1Nobel Institute for Neurophysiology,
Tegnér, Jesper and
Sten Grillner.
Interactive effects of the GABABergic
modulation of calcium channels and calcium-dependent potassium channels
in lamprey. The GABAB-mediated modulation of
spinal neurons in the lamprey is investigated in this study. Activation
of GABAB receptors reduces calcium currents through both
low- (LVA) and high-voltage activated (HVA) calcium channels, which
subsequently results in the reduction of the calcium-dependent
potassium (KCa) current. This in turn will reduce the peak
amplitude of the afterhyperpolarization (AHP). We used the modulatory
effects of GABAB receptor activation on N-methyl-D-aspartate (NMDA)-induced,
TTX-resistant membrane potential oscillations as an experimental model
in which to separate the effects of GABAB receptor
activation on LVA calcium channels from that on KCa
channels. We show experimentally and by using simulations that a direct
effect on LVA calcium channels can account for the effects of
GABAB receptor activation on intrinsic membrane potential oscillations to a larger extent than indirect effects mediated via
KCa channels. Furthermore, by conducting experiments and
simulations on intrinsic membrane potential oscillations, we find that
KCa channels may be activated by calcium entering through
LVA calcium channels, providing that the decay kinetics of the calcium
that enters through LVA calcium channels is not as slow as the calcium entering via NMDA receptors. A combined experimental and computational analysis revealed that the LVA calcium current also contributes to
neuronal firing properties.
Neuromodulators affect the operation of neural
networks (Grillner et al. 1995 The membrane properties of neurons in the lamprey spinal cord were
characterized in some detail (Buchanan 1982 GABA-immunoreactive neurons are present in the lamprey spinal cord
(Brodin et al. 1990 The objective of this study was to investigate the relative importance
of the modulatory effect of GABAB receptor activation on
LVA calcium channels and its indirect effect on KCaAP
channels brought about by its direct action on HVA calcium channels. We examined whether calcium entry through LVA Ca2+ channels
activates KCa channels and what influence LVA calcium currents have on neuronal firing properties. To this end, we used intracellular recordings to examine the effect of GABAB
receptor activation on intrinsic
N-methyl-D-aspartate (NMDA)-induced,
TTX-resistant membrane potential oscillations (NMDA-induced
oscillations). This analysis was extended with computer simulations to
separate the effects of Ca2+ channels from those of
KCa channels. We used the pacemaker oscillations as a model
system for how calcium and calcium-dependent properties interact. Thus
we do not address the possible role of the NMDA-induced oscillations in
the swimming rhythm.
Some of the results were reported previously in abstract form
(Tegnér et al. 1992 Experiments
PROTOCOL.
Experiments were performed with adult lampreys Petromyzon marinus
(n = 2) and Ichthyomyzon unicuspis
(n = 11). The animals were anesthetized with tricaine
methane sulfonate (MS222, 100 mg/l). The spinal cord was isolated in
cooled physiological Ringer and pinned down in a Sylgard-lined chamber
(Rovainen 1974 ANALYSIS OF EXPERIMENTAL DATA.
The threshold for detecting the onset and offset of the plateau phase
was identified as indicated in Fig. 1.
The threshold was manually set between the average plateau potential
and the trough potential. No detectable difference in the results was obtained when this procedure was repeated with the same trace. That the
oscillation period is large and the slope of the voltage curve is
steepest around the threshold accounts for the finding that the results
are not sensitive to exactly where the threshold is taken. The
following parameters were analyzed (Fig. 1): cycle duration
(peak-to-peak duration), the duration of the plateau, and the
hyperpolarized phase, with the hyperpolarized phase defined as the
difference in time between the cycle duration and plateau duration. The
plateau proportion was calculated as the ratio between the duration of
the plateau and the cycle duration. The peak amplitude was defined as
the maximum voltage of the first peak during the plateau phase. The
trough potential refers to the lowest potential during the
hyperpolarized phase. The cycle-to-cycle calculations were done with
DATAPAC (Axon Instruments) in which an appropriate threshold was
defined. MATLAB (MathWorks) was used for calculations of averages
(~20 cycles in all instances) and graphic display.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; Marder and
Calabrese 1996
). A neuromodulator acts as a rule via several
ionic mechanisms. It is therefore of interest to identify the role of
the separate mechanisms and determine the summed effects at the
neuronal and network level. We combine physiological experiments with
computer simulations to investigate the neuromodulatory action of
GABAB receptor activation on the neuronal level in lamprey
(Matsushima et al. 1993
). We focus on the interactive
effects between Ca2+ and KCa channels in spinal neurons.
, 1993
;
Buchanan and Grillner 1987
; Rovainen
1983
; Sigvardt et al. 1985
; Wallén and Grillner 1987
). Both low voltage-activated (LVA) and
high-voltage-activated (HVA) calcium channels are present
(Bacskai et al. 1995
; Christenson et al.
1993
; El Manira and Bussières
1997
; Grillner et al. 1998
; Matsushima et
al. 1993
). The calcium entering through HVA calcium channels
during an action potential (AP) activates apamin-sensitive KCa channels (KCaAP). These channels are
activated during the postspike afterhyperpolarization (AHP) and
determine the degree of spike frequency adaptation (El Manira et
al. 1994
; Gustafsson 1974
; Hill et al.
