NMDA receptor activation inhibits neuronal volume regulation after swelling induced by veratridine-stimulated Na+ influx in rat cortical cultures

Abstract

Neurons and glia experience rapid fluctuations in transmembrane solute and water fluxes during normal brain activity. Cell volume must be regulated under these conditions to maintain optimal neural function. Almost nothing is known, however, about how brain cells respond to volume challenges induced by changes in transmembrane solute flux. As such, we characterized the volume-regulatory mechanisms of cultured cortical neurons swollen by veratridine-stimulated Na+ influx. Exposure of cortical neurons to 100 microM veratridine for 10-15 min caused a 1.8- to 2-fold increase in cell volume that persisted for at least 90 min. This volume increase was blocked by extracellular Na+ removal or by exposure to 5 microM tetrodotoxin, indicating that swelling is a result of Na+ entry via Na+ channels. Treatment of cells with veratridine together with various NMDA receptor antagonists had no effect on the magnitude of swelling. NMDA receptor antagonist-treated cells, however, underwent nearly complete volume recovery within 50-70 min after veratridine exposure. This recovery suggests that NMDA receptor activation disrupts neuronal osmoregulatory pathways. Volume regulation was blocked by Ba2+, quinidine, or 5-nitro-2-(3-phenylpropylamino) benzoic acid, indicating that swelling activates volume regulatory K+ and Cl- channels. Veratridine also caused a rapid, transient increase in intracellular Ca2+. Extracellular Ca2+ removal or intracellular Ca2+ chelation prevented or dramatically reduced veratridine-induced increases in intracellular Ca2+ and completely blocked volume recovery. These findings indicate that increases in Ca2+ during cell swelling induced by Na+ influx are required for activation of neuronal volume-regulatory pathways.

Fig. 1.

Effect of 100 μm veratridine exposure on neuronal somal volume (n = 12;17). Veratridine (VT) was added as indicated by thearrow. Values are mean ± SE.

Fig. 2.

Effect of 5 μm TTX in the presence or absence of 100 μm veratridine (VT) on neuronal somal volume. TTX and VT were added as indicated by the arrows. Values are mean ± SE (TTX, n = 3;3; TTX + VT,n = 3;3).

Fig. 3.

Effect of extracellular Na⁺ removal in the presence or absence of 100 μm veratridine (VT) on neuronal somal volume. Sodium was replaced by NMDG and veratridine was added as indicated by thearrows. Values are mean ± SE (0 Na⁺,n = 2;3; 0 Na⁺ + VT,n = 3;3).

Fig. 4.

Effect of 10 μm MK-801 on veratridine-induced cell swelling. Cells were pretreated with MK-801 for 5 min before 100 μm veratridine (VT) was added to the bath (arrow). Values are mean ± SE (n = 13;16).

Fig. 5.

Representative DIC images of veratridine (VERAT)-treated neurons in the presence or absence of 10 μm MK-801. Cells were exposed to 100 μm veratridine at 0 min. In the absence of MK-801, neurons remain swollen and take on a refractile appearance. Cells treated with MK-801, however, undergo a complete volume recovery and have a normal appearance.

Fig. 6.

Effect of 100 μm CNQX and 1 mm glycine on veratridine-induced cell swelling. Values are mean ± SE (n = 8;13). Cells were pretreated with CNQX and glycine for 5 min before 100 μm veratridine (VT) was added to the bath (arrow).

Fig. 7.

Effect of delayed treatment with 10 μm MK-801 on somal volume of veratridine-treated neurons. MK-801 was added 25 min (filled circles,n = 3;3) or 55 min (open circles,n = 6;6) after the addition of 100 μmveratridine to the bath (arrow). Values are mean ± SE.

Fig. 8.

Effect of K⁺ and Cl⁻channel blockers on MK-801-elicited RVD. RVD is blocked by 50 μm NPPB (n = 7;7), 5 mmBa²⁺ (n = 6;7), or 1 mmquinidine (n = 9;9). Values are mean ± SE. Cells were pretreated with 10 μm MK-801 for 5 min before 100 μm veratridine (VT) was added to the bath (arrow). K⁺ and Cl⁻channel blockers were added 20 min after veratridine.

Fig. 9.

Veratridine-induced changes in intracellular Ca²⁺ concentration. Fura-2 AM emission ratios were measured in neuronal cell bodies. Veratridine (VT) was added to the bath as indicated by the arrow. Cells were treated with 100 μm veratridine alone (solid points) or 100 μm veratridine plus 10 μm MK-801 (open points). MK-801 was added to the bath 5 min before veratridine addition. Values are mean ± SE (VT, n = 5;26; VT + MK-801,n = 5;30).

Fig. 10.

Effect of delayed addition of MK-801 on intracellular Ca²⁺ levels. Cells were exposed to 100 μm veratridine for ∼25 and ∼60 min before addition of 10 μm MK-801 to the bath (arrows). Values are mean ± SE (left, n = 3–5;13–27; right, n = 3;25).

Fig. 11.

A, Effect of Ca²⁺-free medium and intracellular BAPTA loading on MK-801-induced RVD. Values are mean ± SE (Ca²⁺ removal, n = 7;8; BAPTA loading, n = 4;5). B,Effect of Ca²⁺-free medium and intracellular BAPTA loading on intracellular Ca²⁺ levels. Values are mean ± SE (Ca²⁺ removal, n = 1;9; BAPTA loading,n = 2;11). For the experiments shown in both panels, cells were pretreated with 10 μm MK-801 for 5 min before addition of 100 μm veratridine (VT) to the bath (arrows).