Biomedical subjects
C R Rose
Publications and source records attributed to C R Rose.
Neurotrophin-evoked rapid excitation of central neurons.
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Neurotrophin-evoked rapid excitation through TrkB receptors.
Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.
Two-photon Na+ imaging in spines and fine dendrites of central neurons.
Dendritic spines are assumed to be the smallest units of neuronal integration. Because of their miniature size, however, many of their functional properties are still unclear. New insights in spine physiology have been provided by two-photon laser-scanning microscopy which allows fluorescence imaging with high spatial resolution and minimal photodamage. For example, two-photon imaging has been employed successfully for the measurement of activity-induced calcium transients in individual spines. Here, we describe the first application of two-photon imaging to measure Na+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices. Whole-cell patch-clamped neurons were loaded with the Na(+)-indicator dye SBFI (sodium-binding benzofuran-isophthalate). In situ calibration of SBFI fluorescence with ionophores enabled the determination of the actual magnitude of the [Na+]i changes. We found that back-propagating action potentials (APs) evoked Na+ transients throughout the proximal part of the dendritic tree and adjacent spines. The action-potential-induced [Na+]i transients reached values of 4 mM for a train of 20 APs and monotonically decayed with a time constant of several seconds. These results represent the first demonstration of activity-induced Na+ accumulation in spines. Our results demonstrate that two-photon Na+ imaging represents a powerful tool for extending our knowledge on Na+ signaling in fine cellular subcompartments.
K(+)-induced reversal of astrocyte glutamate uptake is limited by compensatory changes in intracellular Na+.
Glutamate uptake is coupled to counter-transport of K+, and high external K+ concentrations can induce reversal of glutamate uptake in whole-cell patch-clamp and isolated membrane preparations. However, high external K+ causes little or no reversal of glutamate uptake in intact astrocytes, suggesting a regulatory mechanism not evident in membrane preparations. One mechanism by which intact cells could limit the effects of altered extracellular ion concentrations on glutamate transport is by compensatory changes in intracellular Na+ concentrations. This possibility was examined using astrocyte cultures treated in two ways to reduce the driving force for glutamate uptake: incubation in high K+ (with reciprocal reduction in Na+), and incubation with metabolic inhibitors to induce ATP depletion. ATP depletion produced a rise in intracellular Na+, a collapse of the membrane sodium gradient and a massive reversal of glutamate uptake. By contrast, incubation in high K+/low Na+ medium did not significantly alter the sodium gradient and did not induce glutamate uptake reversal. The sodium gradient was shown to be maintained under these conditions by compensatory reductions in intracellular Na+ that approximately matched the reductions in extracellular Na+. These findings suggest a mechanism by which astrocytes may limit reversal of glutamate uptake under high K+/low Na+ conditions, and further suggest a general mechanism by which Na(+)-dependent transport processes could be shielded from fluctuating extracellular ion concentrations.
Leech giant glial cell: functional role in a simple nervous system.
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(2+) and H(+) transients mediated by neurotransmitter receptors and ion-driven carriers. Among its numerous properties, the leech giant glial cell prominently expresses a large K(+) conductance, voltage-dependent Ca(2+) channels, ionotropic non-NMDA glutamate receptors, and an electrogenic, reversible Na(+)-HCO(3)(-) cotransporter.
Effects of glucose deprivation, chemical hypoxia, and simulated ischemia on Na+ homeostasis in rat spinal cord astrocytes.
A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.
Regulation of intracellular sodium in cultured rat hippocampal neurones.
