Integrin regulation of cytoplasmic calcium in excitatory neurons depends upon glutamate receptors and release from intracellular stores

Integrins regulate cytoplasmic calcium levels ([Ca²⁺]i) in various cell types but information on activities in neurons is limited. The issue is of current interest because of the evidence that both integrins and changes in [Ca²⁺]i are required for Long-Term Potentiation. Accordingly, the present studies evaluated integrin ligand effects in cortical neurons. Integrin ligands or α5β1 integrin activating antisera rapidly increased [Ca²⁺]i with effects greater in glutamatergic than GABAergic neurons, absent in astroglia, and blocked by β1 integrin neutralizing antisera and the tyrosine kinase antagonist genistein. Increases depended upon extracellular calcium and intracellular store release. Ligand-induced effects were reduced by voltage-sensitive calcium channel and NMDA receptor antagonists, but blocked by tetrodotoxin or AMPA receptor antagonists. These results indicate that integrin ligation triggers AMPA receptor/depolarization-dependent calcium influx followed by intracellular store release and suggest the possibility that integrin modulation of activity-induced changes in [Ca²⁺]i contributes importantly to lasting synaptic plasticity in forebrain neurons.     

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na⁺ channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na⁺ imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na⁺ clearance in dendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na⁺ gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K⁺ currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a 'dual' firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).     

Stromal-cell-derived factor 1alpha /CXCL12 modulates high-threshold calcium currents in rat substantia nigra

Dopaminergic neurons of the substantia nigra constitutively express the CXCR4 receptor for the chemokine stromal-cell-derived factor 1α (CXCL12) but, to date, no direct effect of CXCR4 activation by CXCL12 on membrane conductance of dopaminergic neurons has been demonstrated. We tested the effects of CXCL12 on whole-cell currents of dopaminergic neurons recorded in patch clamp in substantia nigra slices and showed that CXCL12 (0.01–10 n m ) increased the amplitude of total high-voltage-activated (HVA) Ca currents through CXCR4 activation. This effect was reversibly reduced by ϖ-conotoxin-GVIA, suggesting that CXCL12 acted on N-type Ca currents, known to be involved in dopamine (DA) release. We therefore investigated the effects of CXCL12 on DA release from cultured dopaminergic neurons from the rat mesencephalon. In basal conditions, CXCL12 alone had no effect on DA release. When neurons were depolarized with KCl (20 m m ), and thus when HVA Ca currents were activated, low CXCL12 concentrations (1–50 n m ) increased DA release via CXCR4 stimulation. These data strongly suggest that the chemokine CXCL12 can act directly as a neuromodulator of dopaminergic neuronal electrical activity through the modulation of HVA currents.     

Neurosteroid dehydroepiandrosterone sulphate inhibits persistent sodium currents in rat medial prefrontal cortex via activation of sigma-1 receptors

Dehydroepiandrosterone sulphate is one of the most important neurosteroids. In the present paper, we studied the effect of dehydroepiandrosterone sulphate on persistent sodium currents and its mechanism and functional consequence with whole-cell patch clamp recording method combined with a pharmacological approach in the rat medial prefrontal cortex slices. The results showed that dehydroepiandrosterone sulphate inhibited the amplitude of persistent sodium currents and the inhibitory effect was significant at 0.1 microM, reached maximum at 1 microM and decreased with the increase in the concentrations of above 1 microM. The effect of dehydroepiandrosterone sulphate on persistent sodium currents was canceled by the Gi protein inhibitor and the protein kinase C inhibitor, but not by the protein kinase A inhibitor. The effect of dehydroepiandrosterone sulphate on persistent sodium currents was also canceled by the sigma-1 receptor blockers and the sigma-1 receptor agonist could mimic the effect of dehydroepiandrosterone sulphate. Dehydroepiandrosterone sulphate had no significant influence on neuronal excitability but could significantly inhibit chemical inhibition of mitochondria-evoked increase in persistent sodium currents. These results suggest that dehydroepiandrosterone sulphate inhibits persistent sodium currents via the activation of sigma-1 receptors-Gi protein-protein kinase C-coupled signaling pathway, and the main functional consequence of this effect of DHEAS is presumably to protect neurons under ischemia.     

Changes in acetylcholine receptor distribution and binding properties at the neuromuscular junction during aging

Junctional and extrajunctional acetylcholine receptors were characterized in diaphragm muscle obtained from mature adult and aged rats. Rhodamine-conjugated -bungarotoxin was used to visualize receptor localization. At this level of resolution, there were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. Specific binding characteristics were assayed by measuring ¹²⁵I--bungarotoxin binding. Maximal binding to intact junctional and extrajunctional tissue samples was greater in the older rats. The association rate constant in minced tissue decreased in the older animals. Retardation of the initial rate of toxin binding by d-tubocurarine was described by a two-component nonlinear Hofstee plot; values of K_i were about the same for both age groups, but there was a significant shift towards the low-affinity values in the aged rats. Miniature end-plate currents (m.e.p.c.s) were recorded under voltage-clamp conditions before and after AChE inhibition. When AChE activity was inhibited m.e.p.c. amplitudes and decay time-constants increased in both age groups. The magnitude of these increases was larger in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACh-induced membrane noise. Lipid composition was assayed in whole muscle and isolated sarcolemma. Muscle cholesterol concentration rose 15–20 percent, but phospholipid concentrations were maintained. However, neither cholesterol, phospholipid levels, nor membrane fluidity changed significantly with age in isolated sarcolemmal membrane fractions.

