Calcium sensitive non-selective cation current promotes seizure-like discharges and spreading depression in a model neuron
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As described by others, an extracellular calcium-sensitive non-selective cation channel ([Ca2+]o-sensitive NSCC) of central neurons opens when extracellular calcium level decreases. An other non-selective current is activated by rising intracellular calcium ([Ca2+] i ). The [Ca2+]o-sensitive NSCC is not dependent on voltage and while it is permeable by monovalent cations, it is blocked by divalent cations. We tested the hypothesis that activation of this channel can promote seizures and spreading depression (SD). We used a computer model of a neuron surrounded by interstitial space and enveloped in a glia-endothelial “buffer” system. Na+, K+, Ca2+ and Cl− concentrations, ion fluxes and osmotically driven volume changes were computed. Conventional ion channels and the NSCC were incorporated in the neuron membrane. Activation of NSCC conductance caused the appearance of paroxysmal afterdischarges (ADs) at parameter settings that did not produce AD in the absence of NSCC. The duration of the AD depended on the amplitude of the NSCC. Similarly, NSCC also enabled the generation of SD. We conclude that NSCC can contribute to the generation of epileptiform events and to spreading depression.
KeywordsCalcium depletion Calcium dependent current Non-specific cation current Epilepsy Seizure mechanisms Spreading depression
A TTX resistant non-selective cation current (NSCC) that is activated by low [Ca2+]o was first described by Hablitz et al. (1986) in chick dorsal root ganglion cells. Hablitz et al. (1986) suggested that a similar current in central neurons could play a role in the generation of seizures and spreading depression (SD). MacDonald, Xiong, and collaborators (Chu et al. 2003; Xiong and MacDonald 1999; Xiong et al. 1997) detected in mammalian central neurons an NSCC that is activated by the lowering of [Ca2+]o. This channel is maximally open when extracellular calcium concentration is zero, and at the normal [Ca2+]o of 1.2–1.5 mM it permits the flow of a small current which, presumably, adds to the resting conductance of the cell. This channel is not voltage dependent and it is about equally permeable to Na+ and K+ but not to Ca2+ or other divalent and multivalent ions. It is, however, blocked not only by Ca2+ but also by other divalent cations (Xiong et al. 1997). With view of the depolarization caused by the activation of the [Ca2+]o-sensitive NSCC, Xiong et al. (1997) also suggested that it could contribute to the generation of epileptiform seizures.
Swandulla and Lux (1985) discovered a non-selective cation channel in snail neurons that is activated by elevated intracellular Ca2+ concentration ([Ca2+] i ). A [Ca2+] i dependent NSCC (often denoted ICAN) has also been recorded in hippocampal pyramidal neurons (Caeser et al. 1993; Crepel et al. 1994) and substantia nigra inhibitory neurons (Lee and Tepper 2007). The TRPM4b channel, which is expressed in mammalian neurons, has been characterized as a [Ca2+] i -activated, Ca2+-impermeable, monovalent cation-permeable channel (Fleig and Penner 2004; Launay et al. 2002). Since uptake of Ca2+ into neurons results simultaneously in an increase in [Ca2+] i and a decrease in [Ca2+]o, if the two Ca2+ sensitive currents co-exist in the same cell, they are expected to reinforce one another and in fact could perhaps be generated by the same channel (see Section 3).
It has been known for a considerable time that low [Ca2+]o can induce seizure-like spontaneous discharges in intact brain (Sabbatani 1901) as well as in brain tissue slices (Jefferys and Haas 1982; Konnerth et al. 1986). Besides, at the onset of seizures, no matter how induced, [Ca2+]o drops precipitously (Heinemann et al. 1977, 1978; Pumain et al. 1983, 1985). Taken together, these two sets of observations suggest that the drop in [Ca2+]o, as also the increase in [K+]o, may be a link in one of the parallel feedback loops that can promote epileptic seizure discharges. The induction of spontaneous burst discharges by low [Ca2+]o has usually been attributed to reduced screening of neuron membrane surface charges leading to enhancement of voltage dependent inward Na+ current and hence reduced firing threshold (Hille 2001). Discovery of the [Ca2+]o-dependent NSCC adds another mechanism by which depletion of extracellular Ca2+ can promote neuron excitation even more powerfully.
