| Orexin A and orexin B are newly discovered peptides which are synthesized exclusively within lateral hypothalamic area (LHA) and perifornical area (PFA), but the orexinergic fibers project widely to various regions in the brain including the cerebellum. The wide orexinergic projections determine that the central orexinergic system can modulate lots of physiological functions, such as sleep-awake cycle, food intake, emotions, nociception and addiction. Recently, there are some evidence that the orexinergic system may also have an impact on motor behavior. First, the orexinergic system defect animals exhibited a phenotype strikingly similar to human narcolepsy which is a common sleep disorder (excessive daytime sleepiness, premature transitions to REM sleep) accompanied by cataplexy whcih is characterized by sudden skeletal muscle weakness without impairment of consciousness. Second, administration of orexins in vivo induced changes of movements. However, the actions and the underlying mechanisms of the orexinergic system on individual brain motor structures have not been entirely investigated.The cerebellum is one of the most important subcortical motor structures which plays an important role in movement coordination, and the cerebellar nuclei (especially interpositus nucleus) are the final output of the cerebellum, so the cerebellar nuclei play an important role in moter control. Thus, by means of electrophysiological and behavior techniques, the present study was designed to investigate whether the central orexinergic systems can modulate neuronal activities of the IN and through which influence the motor control.First, the effects of orexins on the unitary firing of neurons in IN were investigated by extracellular recordings. The results showed that both orexin A and orexin B could increase the spontaneous firing rate of IN neurons and both of the excitations induced by orexin A and B were not blocked by the low-Ca2+/high-Mg2+ medium, suggesting a direct postsynaptic action of the peptides. Furthermore, highly selective OX2R agonist [Ala", D-Leu15]-orexin B mimicked the excitatory effect of orexins on the cerebellar IN neurons, but SB-334867, a highly selective OX1R antagonist, did not block the excitatory response of cerebellar IN neurons to orexins. These results demonstrate that orexins excite the cerebellar IN neurons through OX2R.However, the extracellular recording method has its own limitation, which can not distinguish different types of IN neurons (there are two types neurons in IN, projection neurons and interneurons) and detect the ion channel mechanisms Therefore, in the next work by means of whole-cell patch clamp techniques, we investigated which type of IN neurons could be modulated by orexins and what ion channel/channels mediated those modulations. Our results showed that:(1) orexin A selectively excited rat IN projection neurons rather than interneruons; (2) orexin A induced a dose-dependent whole-cell inward current in IN neurons and increase the spontaneous firing rate of IN neurons. The excitatory effect of orexin A on IN neurons was not blocked by TTX, suggesting a direct postsynaptic mechanism; (3) [Ala", D-Leu15]-orexin B mimicked the excitatory effect of orexins on the cerebellar IN neurons and SB-334867 partly blocked the excitatory response of the neurons to orexin A, suggesting that orexins can excite the cerebellar IN neurons through both OX1R and OX2R. Considering the rats we used in the whole-cell patch clamp experiment were 2- or 3-week old instead of adults, it suggests that the OX1R may play a more crucial role in the development of animals; (4) Na+-Ca+ exchanger antagonist KB-R7943 or inward rectifier K+ channel blocker Ba2+ could partly block the orexin A-induced inward current, respectively, and KB-R7943 and Ba2+ together totally blocked the orexin A-induced inward current. These results demonstrate that orexin A excites the IN neurons via both activation of Na+-Ca+ exchangers and closure of inward rectifier K+ channels.Finally, we investigated the the effects of microinjection of orexinergic reagents into adult male rats cerebellar IN on motor performance in the open-field, accelerating rota-rod treadmill and balance beam tasks, so as to reveal whether the hypothalamocerebellar orexinergic projections might modulate movements through their actions on the cerebellar IN. The results showed that:(1) Bilateral microinjection of orexin A or orexin B into the cerebellar IN remarkably increased the number of the animals entering squares and exploring, the time that animals balanced steadily on the rota-rod and markedly shortened the duration to pass through the balance beam, whereas normal saline influenced none; (2) Administration of SB-334867 into bilateral cerebellar IN, there was no significant effect on these rats behaviors, suggesting the effect of orexin A on motor behaviors was mainly via OX2R.Combined with our pervious results of electrophysiology, the findings suggest that the hypothalamocerebellar orexinergic projections may not only modulate neuronal activity of the cerebellar IN, but also regulate the cerebellar outputs, and subsequently influence the animals'motor performance. On the other hand, since the hypothalamus is a high center regulating visceral activities, it is suggested that the modulation of hypothalamocerebellar orexinergic projections on cerebellar motor functions may help animal to generate integrated or coordinated somatic-visceral responses. |
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