Biophysics Sector and INFM Unit, International School for Advanced Studies (SISSA), 34014 Trieste, Italy
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ABSTRACT |
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Lape, Remigijus and
Andrea Nistri.
Current and Voltage Clamp Studies of the Spike Medium
Afterhyperpolarization of Hypoglossal Motoneurons in a Rat Brain Stem
Slice Preparation.
J. Neurophysiol. 83: 2987-2995, 2000.
Whole-cell patch clamp recordings were
performed on hypoglossal motoneurons (HMs) in a brain stem slice
preparation from the neonatal rat. The medium afterhyperpolarization
(mAHP) was the only afterpotential always present after single or
multiple spikes, making it suitable for studying its role in firing
behavior. At resting membrane potential (68.8 ± 0.7 mV), mAHP
(23 ± 2 ms rise-time and 150 ± 10 ms decay) had 9.5 ± 0.7 mV amplitude, was suppressed in Ca2+-free medium or by
100 nM apamin, and reversed at
94 mV membrane potential. These
observations suggest that mAHP was due to activation of
Ca2+-dependent, SK-type K+ channels. Carbachol
(10-100 µM) reversibly and dose dependently blocked the mAHP and
depolarized HMs (both effects prevented by 10 µM atropine). Similar
mAHP block was produced by muscarine (50 µM). In control solution a
constant current pulse (1 s) induced HM repetitive firing with small
spike frequency adaptation. When the mAHP was blocked by apamin, the
same current pulse evoked much higher frequency firing with strong
spike frequency adaptation. Carbachol also elicited faster firing and
adapting behavior. Voltage clamp experiments demonstrated a slowly
deactivating, apamin-sensitive K+ current
(IAHP) which could account for the mAHP.
IAHP reversed at
94 mV membrane potential,
was activated by depolarization as short as 1 ms, decayed with a time
constant of 154 ± 9 ms at
50 mV, and was also blocked by 50 µM carbachol. These data suggest that mAHP had an important role in
controlling firing behavior as clearly demonstrated after its
pharmacological block and was potently modulated by muscarinic receptor activity.
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INTRODUCTION |
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Hypoglossal motoneurons (HMs) innervate tongue
muscles (Lowe 1980) and thus are involved in several
functions like respiration, mastication, swallowing, and suckling.
Indeed, considerable interest is centered on these motoneurons because
their dysfunction may result in diseases like obstructive sleep apnea
or sudden infant death syndrome (Gauda et al. 1987
;
Wiegand et al. 1991
; Willinger 1989
).
Studies that have used rodent brain stem slices as models to understand
the basic properties of HMs have demonstrated that these cells possess
several voltage-activated membrane conductances (Haddad et al.
1990; Mosfeldt Laursen and Rekling 1989
;
Viana et al. 1993a
,b
). HMs display characteristic firing
patterns following membrane depolarization (Sawczuk et al. 1995
,
1997
; Viana et al. 1995
), an aspect of special
relevance because it indicates how integration of the electrical
behavior at somatic HM level is eventually translated into output
signals to the tongue muscles.
In response to current step injection adult HMs fire initially at
high-frequency with a multicomponent decay to a much lower discharge
rate (Sawczuk et al. 1995, 1997
). Conversely, the
majority of neonatal HMs exhibits a steady pattern of repetitive firing which is reached after a single, fast period (about 200 ms) of frequency adaptation. A smaller subgroup actually shows rapid acceleration of firing to steady state level (Viana et al.
1993b
, 1995
). As in the case of spinal (Barrett et al.
1980
; Krnjevic et al. 1979
) or facial
(Nishimura et al. 1989
) motoneurons, a Ca2+-dependent afterhyperpolarization of medium
duration (mAHP) is proposed to control adaptation of neonatal
(Viana et al. 1995
) and adult (Powers et al.
1999
) HMs. Such an mAHP is modulated by transmitters like
serotonin (5-HT) or norepinephrine (Bayliss et al. 1995
;
Parkis et al. 1995
), raising the possibility that firing
behavior is a dynamic property susceptible to changes induced by
locally released transmitters. Notwithstanding the progress made by
these studies, full understanding of the mechanisms underlying different degrees of repetitive firing and adaptation is still lacking.
