Department of Physiological Science, University of California, Los Angeles, California 90095-1568
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ABSTRACT |
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del Negro, Christopher A., Chie-Fang Hsiao, and Scott H. Chandler. Outward currents influencing bursting dynamics in guinea pig trigeminal motoneurons. To initiate and maintain bursts (and plateau potentials) in the presence of serotonin, guinea pig trigeminal motoneurons utilize L-type Ca2+ and persistent Na+ inward currents. However, the intrinsic currents that contribute to burst termination and determine the duration of the interburst interval are unknown. Therefore we investigated the roles of outward currents, whose slow activation is coupled to cytosolic cation (Ca2+ and Na+) accumulation. First we examined a Ca2+-dependent K+ current (IK-Ca) with apamin and Ba2+-substituted, low-Ca2+ solution. Blockade of IK-Ca lengthened burst duration and cycle time but did not abolish bursting. Next we studied the Na+/K+-ATPase pump current (Ip) with cardiac glycosides. In the presence of apamin or low-Ca2+/Ba2+ solution, blocking Ip (with ouabain or strophanthidin) decreased both burst duration and cycle time and ultimately transformed bursting into tonic spiking. We conclude that IK-Ca and Ip contribute to burst termination in trigeminal motoneurons. These currents influence temporal bursting properties such as burst duration and cycle time and may help determine the phasic activity of motoneurons during rhythmic oral-motor behaviors.
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INTRODUCTION |
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Neuronal bursting behavior, by a traditional
mechanism (Del Negro et al. 1998), utilizes non- or
slowly inactivating inward currents to generate a region of negative
slope resistance in the steady-state current-voltage
(I-V) relationship. This region of negative slope
resistance creates two stable membrane states on either side of spike
threshold: one quiescent state and another suprathreshold or active
state, at which the cell discharges tonically. These two states are
traversed during the quiescent and active phases of bursting.
To initiate individual bursts, the inward currents underlying the
negative slope resistance cause rapid depolarization, which shifts the
membrane potential from its quiescent to active state (see Fig.
3B of Del Negro et al. 1998). Conversely,
bursts terminate because of much slower membrane processes that
generate repolarizing currents (Bertram et al. 1995
;
Rinzel and Ermentrout 1998
). During the active phase
these steadily growing currents cause a decline in intraburst spike
frequency and ultimately a return to quiescence. The source of the
repolarizing currents can be a distinct set of ion channels,
intramembranous ion pumps, the slow inactivation of inward currents, or
a combination of these factors. The repolarizing currents that
terminate bursts are usually activated by cytosolic cation accumulation
(Gorman et al. 1981
, 1982
; Li and Hatton
1996
; Rose and Ransom 1997
). For example, inward
Ca2+ current can activate Ca2+-dependent
K+ currents during bursts (Legendre et al.
1985
; Tell and Jean 1993
) or cause
Ca2+-dependent Ca2+ current inactivation
(Benson and Adams 1989
; Canavier et al. 1991
; Heyer and Lux 1976
; Kramer and
Zucker 1985
). Na+ currents can activate the
Na+/K+-ATPase pump during bursts
(Johnson et al. 1992
; Li et al. 1996
; Tsai and Chen 1995
) or possibly experience voltage- or
cytosolic Na+-dependent inactivation (Fleidervish et
al. 1996
).
In trigeminal motoneurons that innervate jaw musculature both
Ca2+ and Na+ currents are important for
serotonin-induced bursting (Hsiao et al. 1998).
Therefore intracellular accumulation of Ca2+ and
Na+ ions may activate distinct slow repolarizing currents.
Here we test the hypothesis that an apamin-sensitive,
Ca2+-dependent K+ current
(IK-Ca) and a cardiac glycoside-sensitive
Na+/K+-ATPase pump current
(Ip) activated by intracellular Ca2+
and Na+, respectively, contribute to burst termination and
the overall dynamics of bursting behavior.
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METHODS |
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Trigeminal motoneurons were recorded intracellularly in
450-µm-thick transverse brain stem slices from adolescent guinea pigs (150-250 g), as shown previously (Chandler et al. 1994;
Hsiao et al. 1998
). Slices were perfused in a
gas-interface chamber at 34°C with oxygenated Ringer solution
containing (in mM) 130.0 NaCl, 3.0 KCl, 1.25 KH2PO4, 20.0 NaHCO3, 10.0 D-glucose, 2.4 CaCl2, and 1.3 MgSO4. Microelectrodes were filled with 3 M KCl (15-20 M
). Current-clamp experiments were performed with an Axoclamp 2A
amplifier in bridge mode (Axon Instruments, Burlingame, CA).
