Laboratoire de Neurophysiologie, Faculté de Médecine, Université Laval, Quebec G1K 7P4, Canada
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ABSTRACT |
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Massimini, Marcello and Florin Amzica. Extracellular Calcium Fluctuations and Intracellular Potentials in the Cortex During the Slow Sleep Oscillation. J. Neurophysiol. 85: 1346-1350, 2001. During slow wave sleep the main activity of cortical neurons consists of synchronous and rhythmic alternations of the membrane potential between depolarized and hyperpolarized values. The latter are long-lasting (200-600 ms) periods of silence. The mechanisms responsible for this periodical interruption of cortical network activity are unknown. Here we report a decrease of ~20% in the extracellular calcium concentration ([Ca]out) progressively taking place in the cortex between the onset and the offset of the depolarizing phase of the slow sleep oscillation. Since [Ca]out exerts a high gain modulation of synaptic transmission, we estimated the associated transmitter release probability and found a corresponding 50% drop. Thus the periods of silence occurring in the cortical network during slow wave sleep are promoted by recurrent [Ca]out depletions.
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INTRODUCTION |
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A slow oscillation (<1 Hz) has
been described in virtually all cortical neurons of anesthetized
(Steriade et al. 1993b) and naturally sleeping cats
(Steriade et al. 1996
) and in the sleep electroencephalogram (EEG) or magnetoencephalogram of humans
(Achermann and Borbély 1997
; Amzica and
Steriade 1997
; Simon et al. 2000
). This
oscillation appears as the cyclic fluctuation of the neuronal membrane
potential between two voltage levels: a depolarizing phase made of
synaptic activity and a hyperpolarizing period characterized by the
absence of network activity. The mechanisms governing the switch
between these two states are still poorly understood. It has been shown
that the hyperpolarizing phase is associated with disfacilitation
within the cortical network (Contreras et al. 1996
). As
demonstrated by multiple intra- and extracellular recordings in intact
and lesioned brains, the slow oscillation is cortically generated
(Steriade et al. 1993c
) and takes place as a synchronous network event (Amzica and Steriade 1995
).
Synchronous activities in large populations of neurons are likely to
produce coherent modifications of the ionic composition of the
extracellular space. In particular, Ca2+, given
its relatively low resting level (1-1.3 mM), is in a critical position
since small absolute changes can produce large shifts in its
extracellular concentration. Experimental results (Nicholson 1980; Nicholson et al. 1977
,
1978
; Pumain et al. 1983
; Somjen 1980
) and mathematical models (Egelman and Montague
1998
; Wiest et al. 2000
) converge in
demonstrating that a decrease in [Ca]out occurs
both during physiological and pathological neuronal activations. The
drop of [Ca]out is mainly due to
Ca2+ inflow at the postsynaptic level
(Bollmann et al. 1998
; Borst and Sakmann
1999
; Heinemann and Pumain 1981
; Rusakov
et al. 1999
). Even slight changes of
[Ca]out are known to modulate, with a high gain, transmitter release and therefore the synaptic function (Bootman and Berridge 1995
; Dodge and Rahamimoff
1967
; Katz and Miledi 1970
; Mintz et al.
1995
; Qian et al. 1997
).
We tested the hypothesis that, during the depolarizing phase of the slow sleep oscillation, the occurrence of a simultaneous activation in virtually all neocortical neurons induces a phasic depletion of extracellular Ca2+. As a consequence, some degree of synaptic depression would occur in the cortex, thus favoring the onset of the hyperpolarizing phase. Further, during the ensuing silent epoch, [Ca]out resting levels would be restored together with synaptic efficacy.
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METHODS |
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Experiments were performed on eight adult cats, under general anesthesia with ketamine-xylazine. [Ca]out and DC field potentials (FPs) were measured by means of double-barreled ion-sensitive microelectrodes (ISMs), together with intracellular membrane potentials in neocortical neurons from the suprasylvian gyrus (areas 5 and 7). In some cases the slow oscillation was disrupted by the electric stimulation of the peduncolopontine tegmental (PPT) nucleus. The ISMs were filled with the calcium ionophore I-Cocktail A (Fluka) and were calibrated, before and after recordings, in appropriate solutions with Ca2+ concentrations between 0.2 and 6 mM; the calibration values were fitted with a logarithmic trend. Only electrodes reaching 90% of the response in <20 ms were used. The time course of the response was measured stepping the electrodes trough drops containing different Ca2+ concentrations (0.2, 0.5, 1, 1.5, 2, 4, and 6 mM). The drops were held at close distance by silver rings, which were connected to the ground. Thus ISMs were far faster than the phenomena under investigation. Since ISM potentials could be contaminated through capacitive coupling by FPs, the latter were subtracted from the former, and the resulting signal was linearized and transposed into concentration values using the parameters extracted from the logarithmic fitting of the calibration points.
