Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Ca(2+) binding protein frequenin mediates GDNF-induced potentiation of Ca(2+) channels and transmitter release

Molecular mechanisms underlying long-term neurotrophic regulation of synaptic transmission and plasticity are unknown. We report here that long-term treatment of neuromuscular synapses with glial cell line-derived neurotrophic factor (GDNF) potentiates spontaneous and evoked transmitter release, in ways very similar to presynaptic expression of the Ca(2+) binding protein frequenin. GDNF enhances the expression of frequenin in motoneurons, and inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca(2+) influx into the nerve terminals during evoked transmission by enhancing Ca(2+) currents. The effect of GDNF on Ca(2+) currents is blocked by inhibition of frequenin expression, occluded by overexpression of frequenin, and is selective to N-type Ca(2+) channels. These results identify an important molecular target that mediates the long-term, synaptic action of a neurotrophic factor.     

Calcium-induced transitions between the spontaneous miniature outward and the transient outward currents in retinal amacrine cells

Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between -60 and -40 mV. At more depolarized potentials, a transient outward current (I(to)) appears and SMOCs disappear. Both SMOCs and the I(to) are K(+) currents carried by BK channels. They both arise from Ca(2+) influx through high voltage-activated (HVA) Ca(2+) channels, which stimulates release of internal Ca(2+) from caffeine- and ryanodine-sensitive stores. An increase in Ca(2+) influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca(2+) influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca(2+) influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the I(to) at high levels of Ca(2+) influx. Therefore, low levels of Ca(2+) influx, arising from low levels of activation of the HVA Ca(2+) channels, produce randomly occurring SMOCs within the range of -60 to -40 mV. Further depolarization leads to greater activation of the HVA Ca(2+) channels, larger Ca(2+) influx, and the disappearance of discontinuous SMOCs, along with the appearance of the I(to). Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.     

Nitric oxide inhibits nociceptive transmission by differentially regulating glutamate and glycine release to spinal dorsal horn neurons

Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.     

T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.     

Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.     

Ablation of Ca2+ channel beta3 subunit leads to enhanced N-methyl-D-aspartate receptor-dependent long term potentiation and improved long term memory

The beta subunits of voltage-dependent Ca(2+) channels (VDCCs) have marked effects on the properties of the pore-forming alpha(1) subunits of VDCCs, including surface expression of channel complexes and modification of voltage-dependent kinetics. Among the four different beta subunits, the beta(3) subunit (Ca(v)beta3) is abundantly expressed in the hippocampus. However, the role of Ca(v)beta3 in hippocampal physiology and function in vivo has never been examined. Here, we investigated Ca(v)beta3-deficient mice for hippocampus-dependent learning and memory and synaptic plasticity at hippocampal CA3-CA1 synapses. Interestingly, the mutant mice exhibited enhanced performance in several hippocampus-dependent learning and memory tasks. However, electrophysiological studies revealed no alteration in the Ca(2+) current density, the frequency and amplitude of miniature excitatory postsynaptic currents, and the basal synaptic transmission in the mutant hippocampus. On the other hand, however, N-methyl-d-aspartate receptor (NMDAR)-mediated synaptic currents and NMDAR-dependent long term potentiation were significantly increased in the mutant. Protein blot analysis showed a slight increase in the level of NMDAR-2B in the mutant hippocampus. Our results suggest a possibility that, unrelated to VDCCs regulation, Ca(v)beta3 negatively regulates the NMDAR activity in the hippocampus and thus activity-dependent synaptic plasticity and cognitive behaviors in the mouse.     

BDNF acutely modulates synaptic transmission and calcium signalling in developing cortical neurons.

