Neuronal excitability and conduction velocity changes in hippocampal slices from streptozotocin-treated diabetic rats.

The effects of streptozotocin-induced diabetes on the electrophysiological properties of central neurones were investigated in rat hippocampal slices. No differences in membrane potential, input resistance or action potential parameters could be detected in pyramidal or granule cells. However, a slowing of conduction velocity in granule cells and an increase in excitability of fibre volleys in both the perforant pathway and granule cell mossy fibre projections was observed. The lack of effect on evoked fEPSPs in these pathways is also consistent with diabetes-dependent changes in the voltage-dependence of synaptic strength.

Chloride-dependent pH regulation in connective glial cells of the leech nervous system.

We used double-barreled pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH is regulated in the connective glial cells of the medicinal leech. The experiments indicate that a Cl(-)- and HCO3(-)-dependent mechanism mediates some recovery from intracellular alkalosis even in the absence of external Na+. This suggests the presence of Na(+)-independent Cl-/HCO3- exchange in the connective glial membrane. At alkaline pHi, this exchange most likely operates in the direction of net acid loading (i.e. HCO3- efflux).

The role of glycine in anoxia/aglycaemia-induced potentiation of N-methyl-D-aspartate receptor-mediated postsynaptic potentials in the rat hippocampus.

During brain ischaemia there is a sustained increase in extracellular glycine levels, and the potential role of these changes in modulating N-methyl-D-aspartate (NMDA) receptor-mediated activity following an anoxic/aglycaemic insult was studied in the rat hippocampal slice. Addition of large saturating concentrations of glycine (100 microM and 1 mM) to the superfusate resulted in increased extracellularly recorded NMDA receptor-mediated components of excitatory postsynaptic potentials (EPSPs) recorded in area CA1. The effects of added glycine were strychnine-insensitive and blocked by a competitive NMDA antagonist. Anoxic/aglycaemic insults to the tissue caused persistent increases in NMDA receptor-mediated EPSPs, but the magnitude of the observed upregulation was unaffected by the presence of added saturating concentrations of glycine. The data suggests that alterations in glycine levels after oxygen deprivation are not responsible for the long term modulation of NMDA receptor activity.

Chronic hyperglycaemia increases neuronal sensitivity to high potassium in hippocampal slices from streptozotocin-treated diabetic rats.

The chronic hyperglycaemia associated with diabetes mellitus increases susceptibility to epileptiform-like activity in the central nervous system. Changes in extracellular potassium levels directly influence neuronal excitability and significantly one of the major regulators of extracellular potassium, the Na-K ATPase, is known to be down-regulated by chronic hyperglycaemia. The sensitivity of hippocampal slices, from rats made diabetic for 2-3 weeks with streptozotocin, to increases in the extracellular potassium concentration ([K+]o) was, therefore, investigated. Raising [K+]o to 10 mM increased the number of orthodromically-evoked population spikes (PSs) in area CA1. This recruitment was significantly greater in hippocampal slices from diabetic rats, which also exhibited significantly more spontaneous activity. These findings confirm a diabetes-dependent increase in sensitivity of central neurones to changes in [K+]o and may help to explain the increased susceptibility to epileptiform activity in this disease state.

The effect of extracellular weak acids and bases on the intracellular buffering power of snail neurones.