The acceptance criteria for trigeminal motoneurons included resting
potential less than 50 mV, input resistance of 6-18 M
, and action
potential amplitude >60 mV (Chandler et al. 1994
;
Curtis and Appenteng 1993
).
Bath-applied drugs were effective after ~3.5 min of perfusion;
complete solution change required ~20 min. We used 10 µM serotonin (Sigma Chemical, St. Louis, MO) to elicit bursting; this dose reliably
induces bistability and bursting in trigeminal motoneurons (Hsiao et al. 1998). Other drugs were used at the
following concentrations: apamin (0.2 µM, Sigma), ouabain (6 µM,
Sigma), and strophanthidin (4 µM, Sigma). In some experiments
Ca2+ concentration was lowered to 0.4 mM and substituted
with Ba2+ on an equimolar basis.
Bursting characteristics such as the time of burst onset and termination and intraburst spike frequency were measured with Datapac III v0.1.47 (Run Technologies, Irvine, CA). Burst duration was defined as the mean time from burst onset to termination in three or more consecutive bursts. Burst cycle time was defined as the mean time from burst onset to onset in three or more consecutive cycles. Means are reported as ±SE. Pharmacologically induced changes in bursting characteristics were tested for each cell individually with unpaired Student's t-tests (SYSTAT 7.0, SPSS, Chicago, IL).
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RESULTS |
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In the presence of serotonin, trigeminal motoneurons exhibit
plateau potentials and spontaneous rhythmic bursting (Hsiao et al. 1998). Figure 1 illustrates
these behaviors in a typical cell. At resting potential, the cell was
bistable. Therefore transient stimuli evoked plateau potentials (Fig.
1, A and B, n = 135, including cells from our earlier paper) (Hsiao et al. 1998
). This
behavior could be converted to continuous bursting activity by
injecting depolarizing constant current (Fig. 1, A and
C, n = 90). In all cells recorded, burst
duration ranged from 0.7 to 53 s, the interburst interval ranged
from 4.2 to 123 s, and the cycle time ranged from 4.9 to 178 s.
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Several features of the plateau potentials and bursts suggest that
repolarizing currents were recruited throughout the active phase.
First, during the plateau potentials and bursts, spike frequency
declined monotonically until burst termination (Fig. 1, B
and C, bottom), which is characteristic of a
traditional, negative slope resistance-based bursting mechanism
(Bertram et al. 1995; Del Negro et al.
1998
). Also a hump-like depolarizing afterpotential occurred at
burst termination. This suggests that the voltage trajectory rose after
the previous spike but failed to reach threshold putatively because of
the growing repolarizing current (Fig. 1, B and
C, arrows). Last, after burst termination, the membrane
potential was hyperpolarized by 5 mV compared with burst onset,
suggesting the growth of outward currents during the burst (Fig.
1C, broken line inserted for reference).
Role of Ca2+-dependent K+ current
L-type Ca2+ currents provide the majority of inward
current needed for serotonin-induced bursting (Hsiao et al.
1998). Because Ca2+ may accumulate intracellularly
during bursts and trigeminal motoneurons express an apamin-sensitive,
Ca2+-dependent K+ current (Chandler et
al. 1994
; Kobayashi et al. 1997
), we tested whether IK-Ca was involved in the burst
termination. Specifically, we applied apamin (n = 5) or
Ba2+-substituted, low-Ca2+ solution
(n = 4) to reduce or eliminate
IK-Ca.
Apamin typically prolonged burst duration and extended the cycle time.
In Fig. 2A mean burst duration
increased significantly from 47 ± 2 to 271 ± 30 s
(P < 0.001), and mean cycle time increased from
115 ± 5 to 490 ± 46 s (P < 0.001) in
the presence of apamin. In general, apamin significantly increased
burst duration by 313 ± 76% (n = 5/5,
P < 0.01) and extended burst cycle time by 202 ± 58% (n = 4/5, P < 0.05). In addition
to increasing burst duration and cycle time, apamin also elevated the
intraburst spike frequency-time (f-t)
relationship (Fig. 2C), consistent with its suppression of
the medium-duration afterhyperpolarization (mAHP) in trigeminal motoneurons (Fig. 3B)
(Chandler et al. 1994; Hsiao et al. 1997
; Kobayashi et al. 1997
).
