Block of rapid depolarization induced by in vitro energy depletion of rat dorsal vagal motoneurones

Abstract

1. The ionic mechanisms contributing to the rapid depolarization (RD) induced by in vitro ischaemia have been studied in dorsal vagal motoneurones (DVMs) of brainstem slices. Compared with CA1 hippocampal neurones, RD of DVMs was slower, generally occurred from a more depolarized membrane potential and was accompanied by smaller increases in [K+]o. 2. RD was not induced by elevation of [K+]o to values measured around DVMs during in vitro ischaemia or by a combination of raised [K+]o and 2-5 microM ouabain. 3. Neither TTX (5-10 microM) nor TTX combined with bepridil (10-30 microM), a Na+-Ca2+ exchange inhibitor, slowed RD. Block of voltage-dependent Ca2+ channels with Cd2+ (0.2 mM) and Ni2+ (0.3 mM) led to an earlier onset of RD, possibly because [K+]o was higher than that measured during in vitro ischaemia in the absence of divalent ions. 4. When [Na+]o was reduced to 11.25-25 mM, RD did not occur, although a slow depolarization was observed. RD was slowed (i) by 10 mM Mg2+ and 0.5 mM Ca2+, (ii) by a combination of TTX (1.5-5 microM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D-2-amino-5-phosphonovalerate (AP5, 50 microM) and (iii) by TTX (1.5-5 microM) and AP5 (50 microM). 5. Ni2+ at concentrations of 0.6 or 1.33 mM blocked RD whereas 0.6 mM Cd2+ did not. A combination of Cd2+ (0.2 mM), Ni2+ (0.3 mM), AP5 (50 microM) and bepridil (10 microM) was largely able to mimic the effects of high concentrations of Ni2+. 6. It is concluded that RD is due to Na+ entry, predominantly through N-methyl-D-aspartate receptor ionophores, and to Ca2+ entry through voltage-dependent Ca2+ channels. These results are consistent with known changes in the concentrations of extracellular ions when ischaemia-induced rapid depolarization occurs.

Figure 1. Changes in membrane potential and extracellular K⁺ activity induced by in vitro ischaemia

A and B, changes in membrane potential of dorsal vagal motoneurones (A) and CA1 pyramidal cells (B) induced by switching from a standard superperfusate to one bubbled with 95 % O₂-5 % CO₂ and lacking in glucose. Time zero (arrow) indicates when the new perfusate arrived at the bubble trap in the solution line (see Methods). C and D, potassium ion activity recorded extracellularly at the same time as some of the recordings in A or B, respectively. In studies of dorsal vagal motoneurones these measurements were made in the contralateral dorsal vagal motonucleus, and in studies of CA1 pyramidal cells they were made in the pyramidal cell layer.

Figure 2. Original records of rapid depolarization and recovery in dorsal vagal neurones

Raw data, as recorded with the MacLab system, showing response of two DVMs to in vitro ischaemia, and their subsequent recovery on return to standard perfusate. Arrowheads indicate when the new perfusate arrived at the bubble trap in the solution line (see Methods). Asterisks mark periods of each trace when hyperpolarizing current was injected into the neurones to return membrane potential to initial values for monitoring of input resistance. Positive-going shifts in membrane potential are action potentials, which were often truncated by the slow digitizing rate (4 samples s⁻¹).

Figure 3. Effects of raised [K⁺]_o and ouabain on membrane potential and extracellular K⁺ activity

A, changes in membrane potential of DVMs induced by switching the superperfusate from one containing 3 mM K⁺ to one containing 10 mM KCl (−) or 10 mM K₂SO₄ () at time zero (arrow; see Methods). B, effects on membrane potential of 2–5 μM ouabain when [K⁺]_o was raised to 10 mM. C and D, changes in membrane potential (C) and contralaterally measured extracellular K⁺ activity (D) induced by 25 μM ouabain.

Figure 4. Effects of low concentrations of divalent ions on rapid depolarization and extracellular K⁺ activity

A, changes of membrane potential in DVMs induced by in vitro ischaemia in the presence of 0.2 mM Cd²⁺ and 0.3 mM Ni²⁺. Perfusate switched at time zero (arrow; see Methods). B, in some experiments extracellular K⁺ activity was simultaneously measured in the contralateral dorsal vagal motonucleus.

Figure 6. Rates of ischaemia-induced rapid depolarization during various treatments

The size of the symbols prevents all data points from being visible. Means ± s.e.m. are indicated by filled circles with bars. A one-way ANOVA demonstrated a highly significant effect of drug treatment on the rate of rapid depolarization (P < 0.0001). The asterisks mark those treatments demonstrating significant differences from controls (Dunnett's procedure): low [Na⁺]_o, 0.6 mM Ni²⁺, 1.33 mM Ni²⁺ and combined 0.2 mM Cd²⁺, 0.3 mM Ni²⁺, 50 μM AP5 and 10 μM bepridil ± 3–5 μM TTX.

Figure 5. Effects of TTX, low [Na⁺]_o and glutamate receptor antagonists on rapid depolarization

Changes in membrane potential of DVMs induced by in vitro ischaemia in the presence of: 5–10 μM TTX (−) or 3–5 μM TTX with 10–30 μM bepridil () (A); when the extracellular Na⁺ concentration was reduced to 11.25 (), 18 (−) or 25 mM () (B); in the presence of 3–5 μM TTX and 50 μM AP5, with (−) and without 10 μM CNQX () (C). Perfusate switched at time zero (arrow; see Methods).

Figure 7. Treatments which act at multiple targets slow or eliminate rapid depolarization

Ischaemia-induced changes in membrane potential of DVMs in the presence of: 10 mM Mg²⁺ and 0.5 mM Ca²⁺ (A); 0.6 mM Ni²⁺ () or 1.33 mM Ni²⁺ (−) (B); 0.3 mM Ni²⁺ () or 0.6 mM Cd²⁺ (−) (C); 0.2 mM Cd²⁺, 0.3 mM Ni²⁺, 50 μM AP5 and 10 μM bepridil, with (−) and without () 3–5 μM TTX (D). Perfusate switched at time zero (arrow; see Methods).