Electrophysiological Studies Of HCN Channels In The Globus Pallidus Of Both Normal And MPTP Parkinsonian Mice

The globus pallidus (GP) is an important relay nucleus located centrally in the basal ganglia, which plays a key role in multiple feedback loops in both health and pathological states. Recent studies have reported that hyperpolarization activated cyclic-nucleotide gated (HCN) channels occupies a significant position in globus pallidus pacemaking activity. Morphological studies have shown that HCN channels are expressed in the globus pallidus. Previous in vitro patch clamp recordings revealed that blockade of HCN channels significantly changes the spontaneous firing of globus pallidus neurons.Object:To evaluate the electrophysiological effects of HCN channel blocker ZD7288and/or CsCl on the spontaneous firing of globus pallidus neurons, as well as the expression of HCN channels in both normal and MPTP-treated parkinsonian mice.Methods:In vivo extracellular single unit recordings, MPTP parkinsonian mice and immunohistochemical staining were performed in the present study.Results:1. In normal mice, the spontaneous firing activity of77pallidal neurons were recorded. The average frequency of spontaneous firing was7.9±0.8Hz. Three firing pattern of globus pallidus neurons in mice were recorded, with20(25.4%) regular firing neurons,43(56.3%) irregular firing neurons and14(18.3%) burst firing neurons.2. In normal mice, micro-pressure ejection of0.05mM ZD7288decreased the frequency of spontaneous firing from9.8±1.5Hz to5.0±0.9Hz in30out of the58(51.7%) neurons. The average decrease was54.0±5.1%(P<0.001). Further firing pattern analysis showed that ZD7288increased both CV (Basal:0.82±0.14; ZD7288:1.39±0.19; P＜0.001) and FF (Basal:0.29±0.12; ZD7288:1.57±0.49; P＜0.001), suggesting that ZD7288decreased the firing regularity of neurons. In another22(37.9%) pallidal neurons with the basal firing rate of5.9±1.3Hz, ZD7288increased the firing rate to10.8±2.1Hz. The average increase was90.3±14.8%(P＜0.001). A negative correlation between ZD7288-induced change in firing rate and basal firing level were observed in globus pallidus neurons (r=-0.284, P＜0.05).3. In another group, the spontaneous firing activity of19pallidal neurons was recorded in normal mice. Micro-pressure ejection of1mM CsC1decreased the frequency of spontaneous firing from8.6±1.3Hz to3.9±1.1Hz in6(31.6%) neurons. The average decrease was59.7±9.8%(P＜0.001). In another8(41.1%) out of the19pallidal neurons with the basal firing rate of4.7±1.4Hz, ZD7288increased the firing rate to7.5±2.1Hz. The average increase was81.1±17.1%(P＜0.05).4. In MPTP-treated parkinsonian mice, the basal firing rate was7.5±0.7Hz in28pallidal neurons recorded, which was not significantly different from that of normal mice (P>0.05). Six out of the28pallidal neurons were identified as regular firing neurons,15were irregular firing neurons and7were burst firing neurons. The fraction of irregular firing and burst firing neurons in MPTP parkinsonian mice tends to be more than that in normal mice although there was no statistical significance (x2=0.38,0.10,0.71d.f.=1, P>0.05).5. In MPTP parkinsonian mice, local administration of0.05mM ZD7288decreased the firing rate from10.3±1.6Hz to5.8±1.1Hz in10(35.7%) out of the28pallidal neurons. The average decrease was40.4±6.1%(P<0.01).In another15(53.6%) pallidal neurons, ZD7288increased the spontaneous firing rate from7.0±1.3Hz to13.4±3.1Hz. The average increase was150.9±53.7%(P＜0.05). ZD7288did not change the CV and FF in neurons with both ZD7288-induced decrease and increase of firing rate. A negative correlation between ZD7288-induced change in firing rate and basal firing level were observed in globus pallidus neurons (r=-0.396, P＜0.05).6. Positive expression of HCN1, HCN2, HCN3and HCN4subtypes was observed in the globus pallidus of both normal and MPTP parkinsonian mice. Immunolabeling for HCN1was mainly observed on cell body, while the strength of labeling for HCN2and HCN3on cell body was similar to that on neuropil elements. Weak HCN4subtype labeling was observed on neuropil elements. The number of HCN immunopositive pallidal neurons in MPTP parkinsonian mice was decreased than that in normal mice.Conclusion:The present in vivo electrophysiological studies provided evidence that blockade of HCN channels produced inhibition or excitation on the globus pallidus neurons of both normal and MPTP parkinsonian mice. Furthermore, ZD7288changed the firing pattern of pallidal neurons in normal mice but not parkinsonian mice. Immunostaining showed positive expression of HCN1, HCN2, HCN3and HCN4subtypes in the globus pallidus, with fewer immunopositive neurons in parkinsonian mice. The present results may provide a rationale for further investigations into the involvement of pallidal HCN channels in the pathophysiology of Parkinson’s disease.