Activity-Related Calcium Dynamics in Motoneurons of the Nucleus Hypoglossus From Mouse

Mario B. Lips and Bernhard U. Keller

Zentrum Physiologie und Pathophysiologie, Universität Göttingen, 37073 Göttingen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lips, Mario B. and Bernhard U. Keller. Activity-Related Calcium Dynamics in Motoneurons of the Nucleus Hypoglossus From Mouse. J. Neurophysiol. 82: 2936-2946, 1999. A quantitative analysis of activity-related calcium dynamics was performed in motoneurons of the nucleus hypoglossus in the brain stem slice preparation from mouse by simultaneous patch-clamp and microfluorometric calcium measurements. Motoneurons were analyzed under in vitro conditions that kept them in a functionally intact state represented by rhythmic, inspiratory-related bursts of excitatory postsynaptic currents and associated action potential discharges. Bursts of electrical activity were paralleled by somatic calcium transients resulting from calcium influx through voltage-activated calcium channels, where each action potential accounted for a calcium-mediated charge influx around 2 pC into the somatic compartment. Under in vivo conditions, rhythmic-respiratory activity in young mice occurred at frequencies up to 5 Hz, demonstrating the necessity for rapid calcium elevation and recovery in respiratory-related neurons. The quantitative analysis of hypoglossal calcium homeostasis identified an average extrusion rate, but an exceptionally low endogenous calcium binding capacity as cellular parameters accounting for rapid calcium signaling. Our results suggest that dynamics of somatic calcium transients 1) define an upper limit for the maximum frequency of respiratory-related burst discharges and 2) represent a potentially dangerous determinant of intracellular calcium profiles during pathophysiological and/or excitotoxic conditions.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In mammals, motoneurons of the nucleus hypoglossus have been associated with controlled movements of the tongue and, accordingly, have been linked to vital physiological functions like swallowing, suckling, or mastication (Lowe 1980). A defined pattern of rhythmic hypoglossal motoneuron activity is correlated with the respiratory rhythm generated in close proximity to the nucleus ambiguus in the pre-Bötzinger complex of the lower brain stem (Johnson et al. 1994; Smith et al. 1991, 1992). Rhythmic inspiratory-related activity of hypoglossal motoneurons has been monitored in different experimental systems, including in vivo recordings in various mammals (Kubin et al. 1996; Okabe et al. 1994; Pierrefiche et al. 1997; Richmonds and Hudgel 1996), in vitro recordings of en bloc preparations containing the brain stem and spinal cord (Suzue 1984), and patch-clamp recordings from brain stem slice preparations (Elsen and Ramirez 1998; Frermann et al. 1999; Smith et al. 1991). Both in vivo and in vitro investigations of hypoglossal motoneurons have provided a wealth of information about the underlying electrophysiological parameters, including the passive membrane properties in different postnatal stages of development (Berger et al. 1996; Viana et al. 1994), the functional characteristics of synapses and their modulation by different second-messenger systems (e.g., O'Brien et al. 1997; Umemiya and Berger 1995b), and the functional profile of voltage-dependent conductances (Bayliss et al. 1995; Umemiya and Berger 1994; Viana et al. 1993a,b).

Intracellular calcium signals and associated second-messenger cascades represent a strong determinant of hypoglossal motoneuron activity under physiological conditions. For example, voltage-dependent calcium influx plays a prominent role in regulating action potential frequency during clusters of action potential discharges ("bursts"), where openings of high-voltage-activated calcium channels (HVA channels) have been shown to shape action potential afterhyperpolarizations via opening of calcium-activated K+ channels (KCa+ channels) (Bayliss et al. 1995; Viana et al. 1993a,b). Accordingly, the timing and frequency of action potential discharges is closely linked to cytosolic calcium levels during bursts (Viana et al. 1993a,b). Besides action potential-induced calcium influx, previous investigations have demonstrated at least four types of voltage-activated Ca2+ channels in hypoglossal neurons, including low-voltage activated (LVA) and three different HVA channel types (Bayliss et al. 1995; Umemiya and Berger 1994, 1995a,b). Moreover, the observation of synaptically activated glutamate receptors of the N-methyl-D-aspartate (NMDA) receptor type has suggested that subsynaptic calcium influx also contributes to calcium responses (O'Brien et al. 1997; Vanselow et al. 1998), where the precise role of specific calcium influx pathways for the overall pattern of electrical activity is only little understood.

Although the molecular basis of calcium signaling has been investigated in great detail, much less is known about the integration and superposition of calcium responses in hypoglossal motoneurons in their physiological state. In hypoglossal neurons associated with rhythmic-respiratory activity, underlying calcium signals are particularly interesting because the respiratory network of young mice may operate at breathing rates up to 5 Hz (Jacquin et al. 1996). This leaves only 200 ms to elevate and recover cytosolic calcium levels between inspiratory bursts. Accordingly, one objective of our study was to analyze the cellular parameters of calcium homeostasis that account for rapid calcium signaling. A second objective was to better understand the previously described, selective vulnerability of hypoglossal motoneurons to calcium-related excitotoxic stress (Doble 1995; Krieger et al. 1994; Reiner et al. 1995). In this context, cell-specific adaptations of hypoglossal calcium homeostasis have been discussed as important cellular determinants of neuronal vulnerability (Ho et al. 1996; Kiernan and Hudson 1991; Krieger et al. 1996). Within the frame of the present investigation, the quantitative analysis of calcium homeostasis might further identify specific cellular parameters responsible for selective damage of hypoglossal cells. Part of this work has been published in preliminary form (Lips and Keller 1997).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Functional analysis of hypoglossal motoneurons in brain stem-spinal cord preparations and brain stem slices

