Cav1.2 calcium channels modulate the spiking pattern of hippocampal pyramidal cells

Ca(v)1.2 L-type calcium channels support hippocampal synaptic plasticity, likely by facilitating dendritic Ca2+ influx evoked by action potentials (AP) back-propagated from the soma. Ca2+ influx into hippocampal neurons during somatic APs is sufficient to activate signalling pathways associated with late phase LTP. Thus, mechanisms controlling AP firing of hippocampal neurons are of major functional relevance. We examined the excitability of CA1 pyramidal cells using somatic current-clamp recordings in brain slices from control type mice and mice with the Ca(v)1.2 gene inactivated in principal hippocampal neurons. Lack of the Ca(v)1.2 protein did not affect either affect basic characteristics, such as resting membrane potential and input resistance, or parameters of single action potentials (AP) induced by 5 ms depolarising current pulses. However, CA1 hippocampal neurons from control and mutant mice differed in their patterns of AP firing during 500 ms depolarising current pulses: threshold voltage for repetitive firing was shifted significantly by about 5 mV to more depolarised potentials in the mutant mice (p<0.01), and the latency until firing of the first AP was prolonged (73.2+/-6.6 ms versus 48.1+/- 7.8 ms in control; p<0.05). CA1 pyramidal cells from the mutant mice also showed a lowered initial spiking frequency within an AP train. In control cells, isradipine had matching effects, while BayK 8644 facilitated spiking. Our data demonstrate that Ca(v)1.2 channels are involved in regulating the intrinsic excitability of CA1 pyramidal neurons. This cellular mechanism may contribute to the known function of Ca(v)1.2 channels in supporting synaptic plasticity and memory.     

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.     

A unified model of CA1/3 pyramidal cells: an investigation into excitability

After-depolarisation is a hallmark of excitability in hippocampal pyramidal cells of CA1 and CA3 regions, because it constitutes the subthreshold relation between inward and outward ionic currents. This relationship determines the nominal response to stimuli and provides the necessary conditions for firing a spike or a burst of action potentials. Nevertheless, after-depolarisation is an inherently transient phenomenon that is not very well understood. We study after-depolarisation using a single-compartment pyramidal-cell model based on recent voltage- and current-clamp experimental data. We systematically investigate CA1 and CA3 behaviour and show that changes to maximal conductances of T-type Ca(2+)-current and muscarinic-sensitive and delayed rectifier K(+)-currents are sufficient to switch the behaviour of the model from a CA3 to a CA1 neuron. We use model analysis to define after-depolarisation and bursting threshold. We also explain the influence of particular ionic currents on this phenomenon. This study ends with a sensitivity analysis that demonstrates the influence of specific currents on excitability. Counter-intuitively, we find that a decrease of Na(+)-current could cause an increase in excitability. Our analysis suggests that a change of high-voltage activated Ca(2+)-current can have a similar effect.     

Mechano-perception in Chara cells: the influence of salinity and calcium on touch-activated receptor potentials, action potentials and ion transport

This paper investigates the impact of increased salinity on touch-induced receptor and action potentials of Chara internodal cells. We resolved underlying changes in ion transport by current/voltage analysis. In a saline medium with a low Ca(2+) ion concentration [(Ca(2+))(ext)], the cell background conductance significantly increased and proton pump currents declined to negligible levels, depolarizing the membrane potential difference (PD) to the excitation threshold [action potential (AP)(threshold)]. The onset of spontaneous repetitive action potentials further depolarized the PD, activating K(+) outward rectifying (KOR) channels. K(+) efflux was then sustained and irrevocable, and cells were desensitized to touch. However, when [Ca(2+)](ext) was high, the background conductance increased to a lesser extent and proton pump currents were stimulated, establishing a PD narrowly negative to AP(threshold). Cells did not spontaneously fire, but became hypersensitive to touch. Even slight touch stimulus induced an action potential and further repetitive firing. The duration of each excitation was extended when [Ca(2+)](ext) was low. Cell viability was prolonged in the absence of touch stimulus. Chara cells eventually depolarize and die in the saline media, but touch-stimulated and spontaneous excitation accelerates the process in a Ca(2+)-dependent manner. Our results have broad implications for understanding the interactions between mechano-perception and salinity stress in plants.     

