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

Abstract

NMDA receptors in the prefrontal cortex (PFC) play a crucial role in cognitive functions. Previous research has indicated that angiotensin II (Ang II) affects learning and memory. This study aimed to examine how Ang II impacts NMDA receptor activity in layer V pyramidal cells of the rat PFC. Whole-cell patch-clamp experiments were performed in pyramidal cells in brain slices of 9-12-day-old rats. NMDA (30 μM) induced inward currents. Ang II (0.001-1 µM) significantly enhanced NMDA currents in about 40% of pyramidal cells. This enhancement was reversed by the AT₁ antagonist eprosartan (1 µM), but not by the AT₂ receptor antagonist PD 123319 (5 μM). When pyramidal neurons were synaptically isolated, the increase in NMDA currents due to Ang II was eliminated. Additionally, the dopamine D1 receptor antagonist SCH 23390 (10 μM) reversed the Ang II-induced enhancement, whereas the D2 receptor antagonist sulpiride (20 μM) had no effect. The potentiation of NMDA currents in a subpopulation of layer V pyramidal neurons by Ang II, involving AT1 receptor activation and dopaminergic signaling, may serve as an underlying mechanism for the effects of the renin-angiotensin system (RAS) elements on neuronal functions.

Keywords: AT1 angiotensin receptor; D1 dopamine receptor; NMDA receptor; RAS; neuromodulation; prefrontal cortex.

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).

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).

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.

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 difference from the 1 µM Ang II potentiation group. (B) Immunohistochemical detection of AT₁ receptor protein expression (left panel) was conducted using the MBS151548 anti-AT1R rabbit polyclonal antibody, and fluorescent in situ hybridization analysis of AT1R mRNA expression (right panel) was performed in the mPFC of 10-day-old Wistar rats. For immunohistochemical detection, the MBS151548 anti-AT1R rabbit polyclonal antibody preincubated with the MBS152017 AT1R blocking peptide served as the negative control (left panel, square below). Blue indicates cells counterstained with 4′,6-diamidino-2-phenylindole (DAPI), green indicates cells stained with anti-AT1R rabbit polyclonal antibody (left panel), and cells expressing AT1R mRNA (right panel). L1, L2/3, and L5/6 denote the layers of the mPFC.

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 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 difference from the 1 µM Ang II potentiation group.