Neuron-glia interactions: Indirect effect of GABA on cultured glial cells

Summary A study was made of the action of GABA on the membrane potential and resistance of satellite glial (SG) cells in cultures of rat dorsal root ganglia. GABA (10^-4M) depolarized all SG cells tested without producing significant changes in membrane resistance. Similar results were obtained from astrocytes of cultured rat spinal cord and brain stem, although only half of the cells tested were depolarized by GABA. Bicuculline (10^-5 and 10^-6M) which blocked the GABA-depolarization on cultured dorsal root ganglion (DRG) neurons, also markedly reduced or blocked the action of GABA on SG cells. When GABA was tested in sodium-free bathing solution, the amino acid caused a depolarization of similar shape and amplitude as in normal (137 mM Na⁺) bathing fluid, indicating that uptake processes are probably not involved in producing the depolarization by GABA.It is suggested that the depolarizing action of GABA on glial cells is an indirect effect due to the release of potassium from adjacent neurons during the action of the amino acid.     

Aspartate, glutamate and gamma-aminobutyric acid depolarize cultured astrocytes

Cultures of differentiated, glial fibrillary acidic protein-positive astrocytes from early postnatal rat cerebral hemispheres respond with depolarization of 2-36 mV to glutamate, gamma-aminobutyric acid (GABA) and aspartate but not to glycine or taurine. While GABA resulted in a transient depolarization, the effect of glutamate and aspartate persisted during the application. Since neurons were not present in these cultures a contribution of transmitter-mediated K+ release from adjacent neurons could be excluded. The depolarization triggered by these neurotransmitters is therefore an intrinsic reaction of astrocytes.     

Persisting modification of dendritic calcium influx by excitatory amino acid stimulation in isolated Ca1 neurons.

Spatiotemporal changes of the intracellular calcium ion (Ca2+) were recorded by digital ratio imaging of fura-2 in pyramidal neurons acutely isolated from the adult guinea-pig hippocampus. Increases in calcium were evoked in tetrodotoxin (2 microM) containing saline either by stimulation with the excitatory amino acids, glutamate or N-methyl-D-aspartate, or by depolarization with high potassium (50 mM). Local stimulation with excitatory amino acids, applied from a microelectrode with 1-2-s iontophoretic pulses at the dendrite, induced a rapid increase in intracellular Ca2+ predominantly supported by a Ca2+ influx at the site of stimulation (primary response). Ca2+ levels recovered within 1-2 min in about one-half of the neurons examined. In the remaining neurons the initial exposure to excitatory amino acids induced a non-recovering gradient of Ca2+, highest at the site of stimulation, that lasted for periods of minutes (secondary response). Within the population that showed recovery from the initial agonist exposure, a second, or in some cases, a third application triggered the sustained, secondary response. Pretreatment of neurons with the protein kinase inhibitor sphingosine (10 microM) blocked development of the secondary response but had no effect on the primary response to the excitatory amino acids. There were no Ca2+ increases in Ca(2+)-free medium with either agonist, and responses to N-methyl-D-aspartate were blocked by 2-amino-4-phosphovaleric acid and significantly reduced at physiological concentrations of Mg2+ (1.8 mM). The maintained gradient of Ca2+ was supported by a continuous influx of calcium from outside the cell. In contrast, dendritic gradients of Ca2+ induced by short exposures to high potassium (50 mM, 5 s) collapsed immediately at the end of the stimulus and could be repeatedly evoked. Minute-long exposures to high K, induced large, repeatable changes in Ca2+ but there was always rapid recovery in normal saline. K depolarization applied after excitatory amino acid stimulation produced larger Ca2+ changes than the same K stimulus applied before the cell was stimulated with the excitatory amino acid. Bath application of GABA (10-100 microM) reduced the magnitude of the maintained Ca2+ gradients. The functional significance of the extended, secondary response cannot be directly established from these measurements on isolated neurons, but its properties could give rise, in part, to mechanisms involved in neural plasticity, in kindling epileptogenesis or in glutamate-induced toxicity.     