To analyze the rhythmic respiratory-related activity of hypoglossal neurons in vitro, electrophysiological recordings were performed both in brain stem-spinal cord and isolated slice preparations. The in vitro brain stem-spinal cord preparation was chosen for rhythmic analysis because it permits simultaneous recording of respiratory discharges from hypoglossal nerves and from C1-C4 cervical nerves that innervate the diaphragm (Lips and Keller 1998). Brain stem-spinal cord and slice preparations were obtained from 2- to 6-day-old mice. Rhythmic-inspiratory discharges were recorded with suction electrodes from roots of hypoglossal and cervical nerves according to previously described methods (Brockhaus et al. 1993; Lips and Keller 1998; Smith et al. 1991). Patch-clamp experiments on transverse slices of the brain stem with a thickness of 150-600 µm were performed as previously described (Edwards et al. 1989; Keller et al. 1991; Titz and Keller 1997). For patch-clamp analysis of rhythmic activity, slice preparations with a thickness of 300-600 µm were utilized. If not indicated otherwise, analysis of calcium homeostasis was performed on slices with a thickness of 150-250 µm. All animal experiments were carried out in accordance with the guidelines of the Ethics Committee of the Medical Faculty of the University of Göttingen. Animals were anesthetized with ether and decapitated, and brains were removed and subsequently cooled to 4°C. Slices were maintained at room temperature in continuously bubbled (95% O2-5% CO2) bicarbonate buffered saline (in mM: 118 NaCl, 3 KCl, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4, 1.5 CaCl2, and 20 glucose) at pH 7.4. Before the recordings, slices were incubated for at least 1 h to allow recovery. For whole cell recordings (Hamill et al. 1981), slices were placed in the recording chamber under a Zeiss upright microscope and continuously superfused with the solution described above (>= 2 ml/min). If not indicated otherwise, experiments were carried out at room temperature (22 ± 1°C).

Measurements of breathing rates

Breathing rates of mice were measured as previously described by Erickson et al. (1996). In short, individual animals were placed in a chamber (20 ml) that was connected to a differential pressure transducer (model DP103-14, Validyne Engineering, Northridge, CA). Temperature was monitored and held constant at 31°C while pressure was measured with reference to a second chamber of identical volume. Each chamber was connected to atmospheric pressure through a slow leak (27-gauge hypodermic needle) to minimize pressure differences. Breathing rates were recorded by using the EPC-9 software for data acquisition and analysis.

Patch-clamp recordings

Patch-clamp experiments on slice preparations were performed as previously described (Titz and Keller 1997; Weigand and Keller 1998). The intracellular pipette solution contained (in mM) 140 KCl (alternatively 140 CsCl), 10 HEPES, 2 MgCl2, 4 Na2-ATP, and 0.4 Na-GTP (adjusted to pH 7.3 with KOH or CsOH). Fura-2 was bought from Molecular Probes (Eugene, OR) and used in concentrations in the range of 50 µM to 1 mM in the pipette solution. Patch pipettes were pulled from borosilicate glass tubing (Hilgenberg, Malsfeld, Germany) and heat polished before use. When filled with intracellular solution, they had resistances of 2.0-3.5 MOmega . Voltage-clamp recordings were performed with a patch-clamp amplifier (EPC-9, HEKA, Lambrecht, Germany) employing optimal series resistance compensation (Lips and Keller 1998; Titz and Keller 1997). The series resistance of hypoglossal motoneurons before compensation was typically 8-15 MOmega . Cells with series resistances higher than 15 MOmega were not included in the analysis. Series resistance compensation was set to 50-60%. No compensation was made for liquid junction potentials. When not stated otherwise, whole cell currents were recorded with sampling frequencies of 100 Hz to 5 kHz and filtered (4-pole Bessel filter 2.9 kHz) before analysis.

Microfluorometric calcium measurements

Intracellular calcium concentrations were measured according to previously described methods (Frermann et al. 1999; Lips and Keller 1998; Palecek et al. 1999). In short, fluorescence signals were detected by a photomultiplier mounted to a Viewfinder (Fa. TILL Photonics). Simultaneous patch-clamp and somatic calcium measurements were performed by defining a rectangular field of interest across the soma, allowing to monitor integrated calcium-dependent and -independent fluorescence. Dendrites could be identified under fluorescence optics, but their calcium signals were more difficult to analyze because they displayed prolonged filling time constants for fura-2 and small diameters in the submicrometer domain. Slow recordings of somatic fura-2 fluorescence signals at 360 nm (F360) and 390 nm (F390), membrane current and voltage at a sampling rate of 30 Hz were recorded by the X-chart version of the EPC-9 software. Rapid recordings of membrane current, F360 and F390 were obtained by the Pulse software (EPC-9) at a sampling rate of 5 kHz. For each recording interval lasting up to 70 s, fluorescence signals F390 and F360 were recorded at short intervals of 25 ms each. After that, F390 was collected for the rest of the interval to monitor calcium changes during voltage stimulation protocols. Calculations of intracellular calcium concentrations and further analysis were performed off-line by using the software Pulsefit (HEKA, Lambrecht, Germany) and IGOR (Wavemetrics, Lake Oswego, OR). Calibration constants for fura-2 were determined according to Grynkiewicz et al. (1985) by patch clamping cells with the following intracellular solutions (in mM): Rmin: 140 KCl, 10 HEPES, 2 MgCl2, 4 Na2-ATP, 0.4 Na-GTP, and 10 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) (adjusted to pH 7.3 with KOH); Rmedium: 140 KCl, 10 HEPES, 2 MgCl2, 4 Na2-ATP, 0.4 Na-GTP, 9.9 BAPTA, and 6.6 CaCl2, yielding a final concentration of 450 nM [Ca2+]i; and Rmax: 140 KCl, 10 HEPES, 2 MgCl2, 4 Na2-ATP, 0.4 Na-GTP, 10 CaCl2. Under our experimental conditions, the dissociation constant for fura-2 (Kd) was determined by using the equation
[Ca<SUP>2+</SUP>]<SUB>i</SUB>=<IT>K</IT><SUB><IT>d</IT></SUB>(<IT>R</IT><SUB><IT>max</IT></SUB><IT>/</IT><IT>R</IT><SUB><IT>min</IT></SUB>)(<IT>R</IT><IT>−</IT><IT>R</IT><SUB><IT>min</IT></SUB>)<IT>/</IT>(<IT>R</IT><SUB><IT>max</IT></SUB><IT>−</IT><IT>R</IT>)
Calibration constants Kd, Rmax, and Rmin were adjusted after several days of experiments to account for small fluorescence changes in the microfluorometric system. Typical values for Kd, Rmin, and Rmax were 241 nM, 0.49, and 3.66, respectively. Fura-2 concentrations were checked by spectroscopic analysis demonstrating that nominal fura-2 concentrations reflected real concentrations with a deviation around 10%. To determine the decay time constant a single exponential was fitted from peak to baseline using IGOR software (WaveMetrics). For amplitude determinations, data were filtered with 0.05 kHz to reduce noise. Statistics are given throughout the text as means ± SD.

