive neurons tested for the presence of orexin receptor mRNA, were OX2 mRNA-positive. Immunohistochemical staining for putative orexin receptors on NI neurons, confirmed stronger expression of OX2 than OX1 receptors. Our data demonstrate a strong influence of orexin-A on NI neurons, consistent with an important role for this hypothalamic/tegmental circuit in the regulation of arousal/vigilance and motivated behaviours.
Keywords: 4- aminopyridine (PubChem CID 1727); Arousal; Bicuculline methiodide (PubChem CID 104871); CNQX disodium salt (PubChem CID 6093155); DL-AP5 (PubChem CID 1216); In vitro electrophysiology; Motivated behaviours; Neural tract-tracing; Nickel (II) chloride (PubChem CID 24385); Nifedipine (PubChem CID: 4485); Orexin-A (PubChem CID: 56842143); Orexin/OX(1/2); Relaxin-3/RXFP3; Tetrodotoxin citrate (PubChem CID: 16759596).
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