Figure 5.
6-OHDA application enhances tonic spike firing in BGMT neurons via a reduction of IM K+ conductance. A, Voltage responses to increasing depolarizing step current injections (100, 200, 260, 300 pA) for a representative neuron of control mouse before (left panel) and after 10 min of adding 10–20 μm XE-991 to the bath ACSF (right panel). To block ionotropic glutamatergic and GABAergic synaptic inputs, DNQX (AMPA/kainate receptors antagonist, 10 μm), D-AP5 (NMDA receptor antagonists, 50 μm), and gabazine (GABA-A antagonist, 10 μm) were present in the ACSF throughout. B, Voltage responses to increasing pulse current injections (at amplitude 40, 70, 100, 150 pA) of respresenative neuron from 6-OHDA-treated mouse before (left panel) and after (right panel) 10-min application of XE-991. C, The F-I curve of a representative neuron from a control mouse is shown before and after 10–20 min bath exposure to XE-991 (10–20 μm). The lift shift following XE991 exposure suggests IM inhibition induced hyperexcitability. D, For each individual neuron, a Boltzmann sigmoidal fit was done for the F-I curve and the intercept at 3-Hz firing was determined as rheobase (see Materials and Methods). Neurons after exposure to XE-991 showed significant reduction in the rheobase compared with control ACSF (CON: 295.0 ± 41.19 pA; CON + XE-991: 225.2 ± 38.59 pA, n = 8; paired t test, p = 0.026). Baseline membrane potentials were depolarized up to −58.4 ± 1.2 mV by applying bias current 83.9 ± 26.1 pA to inactivate T-type Ca2+ currents and prevent burst firing. E, Based on Boltzmann sigmoidal fit for the F-I curve, the intercept at 10-Hz firing was determined as current at 10 Hz (see Materials and Methods). Neurons showed a significant reduction in the current required to drive 10-Hz firing after XE-991 application (CON: 325.9 ± 43.4 pA; CON + XE-991: 263.8 ± 40.94 pA, n = 8; paired t test, p = 0.0359). F, Average VT significantly decreased in the presence of XE-991 (CON: −32.89 ± 1.18 mV; CON + XE-991: −35.3 ± 1.28 mV; paired t test, p = 0.0064). G–J, XE-991 did not generate further changes in firing frequency of neurons from 6-OHDA-treated mice (6-OHDA vs 6-OHDA + XE-991) 1–7.1 months post lesion (mean ± SEM: 4.0 ± 1.0 months). G, Example of firing in single 6-OHDA neuron. Neither tonic rheobase (6-OHDA: 87.1 pA [−6.5,385.5]; 6-OHDA + XE-991: 80.0 pA [−16.6,372.7], n = 10; Wilcoxon test, p = 0.084), current at 10-Hz 6-OHDA: 100.3 ± 25.18 pA; 6-OHDA + XE-991: 92.04 ± 21.87 pA, n = 8; paired t test, p = 0.159), nor VT (6-OHDA: −38.03 ± 1.4 mV; 6-OHDA + XE-991: −38.12 ± 1.3 mV, n = 10; paired t test, p = 0.88) showed changes in the presence of XE-991. Box and whisker plots represent medians, quartiles, and 5th–95th percentiles. Individual neurons are shown as line graphs between the whisker plots.
Figure Contributions: Edyta K. Bichler conducted experiments, performed statistical analysis, and prepared figure. Dieter Jaeger reviewed data and analysis.