L. Vyklický

The effect of external pH changes on responses to excitatory amino acids in mouse hippocampal neurones.

1. The whole‐cell and outside‐out configurations of the patch‐clamp technique were used to record responses to excitatory amino acids in mouse hippocampal neurones in cell culture at different pH. The amino acids kainate, quisqualate, N‐methyl‐D‐aspartate (NMDA) and L‐glutamate were applied by a rapid perfusion system. 2. In the whole‐cell recording mode the responses to NMDA or to low concentrations of glutamate, recorded in the absence of Mg2+ and with glycine in the extracellular superfusion solution, were antagonized by acidic pH and potentiated by an alkaline extracellular solution. Decrease in pH from 7.3 to 6.0 reduced NMDA responses to 33 +/‐ 2% and an increase in pH from 7.3 to 8.0 potentiated it to 141 +/‐ 6%. The responses to quisqualate and kainate were only slightly changed by altering the pH from 7.3 to 6.3 or 8.3. 3. The equilibrium dissociation constant (Kd) for H+ antagonism of responses to NMDA, estimated from the fit of a single‐binding‐site adsorption isotherm, was calculated to be 0.25 +/‐ 0.06 microM, corresponding to pH 6.6 +/‐ 0.1. The H+ attenuation of NMDA current was voltage independent at membrane potentials ‐60 to +30 mV. 4. H+ antagonism of responses to NMDA was reduced when the NMDA concentration was lowered. In the pH range 6.3‐8.3 the H(+)‐induced reduction did not vary with the concentration of glycine or Mg2+. The sensitivity of NMDA current to Zn2+ was unchanged in the pH range 6.3 +/‐ 8.0. These results suggest that H+ ions do not directly interfere with the binding of NMDA to its agonist recognition site or with the binding of glycine, Mg2+ and Zn2+ to the specific allosteric sites on the NMDA receptor‐channel complex. 5. In outside‐out patches held at ‐60 mV, unitary NMDA‐activated currents were recorded at pH 7.3 and 6.3. The mean NMDA single‐channel conductance (gamma) obtained for the largest and most frequent openings were: gamma 7.3 = 52.5 +/‐ 0.8 pS and gamma 6.3 = 51.8 +/‐ 0.9 pS. The duration of the mean channel open time, tau o, decreased from 4.75 +/‐ 0.25 ms in the control at pH 7.3 to 3.59 +/‐ 0.21 ms at pH 6.3. The mean burst duration, tau b, was reduced from 8.51 +/‐ 0.78 ms at control pH 7.3 to 5.1 +/‐ 0.34 ms at pH 6.3. The frequency of NMDA channel bursts was reduced by 31%.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes of of extracellular potassium concentration induced by neuronal activity in the spinal cord of the cat

1. Changes of extracellular K+ concentration, [K]e, arising in the spinal cord of the cat in response to an afferent stimulation were studied by means of K+‐specific micro‐electrodes.