1. We studied regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal neurones using fluorescence ratio imaging of the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). 2. In standard CO2/HCO3(-)-buffered saline with 3 mM K+, neurones had a baseline [Na+]i of 8.9 +/- 3.8 mM (mean +/- S.D.). Spontaneous, transient [Na+]i increases of 5 mM were observed in neurones on 27% of the coverslips studied. These [Na+]i increases were often synchronized among nearby neurones and were blocked reversibly by 1 microM tetrodotoxin (TTX) or by saline containing 10 mM Mg2+, suggesting that they were caused by periodic bursting activity of synaptically coupled cells. Opening of voltage-gated Na+ channels by application of 50 microM veratridine caused a TTX-sensitive [Na+]i increase of 25 mM. 3. Removing extracellular Na+ caused an exponential decline in [Na+]i to values close to zero within 10 min. Inhibition of Na+,K(+)-ATPase by removal of extracellular K+ or ouabain application evoked a [Na+]i increase of 5 mM min-1. Baseline [Na+]i was similar in the presence or absence of CO2/HCO3-; switching from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline, however, increased [Na+]i transiently by 3 mM, indicating activation of Na(+)-dependent Cl(-)-HCO3- exchange. Inhibition of Na(+)-K(+)-2Cl- cotransport by bumetanide had no effect on [Na+]i. 4. Brief, small changes in extracellular K+ concentration ([K+]o) influenced neuronal [Na+]i only weakly. Virtually no change in [Na+]i was observed with elevation or reduction of [K+]o by 1 mM. Only 30% of cells reacted to 3 min [K+]o elevations of up to 5 mM. In contrast, long [K+]o alterations (> or = 10 min) to 6 mM or greater slowly changed steady-state [Na+]i in the majority of cells. 5. Our results indicate several differences between [Na+]i regulation in cultured hippocampal neurones and astrocytes. Baseline [Na+]i is lower in neurones compared with astrocytes and is mainly determined by Na+,K(+)-ATPase, whereas Na(+)-dependent Cl(-)-HCO3- exchange, Na(+)-HCO3- cotransport or Na(+)-K(+)-2Cl- cotransport do not play a significant role. In contrast to glial cells, [Na+]i of neurones changes only weakly with small alterations in bath [K+]o, suggesting that activity-induced [K+]o changes in the brain might not significantly influence neuronal Na+,K(+)-ATPase activity.
Gap junctions equalize intracellular Na+ concentration in astrocytes.
Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na+]i in neighboring astrocytes was very similar (12.0 +/- 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na+]i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and alpha-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na+ load induced by removal of extracellular K+, indicating that octanol's effects on baseline [Na+]i were not due to inhibition of Na+, K+-ATPase activity. Under control conditions, increasing [K+]o from 3 to 8 mM caused similar decreases in [Na+]i in groups of astrocytes, presumably by stimulating Na+, K+-ATPase. During octanol application, [K+]o-induced [Na+]i decreases were amplified in cells with increased baseline [Na+]i, and reduced in cells with decreased baseline [Na+]i. This suggests that baseline [Na+]i in astrocytes "sets" the responsiveness of Na+, K+-ATPase to increases in [K]o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na+]i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na+ ions to equalize baseline [Na+]i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion.
Pharmacological characterization of Na+ influx via voltage-gated Na+ channels in spinal cord astrocytes.
Spinal cord astrocytes display a high density of voltage-gated Na+ channels. To study the contribution of Na+ influx via these channels to Na+ homeostasis in cultured spinal cord astrocytes, we measured intracellular Na+ concentration ([Na+]i) with the fluorescent dye sodium-binding benzofuran isophthalate. Stellate and nonstellate astrocytes, which display Na+ currents with different properties, were differentiated. Baseline [Na+]i was 8.5 mM in these cells and was not altered by 100 microM tetrodotoxin (TTX). Inhibition of Na+ channel inactivation by veratridine (100 microM) evoked a [Na+]i increase of 47.1 mM in 44% of stellate and 9.7 mM in 64% of nonstellate astrocytes. About 30% of cells reacted to veratridine with a [Na+]i decrease of approximately 2 mM. Qualitatively similar [Na+]i changes were caused by aconitine. The effects of veratridine were blocked by TTX, amplified by (alpha-)scorpion toxin and usually were readily reversible. Veratridine-induced [Na+]i increases were reduced upon membrane depolarization with elevated extracellular [K+]. Recovery to baseline [Na+]i was unaltered during blocking of K+ channels with 4-aminopyridine. [Na+]i increases evoked by the ionotropic non-N-methyl--aspartate receptor agonist kainate were not altered by TTX. Our results indicate that influx of Na+ via voltage- gated Na+ channels is not a prerequisite for glial Na+,K+-ATPase activity in spinal cord astrocytes at rest nor does it seem to be involved in [Na+]i increases evoked by kainate. During pharmacological inhibition of Na+ channel inactivation, however, Na+ channels can serve as prominent pathways of Na+ influx and mediate large perturbations in [Na+]i, suggesting that Na+ channel inactivation plays an important functional role in these cells.
