Fig 1.
Effect of caffeine on sIAHP in rat CA1 neurons.
(A) Representative sIAHP trace at steady state. (B) sIAHP trace maximally enhanced after 2.5 min into the application of caffeine (0.5 mM). (C) sIAHP reduction after continuous application of caffeine. (D) Time course of action of caffeine (0.5 mM) on the sIAHP amplitude. Data point in black and indicated by arrows correspond to traces (A-C). (E) Summary bar diagram, showing that the sIAHP amplitude first increased by 25.3 ± 2.9% (max: n = 22; P < 0.0001), followed by a decrease by 24.5 ± 3.6% at steady state (ss: n = 16; P < 0.0001) when compared to the current amplitude preceding caffeine application. (F) Representative sIAHP trace recorded at steady state but with the PKA inhibitor Rp-cAMPS (500 μM) applied intracellularly. (G) sIAHP current increase in the presence of Rp-cAMPS and after ~17.5 min application of caffeine (0.5 mM), a time point comparable to (C). (H) Overlay of the sIAHP traces in F and G. (I) Time course of action of caffeine (0.5 mM) in the presence of Rp-cAMPS; (ss-Rp) indicates the trace shown in G. (J) Bar diagram comparing the relative (%) changes in sIAHP amplitude in response to caffeine application at steady state in the absence (ss; of panel E) and presence of the PKA inhibitor Rp-cAMPS (ss-Rp). sIAHP amplitude increased by 29.8 ± 7.9% (ss-Rp: n = 4; P = 0.03) in the presence of Rp-cAMPS after caffeine application when compared to the sIAHP current amplitude preceding caffeine application. The difference between the effect of caffeine with or without Rp-cAMPS is highly significant (P < 10−6).
Fig 2.
Effect of ryanodine on the sIAHP measured at steady state in rat CA1 pyramidal neurons.
(A) Representative sIAHP trace measured at steady state. (B) Reduction of the sIAHP upon application of ryanodine (10 μM). (C) Superimposed traces before and after application of ryanodine. (D) Time course of action of ryanodine (10 μM) on the sIAHP amplitude in the same cell as for the traces shown in panels A-C. (E) Summary bar diagram showing that ryanodine decreases the amplitude of the sIAHP by 23.3 ± 3.7% (n = 5; P = 0.03). Data are normalised to the control, set as 100%.
Fig 3.
CICR contributes to the activity-dependent potentiation of sIAHP in rat CA1 pyramidal neurons.
(A-I) Representative current traces at 0 min, 3 min and 15 min show the potentiation of the sIAHP. (A-C) Control traces recorded in the presence of 0.2% DMSO. (D-F) Reduced potentiation of the sIAHP in the presence of 10 μM ryanodine. (G-I) Reduced potentiation of the sIAHP in the presence of 50 μM CPA. (J) Superimposed time-courses of three representative cells comparing the different degrees of the potentiation of sIAHP amplitude under control conditions, 10 μM ryanodine and 50 μM CPA. (K) Bar chart summarizing the sIAHP amplitude at the beginning of the recording (0 min) and the end of the recording (15 min), (control, 0 min: 23.1 ± 5.1 pA; 15 min: 79.0 ± 7.7 pA; n = 5; paired t-test: P = 0.0017; 10 μM ryanodine, 0 min: 27.4 ± 2.6 pA; 15 min: 58.2 ± 9.3 pA; n = 5; paired t-test: P = 0.02; 50 μM CPA; 0 min: 27.5 ± 6.6 pA; 15 min: 39.2 ± 3.1 pA; n = 5; paired t-test: P = 0.12). (L) Summary of the relative (%) time-courses of potentiation of sIAHP measured in all cells (n = 5 for each condition) under control conditions (0.2% DMSO), in 10 μM ryanodine and in 50 μM CPA. Reduction of the sIAHP amplitude potentiation in the presence of ryanodine (two-way ANOVA with post-hoc Bonferroni test: P < 0.0001) and of CPA (two-way ANOVA with post-hoc Bonferroni test: P < 0.0001). (M) Ratio of the sIAHP amplitude measured at 0 and 15 min under control conditions (mean ± SEM: 3.9 ± 0.7; n = 5), in ryanodine (2.2 ± 0.3; n = 5; P = 0.04) and in CPA (1.9 ± 0.4; n = 5; P = 0.03).
Fig 4.
Lack of type 3 ryanodine receptor (RyR3) affects the firing properties and sIAHP current density in mouse CA1 pyramidal neurons.