These data indicate that the numbers of junctional and extrajunctional receptors increase with age. In the junctional region, this is quite likely due to an expanded field of receptors and not an increased density. This is associated with an increased fraction of receptors with lower binding affinity during aging. These changes apparently are not caused by major changes in membrane fluidity or lipid composition.     

- and -type Calcium Currents Differ in Finch and Rat Ventricular Cardiomyocytes

We compared -type Ca current (I_CaL) and -type Ca current (I_CaT) in finch and rat myocytes, using whole-cell patch clamp techniques. Cell capacitance averaged 50±4 pF in finch (n=25)v145±8 pF in rat (n=38) cells,P<0.001. In cells bathed in 1 m Ca_oat 22°C, peakI_CaLamplitude, during a voltage clamp step (10 m EGTA in pipette) from −45 mV to −5 mV, averaged 10.5±0.3 pA/pF in finchv6.9±0.6 pA/pF,P<0.001 in rat cells.I_CaLinactivation kinetics were faster in finch (4.6±0.3 ms) than in rat (13.4±1.3 ms) cells,P<0.001.I_CaTwas not detectable in rat cells (2 m bathing [Ca]); but in finch cells, a largeI_CaTwhich averaged 6.8±1.4 pA/pF was activated at −30 mV and was relatively insensitive to nitrendipine (0.1μ ). The distinctive features ofI_CaLandI_CaTin finch cells may have a role in the ability of the finch to achieve a very rapid heart rate. They may also facilitate excitation-Ca²⁺release coupling in finch ventricular cells which are devoid of T tubules and have relatively few junctions between the sarcolemma and the sarcoplasmic reticulum.     

An algorithm for eddy currents symmetrization and compensation

Eddy currents, which are induced in the magnet cryostat by pulsed magnetic field gradients in MRI, generate undesired eddy fields within the imaging volume. In this work, an automated and computerized algorithm to compensate these eddy currents is presented. The compensation is done in two steps: (i) Eddy fields are symmetrized electronically with an R-C filter, (ii) The symmetric eddy fields are compensated by another R-C filter. The compensation algorithm is iterative; therefore, errors that remain from one iteration are eliminated in the next iteration. Hence, the compensation process is very robust and accurate. It is shown that all the even harmonics of the eddy fields are eliminated by the symmetrization process, but the odd field harmonics remain. The amplitude of these odd harmonics can be significantly reduced if the gradient coils are designed so that the field they generate is spatially similar to the eddy fields.     

Glycinergic Inhibitory Synaptic Currents and Related Receptor Channels in the Zebrafish Brain

To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio redo) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole-cell recordings of the Mauthner cells, applications of 10^?6 M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl^?-dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca²⁺ and adding Mg²⁺ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mISCs). Applications of 0.5–1 μMM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of-50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81–86 pS and another displaying only one opening level of 41–43 pS. These two mean conductance states had similar mean open times, of 0.6–1 and 4.5–6 ms respectively. In addition, three mean closed times were observed for the 41–43 pS level. The shortest group (0.6–1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14–22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4–5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre-and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation.     

Alterations in Ito1, IKrand Ik1Density in the BIO TO-2 Strain of Syrian Myopathic Hamsters

The BIO TO-2 strain of cardiomyopathic hamster provides a model of dilated low output heart failure. The goal of the study was to determine whether changes in potassium currents occur in this model of heart failure. The densities of I_to1, I_Krand I_K1in 8-month-old myopathic hamsters were not significantly different from their age-matched controls. The half-maximum activation voltage (V¹/₂) of I_Krand I_to1, as well as the voltage-dependence of I_to1inactivation were also similar in both groups at 8 months. I_to1inactivation exhibited a double exponential time-course; the slow component (τ₂), but not the rapid component (τ₁), was larger in the myopathic animals. The densities of I_to1, I_Krand I_K1were not significantly different in the 8- and 10-month-old control animals. However, the densities of I_to1, I_Krand I_K1were all significantly lower in the 10-month-old myopathic hamsters relative to the 10-month-old controls. The V¹/₂for I_Krand I_to1activation was the same in myopathic and control animals.τ₂, but notτ₁, of I_to1inactivation was again larger in the myopathic animals. The voltage-dependence of I_to1inactivation was shifted slightly, but significantly, positive in the myopathic animals. Lastly, a sustained outwardly rectifying current that activated upon depolarization was found to be larger in the myopathic animals at both 8 and 10 months of age. In conclusion, many of the alterations in potassium current densities in the 10-month-old cardiomyopathic hamsters are qualitatively similar to the changes observed in the failing human heart.     

离子通道电流的计算机分析处理─—Pclamp5.5.1版本软件系统的应用

Pclam5．5．1版本软件系统，是国际上用于膜片钳制实验的先进软件系统。我们应用的结果表明：在实验中应用软件采集和分析数据是非常方便、节省时间，且自动化程度高、功能全、应用范围广，它为电生理实验，尤其是离子通道电流研究提供了先进的方法和手段。