Mechanisms of H+ and Na+ changes induced by glutamate, kainate, and D-aspartate in rat hippocampal astrocytes.
The excitatory transmitter glutamate (Glu), and its analogs kainate (KA), and D-aspartate (D-Asp) produce significant pH changes in glial cells. Transmitter-induced pH changes in glial cells, generating changes in extracellular pH, may represent a special form of neuronal-glial interaction. We investigated the mechanisms underlying these changes in intracellular H+ concentration ([H+]i) in cultured rat hippocampal astrocytes and studied their correlation with increases in intracellular Na+ concentration ([Na+]i), using fluorescence ratio imaging with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) or sodium-binding benzofuran isophthalate (SBFI). Glu, KA, or D-Asp evoked increases in [Na+]i; Glu or D-Asp produced parallel acidifications. KA, in contrast, evoked biphasic changes in [H+]i, alkaline followed by acid shifts, which were unaltered after Ca2+ removal and persisted in 0 CI(-)-saline, but were greatly reduced in CO2/HCO3(-)-free or Na(+)-free saline, or during 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) application. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked KA-evoked changes in [H+]i and [Na+]i, indicating that they were receptor-ionophore mediated. In contrast, CNQX increased the [H+]i change and decreased the [Na+]i change induced by Glu. D-Asp, which is transported but does not act at Glu receptors, induced [H+]i and [Na+]i changes that were virtually unaltered by CNQX. Our study indicates that [Na+]i increases are not primarily responsible for Glu- or KA-induced acidifications in astrocytes. Instead, intracellular acidifications evoked by Glu or D-Asp are mainly caused by transmembrane movement of acid equivalents associated with Glu/Asp-uptake into astrocytes. KA-evoked biphasic [H+]i changes, in contrast, are probably attributable to transmembrane ion movements mediated by inward, followed by outward, electrogenic Na+/HCO3- cotransport, reflecting KA-induced biphasic membrane potential changes.
Intracellular sodium homeostasis in rat hippocampal astrocytes.
1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI-AM (acetoxymethylester of sodium-binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the 'Na+' signal. 2. Baseline [Na+]i was 14.6 +/- 4.9 mM (mean +/- S.D.) in CO2/HCO3(-)-containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/- 0.32 mM min-1) followed by a slower phase (0.15 +/- 0.09 mM min-1). 3. Changing from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)-K(+)-2Cl- cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage-gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)-ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min-1, respectively. This indicated that Na+, K(+)-ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o-induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/- 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)-HCO3- cotransport, Na(+)-K(+)-2Cl- cotransport and Na+, K(+)-ATPase, and that both Na+, K(+)-ATPase and inward Na(+)-K(+)-2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.
Extracellular Ca2+ changes during transmitter application in the leech central nervous system.
Changes in extracellular Ca2+ concentration ([Ca2+]e) evoked by transmitters and transmitter agonists, respectively, and by elevation of bath K+ concentration were recorded in isolated segmental ganglia of the leech Hirudo medicinalis using Ca(2+)-selective microelectrodes. A 1-min bath application of kainate (10 microM), glutamate (1 mM), aspartate (1 mM), or carbachol (200 microM) decreased [Ca2+]e by up to 1 mM, whereas the inhibitory transmitters gamma-amino butyric acid (GABA, 100 microM) and serotonin (5-HT, 100 microM) did not change [Ca2+]e. The amplitude of the kainate-induced changes in [Ca2+]e increased with repetitive applications, and changes were blocked by 6-cyano-7-dinitroquinozaline-2,3-dione (CNQX). Elevation of bath K+ concentration from 4 to 40 mM led to a Ni(2+)-sensitive decrease in [Ca2+]e by 0.9 mM. Our results suggest that excitatory transmission in the leech central nervous system might be accompanied by substantial decreases in [Ca2+]e.
pH regulation and proton signalling by glial cells.
