Orexin-A And Adenosine Modulate The Excitability Of Principal Neurons In The Superficial Layers Of Rat Entrohinal Cortex

The entorhinal cortex (EC) has long been regarded as a bidirectional gateway between higher cortical areas and the hippocampus. Stellate and pyramidal neurons are the principal neurons in the superficial layers of the EC. The axons of the stellate neurons in layer II of the EC form the perforant path that innervates the dentate gyrus and CA3, whereas those of the pyramidal neurons in layer III form the temporoammonic pathway that synapses onto the distal dendrites of pyramidal neurons in CA1 and the subiculum. There is strong experimental evidence that the EC not only occupies a key position in the neocortex-hippocampus-neocortex memory circuit, but also is part of a network aiding in the consolidation and recall of spatial memories. It is well known that fulfilling congnitive function depends on the level of wakefulness. Orexins (hypocretins) are wake-promoting neuropeptides which are mainly produced by the neurons within the lateral hypothalamus. Orexin system includes two separate peptides orexin-A and orexin-B proteolytically derived from the same precursor protein and two specific G-protein-coupled receptors OX1R and OX2R. Many studies have indicated that the excitatory effects of orexins involve diverse signaling cascades triggering the multiple ionic and synaptic mechanisms in varying brain regions. There is good agreement that the increase in adenosine levels during prolonged intervals of wakefulness is assumed to be caused by an increase in metabolism and neural activity. In contrast to orexins, adenosine has a general inhibitory effect and thereby reduces excitability throughout the brain. However, the function of orexins and adenosine in the EC is still eclusive. We hepothesize that orexins effects within the EC may increase neuronal excitability and thereby promote EC arousal in physiological condition, while adenosine may play a central role in bringing about the effects of prolonged wakefulness on macroscopic brain activity by decreasing neural excitability.In the present study, we firstly investigated the electrophysiological effects of orexin-A and adenosine on stellate neurons and pyramidal neurons in live brain slices of the EC using whole-cell patch-clamp recordings, and then observed signaling cascades triggering the ionic and synaptic mechanisms involved in orexin-A. The results show as follow:1. Orexin-A depolarizes stellate and pyramidal neurons in the superficial layers of EC through effects on K+ channels and non-selective cation conductancesThe sustained K+ current was first isolated in 1μM TTX to block Na+ currents and 5 mM 4-AP to block the transient K+ current (IK) and was evoked by +20 mV voltage steps (0.5 s) from -60 mV holding potential to +40 mV. Exposure to orexin-A (100 nM) resulted in statistically significant decreases in the sustained IK in response to the larger depolarizing pulses (n = 8), indicating that orexin-A depolarizes principal neurons through the inhibition of sustained IK.We next used slow voltage ramps [-100 to 0 mV (10 s)] following a prepulse to -100 mV (0.5 s) to determine if orexin-A influenced EC neurons as a consequence of activation of such conductances. The data show the difference current obtained by subtracting control ramps from those obtained during orexin-A (100 nM). The difference current was linear throughout the voltage range tested (r2 = 0.99) indicating a lack of voltage dependence to the current. The mean reversal potential of the orexin-A-sensitive current was found to be -41.8±4.8 mV (n = 6). These data demonstrate orexinergic effect on neuronal excitability occurring as a consequence of the modulation of nonselective cationic conductances (NSCC).We also tested the effect of orexin-A on hyperpolarization-activated/cyclic nucleotide-gated channels, G-protein-coupled inwardly rectifying K+ channels, voltage-dependent Ca2+ channels (VDCC) and Na+/Ca2+ exchangers on the principal neurons in the superficial layers of the EC, and found these channels were not involved in.2. The excitatory action of orexin-A involves OX1Rs and PKC mediates both the inhibition of IK and activation of NSCC induced by orexin-ABath application of SB-334867 (5μM), a specific OX1R antagonist, completely blocked the orexin-A (100 nM)-induced depolarization of membrane potential. Entorhinal slices were again preincubated in ACSF containing PKC inhibitor BIS-II for at least 2 h before cell recordings