Calcium signals in motoneurones are shaped by multiple processes, including calcium influx, release, buffering, uptake and extrusion across cellular membranes (McBurny & Neering, 1987; Blaustein, 1988; Baimbridge et al. 1992; Neher, 1995; Lips & Keller, 1999; Palecek et al. 1999). Under pathophysiological conditions, disturbances of calcium homeostasis have been linked to severe motoneurone damage resulting from either excitotoxic stress or related cellular disruptions (DePaul et al. 1988; Choi, 1988; Rohstein & Kuncl, 1995; Krieger et al. 1996; Shaw & Ince, 1997; Roy et al. 1998; Carriedo et al. 2000). For example, in human amyotrophic lateral sclerosis (ALS) and related animal models of the disease, overexcitation of glutamatergic synapses and excess calcium influx have been associated with severe cell damage (Choi, 1987; Meldrum & Garthwaite, 1990; Rothstein et al. 1992, 1995; Carriedo et al. 1996; Medina et al. 1996; Shaw & Ince, 1997; Bar-Peled et al. 1999). Moreover, in genetically determined forms of ALS, mutations in either axonal neurofilaments (NFL) or the enzyme superoxide dismutase (SOD1) have been shown to trigger calcium-related motoneurone degeneration (Tu et al. 1996; Bruijn et al. 1998; Morrison & Morrison, 1998; Siklos et al. 1998; Williamson et al. 1998; Cleveland, 1999). The clinical importance of these mechanisms is illustrated by the finding that blockers of cellular calcium influx provide neuroprotection, where reductions of neurotransmitter- and voltage-dependent calcium influx display beneficial effects (Smith et al. 1992; Gurney et al. 1996; Roy et al. 1998).
An important question is related to the role of individual cellular parameters for calcium-mediated neuronal damage. For example, studies on hippocampal cells have suggested that high calcium buffering enhances neuronal vulnerability, mainly by disrupting calcium-dependent inactivation of voltage-activated calcium channels (Chad, 1989; Abdel-Hamid & Baimbridge, 1997; Nägerl & Mody, 1998). This view has received support from studies of transgenic animals, where genetic ‘knock-out’ of the cytosolic calcium buffer calbindin protected hippocampal cells against ischaemia-related degeneration (Klapstein et al. 1998). On the other hand, several studies have demonstrated that decreased buffer concentrations can enhance neurodegeneration in other model systems (Alexianu et al. 1994, 1998; Tymianski et al. 1994; Reiner et al. 1995; Roy et al. 1998). In those studies, neuroprotective effects of buffer elevation were mainly attributed to reduced peak amplitudes of intracellular free calcium levels for a given calcium influx.
In this report, we investigated the potential role of endogenous calcium homeostasis for selective motoneurone vulnerability. More specifically, our study was motivated by the fact that selected populations of motoneurones in the nucleus hypoglossus or spinal cord are particularly impaired, while others like oculomotor neurones are essentially unaffected. This is a well-known phenomenon in advanced stages of human ALS, but also in associated animal models of motoneurone disease (Ince et al. 1993; Elliot & Snider, 1995; Reiner et al. 1995). By performing a quantitative analysis based on the ‘added buffer’ approach (Neher & Augustine, 1992; Neher, 1995), we were able to compare in detail individual parameters of cellular calcium homeostasis in oculomotor neurones with values in hypoglossal and spinal cells (Lips & Keller, 1998; Palecek et al. 1999). In summary, our measurements indicate that the calcium buffering capacities in oculomotor neurones are comparable to those found in hippocampal and cortical cells (Neher, 1995), but 5- to 6-fold higher relative to those of hypoglossal and spinal motoneurones. Accordingly, our results are in agreement with the view that the remarkable stability of oculomotor neurones during ALS-related motoneurone disease partially results from a specialised calcium homeostasis with high calcium buffering.