Quantitative model of calcium homeostasis in hypoglossal motoneurons

Somatic calcium homeostasis was approximated by a linear, one-compartment model similar to the one previously described (Helmchen et al. 1996, 1997; Neher 1995). In this model, a single "effective" extrusion rate gamma  is assumed, which is justified if somatic calcium transients are described by a monoexponential decay phase. A second important parameter of the model is the endogenous calcium buffering of the cell, which is quantified by binding capacity kappa S (Neher 1995). Both parameters can be determined by loading the cell with a buffer with known calcium-buffering properties, which was the calcium indicator-dye fura-2 under our experimental conditions. For a given concentration of [fura-2], the corresponding "exogenous" binding capacity kappa B' was calculated from the equation (Helmchen et al. 1996, 1997; Lips and Keller 1998; Neher 1995)
&kgr;<SUB>B′</SUB>=[fura-2]<IT>K</IT><SUB><IT>d</IT></SUB><IT>/</IT>{([<IT>Ca<SUP>2+</SUP></IT>]<SUB><IT>i,rest</IT></SUB><IT>+</IT><IT>K</IT><SUB><IT>d</IT></SUB>)([<IT>Ca<SUP>2+</SUP></IT>]<SUB><IT>i,peak</IT></SUB><IT>+</IT><IT>K</IT><SUB><IT>d</IT></SUB>)}
where [Ca2+]i, rest, [Ca2+]i, peak and Kd denote resting, peak calcium concentration and the dissociation constant of fura-2, respectively.

For a linear, one-compartment model, the decay time constant tau  of a given calcium transient is described by the equation
&tgr;=(&kgr;<SUB>B′</SUB>+&kgr;<SUB>S</SUB>+1)/&ggr;
where kappa S, kappa B', and gamma  denote endogenous binding capacity, exogenous binding capacity of fura-2 and effective extrusion rate constant, respectively (Helmchen et al. 1996, 1997; Neher 1995). This equation was used to extrapolate decay time constants of calcium transients under dye-free conditions (kappa B' = 0) from those measured by microfluorometric measurements in the presence of known [fura-2]. In the same model, amplitudes A of calcium transients are given by
1/<IT>A</IT><IT>=</IT>(<IT>&kgr;<SUB>B′</SUB>+&kgr;<SUB>S</SUB>+1</IT>)<IT>/</IT>[<IT>Q</IT><SUB><IT>Ca</IT></SUB><IT>/</IT>(<IT>2</IT><IT>FV</IT><SUB><IT>cell</IT></SUB>)]
where Qca, Vcell, and F denote the calcium-mediated charge influx associated with the calcium transient, the volume of the cell accessible for calcium influx and the Faraday constant, respectively. This equation was utilized to approximate the amount of calcium-mediated charge influx for a given calcium amplitude.

To evaluate the calcium-related energy consumption in hypoglossal neurons, the above equation was utilized to identify the calcium-mediated charge influx during an inspiratory-related burst with calcium elevation A (see also Palecek et al. 1999)
<IT>Q</IT><SUB><IT>Ca</IT></SUB><IT>=2</IT>(<IT>1+&kgr;<SUB>B′</SUB>+&kgr;<SUB>S</SUB></IT>)<IT>AFV</IT><SUB><IT>cell</IT></SUB>
By assuming constant electrochemical gradients, the associated, calcium-related energy consumption ECa necessary to restore resting calcium concentrations after a burst is given by
<IT>E</IT><SUB><IT>Ca</IT></SUB><IT>=</IT>constant(<IT>1+&kgr;<SUB>B′</SUB>+&kgr;<SUB>S</SUB></IT>)<IT>A</IT>
indicating that for this simplified model ECa is proportional to the endogenous binding capacity kappa S of the cell if all other parameters are held constant.

The accessible volume of the cell Vcell was either approximated by measuring the geometric volume from fluorescence images (Fig. 2), or by using the equation (Helmchen et al. 1997; Oliva et al. 1988)
<IT>V</IT><SUB><IT>cell</IT></SUB><IT>=&tgr;<SUB>L</SUB></IT><IT>rD</IT><SUB><IT>fura-2</IT></SUB><IT>/</IT><IT>R</IT><SUB><IT>s</IT></SUB>
where tau L, r, Dfura-2, and Rs denote the fura-2 filling time of the soma, the specific resistance of the solution (70 Omega cm), the diffusion time constant for fura-2 (166 µm2 s-1) (Adler et al. 1991) and the series resistance, respectively.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Simultaneous electrophysiological and microfluorometric measurements

To investigate the temporal profile of respiratory-related activity, its time course was directly measured in vivo (Fig. 1) by using a plethysmographic device to monitor animal ventilation (Jacquin et al. 1996). In 2- to 6-day-old mice later used for electrophysiological experiments, respiratory frequencies ranged from a lower level of 2 Hz to periods of elevated breathing frequencies around 5 Hz (3.5 ± 1.1 Hz, mean ± SD, n = 9), depending on the experimental parameters and the metabolic condition of the animal. During electrophysiological experiments, rhythmic-respiratory activity of hypoglossal and cervical nerves could be maintained under in vitro conditions as exemplified by electrophysiological hypoglossal nerve recordings illustrated in Fig. 1C (XII nerve) (Smith et al. 1991, 1992). Under these conditions, rhythmic-inspiratory activity was represented by repetitive clusters of action potential discharges (bursts). Rhythmic activity persisted for several hours, suggesting that hypoglossal motoneurons could be preserved in a functionally intact state. Under our experimental conditions, average durations of inspiratory-related bursts were found to be 0.86 ± 0.25 s (n = 20).



