Electrophysiological And Behavioral Studies Of HCN Channels In Rat Subthalamic Nucleus

The subthalamic nucleus (STN) is a key component in the basal ganglia, which participates indirect pathway that mediate a variety of motor functions both in health and pathological states. Recent studies have showed that hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are high expressed in the STN and play a regulatory role in neuronal excitability. Previous in vitro patch clamp recordings revealed that HCN channels are involved in the regulation of intrinsic pacemaking of the STN.Object:To investigate the electrophysiological effects of HCN channel blocker ZD7288on the firing rate of subthalamic nucleus neurons in normal rats, the electrophysiological effects of HCN channel agonist8-Br-cAMP and another HCN channel blocker CsCl on the firing rate of subthalamic nucleus neurons in normal rats, the expression of HCN channels in the subthalamic nucleus, and the effects of ZD7288and8-Br-cAMP on the posturing regulation in haloperidol-induced catalepsy rats.Methods:Extracellular single unit recordings in normal rats, immunohistochemical staining, Nissl staining and behavioral test were performed in the present study.Results:1. In the present electrophysiological recordings, total160subthalamic nucleus neurons in71normal rats were recorded. The average spontaneous firing rate was10.2±0.8Hz. According to the ISI histogram and autocorrelogram, the neurons were classified into three different firing types:78(48.8%) regular type,52(32.5%) irregular type and30(18.7%) bursty type.1. In normal rats, the selective HCN channel antagonist ZD7288, produced two opposite effects in all the89subthalamic nucleus neurons recorded. In42out of the89subthalamic nucleus neurons, ZD7288decreased the frequency of spontaneous firing from11.6±1.8Hz to5.7±1.3Hz (n=42, P<0.001). The average decrease was56.7±5.3%(P<0.001). In the remaining47subthalamic nucleus neurons with the basal firing rate of9.5±1.6Hz, ZD7288increased the spontaneous firing rate to16.3±2.4Hz (n=47, P<0.001). The average increase was142.2±29.8%(P<0.001).2. In order to explore the mechanism of ZD7288-induced increase in firing rate,8-Br-cAMP was used in the spontaneous firing activity of another39subthalamic nucleus neurons recorded in normal rats. Micropressure ejection of0.1mM8-Br-cAMP18subthalamic nucleus neurons increased the frequency of spontaneous firing from9.0±1.9Hz to15.6±3.1Hz (n=18, P<0.001). The average increase was126.4±36.8%(P<0.001). In15out of the39subthalamic nucleus neurons,8-Br-cAMP decrease the spontaneous firing rate from6.5±0.8Hz to2.0±0.5Hz (n=15, P<0.001). The average decrease was66.7±6.5%(P<0.001). In the remaining6neurons,8-Br-cAMP did not change the firing rate significantly (basal:11.9±3.2Hz;8-Br-cAMP:12.9±4.0Hz; increase:2.9±5.8%, n=6, P>0.05).3. In another group, the spontaneous firing activity of21subthalamic nucleus neurons were recorded in normal rats. Micropressure ejection of lmM CsCl decreased the frequency of spontaneous firing from7.1±1.0Hz to2.4±0.6Hz in13neurons (n=13, P<0.001). The average decrease was67.0±8.1%(P<0.001). In another8neurons with the basal firing rate of12.0±3.8Hz, CsCl increased the firing rate to20.2±6.0Hz (n=8, P<0.05). The average increase was110.6±48.7%(P<0.05).4. In behaving test, intrapallidal microinjection of ZD7288or8-Br-cAMP could induce different dystonic posturing in rats with haloperidol-induced catalepsy. In7out of the12rats of ZD7288group, ZD7288induced significant contralateral deflection posture (u=47.5, P<0.001compared to normal saline group). In the rest5rats, ZD7288induced significant ipsilateral deflection posture (u=36.000. P<0.001compared to normal saline group). In6out of the12rats of8-Br-cAMP group,8-Br-cAMP induced significant contralateral deflection posture (u=75.4, P<0.001compared to normal saline group). In the rest3rats,8-Br-cAMP induced significant ipsilateral deflection posture (u=61.0, P<0.001compared to normal saline group).5. In immunohistochemical staining, HCN subunits are clearly expressed and distributed throughout the subthalamic nucleus neurons in normal rat.Conclusion:The present in vivo electrophysiological studies provided evidence that blockade of HCN channels by ZD7288produced excitation or inhibition on the subthalamic nucleus neurons of normal rats. HCN channel agonist8-Br-cAMP and another HCN channel blocker CsCl can induce the same bidirectional effects on the subthalamic nucleus neurons of normal rats as well. We hypothesized that the unequal expression of different HCN subunits in individual subthalamic neurons and/or unequal expression of HCN channels on different neuronal subpopulation may be related to its bidirectional modulation observed in present electrophysiological study. Behavioral studies revealed that intrapallidal microinjection of ZD7288could induce different dystonic posture in rats with haloperidol-induced catalepsy, which was related to bidirectional effects of ZD7288. The present immunohistochemistry under light microscope showed that four HCN subunits are clearly expressed and distributed throughout the subthalamic nucleus neurons in normal rat. Based on the present findings, we hypothesize that alterations of the HCN channels in the subthalamic nucleus may contribute to its involvement in some neurological diseases.