Endoplasmic Reticulum and Mitochondrial Calcium Handling Dynamically Shape Slow Afterhyperpolarizations in Vasopressin Magnocellular Neurons

TG block of VP AHPs persists when AHPs are evoked by photolytic Ca²⁺ uncaging. A, Representative example of a Ca²⁺ trace (top) evoked by a single 50 ms pulse of 405 nm light (blue arrow) and the corresponding current clamp AHP evoked in response to the Ca²⁺ uncaging (bottom) under control conditions (left) and after TG incubation (right). B, Summary data of TG's effects on AHPs evoked by Ca²⁺ uncaging. Control n = 11, TG n = 8. 0.1 s amplitude (Mann–Whitney test; p < 0.5), 1 s amplitude (Mann–Whitney test; p < 0.05), duration (unpaired t test; t = 2.47; p < 0.05), area (unpaired t test; t = 2.27; p < 0.05), Ca²⁺ amplitude (unpaired t test; t = 0.01; p > 0.05), Ca²⁺ area (Mann–Whitney test; p > 0.05). C, Replication of current clamp AHPs evoked by 20 spike 20 Hz depolarizing pulse trains and corresponding Ca²⁺ responses using the modified Ca²⁺ uncaging internal solution containing Ca²⁺, a different Ca²⁺ indicator (fluo-4), and DM-Nitrophen which acts as a Ca²⁺ buffer. TG's effect on AHPs persists under these conditions. D, Summary data of TG's effects on AHPs evoked by 20-spike 20 Hz depolarizing pulse trains using the Ca²⁺ uncaging internal. Control n = 11, TG n = 8. 0.1 s amplitude (unpaired t test; t = 2.02; p > 0.5), 1 s amplitude (unpaired t test; t = 3.56; p < 0.01), duration (Mann–Whitney test; p < 0.0001), area (unpaired t test; t = 2.26; p < 0.05), Ca²⁺ amplitude (unpaired t test; t = 1.21; p > 0.05), Ca²⁺ area (unpaired t test; t = 1.60; p > 0.05).

Caffeine bath application potentiates AHPs. A, Representative example of a current-clamp 30-spike 20 Hz AHP (top) and the corresponding Ca²⁺ responses (bottom) before and after caffeine. Current-clamp AHP evoked in response to the uncaging (bottom) under control conditions (left) and after TG incubation (right). B, Summary data of caffeine's effects on current clamp AHPs. Control and caffeine n = 8. 0.1 s amplitude (paired t test; t = 1.87; p > 0.5), 1 s amplitude (paired t test; t = 1.03; p > 0.05), duration (paired t test; t = 2.86; p < 0.05), area (Wilcoxon test; p > 0.05), Ca²⁺ peak (paired t test; t = 3.64; p < 0.01), Ca²⁺ area (paired t test; t = 2.52; p < 0.05). C, Representative spike trains evoked by a 70 pA depolarizing step before and after caffeine bath application. Note the decreased spike frequency in the neuron after caffeine. D, Summary data of AHPs before and after caffeine evoked by depolarizing steps. Control and caffeine n = 8. Statistics performed with a two-way ANOVA and Šídák's multiple-comparisons test. p values for the overall test are provided in parentheses; for multiple comparisons, refer to the figure. Spike Hz (F = 5.2; p < 0.5), AHP amplitude (F = 0.87; p > 0.5), AHP amplitude/spike count (F = 2.21; p = 0.05). Neuronal gain did not significantly differ between groups (control 0.16 ± 0.018 vs caffeine 0.15 ± 0.017; linear regression, F = 0.3, p > 0.05).

Blocking mitochondrial Ca²⁺ buffering enhances I_AHP. A, Representative voltage-clamp trace of I_AHP (left) and corresponding Ca²⁺ responses (right) before and after CCCP bath application. B, Summary data of CCCP's effects on I_AHP. Control and CCCP n = 7. 0.1 s amplitude (paired t test; t = 2.54; p < 0.5), 1 s amplitude (paired t test; t = 5.42; p < 0.01), duration (paired t test; t = 5.79; p < 0.01), area (Wilcoxon test; p < 0.05), Ca²⁺ peak (paired t test; t = 2.61; p < 0.05), Ca²⁺ area (paired t test; t = 4.40; p < 0.01). C, Representative voltage-clamp trace of I_AHP (left) and corresponding Ca²⁺ responses (right) during Ru360 perfusion through the patch pipette at 5, 10, and 15 min and then subsequent bath application of CCCP. D, Summary data of Ru360 + CCCP's effects on I_AHP. n = 10. Statistics performed with a repeated-measures one-way ANOVA and Šídák's multiple-comparisons test. p values for the overall test are provided in parentheses; for multiple comparisons, refer to the figure. 0.1 s amplitude (F = 11.4; p < 0.01), 1 s amplitude (F = 6.59; p < 0.05), duration (F = 20.51; p < 0.0001), area (F = 8.75; p < 0.01), Ca²⁺ peak (F = 5.27; p < 0.05), Ca²⁺ area (F = 9.35; p < 0.01).

Blocking the mitochondrial Ca²⁺ uniporter with Ru360 enhances the AHP and slows firing in current clamp. A, Representative AHPs evoked with 30 spikes at 20 Hz (left) and corresponding Ca²⁺ responses (right) at 1 and 9 min after break-in. Note the enhancement of the AHP and Ca²⁺ signal time course after 9 min of perfusion. B, Summary data of Ru360 effects on AHPs compared with neurons without Ru360. Control n = 4, Ru360 n = 6. Statistics performed with a repeated-measures two-way ANOVA and Šídák's multiple-comparisons test. p values for the group comparison are provided in parentheses; for multiple comparisons, refer to the figure. 0.1 s amplitude (F = 28.72; p < 0.0001), 1 s amplitude (F = 14.52; p < 0.0001), duration (F = 18.86; p < 0.0001), area (F = 10.37; p < 0.0001), Ca²⁺ peak (F = 16.48; p < 0.0001), Ca²⁺ area (F = 13.69; p < 0.0001). C, Representative example of spiking in response to a 70 pA depolarizing step 1 min and 9 min after achieving whole-cell configuration with Ru360 in the pipette. Note the lower spike frequency at 9 min. Summary data of Ru360 effects on AHP spike frequency performed with a one-way ANOVA and Šídák's multiple-comparisons test (n = 5; F = 21.76; p = 0.001). For multiple comparisons refer to the figure.