Figure 3

Electrophysiological alterations of LC neurons in an in vitro model of rotenone toxicity resemble those of αSyn overexpression. (a) Representative whole-cell spike train recording of a spontaneously active LC neuron in an acute brainstem slice derived from a C57BL/6 WT mouse. Wash-in of 1 µM rotenone after 2 min of stable firing tendentially, but not significantly, reduced the discharge rate of LC neurons within 10 min. (b) Example spike train recordings after slices were incubated for 2 h in standard ACSF (control, black) or ACSF + 1 µM rotenone (red) prior to recording. (c) Quantification of spontaneous firing 5 min (n = 6), 10 min (n = 6) and 2 h (n = 8) after exposure to 1 µM rotenone. Pre-incubation of slices in rotenone led to a significant acceleration of the spontaneous firing rate compared to control (n = 6). (d) Representative AP traces of a control LC neuron (black) and an LC neuron pre-incubated in rotenone (red). (e) Quantification revealed a significant reduction of the AHP amplitude of LC neurons in slices incubated with rotenone compared to slices incubated in control ACSF (control: n = 10, rotenone: n = 8). (f) Representative whole-cell voltage clamp recordings of I_A currents activated by a 500 ms voltage step to 0 mV from a holding potential of – 80 mV (as depicted by rhombus in 2a). (g) Peak current densities of K⁺ outward currents were ascertained from the start of the voltage step (indicated by rhombus in f) and did not show a significant difference between control LC neurons and rotenone-exposed LC neurons. (h) CoCl₂-sensitive Ca²⁺ inward currents derived from a voltage step to − 10 mV from a holding potential of – 80 mV as depicted in the inset. Either under control conditions (black) or after 2 h incubation in 1 µM rotenone (red), 1 mM CoCl₂ were washed in to block Cav channels. (i) Quantification of CoCl₂-sensitive current densities revealed no significant difference between control and rotenone-exposed LC neurons. (j) Representative I_AHP currents, activated by the two-pulse voltage clamp protocol. (k) Quantification of I_AHP current densities revealed a drastic reduction of AHP outward currents after pre-incubation in rotenone. (l) In accordance with reduced medium I_AHP currents, the kinetic analysis of the decay time (τ) showed a slowing of the fast and medium decay time constants after 2 h rotenone treatment (control: n = 10, rotenone: n = 11). Box plots display median and 25/75 percentile, whiskers indicate outliers. Rest of the data are presented as mean ± SEM and also as individual data points. *p < 0.05; **p < 0.01; ***p < 0.001. [(c,e,g,l) unpaired Student’s t-test vs. control. (i) Mann–Whitney-U test vs. control. (k) Welch’s t-test vs. control].