Results presented in this study indicate that a large subpopulation (approximately 65%) of hippocampal astrocytes in situ exhibit calcium oscillations in the absence of neuronal activity. Further, the spontaneous oscillations observed within individual hippocampal astrocytes generally developed asynchronously throughout the astrocyte's fine processes and occasionally spread through a portion of that astrocyte as a calcium wave but do not appear to spread among astrocytes as an intercellular calcium wave. Bath application of cyclopiazonic acid and injection of individual astrocytes with heparin blocked astrocytic calcium oscillations. Application of tetrodotoxin or incubation of slices with bafilomycin A1 had no effect on astrocytic calcium oscillations but did block evoked and spontaneous postsynaptic currents measured in CA1 pyramidal neurons. Application of a cocktail of antagonists for metabotropic glutamate receptors and purinergic receptors had no effect on the astrocytic calcium oscillations but blocked the ability of purinergic and metabotropic glutamatergic agonists to increase astrocytic calcium levels. These results indicate that the spontaneous calcium oscillations observed in hippocampal astrocytes in situ are mediated by IP3 receptor activation, are not dependent on neuronal activity, and do not depend on activation of metabotropic glutamate receptors or purinergic receptors. To our knowledge, this is the first demonstration that astrocytes in situ exhibit intrinsic signaling. This finding supports the hypothesis that astrocytes, independent of neuronal input, may act as pacemakers to modulate neuronal activity in situ.

The giant glial cell in the central nervous system of the leech Hirudo medicinalis has been the subject of a series of studies trying to link its physiological properties with its role in neuron-glia interactions. Isolated ventral cord ganglia of this annelid offer several advantages for these studies. First, single giant glial cells can easily be identified and are quite accessible to electrophysiological and microfluorometric studies. Second, only two giant macroglial cells are located in the neuropil of each ganglion, rendering them well suited for studying neuron-glia interactions. Third, many neurons can be identified and are well known with respect to their physiology and their roles in controlling simple behaviors in the leech. This review briefly outlines the major recent findings gained by studying this preparation and its contributions to our knowledge of the functional role of glia in nervous systems. Emphasis is directed to glial responses during neuronal activity and to the analysis of intracellular Ca 2ϩ and H ϩ transients mediated by neurotransmitter receptors and ion-driven carriers. Among its numerous properties, the leech giant glial cell prominently expresses a large K ϩ conductance, voltage-dependent Ca 2ϩ channels, ionotropic non-NMDA glutamate receptors, and an electrogenic, reversible Na ϩ -HCO 3 Ϫ cotransporter.

We have measured the intracellular Ca2+ concentration, [Ca2+]i, and the intracellular Na+ concentration, [Na+]i, with the fluorescent dyes fura‐2 (for Ca2+) and SBFI (for Na+) in situ in giant glial cells of the central nervous system of the leech Hirudo medicinalis. The basal [Ca2+]i was 79 ± 35 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and 75 ± 29 nm (mean ±s.d., n= 82) in unclamped cells at a mean membrane potential of −67 ± 6 mV. Removal of external Na+ evoked a small reversible [Ca2+]i increase of 29 ± 21 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and of 35 ± 18 nm (n= 37) in unclamped cells. This [Ca2+]i increase, and the time constant of the subsequent [Ca2+]i recovery after Na+ re‐addition, did not change significantly with the holding potential between −110 and −60 mV. The basal [Na+]i was 5.6 ± 1.3 mm (n= 18). Increasing [Na+]i by inhibiting the Na+‐K+ pump with 100 μm ouabain had no effect on the [Ca2+]i rise upon removal of external Na+. The time course of recovery from a [Ca2+]i load mediated by voltage‐dependent Ca2+ influx during depolarization in high K+ was unaffected by the removal of external Na+. Cyclopiazonic acid (10 μm), an inhibitor of the endoplasmic reticulum Ca2+‐ATPase, caused a transient increase in [Ca2+]i of 28 ± 11 nm (n= 5), and significantly slowed the recovery from imposed [Ca2+]i loads. Iontophoretic injection of orthovanadate, an inhibitor of P‐type ATPases including the plasma membrane Ca2+‐ATPase, caused a persistent increase in the basal [Ca2+]i of 163 ± 101 nm (n= 5) in standard saline, and of 427 ± 338 nm in Na+‐free saline (n= 5). Vanadate injection significantly slowed the recovery from [Ca2+]i loads. Removal of external Na+ during vanadate injection induced an additional, reversible [Ca2+]i increase of 254 ± 64 nm (n= 3). The results suggest that the low basal [Ca2+]i in these glial cells is predominantly maintained by a Ca2+‐ATPase in the plasma membrane. This ATPase is also the main Ca2+ extruder after an intracellular Ca2+ load, while intracellular stores appear to contribute little to this recovery. A Na+‐Ca2+ exchanger seems to play a minor role in the maintenance of basal [Ca2+]i in these cells, but becomes prominent when the plasma membrane Ca2+‐ATPase is blocked.

The glutamate-agonist kainate evokes Ca2+ transients in both neurones and glial cells. Owing to the membrane depolarization elicited by kainate, a Ca2+ influx could occur through voltage-gated Ca2+ channels or through the kainate-gated cation channels directly. We have measured ratio signals of the calcium indicator dye fura-2, injected into giant glial cells of the leech Hirudo medicinalis, as response to kainate (5-20 microM) in the presence of different divalent cations. The responses to kainate increased during the first 2-4 kainate applications, both in unclamped and in voltage-clamped cells. The fura-2 fluorescence ratio (F350/F380) still increased when Ca2+ was replaced by Ba2+ but was suppressed in Ca(2+)-free saline and in the presence of Ni2+ (2 mM). Co2+ and Mn2+ (2 mM) also reduced the kainate-induced fura-2 fluorescence signals, due to entry of these divalent cations into the cells and subsequent quenching the fluorescence of the intracellular dye. It is concluded that Ni2+ blocks the kainate-induced membrane depolarization and Ca2+ transient but apparently does not enter the cells, while Ba2+, Co2+, and Mn2+ appear to permeate the membrane, presumably through the kainate-gated channels.

We have employed two independent techniques to measure the intracellular pH (pHi) in giant glial cells of the leech Hirudo medicinalis, using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and double-barreled neutral-carrier, pH-sensitive microelectrodes, which also record the membrane potential. We have compared two procedures for calibrating the ratio of the BCECF signal, excited at 440 nm and 495 nm: 1) the cell membrane was H(+)-permeabilized with nigericin in high-K+ saline at different external pH (pHo) values, and 2) the pHi of intact cells was perturbed in CO2/HCO3(-) -buffered saline of different pH, and the BCECF ratio was calibrated according to a simultaneous microelectrode pH reading. As indicated by the microelectrode measurements, the pHi did not fully equilibrate to the pHo values in nigericin-containing, high-K+ saline, but deviated by -0.12 +/- 0.02 (mean +/- SEM, n = 37) pH units. In intact cells, the microelectrode readings yielded up to 0.15 pH unit lower values than the calibrated BCECF signal. In addition, larger dye injections into the cells (> 100 microM) caused an irreversible membrane potential loss indicative of some damage to the cells. The amplitude and kinetics of slow pHi changes were equally followed by both sensors, and the dye ratio recorded slightly higher amplitudes during faster pHi shifts as induced by the addition and removal of NH4+.