86Rubidium release from cultured primary astrocytes: effects of excitatory and inhibitory amino acids

The effects of high K+, glutamate and its analogue, kainate, on K+ release were studied in primary astrocyte cultures prepared from newborn rat brains using 86Rb+ as a tracer for K+. An increase in 86Rb+ release was observed when the extracellular K+ concentration was elevated (10-40 mM). Glutamate and kainate stimulated the release in a dose-dependent manner, 100 microM concentrations being about as equally effective as high K+ (40 mM). Both compounds also caused an increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine, indicating depolarization of the membrane. No significant Na+-dependent uptake of [3H]kainate occurred in the cells, thus excluding the possibility that depolarization was due to electrogenic uptake of amino acid into the cells. GABA and taurine significantly depressed the high K+- and glutamate-induced 86Rb+ release. Taurine itself caused a small increase in 86Rb+ release and the membrane was depolarized, judging from the increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine. No effect of taurine was observed when the Cl- concentration was reduced in the experimental medium. The results suggest that cultured astrocytes respond by membrane depolarization to high external K+ and to glutamate and kainate. The degree of this depolarization can be modified by the inhibitory amino acids GABA, taurine and glycine, the effect of taurine probably being mediated by an increase in Cl- conductance across the cell membrane. The role of functional receptors for amino acid transmitters and the effects observed are discussed.     

Kainic acid evokes a potassium efflux from astrocytes

Cultured astrocytes are depolarized by excitatory amino acids such as kainic acid. In these experiments we tested the hypothesis that the kainic acid-induced depolarization also causes an efflux of K+ from astrocytes in the rat. Using K+-sensitive microelectrodes we measured a K+ efflux from cultured astrocytes during the perfusion of kainic acid. The effects of kainic acid were then tested on glial cells in the neuron-free CA3 region of kainic acid-lesioned hippocampus. Glial cells were depolarized by kainic acid and an efflux of K+ was recorded. These effects are most likely due to direct effects on the glial cells because histological examination of the hippocampus showed this area to be free of neurons. Therefore it is hypothesized that kainic acid and any transmitter that depolarizes glial cells, will increase neuronal excitability indirectly by a K+ efflux from glial cells. This will have widespread implications for iontophoretic studies of transmitter actions.     

GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways

GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca2+ concentration of guinea-pig myenteric neurons (at 35 degrees C) using Fura-2-AM. Neurons were identified by 75 mM K+ depolarization (5 s), which evoked a transient intracellular Ca2+ concentration increase. GABA (10 s) induced a dose dependent (5 nM-1 microM) transient intracellular Ca2+ concentration rise in the majority of neurons (500 nM GABA: 251+/-17 nM, n=232/289). Interestingly, the response to 5 microM GABA (n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly (P<0.001) reduced by GABAA and GABAB receptor antagonists (10 microM) bicuculline and phaclofen. The GABAA agonist isoguvacine (10 microM) and GABAB agonist baclofen (10 microM) induced similar responses as 50 nM GABA, while the GABAC agonist cis-4-aminocrotonic acid (CACA) (10 microM) only elicited small responses in a minority of neurons. Removal of extracellular Ca2+ abolished all responses while depletion of intracellular Ca2+ stores by thapsigargin (5 microM) did not alter the responses to 500 nM GABA (n=13), but reduction of Ca2+ influx through voltage-dependent Ca2+ channels did. The nicotinic antagonist hexamethonium (100 microM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and the 5-HT3 receptor blocker ondansetron only had minor effects. Conclusion: GABA elicits transient intracellular Ca2+ concentration responses in the majority of myenteric neurons through activation of GABAA and GABAB receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5 microM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.     

Studies of axon-glial cell interactions and periaxonal K- homeostasis--I. The influence of Na+, K+, Cl- and cholinergic agents on the membrane potential of the adaxonal glia of the crayfish medial giant axon

The ionic basis for the low (-40 mV) resting membrane potential of glial cells surrounding the giant axons of the crayfish and their hyperpolarization by cholinergic agents (to -55 mV) was studied using standard electrophysiological techniques, ionic substitutions and pharmacological agents. The resting membrane potential of the glial cell was depolarized by increasing [K+]o, but the response was not Nernstian. Na+ depletion caused a small depolarization of the glial resting membrane potential, whereas Cl- depletion resulted in a hyperpolarization comparable to that seen with carbachol at various [K+]o. Both furosemide (1 mM) and bumetanide (0.1 mM) produced an 8-10 mV hyperpolarization as compared to 15-17 mV seen with Cl- depletion or carbachol. Carbachol has no further effect on the potential following furosemide treatment or Cl- depletion. After carbachol administration or Cl- depletion the resting membrane potential of the glial cell responded to [K+]o in a more Nernstian manner. The data indicate that the low resting membrane potential of glial cells is due to a combination of a low [K+]i and an outwardly-directed (depolarizing) Cl- electrochemical gradient. Carbachol acts to decrease Cl- conductance, resulting in the hyperpolarization of the glial cell membrane and a decrease in the outwardly-directed K+ electrochemical gradient by approximately two-thirds. We hypothesize that this mechanism for modulation of the glial cell membrane potential and the K+ electrochemical gradient serves to enhance the uptake of K+ by the glial cell transport system.     