Intracellular recordings were obtained with glass microelectrodes
filled with 3 M potassium acetate and DC resistance between 30 and 40 M. Only stable recordings with resting membrane potentials more
negative than
60 mV, overshooting action potentials and input
resistances between 17 and 24 M
were kept for analysis. To ensure
stability of intracellular recordings, we paralyzed the animals with
gallamine triethiodide (33 mg/kg iv) and ventilated them artificially,
with control of the end-tidal CO2 concentration between 3.5 and 3.7%. Further stability was obtained by cisternal drainage, bilateral pneumothorax, hip suspension, and by filling the
hole in the calvarium with a 4% agar solution. Body temperature was
maintained at 37-38°C. All pressure points were infiltrated with
lidocaine, and a constant state of deep anesthesia was ensured by
continuously monitoring an EEG with slow waves. Additional doses of
ketamine-xylazine were administered at the slightest sign of EEG
activation. A high-impedance Neurodata amplifier with active bridge
circuitry was used to record and inject current into the cells. The
headstage amplifier for ISMs was modified with an ultra ultra low input
current (<25 fA) amplifier (National Semiconductor). Signals were
recorded on tape with band-pass of 0-9 kHz and digitized off-line at
10 kHz for analysis and display.
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RESULTS |
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All recorded neurons (n = 53) and FPs displayed a
spontaneous slow (<1 Hz) oscillatory pattern as previously described
in detail (Steriade et al. 1993b) (see also neuron in
Fig. 2A). When the double-barreled electrode was placed at
the cortical surface, the field and ISM potentials had almost identical
variations and, after subtraction of the field potential signal from
the ISM signal, no Ca2+ phasic activity was
recorded (Fig. 1, A and
C). When the electrode was lowered in the depth of the
cortex (0.5-1.5 mm), FPs displayed reversed activity with respect to
the surface recording (see also Contreras and Steriade
1995
), and, in spite of the similarity between ISM potentials
and FPs, their subtraction disclosed phasic Ca2+
variations (Fig. 1, B and C). From the surface
recording, it is clear that the ISM also picks up FPs. Therefore this
contamination has to be present in the depth recording too, and
justifies the total subtraction of FP signal from the output of the
ISM. Moreover, we were concerned about the possibility that the ISM
reflects a delayed FP. This was ruled out by the flat result after the superficial subtraction (Fig. 1A). The depth
[Ca]out fluctuations ranged from peak values of
1.18 ± 0.03 mM (mean ± SD) down to 0.95 ± 0.05 mM
around an average level of 1.04 ± 0.2 mM. The amplitude of the
fluctuations ranged from 0.18 to 0.27 mM, being, on the average of all
experiments, around 20% of the maximum value.
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To compare the dynamics of Ca2+ observed during
the slow oscillation with the levels occurring during an activated
state, stimulating electrodes were placed in the PPT nucleus of three
cats. PPT electrical stimulation (30-60 Hz) resulted in the disruption
of the slow oscillation and EEG activation as previously described
(Steriade et al. 1993a). In all cases (n = 25), simultaneously with the EEG activation, the phasic fluctuations
of [Ca]out were abolished and
[Ca]out maintained a steady level (1.13 ± 0.05 mM) for the duration of the activation (Fig. 1D). This
Ca2+ concentration was slightly below the maximum
[Ca]out, but higher than the average measured
during the slow oscillation.
Simultaneous recordings with ISMs and intracellular pipettes linked the
[Ca]out variations to the cellular activity
(Fig. 2A). Calcium level
reached its maximum at the end of the hyperpolarizing phase and started
to decrease soon after the onset of the neuronal depolarization. The
slope of calcium fall was not constant, being steeper (0.35 to
0.46
mM/s) during the first 200-300 ms of the neuronal depolarization. The
slope during the last 200-300 ms of the depolarizing phase was milder
(
0.05 to
0.08 mM/s). [Ca]out reached its
minimum just before the end of the depolarization of the neuron and
rose almost linearly during the following hyperpolarizing phase (Fig.
2B) with a slope value in the 0.75- to 0.95-mM/s range.
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DISCUSSION |
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Many factors could account for the drop of
[Ca]out during the depolarizing phase of the
slow oscillation with respect to the baseline of the activated periods.
1) The onset of the depolarizing phase reflects simultaneous
firing of large populations of neurons (Amzica and Steriade
1995). 2) As revealed by intracellular studies in
behaving cats, many neurons, firing during the depolarizing phase of
the slow oscillation, become silent after EEG activation due to
awakening (Steriade et al. 1999
). 3)
Low-threshold Ca2+ currents, expressed in some
(15%) cortical neurons (Paré and Lang 1998
), may
be deinactivated during the hyperpolarizing phase thus contributing to
a larger calcium influx into neuronal elements during the following
depolarization. 4) Ca2+ might also be
uptaken by glia (Pasti et al. 1997
) and at the presynaptic level (Alici and Heinemann 1995
;
Igelmund et al. 1996
). It is not possible to
establish in vivo the exact balance of all these contributions. A
series of studies suggests the preponderance of the postsynaptic uptake
(Bollmann et al. 1998
; Borst and Sakmann 1999
; Heinemann and Pumain 1981
; King et
al. 2000
; Rusakov et al. 1999
).
