1. We have studied extracellular pH (pHe) and intracellular pH (pHi) changes evoked by repetitive electrical side nerve stimulation (20 Hz, 1 min) in segmental ganglia of the leech Hirudo medicinalis using double-barreled, pH-sensitive microelectrodes to elucidate the involvement of neurotransmitters, of carbonic anhydrase, and of active acid/base transport on the extracellular H+ homeostasis. In saline buffered with 5% CO2-24 mM HCO3-, the stimulation induced a small and brief alkalinization followed by an acidification in the extracellular spaces (ECS), whereas neurons acidified and glial cells alkalinized (see previous paper). 2. Blocking synaptic transmitter release by superfusion with 20 mM Mg2+ saline (CO2/HCO3(-)-free) led to a reversible reduction of both activity-induced pHe changes by approximately 90% and to a complete suppression of the intracellular acidification of neurons. After application of the glutamate/kainate receptor blocker 6-cyano-7-dinitroquinozaline-2,3-dione (CNQX, 50 microM) to CO2/HCO3(-)-free saline, the stimulus-evoked pHe changes were reversibly reduced. The gamma-aminobutyric acid-A (GABAA) receptor antagonist picrotoxin (50 microM) led to an amplification of the extracellular alkalinization in the presence of CO2/HCO3-. Bath application of the excitatory transmitter agonists carbachol or kainate to CO2/HCO3(-)-free saline induced biphasic alkaline-acid transients in the ECS; the inhibitory transmitters GABA and serotonin had no detectable effects on the pHe (saline buffered with CO2/HCO3-).(ABSTRACT TRUNCATED AT 250 WORDS)

The intracellular K activity (aKi) and membrane potential of sensory neurons in the leech central nervous system were measured in normal and altered external K+ concentrations, [K+]o, using double-barreled, liquid ion-exchanger microelectrodes. In control experiments membrane potential measurements were made using potassium chloride-filled single-barreled microelectrodes. All values are means +/- SD. At the normal [K+]o (4 mM) the mean aKi of all cells tested was 72.6 +/- 10.6 mM (n = 40) and the average membrane potential was -47.3 +/- 5.2 mM (n = 40). When measured with single-barreled microelectrodes, the membrane potential averaged -45.3 +/- 2.9 mV (n = 12). Assuming an intracellular K+ activity coefficient of 0.75, the intracellular K+ concentration of sensory neurons would be 96.8 +/- 14.1 mM). With an extracellular K+ concentration of 5.8 mM in the intact ganglion compared to the K+ concentration of 4 mM in the bath, the K+ equilibrium potential was -71.5 mV. When the ganglion capsule was opened, the extracellular K+ concentrations in the ganglion were similar to that of the bathing medium and the calculated K+ equilibrium potential was -81 mV. The membrane of sensory neurons depolarized following the changes to elevated [K+]o (greater than or equal to 10-100 mM), whereas aKi changed only little or not at all. At very low [K+]o (0.2, 0 mM) aKi and membrane potential showed little short-term (less than 3 min) effect but began to change after longer exposure (greater than 3 min). Reduction of [K+]o from 4 to 0.2 mM (or 0 mM) produced first a slow, and then a more rapid decrease of aKi and membrane resistance, accompanied by a slow membrane hyperpolarization. Following readdition of normal [K+]o, the membrane first depolarized and then transiently hyperpolarized, eventually returning slowly to the normal membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)

1. We have measured the effect of repetitive electrical nerve root stimulation on the extracellular potassium activity (aKe) and the extracellular pH (pHe) and intracellular pH (pHi) in segmental ganglia of the leech Hirudo medicinalis with double-barreled K(+)- and pH-sensitive microelectrodes. To investigate the influence of CO2/HCO3-, we compared the stimulus-evoked changes in aKe, pHe, and pHi in the presence and absence of 5% CO2-24 mM HCO3- in the saline. 2. An electrical nerve root stimulation at 20-30 Hz for 1 min caused a rapid increase of 1.11 +/- 0.79 (SD) mM in aKe, followed by an aKe undershoot of 0.17 +/- 0.15 mM when the stimulation was discontinued (n = 6). aKe transients were not significantly affected by CO2/HCO3-. 3. In 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered, nominally CO2/HCO3(-)-free saline, low stimulus intensities or stimulus durations up to a few seconds resulted in a fast alkaline pHe transient. This alkalinization was followed by a larger and longer-lasting extracellular acidification when the stimulation was intensified and prolonged. A stimulation at 20 Hz, 5 V for 1 min caused an average alkaline shift of 0.083 +/- 0.055 pH units, followed by an acidosis of 0.079 +/- 0.038 pH units (n = 63). A change from 5 mM HEPES-buffered saline to 20 mM HEPES-buffered saline attenuated the stimulus-evoked pHe transients by 50-60%.(ABSTRACT TRUNCATED AT 250 WORDS)

1. We studied the effects of photoinactivation of neuropil glial (NG) cells of the leech Hirudo medicinalis on neuronal activity and synaptic transmission. Each segmental ganglion contains two of these giant glial cells, which are electrically and dye coupled. 2. One of the two NG cells in an isolated segmental ganglion was filled with the dye Lucifer yellow (LY). Subsequent irradiation of the ganglion with laser light (440 nm) to photolyze LY caused irreversible depolarization of both NG cells. The NG cells that were filled with LY depolarized from -73 +/- 1.1 (SE) mV to -22 +/- 2.4 mV within 25 +/- 2.8 min of continuous irradiation (n = 22). The other NG cell, which was not directly filled with LY, depolarized with some delay. 3. Photoinactivation of the NG cells caused an irreversible depolarization of Retzius neurons and noxious (N) sensory cells by a mean of 14 mV (n = 36) and 9 mV (n = 24), respectively. In addition, the input resistance was reduced by 54% in Retzius cells and by 34% in N cells. Spikes could not be evoked in Retzius cells after the inactivation of the NG cells, either by intracellular current injection or by electrical nerve stimulation. Similarly, anterior pagoda neurons, annulus erector neurons, and the excitor neurons of the ventrolateral circular muscles became inexcitable. However, N cells, heart interneurons, and most of the heart motor neurons, touch cells, and pressure cells could still generate spontaneous or evoked action potentials. 4. Photoinactivation of the NG cells impaired the electrical connection between the two Retzius neurons. The electrical coupling was completely eliminated in six of eight cell pairs and reduced by 66% in two others. 5. Photoinactivation of the NG cells in the 3rd and 4th segmental ganglion caused a complete block of the chemical synapse between reciprocal inhibitory heart interneurons in these ganglia; the bursting rhythm either stopped or changed to a tonic activity, whereas inhibitory postsynaptic potentials could not be recorded in either heart interneuron anymore. 6. Laser irradiation alone had no effect on neuronal activity and synaptic transmission. Addition of glutathione (10 mM) and ascorbic acid (10 mM) to the saline to bind extracellular radicals that might be produced by the irradiation did not suppress the effects caused by photoinactivation of NG cells. 7. Elevation of bath K+ concentration to 12 mM, acidification of the saline to pH 5.5, and alkalinization to pH 8.5 for 6 min each did not mimick the effects on membrane properties of Retzius cells as produced by inactivation of NG cells. The results suggest some role of glial cells in the maintenance of neuronal activity and electrical and chemical synaptic transmission.

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