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In this report, we describe a quantitative analysis of endogenous calcium homeostasis in oculomotor neurones, having performed simultaneous patch-clamp recordings in sliced tissue and microfluorometric calcium measurements. For this, neurones in the oculomotor nucleus (Fig. 1A) from 2- to 6-day-old mice were investigated using infrared differential interference contrast optics (Stuart et al. 1993), where cells could be visually identified by their large somata and their characteristic dendritic arborisation (Fig. 1B). After whole-cell configurations had been established, motoneurones were filled with the calcium indicator dye fura-2 (Fig. 1C) until a stable steady-state situation was reached (usually after 5–6 min). Oculomotor neurones displayed input resistances of 131 ± 13 MΩ (s.e.m.; n= 22 cells) and resting membrane potentials were around −65 mV. To analyse calcium signalling, cells were normally stimulated by somatic depolarisations to +10 mV lasting 500 ms. To provide well-controlled experimental conditions, voltage-clamp protocols started from holding potentials of −70 mV to primarily activate high voltage-activated (HVA) calcium channel types (Snutch & Reiner, 1992).
Figure 2A illustrates stimulus-induced fluorescence responses at 360 nm and 390 nm for different time intervals after establishing the whole-cell recording configuration. F360 provides an estimate of fura-2 loading of cells and downward deflections in F390 (dF390) reflect depolarisation-induced elevations in cytosolic free calcium levels. Exchange of pipette and somatic solutions occurred with a time constant of 2.8 ± 0.8 min (s.e.m.; n= 5 cells) measured by calcium-independent fluorescence F360 (Fig. 2B). An important assumption of our quantitative analysis is that the absolute amount of calcium influx per depolarisation stimulation remains constant during an experiment. To check this, we determined calcium influx from the product of cytosolic calcium amplitudes and decay times (Fig. 2C). Such ‘calcium integrals’ increased only slightly during an experiment, indicating that the assumption was justified. A slight increase in calcium influx was observed for longer whole-cell recording times (Fig. 2C), presumably resulting from indicator dye filling of cells known to reduce calcium-dependent inactivation of voltage-dependent calcium channels (Palecek et al. 1999). Figure 2D further illustrates this effect as a function of the ‘exogenous’ buffering capacity of fura-2 in the cytosol.
Figure 2. Stimulation-induced fluorescence changes during gradual increases in cytosolic fura-2
A, calcium-dependent (F390) and -independent (F360) fluorescence signals during different time intervals after establishing the whole-cell patch-clamp configuration. At different time periods of the filling process depolarising voltage-steps (+10 mV) of 500 ms duration were performed (see arrow). Note the increasingly prolonged decay times of calcium-dependent F390 signals at the different times after cell rupture. Increased deflections dF390 reflected increasing fractions of cytosolic calcium concentrations captured by fura-2 (500 μM in the pipette solution). B, calcium-independent fluorescence F360 in bead units (BU) plotted as function for different time intervals after establishing the whole-cell recording configuration. F360 signal increase represents the increase in cytoplasmic indicator dye (fura 2) concentration. The continous line represents a single exponential least-square fit (time constant 2.54 min). C, integrated calcium responses as a function of whole-cell recording time. Integrated responses slightly increased during dye loading phase after rupture. Data points were connected with a line fit. •, calcium signals shown in Fig. 2A. D, integrated calcium signals plotted versus calcium binding capacity of fura2 (κB‘). κB′ strongly depends on increasing cytoplasmic fura-2 concentration during dye loading phase (see eqn (3)).
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As indicated above, calcium-dependent deflections dF390 were proportional to the relative amount of cytosolic calcium concentrations ‘captured’ by fura-2. Accordingly, dF390 increased with fura-2 concentrations where the maximum value indicated that the indicator dye had largely overruled the endogenous buffers of the cell. In quantitative terms, this process is approximated by the equation (see Methods):
where dFmax denotes the saturating value of dF390 for very high fura-2 concentrations, κS and κB′ denote the endogenous buffering capacity of the cell and the exogenous buffering capacity of the indicator dye, respectively. For our present purposes, this equation permitted an estimate of κS by measuring dF390 for defined depolarisations at different fura-2 loading states. As displayed in Fig. 3, dF390 was plotted as a function κB′ and data points were fitted with eqn (6). This revealed a value dFmax=3.34 ± 0.11 BU and an endogenous buffering capacity κS= 277 ± 24 (n= 14 cells) for oculomotor neurones.