Ion-selective microelectrodes and their use in excitable tissues

Session I. Electrochemistry of the ISM and Ionophores for the ISM.- Theory of Ion-Selective Electrodes.- New Ion Selective Liquid Membrane Microelectrodes.- Sensitivity of K+-Selective Microelectrodes to pH and Some Biologically Active Substances.- Double-Barrel Ion Selective [K+, Ca2+, Cl?] Coaxial Microelectrodes ISCM for Measurements of Small and Rapid Changes in Ion Activities.- Determination of Selectivity Coefficients of Ion-Selective Microelectrodes.- Session II. Intracellular Measurements of Ionic Activity.- Intracellular Potassium and Chloride Measurements in Sheep Cardiac Purkinje Fibers.- Intracellular Electrochemical Potentials: Skeletal Muscle vs. Epithelial Steady-State vs. Kinetics.- Measurement of Intracellular Calcium Activities.- pH Related Potassium Movements in Rabbit Atrium.- Intracellular Free Calcium Affects Electric Membrane Properties. A Study with Calcium-Selective Microelectrodes and with Arsenazo III in Helix Neurons.- Measurements of Free Ca2+ in Nerve Cell Bodies.- Intracellular Recording of Potassium in Neurons of the Motor Cortex of Awake Cats Following Extracellular Applications of Acetylcholine.- Potassium Fluxes of Isolated Nerve Endings Monitored by a Valinomycin-Activated Ion-Selective Membrane electrode in vitr..- Session III. Ion Activities in the Spinal Cord and Their Physiological Significance.- Extracellular K+ Accumulation in the Spinal Cord.- Extracellular Potassium and Calcium Activities in the Mammalian Spinal Cord, and the Effect of Changing Ion Levels on Mammalian Neural Tissues.- Relations Between Extracellular K+ and Ca++ activities and Local Field Potentials in the Spinal Cord of the Rat during Focal and Generalized Seizure Discharges.- Ion Fluxes Across the Membrane of Motoneurons during the Action of Glutamate.- The Ionic Basis of the IPSP in Spinal Motoneurones of the Frog.- Effects of Increased Extracellular Potassium Activity in the Frog Spinal Cord on the Flexor Reflex.- Extracellular K+ Activity During Electrical Stimulation of Rat Sympathetic Ganglia, Vagus and Optic Nerves in vitr..- Extracellular Potassium Activity and Axon Excitability.- Session IV. Ionic Activity Changes in the Brain and their Physiological Significance.- Extracellular Potassium, Calcium and Volume Profiles During Spreading Depression.- Cortical pH and pCa in Relation to DC Potential Shifts During Spreading Depression and Asphyxiation.- Changes of Extracellular Potassium in Rat Cerebellar Cortex Indicate a Reduced Na-K-Pump Activity During Acute and Chronic Li-Application.- Changes in Extracellular Free Ca2+ in the Sensorimotor Cortex of Cats During Electrical Stimulation and Iontophoretic Application of Amino-Acids.- Measurements of Ion Activity in the CNS: Extracellular K+ and Ca in the Hippocampus.- Evoked Ionic Alterations in Brain Slices.- Serotonin and GABA-Induced Fluctuations in Extracellular Ion Concentration in the Hippocampal Slice.- The Slow Component of the Direct Cortical Response and Extracellular Potassium.- Potassium Changes Elicited in the Feline Somatosensory Cortex by a Single Stimulus to the Peripheral Nerve.- The Mechanism and Integrative Role of Extracellular Potassium Clearance in the Rat Brain.- Session V. Ionic Activity Changes in the Heart and Skeletal Muscle.- K+ Accumulation and its Physiological Regulatory Effects in Heart Muscle.- Measurement of Intracellular Ionic Calcium Concentration in Guinea Pig Papillary Muscle.- Intracellular Na Activity During K or Rb Activated Response in Sheep Purkinje Fibres. Correlation with Changes in Membrane Potential.- Interaction between pHi, and pCai, in Cardiac Tissue.- The Use of ISMs in Working Skeletal Muscle and Venous Effluent Blood.- Characteristics of Ca2+-Selective Microelectrodes and Their Application to Cardiac Muscle Cells.- Session VI. Other Techniques for the Study of Ionic Activity Changes in Brain Research.- Ion-Selective Properties of the Nerve Cell Membrane.- Glial Cells as Potassium Detectors.- The Role of Cells in the Dispersal of Brain Extracellular Potassium.- Movement of Potassium into Glial Cells in the Retina of the Drone, Apis Mellifera, During Photostimulation.- Intracellular Calcium and the Regulation of Neuronal Membrane Permeability.- CNS and Na-Pump in in sit. Muscle.- Participants.

Changes of extracellular potassium activity in isolated spinal cord of frog under high Mg2+ concentration

Dorsal root potentials (DRPs) and changes of extracellular K(+) accompanying orthodromic stimulation were studied under high Mg(2+) concentration in isolated spinal cord of the frog. Under 20 mM MgSO(4) when synaptic transmission was blocked the amplitude of DRPs, evoked by a single volley in a dorsal root, decreased to 5-15% of the control and the transient increase of extracellular potassium concentration [K](e) evoked by 100 Hz orthodromic stimulation decreased to 10-15%. It is suggested that the DRPs, resistant to high Mg(2+) concentration, are produced by transient increase of K(+) that leaks from primary afferents.

Effects of picrotoxin on potassium accumulation and dorsal root potentials in the frog spinal cord