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Fig. 1. Rhythmic-respiratory activity of hypoglossal nerves in the mouse brain stem. A: schematic drawing of a transversal section showing hypoglossal regions investigated and important landmarks for orientation. NTS, nucleus tractus solitarius; TS, tractus solitarius; N., nucleus. Hypoglossal nerve endings (XII) emerge on the ventral side of the medulla. B: in vivo plethysmographic measurements (at 31°C in the recording chamber) of neonatal mice under normal conditions indicate a breathing frequency of ~4 Hz. Ventilatory responses could be measured by a high sensitive pressure detector. C, top traces: electrical activity that was recorded extracellularly with conventional glass suction electrodes applied to hypoglossal nerve endings (Lips and Keller 1998). Rhythmic-inspiratory discharges of a nerve (XII) were integrated after electronic band-pass filtering [bottom traces, 1-2 kHz; Int. (XII)].

For simultaneous microfluorometric and electrophysiological recordings, the nucleus hypoglossus was first visually identified in brain stem slices by its location close to the dorsal nucleus vagus and the 4th ventricle (see Fig. 1A). After the whole cell patch-clamp configuration had been established, neurons were filled with pipette solutions containing 200 µM of the ratiometric calcium indicator dye fura-2 (Fig. 2). The dye filling was monitored by fluorometric measurements, thus providing estimates both for the dye concentration in the soma and the accessible volume of the cell (filling time constant 5-12 min; see METHODS). For an average loading time constant of 8 min and a series resistance of 11 MOmega , the somatic volume accessible for calcium concentrations was estimated as 5.1 pl. This value was comparable with 5.6 pl estimated from geometric measurements of the fura-2-filled volume of the cell soma (Fig. 2).



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Fig. 2. Hypoglossal motoneuron filled with fluorescent indicator dye fura-2. An intracellularly labeled hypoglossal motoneuron filled with 200 µM fura-2 during a patch-clamp experiment. Photomicrograph shows fluorescence under ultraviolet light excitation with a ×63 magnification objective. Note the extensive dendritic arborization that extended beyond the area shown. Photomultiplier measurements were performed by defining a rectangular window (schematic drawing) on the somatic surface over which fluorescence signals were integrated (Lips and Keller 1998).

During current-clamp measurements, rhythmic respiratory-related activity of hypoglossal motoneurons was reflected by clusters of excitatory postsynaptic potentials (EPSPs) leading to high-frequency action potential discharges. Bursts were composed of 4-10 action potentials within a time interval of 0.5-1 s, thus resembling the temporal profile of rhythmic burst discharges observed in recordings of hypoglossal nerve activity (Fig. 1). They were accompanied by transient rises in the somatic calcium concentrations as displayed in Fig. 3. Calcium transients displayed amplitudes below 100 nM and a monoexponential decay time constant of 772 ± 262 ms (n = 9; 200 µM fura-2, 28°C). They were absent or notably reduced during clusters of EPSPs that did not evoke trains of action potentials as depicted in Fig. 4. This was not surprising because kinetic properties of EPSPs suggested that mostly alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels were activated. Calcium transients were also suppressed when cells were recorded in voltage-clamp mode (-70 mV, Fig. 4B). In this case, rhythmic activity was represented by clusters of excitatory postsynaptic currents (EPSCs) that could be pharmacologically classified as AMPA receptor-mediated EPSCs. NMDA receptor channels, known to be synaptically activated in hypoglossal motoneurons (O'Brien et al. 1997; Vanselow et al. 1998), were blocked by extracellular magnesium for membrane voltages negative to -50 mV, explaining the absence of notable calcium signals in somatic compartments.



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Fig. 3. Rhythmic calcium elevations in current-clamp mode. Rhythmic burst activity and intracellular calcium changes recorded from hypoglossal motoneurons under current-clamp conditions. Fast calcium measurements (top traces) were obtained by high sampling frequencies of the F390-signal and low-pass filtering at 50 Hz. Note the absence of notable calcium elevations for clusters of excitatory postsynaptic currents (EPSCs) that did not evoke action potential activity. Bath temperature was 28°C; slice thickness was 600 µm; pipette solution contained 200 µM fura-2.



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Fig. 4. Excitatory postsynaptic potentials (EPSPs) and EPSCs in rhythmically active cells. A: whole cell patch-clamp recordings in current-clamp mode during simultaneous microfluorometric calcium measurements in a spontaneously active hypoglossal motoneuron. Top traces: calcium signals measured with the ratiometric dye fura-2. Bottom traces: current-clamp signal. B: corresponding patch clamp recordings in voltage clamp mode. The somatic membrane potential was held at -70 mV. Calcium signals were sampled at 5 kHz and filtered off-line at 50 Hz. Whole cell currents were sampled at 5 kHz and filtered at 3 kHz, respectively. Bath temperature was 26°C; pipette solution contained 200 µM fura-2.