A metabolically stable analog of 1,4,5-inositol trisphosphate activates a novel K+ conductance in pyramidal cells of the rat hippocampal slice

IP(s)3, a metabolically stable analog of 1,4,5-inositol trisphosphate (IP3), inhibited action potential firing when injected into hippocampal pyramidal cells. This effect was associated with decreased input resistance, a more negative resting potential, outward rectification at depolarized potentials, and an afterhyperpolarization. The response to IP(s)3 was unaffected by antagonists of Na+, Ca2+, and Cl- conductances, but was sensitive to changes in extracellular K+ concentration. The IP(s)3-induced conductance was voltage-dependent, was activated in 10 ms with depolarization, and was blocked by extracellular Ba2+ or intracellular Ca2+ chelation. It was not suppressed by other K+ conductance antagonists. Thus, IP(s)3 may activate a novel K+ conductance in CA1 pyramidal cells. IP3 itself did not elicit this conductance, suggesting it may be rapidly metabolized in these cells.     

Acute actions of marine toxin latrunculin A on the electrophysiological properties of cultured dorsal root ganglion neurones

The effects of latrunculin A, isolated from the nudibranch Chromodoris sp., on the excitability of neonatal rat cultured dorsal root ganglion neurones were investigated using patch-clamp recording and Ca(2+) imaging techniques. Under current-clamp conditions, acute application of latrunculin A (100 microM) reversibly induced multiple action potential firing and significantly increased action potential duration. No significant effects on action potential peak amplitude, threshold of action potential firing, resting membrane potential and input resistance were observed. Under voltage-clamp conditions, significant and dose-dependent suppression of K(+) current was seen with 10-100 microM latrunculin A. Additionally, a significant difference between inhibition of the current measured at the peak and the end of a 100 ms voltage step was seen with 100 microM latrunculin A. Fura-2 fluorescence Ca(2+) imaging revealed that latrunculin A (100 microM) significantly inhibited Ca(2+) transients evoked by KCl-induced depolarisation in all neurones. In 36% of DRG neurones, latrunculin A alone had no effect on intracellular Ca(2+). In 64% of neurones, latrunculin A alone evoked a transient rise in intracellular Ca(2+). Moreover, latrunculin A (10-100 microM) significantly inhibited the mean high voltage-activated Ca(2+) current. The effects of latrunculin A on action potential firing and K(+) currents were attenuated by intracellular phalloidin, an indication that these effects are mediated through actin disruption.     

豚鼠背侧海马锥体细胞自发放电特征

实验采用细胞外玻璃微电极采集豚鼠海马神经元放电信号,并将信号转化为峰峰间期(interspike interval,ISI)以研究麻醉和清醒状态海马锥体细胞自发放电线性和非线性特点。实验建立了豚鼠海马锥体细胞与中间神经元电生理鉴别标准;麻醉和清醒状态下豚鼠海马CA1和CA3区锥体细胞自发放电频率、时程、复杂度等无显著区别;麻醉组豚鼠海马锥体细胞ISI序列的复杂度小于清醒组,锥体细胞分型和ISI变异度等表现不同。实验表明,麻醉和清醒状态下豚鼠海马锥体细胞自发放电呈不同线性和非线性特征。传统和非线性研究手段的结合,可能较全面地反映海马锥体细胞自发放电特性。     

Ionic mechanisms and Ca2+ dynamics underlying the glucose response of pancreatic β cells: a simulation study

To clarify the mechanisms underlying the pancreatic β-cell response to varying glucose concentrations ([G]), electrophysiological findings were integrated into a mathematical cell model. The Ca(2+) dynamics of the endoplasmic reticulum (ER) were also improved. The model was validated by demonstrating quiescent potential, burst-interburst electrical events accompanied by Ca(2+) transients, and continuous firing of action potentials over [G] ranges of 0-6, 7-18, and >19 mM, respectively. These responses to glucose were completely reversible. The action potential, input impedance, and Ca(2+) transients were in good agreement with experimental measurements. The ionic mechanisms underlying the burst-interburst rhythm were investigated by lead potential analysis, which quantified the contributions of individual current components. This analysis demonstrated that slow potential changes during the interburst period were attributable to modifications of ion channels or transporters by intracellular ions and/or metabolites to different degrees depending on [G]. The predominant role of adenosine triphosphate-sensitive K(+) current in switching on and off the repetitive firing of action potentials at 8 mM [G] was taken over at a higher [G] by Ca(2+)- or Na(+)-dependent currents, which were generated by the plasma membrane Ca(2+) pump, Na(+)/K(+) pump, Na(+)/Ca(2+) exchanger, and TRPM channel. Accumulation and release of Ca(2+) by the ER also had a strong influence on the slow electrical rhythm. We conclude that the present mathematical model is useful for quantifying the role of individual functional components in the whole cell responses based on experimental findings.     

