Article Figures & Data
Figures
- Figure 1.
Biophysical properties of T-currents in the rat CeM. A, T-current traces from representative CeM neurons generated using a double-pulse protocol with 3.6-s-long prepulses to variable voltages (from –120 to –50 mV in 5-mV increments) and a test potential (Vt) of –50 mV recorded with TMA ATP-free internal solution. B, The average steady-state inactivation (I/Imax) curve; the V50 value is noted on the graph. C, Average current density, as calculated from the steady-state inactivation protocol. D, T-current I–V traces from representative CeM neurons in the voltage range for Vt of –80 to –45 mV from an initial holding potential (Vh) of –90 mV in 2.5-mV increments recorded with TMA ATP-free internal solution. E, The average voltage dependence of a steady-state activation (G/Gmax) curve with V50 value noted on the graph. F, Average current density from multiple I–V curves.
- Figure 2.
ATP regulates current density and relieves voltage-dependent inactivation of T-type calcium channels. A, T-current traces from a representative CeM neuron with respective V50 values generated using a double-pulse protocol with 3.6-s-long prepulses for variable voltages (from −120 to −50 mV in 5-mV increments) and a test potential (Vt) of −50 mV; gray traces recorded with a Cs ATP-free internal solution; and orange traces recorded with an internal solution of Cs with ATP. B, Normalized average steady-state inactivation (I/Imax) curve, depolarizing shift of 7.86 mV in a cohort of neurons with an internal solution of Cs with ATP was statistically significant from a cohort with Cs ATP-free internal solution (unpaired two-tailed t test, t(34) = 6.53, p < 0.001). C, Average current density, as calculated from the steady-state inactivation protocol with Cs-based internal solutions. Addition of ATP significantly increased current density by approximately two-fold [two-way RM ANOVA: interaction (F(13,442) = 12.91, p < 0.001), voltage (F(13,442) = 122.00, p < 0.001), and ATP (F(1,34) = 33.71, p < 0.001, Sidak’s post hoc presented on graph)]. D, The average normalized voltage dependence of steady-state activation (G/Gmax) in internal solution with ATP (orange line) and without ATP (black line) conditions. Note that the two curves overlap. E, Average current density from multiple I–V curves, addition of ATP increases T-current density [two-way RM ANOVA: interaction (F(13,481) = 9.68, p < 0.001), voltage (F(13,481) = 79.44, p < 0.001), and ATP (F(1,37) = 32.76, p < 0.001, Sidak’s post hoc presented on graph)]; *p < 0.05, ***p < 0.001.
- Figure 3.
ATP modulates recovery from inactivation of isolated T-currents. A, T-current traces from a representative CeM neuron generated using a paired-pulse protocol (top). Original traces show recovery from inactivation conditions with ATP at –120 mV, which lasted from 200 to 1000 ms (bottom); orange trace, 200 ms; gray trace, 400 ms; violet trace, 600 ms; blue trace, 800 ms; and green trace, 1000 ms. B, Recovery time courses with an ATP internal solution (orange) and an ATP-free internal solution (gray) fitted with a double exponential function at –120 mV with respective time constants tau (τ). Gray box indicates significantly different data points in two recording conditions. C, Recovery time courses with an ATP internal solution (orange) and ATP-free solution (gray) fitted with a double exponential function at –90 mV with respective time constants (τ). Note that recovery from inactivation was slower with the ATP-free internal solution solution at –120 mV than at –90 mV.
- Figure 4.