The functional implications of calcium transients were further investigated by experiments in current-clamp mode during current injections into the somatic compartment. As illustrated in Fig. 5, current injections of 30 pA were sufficient to evoke a train of action potentials, where the number of action potentials evoked was closely dependent on the duration of current injection. As previously studied in detail with an electrophysiological approach (Bayliss et al. 1995; Viana et al. 1993a,b), action potential-evoked calcium influx through HVA channels was followed by subsequent activation of calcium-activated [KCa]+ channels. The resulting [KCa]+-dependent afterhyperpolarization was a strong determinant of action potential discharge frequency during bursts (Bayliss et al. 1995). In good agreement with these observations, somatic calcium responses were closely correlated with the number of action potentials evoked. As shown in the left panel of Fig. 5 by an averaged response of 60 consecutive stimulations, the occurrence of a single action potential accounted for a somatic calcium rise of ~6 nM. Equivalent stimulation pulses that did not evoke an action potential showed no calcium response (not shown). Averaged responses during longer periods of current injection are exemplified in the middle and right panels of Fig. 5. Peak somatic calcium rises were found to be 25 and 34 nM for a total number of 7 and 14 action potentials, respectively. This corresponded to integral calcium responses of 2.4 nM-s (1 action potential), 20.8 nM-s (7 action potentials), and 29.7 nM-s (14 action potentials), demonstrating notable, action potential-induced calcium influx into the somatic compartment.



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Fig. 5. Calcium elevations during action potential activity. Action potential activity and associated intracellular calcium responses recorded from hypoglossal motoneurons in current-clamp configuration. Fast calcium signals (top traces) were obtained by rapidly sampling F390 (1-2 kHz) and subsequent low-pass filtering at 50 Hz. Left: membrane potential during stimulation exemplified by a single action potential and corresponding averaged calcium response (n = 60) for depolarizing stimulation pulses lasting 9 ms (injected current 70 pA). Middle: current injection of 30 pA for 500 ms and corresponding averaged calcium response (n = 9); right: current injection of 30 pA for 1 s and corresponding averaged calcium response (n = 3). Peak amplitudes of Delta [Ca2+]i were 6, 25, and 34 nM (left to right panel), respectively. Bath temperature was 28°C; slice thickness was 200 µm; pipette solution contained 200 µM fura-2.

Depolarization-induced calcium signals in hypoglossal motoneurons

Voltage-clamp protocols were utilized to analyze somatic calcium dynamics in more quantitative detail. As illustrated in Fig. 6A, steplike voltage pulses starting from a holding potential of -70 mV induced detectable calcium responses for depolarizations positive to -50 mV (39 ± 16 nM at -40 mV, n = 6 cells, 200 µM fura-2; depolarization time, 1 s). Calcium responses increased for positive voltage steps, presumably reflecting the larger open probability of voltage-activated calcium channels. When motoneurons were held at resting potentials of -100 mV, calcium elevations were already observed for membrane depolarizations positive to -60 mV (26 ± 8 nM at -50 mV, n = 6 cells, 200 µM fura-2; depolarization time, 1 s), suggesting that LVA calcium channels were activated. Figure 6B compares calcium responses mediated by different voltage-clamp protocols. For depolarizations from -100 to -50 mV, combined HVA/LVA responses displayed significantly larger amplitudes compared with those starting from holding potentials of -70 mV, indicating a notable contribution of LVA-channel types. With respect to rhythmic hypoglossal activity, these observations demonstrate that both HVA and LVA channels are present in somatic membranes and, under appropriate conditions, could contribute to oscillations in somatic [Ca2+]i.



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Fig. 6. Pulse protocols induce different somatic Ca2+ changes in hypoglossal neurons. To demonstrate the contribution of LVA-Ca2+ currents to the entire Ca2+-flux through voltage-gated channels, 2 different pulse protocols were applied to the hypoglossal motoneurons. A, left traces: Ca2+ changes induced by 4 increasing voltage steps from -70 to -30 mV. Right traces: responses to the same depolarizations after a 2-s prepulse to -100 mV. B: peak calcium elevations plotted against depolarized membrane potential. Note the remarkable amount of calcium entry through LVA-Ca2+ channels after depolarizations to -60 mV. T = 22°C; pipette solution contained 200 µM fura-2.

A potential contribution of intracellular calcium release to somatic calcium oscillations was investigated by a series of experiments correlating calcium elevation with defined voltage-clamp protocols. Figure 7 depicts a series of membrane depolarizations starting from a holding potential of -70 mV to voltages between -60 and +50 mV. As illustrated in Fig. 7A, [Ca2+]i showed a linear dependence on membrane depolarization time for all voltage steps applied, at least for the depolarization interval of 1 s utilized in this experiment. This finding is not easily compatible with a pronounced release of calcium from intracellular stores, expected to lead to notable inhomogeneities in [Ca2+]i once the threshold for calcium release is reached (e.g., Llano et al. 1994; Neering and McBurney 1984). A second argument is provided by the decay of calcium signals directly after the end of the pulse, suggesting that calcium elevations were rapidly terminated after voltage-dependent calcium channels were closed. Similar results were found for n = 16 cells. Furthermore, calcium elevations were also investigated as a function of depolarization time intervals. As illustrated in Fig. 8, calcium amplitudes depended linearly on depolarization times for elevations between 50 and 350 nM. Similar observations were made in n = 7 cells, suggesting that voltage-dependent calcium influx was the dominant process.



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Fig. 7. Depolarization-induced calcium signals in hypoglossal motoneurons. A: voltage protocol starting from a holding potential of -70 mV to demonstrate Ca2+ signals mediated mainly by HVA channels. The higher time resolution demonstrates the linear rise of cytosolic Ca2+ concentration for each depolarizing pulse. B: peak calcium amplitudes plotted as a function of membrane depolarization.



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Fig. 8. Somatic calcium responses for different depolarization time intervals. A: depolarizing pulses of increasing duration induce Ca2+ elevations with growing amplitude in a hypoglossal motoneuron. B: peak Ca2+ elevation plotted against pulse length shows an essentially linear relationship. The fitted line corresponds to the function y = ax + b with parameters a = 434 nM s-1 and b = 31.2 nM.