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We also tested the role of IK-Ca with
Ba2+-substituted, low-Ca2+ solution because
Ba2+ can penetrate Ca2+ channels but does not
activate IK-Ca. Similar to apamin,
low-Ca2+/Ba2+ solution enhanced burst duration
and cycle time. In Fig. 2B mean burst duration increased
significantly from 3.5 ± 0.2 to 11.9 ± 0.6 s
(P < 0.001), and mean cycle time increased from
9.0 ± 0.5 to 22.0 ± 0.8 s (P < 0.001). In general, low Ca2+/Ba2+ significantly
increased burst duration by 357 ± 142% (n = 3/4, P < 0.01) and extended cycle time by 271 ± 76%
(n = 3/4, P < 0.001). Like apamin,
low-Ca2+/Ba2+ solution also elevated the
intraburst f-t curve (Fig. 2D). The elevation in spike frequency could be caused by suppression of the
mAHP, blockade of Ba2+-sensitive K+ channels
(Hsiao et al. 1997), or a combination of both effects.
Although apamin and low-Ca2+/Ba2+ solution upwardly shifted the intraburst f-t relationship, bursts still terminated (in the absence of IK-Ca) when the instantaneous intraburst spike frequency declined to low values similar to control. In Fig. 2C, spike frequency at the time of burst termination was 5 Hz in control and 8 Hz in the presence of apamin. In Fig. 2D, the spike frequency was 3 Hz in control and 5 Hz in low-Ca2+/Ba2+ solution. These data suggest that the net outward current at the time of burst termination was similar in the presence or absence of IK-Ca. Therefore some compensatory mechanism(s) must be present to terminate bursts after the blockade of IK-Ca.
Role of Na+/K+-ATPase pump
If IK-Ca was the only outward current
controlling spike frequency decline and burst termination, we would
expect apamin or low-Ca2+/Ba2+ solution to
induce tonic spiking (because bursts would not be able to terminate).
However, bursting continued in apamin or
low-Ca2+/Ba2+ conditions, suggesting additional
sources of slow repolarizing current. Because transient and persistent
Na+ currents contribute to serotonin-induced bursting in
trigeminal motoneurons (Hsiao et al. 1998), cytosolic
Na+ accumulation might also activate slow repolarizing
current, analogous to Ca2+, or cause slow Na+
current inactivation. The Na+/K+-ATPase pump is
associated with bursting behavior (Johnson et al. 1992
;
Li et al. 1996
; Tsai and Chen 1995
), is
activated by cytosolic Na+, and is electrogenic (because of
pump stoichiometry: 3 Na+ ions extruded for every 2 K+ ions that enter) (Läuger 1991
).
Therefore we tested the potential contribution of the
Na+/K+-ATPase pump current
(Ip) with cardiac glycosides ouabain
(n = 8) and strophanthidin (n = 6),
which block pump activity.
To determine the role of Ip we first applied apamin (n = 4, Fig. 3A) or low-Ca2+/Ba2+ solution (n = 4, Fig. 4A) to block IK-Ca. Once apamin or low Ca2+/Ba2+ was effective, as measured by blockade of the mAHP (Fig. 3B, top), ouabain was added to block Ip. In all trigeminal motoneurons tested, ouabain ultimately abolished bursting and induced tonic spiking. Nevertheless, in three of four cells exposed to apamin + ouabain (Fig. 3C) and two of four cells exposed to low Ca2+/Ba2+ + ouabain (Fig. 4A), bursting activity continued during a transition period that preceded tonic spiking.
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During this transitional bursting phase, ouabain decreased burst duration and cycle time and reduced the amplitude of the postburst afterhyperpolarization (AHP). In Fig. 3, burst duration decreased significantly from 10.1 ± 1.0 to 7.6 ± 0.4 s (P < 0.05), and cycle time decreased from 29.8 ± 5.2 to 10.6 ± 0.3 s (P < 0.001) (measured at the temporal midpoint between the time of ouabain application and the time at which motoneurons assumed tonic spiking). Also ouabain reduced the postburst AHP, as measured during the interburst interval (Fig. 3C, broken line inserted for reference). In low-Ca2+/Ba2+ solution, ouabain similarly decreased burst duration and cycle time and reduced the postburst AHP (Fig. 4A). In general, ouabain significantly decreased burst duration to 54 ± 11% of burst duration in the presence of apamin alone (n = 3/3, P < 0.05) and to 68% of burst duration in low-Ca2+/Ba2+ solution (n = 1/2, P < 0.05). Moreover, ouabain decreased cycle time to 6 ± 2% of cycle time in the presence of apamin alone (n = 3/3, P < 0.001) and to 33% of cycle time in low-Ca2+/Ba2+ solution (n = 2/2, P < 0.001).