In a study of the ion currents that are responsible for hypoxic spreading depression-like depolarization (HSD) in hippocampal tissue slices Müller and Somjen (2000) found that blockade of voltage gated Na+ as well as glutamate-controlled channels by a mixture of TTX, CPP and DNQX, while it postponed HSD, it did not usually prevent it. Adding Ni2+ to the blocking cocktail did, however, reliably suppress HSD (Müller and Somjen 1998). This lead to the conclusion that HSD is generated by the cooperation of several inward currents, one of which could be a non-specific cation current (NSCC) that is insensitive to TTX but is blocked by Ni2+ (Müller and Somjen 1998, 2000). This as yet unidentified current could be the [Ca2+]o sensitive current, which is indeed inhibited by divalent cations.
Earlier we have simulated seizure-like and SD-like processes (Kager et al. 2000, 2002, 2007; Somjen et al. 2008) in a neuron model based on the NEURON simulation environment of Hines, Moore and Carnevale (Hines and Carnevale 1997). It appeared that SD and HSD could be simulated in the presence of either a persistent Na+ current (INa,P) or one that resembled NMDA-controlled current (INMDA). When both were activated, SD occurred sooner and lasted longer than with either alone (Kager et al. 2002). Importantly, both simulated AD and SD required a substantial increase in [K+]o, illustrating pathological positive feedback where shifts in ion concentration influence the very channels through which the ions flowed in the first place (review in Somjen 2004).
We now report computer simulations that demonstrate that, similarly to altered [K+]o/[K+] i ratio, low [Ca2+]o can also promote seizure-like firing and SD-like depolarization due to activation of [Ca2+]o -sensitive NSCC. Some of the data appeared in abstract form (Somjen et al. 2004). A companion paper (Somjen et al. 2008) examines neuron-glia interaction mediated by ion flux.
1 Experimental procedures
The model was created in the simulation environment “Neuron” of Hines and Carnevale (1997). A neuron and a glia-endothelial buffer space were aligned in parallel and communicated with each other through a limited interstitial space (IS). The neuron consisted of a soma with unbranched basal and apical dendrites attached to the opposite ends. The electrical currents were carried by the appropriate ions according to the Goldman–Hodgkin–Katz current equations (Hille 2001) and the ions accumulated in the relevant compartments. Diffusion between compartments was also implemented and charge was conserved and electroneutrality maintained in all compartments. The ions taken into account were Na+, K+, Ca2+, Cl− and A−, the latter representing non-permeating anions in the neuron and in the glia cytosol. Ionic pumps were implemented that exchanged 3 Na+ for 2 K+, that exchanged 3 Na+ for 1 Ca2+ and ones that extruded Ca2+, all under the assumption of unlimited energy. (See Kager et al. 2000, 2002, 2007 and Somjen et al. 2008 for details).
Linear leak currents of Na+, and K+ plus a small NSCC (see below) and the pump currents controlled the resting potential of around −70 mV. Resting calcium concentration was set by adjusting the calcium extrusion mechanisms to reach a stable resting value around 55 nM at −70 mV.
The glial membrane contained leak conductances and pump currents (corresponding to the “passive” glia as defined in Somjen et al. (2008) but no Ca2+ conductance. Calcium was not represented in the glial cytosol. The leak potassium conductance was much more dominating than the one of the neuron membrane, yielding a resting membrane potential in the glial cell of −88 mV (for details see Kager et al. 2007, Somjen et al. 2008).
The invariant Cl− conductance in both neuron and glial compartments allowed maintenance of ion balance during unequal redistribution of cations and of osmotic pressure with associated volume changes. To avoid excessive complication of the model we did not include a Cl− pump. This is justified because GABA induced or voltage dependent Cl− currents were not represented. The resting Cl− concentration gradient was in equilibrium at resting membrane potential. Impermeant anion (A−) concentrations were chosen to balance the intracellular electrical charges. Changes in ionic strengths were translated into “virtual” osmotic pressure and resulted in volume changes of the relevant compartments with resulting concentration changes, according to the set of equations described in detail in Kager et al. (2002).