Figure 3. Determination of the endogenous Ca2+ binding capacity by investigating ΔF390 as a function of κB‘
Depolarisation-induced deflections in calcium-dependent fluorescence ΔF390 as a function of the binding capacity of fura-2. The analysis reveals κS=277.0 ± 23.8 in oculomotor neurones and 3.34 ± 0.11 fluorescence bead units (BU) as the maximum value ΔFmax. The half-maximum value ΔFmax/2 reflects the endogenous binding capacity κS (dashed line).
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Calcium responses depended strongly on membrane depolarisation as illustrated in Fig. 4. Membrane depolarisations positive to −40 mV were necessary to evoke a robust HVA-mediated calcium response. During depolarising intervals, calcium concentrations increased in a linear manner, consistent with the idea that constant, voltage-dependent calcium influx was an essential determinant. Calcium responses reached a saturating level for membrane depolarisations positive to 0 mV with a half-maximum response at −20 mV. For a systematic analysis of calcium responses and homeostasis, stimulation episodes were separated by time intervals of 30–80 s to achieve complete recovery of calcium transients between consecutive stimulations. Under such conditions, resting calcium concentrations were found to be [Ca2+]rest= 77 ± 24 nM (n= 12 cells). Furthermore, reproducible calcium signals were obtained during standard experiments lasting up to 1 h.
Figure 4. Depolarisation-induced calcium responses in oculomotor neurones
Somatic calcium responses during defined voltage steps from −70 to +40 mV (+10 mV step size) lasting 1 s. To isolate high voltage activated calcium channel (HVA) responses from low voltage (LVA) types, the resting membrane potential was held at −70 mV at the beginning of membrane depolarisation. Voltage steps positive to −40 mV led to robust somatic calcium responses. Half-maximum calcium responses were reached at −20 mV. Membrane depolarisations positive to 0 mV led to saturation of the calcium response. At voltages greater than +30 mV calcium response decreased. Recovery from elevated calcium concentrations to basal levels was adequately described by a mono-exponential function (y=A+Bexp(-Ct)); where A, B and C are constants.
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To define the relevant parameters of endogenous calcium homeostasis, amplitudes and decay times of calcium transients were determined for different fura-2 loading states. Figure 5 shows decay times of depolarisation-induced calcium transients as a function of κB‘. As described in Methods, the quantitative model of somatic calcium homeostasis predicts that decay times of calcium transients are a linear function of fura-2 buffering capacity:
(Neher & Augustine, 1992; Helmchen et al. 1997), where γ denotes the effective extrusion rate. Figure 5 displays the analysis of τ as a function of κB‘, where endogenous calcium binding capacities were found to be 264 ± 25 (n= 11 cells). This indicated that only 1 out of 264 calcium ions in the cytosol contributed to the free calcium concentration. An estimate of the ‘effective’ extrusion rate was obtained from the linear slope of the function τ(κB‘), yielding a value γ= 156 ± 20 s−1 (n= 11 cells). As illustrated in Fig. 6, inverse amplitudes of calcium transients provided an independent estimate of the endogenous buffering capacity. This analysis identified a value κS= 258 ± 29 (n= 18), which was in reasonable agreement with previous approximations from decay times and dF390. Measurements of amplitudes as a function of κB′ also provided an estimate for the overall calcium influx into the soma given by (see Methods):
where F denotes the Faraday constant and A calcium amplitude. According to this analysis, 500 ms depolarisations were associated with a calcium-mediated charge influx of 54.9 ± 7.7 pC pl−1 into the somatic compartment.
