Angiotensin II excites paraventricular nucleus neurons that innervate the rostral ventrolateral medulla: an in vitro patch-clamp study in brain slices

Abstract

Neurons of the hypothalamic paraventricular nucleus (PVN) are key controllers of sympathetic nerve activity and receive input from angiotensin II (ANG II)-containing neurons in the forebrain. This study determined the effect of ANG II on PVN neurons that innervate in the rostral ventrolateral medulla (RVLM)-a brain stem site critical for maintaining sympathetic outflow and arterial pressure. Using an in vitro brain slice preparation, whole cell patch-clamp recordings were made from PVN neurons retrogradely labeled from the ipsilateral RVLM of rats. Of 71 neurons tested, 62 (87%) responded to ANG II. In current-clamp mode, bath-applied ANG II (2 muM) significantly (P < 0.05) depolarized membrane potential from -58.5 +/- 2.5 to -54.5 +/- 2.0 mV and increased the frequency of action potential discharge from 0.7 +/- 0.3 to 2.8 +/- 0.8 Hz (n = 4). Local application of ANG II by low-pressure ejection from a glass pipette (2 pmol, 0.4 nl, 5 s) also elicited rapid and reproducible excitation in 17 of 20 cells. In this group, membrane potential depolarization averaged 21.5 +/- 4.1 mV, and spike activity increased from 0.7 +/- 0.4 to 21.3 +/- 3.3 Hz. In voltage-clamp mode, 41 of 47 neurons responded to pressure-ejected ANG II with a dose-dependent inward current that averaged -54.7 +/- 3.9 pA at a maximally effective dose of 2.0 pmol. Blockade of ANG II AT1 receptors significantly reduced discharge (P < 0.001, n = 5), depolarization (P < 0.05, n = 3), and inward current (P < 0.01, n = 11) responses to locally applied ANG II. In six of six cells tested, membrane input conductance increased (P < 0.001) during local application of ANG II (2 pmol), suggesting influx of cations. The ANG II current reversed polarity at +2.2 +/- 2.2 mV (n = 9) and was blocked (P < 0.01) by bath perfusion with gadolinium (Gd(3+), 100 muM, n = 8), suggesting that ANG II activates membrane channels that are nonselectively permeable to cations. These findings indicate that ANG II excites PVN neurons that innervate the ipsilateral RVLM by a mechanism that depends on activation of AT1 receptors and gating of one or more classes of ion channels that result in a mixed cation current.

Fig. 1

Experimental preparation. A: rhodamine-containing microspheres were microinjected (50 nl) into the rostral ventrolateral medulla (RVLM) at a site where prior microinjection of l-glutamate (1 nmol in 100 nl) elicited an increase in arterial pressure (inset). B: retrograde labeling of the ipsilateral paraventricular nucleus (PVN) was observed in the dorsal and ventrolateral subnuclei, but was effectively absent from the central magnocellular (CM) subnucleus. C: recorded neurons were filled with biocytin. Peroxidase staining revealed elliptical cell bodies with multiple primary dendrites. Note: each labeled cell was individually recorded and filled with biocytin. D: infrared-DIC image of PVN slice preparation showing the patch electrode positioned on a recorded neuron and the microinjector pipette positioned adjacent to the recorded neuron (left). Fluorescent image shows that the recorded (and a nearby) neuron contained retrograde tracer (right).

Fig. 2

Effect of bath-applied angiotensin II (ANG II; 2 μM) on PVN-RVLM neuronal discharge. During bath perfusion with normal artificial cerebrospinal fluid (ACSF), the recorded neuron fired slowly (0.2 Hz) and irregularly at its resting membrane potential of −53 mV (Control: top sweep). Switching to ANG II-containing ACSF (2 μM) for 3 min gradually increased discharge (sweep 2), which reached a maximum of 3.0 Hz ~4 min after returning to normal ACSF (sweep 3). More than 20 min after initiating the ANG II washout, discharge had nearly returned to baseline (0.9 Hz; bottom sweep). Dashed line = 0 mV.

