Caffeine mediates cation influx and intracellular Ca2+ release in leech P neurones.

We investigated the effect of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) of leech P neurones by using the fluorescent indicator Fura-2. Caffeine induced a [Ca2+]i increase that was strongly reduced, but not abolished, in Ca(2+)-free solution. The effect of caffeine on [Ca2+]i was dose-dependent: while 5 mM caffeine evoked a persistent [Ca2+]i increase that could be elicited repetitively, 10 mM caffeine or more induced a transient [Ca2+]i increase that was strongly reduced upon subsequent applications at the same concentration. Surprisingly, the cells remained fully responsive to a moderately increased caffeine concentration. The caffeine-induced [Ca2+]i increase was not blocked by millimolar concentrations of La3+, Mg2+, Cd2+, Zn2+, Co2+, Ni2+, or Mn2+. While La3+ and Mg2+ had no effect on the caffeine response, the other cations caused irreversible changes in the Fura-2 fluorescence. The inhibitors of intracellular Ca2+ pumps-thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ)--had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, but they reduced it in Ca(2+)-free solution. Ryanodine had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, and also in Ca(2+)-free solution it seemed to be largely ineffective. Caffeine evoked complete fluctuations of the membrane potential. The effect in Ca2+ free and in Na(+)-free solution suggests that the depolarizing response components were mainly due to Na+ influx, while Ca2+ reduced the Na+ influx and/or activated mechanisms which re- or hyperpolarize the cells. It is concluded that leech P neurones possess caffeine-sensitive intracellular Ca2+ stores, as well as caffeine-sensitive ion channels, in the plasma membrane that are activated by a voltage-independent mechanism. The plasma membrane channels are permeable to various divalent cations including Ca2+, and possibly also to Na+.

A functional alpha-bungarotoxin receptor is present in chick cerebellum: purification and characterization.

It has recently been demonstrated that alpha-bungarotoxin receptors, which behave as functional nicotinic receptors, are present in chick CNS. In this paper, we report the purification and characterization of a functional alpha-bungarotoxin receptor from chick cerebellum, a nervous tissue in which a clear inhibition of induced nicotine effects has been reported in vivo. This receptor contains at least three subunits of apparent mol. wt 52,000, 57,000 and 67,000. The use of monoclonal antibodies specific for the alpha 7 subunit demonstrated that 75% of the molecules present in our purified preparation belong to the alpha 7 subtype and that this antibody labels the 57,000 band in western blot, thus indicating that this is the toxin binding subunit. Reconstruction experiments in planar lipid bilayers show that this alpha-bungarotoxin receptor forms a cation selective channel whose opening is blocked by d-tubocurarine. Binding experiments on immobilized receptors over an alpha-bungarotoxin-Sepharose affinity column show that the ligand binding subunit is present in vivo in two copies per receptor. Immunological, pharmacological and functional experiments show that this purified receptor is very similar, but not identical, to the previously characterized chick optic lobe receptor, thus indicating the heterogeneity of these alpha-bungarotoxin receptors in the CNS.

Localization of carbonic anhydrase in identified glial cells of the leech central nervous system: application of histochemical and immunocytochemical methods.

We localized the enzyme carbonic anhydrase (CA) in frozen sections of the leech (Hirudo medicinalis) central nervous system by two histochemical techniques and the indirect immunofluorescence technique. Hansson's cobalt precipitation method and the use of 1-dimethylamino-naphthalene-5-sulfonamide (DNSA) to build a fluorescent enzyme-substrate complex showed that glial cells are the sites of CA activity in the leech. Neuropil and connective glial cells surrounding the axons had strong CA activity, whereas packet glial cells, which surround neuron cell bodies, and neurons themselves remained unstained. Glial cells reacted markedly with FITC-coupled antibodies against CA isoenzyme II, but experiments with antibodies against CA isoenzyme I showed no reaction.

Ionic mechanism of a hyperpolarizing 5-hydroxytryptamine effect on leech neuropile glial cells.