Ouabain ultimately eliminated bursting. When IK-Ca alone was blocked the intraburst f-t curve shifted upward but remained smooth and monotonic (Figs. 2, C and D, and 3A). When ouabain was added burst duration, burst cycle time, and the postburst AHP all decreased. Later in the experiment ouabain disrupted the smooth monotonic decline in intraburst spike frequency, the hallmark of stable bursting activity. The intraburst f-t curve became erratic and then broke down completely (Fig. 3, Cb-d). Finally, the combined block of IK-Ca and Ip prevented the rhythmic alternation of active and quiescent bursting phases. Instead, trigeminal motoneurons assumed the tonic spiking state shown in Figs. 3Cd and 4A (middle panel). This suggests that cells entered an active phase that could not terminate, putatively caused by blockade of both slow repolarizing currents, IK-Ca and Ip.
During the nonterminating active phase, action potentials declined slightly in amplitude, ~20% (Fig. 3B, bottom), and repolarized more slowly, suggesting that ouabain affected the Na+ and K+ concentration gradients to some extent. In the remaining cells exposed to apamin + ouabain (n = 1/4) and low-Ca2+/Ba2+ + ouabain (n = 2/4) the transition to tonic spiking occurred rapidly, and a transient period of bursting activity was indecipherable (not shown).
To further test the role of Ip, we repeated these experiments with the cardiac glycoside strophanthidin. This drug was applied sequentially, after apamin, with the same criteria as the ouabain experiments (n = 6). Strophanthidin, like ouabain, ultimately abolished bursting and induced a nonterminating active phase (Fig. 4B). Three of six cells exposed to strophanthidin exhibited an analyzable transitional bursting phase. Similar to ouabain, strophanthidin decreased burst duration and cycle time. In Fig. 4B, burst duration decreased significantly from 4.4 ± 0.2 to 1.5 ± 0.2 s (P < 0.01), and cycle time decreased from 11.3 ± 0.8 to 2.9 ± 0.2 s (P < 0.001) (measured at the temporal midpoint between the time of strophanthidin application and the time at which cells assumed tonic spiking). Strophanthidin, similar to ouabain, reduced the postburst AHP (Fig. 4B, broken line inserted for reference). In general, strophanthidin significantly decreased burst duration to 54% of burst duration in the presence of apamin alone (n = 2/3, P < 0.001) and decreased cycle time to 43% of cycle time in apamin alone (n = 3, P < 0.001). One-half of the trigeminal motoneurons exposed to strophanthidin (n = 3/6) rapidly assumed tonic spiking without a discernable transitional bursting phase (not shown).
The nonterminating active phase induced by blockade of
IK-Ca and Ip could be
manually terminated by extrinsic current injection (n = 8). Figure 5 shows a typical cell in the
presence of apamin + strophanthidin. Under these conditions rapid burst
termination only occurred after repolarizing current injection, whereas
bursts initiated spontaneously 10-20 s after readjustment of current bias. These data suggest that the motoneurons still possessed a region
of negative slope resistance in the steady-state
I-V curve and could initiate bursts (the
relationship between the negative slope resistance and burst initiation
is explained by Del Negro et al. 1998) but did not possess sufficient
intrinsic slow repolarizing processes to terminate bursts autonomously.
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To investigate how Ip influences intraburst spike frequency and the membrane potential trajectory after burst termination, we performed the following experiment. In the absence of serotonin, we applied apamin to trigeminal motoneurons and simulated bursts by applying current pulses of variable magnitude (2-4 nA) and duration (0.4-13.6 s). For each simulated burst, we examined 1) the postburst AHP after termination of the stimulus pulse and 2) the intraburst f-t relationship. We then compared these measures in control versus ouabain (n = 6) or strophanthidin (n = 1) conditions. In all cells tested ouabain blocked the postburst AHP (Fig. 6A, inset), suggesting that Ip activated during the simulated burst and produced the ouabain-sensitive AHP at burst termination. Ouabain also shifted the intraburst f-t relationship upward, which further suggests that Ip normally activated during the simulated burst and constrained intraburst spike frequency (Fig. 6B).
