The Modulatory Effects Of Orexin-A On Glutamate Receptor Function In Pyramidal Neurons From Rat Prefrontal Cortex

Orexin(hypocretin) mainly produced by the neurons within the lateral hypothalamus , is a micromolecular neuropeptide discovered in 1998 .Meanwhile, perifornical area,dorsomedial nucleus of hypothalamus as well as posterior hypothalamic also contain orexin neurons. Orexin system includes two separate peptides orexin-A and B proteolytically derived from the same precursor protein and two specific G-protein-coupled receptors OX1R and OX2R. Although the distribution of orexin neurons is limited, the fiber projection and receptor expression of orexin system are extremely extensive, spreading throughout to many brain regions. Orexin system is relative with the regulation of many physiological functions, among these, its regulative role on"sleep-wake"is now considered to be most important. Recent studies have revealed orexin system also extensively participate in the regulation of learning and memory,drug addiction,emotional disorder and so on. As we known, prefrontal cortex (PFC) plays an important role on execution of these higher nervous activity, PFC also receives the orexinergic projections and expresses the orexin receptors.In the previous study, we have shown that orexin-A can directly excite the pyramidal neurons of PFC dose-dependently. Glutamate is one of the most important neurotransmitter distinctly expressed in the PFC, the excitatory synaptic transmission mediated by glutamate is relative closely with the function of PFC. Other neurotransmitters can also modulate the function of PFC by regulating the activity of glutamatergic path in PFC. However, whether orexin system can regulate the function of PFC directly through regulating the activity of glutamatergic system, especially, the signaling pathways involved in this process have received little attention.In the present study, we firstly identified the distribution of orexin receptors in rat PFC by means of immunohistochemistry. Subsequently,we explored the role of orexin-A on glutamatergic system of PFC and the intracellular signaling mechanisms by using whole-cell patch-clamp recordings technique on acutely dissociated pyramidal neurons of rat PFC. The results show as follow:1. Distribution of orexin receptor in PFC of ratImmunohistochemical studies showed that OX1R expressed in layers V and VI pyramidal neurons of rat PFC, and these pyramidal cells were identified as glutamatergic neurons.2. Regulation of glutamate current by orexin-A on PFC pyramidal neurons in ratBoth glumatate and orexin-A dose-dependently evoked the inward transmembrane current.The current was evoked by 10-3M glutamate as a control group(100%).After treatment with 10-6M orexin-A , 10-3M glutamate induced-current was increased to 146.59%±15.19% (n=8, p<0.01 versus control). These data indicate that glutamate receptor-mediated current is markedly potentiated by orexin-A in the freshly isolated pyramidal neurons, which suggests that a synergistic action between orexin and glutamate systems exists in PFC.3. The modulatory effect of orexin-A on glutamate receptor-induced current is mediated by NMDA receptorThe modulatory effect of orexin-A(10-6M) on glutamate receptor-induced current was blocked by a selective NMDA receptor antagonist 2-amino-5-phosphonovaleric acid, (AP-5, 50μM) ( 102.08%±5.95% , n=6; p>0.05 versus the normal current induced by 10-3 M glutamate).Moreover, after treatment with 10-6M orexin-A,NMDA receptor-induced current significantly increased(143.92%±18.72%, n=6; p<0.01 versus the normal current induced by10-4M NMDA) . These results therefore illustrate that the upregulative effects of orexin-A on glutamate-induced current of pyramidal neurons in PFC is mediated by NMDA receptor.4. PKC, but not PKA, is involved in the excitatory modulatory of orexin-ABIS II (10-6M), a PKC inhibitor, abolished the upregulative effects of orexin-A on NMDA receptor channel(97.21%±9.88%,n=6; p>0.05 versus the normal current induced by10-4M NMDA).In contrast, the modulatory effects of orexin-A on NMDA receptor were still observed when the PKA inhibitor peptide (5-24) (10-6M) was included in the pipette (137.29%±9.16%, n=6; p<0.01 versus 10-4M NMDA). Altogether, these findings illuminate that the potentiation effects of orexin-A on NMDA receptor is mediated by PKC, but not by PKA signaling pathway. In summary,the expression of OX1R in PFC pyramidal neuron of rat was firstly successfully established by morphological studies.Moreover, the results of patch-clamp provide evidence that the modulatory effects of orexin with glutamate on acutely dissociated pyramidal neurons in rat PFC are mediated by the activation of PKC signaling cascades and up-regulates the activity of NMDA receptor subsequently.