[Show abstract][Hide abstract] ABSTRACT: Postsynaptic GABAA-responses of cortical neurones may be composed of an early 'somatic' hyperpolarization (h-GABAA) and a 'dendritic' depolarization (d-GABAA). In order to study underlying anion-fluxes the gradient of bicarbonate across the membrane of CA3-neurones in hippocampal slices (guinea-pig) was reduced by several manoeuvres (NH4-prepulse-technique, CO2/bicarbonate-withdrawal-technique, acetazolamide, DIDS). Each manoeuvre resulted in an attenuation of d-GABAA. The inhibition of a Cl-/bicarbonate-exchanger with DIDS could also weaken h-GABAA which was selectively impaired by ethacrynic acid--known to block an ATP-driven somatic Cl(-)-extrusion mechanism. In conclusion, bicarbonate-fluxes contribute to d-GABAA rather than to h-GABAA which is mainly driven by Cl(-)-fluxes. An uneven distribution of Cl(-)-extrusion mechanisms along the adult CA3-neurones is likely to build up the anion-gradients necessary to generate biphasic GABAA-responses.
[Show abstract][Hide abstract] ABSTRACT: In order to analyze the epileptogenic mechanisms of caffeine and related xanthines, putative effects of these drugs were studied on adenosine receptors of CA3 neurons in hippocampal slices. Epileptogenic concentrations of different xanthine derivatives strongly correlated with their affinities for the inhibitory A1 adenosine receptor subtype. The A1 receptor agonists adenosine and R-PIA reversibly depressed xanthine-induced epileptic activity without effects on the resting membrane potential or on spontaneously occurring action potentials. These findings suggest that the epileptogenic potency of xanthines is primarily due to the blockade of the A1 receptors through an abnormal rise of intracellular cAMP and to the excessive transmembrane calcium fluxes underlying paroxysmal depolarization shifts.
Brain Research 04/1994; 640(1-2):140-5. DOI:10.1016/0006-8993(94)91868-6 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epileptogenic actions of convulsants are often attributed to weakened inhibitory synaptic mechanisms. This assumption was tested by studying GABA-induced postsynaptic membrane potential (MP) changes of CA3 neurones (guinea-pig) before and during exposure to bicuculline methoiodide (BMI), pentylenetetrazol (PTZ), penicillin (PEN) and caffeine (CAF). Under control conditions GABA ejections elicited polyphasic MP fluctuations (components I-III). After adding BMI, PTZ, PEN or CAF, early hyperpolarizations (component I) did not change at epileptogenic threshold concentrations. These convulsants, however, exerted differential effects on the depolarizing component II, but only threshold concentrations of penicillin strongly reduced the amplitude of this component. Simultaneously, component III was slightly accentuated. These findings indicate that changes of GABA responses are not an essential prerequisite for the generation of paroxysmal depolarizations.
[Show abstract][Hide abstract] ABSTRACT: Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cells in vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4-5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (pCO2 > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca2+ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.
Calcified Tissue International 07/1993; 53(1):53-60. DOI:10.1007/BF01352015 · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carbamazepine (CBZ) is known to have beneficial effects in the treatment of epilepsies and in the prophylaxis of affective disorders. Since increased transmembrane calcium fluxes and intracellular calcium concentrations play a key role in the generation of epilepsies and possibly also in the development of these psychiatric disorders the effects of CBZ on epileptic discharges (elicited by caffeine, penicillin and low Mg2+) in CA3 neurons of hippocampal slices were compared with those of the organic calcium antagonist verapamil and found to be almost the same.
[Show abstract][Hide abstract] ABSTRACT: The effects of carbamazepine (CBZ) on action potentials and calcium currents in cultured rat sensory spinal ganglion cells were investigated. CBZ was found to reversibly suppress the calcium-dependent components of action potentials and to reduce the amplitude of the after-hyperpolarizations, while the rising phase and the peak amplitude were hardly changed. Furthermore, CBZ caused a marked reduction in the calcium currents, which in some cells was reversible. The present findings confirm that CBZ has calcium-antagonistic properties.