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DISCUSSION |
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We examined slow outward currents activated by cytosolic cation
accumulation that terminate serotonin-induced bursts in trigeminal motoneurons. We found that IK-Ca and
Ip both substantially contribute to burst
termination and influence temporal bursting characteristics such as
burst duration and the interburst interval. Therefore IK-Ca and Ip probably
influence the motoneuronal burst-like patterns that occur during
rhythmic oral-motor behaviors (Goldberg and Chandler
1990; Goldberg et al. 1982
; Gurahian et
al. 1989
).
During bursting by a traditional mechanism (Del Negro et al.
1998) neurons phasically alternate between quiescent and active membrane states. Stable membrane states are formed whenever the sum of
intrinsic inward and outward currents equals zero and slope conductance
is positive. This occurs where the steady-state
I-V relationship intersects its zero-current axis
with positive slope. If such an intersection occurs at a voltage less
than spike threshold that state is quiescent; if it occurs at a voltage
greater than threshold tonic spiking results. To simultaneously possess
two stable states neurons must express a region of negative slope resistance in their steady-state I-V
relationship, which can twice intersect the zero-current axis with
positive slope.
The inward currents underlying the region of negative slope resistance provide the structural framework for bistability (plateau potentials) and bursting by creating stable membrane states on either side of spike threshold, quiescent and tonic spiking, separated in the I-V relationship by the region of regenerative inward current. Therefore how do bistable or bursting cells move from state to state; what controls the dynamics?
To answer this, we address 1) how membrane potential first moves from its quiescent to active state, thereby initiating a plateau potential or burst, and 2) what slow repolarizing processes activate during the burst to diminish intraburst spike frequency and ultimately terminate the active phase.
Burst initiation
Trigeminal motoneurons utilize L-type Ca2+ and
persistent Na+ currents to initiate and maintain plateau
potentials and bursts (Hsiao et al. 1998). Serotonin
transforms membrane properties by inducing the necessary region of
negative slope resistance and negatively shifting the
I-V relationship so that the region of negative
slope resistance often lies below the zero-current axis in the region
of net inward current (Chandler and Trueblood 1995
;
Hsiao et al. 1998
). In this case the only stable
membrane state is the tonic spiking state, at a depolarized membrane
potential. Therefore bursts initiate autonomously (Figs. 1C
and 2 of Del Negro et al. 1998
).
Sometimes the effects of serotonin are insufficient to shift the region of negative slope resistance fully into the region of net inward current. In this case, the I-V curve intersects the zero-current axis three times (twice with positive slope), and the cell is bistable. Then from the resting state plateau potentials can be initiated by transient stimuli (e.g., Fig. 1B). Adding depolarizing current bias can extrinsically shift the I-V relationship so that the negative slope region lies in the area of net inward current, transforming bistability into bursting, as shown in Fig. 1A.
Burst termination: the role of IK-Ca
Of the slow repolarizing processes that terminate plateau
potentials and bursts in trigeminal motoneurons, we first
identified IK-Ca. Because the blockade of
IK-Ca (by using apamin or
low-Ca2+/Ba2+ solution) lengthened
burst duration and upwardly shifted the intraburst
f-t curve, we propose that
IK-Ca normally activates during bursts caused by
Ca2+ influx and accumulation, constrains intraburst spike
frequency, and helps expedite burst termination. These effects are
consistent with how IK-Ca is recruited and
affects spike frequency in a variety of mammalian motoneurons
(Hounsgaard et al. 1988b; Mosfeldt Laursen and
Rekling 1989
; Nishimura et al. 1989
;
Takahashi 1990
; Viana et al. 1993
;
Zhang and Krnjevic 1987
), including trigeminal
motoneurons (Chandler et al. 1994
; Kobayashi et
al. 1997
). IK-Ca similarly contributes
to burst termination during conditional bursting in several other cell
types (el Manira et al. 1994
; Hu and Bourque 1992
; Legendre et al. 1985
; Tell and Jean
1993
; Wallen and Grillner 1987
), including
N-methyl-D-aspartate (NMDA)-induced bursting in
trigeminal motoneurons (Kim and Chandler 1995
).