Effects of bicarbonate on glial cell membrane potential in Necturus optic nerve

Intracellular electrodes were used to continuously monitor the membrane potential of glial cells in the isolated Necturus optic nerve. Addition of up to 10 mM extracellular bicarbonate (with CO2), at constant pH, produced a hyperpolarization of up to 10 mV (with a time course almost as fast as that of a K+ depolarization) that returned toward baseline during the following 2-15 min. Upon bicarbonate withdrawal, the potential transiently became more positive. The bicarbonate effects were magnified when the K+ conductance was decreased and the cell depolarized by the addition of barium. Similar bicarbonate effects were observed in Cl- free solutions. These results suggest to us that: glial cells have a bicarbonate permeability of the same order as that to K+ and glial cells buffer transient changes in acid base balance in the neuronal microenvironment at the expense of their internal pH.     

Action of baclofen, GABA and antagonists on the membrane potential of cultured astrocytes of rat spinal cord

The action of gamma-aminobutyric acid A (GABAA) and B (GABAB)-agonists has been studied on the membrane potential of astrocytes in explant cultures of rat spinal cord by means of intracellular microelectrode recordings. The GABAB-agonists (-)-baclofen and CGP 27 492 (3-aminopropyl phosphonous acid; 10(-6) to 10(-4) M) caused a hyperpolarization of the majority of astrocytes studied. On approximately 25% of the cells, the compounds had no effect. The hyperpolarization by baclofen (10(-4) M) was reversibly antagonized by the GABAB-antagonist 5-hydroxysaclofen (10(-4) M). GABA and the GABAA-agonist muscimol (10(-4) and 10(-3) M) depolarized approximately two thirds of the glial cells tested, whereas the remaining third remained unaffected. The GABAA-antagonist bicuculline (10(-4) and 10(-3) M) only reduced the depolarization by GABA (10(-4) M) but did not completely block it. On half of the cells tested, the depolarization by GABA was not affected by bicuculline, suggesting that the glial GABAA-receptor is different from the neuronal GABAA-receptor. Our electrophysiological investigations together with recent autoradiographic binding studies strongly suggest the existence of GABAB-receptors on astrocytes whereas there is less evidence for GABAA-sites on these cells.     

Dihydropyridines modulate K+-evoked amino acid and adenosine release from cerebellar neuronal cultures

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, gamma-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 microM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.     

Na(+) and K(+) concentrations, extra- and intracellular voltages, and the effect of TTX in hypoxic rat hippocampal slices

Severe hypoxia causes rapid depolarization of CA1 neurons and glial cells that resembles spreading depression (SD). In brain slices in vitro, the SD-like depolarization and the associated irreversible loss of function can be postponed, but not prevented, by blockade of Na(+) currents by tetrodotoxin (TTX). To investigate the role of Na(+) flux, we made recordings from the CA1 region in hippocampal slices in the presence and absence of TTX. We measured membrane changes in single CA1 pyramidal neurons simultaneously with extracellular DC potential (V(o)) and either extracellular [K(+)] or [Na(+)]; alternatively, we simultaneously recorded [Na(+)](o), [K(+)](o), and V(o). Confirming previous reports, early during hypoxia, before SD onset, [K(+)](o) began to rise, whereas [Na(+)](o) still remained normal and V(o) showed a slight, gradual, negative shift; neurons first hyperpolarized and then began to gradually depolarize. The SD-like abrupt negative DeltaV(o) corresponded to a near complete depolarization of pyramidal neurons and an 89% decrease in input resistance. [K(+)](o) increased by 47 mM and [Na(+)](o) dropped by 91 mM. Changes in intracellular Na(+) and K(+) concentrations, estimated on the basis of the measured extracellular ion levels and the relative volume fractions of the neuronal, glial, and extracellular compartment, were much more moderate. Because [Na(+)](o) dropped more than [K(+)](o) increased, simple exchange of Na(+) for K(+) cannot account for these ionic changes. The apparent imbalance of charge could be made up by Cl(-) influx into neurons paralleling Na(+) flux and release of Mg(2+) from cells. The hypoxia-induced changes in interneurons resembled those observed in pyramidal neurons. Astrocytes responded with an initial slow depolarization as [K(+)](o) rose. It was followed by a rapid but incomplete depolarization as soon as SD occurred, which could be accounted for by the reduced ratio, [K(+)](i)/[K(+)](o). TTX (1 microM) markedly postponed SD, but the SD-related changes in [K(+)](o) and [Na(+)](o) were only reduced by 23 and 12%, respectively. In TTX-treated pyramidal neurons, the delayed SD-like depolarization took off from a more positive level, but the final depolarized intracellular potential and input resistance were not different from control. We conclude that TTX-sensitive channels mediate only a fraction of the Na(+) influx, and that some of the K(+) is released in exchange for Na(+). Even though TTX-sensitive Na(+) currents are not essential for the self-regenerative membrane changes during hypoxic SD, in control solutions their activation may trigger the transition from gradual to rapid depolarization of neurons, thereby synchronizing the SD-like event.     