Abstract The effects of picrotoxin on the changes of extracellular potassium concentration (Δ[K + ] e ), field potentials and dorsal root potentials evoked by afferent stimulation, were studied in the isolated spinal cord of the frog. Δ[K + ] e was measured with potassium selective micro-electrodes. In a normal Ringers solution the Δ[K + ] e evoked by a single pulse applied to a dorsal root did not exceed 0.05 mM. In solutions containing picrotoxin (10 −7 −10 −5 m ) the Δ[K + ] e increased to 0.06–0.1 m m . At higher concentrations (10 −4 −10 −3 m ) of picrotoxin the Δ[K + ] e reached 3–6 m m and spontaneous elevations of [K + ] e were observed synchronously with the dorsal root potentials. The latter were depressed by 20–40% and considerably prolonged. The time constant of their ascending phase increased from 9 to 10 ms to 30–40 ms. The second component of the negative field potential, recorded from the intermediate region, increased and its time course corresponded to that of the evoked dorsal root potentials. Impulse activity of motoneurones and interneurones evoked by afferent stimulation was greatly enhanced. Picrotoxin (10 −4 −5.10 −4 m ) was found to have no effect on the ‘asynaptic’ component of evoked dorsal root potentials, which is resistant to 20 m m MgSO 4 and to the absence of Ca 2+ . It is therefore unlikely that the depressant effect of picrotoxin on the evoked dorsal root potentials is produced by its direct action on the potassium conductance of primary afferents. The findings are consistent with a dual mechanism of dorsal root potentials. The fast component of evoked dorsal root potentials which is depressed by picrotoxin is apparently produced by activation of axo-axonic synapses at the primary afferents, while the slow component is due to transient accumulation of extracellular K + . The potassium component of the evoked dorsal root potentials becomes dominant in solutions with high concentrations of picrotoxin (10 −4 −10 −3 m ) when impulse transmission is greatly enhanced.

The effects of increased extracellular potassium in the isolated spinal cord on the flexor reflex of the frog.

The effects of superfusing the isolated frog spinal cord with various concentrations of K+ on flexor reflexes were examined. The reflexes were evoked by pinching or by electrical stimulation. An increase in [K+]e from 2 to 6 mmol/l facilitates the flexor reflex. Higher [K+]e produces inhibition. It is suggested that the increase in [K+]e which may occur under physiological conditions facilitates the impulse transmission, and that larger increases in [K+]e which likely occur only under extreme conditions, result in inhibition.

Spider venom of Araneus opens and desensitizes glutamate channels in chick spinal cord neurones

Whole cell currents induced by the excretions of venom glands from the spider Araneus diadematus (ASV) were tested in neurones from the embryonic spinal cord of the chick in culture by the patch clamp technique. ASV in a dose of one bite in 1 ml induced a large membrane current of the same polarity as the excitatory amino acids, which decreased during long-lasting application. The effects were, to a large extent, rapidly reversible. It is suggested that ASV activates and desensitizes the receptor channels for excitatory amino acids.

Primary afferent depolarization and changes in extracellular potassium concentration induced by L-glutamate and L-proline in the isolated spinal cord of the frog

To test the hypothesis that L-proline acts as an antagonist on glutamate receptors [17, 18], the interaction between L-glutamate and L-proline was studied in the isolated spinal cord of the frog. Glutamate at concentrations of 10(-6) -5 x 10(-3) mol/l depolarized the primary afferent fibres and increased extracellular potassium concentration, [K+]e, by 0.3-4 mmol/l. Repeated applications lead to inactivation of the response. L-Proline at 5 x 10(-3) -10(-2) mol/l, also depolarized the primary afferents and increased [K+]e by 0.5-2 mmol/l, but there was only a slight decrease of the effects after repeated application. The effects were additive when the amino acids were applied simultaneously. The effect of L-proline was still present when it was applied during inactivation of the glutamate receptors. This suggests that L-glutamate and L-proline act on different receptors.

Projection of tooth pulp afferents to the brainstem and to the cortex in the cat

The projection of tooth pulp afferents in the spinal trigeminal nucleus (N.V.sp.) and the effect of dorsolateral medullotomy on cortical potentials evoked by electrical stimulation of Adelta tooth pulp nerve fibres were studied in cats. It was confirmed that antidromic responses were recorded in the tooth pulp nerve to stimulation of the ipsilateral but not the contralateral N.V.sp.[11]. Dorsolateral medullotomy at the level of the obex did not alter the cortical potentials induced by single pulses applied to the tooth pulp. It is concluded that Adelta tooth pulp afferents project exclusively to the ipsilateral trigeminal nucleus and that the impulse transmission to the cortex persists after transection of the pars caudalis of the N.V.sp.

Glutamine-induced membrane currents in cultured chick spinal cord neurons

The effects of L-glutamine (GLN) on cultured spinal cord neurons from the chick were studied in the whole cell mode of the patch clamp technique. GLN induced membrane currents rectified at positive membrane potentials (m.p.) and reversed polarity close to zero m.p. The dose-response curve was nearly linear at a semilogarithmic scale for concentrations of 10(-5) M-10(-2) M. Summation of the responses evoked by GLN (10(-3) M) and glycine (10(-3) M) was observed when these two amino acids were applied together, while no significant increase of the responses was present when GLN was applied together with L-glutamate (10(-3) M) or kainate (10(-3) M). It is suggested that GLN binds to the glutamate receptors and activates the same type of ionic channels as glutamate and kainate.