The ionic mechanism of a membrane effect of 5-hydroxytryptamine (5-HT) on neuropile glial (NG) cells in ganglia of the medicinal leech was investigated with conventional single-barrelled microelectrodes. Control experiments were made with double-barrelled ion-selective microelectrodes. 5-Hydroxytryptamine hyperpolarized the NG-cell membrane and increased the conductance considerably. Methysergide, a potent 5-HT antagonist, blocked the 5-HT-induced hyperpolarization completely. When leech ganglia were superfused with physiological bathing media free of 5-HT, the NG-cell membrane conductance returned to the original value, but the membrane potential recovered only partially from the hyperpolarization in most experiments. In glial membranes artificially depolarized by means of constant-current injection, the amplitude of the 5-HT response increased. The amplitude decreased with membrane hyperpolarization and reversed at - 73 mV, close to the potassium equilibrium potential. The reversal potential changed by 52 mV when the extracellular potassium concentration was altered by a factor of 10. We conclude that 5-HT increases the potassium conductance of NG-cell membranes.

External ions and membrane potential of leech neuropile glial cells.

In ion-substitution experiments supplemented by measurements of the membrane conductance, the membrane potential of neuropile glial (NG) cells in the CNS of th medicinal leech has exhibited a dependence of the external concentration of both potassium and chloride. The membrane potential was largely dependent on the external potassium concentration, as may be inferred from the change in potential as the potassium concentration of the bathing solution was changed. The external potassium concentrations had been corrected to allow for the discrepancy between intra- and extraganglionic levels found with ion-selective electrodes. A transient membrane depolarization was recorded when the chloride in the bathing medium was replaced by sulphate or glucuronate. The restoration of the normal membrane potential following the return to chloride-based saline was preceded by a transient hyperpolarization. After transfer to low-chloride solutions, the transient depolarization of the NG cell membrane was followed by a steady-state hyperpolarization. The amplitude of the steady-state hyperpolarization depended on the concentration of chloride in the bathing medium. The membrane conductance decreased in low-chloride solutions.

Intracellular acidosis of identified leech neurones produced by substitution of external sodium.

The intracellular pH, pHi, of identified neurones of the central nervous system of the leech Hirudo medicinalis L. was measured with double-barrelled neutral carrier pH-sensitive microelectrodes. The active regulation of pHi of these neurons is due to amiloride-sensitive Na-H exchange and hence requires extracellular Na, Nao. We have measured a decrease of pHi following the removal of Nao. The rate of intracellular acidification in Na-free saline was similar to that in the presence of 2 mM amiloride suggesting that the acidification was due to inhibition of the Na-H exchange. The rate of intracellular acidification depended on the Na substitute chosen; it was 0.02 +/- 0.005 pH units/min (+/- S.D., n = 17) when Na was replaced by N-methyl-D-glucamine. A similar rate of acidification occurred with tris-hydroxymethyl-aminomethane (Tris) while the rate of acidification was higher with bis-2-hydroxymethyl-dimethylammonium (BDA, 0.033 +/- 0.016 pH units/min (+/- S.D., n = 7) and tetramethylammonium (TMA, 0.046 +/- 0.017 pH units/min (n = 3) as Na substitutes. A high, non-linear rate of intracellular acidification was observed, when Li, K or choline were used as Na substitute. The recovery of pHi from acidification upon readdition of Nao was fast, only when Li had replaced Na was the pHi recovery considerably delayed. In conclusion, in all experiments using different Na substitutes the removal of Nao caused a substantial intracellular acidification presumably due to inhibition of Na-H exchange. These changes in pHi might be relevant for results obtained by experiments in which Na-free solutions are used.

Potassium activity in leech neuropile glial cells changes with external potassium concentration.

