Responses of Regularly-Firing Aplysia R₃-R₁₃ Neurones to Normoxia and Hypoxia

In L₂-L₆ neurones otAplysia californica, hypoxia has been observed to cause reversible increases in intracellular potassium ion activities and concomitant membrane hyperpolarizations (Coyer, Halsey & Strong, 1983). It was concluded that these reversible changes seem to be brought about by augmentation of the sodium pump, possibly as a result of stimulation of ATP synthesis via glycolytic feedback. For regularly-firing neurones of the upper right quadrant R₃-R₁₃, Chaplain (1976, 1979), Chen, von Baumgarten & Takeda (1971), and Chen, von Baumgarten & Harth (1973) have shown that signalling patterns of these neurones are altered by compounds whose function is to regulate cellular metabolism either by inhibiting glycolysis or by disrupting certain steps in this process such as the catalysis of fructose-6-phosphate (F-6-P) by phosphofructokinase (PFK).

In the present paper, hypoxia is shown to cause irreversible decreases in intracellular potassium ion activity ⁠, membrane potential (E_M), and membrane slope resistance of R₃-R₁₃ neurones. Computations of the relative permeability ratios (P_Na/P_K), based upon a modification of the Goldman equation, are also expressed for the responses of these neurones to normoxia and hypoxia (see Coyer, 1981; Coyer et al. 1983). Irreversible effects of hypoxia upon membrane potential and input resistance have been given in a preliminary report (Coyer, Halsey & Strong, 1981).

Neurones within the right rostral ‘white cell’ quadrant were recognized visually as R₃–R₁₃ using established criteria (Frazier et al. 1967; Kandel, Frazier, Waziri & Coggeshall, 1967; Koester & Kandel, 1977). Standard electrophysiological techniques were E_Mployed to measure the membrane potentials of 10 cells during normoxia, hypoxia and reoxygenation. These neurones were exposed to 30 min of normoxia corresponding to a between 130 and 150 Torr, 115 min of hypoxia (⁠⁠, and 55 min of reoxygenation. Intracellular potassium ion activity was measured using double-barrelled, K⁺-selective microelectrodes (Walker, 1971; Khuri, Hajjar & Agulian, 1972; Vyskočil & Kří ž, 1972). The construction of these microelectrodes has been adequately described by Vyskočil & Kříž (1972), Schlue & Deitmer (1980) and Deitmer & Schlue (1981), and their usage in making intracellular measurements in Aplysia neurones has been described by Coyer et al. (1983). Dynamic current voltage curves were constructed by passing a linear, depolarizing current across the neural membrane (see Coyer et al. 1983). The outputs of high input impedance d.c. amplifiers were displayed on high fidelity penwriters (Gould) and recorded on a four-channel FM magnetic tape recorder (Hewlett-Packard). Oxygen microelectrodes and determinations were made following the techniques described by Coyer et al. (1983). Normal Aplysia saline consisted of: 425mm-NaCl, 10mm-KCl, L₀mM-CaCl₂, 22mM-MgC1₂, 26 mM-MgSO₄, 2·5 mM-NaHCO₃, L₀mM-Tris-HCl (pH = 7·3). Saline was delivered constantly at a rate of 10 ml min⁻¹ to the experimental bath from a reservoir located above it. Hypoxia was arbitrarily defined as a suffusate below 20 Torr, which probably corresponds to an intracellular of 5–7 Torr (Coyer et al. 1978). The calculation used in determining the relative permeability ratios (P_Na/P_K) from the values of the intracellular potassium ion activities was as described elsewhere (Coyer, 1981; Coyer et al. 1983). Means and standard errors (X±S.E.) of E_M and were computed using sample statistical procedures. Mean values of these variables were used in solving the equations for E_K, the potassium equilibrium potential and the P_Na/P_K ratio.

Hypoxia produced a depolarization of the membrane potential (EM) and loss of intracellular potassium activity that continued during reoxygenation (Fig. 1A). In 10 cells, a 115-min period of hypoxia produced a depolarization of their membrane potentials (EM) from a mean of −46 ·9 ± 3 ·1 to −20·8 ± 4·4mV (S.E.). In addition, intracellular potassium ion activities decreased significantly from 118·9 ± 5·1 to 67·7 ± 8·5 mM-K⁺. Computed values of the potassium equilibrium potential (E_K) were −70·9 mV and −54·5 mV respectively. During reoxygenation of the saline surrounding the ganglion, there was a continued depolarization of E_M to −11·5 ± 3·2 mV and progressive fall in to 49·2 ± 4·9 mm. During hypoxia, there was a depolarization of approximately 26 mV as opposed to a change in E_K of 16 mV. Therefore, the extent of the depolarization is greater than could be accounted for by the change in E_K. The computed P_Na/P_K ratios (Fig. 2, solid circles) increased from a value of 0·028 –0·031 to 0·045. The irreversibility of this increase in the P_Na/P_K ratio and thfl continuing depolarization and loss of are shown in the right portion of Fig. 1A as the P_Na/P_K ratio reaches a value of 0 ·183.