Figure 5. Decay times of calcium transients as a function of calcium binding capacity of fura-2 (κB‘)
A, calcium transients evoked by 500 ms voltage steps to +10 mV. Decay phases of calcium transients were approximated by a single exponential function. Left trace illustrates a Ca2+ response soon after establishing the whole cell configuration corresponding to low exogenous Ca2+ binding capacity of fura-2 (κB‘) and rapid decay time. Right trace illustrates a Ca2+ response after complete filling of the neurone with fura-2, corresponding to high exogenous Ca2+ binding capacity (κB‘) and slow decay time. Pipette solution contained 1000 μM fura 2. B, decay time constants were plotted against Ca2+ binding capacities, κB‘, of fura-2 and mag-fura-5. Concentrations and binding capacities of indicator dyes were estimated as described in Methods. A linear regression of τversusκB′ was determined according to a least-square fit. The negative intercept of the regression line yielded a calcium binding capacity of 263.8 ± 25.1 (11 cells) for oculomotor neurones. Calcium decay time constant at κB′= 0 was extrapolated to 1.7 ± 0.1 s (11 cells).
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Figure 6. Amplitude of calcium transients as determinants of the endogenous Ca2+ binding capacity (κS)
The inverse amplitude of calcium transients was plotted as a function of calcium binding capacity of fura-2 (κB‘). The straight line shows a linear regression and the negative x-axis intercept reflects κS+ 1, thus revealing an endogenous Ca2+ binding capaciy of 258.3 ± 28.7 (n= 18). The intercept with the y-axis identified a value A0=1.08 ± 0.11μM, thus providing an estimate of calcium amplitudes under dye-free conditions.
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During patch-clamp experiments, fura-2 loading of cells modified calcium kinetics by retarding recovery phases of calcium transients and reducing peak amplitudes. To approximate calcium dynamics in the absence of indicator dye, we used the quantitative model of somatic calcium homeostasis illustrated in Fig. 7. A linear extrapolation of Fig. 5 to κB′= 0 identified a recovery time constant of 1.7 ± 0.2 s (21°C) in the absence of fura-2. Fluorescence measurements also indicated that a depolarisation to +10 mV for 500 ms (500 μM fura-2 corresponding to κB′= 493) resulted in a somatic calcium elevation of 0.37 ± 0.04μM (Fig. 6). By performing a linear extrapolation of calcium amplitudes to κB= 0 (see Fig. 6), we estimated an average elevation of 1.08 ± 0.11μM for depolarisation-induced calcium transients under dye-free conditions.
Figure 7. Quantitative model of calcium homeostasis in oculomotor neurones
A, scheme of the one compartment model and the main parameters that determine somatic calcium responses. κB′ and κS denote the exogenous and endogenous buffering capacities of the cell, respectively. The ‘effective’ extrusion rate γ provides a quantitative description of combined calcium extrusion across the plasma membrane and calcium uptake in intracellular stores. B, simulation of a somatic calcium response for a calcium influx corresponding to 54.9 pC pl−1 (500 ms lasting depolarisation to +10 mV) in the absence of indicator dye κB′= 0) for oculomotor neurones. C, corresponding simulation for spinal motoneurones (calcium influx 54.9 pC pl−1), values for κS, γ and τ were taken from measurements previously described by Palacek et al. 1998.
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One of the most interesting aspects of cellular calcium homeostasis is related to the parameters that specifically protect oculomotor neurones against calcium-related degeneration and motoneurone disease (Ince et al. 1993; Elliot & Snider, 1995; Reiner et al. 1995; Shaw & Ince, 1997; Morrison & Morrison, 1998). As illustrated in Fig. 7, we utilised the quantitative model of calcium homeostasis to evaluate specific scenarios of calcium signalling. Figure 7B displays the calculated amplitude and time course of a somatic calcium transient in an oculomotor neurone evoked by a 500 ms depolarisation to +10 mV (influx of 54.9 pC pl−1). For comparison, Fig. 7C shows a transient evoked by the same influx into a spinal motoneurone, which was calculated from a corresponding model previously described (Palecek et al. 1999). Transients in spinal neurones were characterised by 5.3-fold larger amplitudes, but rapidly recovering calcium transients, resulting from comparable extrusion rates and lower buffering capacities. In more general terms, parameters of calcium responses are plotted in Fig. 8A as a function of endogenous buffering capacity. In this case, extrusion rates were assumed to be 145 s−1, which was similar to values found in the somata of oculomotor and spinal motoneurones. It is interesting to note that for a given calcium influx, amplitudes of calcium transients display a hyperbolic increase for reduced endogenous buffering, and that low capacities (500 ms depolarisation; influx of 54.9 pC pl−1) are associated with an average somatic calcium elevation of several micromolar.