Action of GABA on neurones and satellite glial cells of cultured rat dorsal root ganglia

The action of GABA was tested on the membrane potential of neurones and satellite glial cells of rat dorsal root ganglia (DRG) in tissue culture. GABA added to the bathing fluid at concentrations of 10^?4 and 10^?5 M caused a depolarization of neurones which was blocked by bicuculline (10^?5 and 10^?6 M). The depolarizing action of GABA clearly showed signs of receptor desensitization. Similarly, GABA caused a depolarization of satellite glial (SG) cells which also revealed desenitization was blocked by bicuculline.     

Membrane properties underlying patterns of GABA-dependent action potentials in developing mouse hypothalamic neurons

Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons (n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2-9 (P2-9) mice. With conventional patch pipettes (containing 29 mM Cl-), action potentials were also elicited by GABA from neurons of 2-13 days in vitro (2-13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 microM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 microM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd(2+) (200 microM) and Ni(2+) (300 microM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl-] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl-]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current (E(GABA)) was positive to the resting membrane potential, suggesting a high intracellular [Cl-] in developing mouse neurons. Varying the holding potential from -80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca(2+) from the extracellular solution did not block spikes, indicating GABA-evoked Na+ -based spikes. Although E(GABA) was more positive within 2-5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the E(GABA) is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl-, not bicarbonate, was primarily responsible for generating multiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.     

Depolarization of cultured astrocytes by glutamate and aspartate

The excitatory transmitter substances glutamate and aspartate are known to have a depolarizing action on cultured CNS neurones, the depolarization being associated with an increase in membrane conductance. When the effects of these amino acids (at a concentration of 10^?4 M) were studied on the membrane potential and resistance of cultured glial cells, they also caused a depolarization of many astrocytes but without producing significant changes in membrane resistance. The majority of glial cells depolarized by glutamate and aspartate were lying in the vicinity of neurones in the dense zone of the cultures, whereas isolated astrocytes in the outgrowth zone were usually not affected by the amino acids. 4-Aminopyridine (5 mM), a substance known to block K⁺-conductance in various excitable membranes, reversibly reduced or abolished the depolarization caused by glutamate and aspartate on glial cells, but had no or only a small effect on the depolarization of neurones caused by these amino acids.These results suggest that the depolarization of glial cells by glutamate and aspartate is caused by an increase in the concentration of extracellular K⁺ which is released from neighbouring neurones during their activation by the amino acids.     

Some functional consequences of GABA uptake by brain cells

Recent electrophysiological studies on the rat hippocampus (in vivo and in vitro) provide further evidence that neuronal and glial uptake of the inhibitory transmitter gamma-aminobutyric acid (GABA) limits the intensity and the duration of effects not only of locally applied exogenous GABA but also of GABAergic inhibitory synaptic potentials (IPSPs). There is good reason to believe that such uptake is at least partly responsible for the 'fading' of GABA action. Moreover, because it is probably driven by the transmembrane Na+ electrochemical gradient and is accompanied by Na+ influx, GABA uptake is potentially electrogenic and therefore may have a depolarizing effect on both neurons and glia.     

