Fig 1.
(A) 2P reconstruction of SNc DA neuron. (B) Left, normal pacemaking of a SNc DA neuron. Middle, TTX (1 μM) application uncovered slow oscillatory potential (SOP). Right, 5 μM baclofen application hyperpolarized the cell. (C) Summary of hyperpolarization due to application of 5 μM baclofen (n = 8, median = -25.24 mV). (D) Sustained uncaging of 5 μM RuBi-GABA in the presence of 25 μM gabazine (to block GABAA receptors) hyperpolarized cells in a manner similar to that seen following baclofen application. (E) Summary of hyperpolarization due to sustained 5 μM RuBi-GABA uncaging (n = 7, median = -11.71 mV).
Fig 2.
(A) Top, plot of the normalized firing rate before during and after a 60 s uncaging pulse (blue bar) of 5 μM RuBi-GABA in the presence of 25 μM gabazine (n = 4). Bottom, plot of normalized firing rate (black line) and running standard deviation (grey area) before, during and after a 50 ms uncaging pulse in the presence of 5 μM RuBi-GABA and 25 μM gabazine (n = 12); application of 2 μM CGP 55845 blunted the changes in spiking induced by RuBi-GABA uncaging (orange line, n = 4). Example raster plots are shown at the top of the panel. (B) Left, two different time scales showing action potentials just prior to and after GABA uncaging. Right, overlaid action potentials from just prior to and after GABA uncaging showing a clear reduction in the mAHP. (C-D) As in panel A (bottom) and B, but in the absence of gabazine (n = 9).
Fig 3.
(A) Example SK voltage-clamp recordings showing baseline (black), and responses to 200 nM apamin (blue) and 5 μM baclofen (orange). Right, summary of normalized response to baclofen (n = 8, Wilcoxon signed rank test, p = 0.0078). (B) SOPs (black) are greatly slowed and increase in amplitude with exposure to 200 nM apamin (blue). Right, summary of the normalized variance (n = 9, Wilcoxon signed rank test, p = 0.0151).
Fig 4.
(A) Application of 10 μM isradipine (orange) to inhibit CaV1 channels did not reduce total SK charge (n = 8, Wilcoxon signed rank test, p = 0.3828), while inhibiting CaV3 channels with 10 μM mibefradil (blue) inhibited roughly half the charge (n = 8, Wilcoxon signed rank test, p = 0.0078). (B) 5 μM baclofen (green) application did not inhibit T-type calcium current (n = 12, Wilcoxon signed rank test, p = 0.1099).
Fig 5.
PKA activation prevents GABAB modulation of SK.
(A) Schematic diagram showing the hypothesized signaling pathway from GABAB receptor activation to SK channels, and the site of action of 8-bromo-cAMP and H-89 in that pathway. (B) A 50 ms uncaging pulse elicited an immediate and significant change in SOP variance (black, n = 10, Wilcoxon signed rank test, p = 0.002). (C) The same was not seen when cells were incubated in 1 μM 8-bromo-cAMP (orange, n = 4, Wilcoxon signed rank test, p = 0.875). (D) Directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SOP variance (n = 4, Wilcoxon signed rank test, p = 0.75). (E) Inhibiting PKA with 10 μM H89 increases SOP variance (n = 6, Wilcoxon signed rank test, p = 0.0938). (F) Summary data for panels A-B. (G) Summary data for panels C-D. (H) Top, inhibiting PKA with 10 μM H89 significantly decreases SK current (n = 6, Wilcoxon signed rank test, p = 0.0313). Bottom, directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SK current (n = 5, Wilcoxon signed rank test, p = 1.00). (I) Summary data for PKA modulators from panel H and Rp-8-CPT-cAMPS (n = 3, Wilcoxon signed rank test, p = 0.25).
Fig 6.
ER Ca2+ contribution to SK current.
(A) Application of CPA to empty ER Ca2+ stores significantly reduced SK current (orange, n = 6, Wilcoxon signed rank test, p = 0.0313) and further application of 10 μM H89 to inhibit PKA activity further significantly reduced SK current (brown, n = 6, Wilcoxon signed rank test, p = 0.0313), but does not reduce it any further than H89 alone (Mann-Whitney U test, p = 0.1320). Inhibiting ryanodine receptors with 10 μM dantrolene did not change SK current (black, n = 4, Wilcoxon signed rank test, p = 1.00). (B) Left, an example experiment showing the effect of DHPG application on ER Ca2+ levels. Right, summary of normalized data showing a small, but not significant change in induced ER Ca2+ release after application of H89 (n = 4) compared to control (n = 7) recordings (Mann-Whitney U test, p = 0.2303).
Fig 7.
Schematic diagram depicting hypothesized signaling pathways involved in the GABAB receptor-mediated inhibition of SK channels.
GABAB receptor inhibition of AC by Gi signaling is hypothesized to be responsible for reduced cAMP levels and PKA signaling. The reduction in PKA activity is hypothesized to reduce SK channel opening through mechanism that are independent of either plasma membrane Ca2+ channels or release from intracellular stores.