Another interesting aspect of differential calcium homeostasis in motoneurones is related to the volume of localised calcium elevations around open calcium channels. To provide a quantitative estimate, we determined the diameter of a ‘shell’ of ‘unbuffered’ calcium concentrations in the vicinity of an open calcium channel (Neher, 1998; see also Methods). Figure 8B displays this parameter as a function of endogenous buffering capacity by assuming that endogenous buffers are represented by large proteins with a kon rate of 5 × 108μm s−1 and a low affinity for calcium according to Kd= 10 μM >> [Ca2+]i (see also Methods, and eqn (8)). High endogenous buffering with κS= 264 is associated with small calcium domains with an estimated diameter L= 26 nm, while κS= 50 found for spinal neurones accounts for L= 60 nm (see also Table 1). This provides one example for the more general principle that higher buffers facilitate the ‘equilibration’ of local gradients (Roberts, 1994; Neher, 1995; Klingauf & Neher, 1997). One underlying assumption is that calcium influx rates are small, so that ‘buffer depletion’ is negligible. For higher rates where calcium influx ‘depletes’ local concentrations, the relative fraction of calcium ions contributing to [Ca2+]i is significantly increased (Roberts, 1994; Klingauf & Neher, 1997; Neher, 1998). Again, this effect is particularly prominent under low buffering conditions, further magnifying the relatively large size of local calcium domains in spinal and hypoglossal motoneurones compared with oculomotor cells.
Table 1. A quantitative comparison of calcium homeostasis in selectively vulnerable and resistant motoneurones
|Cell type||OMN selectively resistant||SMN* selectively vulnerable||HMN* selectively vulnerable|
|L||26 nm||60 nm||66 nm|
|τ||1.7 s||0.4 s||0.7 s|
|A||1.08 μM||5.72 μM||6.96 μM|
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The ‘added buffer’ approach (Neher & Augustine, 1992) permitted us to address this question in a quantitative way. We found endogenous calcium binding ratios of κS= 264 in oculomotor neurones, which was 5–6 times larger compared with binding ratios κS= 41 and κS= 50 in vulnerable hypoglossal and spinal motoneurones, respectively (see also Table 1; Lips & Keller, 1998; Palecek et al. 1999). This value was 3.5 times lower compared with those found in Purkinje cells (κS= 900; 6-day-old mice; Fierro & Llano, 1996), but comparable to κS= 160–207 in hippocampal CA1 neurones (Helmchen et al. 1996). Extrusion rates in oculomotor neurones were found to be γ= 156 s−1, which was similar to γ= 140 s−1 found in spinal motoneurones. These rates were small, relative to those found in the calyx of Held (400 s−1, 21°C; Helmchen et al. 1997), but several times faster compared with hypoglossal motoneurones (60 s−1; Lips & Keller, 1998) and adrenal chromaffin cells (13 s−1, Neher & Augustine, 1992). Calcium influx during membrane depolarisation was also heterogenous in different motoneurone populations. For a 500 ms depolarisation to +10 mV, we found an influx of 55 ± 8 and 21 ± 5 pC pl−1 in oculomotor and spinal motoneurones, respectively. Our results therefore indicate that differences in buffering capacity, but not in ‘effective’ extrusion rates or voltage-dependent calcium influx, provide a plausible cellular explanation for selective vulnerability (Lips & Keller, 1998; Palacek et al. 1999).
High calcium buffering capacities in ALS-resistant motoneurones are consistent with earlier expression studies of calcium binding proteins (Baimbridge et al. 1992; Ince et al. 1993; Alexianu et al. 1994; Elliot & Snider, 1995; Reiner et al. 1995). In a comparative investigation, DePaul et al. (1988) demonstrated high expression of calcium binding proteins in oculomotor neurones in an immunocytochemical study of selective motoneurone vulnerability. In this case, high buffering in oculomotor neurones has been associated with elevated expression of parvalbumin, a calcium binding protein that is expressed in low concentrations in hypoglossal and spinal motoneurones. Moreover, several in vitro cell culture studies have demonstrated that elevated calcium buffering reduces ALS-related motoneurone damage, providing support for the idea that increased buffer concentrations display beneficial protection (Tymianski et al. 1994; Ho et al. 1996; Roy et al. 1998).