Values for E_M were replotted as a function of (Fig. 3). Again the calculated slope of 89·3 mV shows that the depolarization cannot be explained by changes in E_K only.

Membrane slope resistance decreased during hypoxia and reoxygenation (Fig. IB).

The results suggest that hypoxia causes irreversible depolarization of R₃–R₁₃ neurones occupying the Aplysia abdominal ganglion, together with a decrease in and E_K. The calculated P_Na/P_K ratio increased as the neurones continued to depolarize at a level which was not commensurate with changes in E_K. Thus, the depolarization could be caused by a change in together with a change in P_Na/P_K. An important, alternative explanation which could account for the depolarizations could be that these cells have an electrogenic sodium pump whose contribution is important in maintaining the resting membrane potential (see Discussion below). Similar changes in membrane potential, which were found to exist in Rz by Carpenter & Alving (1968) during inhibition of the sodium pump, may account for the discrepancy between changes in membrane potential and E_K in the R₃–R₁₃ neurones.

The depression in the membrane resistance and associated decreases in and E_M indicate that the membranes of these cells become more permeable to either sodium, chloride, potassium or a combination of these ions during hypoxia. Substantial increases in the P_Na/P_K ratios during hypoxia are shown in Fig. 2 (closed circles). The decrease in membrane resistance during hypoxia is consistent with these observed increases in the P_Na/P_K ratios. The observed irreversible nature of membrane depolarization, loss of ⁠, and depression of the membrane resistance might be expected from changes in ionic permeabilities.

Normally, the metabolically-dependent sodium pump contributes to the membrane potential by maintaining the asymmetric distribution of ions across the neural membrane. It has been suggested that molluscan neurones have ATP reserves sufficient for 20–30 min of normal sodium pumping providing the intracellular is higher than 20 Torr, as exists under normal, aerated conditions of intracellular recording (Kerkut & York, 1969; Junge & Oritz, 1978). Kerkut & York (1969) demonstrated the oxygen sensitivity of the electrogenic sodium pump in brain neurones of the snail Helix by injecting cells with sodium and potassium ions. Finding that sodium-injected neurones had membrane potentials which were more dependent upon than did potassium-injected neurones, Kerkut & York (1969) concluded that the sodium pump relies heavily upon the process of oxidative phosphorylation to supply energy in the form of ATP or another high-energy phosphate containing compound. More than likely, the metabolic reserves of the R₃-R₁₃ neurones ara consumed during a 115-min period of hypoxia under correspondingly low intracellular conditions.

Tonic release of neurotransmitter has been mentioned as a possible effect of hypoxia on the L₂-L₆ neurones (Coyer et al. 1983). Pinsker & Kandel (1969) have demonstrated the sensitivity of the pump current in L₅ to the presynaptic influence of interneurone L₁₀.It is possible either that the sodium pump of R₃-R₁₃ neurones is modulated by presynaptic sources and neurotransmitter release during hypoxia, or that these permeability changes result from synaptic inputs. In addition, Mayeri, Brownell, Branton & Simon (1979a) and Mayeri, Brownell & Branton (1979b) have shown the tonic influences resulting from release of egg-laying hormone (ELH) contained in bag cells on these and other neurones within the ganglion. Since the bag cells were not isolated from these R₃-R₁₃ cells, this may be a factor worthy of consideration. However, during current stimulation of the bag cells (i.e. to release ELH), only slow inhibition and some instances of transient excitation were observed (Mayeri et al. 1979b). Depolarization and release of intracellular potassium were observed in these studies of hypoxia which is in opposition to those which were shown to be mediated by ELH (Mayeri et al. 1979b).

Both groups of neurones are oxygen sensitive although prolonged hypoxic exposure has opposite effects on the membrane potentials and intracellular potassium ion activities of these two groups of cells. One can only speculate that the R₃-R₁₃ neurones have a greater pump dependence for the membrane potential and that hypoxia steadily upsets the ionic imbalance of sodium, potassium and chloride ions across the neural membrane which contributes to the membrane potential. This difference may reflect an intrinsic difference in the active pump sites or possibly the metabolism of each cell type and its capability to provide ATP for operation of the pump. As suggested in an earlier paper (Coyer et al. 1983), activity of the sodium pump in L₂-L₆ neurones may be stimulated by decreasing oxygen conditions through glycolytic feedback and ATP synthesis. In R₃-R₁₃ neurones this phenomenon may not occur due to differences in the metabolic machinery of these cells. Future experiments are planned to test this hypothesis by testing their pump sensitivities to ouabain and also by injecting intermediary compounds, such as ADP and citrate, into R₃-R₁₃ and L₂-L₆ neurones using a pressure microinjection system. In each of these groups of neurones, these experiments will test how prevalent the sodium pump is and whether or not it can be stimulated by intermediary compounds which may be thought to stimulate ATP synthesis through glycolytic feedback mechanisms.

This research was supported in part by Grants NINCDS NS 08802 and NS 07123.