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation(LTP). We found that BDNF rapidly induced potentiation of synaptic activity and an increase in the intracellular Ca2+ concentration in cultured cortical neurons. Within minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as was the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). Fura-2 recordings showed that BDNF acutely elicited an increase in intracellular calcium concentration ([Ca2+]i). This effect was partially dependent on extracellular Ca2+. In calcium-free perfusion medium a substantial calcium signal remained which disappeared after loading of cortical neurons with 5 microM U-73122. BDNF-induce Ca2+ transients were completely blocked by K252a and partially blocked by Cd2+. The results demonstrate that BDNF can enhance synaptic transmission and induce directly a rise in [Ca2+]i that require two routes: the release of Ca2+ from intracellular calcium stores and influx of extracellular Ca2+ mainly through voltage-dependent Ca2+ channels in cultured cortical neurons.     

Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells.

Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin-Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation.     

Ca2+ influx into identified leech neurons induced by 5-hydroxytryptamine

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca(2+)](i)) in leech neurons. As [Ca(2+)](i) plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca(2+)](i) (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca(2+)](i) increase which was predominantly due to Ca(2+) influx since it was abolished in Ca(2+)-free solution. The 5-HT-induced Ca(2+) influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca(2+) channels. In P and N1 neurons, the membrane depolarization was due to Na(+) influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca(2+) channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca(2+)](i).     

Acute modulation of calcium currents and synaptic transmission by gabapentinoids

Gabapentin and pregabalin are anticonvulsant drugs that are extensively used for the treatment of several neurological and psychiatric disorders. Gabapentinoids (GBPs) are known to have a high affinity binding to α2δ-1 and α2δ-2 auxiliary subunit of specific voltage-gated calcium channels. Despite the confusing effects reported on Ca (2+) currents, most of the studies showed that GBPs reduced release of various neurotransmitters from synapses in several neuronal tissues. We showed that acute in vitro application of pregabalin can reduce in a dose dependent manner synaptic transmission in both neuromuscular junctions and calyx of Held-MNTB excitatory synapses. Furthermore presynaptic Ca (2+) currents treated with pregabalin are reduced in amplitude, do not show inactivation at a clinically relevant low concentration of 100 μM and activate and deactivate faster. These results suggest novel modulatory role of acute pregabalin that might contribute to better understanding its anticonvulsant/analgesic clinical effects.     

Nitric oxide augments voltage-gated P/Q-type Ca(2+) channels constituting a putative positive feedback loop

P/Q-type Ca(2+) channels, which are postulated to play major roles in synaptic transmission, are regulated in a variety of ways. Ca(2+) currents through P/Q-type Ca(2+) channels (Ca(v)2.1/beta(1a)/alpha(2)delta) heterologously expressed in mammalian cells were recorded using the whole-cell patch clamp method. The oxidant H(2)O(2) increased the current amplitude and the effect was reversed by the reducing agent dithiothreitol (DTT). The stimulatory effect of H(2)O(2) on the Ca(2+) current was mimicked by the NO donors, SNAP, and diethylamine NONOate, and reversed by the reducing agent DTT. The presence of a soluble guanylate cyclase inhibitor did not abolish the ability of SNAP to increase the Ca(2+) current. Adenovirus-mediated overexpression of nitric oxide synthase in combination with application of the Ca(2+) ionophore A23187 also increased the Ca(2+) current amplitude and the effect was again reversed by DTT. The NOS inhibitor L-NAME abolished the stimulatory effect of A23187, and A23187 did not change the Ca(2+) currents in the cells treated with control adenovirus particles. The time course of the decline of the Ca(2+) current, but not of the Ba(2+) current, in response to repeated depolarization was markedly slowed by adenovirus-mediated overexpression of nitric oxide synthase. The results demonstrate that nitric oxide enhances the channel activity by promoting oxidation and suggest that Ca(2+), nitric oxide synthase, and nitric oxide could constitute a positive feedback loop for regulation of voltage-gated P/Q-type Ca(2+) channels.     