The effect of the external K+ concentration on the intracellular K+ activity of neuropile glial cells and of sensory neurons in the central nervous system of the leech (Hirudo medicinalis L.) was determined directly with double-barreled ion-sensitive microelectrodes. As the external K+ concentration was raised, the intracellular K+ activity of the neuropile glial cells increased considerably, and was accompanied by the uptake and/or intracellular synthesis of an, as yet, unidentified substance. In contrast, the intracellular K+ activity of sensory neurons was unchanged when the external K+ concentration was increased. These neurons appeared not to accumulate a second substance.

The ouabain-induced [Ca2+]i increase in leech Retzius neurones is mediated by voltage-dependent Ca2+ channels.

In leech Retzius neurones the inhibition of the Na+/K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+/K+ pump in bathing solutions of different ionic composition. The results show that Na+/K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+/K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+/K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels.

Macroscopic and single-channel chloride currents in neuropile glial cells of the leech central nervous system.

In patch-clamp experiments we characterized four Cl- channels (42 pS, 70 pS, 80 pS and 229 pS) underlying the large Cl- conductance of leech neuropile glial cells. They differed with respect to their gating, their rectification and their activity in the cell-attached configuration, showed the selectivity sequence I->Cl->/=Br->F- and were impermeable to SO42-. The four channels were blocked by NPPB, DPC, niflumic acid and DIDS and exhibited either three or four sublevel states. The outward rectifying 42 pS, 70 pS and 80 pS Cl- channels were classified as intermediate conductance Cl- channels and they could contribute to the high Cl- conductance of the glial membrane, which stabilizes the glial membrane potential. The inward rectifying 229 pS Cl- channel is very similar to vertebrate high conductance Cl- channels, which are assumed to be part of an emergency system that is activated under pathophysiological conditions. In voltage-clamp experiments we calculated that the Cl- conductance amounts to one-third of the total membrane conductance. Reduction of this Cl- conductance by Cl- channel inhibitors markedly depolarized the glial cell membrane. These prominent depolarizations depended on Na+ influx and in most cases the glial cells failed to regulate their membrane potential following wash-out of the inhibitors.

Mechanism of the kainate-induced intracellular acidification in leech Retzius neurons.

We examined the effect of the glutamatergic agonist kainate on the membrane potential, the intracellular Na+ concentration ([Na+]i), the intracellular-free Ca2+ concentration, and on the intracellular pH of Retzius neurons of the medicinal leech, Hirudo medicinalis, in order to investigate the mechanism responsible for the intracellular acidification caused by glutamatergic stimulation. The recordings were made with Na+- and pH-sensitive microelectrodes and iontophoretically injected Fura-2. Bath application of kainate evoked a marked membrane depolarization, a [Na+]i increase, and an intracellular acidification. The intracellular acidification was unaffected by reversal of the electromotive force for H+, suggesting that an influx of H+ from the interstitial space does not contribute to the acidification. While the Ca2+ channel blockers La3+ and Co2+ had no effect on the kainate-induced intracellular acidification, suggesting that a Ca2+ influx via voltage-dependent Ca2+ channels was not relevant, the acidification was reduced in Ca2+-free saline solution. In Na+-free saline solution the kainate-induced intracellular acidification was absent, suggesting the involvement of Na+ influx in generating the acidification. When injected iontophoretically Na+ induced an intracellular acidification but Li+, K+, Rb+ or Cs+ did not. Furthermore, a [Na+]i increase induced by blocking the Na+/K+ pump also led to an intracellular acidification. We conclude that the [Na+]i increase is the crucial event underlying the kainate-induced intracellular acidification. Possible mechanisms linking the [Na+]i increase to the intracellular acidification are discussed.

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).

Single potassium channels in neuropile glial cells of the leech central nervous system.

We performed patch-clamp experiments to identify distinct K+ channels underlying the high K+ conductance and K+ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K+ channels were characterized, which were found to be distributed in clusters. Since no other types of K+ channels were observed, these appear to be the complete repertoire of K+ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K+ channel could markedly contribute to the high K+ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K+ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca2+ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K+ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K+ content. The 40 pS K+ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than -60 mV, flickering events indicated voltage-dependent gating.