Quantitative analysis of somatic calcium homeostasis

Several processes have been implicated in physiological calcium control including Na+/Ca2+ exchange across the plasma membrane, calcium uptake into intracellular stores like endoplasmic reticulum or mitochondria or ATP-dependent calcium extrusion across the plasma membrane. In respiratory-related neurons of fast breathing mammals such as the mouse, the dynamics of calcium homeostasis are particularly interesting as rhythmic-inspiratory bursts and associated calcium transients occur at breathing rates up to 5 Hz (Fig. 1A). To approximate calcium dynamics under dye-free conditions from microfluorometric measurements, it is important to consider their retardation by the calcium indicator-dye (Neher 1995; Neher and Augustine 1992; Palecek et al. 1999). To find an estimate, the temporal profile of calcium recovery was quantified by voltage-clamp protocols illustrated in Fig. 9. For calcium elevations around 100 nM above resting levels, [Ca2+]i decayed according to a single exponential function with a decay time constant of 3.5 s (Delta [Ca2+]i = 159 nM, 200 µM fura-2, 22°C, Fig. 9B). This time constant was prolonged to 5.2 s for calcium elevations of 476 nM under identical experimental conditions (2.4 ± 1.3 s for Delta [Ca2+]i = 60 nM; 4.3 ± 1.5 s for Delta [Ca2+]i = 290 nM; n = 7 cells). As described in METHODS, these values can be utilized to determine the effective extrusion rate gamma  by using the equation
&ggr;=(&kgr;<SUB>S</SUB>+&kgr;<SUB>B′</SUB>+1)/&tgr;
where kappa S, kappa B', and tau  have the above-mentioned meaning (Helmchen et al. 1996, 1997; Neher 1995). With an endogenous calcium-binding capacity (kappa S) of 41 characteristic for hypoglossal motoneurons (Lips and Keller 1998), decay time constants of calcium transients were transformed into effective extrusion rate constants of 131 ± 70 s-1 and 51 ± 18 s-1 for elevations of 60 and 290 nM, respectively (n = 7 cells). This result was remarkable in the light of the previously noted vulnerability of hypoglossal motoneurons to metabolic stress and excitotoxic conditions (DePaul et al. 1988; Lips and Keller 1998; Reiner et al. 1995), suggesting that reduced extrusion rates at elevated concentrations could represent an important determinant of intracellular calcium levels during pathophysiological states.



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Fig. 9. Calcium transients during gradual increase in cytosolic concentration of calcium. A: Ca2+ elevations were induced by different voltage steps from -70 mV to -50, -30, -10, 0, 10, and 30 mV. Recovery times to the baseline Ca2+ levels were fitted by a monoexponential function. B: scatter plot showing the correlation between calcium elevation and decay time constant. The fitted line corresponds to the function y = a x + b with parameters a = 6.36 s µM-1 and b = 2.27 s. Decay time constants of calcium transients could be recalculated to "effective" extrusion rate constants (gamma ) of 76 s-1 and 35 s-1 for elevations of 159 and 476 nM, respectively (see text).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rhythmic motoneuron activity and associated calcium signaling

In this report, we demonstrate simultaneous electrophysiological patch-clamp and microfluorometric calcium recordings from motoneurons in the nucleus hypoglossus from mouse. Recording conditions were optimized to preserve motoneurons in a functionally intact state monitored by rhythmic inspiratory-related activity of hypoglossal nerves (Smith et al. 1991, 1992). During whole cell measurements, rhythmic electrical activity was represented by repetitive EPSPs, leading to high-frequency action potential discharges. Parallel calcium transients displayed amplitudes below 100 nM and decay time constants around 800 ms (28°C, 200 µM fura-2). These parameters were similar to those previously observed for spontaneous calcium oscillations in respiratory-related interneurons under comparable experimental conditions (Frermann et al. 1999). Differences were observed for calcium amplitudes, which were notably larger in interneurons (220 nM). This was most likely explained by differences in the activation profile of voltage-dependent calcium currents, displaying higher calcium influx rates at negative membrane potentials in interneurons compared with motoneurons (Frermann et al. 1999; Lips and Keller 1998).

In hypoglossal motoneurons, spontaneous elevations in [Ca2+]i were suppressed when membrane potentials were held in voltage-clamp mode. At -65 mV, rhythmic activity was represented by clusters of EPSCs, which could be kinetically and pharmacologically classified as AMPA receptor-mediated EPSCs. NMDA receptor channels, known to be synaptically activated in interneurons and hypoglossal motoneurons, contributed little to somatic signals because they were blocked by extracellular magnesium in the negative voltage range (O'Brien et al. 1997; Vanselow et al. 1998). Similar results were obtained for EPSPs measured in current-clamp mode, suggesting that opening of synaptic receptors alone was not sufficient to induce notable somatic calcium elevations during spontaneous rhythmic activity. It is interesting to note, however, that occurrence of action potential activity could temporally relieve activated NMDA receptor channels from magnesium block, potentiating calcium influx through synaptic channels during spontaneous bursts (Stuart and Sakmann 1995; Yuste and Tank 1996).