(A-B) action potentials were induced by current injections from 40 pA to 160 pA (in 40 pA step-increments) for 1 s in RyR3 +/+ and RyR3 −/− CA1 pyramidal neurons. The membrane resting potential was -68 mV (A) and -69 mV (B). (C) Action potential frequency was higher in RyR3 +/+ compared to RyR3 −/− CA1 pyramidal neurons at the same stimulation strength (P = 0.004, two-way ANOVA with Bonferroni’s test). At 40 pA action potentials could not be elicited in either RyR3 +/+ or RyR3 −/− CA1 pyramidal neurons. The action potential frequency did not differ significantly (P = 0.5) for current injections at 80 pA for RyR3 +/+ (0.7 ± 0.2 Hz, n = 32) and RyR3 −/− (0.2 ± 0.1 Hz, n = 37). However, at higher current injections action potential frequencies were substantially different (120 pA: RyR3 +/+ = 1.9 ± 0.4 Hz, n = 27 vs RyR3 −/− = 1.1 ± 0.2 Hz, n = 34, P = 0.0003; and 160 pA RyR3 +/+ = 2.8 ± 0.8 Hz, n = 12 vs RyR3 −/− = .1 ± 0.3 Hz, n = 26, P = 0.001). (D-E) Representative sIAHP traces obtained from RyR3 +/+ and RyR3 −/− mouse CA1 pyramidal neurons. (F-I) Properties of the sIAHP recorded at steady-state in RyR3 +/+ and RyR3 −/− mice ~15 minutes after the onset of the recording. (F) No significant difference was observed for current amplitude (RyR3 +/+ = 42.5 ± 8.1 pA, n = 10 vs RyR3 −/− = 30.5 ± 6.1 pA, n = 7; P = 0.2). (G) Similarly, there was no significant difference for sIAHP charge transfer (RyR3 +/+ = 172.9 ± 34.6 pC, n = 10 vs RyR3 −/− = 120.6 ± 22.8 pC, n = 7; P = 0.3) and (H) the deactivation time constant of sIAHP (RyR3 +/+ = 3.6 ± 0.3 s, n = 10 vs RyR3 −/− = 3.5 ± 0.3 s, n = 7; P = 0.9). (I) sIAHP current density was reduced in RyR3 -/- neurons (RyR3 −/− = 0.8 ± 0.1 pA/pF, n = 7 vs RyR3 +/+ = 1.7 ± 0.5 pA/pF, n = 10; P = 0.03, Mann-Whitney test).
Fig 5.
Inhibition of CICR by ryanodine reduced the sIAHP amplitude at steady state in RyR3 +/+ and RyR3 −/− mouse CA1 pyramidal neurons.
Representative sIAHP traces obtained from RyR3 +/+ (A) and RyR3 −/− (D) mouse CA1 pyramidal neurons at steady-state, at the end of the run-up phase. Current traces from the same neurons 15 minutes after the application of 10 μM ryanodine (RyR3 +/+ (B) and RyR3 −/− (E)). (C and F) Superimposed traces showing the reduction of the sIAHP amplitude. Scale bars in (F) apply to all panels, (A-F). (G) Time course of sIAHP amplitude in the same RyR3 +/+ neuron as shown in A-C. (H) Time course of the sIAHP amplitude in the same RyR3 −/− neuron as shown in D-F. (I) Overall decrease in the sIAHP amplitude summarized in a box-and-whiskers plot, showing comparable reduction by ryanodine in RyR3 +/+ (mean ± SEM: 39.0 ± 6.9%; median = 36.2%, n = 7) and RyR3 −/− neurons (mean ± SEM: 36.9 ± 9.4%; median = 33.4%, n = 5) (P = 0.9).
Fig 6.
CA1 pyramidal neurons lacking type 3 ryanodine receptor (RyR3 −/−) have a faster and reduced activity-dependent potentiation of sIAHP.
(A, B) Superimposed traces of the sIAHP recorded at 0 and 15 minutes from RyR3 +/+ and RyR3 −/− CA1 pyramidal neurons. (C) Box-and-whiskers plot summarizing the sIAHP amplitudes recorded before the run-up phase (0 min) and at the end of potentiation (15 min). At 0 min the sIAHP peak amplitude in RyR3 +/+ (mean ± SEM: 4.2 ± 1.8 pA; median = 2.3 pA; n = 10) and in RyR3 −/− CA1 pyramidal neurons (mean ± SEM: 9.2 ± 3.4 pA; median = 4.7 pA; n = 7) was similar (P = 0.07, Mann-Whitney test). At steady state (15 min) the amplitude of sIAHP was similar in RyR3 +/+ neurons (mean ± SEM: 45.2 ± 8.1 pA; median = 37.7 pA; n = 10) and in RyR3 −/− (mean ± SEM: 30.5 ± 6.1 pA; median = 27.1 pA; n = 7) (P = 0.2). This shows that a clear potentiation was observed for both RyR3 +/+ (P = 0.0006, paired t-test) and RyR3 −/− (P = 0.006, paired t-test) neurons, because the sIAHP amplitude at steady state was clearly larger when compared with sIAHP amplitude at the beginning of the recording. (D) Time course of relative amplitude increase of sIAHP during the first 15 min of the recording, with current amplitudes normalised to the starting current measured at 0 min. The sIAHP potentiation was overall larger in RyR3 +/+ than in RyR3 −/− CA1 pyramidal cells (two-way ANOVA with Bonferroni’s test, P < 0.001). (E) The time constant (τ) of sIAHP potentiation was obtained by fitting a mono-exponential function to the sIAHP amplitude during the run-up phase of each individual experiment. The time constant of potentiation was faster in RyR3 −/− (mean ± SEM: 3.1 ± 0.7 min; median = 3.2 min; n = 7) compared to RyR3 +/+ CA1 neurons (mean ± SEM: 6.7 ± 1.1 min; median = 5.6 min; n = 10) (P = 0.043).