However, additional slow processes must influence burst termination in trigeminal motoneurons. When IK-Ca was blocked bursting activity continued. In the absence of IK-Ca spike frequency was 5-8 Hz at the time of burst termination, which was similar to the spike frequency at the time of burst termination in control (3-5 Hz, Fig. 2). This suggests that in all conditions the net outward current was comparable at the end of the active phase. Although IK-Ca normally contributes to burst termination, additional repolarizing processes compensate for IK-Ca loss under apamin or low-Ca2+/Ba2+ conditions and generate sufficient repolarizing current to terminate the active phase. However, when IK-Ca is reduced or eliminated the remaining slow processes require more time to generate the requisite outward current, consequently lengthening burst duration. The extended cycle time during blockade of IK-Ca most likely reflects the slower time course of relaxation of these compensatory slow repolarizing processes.
Burst termination: the role of Ip
The other slow repolarizing process, which assists IK-Ca to terminate plateau potentials and bursts, is constituted primarily by Ip because removal of Ip ultimately blocks serotonin-induced bursting. If slow inactivation of either the persistent Na+ or the L-type Ca2+ channels contributed substantially to burst termination we would expect bursts to terminate even when IK-Ca and Ip are both blocked. However, the combined block of IK-Ca and Ip always transformed rhythmic bursting into a nonterminating active phase of tonic spiking (Figs. 3 and 4).
We propose that the role of Ip is multifaceted
and that the pump current is composed of both steady-state and dynamic
components. We base our conclusions on the effects of the cardiac
glycosides during the transitional bursting phase that preceded tonic
spiking in some cells. First, cardiac glycosides dramatically decreased burst cycle time. Neurons constantly extrude Na+ via the
Na+/K+ pump (Läuger 1991).
Therefore the electrogenic nature of the pump normally creates a
"background" outward current. This steady-state component of
Ip will affect the voltage trajectory most
effectively when membrane conductance is low, which corresponds to the
interburst interval. In trigeminal motoneurons, blocking this
maintained outward current (i.e., the steady-state component of
Ip) with cardiac glycosides shortens cycle time
by effectively increasing the contribution of the inward currents
during the interburst interval and thereby hastens burst onset. Sakai
et al. (1996)
similarly argue that the steady-state component of
Ip in rabbit sinoatrial node cells influences
the pacemaker depolarization phase, the cardiac equivalent of the
interburst interval.
In addition to the steady-state component of Ip that mostly influences the interburst interval, additional pump current activates specifically during the burst because of spike-mediated enhanced Na+ influx. This dynamic component of Ip constrains intraburst spike frequency and generates a transient postburst AHP (Fig. 6). We propose that the dynamic component of Ip is responsible for burst termination in trigeminal motoneurons under apamin and low-Ca2+/Ba2+ conditions. Consequently, during the ouabain-induced transitional bursting phase, the intraburst f-t relationship became erratic (and ultimately broke down), and the postburst AHP consistently declined in amplitude (Figs. 3 and 4). Once Ip was fully blocked by the cardiac glycosides, every trigeminal motoneuron tested locked into the tonic spiking state that represents a nonterminating active phase.
A similar dynamic component of Ip was
characterized by Johnson et al. (1992) in rat midbrain dopaminergic
neurons. Similar to our data, eliminating Ip
caused cessation of NMDA-induced bursting in midbrain dopaminergic
neurons. These neurons, like trigeminal motoneurons, entered a
nonterminating active phase of tonic spiking. Also similar to
trigeminal motoneurons, the dynamic component of
Ip (the component of Ip
activated specifically during bursts) in dopaminergic neurons was
sufficient to terminate bursts when IK-Ca was
blocked pharmacologically (Johnson and Seutin 1997
; Seutin et al. 1993
). Ip is also
crucial for burst termination in several other neuron types
(Angstadt and Friesen 1991
; de Waele et al.
1993
; Tsai and Chen 1995
). In these cells, too,
blocking Ip induced tonic spiking.
Although burst termination does not critically depend on the slow
inactivation of the persistent Na+ and L-type
Ca2+ currents, these processes probably still occur and
influence burst characteristics. We showed earlier that bath
application of Bay K 8644 prolongs burst duration during
serotonin-induced bursting (Hsiao et al. 1998), most
likely by slowing the kinetics of the L-type Ca2+ channels.