1. Intracellular pH (pHi) was measured in snail neurones using pH-sensitive glass microelectrodes. The influence of externally applied weak acids and bases on the total intracellular buffering power (beta T) was investigated by monitoring the pHi changes caused by the intracellular ionophoretic injection of HCl. 2. In the absence of weak acids or bases a reduction in the extracellular HEPES concentration had no effect on pHi or on beta T. It did, however, reduce slightly the rate of pHi recovery following HCl injection. 3. The presence of CO2 greatly increased beta T. However, as predicted for an open buffer system, the contributions to intracellular buffering by CO2 (beta CO2) decreased as pHi decreased. 4. When added to the superfusate, procaine, 4-aminopyridine, trimethylamine and NH4Cl (1-10 mM) all increased steady-state pHi. Procaine was fastest at increasing pHi and 4-aminopyridine the slowest. All four of these weak bases increased beta T. 5. The intracellular buffering action by these weak bases varied. HCl injection in the presence of procaine usually resulted in steady-state pHi changes with no pHi transients. In the presence of the other three weak bases HCl injections resulted in intracellular acidifications which were followed by pHi recovery-like transients. However, these were not blocked by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) or by CaCl2 and I thus conclude that these transients were as a result of slow or incomplete intracellular buffering by the weak bases. 6. In many cells there was a good correlation between the measured contributions to intracellular buffering by the weak bases (beta base) and those predicted assuming a simple two-compartment open system. In all cases, as predicted, beta base increased as pHi decreased. 7. I found a clear relationship between the concentration of external buffer (HEPES) and the rate at which weak bases, applied to the superfusate, were able to increase pHi. The greater the extracellular buffer concentration the greater was the speed of intracellular alkalinization. 8. Lowering the extracellular buffer concentration reduced the efficiency of intracellular buffering by weak bases in response to an intracellular acid load. HCl injection in the presence of weak base caused a larger initial intracellular acidification if the extracellular HEPES concentration was reduced. 9. In conclusion, both weak acids and weak bases can make very large, pHi-dependent contributions to intracellular buffering by way of open buffer systems.(ABSTRACT TRUNCATED AT 400 WORDS)

The intrinsic intracellular H+ buffering power of snail neurones.

1. We measured intracellular pH (pHi) in snail neurones using pH-sensitive glass microelectrodes. We then calculated the intracellular buffering power (beta i) from the pHi changes associated with the influx or efflux of a variety of weak acids or bases. 2. The weak acid anions butyrate and propionate (20 mM) gave similar values for beta i but those measured using 20 mM-acetate were on average twice as great. 3. Although solutions were nominally CO2-free, blockage of pHi regulation with SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) increased the sizes of the pHi changes upon weak acid addition and removal. The corresponding measured values of beta i were on average 26% lower with SITS than without. 4. With pHi regulation blocked, the use of 2.7% CO2 to measure beta i gave beta i values similar to those measured with butyrate or propionate. These values were about 50% less than those previously measured in snail neurones using CO2. 5. beta i values calculated from the pHi changes due to the removal of 5 mM of the weak bases trimethylamine, procaine and NH4Cl were all similar and comparable to those measured using butyrate or propionate. Removing the influence of pHi regulation on the undershoots after NH4Cl removal was found to decrease the apparent measured values of beta i by 10%. 6. Combining all the data (except the values obtained using CO2 and acetate), and adjusting for the errors due to pHi regulation reducing the sizes of the pHi changes, we found that the mean value for beta i was 10.4 +/- 0.6 mM (+/- S.E.M.) at a mean pHi of 7.36 +/- 0.05. 7. We also investigated the relationship between beta i and pHi using ionophoretic acid injection. By means of step-wise injections, with pHi regulation blocked, we found that at normal pHi levels beta i remained relatively constant. However, at a pHi of less than about 6.8 beta i increased with decreasing pHi.

New method for calculating pHi from accurately measured changes in pHi induced by a weak acid and base.

From the combined Henderson-Hasselbalch relations for a weak acid and a weak base, we derive an expression for steady-state intracellular pH(pHi). The derivation requires only accurate measurements of the pHi changes when the two electrolytes are applied, rather than absolute values. The method is based on the assumption of equilibrium of the neutral forms of the two compounds across the plasma membrane, that is, absence of permeation of the charged forms. It is also assumed that no significant pHi regulation takes place, that there is no significant permeability to intracellular buffers and that intracellular buffering power, over the measured span of pHi, is the same. This precludes the use of CO2/bicarbonate buffered systems. We find that under our experimental conditions steady-state pHi values calculated in this way agree closely with those measured directly with pH-sensitive microelectrodes both in snail neurones and crab muscle. The method would allow the intracellular calibration of other pHi measuring techniques.