产前应激对子代大鼠海马CA3区神经元Ca2+和K+通道的影响

本研究的目的在于探讨产前应激对子代大鼠海马CA3神经元高电压激活(HVA)钙通道、延迟整流钾电流(delayedrectifierpotassiumcurrents,IKD)的影响。产前应激(prenatalstress,PNS)组孕鼠孕晚期给予束缚应激,应用全细胞膜片钳技术进行研究。结果显示产前应激增加了子代海马CA3神经元HVA钙通道峰电流幅值,对照组和产前应激组子代CA3神经元平均最大HVA钙电流峰值分别为-576.52±7.03pA和-702.05±6.82pA(P<0.01)。同时未改变其电导-电压关系,也未改变延迟整流钾通道电流-电压关系、电导-电压关系。结果提示,在胎儿发育的关键时期,给予母体产前应激,引起子代海马神经元HVA钙电流增加,其机制一方面PNS导致皮质酮升高,从而可能增加HVA钙通道mRNA表达;另一方面PNS所致反应性氧化产物(reactiveoxygenspecies,ROS)增多,后者可能通过磷酸化HVACa2 通道亚单位,从而提高HVA钙电流幅值。     

Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus.

Presynaptic inhibition of neurotransmitter release is thought to be mediated by a reduction of axon terminal Ca2+ current. We have compared the actions of several known inhibitors of evoked glutamate release with the actions of the Ca2+ channel antagonist Cd2+ on action potential-independent synaptic currents recorded from CA3 neurons in hippocampal slice cultures. Baclofen and adenosine decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Cd2+ blocked evoked synaptic transmission, but had no effect on the frequency or amplitude of either mEPSCs or inhibitory postsynaptic currents (IPSCs). Inhibition of presynaptic Ca2+ current therefore appears not to be required for the inhibition of glutamate release by adenosine and baclofen. Baclofen had no effect on the frequency of miniature IPSCs, indicating that gamma-aminobutyric acid B-type receptors exert distinct presynaptic actions at excitatory and inhibitory synapses.     

cAMP和cGMP对棉铃虫神经细胞高电压激活钙通道的调节作用

用全细胞膜片钳法研究了cAMP和cGMP对棉铃虫Helicoverpa armigera 3龄幼虫胸腹神经节细胞高电压激活钙通道的调节作用。细胞外液中加入腺苷酸环化酶（AC）激活剂福斯克林（forskolin） 0.1 mmol/L,对于Ba²⁺介导的钙通道电流激活电压、峰电压、峰电流变化以及通道激活和电流达到峰值的时间无影响。电极内液中加入1 mmol/L的cGMP则明显抑制峰电流,且抑制作用呈时间依赖性和浓度依赖性,而对激活电压、峰电压无影响。结果提示,棉铃虫神经细胞高电压激活钙通道的活动可能不受细胞内cAMP水平提高的影响,但被cGMP抑制。     

Platelet-derived growth factor receptor-induced feed-forward inhibition of excitatory transmission between hippocampal pyramidal neurons.

Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity of N-methyl-D-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-gamma, and by blocking the intracellular release of Ca(2+). Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca(2+)- and PKA-dependent inhibition of their modulation by c-Src.     

Regulation of presynaptic Ca(V)2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity

Short-term synaptic plasticity shapes the postsynaptic response to bursts of impulses and is crucial for encoding information in neurons, but the molecular mechanisms are unknown. Here we show that activity-dependent modulation of presynaptic Ca(V)2.1 channels mediated by neuronal Ca(2+) sensor proteins (CaS) induces synaptic plasticity in cultured superior cervical ganglion (SCG) neurons. A mutation of the IQ-like motif in the C terminus that blocks Ca(2+)/CaS-dependent facilitation of the P/Q-type Ca(2+) current markedly reduces facilitation of synaptic transmission. Deletion of the nearby calmodulin-binding domain, which inhibits CaS-dependent inactivation, substantially reduces depression of synaptic transmission. These results demonstrate that residual Ca(2+) in presynaptic terminals can act through CaS-dependent regulation of Ca(V)2.1 channels to induce short-term synaptic facilitation and rapid synaptic depression. Activity-dependent regulation of presynaptic Ca(V)2.1 channels by CaS proteins may therefore be a primary determinant of short-term synaptic plasticity and information-processing in the nervous system.