Angiotensin IV Receptors in the Rat Prefrontal Cortex: Neuronal Expression and NMDA Inhibition

Abstract

Background: N-methyl-D-aspartate type glutamate receptors (NMDARs) are fundamental to neuronal physiology and pathophysiology. The prefrontal cortex (PFC), a key region for cognitive function, is heavily implicated in neuropsychiatric disorders, positioning the modulation of its glutamatergic neurotransmission as a promising therapeutic target. Our recently published findings indicate that AT₁ receptor activation enhances NMDAR activity in layer V pyramidal neurons of the rat PFC. At the same time, it suggests that alternative angiotensin pathways, presumably involving AT₄ receptors (AT4Rs), might exert inhibitory effects. Angiotensin IV (Ang IV) and its analogs have demonstrated cognitive benefits in animal models of learning and memory deficits.

Methods: Immunohistochemistry and whole-cell patch-clamp techniques were used to map the cell-type-specific localization of AT4R, identical to insulin-regulated aminopeptidase (IRAP), and to investigate the modulatory effects of Ang IV on NMDAR function in layer V pyramidal cells of the rat PFC.

Results: AT4R/IRAP expression was detected in pyramidal cells and GABAergic interneurons, but not in microglia or astrocytes, in layer V of the PFC in 9-12-day-old and 6-month-old rats. NMDA (30 μM) induced stable inward cation currents, significantly inhibited by Ang IV (1 nM-1 µM) in a subset of pyramidal neurons. This inhibition was reproduced by the IRAP inhibitor LVVYP-H7 (10-100 nM). Synaptic isolation of pyramidal neurons did not affect the Ang IV-mediated inhibition of NMDA currents.

Conclusions: Ang IV/IRAP-mediated inhibition of NMDA currents in layer V pyramidal neurons of the PFC may represent a way of regulating cognitive functions and thus a potential pharmacological target for cognitive impairments and related neuropsychiatric disorders.

Keywords: AT4 angiotensin receptor; N-methyl-D-aspartate receptor; insulin-regulated aminopeptidase; neuromodulation; prefrontal cortex; renin–angiotensin system.

Figure 1

Inhibitory effect of Ang II on NMDA-induced currents in layer V pyramidal neurons of the medial prefrontal cortex in 9–12-day-old rats. Whole-cell patch-clamp recordings were performed at a holding potential of −70 mV. NMDA (30 µM) in aCSF was applied three times (T₁, T₂, T₃) for 1.5 min, with 10 min intervals between applications. Ang II (3 μM) was superfused for 5 min before and during T₃. (A) Inhibitory effects of Ang IV on the NMDA current at T₃ are expressed as the percentage inhibition of the response measured at T₂. Data are presented as mean ± SEM. Red columns represent the percentage inhibition of NMDA-induced current responses (T₃/T₂) in cells where Ang II inhibited NMDA receptor-mediated ion currents. The number of responsive cells out of the total number of cells tested is shown in the red columns. * p < 0.01, indicating a significant difference from controls. (B) Representative tracing of a current response to 30 μM NMDA at T₂ and T₃, in the presence of 3 μM Ang II superfused for 5 min before and during T₃.

Figure 2

Protein expression of IRAP (green) in distinct layers of the medial prefrontal cortex in young (10-day-old) (A) and adult (6-month-old) (B) rats. The receptor is highly expressed in the cells of layers II–III and V–VI at both ages. DAPI (blue) was used as a counterstain. Scale bars: 200 μm (left panels in (A,B)) and 50 μm (right panels in (A,B)).

Figure 3

Representative images of the immunofluorescent detection of IRAP in distinct cell types in layer V of the mPFC in (A) young (10-day-old) and (B) adult (6-month-old) rats. The receptor is highly expressed in cells with a pyramidal morphology (green arrows) and in GABAergic interneurons (yellow arrows) but not in microglia or astrocytes (red arrows). GAD67, IBA1, and GFAP were used as markers. DAPI was used as a counterstain (blue). Notably, IRAP staining is weaker in 10-day-old animals compared to 6-month-old rats. Furthermore, this reduction is particularly pronounced in GABAergic interneurons but to a lesser extent in pyramidal cells in the 10-day-old rats. Scale bar: 50 μm.