This suggests that L-channel inactivation normally influences burst
duration. Persistent Na+ currents can also inactivate
(Fleidervish et al. 1996
), which could modify burst
duration. Also during simulated bursts in this study (Fig. 6) the
intraburst f-t relationship still declined monotonically in the presence of apamin + ouabain. This suggests some
temporal inactivation of the inward currents or recruitment of an
unidentified outward current other than IK-Ca
and Ip during the simulated burst.
We propose that inactivation of the inward currents indirectly causes shorter burst duration during blockade of Ip. If the inward currents (L-type Ca2+ or persistent Na+ current) inactivate to some extent during bursts they normally de-inactivate during the relatively hyperpolarized interburst interval. Because removal of the tonic component of Ip significantly shortens the interburst interval (described previously), the inward currents may not fully recover from inactivation between bursts. The magnitude of inward current available to generate subsequent bursts would then be compromised. These "compromised" bursts would then require less repolarizing current and therefore less time to terminate.
This proposal posits that less inward current underlies bursts after blockade of Ip. If this is true we would also expect an overall reduction in intraburst spike frequency after blockade of Ip, and specifically we would expect a downward shift in the intraburst f-t curve. In fact, the intraburst f-t curve in the presence of apamin + ouabain (Fig. 3C) was notably depressed compared with the intraburst f-t curve during apamin alone (Fig. 3A). This suggests that the inward currents inactivated to some extent after application of cardiac glycosides, consequently depressing the intraburst f-t curve and shortening burst duration.
In addition to blocking Ip, cardiac glycosides
can induce "slip-mode conductance" in cardiac Na+
channels, making them Ca2+ permeable (Santana et al.
1998). If this effect occurs in neuronal Na+
channels it could potentially enhance inward currents during cardiac
glycoside-challenged bursting and cause tonic spiking. However, we
believe this effect cannot explain the current results because
intraburst spike frequency clearly decreases during the ouabain and
strophanthidin experiments (e.g., Fig. 3C). Decreased intraburst spike frequency is inconsistent with enhancement of inward current.
The tonic spiking state induced by cardiac glycosides is unlikely to represent general rundown in trigeminal motoneurons caused by large shifts in the Na+ and K+ concentration gradients for the following reasons. In the presence of apamin + strophanthidin bursts could initiate normally (Fig. 5), and the evoked action potential was close to full amplitude (Fig. 3B, bottom).
Physiological significance
Trigeminal motoneurons exhibit bistability and bursting in the
presence of serotonin (Hsiao et al. 1998), an endogenous
neuromessenger associated with oral-motor behaviors (Fornal et
al. 1996
; Hsiao et al. 1997
; Veasey et
al. 1995
). Although rhythmic oral-motor behaviors are produced
by brain stem central pattern generators (CPGs) that project to
trigeminal motoneurons (Goldberg and Chandler 1990
;
Lund 1976
; Nakamura and Katakura 1995
),
the intrinsic properties of the motoneurons shape the final output
pattern. In trigeminal (Hsiao et al. 1998
) and spinal
motoneurons (Hounsgaard et al. 1988a
; Kiehn
1991
; Kiehn and Eken 1997
; Kiehn et al.
1996
) bistability and bursting are putatively important for
motor behaviors; these properties can amplify motoneuronal output in
the absence of maintained synaptic excitation. Because trigeminal
motoneurons possess both IK-Ca and
Ip and these two currents influence the temporal
characteristics of serotonin-induced bursting, neuromodulation of
IK-Ca and Ip could
regulate rhythmic motoneuronal spike output to match CPG activity. The
regulation of Ip may simultaneously influence
the phasic activity of the CPG because cardiac glycosides disrupt spinal networks that generate rhythmic motor patterns (Ballerini et al. 1997
). In this way the intrinsic properties of
trigeminal motoneurons would complement and assist the expression of
ongoing spatiotemporal activity in the brain stem networks generating mastication or other rhythmic oral-motor behaviors, such as suckling, grooming, or lapping.
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ACKNOWLEDGMENTS |
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We thank Dr. Jens C. Rekling for a critical reading of the manuscript and M. Z. Castillo for technical contributions.
This work was funded by National Institute of Dental Research Grant RO1 DE-06193.
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FOOTNOTES |
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Address for reprint requests: S. H. Chandler, Dept. of Physiological Science, 2851 Slichter Hall, Los Angeles, CA 90095-1568.
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 22 September 1998; accepted in final form 1 December 1998.
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