Fig 7.
Type 3 ryanodine receptor (RyR3) is a main player in the CICR that mediates the activity-dependent potentiation of sIAHP in mouse CA1 pyramidal neurons.
(A, B) Superimposed traces of the sIAHP recorded at 0 and 15 minutes from RyR3 +/+ (A) and RyR3 −/− (B) CA1 pyramidal neurons in the presence of 10 μM ryanodine from the outset of the recordings. (C) Summary box-and-whiskers plot of the sIAHP amplitudes recorded at 0 min and at 15 min in the presence of ryanodine (10 μM). At 0 min the sIAHP amplitudes in RyR3 +/+ CA1 neurons (mean ± SEM: 12.1 ± 3.7 pA; median = 10.4 pA; n = 5) and in RyR3 −/− CA1 pyramidal neurons (mean ± SEM: 39.9 ± 12.4 pA; median = 21.2 pA; n = 7) were not significantly different (P = 0.06, Mann-Whitney test). At 15 min the current in RyR3 +/+ (mean ± SEM: 45.6 ± 12.1 pA; median = 36.0 pA; n = 5) and in RyR3 −/− (mean ± SEM: 61.0 ± 16.3 pA; median = 41.6 pA; n = 7) was similar (P = 0.4, Mann-Whitney test). In the presence of ryanodine (10 μM) from the onset, RyR3 +/+ cells had a larger sIAHP at 15 minutes than at 0 minute (P = 0.047, n = 5, t-test with Welch correction), while RyR3 −/− neurons had similar sIAHP amplitudes at 0 and 15 minutes (P = 0.3, n = 7, Mann-Whitney test). (D) Time course of relative increase of sIAHP during the first 15 min of the recording in the presence of ryanodine (10 μM), with current amplitudes normalized to the starting current measured at 0 min. The sIAHP potentiation was overall similar in RyR3 +/+ and RyR3 −/− CA1 cells (P = 0.17, two-way repeated measures ANOVA). (E) The time constant (τ) of sIAHP potentiation in RyR3 +/+ CA1 neurons (mean ± SEM: 3.4 ± 0.9 min; median = 2.6 min; n = 5) and in RyR3 −/− (mean ± SEM: 2.0 ± 0.6 min; median = 1.3 min; n = 7) was similar (P = 0.2). (F) Summary bar chart comparing the sIAHP amplitude from RyR3 +/+ and RyR3 −/− mice in the presence and absence of ryanodine. Ryanodine increased the initial amplitude of sIAHP in RyR3 +/+ (P = 0.02, Mann-Whitney test) and RyR3 -/- CA1 neurons (P = 0.045, t-test with Welch correction), but it did not affect the current measured at 15 minutes in either RyR3 +/+ (P > 0.9) or RyR3 −/− CA1 neurons (P = 0.1, Mann-Whitney test). (G) Summary bar chart comparing ratios of the sIAHP amplitude measured at 0 and 15 min from RyR3 +/+ and RyR3 −/− mice with or without ryanodine. Ryanodine did not significantly decrease the sIAHP ratio in RyR3 −/− CA1 neurons, and did not affect the ratio in RyR3 +/+ CA1 neurons, in spite of an apparent trend (one-way ANOVA-Kruskal-Wallis test with Dunn’s multiple comparison test, P > 0.05 for both comparisons). In the presence of ryanodine from the onset, the sIAHP ratio was not significantly different between RyR3 +/+ CA1 cells (5.5 ± 1.8, n = 5) and RyR3 −/− CA1 neurons (3.2 ± 1.8, n = 7) (one-way ANOVA-Kruskal-Wallis test with Dunn’s multiple comparison test, P > 0.05 for this comparison).