Contributions of voltage- and Ca2+-activated conductances to GABA-induced depolarization in spider mechanosensory neurons

Activation of ionotropic gamma-aminobutyric acid type A (GABA(A)) receptors depolarizes neurons that have high intracellular [Cl(-)], causing inhibition or excitation in different cell types. The depolarization often leads to inactivation of voltage-gated Na channels, but additional ionic mechanisms may also be affected. Previously, a simulated model of spider VS-3 mechanosensory neurons suggested that although voltage-activated Na(+) current is partially inactivated during GABA-induced depolarization, a slowly activating and inactivating component remains and may contribute to the depolarization. Here, we confirmed experimentally, by blocking Na channels prior to GABA application, that Na(+) current contributes to GABA-induced depolarization in VS-3 neurons. Ratiometric Ca(2+) imaging experiments combined with intracellular recordings revealed a significant increase in intracellular [Ca(2+)] when GABA(A) receptors were activated, synchronous with the depolarization and probably due to Ca(2+) influx via low-voltage-activated (LVA) Ca channels. In contrast, GABA(B)-receptor activation in these neurons was previously shown to inhibit LVA current. Blockade of voltage-gated K channels delayed membrane repolarization, extending GABA-induced depolarization. However, inhibition of Ca channels significantly increased the amplitude of GABA-induced depolarization, indicating that Ca(2+)-activated K(+) current has an even stronger repolarizing effect. Regulation of intracellular [Ca(2+)] is important for many cellular processes and Ca(2+) control of K(+) currents may be particularly important for some functions of mechanosensory neurons, such as frequency tuning. These data show that GABA(A)-receptor activation participates in this regulation.     

The release of (3H)-GABA from the rabbit retina

The neuronal and glial release of (3H)-GABA from rabbit retina has been studied. The results indicate, that neither are there any glutamate, aspartate or glycine receptors on the GABA accumulating neurons, nor any GABA autoreceptors. (3H)-GABA was found to be released by 40 mM K+ from retinal neurons, but not from glia, and the release was not dependent on extracellular Ca++. This indicates a release from a non vesicular transmitter pool. Ouabain has been proposed as a pharmacological tool for studying the release of (3H)-GABA located in neuronal cytoplasm. However, it induced an increased release of (3H)-GABA from both neurons and glia and it is therefore unlikely that it can be used for the specific purpose of studying release from neuronal cytoplasm.     

Role of GABAA and GABAC receptors in the biphasic GABA responses in neurons of the rat major pelvic ganglia.

The role of gamma-aminobutyric acid-A (GABAA) and GABAC receptors in the GABA-induced biphasic response in neurons of the rat major pelvic ganglia (MPG) were examined in vitro. Application of GABA (100 microM) to MPG neurons produced a biphasic response, an initial depolarization (GABAd) followed by a hyperpolarization (GABAh). The input resistance of the MPG neurons was decreased during the GABAd, whereas it was increased during the GABAh. The GABAd could be further separated into the early component (early GABAd) with a duration of 27 +/- 5 s (mean +/- SE; n = 11) and the late component (late GABAd) with a duration of 109 +/- 11 s (n = 11). The duration of the GABAh was 516 +/- 64 s (n = 11). The effects of GABA (5-500 microM) in producing the depolarization and the hyperpolarization were concentration-dependent. GABA (5-30 microM) induced only late depolarizations. The early component of the depolarization appeared when the concentration of GABA was >50 microM. Muscimol produced only early depolarizing responses. Baclofen (100 microM) had no effect on the membrane potential and input resistance of MPG neurons. Bicuculline (60 microM) blocked the early GABAd but not the late GABAd and the GABAh. Application of picrotoxin (100 microM) with bicuculline (60 microM) blocked both the late GABAd and the GABAh. CGP55845A (3 microM), a selective GABAB receptor antagonist, did not affect the GABA-induced responses. cis-4-Aminocrotonic acid (CACA, 1 mM) and trans-4-aminocrotonic acid (TACA, 1 mM), selective GABAC receptor agonists, produced late biphasic responses in the MPG neurons. The duration of the CACA responses was almost the same as those of the late GABAd and GABAh obtained in the presence of bicuculline. Imidazole-4-acetic acid (I4AA, 100 microM), a GABAC receptor antagonist, depressed the late GABAd and the GABAh but not the early GABAd. I4AA (100 microM) and picrotoxin (100 microM) also suppressed the biphasic response to CACA. The early GABAd and the late GABAd were reversed in polarity at -32 +/- 3 mV (n = 7) and -38 +/- 2 mV (n = 4), respectively, in the Krebs solution. The reversal potential of the GABAh was -34 +/- 2 mV (n = 4) in the Krebs solution. The reversal potentials of the late GABAd and the GABAh shifted to -20 +/- 3 mV (n = 5) and -22 +/- 3 mV (n = 5), respectively, in 85 mM Cl- solution. These results indicate that the late GABA(d) and the GABAh are mediated predominantly by bicuculline-insensitive, picrotoxin-sensitive GABA receptors, GABAC (or GABAAOr) receptors, in neurons of the rat MPG.     