As our investigations were carried out at room temperature, an important question is related to the changes in calcium signalling under physiological temperatures. Previous studies have indeed demonstrated that extrusion rates are strongly temperature dependent, with a Q10 around 2 (Helmchen et al. 1997; Lips & Keller, 1999). This suggested a 3.2-fold acceleration of our experimentally determined transport rates under physiological conditions. As previously shown (Helmchen et al. 1997), cytosolic calcium buffering capacities display only little temperature dependence, indicating that values determined under our experimental conditions were similar to those found in more physiological states. Since our prime objective was a comparison between selectively vulnerable and resistant motoneurones, the most important precondition for the validity of our analysis was that experimental parameters were identical.
Heterogenous effects of calcium buffers in different models of neurodegeneration
In a different line of research, earlier studies have provided evidence that reduced calcium buffering capacities protect neurones against calcium-mediated damage (‘neuroprotection type I’) (Chad, 1989; Abdel-Hamid & Bambridge, 1997; Nägerl & Mody, 1998). In the well-studied example of hippocampal neurones, lowered buffering protected cells against ischaemia-induced degeneration, presumably by enhancing local accumulation of [Ca2+]i. For hippocampal calcium channels characterised by rapid, calcium-dependent inactivation, local accumulation provides a negative feedback mechanism that prevents excess calcium influx during persistent depolarisations. This concept has received support from studies on transgenic animals (Klapstein et al. 1998), where ‘knock-out’ of the calcium-binding protein calbindin protected hippocampal cells during ischaemic episodes. In general, this particular result is explained by several models. One states that calcium calbindin itself mediates toxic effects of large calcium loads, a possibility that cannot be completely excluded based on the data presently available. Electrophysiological studies of voltage-dependent calcium influx, however, supported the notion that reduced calcium buffering enhances calcium channel inactivation and diminishes net calcium loads, thus providing neuroprotective effects in hippocampal neurones (Klapstein et al. 1998).
This concept was inappropriate for interpretation of the results presented in this report, where motoneurone populations with high buffering capacities were best protected (‘neuroprotection type II’; Ince et al. 1993; Elliot & Snider, 1995; Reiner et al. 1995; McMahon et al. 1998). A plausible explanation is that differences in ‘type I’ and ‘type II’ neuroprotection depend on the net effect of variation in buffering capacity. In highly buffered cells like hippocampal neurones, increased buffering magnifies the risk for disruptions in localised calcium regulation (Fig. 8B). In hypoglossal and spinal motoneurones characterised by low endogenous buffering, elevated buffer concentrations apparently prevent excess accumulation of calcium levels around open calcium channels, thus keeping [Ca2+]i in a subcritical range (Fig. 8A). During ‘excitotoxic’ conditions, this reduces the risk for activation of ‘apoptotic’ second messenger cascades including calcium-dependent lipases and phosphatases, which are known to depend on calcium elevations above the micromolar domain (Choi, 1988; Baimbridge et al. 1992; Alexianu et al. 1994; Krieger et al. 1994). Prevention of local calcium accumulation could also reduce the risk of mitochondrial calcium overload and production of reactive oxygen species (ROS), which have been shown to occur in vulnerable motoneurone populations during excess calcium influx through glutamate receptor channels (Carriedo et al. 1996, 2000).