1992
; Meer and Buchanan 1992
;
Wallén et al. 1989
), whereas LVA calcium channels
allow neurons to fire rebound spikes (Matsushima et al.
1993
; Tegnér et al. 1997a
). Subtypes of
these channels can be influenced by modulators such as
5-hydroxytryptamine (El Manira et al. 1997
;
Wallén et al. 1989
), dopamine (McPherson and Kemnitz 1994
; Schotland et al. 1995
), and
GABA (El Manira and Bussières 1997
;
Matsushima et al. 1993
). These modulators either reduce
the conductance through KCa channels directly or reduce it
indirectly through an effect on Ca2+ channels.
). GABAB receptors act
via G proteins in the lamprey spinal cord (Alford and Grillner
1991
; Alford et al. 1991
; El Manira and
Bussières 1997
; Matsushima et al. 1993
). The effects include 1) a reduction of LVA calcium currents
(Matsushima et al. 1993
; Tegnér et al.
1997a
) leading to 2) a decreased postinhibitory rebound depolarization (Matsushima et al. 1993
;
Tegnér et al. 1997a
) and 3) a reduced
current through HVA calcium channels (Matsushima et al.
1993
), which decreases 4) the amplitude of the
apamin-sensitive KCa channels (KCaAP) and
thereby the peak amplitude of the slow AHP (Matsushima et al.
1993
; Tegnér et al. 1997a
).
GABAB receptor activation also results in presynaptic
inhibition, 5) which subsequently decreases inhibitory and
excitatory postsynaptic potentials from premotor interneurons
(Alford and Grillner 1991
; Alford et al. 1991
).
, 1997b
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; Wallén et al.
1985
). The preparation was perfused with oxygenated saline containing (in mM) 91 NaCl, 2.1 KCl, 2.6 CaCl2, 1.8 MgCl2, 20 NaHCO3, and 4 glucose (pH 7.4), with
the temperature held at ~9°C. Intracellular recordings were
performed with sharp microelectrodes. Discontinuous current clamp (DCC)
was used to control the membrane potential. Drugs were added directly
to the saline: NMDA (Tocris Neuramin), TTX, baclofen, and 2-OH saclofen
(Sigma) (Grillner et al. 1981
). Dorsal cells and edge
cells were not included in this study. Neurons exhibiting pacemaker
oscillations were not identified because TTX was used here. Lateral
interneurons do not oscillate in NMDA and TTX (Wallén and
Grillner 1987
). Data were acquired with a personal computer
(486 PC type) with AD/DA interface programs (AXOTAPE, Axon Instruments).
View larger version (18K):
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Fig. 1.
Threshold for detecting the onset and termination of the plateau phase
was set approximately as indicated. Peak-to-peak duration consists of a
plateau (ii) and a hyperpolarized phase (iv) indicated by the dashed
and dotted lines, respectively. The rapid depolarization and
repolarization are indicated by i and iii, respectively. The trough
potential refers to the lowest potential during the hyperpolarized
phase.
Computer simulations
SINGLE CELL MODEL.
We used a compartmentalized model neuron (Koch and Segev
1989; Rall 1977
) with Hodgkin-Huxley-type
membrane properties (Hodgkin and Huxley 1952
). The
neuron is represented in the simulations by a soma, a small initial
segment compartment, and a dendritic tree of three compartments
connected in series to the soma compartment (Grillner et al.
1988
; Wallén et al. 1992
). Parameters of
the cell model were previously matched to the properties of lamprey spinal neurons (Brodin et al. 1991
; Ekeberg et
al. 1991
; Tråvén et al. 1993
). The soma
is equipped with Na+, K+, and calcium channels
of both HVA and LVA type (Tegnér et al. 1997a
).
Calcium-dependent potassium channels (KCa) are also
included in the soma of the model. The initial segment has
Na+ and K+ conductance. In addition to the
passive properties, the three dendritic compartments also have ion
channels to represent input synapses. Inhibitory synapses are located
on the dendritic compartment adjacent to the soma, whereas excitatatory
synapses are placed on the second dendritic compartment (Ekeberg
et al. 1991
; Russell and Wallén 1983
).
Excitatory and inhibitory synaptic effects are modeled as conductance
changes in the dendritic compartment. Excitatory synapses have
voltage-dependent NMDA receptor channels as well as fast
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate
synapses, both of which are saturating, accounting for the fact that
the synaptic conductance cannot grow without limit as the firing
frequency of the presynaptic cell increases (Tråvén
et al. 1993
).
MODEL OF INTRACELLULAR CALCIUM AND INTERACTION WITH KCA
CHANNELS.
Like most other neurons lamprey spinal neurons exhibit spike frequency
adaptation caused by opening of KCa channels during long-lasting activation. During continuous firing the intracellular calcium level increases (Bacskai et al. 1995), leading
to activation of a KCa current. It was therefore important
to model intracellular calcium dynamics in addition to the
KCa channels. Intracellular calcium is modeled by two
different "intracellular pools." A CaAP pool represents
the calcium entering during the AP, and a CaNMDA pool
represents the calcium inflow through NMDA channels (Brodin et
al. 1991
; Ekeberg et al. 1991
). The
KCa channels activated by these two calcium pools are
referred to as KCaAP and KCaNMDA channels,
respectively. Both the CaAP and CaNMDA calcium
pools have parameters for calcium ion influx and decay. The parameter values used in this study are based on those determined in an earlier
study (Brodin et al. 1991
). Moreover, the
CaNMDA pool has a variable, ranging from zero to one,
representing the Mg2+ block of the channel. When the cell
model was extended with an LVA calcium channel (Tegnér et
al. 1997a
) an intracellular calcium pool was included to
represent the calcium entering via LVA calcium channels. We
investigated whether experimental data are consistent with the notion
that a KCa conductance could be activated by calcium entering via LVA-channels, thus referred to as KCaLVA
channel. Moreover, the LVA pool has parameters for the intracellular
calcium kinetics, which we investigated.
