The Role Of HCN Channels In The Process Of Orexin A Playing Its Excitatory Effect On Pyramidal Neurons From Mice Prefrontal Cortex

Orexin (hypocretin) which mainly produced by the neurons within the lateral hypothalamus, is a micromolecular neuropeptide discovered in 1998. 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. 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. As we known, prefrontal cortex (PFC) plays an important role on execution of higher nervous activity, and its activity is correlated with level of wakefulness. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel mainly locates on the dendritic spines of pyramidal neurons in PFC, inducing hyperpolarization -activated currents (Ih) when it is activated. HCN channel participate in the integration of excitatory synaptic input in pyramidal neurons, and thereby influences the excitability of neural network in PFC. Importantly, some transmitters and drugs can influence the neuronal excitability via regulation of HCN channels. Orexin A, as a classical wake-promoting neuropeptide, can enhance the activity of pyramidal neurons in prefrontal cortex effectively, while whether this effect of orexin A on prefrontal cortex pyramidal neurons involves its regulation on HCN currents is still little known.In the present study, using whole-cell voltage-clamp recordings on layer V pyramidal neurons of mouse prelimbic cortex (PL), one important part of the medial prefrontal cortex, we firstly observed the regulation of HCN channel on neuronal excitability, and then the interaction between orexin A and HCN channel was studied systematically. The results show as follow:1. HCN channels regulates the excitability of pyramidal neurons in PL of miceThe membrane potential was initially adjusted to-50 to -53mv using intracellular injection of direct current, and the recording neurons (n=6) fired spontaneously under this condition. Bath application of HCN channel blocker ZD7288 produced a modest depolarization in the membrane potential and dramatically increased spike firing. Adversely, the membrane potential was initially adjusted to -46 to -50mv in order that PL pyramidal neurons (n=5) fired at much higher frequency. Bath application of HCN channel enhancer 8-Br-cAMP dramatically decreased the spike firing. These data indicate that HCN channel can influence the excitability of pyramidal neurons in PFC. Increased HCN current decreases excitability, blockade of HCN channel enhances excitability.2. Relation between the excitatory effect orexin A and the production Ih current on pyramidal neurons in PLNot all recording pyramidal neurons in PL reacted to orexin A, the total reaction rate of orexinA on 54 recorded pyramid neurons was 51.9%, furthermore, the reaction rate of orexinA on 36 Ih (+) pyramid neurons was 66.7%, while the other18 Ih (-) pyramid neurons was 22.2%. These data indicate that the excitatory effect orexin A is much more noticeable in pyramid neurons with HCN channel.3. Orexin A can suppress HCN channel in PL pyramid neurons, which is mediated by OX1R and PKCIn current-clamp model, applying orexinA produced a reduction of voltage sag in recording cells, and the voltage sag ratio induced by Ih current was significantly decreased after application of orexin A (from 1.31±0.09 to 1.08±0.07, P<0.05, n=7). In voltage -clamp model, orexin A produced a significant decrease in the amplitude of Ih current evoked by voltage ramp protocol, and the suppression of orexin A on Ih current displayed in almost all hyperpolarized voltage steps (I-V:-70~-120mv, n=14). In addition, the activation curve of Ih current which was calculated by using the equation Itail.max-Itail/Itail.max had a negative shift of the half-activation voltage (V1/2) from 84.7+/-1.1 to 92.2+/-2.7mV after applying orexin A.The protocol was repeated in the presence of the OXR1 antagonist SB334867 and PKC inhibitor BIS-II. The amplitude of Ih current was decreased to 51.6±6.7 pA by 4 orexin A, and then recovered to 75.4±8.2pA by pre-application of SB334867 (P<0.05, n=3). Similarly, after bath incubation with BIS-II for from 30 to 90 minutes, the amplitude of Ih current respectively recovered to 65.4±5.5% at 60min point (P<0.05, n=7) and to 83.6±6.7% at 90min point (P<0.01, n=5). These data indicate that the role orexin A on HCN channel can be mediated by OXR1 and PKC.4. The state of HCN channel influences excitatory effect of orexin A on PL pyramidal neuronsFirstly, the neurons which could be excited by orexin A effectively were selected. Then, 8-Br-cAMP was bathed in recording solution before orexin A application again. As result, the spike firing induced by orexinA was attenuated modestly compared with the former (from 5.83±0.45Hz; to 3.78±0.66Hz, P<0.05, n=4). This data indicate that the state of HCN channel can influence excitatory effect of orexin A on PL pyramidal neurons.In summary, the studies reported here provide evidence that HCN channels have powerful influence on the firing properties of PL pyramidal neurons. Blockade of HCN channels increases the activity of PL pyramidal neurons, conversely, up-regulation of Ih current dramatically decreases the activity of PL pyramidal neurons. Most importantly, the present studies indicate that orexin A can decrease the amplitude of HCN currents and shift the currents activation curve to more negative level in PL pyramidal neurons, this effect is mediated by OX1 receptor and PKC signalling pathway. Furthermore, the excitatory effect of orexin A on PL pyramidal neurons is attenuated when HCN currents were enlarged.