Mechanisms of T-current inhibition by TTA-P2. A, Traces of inward calcium current in a representative CeM neuron in control conditions recorded with TMA ATP-free internal solution using a double-pulse protocol with 3.6-s-long prepulses to variable voltages; left panel, from –120 to –95 mV in 5-mV increments (control); right panel, traces from the same cell using the identical voltage-protocol during an apparent steady-state inhibition of T-current in the presence of 5 μM TTA-P2. B, Average normalized current density, as calculated from the steady-state inactivation protocol. The presence of TTA-P2 (violet line and data points) decreased current density by 60–65% in comparison to the control conditions (gray line and data points). Data were analyzed with two-way RM ANOVA [interaction (F(12,96) = 36.09, p < 0.001), voltage (F(12,96) = 98.41, p < 0.001), and TTA-P2 (F(1,8) = 41.34, p < 0.001, Sidak’s post hoc presented on figure)]. C, Average normalized steady-state inactivation (I/Imax) curves in control conditions and after application of TTA-P2 in the same cells. TTA-P2 induced a large hyperpolarizing shift in V50 of 15.26 mV (paired two-tailed t test, t(8) = 6.82, p < 0.001). D, Average normalized current density, as calculated from the steady-state inactivation protocol recorded with Cs with ATP internal solution before (orange line and data points) and after application of TTA-P2 (violet line and data points). TTA-P2 reduced current density by 65% (two-way RM ANOVA: interaction (F(13,104) = 55.40, p < 0.001), voltage (F(13,104) = 114.40, p < 0.001), and TTA-P2 (F(1,8) = 73.38, p < 0.001, Sidak’s post hoc presented on figure)]. E, Average steady-state inactivation (I/Imax) curves in control conditions and after TTA-P2 recorded with Cs-based internal solution. TTA-P2 induced a significant hyperpolarizing shift in V50 of 4.55 mV (paired two-tailed t test, t(8) = 2.77, p = 0.024). F, Averaged representative traces recorded with Cs with ATP internal solution under control conditions (orange trace) and after application of TTA-P2 (violet trace) using a protocol depicted on the top of traces (Vt = –50 mV, Vh = –90 mV). G, Averaged normalized current density show reduction in current density by 84% after application of TTA-P2 (paired two-tailed t test, t(10) = 21.51, p < 0.001). H, Averaged current amplitude under control condition was 572.75 ± 70.66 pA and after application of TTA-P2 it was 89.30 ± 24.21 pA (paired two-tailed t test, t(10) = 7.01, p < 0.001); ***p < 0.001.
- Figure 5.
TTA-P2 reduced tonic and rebound burst firing in CeM neurons. A, Original traces from a representative neuron in the CeM before application of TTA-P2 (left panel, orange trace), after application of TTA-P2 (middle panel, violet trace) and after wash (right panel, orange trace). Resting membrane potentials (shown in the lower left corner of each panel) show active membrane responses to a depolarizing (100 pA) current injection. B, TTA-P2 reduced tonic AP firing frequency across all current pulses (from 50 to 200 pA); two-way RM ANOVA (both factors): interaction (F(6,78) = 1.02, p = 0.418), current injection (F(6,78) = 39.61, p < 0.001), and effect of TTA-P2 (F(1,13) = 10.91, p = 0.006). C, TTA-P2 had very little effect on average resting membrane potential (paired two-tailed t test, t(13) = 0.91, p = 0.380). D, TTA-P2 also had little effect on average input resistance of CeM neurons (paired two-tailed t test, t(13) = 0.39, p = 0.702). E, Original traces from a representative CeM neuron showing postinhibitory rebound burst-firing before (orange trace) and after application of TTA-P2 (violet trace). Burst-firing was induced by injection of a hyperpolarizing (–75 pA) current during 400 ms. Note that TTA-P2 completely abolished active membrane response to the current injection. F, Graph of averaged traces of CeM neurons shows LTS amplitude of LTSs was almost completely abolished by the application of TTA-P2 across all hyperpolarizing current pulses from –50 to –225 pA [two-way RM ANOVA (both factors): interaction (F(7,84) = 1.71, p = 0.116), current injection (F(7,84) = 4.32, p < 0.001), and effect of TTA-P2 (F(1,12) = 51.03, p < 0.001)]. G, Bar graph showing TTA-P2 significantly reduced the number of APs in rebound burst (paired two-tailed t test, t(6) = 2.94, p = 0.026). H, Bar graph showing TTA-P2 significantly increased the threshold for the occurrence of LTS (paired two-tailed t test, t(5) = 3.44, p = 0.018). I, Graph showing the average latency to LTS increased significantly with TTA-P2 (paired two-tailed t test, t(6) = 2.46, p = 0.049); *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 6.
Cytosolic ATP regulates neuronal excitability. A, Original traces from representative neurons in the CeM recorded with ATP (left panel, orange trace) and ATP-free internal solutions (right panel, gray trace) show active membrane responses (lower left corner of panels) to a depolarizing (100 pA) current injection. B, Graph of averages of tonic AP firing frequency and current injections of 50–200 pA from multiple experiments shows that ATP-free internal solution significantly reduced firing frequency during smaller depolarizing current injections [two-way RM ANOVA: interaction (F(6120) = 3.18, p = 0.006), current injection (F(6120) = 74.67, p < 0.001), and ATP (F(1,20) = 2.13, p = 0.159; shaded area indicates uncorrected Fisher’s LSD test presented on graph)]. C, Representative traces of the CeM neurons show active membrane responses to hyperpolarizing (–50 pA) current injections in conditions with an ATP internal solution (orange trace) and with ATP-free internal solution (gray trace). D, Graph of averages of LTS amplitudes from multiple experiments following escalating current injections ranging from –50 to –225 mV. Recordings with ATP-free internal solution (gray line) show a significant decrease in LTS amplitude as a response to –50-pA hyperpolarizing current injection [two-way RM ANOVA: interaction (F(7133) = 5.57, p < 0.001), current injection (F(7133) = 15.76, p < 0.001), and ATP (F(1,19) = 0.50, p = 0.488, Sidak’s post hoc presented on figure)]. E, Bar graph shows addition of ATP in the internal solution lowers the threshold for LTS occurrence [unpaired two-tailed t test, t(17) = 2.12, p = 0.048]. F, Graph showing latency to LTS (expressed in ms) in conditions with ATP (orange color) and without ATP (gray color) after escalating hyperpolarizing current injections; numbers in parentheses indicate respective number of cells in each group. G, Bar graph showing exclusion of ATP from the internal solution during hyperpolarizing current injection (from –125 to –225 pA) significantly extended time for LTS formation by increasing the cumulative average latency to LTS (unpaired two-tailed t test, t(19) = 2.10, p = 0.049). H, Bar graph indicates very little difference between the average resting membrane potential recorded with and without ATP (unpaired two-tailed t test, t(20) = 1.78, p = 0.090); *p < 0.05, **p < 0.01.