Loss of the cellular prion protein affects the Ca2+ homeostasis in hippocampal CA1 neurons

Previous neurophysiological studies on prion protein deficient (Prnp(-/-)) mice have revealed a significant reduction of slow afterhyperpolarization currents (sI(AHP)) in hippocampal CA1 pyramidal cells. Here we aim to determine whether loss of PrP(C.) directly affects the potassium channels underlying sI(AHP) or if sI(AHP) is indirectly disturbed by altered intracellular Ca(2+) fluxes. Patch-clamp measurements and confocal Ca(2+) imaging in acute hippocampal slice preparations of Prnp(-/-) mice compared to littermate control mice revealed a reduced Ca(2+) rise in CA1 neurons lacking PrP(C) following a depolarization protocol known to induce sI(AHP). Moreover, we observed a reduced Ca(2+) influx via l-type voltage gated calcium channels (VGCCs). No differences were observed in the protein expression of the pore forming alpha1 subunit of VGCCs Prnp(-/-) mice. Surprisingly, the beta2 subunit, critically involved in the transport of the alpha1 subunit to the plasma membrane, was found to be up-regulated in knock out hippocampal tissue. On mRNA level however, no differences could be detected for the alpha1C, D and beta2-4 subunits. In conclusion our data support the notion that lack of PrP(C.) does not directly affect the potassium channels underlying sI(AHP), but modulates these channels due to its effect on the intracellular free Ca(2+) concentration via a reduced Ca(2+) influx through l-type VGCCs.     

Further studies of the effects of somatostatin and related peptides in area CA1 of rabbit hippocampus

1. In slice studies of mature and immature CA1 hippocampal pyramidal cells from rabbit, somatostatin 14 (SS14), the related peptide somatostatin 28(1-12) [SS(1-12)], and the synthetic analogue of somatostatin 14, SMS-201995 (SMS), had similar effects. When pressure-ejected onto cell somata, these peptides elicited depolarizations, often accompanied by action potential discharge. When applied to dendrites, the peptides produced depolarizations or hyperpolarizations. 2. When a large amount of one of the three somatostatin-related (SS) peptides was applied to the slice at some distance from the impaled cell, hyperpolarizations were observed that were not always blocked by tetrodotoxin (TTX) or low Ca2+. Since SS peptides were also found to depolarize interneurons in area CA1, it seems likely that the hyperpolarizations that were blocked by TTX or low Ca2+ were mediated via excitation of interneurons that in turn hyperpolarized pyramidal cells. 3. All SS peptides also had long-lasting effects on CA1 pyramidal cells that led to spontaneous firing of action potentials and an increase in the number of action potentials discharged in response to a given depolarizing current pulse; the spontaneous discharge effect was blocked by TTX or low Ca2+ plus Mn2+ and, thus, appeared to have a presynaptic mechanism. However, the increase in discharge in response to a constant depolarizing current pulse was not dependent on intact synaptic transmission and, therefore, was attributable to a direct postsynaptic effect of the SS peptides.     

Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K(+) (K(v)) channels are crucial steps in this process. In the present study, K(v) currents (I(Kv)) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I(Kv). This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I(Kv) was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 microM) and XE-991 (10 microM) and enhanced by the KCNQ channel activator flupirtine (10 microM). BaCl(2) (100 microM) or linopirdine (5 microM) mimicked the effects of AVP on K(+) currents, AP generation, and Ca(2+) spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I(Kv) in A7r5 cells. I(Kv) was also inhibited in response to the PKC activator 4beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I(Kv) by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca(2+) spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.     