[Show abstract][Hide abstract] ABSTRACT: In guinea pig hippocampal slices, relations between morphology and spontaneous bioelectric activity of neurons were studied in control saline and with exposure to the epileptogenic drug pentylenetetrazole (PTZ) for 2-3 h. Light and electron microscopic structures of the CA3 region were analysed after recording the membrane potential. Neurons in slices kept in control saline exhibited spontaneous aperiodic bioelectric activities partly mixed with rhythmically occurring burst discharges. In slices exposed to PTZ, these periodic burst discharges and/or paroxysmal depolarization shifts (PDS) predominated. Light microscopic comparison focussing on tissue preservation showed no significant differences between control and PTZ-treated slices. Ultrastructural morphology revealed, on the one hand, no differences regarding spine and synaptic densities, and on the other hand, significantly more irregular electron translucent vacuoles within dendrites of PTZ-treated slices being either randomly distributed or clustered. The vacuoles are interpreted as early changes during epileptic activity.
Brain Research Bulletin 02/1993; 32(4):329-38. DOI:10.1016/0361-9230(93)90196-I · 2.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carbamazepine (CBZ) is used in neurology for the treatment of epilepsies and trigeminal neuralgia and in psychiatry for the prophylactic treatment of affective and schizoaffective psychoses. Since a common mechanism of epilepsies and affective psychoses might be increased intracellular calcium ion levels, CBZ action was analyzed in penicillin, caffeine and low Mg2+ induced model epilepsies which have been shown to be suppressed specifically by organic calcium antagonists. In CA3 and CA1 areas of hippocampal slice preparations of guinea pigs CBZ reduced paroxysmal depolarizations and extracellular field potentials (EFP) in a typical time and concentration dependent manner as it is known from calcium antagonists. Furthermore, subthreshold concentrations of the organic calcium antagonist verapamil intensified the action of CBZ. NMDA induced increases of the discharge rate of EFP were, however, unaffected by CBZ.
European Neuropsychopharmacology 01/1993; 2(4):455-62. DOI:10.1016/0924-977X(92)90009-W · 4.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4-5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.
Calcified Tissue International 11/1992; 51(4):285-90. DOI:10.1007/BF00334489 · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Electrophysiological studies were carried out on long term cultured brainstem tissue taken from neonatal rats with the object of investigating mechanisms underlying respiratory rhythm generation. The preparations were derived from 360 microns thick horizontal medullary slices which were explanted into a chemically defined nutrient medium and which remained organotypically intact for ca. 1 month. In 44 of the 50 explants examined both periodic and aperiodic bioelectric activity was detected, the cycle length of the former ranging from 0.5 to 10 s (mean, 2.7 s) at a pH of 7.4 and bath temperature of 32 degrees C. Periodic activity could take several forms, but commonly consisted of regularly repeated, 100-300 ms long, depolarizing (D-) waves or sequences of inhibitory and/or excitatory postsynaptic potentials. Lowering the pH of the superfusate by lowering the bicarbonate concentration, increasing the pCO2 or adding H+ shortened the interval between periodic events, and increased both the amplitude and duration of the D-waves. The interval was also shortened when the bath temperature was increased (Q10: ca.2.5). The mean resting membrane potential of neurons exhibiting periodic activity was -49 mV (n = 62) and not significantly different from that of aperiodically discharging neurons either in the same preparations or in cultured explants from the neocortex. These observations suggest that brainstem cultures constitute a useful 'model' system for studying pH-dependent rhythm generation in small neuronal networks of the medulla.
[Show abstract][Hide abstract] ABSTRACT: Long-term cultures of organotypic neonatal rat neocortex slices were maintained in a serum-free medium supplemented with 25 mM potassium. Such cultures continue to be bioelectrically silent upon return to a 5 mM potassium medium. The absence of responses to pressure ejected gamma-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) from neurons in treated explants suggests that one consequence of chronic depolarization is a reduction in the density of postsynaptic transmitter receptors. [3H]Muscimol binding to neocortical membrane preparations shows a large reduction in the binding of this agonist to GABAA receptors. These data show that the quantitative expression of at least one neurotransmitter receptor, the GABAA receptor, relies on voltage-dependent activity in developing neocortical neurons in vitro.
