Potentiation of NMDA Receptors by AT1 Angiotensin Receptor Activation in Layer V Pyramidal Neurons of the Rat Prefrontal Cortex

Figures

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  Figure 1. NMDA-evoked inward currents in layer V pyramidal neurons of the rat prefrontal cortex. Whole-cell patch-clamp measurements at a holding potential of −70 mV. Correlation of concentration–response amplitudes for NMDA-induced currents. Each data point corresponds to measurements taken from n cells for each NMDA concentration (10 µM, n = 3; 30 µM, n = 6; 100 µM, n = 7; 300 µM, n = 6).

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  Figure 2. 30 µM NMDA-induced inward currents in layer V pyramidal neurons of the rat PFC. (A) Diagram of the experimental patch-clamp protocol. After the whole-cell configuration was established, 200 µm thick mPFC slices were superfused with aCSF for 10 min to achieve diffusion balance between the patch pipette and the cell interior. Then, 30 μM NMDA was applied three times for 1.5 min (T1, T2, T3), separated by superfusion periods of 10 min with drug-free aCSF. The membrane currents were measured using the amplifier in voltage-clamp mode at a holding potential of −70 mV. The amplitude of the NMDA-induced ion currents was quantified. (B) Representative tracing of the current response to NMDA after three applications of 30 μM NMDA (T1, T2, T3).

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  Figure 3. Effect of 1 nM–1 µM Ang II on NMDA-induced inward currents in layer V pyramidal neurons of the rat prefrontal cortex. Whole-cell patch-clamp measurements were conducted at a holding potential of −70 mV. A total of 30 µM of NMDA was applied three times for 1.5 min (T1, T2, T3) with a 10 min interval between applications. Under these conditions, current responses were consistent at T2 and T3. Ang II at concentrations of 1 nM–1 µM was applied for 5 min before and during T3. (A) Mean ± SEM of n experiments, showing the effects of 0 µM (control) (n = 19), 1 nM (n = 11/28), 10 nM (n = 9/22), and 1 µM (n = 21/47) Ang II on NMDA currents with respect to the response measured at T2. Green bars represent the normalized NMDA-induced current responses (%) in the subpopulation of mPFC layer V pyramidal cells in which Ang II potentiated ion currents. * p < 0.05, a significant difference from the normalized current responses to NMDA under control conditions. (B) Representative tracing of a current response to 30 μM NMDA after T2 and T3, and in the presence of 1 µM Ang II for 5 min before and during T3.

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  Figure 4. Role of AT1 and AT2 receptors in the potentiation of NMDA receptor function by Ang II and protein/mRNA expression of AT1 receptors (AT1R) in the rat mPFC. (A) NMDA-induced current responses in mPFC layer V pyramidal cells were detected using whole-cell patch-clamp measurements at a holding potential of −70 mV. A 30 µM concentration of NMDA was administered three times for 1.5 min each (T1, T2, T3) with a 10 min interval between applications. A 1 µM concentration of Ang II was applied for 5 min before and during T3. In separate experiments, aCSF contained either 1 µM eprosartan (AT1 antagonist) or 5 µM PD 123319 (AT2 antagonist) throughout the entire measurement period. The data are presented as the mean ± SEM of n experiments: effects of 1 µM Ang II (n = 21/47), 1 µM Ang II + 1 µM eprosartan (n = 8), and 1 µM Ang II + 5 µM PD 123319 (n = 5/11) on NMDA currents at T3, normalized with respect to the response measured at T2. Green bars indicate normalized NMDA-induced current responses (T3/T2 %) in cells where Ang II potentiated NMDA receptor-mediated ion currents. If potentiation cannot be observed, all cells in the group are represented. * p < 0.05 indicates a significant…

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  Figure 5. The effect of synaptic isolation and the role of D1 and D2 dopaminergic receptors in Ang II-induced NMDA receptor potentiation. NMDA-induced current responses in mPFC layer V pyramidal cells were detected using whole-cell patch-clamp measurements at a holding potential of −70 mV. A total of 30 µM of NMDA was administered three times for 1.5 min (T1, T2, T3) with a 10 min interval between applications. A total of 1 µM of Ang II was applied for 5 min before and during T3. To test the effect of synaptic isolation, 0.5 µM tetrodotoxin (TTX) was added to the aCSF, or Ca2+-free aCSF was used throughout the entire experiment. To detect D1 receptor and D2 receptor involvement in Ang II-induced potentiation of the NMDA receptor, 10 µM SCH-23390 (D1 receptor antagonist) or 20 µM sulpiride (D2 receptor antagonist) was added to the aCSF throughout the measurements. Mean ± SEM of n experiments: effects of 1 µM Ang II (n = 21/47), 1 µM Ang II + 0.5 µM TTX (n = 8), 1 µM Ang II + Ca2+-free aCSF (n = 15), 1 µM Ang II + 10 µM SCH-23390 (n = 8), and 1 µM Ang II + 20 µM sulpiride (n = 8/20) on NMDA currents at T3 normalized with respect to the response measured in T2. Green bars indicate normalized…