Subthreshold oscillations generated by TTX-sensitive sodium currents in dorsal cochlear nucleus pyramidal cells

During intracellular recordings in rodent brainstem slice preparations, dorsal cochlear nucleus (DCN) pyramidal cells (PCs) exhibit characteristic discharge patterns to depolarizing current injection that depend on the membrane potential from which the responses are evoked. When depolarized from hyperpolarized potentials, PCs can respond with a short-latency action potential followed by a long silent interval (pauser) or a train of action potentials with a long latency (buildup). During the silent intervals in a pauser or a buildup response, the membrane potential slowly depolarizes towards spike threshold, often exhibiting distinct voltage oscillations of 1–2 mV before the first spike. The subthreshold voltage oscillations were investigated using whole cell recordings from DCN PCs in rat pup (P10–14) brainstem slices. The oscillations were unaffected by excitatory and inhibitory neurotransmitter antagonists, and were not temporally locked to the onset of the depolarization. The oscillations typically became larger as spike threshold was approached, and had a characteristic frequency between 40 and 100 Hz. In the presence of tetrodotoxin (TTX, 500 nM), the oscillations were significantly suppressed, and could not be evoked at any voltage below or above spike threshold. The oscillations were not blocked by phenytoin or Cd²⁺, but they were affected by prior activity in the neuron for approximately 1 s. We conclude that voltage-gated Na⁺ channels are required to generate membrane oscillations during the buildup phase. We suggest that the subthreshold oscillations play a role in controlling spike timing in PCs when the membrane potential slowly approaches, or hovers near, spike threshold.     

The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (I _Cl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation ofI _Cl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (–)-indolactam did not affectI _Cl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 mol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19–31 pseudosubstrate peptide, did not influenceI _Cl,vol. Trifluoperazine and tamoxifen fully blockedI _cCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 mol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca²⁺] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 ol/1) and genistein (100 ol/1) did not affectI _Cl,vol Exposing CPAE cells to lysophosphatidic acid (1mol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125^FAK in endothelial cells, neither evoked a Cl^– current nor affectedI _Cl,vol Neither wortmannin (10 mol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affectedI _Cl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl^– current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl^– current induced by hypotonicity.     

Ca2+ and K+ current in cultured vascular smooth muscle cells from rat aorta

Ca²⁺ (I_Ca) and K⁺ (I_K) currents were recorded in single cultured cells from rat aorta using the whole cell clamp technique with patch electrodes. I_Ca was detected at–30 mV, and at 20 mV it reached a peak in about 10 ms and decayed with a t_1/2=50 ms. The mean maximum slope conductance (G_Ca) was 30 S/cm². I_K was detected at–10 mV and at 20 mV reached its maximum with a t_1/2=12 ms. For I_K, G_K=200 S/cm². These channels can be activated during action potentials and play a role in the excitation and contraction of vascular smooth muscle cells.Doctoral training program UAM-1     

Effects of folic and kainic acids on synaptic responses of hippocampal neurones

The actions of the neurotoxic amino acids folate and kainate have been compared on ortho-and antidromic responses evoked in CA1, CA3 and the dentate gyrus of slices of rat hippocampus maintained in vitro. Both in CA1 and the dentate gyrus superfusion of these acids caused an increase in amplitude of the population spike discharging from an excitatory postsynaptic potential which either remained unaffected or was reduced. In the CA3 region kainate and folate had broadly similar actions to enhance the probability of cell firing to synaptic excitation, and also caused epileptiform discharges to occur spontaneously or in response to electrical stimulation. Spontaneous and evoked population bursts in CA3 did not persist in low calcium/high magnesium medium indicating their dependence on intact synaptic transmission; spontaneously occurring bursts in CA1 were eliminated with the latter treatment or when the axonal connections between it and CA3 were cut. Following folate superfusion the commissural-evoked response in CA3 showed large and variable shifts of the latency which were dependent on the stimulus intensity and its timing after a spontaneous population discharge. Although all of the effects of folate were reproduced by bicuculline, no evidence for a decreased recurrent inhibition in CA1 was obtained although this was observed with kainate. The finding that folate and kainate produced their effects in the absence of a detectable effect on the antidromic population spike suggests a mechanism of action other than neuronal depolarization. The implications of these data for the neurotoxic mechanism(s) and the receptor homologies of folate and kainate are discussed.     