were obtained. When these neurons were maintained in 1μM BIS II, orexin-A had no effect on the membrane potential or sustained IK (n = 8) and NSCC (n = 9). Thus, The excitatory action of orexin-A involves OX1Rs and PKC mediates both inhibition of IK and the activation of NSCC induced by orexin-A.3. Orexin-A enhances both excitatory and inhibitory synaptic transmissions in the superficial layers of EC via OX1RsSEPSCs mediated by glutamate and sIPSCs mediated by GABA were recorded in stellate and pyramidal neurons in the presence of the GABAA receptor antagonist bicuculline (10μM) or ionotropic glutamate receptor antagonist CNQX (10μM) and AP-V (50μM). Application of orexin-A (100 nM) significantly increased the frequency (sEPSCs: 202±56 % of control, n = 29, P < 0.001, K-S test; sIPSCs: 199±45 % of control; n = 22; P < 0.001; K-S test) and amplitude (sEPSCs: 138±29 % of control, n = 29, P < 0.001, K-S test; sIPSCs: 114±11 % of control, n = 22, P < 0.001, K-S test) of sEPSCs and sIPSCs. In the presence of bicuculline (10μM) and TTX (1μM) mEPSCs were recorded, and mIPSCs were recorded in the presence of CNQX (10μM), AP-V (50μM) and TTX (1μM). Application of orexin-A significantly increased the frequency (mEPSCs: p < 0.001,n = 6,K-S test; mIPSCs: p < 0.001,n = 5,K-S test) and amplitude (mEPSCs: p < 0.001,n = 6,K-S test; mIPSCs: p < 0.001,n = 5,K-S test) of mEPSCs and mIPSCs. It is noteworthy that in the presence of SB-334867 (5μM) orexin-A did not induce any changes in the frequency (mEPSCs: p = 0.17, n = 6, K-S test; mIPSCs: p = 0.14, n = 5, K-S test) and amplitude (mEPSCs: p = 0.22, n = 6, K-S test; mIPSCs: p = 0.10, n = 5, K-S test) of mEPSCs and mIPSCs, respectively. Together, these results indicate that orexin-A increases glutamatergic and GABAergic synaptic transmission pre- and postsynaptically through activation of OX1Rs in the EC.4. Orexin-A translocates NMDA receptors to the membrane surfaceIn Mg2+-free superfusion medium and at a holding potential of -60 mV, NMDA receptor agonist NMDA (100μM) to principal neurons caused an inward current (INMDA). The amplitude of INMDA was 824.9±163.7 pA (n = 7). In the presence of MK-801 (10μM), an irreversible NMDA receptor antagonist, INMDA was completely blocked. We then washed out the unbound MK-801. During this washout period orexin-A (100 nM) was applied for 15 ~ 30 min, and subsequently, the third application of NMDA induced INMDA again (41.5±26.3 pA, n =7). These data indicate that orexin-A stimulates movement of intracellular NMDA receptors to the membrane surface.5. Adenosine reduces the excitability of principal neurons in the superficial layers of ECIn voltage-clamp model, all cells fired action currents at a holding potential of -40 mV. Application of adenosine (100μM) led to abolish firing activity in tested neurons (n = 31). In a separate set of experiments, excitability was measured as the number of action potentials evoked by a fixed amplitude of current injection (700 ms duration) from resting potential. After adenosine was applied there was a significant decrease in the number of evoked action potentials (control, 5.5±1.4; post-adenosine, 2.4±1.3; washout, 5.2±1.3; n = 16; P < 0.001; paired t test). In current-clamp model, adenosine at concentrations of 50, 100 and 200μM caused a mean hyperpolarization of 1.5±0.3 (n = 6), 2.8±0.5 (n = 20), 4.4±0.6 (n = 12) mV. Interestingly, the inhibitory effect was blocked by 3μM DPCPX (n = 6), a selective A1 receptor antagonist, but not by 10μM DMPX (n = 6), a selective A2 receptor antagonist. In the presence of 8-Br-cAMP, a HCN channel agonist, the amplitude of hyperpolarization-induced by adenosine was reduced; conversely, in the presence of ZD-7288, a HCN channel antagonist, adenosine could not hyperpolarize stellate neuron. These results demonstrate that adenosine reduces the excitability of principal neurons by modulating HCN channels.In summury, the studies reported here provide evidence that the direct excitatory effects of orexin-A on principal neurons in the superficial layers of rat EC are mediated by the activation of OX1Rs and PKC signaling cascades, which inhibit sustained K+ current and activate NSCC. In addition, orexin-A enhances both excitatory and inhibitory synaptic transmissions in the EC via OX1Rs. Moreover, orexin-A produces long-term potention of synaptic strength by movement of NMDA receptors into the membrane surface. Finally, activation of adenosine A1 receptors reduces the excitability of principal neurons involving HCN channels.