DPP6 establishes the A-type K(+) current gradient critical for the regulation of dendritic excitability in CA1 hippocampal neurons

Subthreshold-activating A-type K(+) currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K(+) current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability.     

EPSP amplification and the precision of spike timing in hippocampal neurons

The temporal precision with which EPSPs initiate action potentials in postsynaptic cells determines how activity spreads in neuronal networks. We found that small EPSPs evoked from just subthreshold potentials initiated firing with short latencies in most CA1 hippocampal inhibitory cells, while action potential timing in pyramidal cells was more variable due to plateau potentials that amplified and prolonged EPSPs. Action potential timing apparently depends on the balance of subthreshold intrinsic currents. In interneurons, outward currents dominate responses to somatically injected EPSP waveforms, while inward currents are larger than outward currents close to threshold in pyramidal cells. Suppressing outward potassium currents increases the variability in latency of synaptically induced firing in interneurons. These differences in precision of EPSP-spike coupling in inhibitory and pyramidal cells will enhance inhibitory control of the spread of excitation in the hippocampus.     

One reason for increased seizure susceptibility of hippocampus compared with neocortex

In this study we compared the membrane resting potential and action potential (AP) activation thresholds of neocortical layer 2/3 and CA1 hippocampal pyramidal cells in brain slices from 6–8-day old mice. The activation threshold was −37 ± 2 mV in the neocortical pyramids (5 cells), and −50 ± 1 mV in the CA1 ones (5 cells). The observed difference in the AP activation thresholds may account for a higher excitability of hippocampus as compared to neocortex. The article is submitted by the author in English.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

The ionic basis of cardiac activity in the bivalve mollusc Perna perna

The ionic basis of cardiac activity and aspects of excitation-contraction (E-C) coupling were investigated in the isolated heart of the bivalve mollusc Perna perna, using the sucrose-gap technique. The role of the principal ions was established employing artificial seawater, in which specific ion concentrations were modified, and ion channel blockers. The mean membrane resting potential (MP) and the action potential (AP) were -33+/-0.7 mV (n=89) and 13+/-0.3 mV (n=71), respectively. The MP potential was primarily dependent on K(+) ions. Three types of cardiac APs were identified: fast, slow and spike-plateau potentials. Cardiac activity was maintained in Na(+)- or Ca(2+)-free salines but ceased when either Cd(2+) or EDTA was added to these salines. Other Ca(2+) channel blockers reduced the amplitude and increased duration of the cardiac APs. Tetrodotoxin (TTX) and procaine did not alter the AP. The data showed that the depolarizing phase of the AP was dependent on Ca(2+) influx while the plateau phase, when present, resulted from Na(+) influx that was modulated by Ca(2+). The mechanical responses were more sensitive to changes in extracellular Ca(2+) concentration than were the electrical responses.     

Profound alterations in the intrinsic excitability of cerebellar Purkinje neurons following neurotoxin 3-acetylpyridine (3-AP)-induced ataxia in rat: new insights into the role of small conductance K+ channels

Alterations in the intrinsic properties of Purkinje cells (PCs) may contribute to the abnormal motor performance observed in ataxic rats. To investigate whether such changes in the intrinsic neuronal excitability could be attributed to the role of Ca(2+)-activated K(+) channels (K(Ca)), whole cell current clamp recordings were made from PCs in cerebellar slices of control and ataxic rats. 3-AP induced profound alterations in the intrinsic properties of PCs, as evidenced by a significant increase in both the membrane input resistance and the initial discharge frequency, along with the disruption of the firing regularity. In control PCs, the blockade of small conductance K(Ca) channels by UCL1684 resulted in a significant increase in the membrane input resistance, action potential (AP) half-width, time to peak of the AP and initial discharge frequency. SK channel blockade also significantly decreased the neuronal discharge regularity, the peak amplitude of the AP, the amplitude of the afterhyperpolarization and the spike frequency adaptation ratio. In contrast, in ataxic rats, both the firing regularity and the initial firing frequency were significantly increased by the blockade of SK channels. In conclusion, ataxia may arise from alterations in the functional contribution of SK channels, to the intrinsic properties of PCs.     

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.