An important question is related to the modification of endogenous calcium signaling in hypoglossal motoneurons resulting from slice preparations of central nervous tissue. For example, removal of synaptic inputs could alter integrative calcium responses both in dendritic and somatic compartments. With respect to EPSC-mediated bursts of action potentials and associated calcium signals investigated in this report, magnitudes of calcium responses in slice preparations probably represent a lower boundary for corresponding in vivo signals. Another possibility is that cutting of neuronal processes during slice preparation could lead to uncontrolled calcium influx, thereby disrupting endogenous calcium homeostasis. In this case, several arguments supported the view that such mechanisms were not significantly influencing the results of our analysis. First, hypoglossal motoneurons that were physically damaged disintegrated rapidly, most likely as a result of uncontrolled calcium influx and associated cell damage. Such cells could be easily identified on the surface of slice preparations by their disintegrated somatic shape, and they were not included in the patch-clamp analysis. In contrast, cells used for analysis of calcium homeostasis displayed basal calcium levels below 100 nM, indicating that the sensitive regulation of basal levels was still intact and that the physical damage during slice preparation was minimal. Another argument resulted from patch-clamp recordings from hypoglossal motoneurons in thick slice preparations (>500 µm) (Ladewig and Keller 1998), showing that the parameters of cellular calcium homeostasis were similar to those observed in thin slices (150 µm).

Voltage-dependent calcium influx

Voltage-dependent calcium influx induced by action potential discharges has previously been shown by electrophysiological measurements to control afterdepolarizations and afterhyperpolarizations in hypoglossal motoneurons (Bayliss et al. 1995; Viana et al. 1993b). In this system, action potential-induced afterhyperpolarizations resulted from a colocalization of N-type calcium channels and [KCa]+ channels, closely coupling calcium entry with potassium efflux and membrane hyperpolarization. By using this mechanism, [KCa]+ channel-mediated afterhyperpolarizations control the temporal profile of action potential activity, accounting for a defined discharge pattern during bursts (Bayliss et al. 1995; Viana et al. 1993b) (see also Fig. 5). Several observations supported the view that action potential-induced calcium transients observed during microfluorometric measurements represented the superposition of localized calcium responses responsible for [KCa]+ channel-mediated afterhyperpolarizations. First, somatic calcium transients were evoked by a single action potential, suggesting that a unitary, short depolarization was sufficient to induce a notable calcium response. Second, somatic calcium responses were closely correlated with the number of action potentials evoked. Another argument was provided by the result that calcium elevations rapidly returned to basal levels after the end of a burst. This suggested that secondary processes like calcium-induced calcium release did not significantly shape the response. It was interesting to note that the gradual buildup of basal calcium concentrations during bursts magnified the probability for basal activation of [KCa]+ channels, providing increasingly more favorable conditions for membrane repolarization. During rhythmic-respiratory discharges, this process represents an elegant feedback mechanism to limit the duration of bursts and, more generally, adjust the overall excitability of hypoglossal neurons (Viana et al. 1993a,b).

Earlier electrophysiological studies have investigated in detail different types of calcium influx pathways in hypoglossal motoneurons and their role during action potential activity (Bayliss et al. 1995; Umemyia and Berger 1994, 1995a; Viana et al. 1993a,b). In the present report, we found that both LVA and HVA calcium channels were present in somatic membranes, suggesting that they could potentially contribute to somatic calcium transients. Spontaneous depolarizations from resting potentials of -65 mV presumably activated few or none LVA channels, known to be inactivated in this voltage range (Viana et al. 1993a). In rhythmically active cells, however, hyperpolarizations resulting from spontaneous inhibitory synaptic activity could remove LVA channels from inactivated states, allowing LVA channel-mediated calcium influx under physiological conditions. Another mechanism to remove LVA channels from inactivation could be the transient activation of voltage- or calcium-dependent K+ channels, known to be present and functionally important in hypoglossal motoneurons (Bayliss et al. 1995; Viana et al. 1993a,b) (see also Fig. 5). Other observations suggested that openings of HVA calcium channels were essential. For example, moderate depolarizations from -65 mV induced notable calcium transients, demonstrating the efficiency of HVA channel-mediated calcium influx.

Quantitative model of calcium homeostasis in hypoglossal motoneurons

Under physiological conditions, the spatial-temporal profile of intracellular calcium concentrations is critically determined by calcium buffering, extrusion, and uptake into intracellular stores (Neher 1995). For respiratory-related motoneurons in young mice, effective calcium homeostasis is particularly important because rhythmic-inspiratory bursts and corresponding calcium oscillations occur at maximum breathing frequencies around 5 Hz (Fig. 1). This leaves an interburst interval of 200 ms for a single calcium transient composed of calcium influx, elevation, and subsequent recovery to basal levels. A quantitative model to approximate calcium signaling under physiological conditions has been formulated by Neher and Augustine (1992). For a linear, one-compartment model of calcium homeostasis (see METHODS), the amount of calcium-mediated charge influx Qca can be estimated for a given calcium transient by using the equation (Neher 1995)
<IT>Q</IT><SUB><IT>ca</IT></SUB><IT>=</IT><IT>A</IT>(<IT>&kgr;<SUB>B′</SUB>+&kgr;<SUB>S</SUB>+1</IT>)<IT>2</IT><IT>FV</IT><SUB><IT>cell</IT></SUB>
where A, Vcell, and F denote the amplitude of the transient, the volume of the cell accessible for calcium influx, and the Faraday constant, respectively. With a somatic cell volume of 5.1 pl characteristic for hypoglossal motoneurons, an endogenous calcium-binding capacity of kappa S = 41 (Lips and Keller 1998) and a calcium elevation of 6 nM/AP (200 µM fura-2, kappa B' approx  310), this equation identifies a somatic, calcium-mediated charge influx of 2.1 pC in response to a single action potential.

For the same model, decay time constants of calcium transients are determined by the equation (Neher 1995)
&tgr;=(&kgr;<SUB>S</SUB>+&kgr;<SUB>B′</SUB>+1)/&ggr;
where kappa S and kappa B' have the above-mentioned meaning, and gamma  represents the effective extrusion rate of the cell. For our purposes, this equation can be used to approximate the temporal profile of calcium transients under dye-free conditions (kappa B' = 0) by back-extrapolating the experimentally observed decay time constants of calcium transients. With experimentally determined decay time constants of 800 ms in 200 µM fura-2, decay times of calcium transients were extrapolated to tau  = 140 ± 40 ms (28°C; kappa S = 41) under dye-free conditions. For a Q10 of ~2 found for extrusion rates in hypoglossal motoneurons and comparable neuronal systems (Helmchen et al. 1997; Lips and Keller 1997; Regehr et al. 1994), these data indicated that endogenous calcium transients displayed decay time constants around 70 ms in a living animal (37°C).