Ionic mechanism of 4-aminopyridine action on leech neuropile glial cells.

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K+ channels, but also induced Na+ and Ca2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was -5 mV. Application of 5 mM Ni2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K+ and Cl- channels and 4-AP-induced Ca2+ influx could activate Ca2+-sensitive K+ and Cl- channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K+ conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.

The Na+-K+ pump in neuropile glial cells of the medicinal leech.

The membrane potential of neuropile glial (NG) cells in the central nervous system of the medicinal leech and the K+ concentration in extracellular spaces (ECS) of the neuropile were measured under various experimental conditions to determine properties of a glial Na+-K+ pump. The ganglia were exposed to K+-free saline thereby loading the NG cells with intracellular Na+. Their membranes hyperpolarized transiently when the K+-free solution was replaced by a bathing medium with normal (= 4 mM) K+ concentration. The hyperpolarization increased in amplitude with time of exposure to K+ -free solution and could be abolished by ouabain or by replacing Na+ with Li+. The transient membrane hyperpolarization could not be attributed to K+ depletion in the ECS of the neuropile or to changes in membrane input conductance. In a (bathing) medium containing 5 X 10(-4) M ouabain, the K+ concentration in the ECS increased transiently, and the NG cell membrane depolarized rapidly. This short-term depolarization (duration 2-3 min) was followed by a second long-term depolarization (duration 15 min) of the NG cell membrane, which reached a steady-state 20 min after ouabain application. In a bathing medium with elevated external K+ concentrations, the amplitude of the membrane depolarization was enhanced by ouabain. This depolarizing ouabain effect was a result of K+ accumulation in the ECS. We conclude that the Na+-K+ pump does not contribute directly to the resting membrane potential of NG cells and is not directly involved in K+ homeostasis at the cellular level.

Glutaminergic responses of neuropile glial cells and Retzius neurones in the leech central nervous system.

The effects of glutaminergic agonists on neuropile glial cells and on Retzius neurones in the central nervous system (CNS) of the leech, Hirudo medicinalis were investigated using double-barrelled ion-sensitive microelectrodes. In both types of cells, bath-application of L-glutamate (Glu), kainate (Ka) and quisqualate (Qui) elicited substantial membrane depolarizations which were accompanied by increases of the intracellular Na+ activity aiNa and by concomitant decrease of the intracellular K+ activity aiK. In the glial cells, these alterations of aiNa and aiK were preceded by a transient decrease of aiNa and an increase of aiK upon administration of Ka and Qui. In both glial cells and neurones, N-methyl-D-aspartate (NMDA) did not affect Em, aiK and aiNa. As found for Ka, the neuronal as well as the glial responses to glutaminergic agonists persisted during inhibition of synaptic transmission in high Mg2+, low Ca2+ solutions. The results indicate that leech neuropile glial cells have a Ka/Qui-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones.

Effect of extracellular K+ on the intracellular free Ca2+ concentration in leech glial cells and Retzius neurones.

The effects of extracellular K+ on the intracellular free Ca2+ concentration ([Ca2+]i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the leech Hirudo medicinalis were investigated by using iontophoretically injected fura-2. In both cell types, an elevation of the extracellular K+ concentration ([K+]o) caused an increase in [Ca2+]i, which was blocked by Co2+, Ni2+ and menthol, whereas nicardipine, flunarizine, omega-conotoxin GVIA and omega-agatoxin IVA were ineffective. In Ca(2+)-free solution, the K(+)-induced [Ca2+]i increase was largely suppressed in neuropile glial cells and completely abolished in Retzius neurones. The results indicate that the K(+)-induced [Ca2+]i increase was mainly due to Ca2+ influx through voltage-dependent Ca2+ channels. The Ca2+ channels of the two cell types were activated at different membrane potentials but at the same [K+]o. In both cell types, the recovery from a K(+)-induced [Ca2+]i increase was unaltered in Na(+)-free solution, indicating that active Ca2+ transport across the plasma membrane is mediated by Na(+)-independent mechanisms.