The main expansion of our model compared to its previous versions was the implementation of the Ca-sensitive nonselective cation conductance, gNSCC, (based on data given in Chu et al. 2003; Xiong et al. 1997 and Xiong, personal communication). The nonspecific cation channel senses the extracellular calcium concentration (Fig. 1(a)) and changes its conductance gNSCC for the monovalent cations K+ and Na+.
We assumed gNSCC to be independent of voltage (Xiong et al. 1997) and gave it equal permeability for Na+ and K+. We implemented the Ca2+ gating as a fourth order gating process which yielded an empirical opening time constant of around 166 ms and a closing time constant of 25 ms (Fig. 1(b)). These time constants were independent of current magnitude. The NSCC contributed to the resting conductance of the neuron. Whenever the conductance of the NSCC was altered, the leak conductances had to be slightly adjusted to maintain the balance of the total (net) membrane current and hence keep Vm steady at rest. The code for a representative selection of simulations presented in this paper will be added to the NEURON data base.
The role of the currents generating the voltage response of Fig. 2(b) are plotted in Fig 3 (g–i). As [Ca2+]o decreased in the interstitial space around the soma (Fig. 3(d,e)), the NSCC was turned on (Fig. 3(g,h)). During each action potential the NSCC showed brief positive spikes. Since the non-specific current is generated by the opposing simultaneous fluxes of Na+ and K+, the reversal potential of the NSCC is slightly negative relative to zero, (similalrly to EPSCs), and therefore during the positive overshoot of the action potentials the NSCC briefly reversed from inward to outward. In between spikes, the NSCC provided a sustained inward current. While the amplitude of the NSCC was small compared to the large surges of the transient Na+ current, INa,T, the NSCC provided just enough depolarization between action potentials to enable re-activation of the INa,T and hence the continuation of the AD (Fig. 3(h)). The cooperative action of the INSCC and the INa,T generated the pacemaker potentials that kept the firing going. It should be noted that stronger stimuli could trigger AD even in the absence of the NSCC, assisted in that case by INa,P (see Kager et al. 2007), but the presence of the NSCC lowered the threshold for AD considerably. The AD was self-limiting (Fig. 3(i)) because as EK subsided (Fig. 2(b)), the neuron began to repolarize, while at the same time [Ca2+]o rose toward its rest level (Fig. 3(i)) de-activating the NSCC, and these two shifts prevented re-activation of INa,T. EK began to recover as the ratio [K+]o/[K+] i was being restored by the combined actions of the neuron 3Na/2K pump and the glial buffer (see also Somjen et al. 2008).
We also simulated cerebral hypoxia. In order to explore whether INSCC by itself is capable of supporting hypoxic SD-like depolarization (HSD) we imitated the actions of TTX and glutamate antagonists (cf. experiments by Müller and Somjen 1998, 2000) by setting the conductances of I Na,T, I Na,P and I NMDA to zero. To simulate severe hypoxia, the 3Na+/2K+ pump of the neuron and the glial cell as well as the Ca pump of the neuron were turned off; similarly to our earlier study of HSD (Kager et al. 2002). In this condition SD-like depolarization was generated without applying a depolarizing current stimulus, provided that g NSCC was present (not illustrated). In a subsequent simulation, I NSCC was also eliminated, imitating a condition in which all major Na+ currents were blocked, and now turning off the pump simulating the “hypoxic” condition did not induce SD. Instead, the cell depolarized slowly due to the gradual increase of the [K+]o/[K+] i ratio while the dendritic membrane current (I mem) remained weakly outward, carried mainly by I K,DR and I K,SK. (not illustrated). This behavior is similar to that seen in hippocampal tissue slices after oxygen deprivation in the presence of TTX, glutamate receptor antagonists plus Ni2+ (Müller and Somjen 1998).
The feedback loops operating in these pathological conditions are schematically shown in Fig. 6. With view of the inherent potential for runaway excitation one can ask, as Jung and Tönnies (1950) have, how it is that all of us do not convulse every time we get out of bed. The processes that normally preserve the well controlled operation of cerebral tissue and those that upset its stability in diseased brains fall outside the present study.