Given the broad diversity of molecular mechanisms associated with motoneurone degeneration (Choi, 1988; Smith et al. 1992; Reiner et al. 1995; Rothstein et al. 1995; Abdel-Hamid et al. 1997; Bruijn et al. 1998; Morrison & Morrison, 1998; Cleveland, 1999), it is clear that amyotrophic lateral sclerosis represents a multifactorial disease. It is interesting to note, however, that most mechanisms that have been linked to ALS share a calcium-dependent signal component. For example, several studies have shown that calcium-permeable glutamate (AMPA) receptors are highly expressed in motoneurone populations that are particularly impaired (Shaw & Ince, 1997; Roy et al. 1998; Bar-Peled et al. 1999; Shaw et al. 1999). Although such receptors have also been demonstrated in other neurone types (Shaw & Ince, 1997; Carriedo et al. 2000; Vandenberghe et al. 2000), low buffering in vulnerable motoneurones accounts for a 5- to 6-fold larger AMPA-receptor-mediated calcium response compared with those in oculomotor, cortical or hippocampal cells (Fig. 8). Another risk factor for ALS is an impairment of synaptic glutamate transport (Rothstein et al. 1992, 1995; Trotti et al. 1999), where resulting overactivation of excitatory synapses is thought to be the key trigger for calcium-dependent degeneration of vulnerable cells. Although such a large spectrum of calcium-dependent mechanisms has been associated with ALS (Alexianu et al. 1994; Shaw & Ince, 1997; Morrison & Morrison, 1998; Cleveland, 1999), the resulting, selective pathology of motoneurones is surprisingly similar. Based on the quantitative investigation of calcium homeostasis presented in this report, this similarity is well explained by the unifying model that low endogenous calcium buffers represent a dominating risk factor.
Implications of differential calcium homeostasis for physiological and pathophysiological conditions
Given the increased risk of neuronal damage imposed by low endogenous buffering, it is important to note that low buffering also provides a valuable functional advantage in rhythmically active cells. As pointed out in Fig. 8A, low buffering directly accounts for rapid recovery times of calcium transients if all other parameters are held constant (Neher & Augustine, 1992). In rhythmically active hypoglossal or spinal motoneurones, where action-potential related calcium oscillations occur at maximum frequencies up to 10 Hz (Ladewig & Keller, 1998; Lips & Keller, 1999; Palecek et al. 1999), rapid recovery of calcium transients is an essential requirement for physiological cell function. Indeed, the recently demonstrated neuroprotective effect of ATP-generating creatine in a mouse model of human ALS might partially be explained by an acceleration of recovery rates of activity-related calcium transients (Klivenyi et al. 1999). Although fast recovery times can also be achieved for high buffering by more effective extrusion, this strategy is associated with higher energy consumption in a permanently oscillating system (Lips & Keller, 1999; Palecek, 1999). Taken together, our results therefore indicate that cellular adaptations that account for rapid calcium signalling at relatively low energy cost also enhance a selective vulnerability of spinal and hypoglossal motoneurones during pathophysiological states and ALS-related motoneurone degeneration.
With respect to clinical motoneurone neuroprotection, our observations provide several arguments. First, elevation of cytosolic buffers could achieve neuroprotection by approximating calcium buffering conditions of ALS-resistant cells. This could reduce amplitudes of ‘excitotoxic’ calcium transients and thus prevent initiation of neurodegenerative cascades (Fig. 8A). However, buffer elevation is also associated with a retardation of calcium oscillations during rhythmic activity, which could lead to increased accumulation of ‘excitotoxic’ calcium levels in rapidly bursting hypoglossal or spinal cells (Ladewig & Keller, 1998; Palecek et al. 1999; Lips & Keller, 1999). As mentioned above, this difficulty could be partially overcome by increasing cellular ATP levels and thus accelerating calcium recovery, but more detailed studies are necessary to further validate this hypothesis. Another argument results from the alteration of spatial calcium profiles after buffer elevation. As indicated in Fig. 8B, increased buffering reduces the size of localised calcium domains (Roberts, 1994; Klingauf & Neher, 1997; Neher, 1998), which presumably supports neuroprotective conditions in oculomotor neurones and cell-culture models of motoneurone disease (Alexianu et al. 1994; 1998; Shaw & Ince, 1997; Roy et al. 1998). Under more complex in vivo conditions characteristic of ALS patients, however, reduction of calcium domains could also disrupt important local regulatory mechanisms and thus even enhance degeneration. In summary, it is necessary to perform a detailed analysis of spatio-temporal profiles of physiologically relevant calcium signals in selectively vulnerable and resistant motoneurones before a final conclusion about the clinical value of elevated buffer concentrations can be drawn.