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DATA COLLECTION AND ANALYSIS.
The simulations were run with the UNIX-based SWIM software
(Ekeberg et al. 1994). The initial transients were not
analyzed in the single cell simulations. MATLAB (MathWorks) was used
for analysis and display of the single cell simulations.
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RESULTS |
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Effects of GABAB receptor activation on NMDA-induced, TTX-resistant, membrane potential oscillations
GABAB receptor activation reduces the calcium current
entering through HVA and LVA calcium channels (Matsushima
et al. 1993). The amplitude of the AHP following an AP is
reduced because of the reduction of the HVA calcium current and
consequently also the KCa current. Furthermore, the
tendency for a rebound depolarization and firing caused by an LVA
Ca2+ current is also decreased. To examine the summed
action of these mechanisms, we examined the effects of
GABAB receptor activation with baclofen on NMDA-induced
oscillations. These oscillations (Fig. 1) are characterized by four
different phases (Brodin et al. 1991
; Grillner
and Wallén 1985
; Sigvardt et al. 1985
;
Wallén and Grillner 1987
):
1) an initial rapid depolarization (Fig. 1) caused by a
regenerative opening of the voltage-dependent NMDA conductance,
followed by 2) a plateau depolarization when the inward
NMDA current is matched by an outward current through voltage-dependent K+ channels. During the plateau a gradual Ca2+
entry takes place, leading to the activation of KCa
channels and a graded repolarization. At a certain potential the NMDA
channels close 3), and a rapid repolarization (Fig. 1)
follows. Thereafter, 4) a slow, gradual depolarization
follows (Fig. 1) until the NMDA channels open again. Moreover, apamin,
which blocks the KCa channels, markedly prolongs the
duration of the plateau (El Manira et al. 1994
), thus
providing further support for the mechanisms described previously.
Membrane potentials were recorded intracellularly in the presence of
150 µM NMDA and 3 µM TTX (Fig.
2A1). Addition of baclofen (40 µM, Fig. 2A2) increased the cycle duration of the
oscillations (Fig. 2B1, - - - , P < 0.001). Because the shape of the oscillations is strongly voltage
dependent, DCC techniques were used. Thus, by varying the amount of
current injected continuously, the effects of baclofen can be compared
over a range of trough potentials to be certain that the effects
observed were not affected by changes in the trough potential. All
cells (n = 8) were analyzed quantitatively with
this paradigm. Figure 2 shows one example where an activation of
GABAB receptors decreased the plateau duration (Fig.
2B2, - - - , , P < 0.001, n = 20) and increased the duration of the hyperpolarized phase (Fig. 2B3, - - - ,
,
P < 0.001, n = 20) and the
cycle duration (Fig. 2B1, - - - ,
,
P < 0.001, n = 20). Baclofen
decreased the plateau proportion (Fig. 2B4, - - - ,
, P < 0.001, n = 20),
whereas the peak amplitude remained unchanged (Fig. 2B5,
- - - , 4/8 cells,
, P > 0.1, n = 20). The increase in the duration of the
hyperpolarized phase and the decrease of the plateau proportion was
observed in all cells (8/8 cells) and for all trough potentials
investigated.
|
However, some variations were observed. In the cell shown in Fig.
3, double-peaked oscillations occurred in
controls when the average trough potential was 72 mV (Fig.
3A1). Addition of baclofen (Fig. 3A2)
increased the duration of the hyperpolarized phase (Fig.
3C2, - - - , P < 0.001, n = 20). The cycle duration, however, remained
unchanged at the
72-mV trough potential when double-peaked
oscillations occurred (n = 3) (Fig.
3C1, indicated by *, P > 0.1, n = 20). Moreover, the plateau duration was also decreased by baclofen. Finally, the peak amplitude was reduced by GABAB receptor activation for all trough potentials
(Fig. 3C3, - - - , 4/8 cells, P < 0.001, n = 20).
|
To summarize, activation of GABAB receptors induces a shorter plateau and an increase of the hyperpolarized phase. The significance of this result is that it provides constraints on the interactive effects between the GABABergic modulation of calcium and calcium-dependent potassium channels as detailed in the two following sections. First we address the role of LVA calcium channels in generating pacemaker oscillations, and in the following section the possibility that a KCa channel is activated by the LVA calcium is examined.
Role(s) of LVA calcium channels during NMDA-induced, TTX-resistant membrane potential oscillations
If the main effect of an activation of GABAB receptors
is an opening of KCa channels an increase in both the
duration of the plateau and the hyperpolarized phases would be expected
(El Manira et al. 1994; Tegnér et al.
1998
). Baclofen, however, reduced the duration of the plateau
phase, thus motivating an analysis of the influence of the LVA calcium
component during pacemaker oscillations. To this end, a previously
developed model of LVA calcium channels (Tegnér et al.