The regulation of H+ in nervous systems is a function of several processes, including H+ buffering, intracellular H+ sequestering, CO2 diffusion, carbonic anhydrase activity and membrane transport of acid/base equivalents across the cell membrane. Glial cells participate in all these processes and therefore play a prominent role in shaping acid/base shifts in nervous systems. Apart from a homeostatic function of H(+)-regulating mechanisms, pH transients occur in all three compartments of nervous tissue, neurones, glial cells and extracellular spaces (ECS), in response to neuronal stimulation, to neurotransmitters and hormones as well as secondary to metabolic activity and ionic membrane transport. A pivotal role for H+ regulation and shaping these pH transients must be assigned to the electrogenic and reversible Na(+)-HCO3-membrane cotransport, which appears to be unique to glial cells in nervous systems. Activation of this cotransporter results in the release and uptake of base equivalents by glial cells, processes which are dependent on the glial membrane potential. Na+/H+ and Cl-/HCO3-exchange, and possibly other membrane carriers, accomplish the set of tools in both glial cells and neurones to regulate their intracellular pH. Due to the pH dependence of a great variety of processes, including ion channel gating and conductances, synaptic transmission, intercellular communication via gap junctions, metabolite exchange and neuronal excitability, rapid and local pH transients may have signalling character for the information processing in nervous tissue. The impact of H+ signalling under both physiological and pathophysiological conditions will be discussed for a variety of nervous system functions.
Activity-induced Ca2+ transients in nerve and glial cells in the leech CNS.
We have measured activity-induced Ca2+ transients in Retzius neurones, neuropile glial cells, and extracellular spaces of isolated ganglia of the leech Hirudo medicinalis using the fluorescent dye fura-2 and Ca(2+)-sensitive microelectrodes. Neuronal activity, induced by electrical side nerve stimulation (20 Hz/1 min), elicited transient rises of intracellular Ca2+ in both neurones and glial cells, which amounted to 24 +/- nM (n = 15) and 17 +/- 14 nM (n = 7), respectively. The extracellular Ca2+ declined by 160 +/- 73 microM (n = 6) during stimulation. Intra- and extracellular Ca2+ transients were reduced by the glutamate/kainate receptor blocker CNQX (6-cyano-7-dinitroquinozaline-2,3-dione; 50 microM). Our results show that neuronal activity evokes Ca2+ signals not only in neurones, but also in glial cells and suggest that these Ca2+ transients are partly mediated via activation of glutamate/kainate receptors.
Stimulus-evoked changes of extra- and intracellular pH in the leech central nervous system. I. Bicarbonate dependence.
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)
Stimulus-evoked changes of extra- and intracellular pH in the leech central nervous system. II. Mechanisms and maintenance of pH homeostasis.
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)
Evidence that glial cells modulate extracellular pH transients induced by neuronal activity in the leech central nervous system.
1. The role of the giant neuropile glial cells in the buffering of activity-related extracellular pH changes was studied in segmental ganglia of the leech Hirudo medicinalis L. using pH-sensitive microelectrodes and a slow, two-electrode voltage-clamp system. Neuronal activity was induced by electrical stimulation of a ganglionic side nerve (20 Hz, 1 min). 2. In CO2-HCO3(-)-buffered saline the glial cells were depolarized by 6.5 +/- 2.3 mV and alkalinized by 0.024 +/- 0.006 pH units (mean +/- SD) during the stimulation. The stimulation induced an acidification of 0.032 +/- 0.006 pH units in the extracellular spaces (ECS). 3. Voltage clamping the glial cells suppressed the stimulus-induced glial depolarization and turned the intraglial alkalinization into an acidification of 0.045 +/- 0.021 pH units (n = 6) that closely resembled the acidification observed in the presence of the anion transport blocker DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 0.5 mM), and in CO2-HCO(3-)-free saline. 4. Voltage clamping the glial cell resulted in the appearance of a distinct stimulus-induced extracellular alkalinization of 0.024 +/- 0.013 pH units at the onset of the stimulation, as also observed during DIDS application and in the absence of CO2-HCO3-. 5. The results suggest that glial uptake of bicarbonate is mediated by depolarization-induced activation of the electrogenic Na(+)-HCO3- cotransport, which suppresses the profound alkalinization of the ECS during neuronal activity. This is the first direct evidence the glial cells actively modulate extracellular pH changes in a voltage-dependent manner.