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HCN Channelopathy in External Globus Pallidus Neurons in Models of Parkinson’s Disease

C. Savio Chan, Kelly E. Glajch, Tracy S. Gertler, Jaime N. Guzman, Jeff N. Mercer, Alan S. Lewis, Alan B. Goldberg, Tatiana Tkatch1, Ryuichi Shigemoto, Sheila M. Fleming, Dane M. Chetkovich, Pavel Osten, Hitoshi Kita, D. James Surmeier

Figure S1 | GABAergic signaling is not involved in silencing of GPe neurons. (a) Plot of spike regularity of GPe neurons as a function of rate in a control solution. Only neurons with activity were included in the data set. (b) Plot of spiking variability of GPe neurons as a function of rate in the presence of the GABAA receptor antagonist SR95531. (c) Firing rate of GPe neurons before and after SR95531 application. SR95531 has no consistent effect on spiking of GPe neurons from either naïve or reserpine-treated mice. There were no cases in which silent neurons restarted pacemaking (n=8).

Nature Neuroscience: doi:10.1038/nn.2692

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Figure S2 | Nav channel α-subunit mRNA abundance and channel current amplitudes are not altered following dopamine depletion. (a) Voltage protocols (bottom) and representative traces (top) of transient, persistent and resurgent Na+ currents recorded from typical reserpine treated GPe neurons. (b) Population data on Na+ current amplitudes from control and reserpine treated animal groups. These parameters are not significantly different from their corresponding controls (P>0.05, Mann-Whitney). Transientmedian: naïve=677 pA (n=31), reserpine=830 pA (n=39). Persistentmedian: naïve =70 pA (n=27), reserpine=69 pA (n=35). Resurgentmedian: naïve =255 pA (n=36), reserpine=218 pA (n=39). (c) Single-cell RT-PCR serial dilution revealed no detectable changes in Nav α-subunit mRNA abundance in GPe neurons following reserpine treatment. No significant difference was found in the threshold of detection for both Nav1.1 and Nav1.6 α-subunits following reserpine treatment (p values > 0.5, Kolmogorov-Smirnov test). Smooth lines represent best fits of the threshold distrubiton with a log normal function, yielding the modal thresholds as follows: Nav1.1control=0.11 (n=26), Nav1.1reserpine=0.16 (n=24), Nav1.6control=0.14 (n=24), Nav1.6reserpine=0.12 (n=21). Right, cumulative probability analysis reveals identical distribution of detection threshold for Nav α-subunit mRNA abundance in GPe neurons from naïve (black) and reserpine treated (red) animals.

Nature Neuroscience: doi:10.1038/nn.2692

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Figure S3 | Firing capacity of GPe neurons is retained following dopamine depletion. Intracellular current injection into silenced GPe neurons from resperine-treated mice evoked repetitive spiking.

Nature Neuroscience: doi:10.1038/nn.2692

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Figure S4 | Activation kinetics of HCN currents in GPe neurons is unaltered. Current traces from –130 mV were well fit with a bi-exponential function yielding fast and a slow time constants from both naïve (τfast = 164.6 ms, τslow = 918.5 ms n = 20) and reserpine-treated (τfast = 189.1 ms, τslow = 923.8 ms n = 25) animals. No apparent change (P>0.05, Mann-Whitney) in the activation kinetics at this test voltage could be seen. This is summarized in a box plot format (right).

Nature Neuroscience: doi:10.1038/nn.2692

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Figure S5 | Expression of HCN channel transcripts in the GPe following chronic 6-OHDA lesions and HCN2-AAV injection. qPCR expression analyses of HCN1–4 and TRIP8b were performed on GPe from chronic 6-OHDA lesion and HCN2-AAV injected animals. Except for HCN3, the expression levels of HCN1, HCN2, HCN4 and TRIP8b were markedly reduced in GPe, similar to those seen in reserpine-treated animals.

Nature Neuroscience: doi:10.1038/nn.2692

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Figure S6 | AAV-mediated HCN2 delivery did not ameliorate of sensoirmotor deficits in 6-OHDA lesioned mice. (a–c) A challenge beam traverse task assesses overall motor function and coordination. (d–e) Spontaneous behavior was evaluated by monitoring movement in a transparent cylinder. (f) A adhesive removal task was used to measure sensorimotor impairment. (g) Gait impairment was measured (h–i) Pole test was use to examine motor impairment (j) A step adjustment test was used to assess motor impairment. In all behavioral paradigms tested no significant improvement was found with virus treatment (P>0.05, Mann-Whitney).

Nature Neuroscience: doi:10.1038/nn.2692

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Figure S7 | Working model of the cascade of events underlying the downregulation of HCN channels in GPe neurons. GPe (GABAergic) and STN (glutamatergic) are reciprocally connected to each other. Following dopamine depletion STN neurons increase burst firing. This increases bursting in GPe neurons and the associated Ca2+ influx through Cav/L-type Ca2+ channels. Through homeostatic machinery, the rise in Ca2+ level leads to a decrease in HCN channel expression. This leads to a loss of pacemaking in GPe neurons. This process is exacerbated by an increase in the ability of repetitive STN spiking to evoke burst spiking in GPe neurons, as a consequence of the loss in HCN channels, thus an increased coupling of GPe and STN and network synchrony.

Nature Neuroscience: doi:10.1038/nn.2692