1997a
) was used, which allows an independent analysis of the
LVA component and the KCa channels, respectively.
When the LVA calcium current was added to the simulation model, NMDA-induced, TTX-resistant membrane potential oscillations still occurred (Fig. 4A1, solid line). The net LVA conductance, the m3hGLVA factor (Fig. 4A2, solid line, see METHODS), is largest during the rising and initial part of the plateau phase and declines rapidly thereafter. LVA calcium channels become deinactivated during the hyperpolarized phase (increasing the value of the h-state variable, Fig. 4A3, solid line), thus allowing a non-0 current as the neuron depolarizes caused by rapid activation of the LVA calcium current (Fig. 4A4, solid line). The fast voltage-dependent inactivation of the LVA calcium current occurring during the plateau phase (Fig. 4A3, solid line) is responsible for the rapid decline of the LVA calcium conductance (Fig. 4A2, solid line).
|
A reduction of the LVA calcium current (80%, Fig.
4A1, dashed line) shortens the plateau phase and increases
the hyperpolarized phase slightly (see Fig. 4A1), as
indicated by the shaded scale bar that represents the duration of the
hyperpolarized phase in control (solid line). The prolongation of the
plateau phase by an LVA calcium conductance is due to the addition of
the net inward current carried by the LVA calcium (Fig. 4A2,
solid line). The slight increase of the hyperpolarized phase when the
LVA conductance is reduced is the result of two opposing effects. On
the one hand a decreased net inward conductance (LVA) occurring during
the rising phase would tend to increase the duration of the
hyperpolarized phase. The duration of the hyperpolarized phase is,
however, proportional to the level of calcium entering through NMDA
channels, the CaNMDA pool, which in turn activates a
separate set of KCa channels, referred to as
KCaNMDA (Tegnér et al. 1998). The
level of calcium in the CaNMDA pool, which decays slowly,
builds up during the NMDA-induced oscillations, thus activating
KCaNMDA channels, which have a hyperpolarizing influence
during the hyperpolarized phase (Fig. 4A5, solid line). The
peak level of the calcium in the CaNMDA pool during the
plateau phase is reduced when the LVA conductance is reduced (Fig.
4A5, dashed line). This reduction of the Ca2+
level tends to decrease the duration of the hyperpolarized phase. This
was reproduced by simulating a reduced conductance of the KCaNMDA channels (10%, Fig. 4B, solid line)
under control conditions to compensate for the increased
Ca2+ inflow during the plateau phase and thus isolate the
effect of removing the LVA conductance on the duration of the
hyperpolarized phase. This permits a qualitative evaluation of the role
of the LVA calcium channels during the hyperpolarized phase (Fig.
4B, solid line). A reduction of the LVA conductance under
these conditions increases the duration of the hyperpolarized phase
(compare Fig. 4B, dashed and solid lines; the shaded scale
bar is of the same length as that in Fig. 4A1).
To further elucidate the role of the LVA conductance on the duration of
the hyperpolarized phase, the activation and deactivation curves of the
LVA calcium current were shifted in the simulations between 1 and 5 mV
(see Fig. 5 legend) in
the hyperpolarizing direction. Because the LVA
calcium current is modeled with an m3h form (Tegnér et al.
1997a), this factor is calculated from the translated
m and h components, as illustrated in Fig.
5A1. The duration of the plateau and hyperpolarizing phase
of the corresponding m3h curves
during NMDA-induced oscillations are shown in Fig. 5A2. Thus, as the "LVA calcium window" (the non-0
m3h factor) is shifted toward more
negative potentials, the duration of the plateau phase and the
hyperpolarized phase decreases (Fig. 5A2). Figure
5A3 shows that a reduction of an LVA calcium conductance has
a clear effect on the duration of the hyperpolarized phase when the
m3h factor is translated by
3 mV
(compare with Fig. 4A1). The underlying mechanism is
illustrated in Fig. 5B, in which the
3- and
5-mV example
are compared (
and - - - , respectively). The
m3h factor is larger during the
hyperpolarized phase as the m3h
factor is shifted toward the hyperpolarized potential (see Fig. 5B, arrow). The level of calcium in the CaNMDA
pool is unchanged (compare Fig. 5B,
and - - - ), thus
ruling out that the effect on the hyperpolarized phase could be
due to an effect on the KCaNMDA channels as in Fig.
4A.
|
In summary, a reduction of the conductance of the LVA calcium channel decreases the duration of the plateau phase, whereas the duration of the hyperpolarized phase is increased during the NMDA-induced oscillations. The increased duration of the hyperpolarized phase produced by GABAB receptor activation can be accounted for if the m3h curve is translated a few millivolts in the hyperpolarized direction (Fig. 5A1).