We have attempted to reconstruct the firing behavior of neonatal HMs by
starting with a quantitative description of their voltage-dependent
K+ conductances to be used for computer-based
modeling of action potential discharges. For this purpose we recently
characterized two relatively fast,
Ca2+-independent K+
currents (Lape and Nistri 1999). The main goals of the
present study were to investigate the kinetic properties of the current underlying the mAHP, to assess its contribution to firing behavior by
using the selective blocker apamin, and to ascertain the HM firing
properties recorded under patch clamp conditions, as previous work had
relied on sharp electrode recording (Viana et al. 1993b
, 1995
). Furthermore, as muscarinic receptor activity is known to affect repetitive firing probably by modulating the
afterhyperpolarization (AHP) of cortical (Schwindt et al.
1988
) or hippocampal (Storm 1989
) neurons, we
tested if this phenomenon is also applicable to HMs that apparently
possess high levels of muscarinic binding sites (Rotter et al.
1979
; Walmsley et al. 1981
). It is presently unclear if there are functional postsynaptic muscarinic receptors on
HMs as in this area there is only a report of presynaptic muscarinic action in depressing transmitter release (Bellingham and Berger 1996
). A preliminary description of our work has recently
appeared in abstract form (Lape et al. 1999
).
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METHODS |
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Slice preparation
Experiments were carried out using brain stem slices obtained
from 0 to 6 day old rats. Thin slices were prepared following the
procedure described earlier by Viana et al. (1994) and
Lape and Nistri (1999)
. The brain stem was isolated from
neonatal rats and placed into modified ice-cold Krebs solution (see
Solutions). A tissue block containing the lower
medulla was then fixed (with insect pins) onto an agar block inside a
Vibratome chamber filled with ice-cold Krebs solution (bubbled with
O2-CO2) to obtain
200-µm-thick slices. Slices were first transferred to an incubation
chamber for 1 h at 32°C under continuous oxygenation and
subsequently maintained at room temperature for at least 1 h
before use. During recording (at room temperature) slices were
continuously superfused with HEPES-buffered solution (see
Solutions) saturated with
O2-CO2 mixture.
Recording
Brain stem slices placed in a small recording chamber were
viewed with an infrared video camera to identify single hypoglossal motoneurons within the XII nucleus. The conventional whole-cell patch-clamp recording technique (Hamill et al. 1981) was
employed by using either an EPC-7 patch-clamp amplifier [for
voltage-clamp (VC) experiments] or an Axoclamp-2B amplifier [for
current clamp (CC) experiments]. For VC experiments patch electrodes
had 3-5 M
DC resistance, whereas those pulled for CC patch
experiments had 10-18 M
. Seal resistance was usually higher than 2 G
. After seal-rupture series resistance,
Rs (5-15 M
), was routinely monitored and compensated (usually by 40%, range 20-60%) in VC experiments. The VC recordings were performed only when
Rs stabilized and the cells were chosen
for analysis if changes in Rs did not
exceed 10%. The bridge was balanced routinely in CC experiments.
Voltage and current pulse generation and data acquisition were
performed with a PC running pClamp 6.1 software. Currents elicited by
voltage steps were filtered at 3 kHz and sampled at 5-10 kHz.
Solutions
The Krebs solution for slice preparation and maintenance was as follows (mM): 130 NaCl, 3 KCl, 26 NaHCO3, 1.5 Na2HPO4, 1 CaCl2, 5 MgCl2, 10 glucose, 0.0004 l-ascorbic acid (290-310 mOsm). Extracellular solution for electrophysiological recording was as follows (mM): 140 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4, 290-310 mOsm). Patch pipette solution was as follows (mM): 110 K-methyl-SO4, 20 KCl, 10 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATP-Mg (pH 7.2, 260-270 mOsm). To prepare Ca-free Co solution CaCl2 was completely substituted with CoCl2. In various experiments muscarine chloride (muscarine), carbamylcholine chloride (carbachol), atropine methylnitrate, and apamin were applied via the bathing solution (continuously superfused at 2-5 ml/min). Drugs were added by switching to an appropriate extracellular solution which was applied for 5-10 min for equilibration.