[Show abstract][Hide abstract] ABSTRACT: In order to analyze the elementary mechanisms underlying caffeine-induced epileptiform discharges, hippocampal slices of guinea pigs were exposed to this drug. When the bath concentration of caffeine exceeded 0.2 mM, periodically occurring paroxysmal depolarizations (PD) in CA3 neurons appeared. They were accompanied by declines of extracellular free calcium concentration and were suppressed by the organic calcium antagonists verapamil and flunarizine. PD-like fluctuations of the membrane potential could be evoked also in CA3 neurons functionally isolated by tetrodotoxin (TTX). The observations indicate that caffeine-induced PD are generated endogenously and that transmembranous calcium currents contribute to these mechanisms.
[Show abstract][Hide abstract] ABSTRACT: In order to assess the effect of elevated magnesium, neuronal morphology and physiology was studied in chronically cultured organotypic neonatal rat occipital neocortex. Explants grown in 10 mM magnesium were found to experience an approximate 30% cell loss (as shown by cell count and DNA-protein analysis), while 12.5 and 15 mM magnesium showed ca. 47 and 60% cell losses, respectively. Intracellular recording from 10 mM magnesium explants revealed that measurable postsynaptic potentials and action potentials could occur, apparently depending on the type of cell examined. All post-synaptic activities ceased in 12.5 mM magnesium cultures, though action potentials could be elicited by current stimulation. The effects of known depolarizing agents, viz. potassium and N-methyl-D-aspartate, on 12.5 mM magnesium-grown explants were also examined. Explants grown in the presence of 12.5 mM magnesium plus 10 mM potassium showed a dramatic increase in the loss of neurons. The simultaneous addition of 6,7-dinitro-quinoxaline-2,3-dione showed this to be due to an increase in non-N-methyl-D-aspartate mediated cell death in response to glutamate release brought about by the depolarizing effects of the potassium. The addition of 10 microM N-methyl-D-aspartate to 12.5 mM magnesium-grown cultures, on the other hand, improved cell survival to control levels. The mechanism of this reciprocal neuroprotective effect of N-methyl-D-aspartate against magnesium has yet to be elucidated. We conclude that these findings are consistent with regard to the opposing actions of N-methyl-D-aspartate and magnesium on calcium influx and various metabolic processes within the explants.
International Journal of Developmental Neuroscience 02/1991; 9(6):597-606. DOI:10.1016/0736-5748(91)90021-D · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic depolarization is inimical to neuronal growth and synaptogenesis so that spontaneous action potential generation appears to be required for the normal cytomorphological maturation of neocortical networks. The efficacy of 25 mM K in suppressing spontaneous bioelectric activity was monitored by extra- and intracellular recording from the explants. Intracellular recording from individual neurons showed that membrane potentials were reduced to ca -30 mV in potassium cultures but rapidly repolarized to ca -50 mV when returned to normal growth medium. Though action potentials could be readily evoked from these explants, spontaneous discharges and postsynaptic potentials were absent from potassium-treated cultures. Both spontaneous bioelectric activity and postsynaptic potentials returned to the cultures by 5 days after returning the explants to normal growth medium. Extracellular recordings also showed that the explants were bioelectrically silent in the presence of 25 mM K or 25 mM K plus tetrodotoxin. In contrast to tetrodotoxin alone, bioelectric activity was absent when the cultures (with or without tetrodotoxin) were returned to normal growth medium. The explants gradually began to evince spontaneous bioelectric activity between 3 and 5 days after being returned to normal growth medium. Massive cell death induced by chronic exposure to tetrodotoxin was totally prevented by concomitant addition of 25 mM potassium, though these explants were significantly thinner than controls due to a large decrease in neuropil. We conclude that chronic depolarization of neonatal cortical explants by potassium results in a delayed return of spontaneous bioelectric discharges. Chronic depolarization results in a retardation of network formation in these explants apparently due to a lack of neurite and/or synapse formation.(ABSTRACT TRUNCATED AT 250 WORDS)