Simulation of KCa channels activated by the calcium entering through LVA calcium channels
Here we analyze whether an activation of a separate group of
KCa channels (KCaLVA) via LVA calcium channels
is consistent with the effects of baclofen on the pacemaker
oscillations. An LVA calcium pool (see METHODS and
Tegnér et al. 1997a), which in turn activates a
separate KCa conductance through a separate population of
channels, is used. Figure 6 shows a
parameter plot in which the following parameters are investigated
independently of the level of LVA activation: 1) the
strength of the coupling between the LVA calcium and the
KCa channel interpreted either a) as the degree
of activation of the KCa by the LVA calcium or b) simply the maximal conductance of the KCaLVA
channel (GK(CaLVA)), which was actually the
parameter modified during the simulations. The contribution of the LVA
calcium channel to the total KCa current is given by
Eq. 5 (METHODS).
|
The second parameter analyzed was 2) the decay time of the
calcium in the intracellular calcium pool and finally 3) the
NMDA levels [1.0, 2.0 arbitrary units (A. U.)]. A sample trace of the stimulated NMDA-induced oscillations during 2 s is shown in each box (Fig. 6). The kinetics of the decay of the intracellular calcium in
the LVA pool (LVA, see Eq. 2) is referred to
as slow (2 s
1), medium (10 s
1), or fast (20 s
1). The numbers indicate the different values of the
parameter for the decay of the intracellular calcium in the LVA pool.
For example, in the Fig. 6, topmost left box, the following
parameter values apply: NMDA level is 1.0 (A. U.), kinetics of the
decay of the intracellular calcium is 2 (s
1), and the
conductance of the KCaLVA is 0; thus G = 0. This example will be referred to as [KCa(slow), G(0),
N(1.0)]. By using this notation we make the following
observations (Fig. 6). 1) Addition of a
GK(CaLVA) conductance from 0 to 0.8 shortened
the duration of the plateau phase irrespective of the kinetics of the
intracellular calcium pool and the NMDA level. For example, compare the
records in the left and right columns of Fig. 6 at different NMDA
levels. The KCaLVA conductance thus acts as a plateau
terminating factor because, for a given level of intracellular calcium,
the influence of a KCa channel became stronger when the
GK(CaLVA) conductance was increased. Moreover,
2) with a slow decay of the intracellular calcium, the
duration of the hyperpolarized phase increases as the
GK(CaLVA) conductance increases (compare
[KCa(slow), G(0.2), N(1.0)] with [KCa(slow),
G(0), N(1.0)]). Thus, although the duration of
the plateau is decreased, the effective inhibition during the hyperpolarized phase is increased because of the slow decay rate of the
intracellular LVA calcium. 3) If instead the decay rate of
the intracellular calcium is increased to a medium or fast rate, the
duration of the hyperpolarized phase may instead decrease by an
addition of a GK(CaLVA) conductance (compare
left and right columns, Fig. 6). As shown by comparing the middle and
right columns a decrease in the duration of the hyperpolarized phase is
obtained with an increased conductance of the
GK(CaLVA). Note also 4) that an
increased conductance of the GK(CaLVA) reduces
the peak depolarization (compare middle and right column, Fig. 6).
Finally, 5) the effects of increasing the KCaLVA
are more clear at a lower level of NMDA because the level of NMDA had a
weaker depolarizing influence compared with the higher levels of NMDA.
Activation of GABAB receptors increases the duration of the hyperpolarized phase (Figs. 2B3, - - - , and 3C3, - - - ), whereas a decreased conductance of the KCaLVA channels would decrease the duration of the hyperpolarized phase if the kinetics are slow (compare [KCa(slow), G(0.8), N(2.0)] with [KCa(slow), G(0.2), N(2.0)] and [KCa(slow), N(2.0)] in Fig. 6). Thus it is not consistent with the experimental data to assume that the kinetics of the intracellular LVA calcium pool is of the slow type if the LVA calcium activates a KCaLVA channel. The simulations suggest a weaker coupling between the LVA calcium and the corresponding KCaLVA channel (or smaller maximal conductance of KCaLVA) compared with the KCaAP and KCaNMDA channels. The effect of a modulation of the GK(CaLVA) conductance would be too marked if, for example, G = 0.8 is taken as a reference value (see DISCUSSION) because of the finding that GABAB receptor activation decreases the duration of the plateau phase (Figs. 2B2, - - - , and 3C2, - - - ). In conclusion, fast kinetics and low conductance of the putative KCaLVA channel is consistent with the experimental data obtained on the pacemaker oscillations.
Modulation of firing properties in the absence of pacemaker oscillations
It is not clear what overall effect GABABergic modulation will have on neuronal firing as GABAB receptor activation reduces both LVA and HVA calcium currents and KCa currents. Here we examine the influence of GABABergic modulation of the firing properties in the absence of the NMDA-induced oscillations.
The occurrence of a spike, activated by a square current pulse in
controls at a hyperpolarized membrane potential (80 mV) level, could
be blocked by GABAB receptor activation (Fig. 9) (Matsushima et al. 1993
). Figure
7A1 shows a simulated spike
elicited by a long-lasting current pulse in a hyperpolarized neuron
equipped with an LVA calcium conductance. A reduction of the LVA
calcium conductance to GLVA = 0 suppresses the
occurrence of a spike (Fig. 7A2). This result suggests an
important role for LVA calcium channels in subthreshold behavior.
|
A subthreshold current pulse may elicit an AP in the presence of the
KCa channel blocker apamin (see El Manira et al.
1994). To identify possible mechanisms involved in this effect
we simulated that experiment (Fig. 7B). The induction of an
AP by apamin could not be simulated if it was assumed that it only
suppressed the KCaAP channels. It was sufficient to include
a KCaLVA channel with a low conductance, as determined
earlier (Fig. 6), which became activated by the LVA calcium during the
subthreshold current pulse (Fig. 7B1). The simulated
reduction of the KCaLVA (and KCaAP) channels by
apamin induced a spike (Fig. 7B2), thus reproducing the
earlier experimental result (El Manira et al. 1994
).