Analysis
Cell input resistance (Rin) was calculated from small (5 mV or 10 pA) hyperpolarizing voltage or current commands or from the linear portion of the I-V line (ramp test) near the cell resting potential (Vrest). To quantify the spike AHP we measured its peak amplitude (from baseline), area, time constants for monoexponential rise and decay. As these measurements are largely influenced by membrane potential, the cell resting potential was kept at the same level by intracellular current injection throughout the recording session. To measure the amplitude and decay time constants of tail currents, these were fitted with a mono- or biexponential function. The initial 5-ms record after the end of the voltage step was discarded from fitting to avoid contamination by uncompensated capacitance transients. Tail current amplitude was then obtained by extrapolating fitted curves back to the end of the voltage step. Sigma Plot and Clampfit softwares were used for exponential fitting of membrane currents and for linear regression analysis of experimental data. Data are presented as mean ± SE. All potential values were corrected off-line for the liquid junction potential, which was measured as 10 mV. Current leak subtraction was performed by using the Clampfit module. Data were analyzed statistically by using Student's t-test or analysis of variance (ANOVA) test. Significance was accepted when P < 0.05.
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RESULTS |
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Some general characteristics of the action potential (AP)
and its afterpotentials elicited by intracellular current injection are
presented in Fig. 1. Figure 1A
shows that, in analogy with the report by Viana et al.
(1993b), the decay phase of a single AP [typically
evoked by a short (5 ms) current pulse; 0.5 nA] comprised a fAHP (fast
afterhyperpolarization) and a fast afterdepolarization (fADP).
These early afterpotentials were followed by an mAHP which undershot
the baseline for about 250 ms (Fig. 1B, same cell). An
analogous mAHP (about 300-ms long) was also observed at the end of a
spike train induced by a 2-s current pulse (200 pA; see Fig.
1C, different cell from A-B). In a few cells
only (9% of total recorded cell number) a slow afterhyperpolarization
(sAHP) (lasting 2-5 s) appeared after the mAHP (Fig. 1D;
see also Viana et al. 1993b
). In a larger group of cells
(~40%) the mAHP was followed by a slow afterdepolarization (sADP;
1-3-s long; Fig. 1E). Because the mAHP was the most
consistently observed afterpotential (present in all cells recorded;
n = 59) and is known to be the target for transmitter
modulation (see INTRODUCTION), the present report is mainly
concerned with a characterization of the mAHP.
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Basic properties of mAHP
At 68.8 ± 0.7 mV resting membrane potential
(Vrest; n = 59 cells)
the mAHP following a single AP reached peak amplitude of 9.5 ± 0.7 mV (measured from Vrest) with a
monoexponential rise time constant (23 ± 2 ms) from which it
decayed monoexponentially (decay time constant =150 ± 10 ms).
When extracellular Ca2+ was replaced by the same
concentration of Co2+, the mAHP was abolished
(Fig. 2A) but it recovered
when standard external solution was reapplied (n = 4).
Similar results were obtained by application (via the patch electrode)
of
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), a selective chelator of Ca2+, to
block the action of intracellular Ca2+ raised
through trans-membrane influx or release from cytoplasmic stores. In
fact, out of six cells recorded with a patch electrode filled with 1 mM
BAPTA and held at
68 mV, four showed no mAHP and two showed a rather
small mAHP (3 and 1.5 mV, respectively).
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The example of Fig. 2B indicates that, under standard
recording conditions, the mAHP decreased with membrane
hyperpolarization until it disappeared at 94 mV membrane potential.
The inset to Fig. 2B is the plot (for 2 HMs) of the relation
between membrane potential and mAHP amplitude, yielding a reversal
potential of
94 mV, which coincides with the calculated equilibrium
potential for K+ on the basis of the Nernst
equation. This result suggests that the mAHP is a response due to
increased permeability to K+. As the mAHP was
fully and irreversibly blocked by 100 nM apamin without concomitant
suppression of the fAHP (Fig. 2C; similar data were observed
on 9 cells), it seems likely that the K+
conductance responsible for generating the mAHP was mediated by SK
Ca2+-activated channels (Sah
1996
).