With respect to rhythmic respiratory-related activity in vivo, these results have several interesting implications. First, a calcium-mediated charge influx of ~2 pC per action potential is more than two orders of magnitude larger compared with 0.01 pC associated with calcium influx during a single, NMDA-receptor mediated EPSC in hypoglossal motoneurons (Vanselow et al. 1998). Accordingly, a coordinated activity of several hundred NMDA receptor-mediated EPSCs is necessary to achieve the same amount of calcium influx induced by a single action potential. A second interesting observation is related to the temporal profile of calcium transients. In general, two to three monoexponential decay time constants are required to reach basal calcium levels, therefore the recovery phase of calcium transients (140-210 ms) is comparable to the interburst interval of 200 ms found for maximum breathing rates (5 Hz, Fig. 1B). These observations are in agreement with a model where recovery times of calcium transients provide an upper limit for the frequency of inspiratory-related bursts during intervals with high breathing rates. A third interesting implication is that for the linear model presented above, calcium decay time constants are directly proportional to endogenous binding capacities. Correspondingly, the exceptionally low value of kappa S = 41 in hypoglossal motoneurons, which is 20 times smaller compared with that found for cerebellar Purkinje cells (kappa S = 900) (Fierro and Llano 1996; juvenile rats), achieves high-speed recovery of calcium transients if all other parameters are comparable (Lips and Keller 1998; Palecek et al. 1999). Obviously, rapid decays of calcium transients can also be realized for high binding capacities by accelerated extrusion rates, but presumably at the cost of elevated ATP-dependent energy consumption needed for rapid recovery of a given calcium transient in the presence of high buffer concentrations (see METHODS). Such costs could be significant in a permanently oscillating network like the respiratory system of mouse operating at frequencies of several hertz.

Significance of endogenous calcium homeostasis for neuroprotective strategies

Significant disruptions of calcium homeostasis have been associated with neuronal degeneration in different parts of the CNS, in particular with a selective vulnerability of different motoneuron populations to excitotoxic stress and calcium-mediated neuronal damage (Appel et al. 1995; Doble 1995; Krieger et al. 1994; Reiner et al. 1995). Hypoglossal motoneurons are among the neuronal populations most heavily affected (DePaul et al. 1988; Reiner et al. 1995), identifying them as a valuable model system to define the underlying cellular events. Interestingly, motoneuron populations that are particularly affected also display low endogenous binding capacities as determined both by quantitative analysis of endogenous calcium homeostasis and immunocytochemical techniques (Alexianu et al. 1994; Lips and Keller 1998; Palecek et al. 1999; Reiner et al. 1995). Accordingly, an increase of intracellular calcium buffers like calbindin-D28K has been suggested as a neuroprotective strategy to protect vulnerable motoneuron populations against excitotoxic damage (Alexianu et al. 1994; Ho et al. 1996).

In the light of the present report, several parameters concerning hypoglossal calcium homeostasis were identified that could be important for adequate neuroprotection. First, notable calcium elevations were associated with bursts of action potential discharges, indicating that repetitive calcium elevations were part of the normal, physiological activity cycle of hypoglossal motoneurons. Accordingly, reduction in action potential activity could efficiently reduce the overall calcium load of cells. A second argument was provided by the earlier observation that low concentrations of endogenous buffers support highly localized calcium transients in calcium microdomains (Klingauf and Neher 1997; Roberts 1994), mainly by reducing the effective diffusion constant of calcium ions in the cytosol compared with high-buffering conditions (see also Lips and Keller 1998; Palecek et al. 1999). For excess calcium influx commonly associated with excitotoxic stress and neuronal damage (Choi 1988), this predicts a particularly high risk for an uncontrolled elevation of localized calcium levels for cells like hypoglossal neurons with low endogenous binding capacities. A third argument was provided by the finding that the combined action of calcium extrusion mechanisms resulted in retarded extrusion rates at higher calcium concentrations (Fig. 9). Under pathophysiological conditions, this could represent a potentially dangerous mechanism by gradually increasing initial calcium elevations associated with excitotoxic events or neuronal damage (Alexianu et al. 1994; Doble 1995).

With respect to exogenous buffers as neuroprotective agents, our quantitative model predicts that even small amounts of exogenously added buffers significantly prolong recovery times of calcium transients. For example, concentrations as low as 50 µM fura-2 account for a twofold retardation of somatic calcium decay times in hypoglossal motoneurons. Obviously, this could represent an increased risk for gradual accumulation of basal calcium levels during high-frequency rhythmic activity. Taken together, our measurements therefore indicate that, under physiological conditions, low endogenous calcium binding capacities provide hypoglossal motoneurons with a high-speed, energy-conserving calcium homeostasis during bursts of respiratory-related electrical activity. Under pathophysiological conditions, they presumably represent an increased risk for neuronal damage resulting from low protection against uncontrolled calcium influx and excitotoxic disturbances.


    ACKNOWLEDGMENTS

We thank D. Crzan and U. Lange for support with slice preparations and excellent technical assistance. We also thank Drs. J. Brockhaus and K. Ballanyi for help with experiments and electrophysiological recordings.

This research was supported by Deutsche Forschungsgemeinschaft Grants Ke 403/5-2 and Ke 403/6-1, the Graduiertenkolleg "Organization and Dynamics of Neuronal Nets," and Sonderforschungsbereich 406.


    FOOTNOTES

Address for reprint requests: B. U. Keller, Zentrum Physiologie und Pathophysiologie, Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 February 1999; accepted in final form 3 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society