Tight-seal whole-cell voltage-clamp recordings were obtained from somata of CA1 pyramidal cells visualized by differential interference contrast using an EPC-9 patch-clamp amplifier (HEKA Electronics, Lambrecht, Germany). Patch-clamp electrodes (3.5–5 M) were filled with an intracellular solution containing in mM: 135 potassium methylsulfate, 8 NaCl, 10 HEPES, 2 Mg2-ATP, and 0.3 Na3-GTP, 0.5 Calcium Green-5N (Calcium Green-5N was only included when performing Ca2+ imaging), pH 7.3 with KOH (osmolarity 300 mOsm). The intracellular solution used for the measurement of VGCCs contained (in mM): 120 CsCl2, 20 CsFl, 4 MgCl2, 1 EGTA, 4 Mg2-ATP, 10 HEPES, pH 7.3 with CsOH (320 mOsm). Recordings were performed at 23°C in a submerged recording chamber with a constant flow of ACSF (1 mL/min). When VGCCs were studied, bath solution contained (in mM): 148 TEA-Cl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 0.001 TTX, and 0.01 nifedipine (where indicated), pH 7.3 with TEA-OH (320 mOsm). Bath and intracellular solution were devoid of sodium to avoid measurement of sodium currents. Additionally TTX was added to block sodium currents. Neurons were voltage-clamped at −60 mV, and 200 ms depolarizing pulses to +20 mV were delivered every 30 s. Depolarizing pulses were sufficient to activate AHP currents. Analysis was carried out using the HEKA software PulseFit (Heka Electronics, Lambrecht, Germany). The decays of IAHP and sIAHP currents were determined by fitting the trace with a single exponential equation. The amplitude of IAHP was determined at the peak of the current and the sIAHP amplitude was measured 700 ms post stimulus end. For whole-cell patch-clamp recordings of voltage-gated Ca2+ currents depolarizing pulse commands 200 ms in duration were applied from a holding potential of −70 mV. The pulse command was stepped in 10 mV steps from −100 to +30 mV. Between each sweep a 1 s interval was inserted. Current-voltage (I-V) relationships were determined by measuring the peak amplitude value at the different command potentials. The inactivating component of the current was determined for the maximum amplitude by calculating the percent difference between the peak amplitude value and the amplitude value at the end of the command potential step (Fig. 4A, ΔX).
Figure 4. Nifedipine-sensitive voltage-gated l-type Ca2+-channel currents are diminished in hippocampal CA1 neurons of Prnp–/– mice. (a, b) Mean I-V relationships of Prnp+/+ (n = 10, 3 animals) and Prnp–/– (n = 10, 3 animals) hippocampal CA1 neurons treated with or without 10 µm nifedipine. Nifedipine blocked the l-type mediated part of the measured whole-cell current. Insets show representative example current traces evoked by a 200 ms depolarizing voltage step from a holding potential of −70 mV to − 20 mV that were partially blocked by the application of nifedipine. The symbol ΔX indicates the value of the inactivating component of the current. (c) The maximum current amplitude of Prnp–/– CA1 neurons (n = 9, 3 animals) is significantly reduced compared to Prnp+/+ (n = 9, 3 animals, *p < 0.05; Student's t-test). Application of nifedipine reduced current amplitudes of both Prnp+/+ (n = 9, 3 animals) and Prnp–/– (n = 9, 3 animals) CA1 neurons to a similar level (n.s. p > 0.05; Student's t-test). (d) Percent inhibition of the maximum current amplitude by nifedipine. Percent inhibition by nifedipine is significantly higher in Prnp+/+ (n = 9, 3 animals) CA1 neurons compared to Prnp–/– (n = 9, 3 animals, *p < 0.05; Student's t-test). (e) Inactivation of the maximum current amplitude of Prnp+/+ and Prnp–/– CA1 neurons treated with or without nifedipine (10 µm). The percent inactivation of Prnp+/+ (n = 9, 3 animals) CA1 neurons is significantly higher than in Prnp–/– (n = 9, 3 animals, *p < 0.05; Student's t-test) CA1 neurons. Application of nifedipine reduced the percent inactivation of Prnp–/– and Prnp+/+ to a comparable value (n.s. p > 0.05; Student's t-test). Box plot diagrams contain 10, 25, 75 and 90% percentiles; red/grey bar: mean; black bar: median.
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