GABA antagonists increase the release of [3H]acetylcholine from the chick retina

The release of [3H]acetylcholine from the chick retina was studied. A 5 mM increase in K+-concentration caused an increased release, which was Ca2+-dependent. The effect of 5 mM K+ was neither potentiated by bicuculline nor inhibited by isoguvacine or muscimol. This indicates that the K+-induced release is not controlled by GABA. However, bicuculline and picrotoxin increased the spontaneous efflux of radioactivity, whereas GABA had no significant effect. The results suggest that cholinergic neurons are tonically inhibited by a continuous release of endogenous GABA. Neither glycine or strychnine, nor dopamine or haloperidol had any effect on the spontaneous release.     

Effects of chloride transport inhibition and chloride substitution on neuron function and on hypoxic spreading-depression-like depolarization in rat hippocampal slices

Chloride fluxes play a crucial role in synaptic inhibition, cell pH regulation, as well as in cell volume control. In many neuropathological processes, cell swelling is a pivotal parameter, since cell volume changes and the dimension of the interstitial space critically modulate synchronized neuronal activity as well as the tissue's susceptibility to seizures or spreading depression. This study therefore focuses on the effects of different Cl(-) transport inhibitors and Cl(-) substitution on neuronal function and hypoxia-induced changes in rat hippocampal tissue slices. Orthodromically evoked focal excitatory postsynaptic potentials were depressed by furosemide (2mM), 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (1mM) and Cl(-) substitution by methylsulfate, but were enhanced by 4,4'-dinitrostilbene-2,2'-disulfonic acid (1mM). All four treatments induced multiple population spike firing in response to single orthodromic volleys, suggesting reduced synaptic inhibition. Antidromic population spikes increased following Cl(-) withdrawal, were unaffected in the presence of furosemide and 4, 4'-dinitrostilbene-2,2'-disulfonic acid, but were abolished by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The amplitude of the hypoxic spreading-depression-like extracellular potential shift was reduced by furosemide, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and Cl(-) withdrawal, i.e. by the same treatments that depressed orthodromically evoked postsynaptic potentials. Furosemide prolonged the time to onset and the duration of the spreading-depression-like extracellular potential shift, while 4, 4'-dinitrostilbene-2,2'-disulfonic acid shortened the time to onset. Spreading-depression-related cell swelling was recorded as the shrinkage of relative interstitial space, which was measured as tetramethylammonium-chloride space. Neither the Cl(-) transport inhibitors nor Cl(-) withdrawal had any detectable effect on spreading-depression-related cell swelling. CA1 pyramidal neurons usually hyperpolarized during drug application and their input resistance decreased. Cl(-) withdrawal increased their input resistance and caused spontaneous burst firing. Hypoxia caused the expected spreading-depression-like rapid, near complete depolarization of single pyramidal neurons and drastically reduced their input resistance. The three Cl(-) transport inhibitors and Cl(-) withdrawal delayed the onset of the hypoxic depolarization. In low Cl(-) solutions, the apparent threshold potential at which spreading depression was triggered shifted to more positive membrane potentials. The final voltage of the hypoxic depolarization was, however, not affected.It appears from these results that the reduction in the hypoxic spreading-depression-like extracellular potential shifts by Cl(-) transport inhibitors is at least partially attributable to desynchronization of depolarization, not to decreased depolarization in individual cells. Other contributing factors could be changes in recording conditions, depression of swelling-induced amino acid release from glial cells and unspecific side-effects of the applied drugs. Desynchronization could also account for the delayed spreading-depression onset. It is concluded that Cl(-) fluxes play a role in the triggering of spreading depression, but the spreading-depression-like depolarization itself or its self-regenerative character is not mediated by Cl(-).