- Figure 7.
TTA-P2-induced inhibition of tonic and rebound burst-firing in CeM neurons is diminished in recordings with ATP-free internal solution. A1, Bar graph showing TTA-P2 significantly reduced tonic firing frequency of APs with an internal solution containing ATP (paired two-tailed t test; t(13) = 2.96, p = 0.011, n = 14). A2, Bar graph showing TTA-P2 had a smaller effect on tonic firing frequency in recording conditions with an ATP-free internal solution (paired two-tailed t test; t(5) = 14, p = 0.085, n = 6). B1, Bar graph showing TTA-P2 significantly reduced LTS amplitude of CeM neurons in conditions with ATP in the internal solution (paired two-tailed t test; t(12) = 7.14, p < 0.001, n = 13). B2, Bar graph showing TTA-P2 had no significant effect on LTS amplitude in recording conditions with an ATP-free internal solution (paired two-tailed t test; t(4) = 2.53, p = 0.065, n = 5); *p < 0.05, ***p < 0.001.
Tables
- Table 1.
The molarity and components of TMA-based and Cs-based internal solutions used for voltage-clamp recordings and internal solutions used for current-clamp recordings
TMA ATP-free TMA with ATP Cs ATP-free Cs with ATP Internal for current clamp ATP-free Internal for current clamp Molarity Component Molarity Component Molarity Component Molarity Component Molarity Component Molarity Component 135 mM TMA-OH × 5 H2O 135 mM TMA-OH x 5 H2O 110 mM Cs methane sulfonate 110 mM Cs methane sulfonate 130 mM K-D-gluconate 130 mM K-D-gluconate 40 mM HEPES 40 mM HEPES 10 mM HEPES 10 mM HEPES 5 mM EGTA 5 mM EGTA 10 mM EGTA 10 mM EGTA 9 mM EGTA 9 mM EGTA 4 mM NaCl 4 mM NaCl 2 mM MgCl2 × 6 H2O 14 mM Phosphocreatine di-tris 14 mM Phosphocreatine di-tris 0.5 mM CaCl2 x 2 H2O 0.5 mM CaCl2 × 2 H2O 5 mM Mg-ATP 5 mM Mg-ATP 10 mM HEPES 10 mM HEPES 0.3 mM Tris-GTP 0.3 mM Tris-GTP 2 mM Mg-ATP 0.5 mM Tris-GTP Abbreviations for different components of internal solutions are defined in Materials and Methods.
- Table 2.
Average V50 (mean ± SEM) values for steady-state activation and inactivation with different internal solutions in CeM neurons
TMA ATP-free TMA with ATP Cs ATP-free Cs with ATP Cs with ATP GTP-free V50 activation (number of cells) –63.31 ± 0.31 (17) –63.25 ± 0.42 (18) –63.72 ± 0.40 (18) –63.16 ± 0.39 (21) –63.88 ± 0.64 (14) V50 inactivation (number of cells) –95.16 ± 0.62 (17) –89.86 ± 0.41 (22) –81.56 ± 1.11 (15) –73.70 ± 0.64 (21) –75.15 ± 0.58 (15) Unpaired t test V50 inactivation t(37) = 7.36, p < 0.001* t(34) = 6.53, p < 0.001*
t(34) = 1.61, p = 0.117ns*TMA ATP-free vs TMA with ATP *Cs ATP-free vs Cs with ATP
nsCs with ATP GTP-free vs Cs with ATPNote that inclusion of ATP shifted V50 for inactivation toward more depolarized potentials in both Cs-based and TMA-based solutions. In contrast, V50 for activation was minimaly affected in the same cells.
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