The screening of negative surface charges by Ca2+ (see Hille 2001) was not represented in the model. When [Ca2+]o decreases, removal of the screen has the same effect as a decrease in the membrane potential and hence it augments the excitability of neurons and adds to the epileptogenic effect. Incorporating surface screening in a computer model requires an other, major investigation. Other variables remaining equal, decrease in [Ca2+]o would also diminish all inward Ca2+ currents. When, as in our simulations, the decrease in [Ca2+]o is the consequence of influx of Ca2+ into neurons, the accompanying increase in [Ca2+] i is expected to influence a number of calcium dependent processes. For our discussion the most important is the seemingly distinct NSCC that appears to be activated in hippocampal neurons, not by low [Ca2+]o, but by high [Ca2+] i (Swandulla and Lux 1985; Caeser et al. 1993; Crepel et al. 1994; Lee and Tepper 2007). Removing Ca2+ from the bathing fluid depresses the [Ca2+] i -sensitive NSCC, presumably because less Ca2+ is available to flow into cells and raise [Ca2+] i , whereas low [Ca2+]o enhances the [Ca2+]o-sensitive current. In intact brain, however, during high frequency firing or SD, whenever Ca2+ flows into cells [Ca2+] i increases at the same time as [Ca2+]o decreases and, apparently, both signals activate a current carried by monovalent cations. It is an open question whether they flow through two different channels or through the same one. Our simulations of the NSCC did not take into account the effect of the changes in [Ca2+] i .Had we incorporated the added effect of [Ca2+] i , it would presumably have changed only the scaling of gNSCC.
Besides AD, the NSCC also facilitated the ignition of simulated SD and hypoxic SD-like depolarization. HSD was induced even when the transient and persistent Na+ currents (I Na,T and I Na,P) as well as the NMDA-controlled current were turned off. Eliminating these major inward currents imitated the administration of TTX, CPP and DNQX to brain tissue slices. In this condition withdrawing O2 from hippocampal slices induced HSD of greatly delayed onset indicating the operation of an unidentified TTX insensitive and glutamate-independent current. Adding Ni2+ to the “blocking cocktail” did prevent HSD (Müller and Somjen 1998, 2000). The simulations supports the idea that the Ni2+-sensitive “missing” current could be the Ca2+ sensitive NSCC.
According to Xiong et al. (1997) the Ca2+ dependence of the NSCC can be described by a function with Hill coefficient close to unity. This value was derived from data recorded in neurons cultured from the brains of newborn rats. When we tested the model using 1.4 for this parameter, we found that at the resting potential of −70 mV a rather large NSCC was flowing. Since there is no published evidence for a large NSCC in normal adult cerebral neurons at rest, we used a larger value, 3.4, for the Hill coefficient for most of the simulations (Fig. 1). With this setting the NSCC in the un-stimulated neuron was small. Since, however, this choice was arbitrary, we also performed a number of simulations with a Hill coefficient of 1.4. The results were qualitatively similar with both settings of the Hill coefficient, but the less steep calcium-dependence resulted in greater readiness for AD generation (Fig. 4). Incidentally, the Hill coefficient of the [Ca2+] i -activated NSCC generated by TRPM4b channels was either 4 or 6 depending on the treatment of the preparation (Launay et al. 2002).
The model was, of course, simplified not just morphologically but also functionally. Some known ion currents were missing, as were the effects of pH change and of metabolic and other biochemical processes. We ignored these factors, because the questions asked were limited to a possible role of the [Ca2+]o sensing NSCC. Also absent were voltage controlled currents in the glial membrane. We did explore the effect of glial voltage gated K+ currents in a companion paper (Somjen et al. 2008). Lian and Stringer (2004) found some experimental support for extracellular calcium being regulated by astrocytes, and this also deserves further exploration.
Epileptic conditions can have many different causes, and by no means all have been discovered. It is possible that a shift in the [Ca2+] dependence, or increased conductance, or changing steepness of the activation function of either the [Ca2+]o- or the [Ca2+] i -sensitive NSCC, due either to genetic or to acquired anomaly, is one of them.
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