International Journal of Developmental Neuroscience 02/1991; 9(4):339-45. DOI:10.1016/0736-5748(91)90055-Q · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Time courses of effects of lidocaine on sodium currents and sodium dependent action potentials were studied in somata of small and large neurons. Cultured rat sensory spinal ganglion cells (diameter: 30 microns) and neurons of the buccal ganglion of Helix pomatia (diameter: 150 microns) served as the test cells. The latency of the suppressive action of lidocaine was the longer the larger the of the cells was. Maximal blocking effects occurred within 10 min in sensory spinal ganglion cells and within 40 min in snail neurons. Model calculations based on the assumptions (i) that lidocaine is distributed in the extra- and intracellular space by simple diffusion and (ii) that the drug concentration at the outer surface of the cells is elevated stepwisely, revealed a strong dependency of intracellular concentration changes on the size of the cells. From these findings it is concluded that lidocaine blocks sodium channels primarily from the intracellular side.
General Physiology and Biophysics 09/1990; 9(4):331-42. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The membrane potentials of bone cells derived from calvaria of new born rats was shown to be strongly dependent on temperature. When we lowered the temperature from 36 degrees C to 26 degrees C, cells with spontaneous resting membrane potentials (MP) of -80 to -50 mV depolarized (mean amplitude 8 mV; n = 33), and the membrane resistance increased by approximately 80% (n = 20). The temperature response depended on the actual MP, the reversal potential being in the range of -80 to -90 mV. With the application of ouabain (0.1-1 mmol/liter; n = 12), cells depolarized. Simultaneously, the reversal potential of the temperature response was shifted towards more positive values and approached the actual MP level of the cells. Consequently, the depolarization amplitudes induced by lowering temperature were reduced at spontaneous MP levels. The rise of the membrane resistance during cooling was unaffected. When the extracellular chloride concentration was reduced from 133 to 9 mmol/liter, temperature-dependent depolarizations persisted at spontaneous MP values (n = 5). The findings indicate that the marked effects of temperature changes on the MP of bone-derived cells are mainly determined by changes of the potassium conductance.
[Show abstract][Hide abstract] ABSTRACT: Organic calcium antagonists have been reported to abolish epileptic neuronal discharges elicited by pentylenetetrazol and penicillin. It was tested whether the organic calcium antagonist verapamil is able to suppress also paroxysmal depolarization shifts (PDS) induced by bicuculline. This is of special interest, since bicuculline is assumed to produce PDS by blocking GABAergic synaptic inhibition. The experiments were performed on CA3 neurons of hippocampal slices (guinea pig). Verapamil (40, 60, and 80 microM) reduced amplitude, duration and frequency of appearance of PDS until the generation of PDS failed. The results indicate that calcium currents are also involved in bicuculline PDS, and that bicuculline exerts its epileptogenic action, at least in part, on extrasynaptic sites.
[Show abstract][Hide abstract] ABSTRACT: The influence of the inhibitory transmitter gamma-aminobutyric acid (GABA) on depolarizations elicited by the excitatory amino acid N-methyl-D-aspartate (NMDA) was tested in neurons of organotypic neocortical tissue cultures (newborn rat) and in CA3 neurons of the hippocampal slice (guinea pig). Drugs were applied through a 3-barrelled micropipette by pressure ejection. Applications of GABA before the ejection of NMDA increased the amplitude of the depolarizations induced by the excitatory amino acid. It is suggested that the enhancement of NMDA responses by GABA may be mainly mediated by an intracellular common pathway.
Brain Research 03/1990; 510(1):127-9. DOI:10.1016/0006-8993(90)90737-V · 2.84 Impact Factor