Excitatory orexinergic innervation of rat nucleus incertus--Implications for ascending arousal, motivation and feeding control.
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.
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland. Electronic address: anna.blasiak@uj.edu.pl.
Anna
A
Blasiak
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland.
Marcin
M
Siwiec
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland.
Agnieszka
A
Grabowiecka
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland.
Tomasz
T
Blasiak
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland.
Anna
A
Czerw
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
Ewa
E
Blasiak
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland.
Alan
A
Kania
Faculty of Physics, Astronomy and Applied Computer Science, Institute of Physics, Jagiellonian University, 30-348 Krakow, Poland.
Zenon
Z
Rajfur
Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University, 30-387 Krakow, Poland.
Marian H
MH
Lewandowski
The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria 3052, Australia; Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010, Australia; Department of Anatomy and Neuroscience, The University of Melbourne, Victoria 3010, Australia. Electronic address: andrewlg@unimelb.edu.au.
Andrew L
AL
Gundlach
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism; D056347:Neuroanatomical Tract-Tracing Techniques; D056325:Neuronal Tract-Tracers; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D064446:Orexin Receptors / Q000378:metabolism; D000068797:Orexins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011903:Raphe Nuclei / Q000033:anatomy & histology* / Q000187:drug effects / Q000502:physiology*; D017208:Rats, Wistar; D012065:Relaxin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods; D046509:Tissue Culture Techniques
D020746:Calcium Channels, L-Type; D009419:Nerve Tissue Proteins; D056325:Neuronal Tract-Tracers; D064446:Orexin Receptors; D000068797:Orexins; C466742:RLN3 protein, rat; D012065:Relaxin
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)