Removal of GABAergic inhibition alters subthreshold input in neurons in forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) after digit stimulation

Our objective was to test the hypothesis that suppression of GABAergic inhibition results in an enhancement of responses to stimulation of the surround receptive field. Neurons in the forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) receive short latency suprathreshold input from a principal location on the forepaw and longer latency subthreshold input from surrounding forepaw skin regions. Input from principal and surround receptive field sites was examined before, during, and after administration of the GABA(A) receptor blocker bicuculline methiodide (BMI) (in 165 mM NaCl at pH 3.3-3.5). In vivo extracellular recording was used to first identify the location of the glabrous forepaw digit representation within the FBS. In vivo intracellular recording and labeling techniques were then used to impale single FBS neurons in layer IV as well as neurons in layers III and V, determine the receptive field of the cell, and fill the cell with biocytin for subsequent morphological identification. The intracellular recording electrode was fastened with dental wax to a double-barrel pipette for BMI iontophoresis and current balance. A stimulating probe, placed on the glabrous forepaw skin surface, was used to identify principal and surround components of the receptive field. Once a cell was impaled and a stable recording was obtained, a stimulating probe was placed at a selected site within the surround receptive field. Single-pulse stimulation (1 Hz) was then delivered through the skin probe and the percentage of spikes occurring in 1-min intervals before BMI onset was used as a baseline measure. BMI was then iontophoresed while the periphery was simultaneously stimulated, and spike percentage measured during and after BMI ejection was compared with the pre-BMI baseline. The major findings are: (1) suppression of GABAergic inhibition enhanced evoked responses to firing level from sites in surround receptive fields in 65% of the cells ( n=17); (2) evoked responses were rapidly elevated (within 1 min) to suprathreshold firing in the presence of BMI in 31% of the cells; (3) GABAergic inhibition was reversible [suprathreshold spiking gradually reversed to subthreshold excitatory postsynaptic potentials (EPSPs) in 45% of the cells tested]; (4) BMI altered the stimulus-evoked and non-stimulus-evoked firing pattern in SI neurons from single spikes to burst patterns in all tested cells; and (5) iontophoresis of NaCl (165 mM) without BMI was ineffective in altering evoked responses in control cells ( n=4). The present findings support the notion that subthreshold input from surround receptive fields is one possible mechanism for rapid cortical reorganization in barrel cortex and that GABAergic inhibition may regulate its expression. Possible corticocortical and thalamocortical substrates for subthreshold input to reach barrel neurons are discussed.     

Synaptic actions of the ventral root afferents on cat hindlimb motoneurons

Postsynaptic potentials (PSPs) with long latencies were evoked in cat hindlimb motoneurons by stimulation of the distal stump of a cut ventral root. Measurements of their latency and the threshold in the responsible afferent fibers showed that they were produced mainly by the activity of the unmyelinated fibers in the ventral root that enter the spinal cord through the dorsal root. Patterns of PSPs evoked in flexor and extensor motoneurons by ventral root stimulation were similar to those observed in the flexion reflex.     

Medullary locomotor strip and column in the cat

Responses of lateral medullary neurons to microstimulation of two points in the locomotor strip—rostral and caudal to the obex—were recorded intracellularly in mesencephalic decerebellated uncurarized cats. Excitatory and inhibitory postsynaptic potentials and orthodromic action potentials occurred up to 20 ms after a single stimulus. A number of cells responded to stimulation of a locomotor point by a repetitive discharge, and in some cells synaptic responses were evoked by contralateral stimulation. The responsive neurons were scattered among other cells in the lateral medullary tegmentum. At least one-third of neurons with synaptic responses to stimulation of the rostral locomotor point were antidromically invaded from the caudal one. The characteristic length of these descending axons was between 4 and 9 mm, although there were longer axons too.The lateral medullary cells which give synaptic responses to stimulation of the locomotor strip form the locomotor column located medial to the strip, and a portion of these cells send their axons to the strip. It is suggested that the activity is propagated polysynaptically along the column through axonal collaterals of its neurons. One can assume that when repetitive stimulation achieves a threshold for locomotion such a propagation occurs without decrement. As a result, spinal stepping generators are activated.     

