The Cellular Mechanisms Of Excitatory Effects Of Orexin A On Cortical Neurons In Rat

Orexins localized specifically in neurons within the lateral hypothalamus and perifornical area are the novel neuropeptides discovered in 1998 by two groups respectively. Orexin system includes two separate peptides orexin A and B (ORX-A and ORX-B, also known as hypocretin 1 and 2) proteolytically derived from the same precursor protein. The actions of orexins are mainly transduced by orexin-1 and orexin-2 receptors (OX1R and OX2R) belonging to G-protein-coupled receptors superfamily. Much evidence has shown that these peptides are importantly implicated in the regulation and promotion of wakefulness. And this role may be mainly fulfilled by the excitatory action of orexins on multiple subcortical arousal systems, including noradrenergic locus coeruleus, serotoninergic dorsal raphe nuclei, cholinergic mesopontine and basal forebrain neurons, histaminergic tuberomammillary nuclei, and the nonspecific thalamo-cortical projection system. In the previous study, we have shown that ORX-A can directly excite the pyramidal neurons of prefrontal cortex (PFC) dose-dependently. Therefore, these findings have led to the conclusion that the direct hypothalamic-cortical pathway, in addition to the indirect subcortical pathway, may also mediate the roles of orexin system in wakefulness control. Nowadays, much attention has been paid to the studies of mechanisms of excitatory effects induced by orexins in both neurons and non-neurons. And these studies have indicated that the excitatory effects of orexins involve diverse signaling cascades triggering the multiple ionic mechanisms in varying brain areas. However, the mechanisms and, in particular, intracellular signaling pathways of orexins-induced excitability on neurons in the cerebral cortex have received little attention. In addition, several lines of evidence also suggest that the main response to the OXRs activation in neurons appears to be intracellular Ca²⁺ elevation involved different mechanisms. Briefly, orexins are known to increase cytosolic Ca²⁺ concentration ([Ca²⁺]_i) through activation of extracellular Ca²⁺ influx, intracellular stores, or through both mechanisms. Despite the fact that orexins have been shown to elevate intracellular Ca2+ of cultured cortical neurons, it is not sure whether it comes from extracellular Ca2+ or intracellular Ca2+ stores. In the present study, we examined the basic mechanisms underlying the excitatory effects of ORX-A on cortical neurons by using whole-cell patch-clamp recordings and [Ca2+]i imaging. Firstly, we explored the intracellular signaling mechanisms of the ORX-A-induced excitation on acutely dissociated pyramidal neurons of rat PFC by using whole-cell patch-clamp recordings technique. Subsequently, the mechanisms underlying cytosolic Ca2+ elevation induced by ORX-A was investigated by using calcium imaging in cultured cortical neurons and acutely dissociated pyramidal neurons of PFC. 1. Involvement of G-protein in the excitatory effects of ORX-A on PFC pyramidal neurons Current-clamp recordings from a total of 23 pyramidal neurons of PFC showed that 92 % (n=23, 23 of 25) of this population responded to puff application of ORX-A (10-5 M) by a rapid increase in membrane potentials and firing frequency of action potentials. Inclusion of GDP-β-S (0.5mM) in the internal pipette solution, a nonhydrolysable GDP analog that inhibits G-protein-mediated intracellular signaling, abolished the effect of ORX-A (n=15; p<0.05 versus 10-5 M ORX-A), confirming that the actions of ORX-A on PFC pyramidal neurons are also mediated by activation of G-protein. 2. PLC and PKC, but not PKA, are involved in the excitatory effects of ORX-A The responses to ORX-A were also blocked by a phospholipase C (PLC) inhibitor, D609 (10μM) (0.68±0.45 mV, n=9; p<0.05 versus 10-5 M ORX-A). In contrast, the depolarizing effects of ORX-A were still observed when the PKA inhibitor peptide (5-24) (1μM) was included in the pipette (mean depolarization, 24.74±8.79 mV, n=14 of 15; p>0.05 versus 10-5 M ORX-A). Moreover, 12-O-tetradecanoylphorbol-13-acetate (TPA) (a PKC agonist) (100nM) depolarized pyramidal neurons of PFC (mean depolarization, 25.43±7.23 mV, n=6, 6 of 7; p>0.05 versus 10-5 M ORX-A), and BIS II (1μM), a PKC inhibitor, abolished the depolarizing effects of ORX-A (mean depolarization, 0.62±0.34 mV, n=11; p<0.05 versus 10-5 M ORX-A). Altogether, these data indicate that the excitatory effects of ORX-A on PFC pyramidal neurons are mediated by PLC and PKC, but not by PKA signaling pathway.3. Inhibition of whole cell K+ currents in ORX-A-induced excitatory effects is mediated by activation of PLC-PKC pathway The voltage-clamp experiments showed that exposure to ORX-A (10-5M) resulted in statistically significant decline of the whole cell K+ currents (n=21; p<0.05 versus control). Then, the inhibitory effects on K+ currents induced by ORX-A were not observed in the presence of PKC inhibitor, BIS II (1μM) (n=9; p<0.05 versus 10-5 M ORX-A), or PLC inhibitor, D609 (10μM) (n=7; p<0.05 versus 10-5 M ORX-A) in aCSF, respectively. These results therefore illustrate that the excitatory effects of ORX-A on pyramidal neurons of PFC are mediated through inhibiting K+ currents by activation of the PLC and PKC signaling pathways. 4. Extracellular calcium influx contributes to the ORX-A-induced elevation of [Ca2+]i of cultured cortical neurons The [Ca2+]i imaging studies demonstrated that [Ca2+]i in about 25.64% (n=20, 20 of 78) of the cultured cortical neurons was increased in a dose-dependent manner in the presence of ORX-A at varying concentrations (10-7 M, 5×10-7 M and 10-6 M). The increase in peak of Ca2+ fluorescence intensity induced by ORX-A was abolished in Ca2+-free DMEM (n=56; p>0.05 versus control) , but was still observed when pre-incubation of thapsigargin (2μM) (17.24%, n = 5 , 5 of 29; p<0.05 versus control) was applied, suggesting that ORX-A-induced [Ca2+]i elevation does not depend on intracellular Ca2+ stores. These findings first illuminate that ORX-A-induced increase in [Ca2+]i is mainly from extracellular calcium influx in cortical neurons. In addition, we also observed the [Ca2+]i elevation in a few of acutely dissociated neurons of PFC in the presence of ORX-A (10-6M). The responses to ORX-A were similar to that of cultured cortical neurons. In conclusion, these observations provide evidence that the direct excitatory effects of ORX-A on acutely dissociated pyramidal neurons in rat PFC are mediated by the OXR-coupled activation of Gq-protein, which activates the PLC-PKC signaling cascades and inhibits whole cell K+ currents subsequently. Moreover, the increase in [Ca2+]i induced by ORX-A in cortical neurons may be attributed to the extracelluar calcium influx.