Fig. 3

Effect of locally applied ANG II on PVN-RVLM neurons. A: example traces in each column show cell discharge (left), membrane potential (middle), and membrane current (right) responses to locally applied ANG II (2 pmol, 5 s). Bar above each sweep denotes the period of ANG II application. In each recording mode, low-pressure application of ANG II produced a rapid and reproducible response (top 2 sweeps). During each ANG II application, discharge increased to a peak of ~20 Hz and exhibited clear frequency adaptation. Each ANG II–induced burst was followed by slight membrane afterhyperpolarization (left). In the presence of 0.5 mM TTX, local application of ANG II induced an obvious membrane potential depolarization of ~23 mV. Note that current-clamp responses in the absence and presence of TTX (left and middle columns) are from different cells. In both cases, responses to ANG II were elicited at resting V_m (−54 and −53 mV, respectively). In voltage-clamp mode, local ANG II induced a rapid inward current that reached a peak of −65 pA, with V_m held at −60 mV. Note that I_m decayed toward baseline even during the continued application of ANG II. Local application of vehicle (ACSF) for 5 s (sweep 3) was without effect on either membrane potential or current and did not alter the subsequent response to locally applied ANG II (sweep 4). The interval separating each application (ANG II or vehicle) was 60 s. B: summary voltage-clamp data for a group of 14 cells showing that the transient inward current response to low-pressure application of ANG II is dose-dependent. Current response was maximal at a dose of 2.0 pmol. *P < 0.001 compared with vehicle (data not shown).

Fig. 4

Effects of locally applied ANG II depend on AT1 receptors. A: locally applied ANG II (2 pmol, 5 s) increased cell discharge (left), depolarized membrane potential (middle), and induced an inward membrane current (right). Bar above each sweep denotes the period of ANG II application. Top 2 sweeps in each column show the rapid and reproducible response induced by local ANG II. The 3rd sweep shows that effects of ANG II are effectively prevented when ANG II (2 pmol) and the peptide AT1 receptor antagonist [Sar,Ile]-ANG II (4 pmol) are delivered concurrently for 5 s as a cocktail. Responses to local ANG II recovered within ~3 min of antagonist application (bottom sweeps). B: summary graphs showing that cell discharge (n = 5, left), membrane potential (n = 3, middle), and membrane current (n = 11, right) responses to a cocktail containing ANG II (2 pmol) and the AT1 receptor antagonist [Sar,Ile]-ANG II (4 pmol; gray bars) were significantly reduced compared with prior responses induced by ANG II (2 pmol) alone (open bars). Responses to ANG II recovered within 2–3 min following exposure to the ANG II-AT1 receptor antagonist cocktail (black bars). **P < 0.001 and *P < 0.01 vs. ANG II alone.

Fig. 5

Effect of locally applied ANG II on membrane input conductance. A: holding potential was repeatedly stepped (50 ms, 4 Hz) from −60 to −80 mV, and the inward current response was determined before (1), during (2), and after (3) locally applied ANG II (2 pmol). Note: initial current response to each voltage step is a capacitive current transient. Positive-going transients have been truncated for clarity. B: current response to step hyperpolarization increased during application of ANG II, indicating that membrane input conductance increased. Note that sweeps in B are taken from A. Baselines are aligned, and time and amplitude scales are expanded to emphasize the response, which is denoted by the 2 horizontal dashed lines. Capacitive artifacts have been truncated to emphasize the change in ionic current. C: summary data indicate that ANG II significantly increased input conductance of PVN-RVLM neurons by an average of ~80%. *P < 0.001 compared with control.

Fig. 6

Local ANG II induces a nonselective inward cation current in PVN-RVLM neurons. A: summary I-V relationship of the ANG II current from a group of 9 PVN-RVLM neurons. Holding potential was ramped (0.14 mV/ms) from −40 to +20 mV in the absence and presence of ANG II (2 pmol). The ANG II current was determined as the difference of the 2 current responses and was plotted as a function of applied potential. The I-V relationship revealed that the ANG II current was voltage-insensitive (nonrectifying) and had an average reversal potential of +2.2 ± 2.2 mV. Value of the current response to local ANG II at −60 mV averaged −48 ± 16 pA in these cells. B: traces (top) show that the response of an individual PVN-RVLM to locally applied ANG II (2 pmol; left sweep) was reduced in a time-dependent manner during perfusion with ACSF containing Gd³⁺ (100 μM; middle 3 sweeps). Current response was effectively eliminated 8 min after switching to Gd³⁺-containing ACSF and recovered toward the control amplitude ~20 min after returning to normal ACSF (right sweep). Summary graph (bottom) shows that bath application of 100 μM Gd³⁺ significantly reduced the amplitude of the ANG II-induced inward current in a group of 8 PVN-RVLM neurons. *P < 0.01 compared with control.