Expression of voltage-dependent sodium and transient potassium currents in an identified sub-population of dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos

The electrophysiological properties of a subset of dorsal root ganglion (DRG) neurons microdissected from 12-day-old (E12) mouse embryos and acutely isolated were analyzed as soon as 3 after their isolation. Two classes of neurons were defined according to their mean diameter. The larger diameter class was examined in this study. They display uniform cytoskeletal properties with co-expression of vimentin and neurofilament triplet proteins. Patch-clamp methods also revealed a homogeneous and limited repertoire of ionic channels that included (1) a TTX-sensitive Na⁺ current whose properties are similar to that reported in mature mammalian neurons, and (2) two types of K⁺ currents that can be compared with the delayed rectifier (I _k ) and the transient (I _a) potassium currents found in other mammalian preparations. It may be possible to use this in vitro model to examine the development of new types of currents, such as Ca²⁺ currents during neuronal growth and differentiation.     

Effects of acetylcholine on time-dependent currents in sheep cardiac Purkinje fibers

Voltage clamp experiments were carried out on sheep Purkinje fibers to determine the effect of Ach on the time-dependent currents.On the pacemaker current (i _{K 2}) Ach 10^–6 mol·l^–1 had the following effects: shift of the activation curve by a few mV in the depolarizing direction, without change in the rectifier ratio. The potential dependence of the time constants for activation and deactivation was influenced in a similar way as the activation curve.Ach had no effect on the positive dynamic current (i _qr ) or the late plateau outward current (i _x ).The slow inward current (i _si ) as well as the transient inward current (T.I.) were reduced in amplitude and slowed in time course by Ach.The changes in pacemaker current are important in explaining the increased rate of diastolic depolarization in the presence of Ach. The decrease of slow inward current by Ach cannot be made responsible for the plateau shift or the prolongation of the action potential.Supported by F.G.W.O. Belgium 3.0087.74     

The hyperpolarisation-activated current,I f,is not required for pacemaking in single cells from the rabbit atrioventricular node

Using electrophysiological and radiotracer studies in parallel, we have investigated the characteristics of the endogenous Na⁺-dependent amino acid transporter (system B^0,+) in Xenopus oocytes with regard to ion dependence, voltage dependence and transport stoichiometry. In voltage-clamped oocytes (–60 mV) superfusion with saturating concentrations of amino acids (1 mM) in 100 mM NaCl resulted in reversible, inward currents (mean±SEM): alanine, 1.83±0.09 nA (n=21); arginine, 2.54±0.18 nA (n=17); glutamine, 1.73±0.10 nA (n=19). Only arginine evoked a current in choline medium (0.50±0.13 nA, n=10), whereas Cl^– replacement had no effect on evoked currents. The glutamine-evoked current was saturable (I _max=1.73 nA, glutamine K _m=0.12 mM) and linearly dependent upon voltage between –90 and –30 mV. Using direct and indirect (activation) methods, we found that transport can proceed with Na⁺/amino acid coupling stoichiometry of either 11 or 21, but coupling was the same for each amino acid tested (alanine, arginine and glutamine) within a batch of oocytes (i.e. from a single toad). Despite the net single positive charge on arginine, the magnitude of the net transmembrane charge movement during Na⁺-coupled arginine transport was identical to that for the zwitterionic neutral amino acids glutamine and alanine; this may be explained by a concomitant stimulation of K⁺ efflux during arginine transport with a putative coupling of 1 K⁺1 arginine.