On the other hand, overshooting extracellular calcium levels at the end
of the hyperpolarizing phase with respect to the steady value measured
during activated periods may result from Ca2+
extrusion by neurons (DiPolo and Beaugé 1983)
during a period characterized by poor ongoing cortical activity.
The relationship between [Ca]out and
transmitter release is known from in vitro studies performed on both
central and peripheral synapses. We estimated the probability of
transmitter release as a power function of the percentage variation of
[Ca]out with respect to the maximum
concentration ([Ca]max). The exponents of the
function () were ranging from 2.5 (Mintz et al. 1995
) to 4 (Qian et al. 1997
). These two limits provide an
inclusive range of release probability for each measured level of
calcium concentration. During the depolarizing-hyperpolarizing
sequences of the slow oscillation, the release probability was maximal
at the beginning of the depolarizing phase and progressively dropped, reaching a value between 0.55 (
= 2.5) and 0.4 (
= 4)
just before the beginning of the hyperpolarizing phase (Fig.
2C). The probability of synaptic release recovered almost
linearly returning to the full value at the end of the hyperpolarizing phase.
A global disfacilitation in the cortical network has been shown to
underlie the long-lasting hyperpolarization of the slow oscillation
(Contreras et al. 1996). In particular, a progressive running down of synaptic activity during the preceding depolarizing phase is consistent with two observations. First, when slowly oscillating neurons are hyperpolarized with DC current, the last part
of the depolarizing phase decreases in amplitude (see Fig. 3 in
Steriade et al. 1993b
), suggesting that it involves more of intrinsic (depolarization-activated) currents than synaptic ones.
Second, the somatic input resistance has its lowest value at the
beginning of the depolarizing phase and progressively increases for the
rest of the cycle (Contreras et al. 1996
), consistent with the progressive closure of synaptically driven conductances.
An active inhibition is unlikely to be a major factor inducing the
long-lasting hyperpolarizations. Local circuit inhibitory cells are
equally silent during the hyperpolarizing phase of the slow sleep
oscillation and discharge in synchrony with pyramidal neurons
(Steriade et al. 1994). Thus they undergo the same
disfacilitatory action of [Ca]out as
long-axoned cells, and their inhibitory action would diminish toward
the end of the depolarizing phase. Long-range active inhibition is
ruled out by the existence of the slow oscillation in cortical slices
(Sanchez-Vives and McCormick 2000
).
Taking into account the measured calcium extracellular dynamics and
their estimated reflections on synaptic function, we propose the
following sequence of events as a possible scenario for the cyclic
occurrence of silent periods in the cortex during the slow sleep
oscillation: during the depolarizing phase, the synchronous activation
of cortical neurons leads to a progressive depletion (~20% drop) of
external calcium, mainly through a calcium entry at the postsynaptic
sites. At the presynaptic sites, the high sensibility of transmitter
release to extracellular calcium concentration would determine a
progressive decrease (45-60% drop) of synaptic efficacy. In the
network, this effect would be accumulated at each synaptic station in a
cascade reaction leading to an increasing degree of functional
disconnection. At the single neuron level, a reduced synaptic
depolarizing pressure would reduce the contribution of persistent
sodium currents (Stafstrom et al. 1985), leaving Ca2+-dependent K+ currents
(Schwindt et al. 1988
) to shape the offset of the
depolarizing phase. During the silent period of the hyperpolarizing
phase, neurons are able to restore calcium extracellular concentration. At the end of this phase, high extracellular calcium levels and full
synaptic function recovery would provide favorable conditions for the
onset of a new cycle.
In conclusion, our results show that [Ca]out
fluctuations are phasically associated with the depolarizing and
hyperpolarizing phases of the slow sleep oscillation. They bring new
insights into the oscillatory mechanisms of cortical neurons. As the
slow sleep oscillation is a precursor of nocturnal spike-wave seizures (Steriade and Amzica 1994), they may be extended and
included in the models of normal and paroxysmal sleep patterns.
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ACKNOWLEDGMENTS |
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We thank P. Giguère and D. Drolet for technical assistance. F. Amzica is a scholar of Fonds de la Recherche en Santé de Québec; M. Massimini is a doctoral student.
This study was supported by Medical Research Council of Canada Grant MT-15681.
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FOOTNOTES |
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Address for reprint requests: F. Amzica (E-mail: florin.amzica{at}phs.ulaval.ca).
Received 13 October 2000; accepted in final form 8 December 2000.
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REFERENCES |
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