ATP-inhibited K+ channels and membrane potential of identified leech neurons.

The effect of the ATP-inhibited K+ channel on the membrane potential of leech Retzius neurons was analyzed using electrolyte-filled single-barrelled microelectrodes. The membrane potential was independent of the external nutrient supply during a period of 11 h, probably because the internal energy reserves were sufficient. The K+ channel activator HOE 234 ((3S,4R)-3-hydroxy-2, 2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6-phenylsulfonylchromane hemihydrate, 500 microM) induced a membrane hyperpolarization. In the presence of HOE 234, action potentials occurred with a reduced after-hyperpolarization and were discharged in bursts, possibly because of an inhibition of Ca2+ channels. The blocker of ATP-inhibited K+ channels tolbutamide did not significantly alter the membrane potential. In the absence of tolbutamide, the metabolic inhibitors iodoacetate, azide and cyanide (10 mM) evoked membrane hyperpolarizations, but in the presence of 1 mM tolbutamide their hyperpolarizing actions were reduced or abolished while membrane depolarizations were intensified. We conclude that ATP-inhibited K+ channels in the soma membrane of leech Retzius neurons provide coupling of cellular metabolism to electrical activity and ionic fluxes.

Effects of glutamatergic agonists and antagonists on membrane potential and intracellular Na+ activity of leech glial and nerve cells.

The membrane potential of neuropile glial cells and Retzius neurones in the central nervous system of the leech Hirudo medicinalis was measured using electrolyte-filled single-barreled microelectrodes. Intracellular Na+ activity (aNai) was recorded with Na(+)-sensitive double-barreled microelectrodes. Bath-application of kainate, quisqualate and L-glutamate elicited concentration-dependent membrane depolarizations in both cell types as demonstrated by dose-response curves. The competitive quinoxalinedione antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the non-NMDA glutamate receptor inhibited the membrane depolarizations in neuropile glial cells completely, but in Retzius neurones only partially. These results confirm that leech neuropile glial cells have a kainate- and quisqualate-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones. The initial decrease in aNai in neuropile glial cells in kainate- or quisqualate-containing solutions and the afterhyperpolarization in these glial cells and the Retzius neurones following the removal of both glutamate antagonists, were blocked in the presence of the cardiac glycoside ouabain (10(-4) M). In saline solutions containing 42.5 mM Li+ instead of Na+ the afterhyperpolarizations were blocked in neuropile glial cells and Retzius neurones. We conclude that the initial aNai changes and the afterhyperpolarization could be due to the stimulation of the electrogenic Na+/K+ pump in the glial and neuronal membranes.

Voltage-dependent Ca2+ influx into identified leech neurones.

We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K+ concentration ([K+]o) up to 89 mM caused a persistent increase in [Ca2+]i, which was abolished in Ca(2+)-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between -40 and -25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and EM were partly due to the cell-specific generation of action potentials. In Na(+)-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K(+)-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K(+)-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca2+ channels with similar properties.

Self-sustained spreading depressions in the chicken retina and short-term neuronal-glial interactions within the gray matter neuropil.

The chicken retina is an accessible piece of intact gray matter in which a self-sustained form of the 'Spreading Depression' (SD) wave can be easily elicited and recorded for many hours with double barrel ion-sensitive electrodes in the extracellular space. The blockade of glial (Müller) cell potassium channels with barium chloride added to the perfusing Ringer depressed both the negative potential shift typical of SDs and the velocity of spread. Moreover, there was separation of the extracellular increase of potassium and the drop in the extracellular potential: the peak of the potassium wave was increased, as well as its duration whereas the potential wave could be depressed to zero or even inverted to positive. By contrast the transient extracellular calcium drop could not be separated from the extracellular potential wave but appeared related to it: no transient calcium drop was observed when the negative potential was completely depressed or inverted. Both, the amplitude of the extracellular potential and extracellular calcium activity appeared to be important factors controlling the velocity of spread.