Apamin-sensitive KCaAP channels are the main determinant of
the spike frequency regulation in lamprey neurons (Hill et al. 1992; Meer and Buchanan 1992
). It is less clear
to what extent LVA calcium channels contribute to the firing properties
of these neurons. In the simulations described subsequently we studied the influence of the LVA calcium itself without an accompanying KCaLVA channel to identify a possible role of LVA calcium
channels per se. Figure 8A1
shows the firing response to a current pulse for a model neuron without
an LVA calcium channel. The first spike interval (Fig. 8A1,
- - - ) is shorter than the second and third interval in controls,
which is similar to experimental results (Buchanan 1993
;
Wallén et al. 1989
). If an LVA conductance is added to the model neuron (Fig. 8B1), everything else being
equal, the first interspike interval became shorter, as did the
following interval, albeit to a lesser extent. As the neuron is
depolarized, the LVA calcium conductance rapidly activates, whereas it
inactivates during the plateau phase on which the spikes are fired.
Thus the m3h factor rises initially
but declines during the spike train (Fig. 8B2). A simulated
reduction of the KCaAP channel increases the number of
spikes and decreases the first spike interval (Fig. 8A2) in
agreement with earlier experiments (El Manira et al.
1994
; Meer and Buchanan 1992
). The simulations
suggest that, if the main action of baclofen were mediated via LVA
calcium channels (Figs. 1-4), the number of spikes would tend to
decrease, and the first spike interval would increase if
GABAB receptors were activated. On the other hand, if the
indirect effects on the KCaAP channels were dominant, an
increased number of spikes would be expected. To further examine this,
spinal neurons were stimulated by square-wave current pulses (Fig.
9). In all cells (n = 5)
recorded we observed the following. 1) The number of spikes
was reversibly reduced when baclofen was added (compare Fig. 9,
A1 and A2), and 2) a significant
(P < 0.0001) increase in the duration of the first spike interval occurred (Fig. 9, A1, A2, and
B). Thus the effects of baclofen on the spikes (Fig. 9)
correspond well to a simulated reduction of the LVA conductance (Fig.
8).
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In summary, both the simulations and the experiments on the GABABergic modulation of the firing properties in the absence of pacemaker oscillations support the notion that the primary mechanism accounting for the effects of baclofen is the calcium entry through LVA channels.
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DISCUSSION |
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The objective of this study was to clarify the relative contribution of LVA calcium channels and KCa channels in the modulatory actions of GABAB receptors. To this end we used the NMDA-induced oscillations as an experimental and computational model to study the interactive effects between LVA and HVA calcium channels and KCa channels activated by either LVA or HVA calcium channels. We have shown that the direct effect on the LVA calcium channels could account for the modulatory effects of baclofen on the NMDA-induced oscillations. The indirect effect on the KCaAP channels via HVA calcium channels could not account for the modulation of the depolarized and hyperpolarized phase by baclofen. Both simulations and experiments revealed that the LVA calcium conductance could contribute to the duration of the first spike interval.
Role of calcium and calcium-dependent conductances during NMDA-induced oscillations
The importance of the NMDA-induced oscillations in this context is
essentially twofold. 1) It provides an
experimental/computational model allowing an analysis of the
interactions between various intrinsic membrane properties and
2) a source for intrinsic oscillations that could be of
importance in the generation of rhythmic patterns underlying locomotion
in the lamprey spinal cord. Thus, to understand the mechanisms
underlying the generation of locomotion at the cellular level, it is
important to understand how the different conductances interact to
produce membrane potential oscillations. We focused on the cellular
interplay between calcium conductances and their respective
KCa channels without considering their role in the
operation of the neural network. Earlier studies have shown that both
mechanisms can modulate the burst rate in the spinal segmental network
(El Manira et al. 1994; Tegnér et al.
1997a
, 1998
).
Figure 10 summarizes the influences of
calcium channels (HVA and LVA type) and KCa conductances
with either fast or slow intracellular calcium dynamics. It shows their
influence on the duration of the plateau and the hyperpolarized phase.
The duration of the plateau phase is determined by a balance between
Ca2+ channels of either the HVA or the LVA type, which tend
to increase the duration, and a KCa conductance, which
decreases the duration of the plateau phase irrespective of the
dynamics of the intracellular calcium activating the KCa
channel. The duration of the hyperpolarized phase is decreased by the
addition of an LVA calcium channel to the model neuron (Figs. 4 and 5).
This effect becomes more dramatic the larger the
m3h factor is at the potential at
which the transition between hyperpolarization and depolarization
occurs (see Fig. 5A1, left - - - ). Moreover, the
introduction of a KCa channel with slow dynamics, such as the KCaNMDA channel (Brodin et al. 1991;
Ekeberg et al. 1991
), increases the duration of the
hyperpolarized phase. Thus a given level of NMDA has a depolarizing
influence, which activates the KCaNMDA channels in
proportion to the calcium entering via the NMDA channels. These factors
together determine the depolarized and hyperpolarized phase in a
voltage-dependent manner because of the voltage dependence of the NMDA
receptor. The modulatory action of the HVA, LVA, KCaAP, and
KCaLVA channels further contributes to the regulation of
the depolarized and hyperpolarized phases as described previously. It
is now important to further characterize different subtypes of HVA
Ca2+ channels (El Manira and Bussières
1997
) and investigate their influence on NMDA-induced
oscillations experimentally.
