Zentrum Physiologie und Pathophysiologie, Universität Göttingen, 37073 Göttingen, Germany
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ABSTRACT |
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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.
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INTRODUCTION |
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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
).
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METHODS |
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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 M
. 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 M
. Cells with series resistances higher than 15 M
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
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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
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
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
B' was
calculated from the equation (Helmchen et al. 1996
,
1997
; Lips and Keller 1998
; Neher
1995
)
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For a linear, one-compartment model, the decay time constant of a
given calcium transient is described by the equation
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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)
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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
)
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RESULTS |
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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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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 M, 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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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
-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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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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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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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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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 (
[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
[Ca2+]i = 60 nM;
4.3 ± 1.5 s for
[Ca2+]i = 290 nM;
n = 7 cells). As described in METHODS,
these values can be utilized to determine the effective extrusion rate
by using the equation
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DISCUSSION |
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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
)
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For the same model, decay time constants of calcium transients are
determined by the equation (Neher 1995)
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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
S = 41 in
hypoglossal motoneurons, which is 20 times smaller compared with that
found for cerebellar Purkinje cells (
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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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