| Orexins (ORXs, also named as hypocretins) are novel excitatory neuropeptides in lateral hypothalamic area (LHA) discovered in 1998. Orexin system includes two separate peptides (ORX-A and ORX-B) proteolytically derived from the same precursor protein and corresponding two G-protein coupled receptors (OX1R and OX2R). Evidences indicate that the orexin system plays a key role in regulation of sleep-waking cycle. To date, studies on arousal effect of orexin system mainly concentrate on subcortical sleep control areas within brainstem and hypothalamus such as locus coeruleus (LC), dorsal raphe nucleus (DRN), basal forebrain (BF), tuberomammillary nucleus (TMN) and pedunculopontine nucleus (PPT). It is suggested by recent evidence that hypothalamus-cortical orexinergic direct pathway may play an important role in sleep-waking regulation. It has been shown that there are diffuse distribution of orexinergic fibers and receptors in the cerebral cortex, especially in layers Ⅴ and Ⅵ of frontal cortex (FCX). There is a substantial loss of hypothalamic orexin-containing neurons that project to numerous areas of the brain including the cortex in patients with narcolepsy. However, only limited information exists about the cellular action of these two peptides on cortical neurons. In the present study, we examined the basic electrophysiology of acute dissociated pyramidal cells from layers Ⅴ and Ⅵ in FCX of Wistar rat using whole-cell patch-clamp techniques. Then we observed the action of orexin A, glutamate, and GABA on the excibability of these cells. We also inspected the interaction between orexin A and glutamate/GABA. The main results are as follows:1. The distinct morphology of the pyramidal cells of FCX, with triangular or pyramidal phase-bright soma, a 50~100μm apical dendrite and several thin basal dendrites, were readily distinguished.2. A total of 181 FCX pyramidal cells displayed normal electrophysiology characters including resting membrane potential (RMP) of -51.98±9.55mV, series resistance (Rs) of 12.47±3.96ΩM, membrane capacitance (Rc) of 11.71±1.97pF. Most recorded neurons (95%, n=140) had action potentials when evoked by 100 msec or 200 msec hyperpolarizingand depolarizing step currents. About 75% (n=124) of recorded cells showed spontaneous action potentials at RMP during long continuous recording. 3. Glutamate (1mM) evoked depolarization and increasing spontaneous activities of isolated cells (18/18), whereas GABA (0.1mM) induced hyperpolarization and suppressed firing activity (8/8).4. Orexin A directly excited FCX pyramidal cells by dose dependent. The percentage of cells responded to 10, 5, 2, 1, 0.1μM orexin A were 100%, 81%, 63%, 50%, 32% respectively. The amplitude of depolarization evoked by the different concentration of orexin A were 29.6mV,21.5mV,5.2mV,4.6mV respectively under current clamp, and the firing activites increased 867%,354%,161%,154%,83% respectively. Orexin A also evoked inward current under voltage clamp.5. The excitatory effect of orexin A did not show difference when the pyramidal cells were perfused with Ca2+-free ACSF compared to normal ACSF (n=10). However, orexin A resulted in no action on pyramidal cells perfused with Na+-free ACSF (n=5). The results demonstrates that Na+ influx, not Ca2+ is involved in the excitatory effect of orexin.6. Orexin A regulated the excitability induced by glutamate and GABA. Only a weak response could be found when application of orexin A (0.1 μM) or glutamate (0.1 mM) on dissociated cells respectively, but the more significant depolarization and more increasing action potentials were evoked when orexin A (0.1 μM) or glutamate (0.1 mM) were applicated at the same time (5/5). Orexin A also depolarized recorded cells and evoked firing activites (6/6) in presence of GABA (0.1mM). In summary, these findings demonstrated that orexin A may directly induce Na+ influx through orexin receptors activation, and evoked excitatory effect on acutely dissociated FCX pyramidal cells. Furthermore, orexin A may al... |
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