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LVA calcium and the KCaAP contribution to the modulatory effects of GABAB receptor activation
Transmitters act as a rule via several subtypes of receptors
(Nicoll 1988). Moreover, a given receptor subtype may
mediate its effects through one of a wide variety of mechanisms. It is therefore important to investigate the relative contribution of the
different components in the effects mediated by a given transmitter. Activation of GABAB receptors in the lamprey reduces the
LVA and HVA calcium current, which in turn reduces the peak amplitude of the slow AHP (Matsushima et al. 1993
). Thus the
arguments that indicate a predominant influence of the LVA calcium
channels compared with the indirect effect on the KCaAP
channels in lamprey are as follows. 1) During the
NMDA-induced oscillations baclofen decreases the duration of the
plateau phase, whereas the duration of the hyperpolarized phase is
increased (Figs. 2 and 3). 2) A simulated reduction of the
LVA calcium conductance can account for the effects of baclofen during
the NMDA-induced oscillations (Figs. 4 and 5), and 3) apamin
(Blatz and Magleby 1986
), which directly depresses the
KCa current, increases both the duration of the plateau and the hyperpolarized phase (El Manira et al. 1994
).
4) A simulated reduction of the KCaAP
conductance can account for the effects of apamin on the NMDA-induced
oscillations (Tegnér et al. 1998
). 5)
GABAB receptor activation decreases the number spikes
elicited by a current pulse (Fig. 9A1 and A2).
This could be simulated by reducing the LVA calcium current (Fig. 8,
A1 and B1) but not by reducing the
KCaAP component (Fig. 8A2). Thus the modulatory effects of baclofen, which also reduces the HVA calcium current and
thereby the KCaAP current indirectly, differ from those of apamin, with respect to membrane potential oscillations and neuronal firing properties. Moreover, on the network level, 6)
baclofen, reduces the locomotor burst rate (Tegnér et al.
1993
) and also has a clear effect on the burst rate in the
higher NMDA range (Tegnér et al. 1997a
), whereas
7) the effect of an apamin-mediated reduction of the
KCaAP current on the burst rate in the higher NMDA range is
small, as shown experimentally (El Manira et al. 1994
)
and with computer simulations (Tegnér et al.
1998
). 8) A simulated decrease of the LVA calcium
conductance reduces the burst rate in the lower and higher NMDA range
(Tegnér et al. 1997a
). Thus taken together
[6)-8)] this provides evidence from the
network level indicating that LVA calcium channels are more important
for the regulation of the burst rate at the higher NMDA level than are
KCa channels.
In summary, both experiments and computer simulations on the single cell and network level suggest that the modulatory effects of GABAB receptor activation are mainly mediated through LVA calcium channels.
LVA, KCaLVA channels, and interactive effects contributing to firing properties
It is often difficult to characterize the kinetics and conductance
of a KCa channel because the level of intracellular
Ca2+, the contribution of Ca2+ from various
Ca2+ channels, and the intracellular kinetics are generally
not known in sufficient quantitative detail. A simplifying step is to
lump the open-closed transitions of channels and intracellular
transition schemes into differential equations (Ekeberg et al.
1991). In accounting for the effects of LVA calcium channels at
the single cell and network level described by Tegnér et al.
(1997a)
, it was not necessary to assume that the LVA calcium activated
a KCa channel.
The modulatory effects of GABAB receptors on NMDA-induced oscillations, however, provide additional experimental constraints on the effects of the LVA calcium. We used these data to assess whether a KCaLVA channel was activated and to determine the basic characteristics of the calcium kinetics involved. We found that it was not consistent with experimental data on NMDA-induced oscillations (Figs. 2 and 3) to have slow intracellular calcium kinetics for the LVA calcium. Moreover, the maximal conductance of the KCaLVA appears to be small compared with the conductance of KCaAP and KCaNMDA (Fig. 6). Thus fast decay kinetics of the intracellular LVA Ca2+ together with a small conductance of the KCaLVA is consistent with the experiments on NMDA-induced oscillations. However, these conclusions are based on experiments with the modulatory effects of baclofen on the slow NMDA-induced oscillations.
Recent studies (McCobb and Beam 1991; Olsen and
Calabrese 1996
) have shown that the amount and characteristics
of LVA and HVA calcium currents also depend on the envelope of the
voltage curve. In a model of leech heart interneurons it was predicted that LVA calcium currents were partially inactivated during a slow
voltage ramp to a plateau potential (Nadim et al. 1995
;
Olsen et al. 1995
). A similar result (a reduced
m3h factor) was obtained when the
effects of GABAB receptors were simulated with a sinusoidal
current stimulation with a low frequency (Fig. 9) (Tegnér
et al. 1997a
). Furthermore, as illustrated in Fig.