Modulation of mAHP by activation of muscarinic ACh receptors
The cholinergic agonist carbachol largely attenuated the mAHP as
exemplified in Fig. 3A. On
average 50 µM carbachol reduced the mAHP to 37 ± 4% of control
(n = 17 cells; P < 0.001). The action
of carbachol developed quite slowly, taking about 5 min to manifest
fully, was completely reversible on washout, and was dose dependent. In
fact, 10 µM carbachol decrease the mAHP to 76 ± 5% in four
cells, whereas 100 µM concentration reduced it to 20 ± 10% in
two cells. Note that carbachol depressed the peak amplitude of the mAHP
without affecting its rise or decay time course (changes in
rise and
decay were
90 ± 10% and 81 ± 9%, respectively; n = 17 cells). The action by carbachol was not accompanied by any
significant change in spike amplitude (90 ± 2%), duration (107 ± 6%), or threshold (99 ± 6%).
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Together with the depression of the mAHP carbachol (10-100 µM) also produced a dose-dependent, slowly developing and reversible depolarization of all HMs tested. For example, a 50-µM concentration induced a mean depolarization of 12 ± 1 mV without significant change (96 ± 4%) in Rin (n = 17 cells). The carbachol depolarization persisted in the presence of TTX, thus suggesting that it had a direct action on HMs (11 ± 2 mV; n = 3).
The carbachol inhibitory action on the mAHP was prevented by 10-15 min pretreatment with 10 µM atropine (n = 7 cells), a selective antagonist on muscarinic ACh receptors (Fig. 3B). The mAHP however retained its sensitivity to apamin (100 nM; Fig. 3B), indicating distinctive modes of action for carbachol and this toxin in inhibiting the mAHP. Note that on average atropine per se increased the mAHP by 29 ± 7% (n = 7; P < 0.05). The selective muscarinic receptor agonist muscarine (50 µM) also produced HM depolarization (10 ± 4 mV) and attenuated the mAHP (Fig. 3C; on average to 40 ± 20%; n = 3.
mAHP involvement in repetitive firing
The duration and magnitude of the mAHP would predict this response
to be a potent regulator of the discharge properties of HMs,
particularly in view of a similar role of the mAHP in firing adaptation
of hippocampal cells (Storm 1990). It was thus
surprising to observe that long current pulses evoked repetitive firing
of HMs with slight adaptation only despite the mAHP presence (see Fig.
4A). This cell is
representative of the most commonly observed firing pattern in control
solution. In fact, out of a group of 59 motoneurons which were able to
fire repetitively, 37 had exclusively very fast adaptation (lasting up
to 50 ms and affecting the first 2-5 spikes only), whereas 17 cells
showed no measurable adaptation and five possessed slower adaptation
lasting ~200 ms. One possibility for the rather modest (or even
absent) process of firing adaptation might be that the action of the
mAHP was opposed by coactivation of other conductances. To reveal the
influence of the mAHP on spike firing, we applied the selective blocker
apamin (100 nM) as depicted in Fig. 4A (right).
In the presence of apamin the firing properties of the cell were
radically transformed with a large rise in frequency affecting both the
early and late action potentials in the train (see Fig. 4B).
Effects similar to those of apamin were also observed by applying
Ca2+-free Co2+ solution
(n = 4; not shown). Furthermore, in apamin solution firing adaptation was manifested as indicated by the continuous decline
in firing frequency over time (compare time course of filled and open
circle graphs in Fig. 4B). The relation between spike
discharge frequency and injected current
(f-I relation) was further investigated to
determine the frequency coding properties of these cells. Repetitive AP
discharges were observed in all neurons tested with 1-s constant
current pulses of varying intensities. An example of instantaneous
firing frequency (1/interspike interval) versus injected current plot
is shown for the first and last interspike interval (Fig.
4C). Although in control solution the difference between
these values was small and skewed toward large injected currents, in
apamin solution there was a much more substantial difference throughout
the injected current range. The f-I relation slope calculated for the first interspike interval (100 ± 10 Hz/nA) and for steady state firing (58.1 ± 0.7 Hz/nA) evoked by
50-200 pA currents increased, in apamin solution, to 320 ± 20 Hz/nA and 160 ± 20 Hz/nA, respectively (n = 12 cells).
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Application of carbachol (50 µM) also increased firing frequency of HMs in response to current injection (Fig. 4D). Spike frequency adaptation was manifested in the presence of carbachol (Fig. 4E), leading to a stronger separation between early and late firing frequencies (Fig. 4F).
