Electrophysiological And Behavioral Studies Of HCN Channels In Rat Globus Pallidus

As an important relay nucleus in the indirect pathway of the basal ganglia, the globus pallidus plays a significant role in motor control under both health and pathological states. Recent studies have revealed that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels occupies a critical position in globus pallidus pacemaking activity. Morphological studies have shown the expression of HCN channel in the globus pallidus. Previous in vitro patch clamp recordings revealed that HCN channels are involved in the regulation of spontaneous firing of the globus pallidus. Object: To evaluate the electrophysiological effects of HCN channel blocker ZD7288 on the firing rate of globus pallidus neurons in normal and parkinsonian rats, the posturing regulation in haloperidol-induced catalepsy rats as well as the expression of HCN channels in the globus pallidus. Methods:In vivo extracellular single unit recordings,6-OHDA-lesioned parkinsionian rats, behavioral test and immunohistochemical staining were performed in the present study. Results:1. In normal rats, the spontaneous firing activity of 35 pallidal neurons were recorded. Micropressure ejection of 0.5mM ZD7288 decreased the frequency of spontaneous firing from 12.0±2.1 Hz to 6.5±1.3 Hz in 16 (45.7%) out of the 35 pallidal neurons. The average decrease was 53.9±6.7%(P<0.001). In another 16 (45.7%) out of the 35 pallidal neurons with the basal firing rate of 8.9±0.9 Hz, ZD7288 increased the firing rate to 16.6±1.9 Hz. The average increase was 90.3±14.8%(P<0.001). 2. On the lesioned side of 6-OHDA-lesioned parkinsonian rats, local administration of 0.5mM ZD7288 decreased the firing rate from 7.8±2.4 Hz to 3.1±1.3 Hz in 10 (37.0%) out of the 27 pallidal neurons. The average decrease was 70.2±7.5%(P<0.001). In another 15 (55.6%) out of the 27 pallidal neurons, ZD7288 increased the spontaneous firing rate from 7.8±1.4 Hz to 17.1±3.0 Hz. The average increase was 149.8±29.8%(P<0.001).3. On the unlesioned side of 6-OHDA-lesioned parkinsonian rats, ZD7288 significantly decreased the firing rate in 12 (42.9%) out of the 28 neurons (basal:12.8±2.4 Hz; ZD7288:7.1±1.4 Hz; average decrease:46.1±4.6%, P<0.001). In 10 (35.7%) out of the 28 neurons, ZD7288 increased the firing rate from 7.9±2.4 Hz to 14.±3.0 Hz (P<0.01). The average increase was 130.7±34.2%. The ZD7288-induced decrease of firing rate on the lesioned side of pallidal neurons was stronger than that on the unlesioned side (P<0.05) 4. In order to explore the mechanism of ZD7288-induced increase in firing rate, mixture of D-AP5 (0.8mM) and CNQX (1.OmM) was used to identify the possible involvement of glutamatergic transmission. Total 17 pallidal neurons were recorded in 11 normal rats. Micropressure ejection of D-AP5 and CNQX did not induce any change in the spontaneous firing rate of pallidal neurons. In the presence of D-AP5 and CNQX, ZD7288 only increased the spontaneous firing in 3 out of the 17 pallidal neurons. The excitatory rate (17.7%) was significantly lower than that in the absence of D-AP5 and CNQX (45.7%, P<0.05). In 8 out of the 17 pallidal neurons, ZD7288 with D-AP5 and CNQX did not alter the firing rate significantly. The percentage of neurons with no response to ZD7288 (47.0%) was significantly different from that without D-AP5 and CNQX (8.6%, P<0.01).5. In behaving test, intrapallidal microinjection of ZD7288 could induce different dystonic posturing in rats with haloperidol-induced catalepsy. In 4 out of the 9 rats, ZD72884 induced significant contralateral dystonic posturing (u=132.500, P<0.001 compared to normal saline group). In the rest 5 rats, ZD72884 induced significant ipsilateral dystonic posturing (u=9.000, P<0.001 compared to normal saline group).6. In the globus pallidus of both normal and 6-OHDA-lesioned rats, positive immunolabeling for HCN1, HCN2 and HCN4 was observed in the medium-size multipolar neurons, with more intense for HCN2 and weak to moderate for HCN1 and HCN4. Immunolabeling for HCN1 was mainly observed in the cell body and more weakly in the neuropil. However, the perikaryal labeling for HCN2 was less intense than that for HCN1, with stronger HCN2 labeling observed in the neuropil. Immunolabeling for HCN4 was also present in the globus pallidus, where the strength of labeling on the cell bodies and neuropil elements was similar. Conclusion:The present in vivo electrophysiological studies provided evidence that blockade of HCN channels produced excitation or inhibition on the globus pallidus neurons of both normal and 6-OHDA parkinsonian rats. Glutamaterigic transmission may be involved in ZD7288-induced excitation. Behavioral studies revealed that intrapallidal microinjection of ZD7288 could induce different dystonic posturing in rats with haloperidol-induced catalepsy, which was related to the different electrophysiological effects of ZD7288. Finally, immunostaining showed positive expression of HCN1、HCN2 and HCN4 subtypes in the globus pallidus of both normal and parkinsonina rats. As the globus pallidus and HCN channels are involved in the pathophysiology of basal ganglia movement disorders, our results may provide a rationale for further investigations.