Figure 3

Representative images of the immunofluorescent detection of IRAP in distinct cell types in layer V of the mPFC in (A) young (10-day-old) and (B) adult (6-month-old) rats. The receptor is highly expressed in cells with a pyramidal morphology (green arrows) and in GABAergic interneurons (yellow arrows) but not in microglia or astrocytes (red arrows). GAD67, IBA1, and GFAP were used as markers. DAPI was used as a counterstain (blue). Notably, IRAP staining is weaker in 10-day-old animals compared to 6-month-old rats. Furthermore, this reduction is particularly pronounced in GABAergic interneurons but to a lesser extent in pyramidal cells in the 10-day-old rats. Scale bar: 50 μm.

Figure 4

Inhibitory effects of Ang IV on the NMDA-induced current in layer V pyramidal neurons of the mPFC in young (9–12 days old) rats. Whole-cell patch-clamp recordings were performed at a holding potential of −70 mV. NMDA (30 µM) in aCSF was applied three times for 1.5 min (T₁, T₂, T₃), with a 10 min interval between applications. Ang IV (1 nM to 1 μM) was superfused 5 min before and during T₃. (A) Inhibitory effects of Ang IV on the NMDA current at T₃ are expressed as the percentage inhibition of the response measured at T₂. Data are presented as mean ± SEM. Red columns represent the percentage inhibition of NMDA-induced current responses in cells where Ang IV inhibited NMDA receptor-mediated ion currents. The number of responsive cells out of the total number of cells tested is indicated in the red columns. * p < 0.01; significant difference from controls. (B) Representative tracing of a current response to 30 μM NMDA after T₂ and T₃ and in the presence of Ang IV (100 nM) for 5 min before and during T₃.

Figure 5

The inhibitory effect of Ang IV persisted during synaptic isolation by a Ca²⁺-free solution or TTX-containing aCSF on the NMDA-induced current in layer V pyramidal neurons of the mPFC in young (9–12 day-old) rats. Whole-cell patch-clamp recordings were performed at a holding potential of −70 mV. NMDA (30 µM) was applied three times for 1.5 min (T₁, T₂, T₃), with a 10 min interval between applications. Ang IV (100 nM) was superfused 5 min before and during T₃, while the Ca²⁺-free solution or TTX (0.5 μM) was superfused throughout the experiment. The inhibitory effects of Ang IV on the NMDA current at T₃ are expressed as the percentage inhibition of the response measured at T₂. Data are presented as mean ± SEM. Red columns represent the percentage inhibition of NMDA-induced current responses in cells where Ang IV inhibited NMDA receptor-mediated ion currents. The number of responsive cells out of the total number of cells tested is indicated in the red columns. * p < 0.01; significant difference from controls.

Figure 6

The inhibitory effect of Ang IV was reproduced by the IRAP inhibitor LVVYP-H7 on the NMDA-induced current in layer V pyramidal neurons of the mPFC in young (9–12 days old) rats. Whole-cell patch-clamp recordings were performed at a holding potential of −70 mV. NMDA (30 µM) in aCSF was applied three times for 1.5 min (T₁, T₂, T₃), with a 10 min interval between applications. LVVYP-H7 (1 nM–100 nM) was superfused for 5 min before and during T₃. The inhibitory effects of LVVYP-H7 on the NMDA current at T₃ are expressed as the percentage inhibition of the response measured at T2. Data are presented as mean ± SEM. Red columns represent the percentage inhibition of NMDA-induced current responses in cells where LVVYP-H7 inhibited NMDA receptor-mediated ion currents. The number of responsive cells out of the total number of cells tested is indicated in the red columns. * p < 0.01, indicating a significant difference from the control.