4A2, the
m3GLVA factor declines
rapidly after the fast voltage upstroke, whereas a non-0 component
remains during the plateau phase. Olsen and Calabrese (1996)
confirmed
experimentally the suggested dependence on the waveform in leech in
that either a faster rise time or a more hyperpolarized holding
potential increased the LVA calcium current. McCobb and Bean (1991)
demonstrated that the size of HVA currents was more sensitive to an
increased duration of the AP compared with an LVA calcium current
because of a rapid inactivation of the LVA calcium current at a
depolarized potential. Moreover, the LVA calcium component evoked at a
low holding potential with a brief AP was comparable with that of the
HVA calcium channels. This suggests that the modulatory influence of
the KCaLVA channels in lamprey will depend on the waveform
in a manner similar to that of an LVA calcium current. Thus the amount
of calcium entering through the LVA channels during the NMDA-induced
oscillations is small compared with that from HVA calcium channels
activating KCaAP channels, thus potentially masking the
effects of KCaLVAchannels. Taken together, this could mean
that our estimation of the GK(CaLVA) conductance
is an underestimation.
Another line of evidence that supports the involvement of
KCaLVA channels is the finding that application of apamin
induces an AP on a previously subthreshold pulse (Fig. 2, C
and D, in El Manira et al. 1994). It proved
necessary to include an apamin-sensitive KCaLVA channel in
the model to account for this finding (Fig. 7, B1 and
B2). Thus LVA calcium enters the neuron during a
subthreshold pulse (Fig. 7B1), activating a
KCaLVA channel that prevents the occurrence of a spike
elicited by a current pulse close to threshold (Fig. 9)
(Matsushima et al. 1993
), a finding that was simulated here (Fig. 7, A1 and A2) by reducing the LVA
calcium conductance. Another result that supports the inclusion of a
KCaLVA channel in the neuron model is the finding that
apamin reduces the latency of the first spike [contained in the
material presented by El Manira et al. (1994)
and Hill et al. (1992)
].
This was most clear when the current pulse only elicited a few spikes
in control experiments. This finding could be simulated if a
KCaLVA channel activated by the LVA calcium was added to
the model (not illustrated). A conductance on the order of 80% of the
KCaAP was sufficient. Thus both the effect of apamin on the
latency of the first spike as well as the induction of a spike depend
on the presence of KCaLVA channels, which therefore are
presumed to be sensitive to apamin.
Apamin-sensitive KCaAP channels contribute significantly to
spike frequency adaptation (El Manira et al. 1994);
Hill et al. 1992
; Meer and Buchanan 1992
)
(Fig. 8, A1 and A2). We showed that the LVA
calcium current itself contributes to the firing properties in the
absence of pacemaker oscillations (Fig. 8, A1 and
B1). The presence of an LVA conductance in the model reduced
the duration of the first spike interval and increased the number of
spikes elicited by a current pulse (Fig. 7, A1 and
B1). This is due to the fact that the inactivation of LVA
calcium channels is removed to a large extent at hyperpolarized
potentials, and they are subsequently activated when the neuron fires,
thus providing an inward current during the early part of the spike
train (Fig. 8B2). The finding that an experimental
activation of GABAB receptors reduced the number of spikes
and increased the duration of the first spike interval (Fig. 9,
A1, A2, and B) supports this
simulation result.
A subpopulation of the neurons within the dorsal horn of the turtle
spinal cord can generate burst activity (Russo and Hounsgaard 1996a). This is due to the presence of LVA calcium channels,
and here it has also been shown that the first spike interval is the shortest, the firing increases as the holding potential is
hyperpolarized, and the duration of the first spike interval decreases
accordingly. Moreover, in another group of neurons that generate
plateau potentials (Russo and Hounsgaard 1996b
),
apamin increases the firing rate and decreased the duration of the
first spike interval, a finding that also applies to motorneurons
(Hounsgaard et al. 1988
). Finally, a blockade of the
L-type calcium channel decreased the firing rate and increased the
duration of the first spike interval compared with the spike train
elicited when apamin was applied (Hounsgaard and Mintz
1988
). Thus the inward calcium current itself influences the
first spike interval in the turtle spinal cord, similar to the role of
LVA calcium channels suggested in this study. Furthermore, both in the
turtle and lamprey spinal cord neurons, the presence of an inward
calcium current affects the number of spikes elicited. In conclusion,
the results on spinal neurons in the turtle are qualitatively similar
to the firing properties of lamprey neurons presented here (Fig. 7-9)
and in earlier studies (Buchanan 1993
; El Manira
et al. 1994
; Meer and Buchanan 1992
).
This study shows that a modulation of the LVA calcium current can account for the effects of baclofen on NMDA-induced oscillations and firing properties of lamprey spinal neurons. Furthermore, the experiments and simulations taken together suggest that a low-conductance KCaLVA channel of the apamin-sensitive type could be activated by the calcium (with a fast decay) entering through LVA calcium channels.
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ACKNOWLEDGMENTS |
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We are indebted to Dr. Örjan Ekeberg for the development of the SWIM simulation software (URL http://www.nada.kth.se/sans/). We express our gratitude to Drs. Jeanette Hellgren-Kotaleski, David Parker, and Richard Thompson for helpful discussions and critical reading.
This work was supported by Medical Research Council project no. 3026, Swedish Natural Science Research Council project no. B-AA/BU03531, Swedish National Board for Industrial and Technical Development, NUTEK, project no. 8425-5-03075, and the Swedish Society for Medical Research.
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FOOTNOTES |
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Address for reprint requests: J. Tegnér, Nobel Institute for Neurophysiology, Dept. of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden.
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 7 May 1998; accepted in final form 6 November 1998.
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REFERENCES |
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