In the presence of 100 nM apamin, carbachol (50 µM) had little effect on firing properties (Fig. 5A) as quantified in Fig. 5B where data points for apamin or carbachol tests overlap. The analysis of f-I plots (Fig. 5C) indicates strong similarity between the effects of apamin and carbachol on the AP frequency rise and firing adaptation. Nevertheless, despite occlusion by apamin of carbachol effects on firing, this latter substance was still able to depolarize HMs (8 ± 2 mV; n = 5) without significant change in Rin (89 ± 5%; n = 5). The present data suggest that muscarinic receptor activation could differentially modulate membrane potential and firing properties of HMs.
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Membrane current sensitive to apamin and carbachol
Apamin-sensitive membrane currents were investigated under voltage
clamp conditions. Outward membrane currents were recorded, in the
presence of TTX, by delivering depolarizing voltage steps (1-s
duration) in the range 40-+20 mV, whereas
Vh was usually
50 mV to minimize
contamination by low threshold K+ currents such
as Ifast (Lape and Nistri
1999
). However, tests (n = 8 cells) carried out
with
60 or
70 mV Vh yielded similar results to those from the less negative
Vh. The standard depolarizing pulse
protocol generates a heterogeneous, voltage-dependent outward current
(Lape and Nistri 1999
), a typical example of which is shown in Fig. 6A. The outward
current did not inactivate during the 1-s-long voltage steps and was
followed by a tail current. The contribution of the apamin-sensitive
current to the total membrane current was examined by adding 100 nM
apamin to the extracellular solution. Apamin (which did not change leak
conductance; 96 ± 3%) reduced by 36 ± 1% the outward
current (Fig. 6B; n = 10) measured 10 ms
before the voltage step termination. The apamin-sensitive outward
current could then be obtained by subtracting the current recorded in
apamin solution from the control one. The average I-V
relation for the apamin sensitive current is plotted in Fig. 6C. Its apparent activation threshold was
40 mV from which
the current grew monotonically with increasing membrane potential. Note
that at +20 mV, the value which corresponds to the peak of an action
potential, the apamin current had an average cord conductance of 7 ± 2 nS. Of course, this value was calculated when the current was
under apparent steady-state conditions following a relatively long
depolarization, an experimental condition which cannot approximate the
rapid voltage change generated by a single AP.
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To characterize the apamin-sensitive current deactivation we studied
tail currents following voltage steps to +20 mV in the absence or in
the presence of apamin (see Fig. 6B). In control conditions
tail currents could be fitted by two exponentials with decay time
constants of 24 ± 3 ms and 154 ± 9 ms at 50 mV,
respectively (n = 11 cells). In the presence of apamin
only a fast monoexponential component remained (decay time constant
=19 ± 3 ms; n = 5 cells). After current
subtraction, the apamin-sensitive tail current was shown to have a
monoexponential decay (140 ± 20 ms), which was voltage
independent in the range between
40 and
120 mV (not shown). This
observation suggests that deactivation of the apamin-sensitive current
was relatively slow and unaffected by membrane potential. Apamin-sensitive tail currents reversed at
91 ± 1 mV
(n = 5) membrane potential, a value very near
EK+ (
95 mV).
Studying the activation kinetics of the apamin-sensitive current was difficult because of its contamination by the concomitant development of other voltage-dependent currents. To partially circumvent this problem we studied the kinetics of generation of the apamin-sensitive tail currents by applying voltage steps of different lengths. In this case the current flowing at the end of each voltage command should have represented the activation of a certain fraction of apamin-sensitive channels for a given membrane potential. The protocol therefore consisted of delivering fixed-amplitude voltage steps of increasing duration (from 1 to 50 ms) in the absence or the presence of apamin and in measuring the tail currents obtained after current subtraction. The subtracted tail currents were normalized with respect to the one obtained after a 50-ms step and then plotted (Fig. 6E) versus the voltage-step duration (step command to 20 mV). The time course of tail current development was fitted by two exponentials with time constants of 0.7 ± 0.1 and 24 ± 3 ms (n = 5 cells). Note that even 1-ms voltage command was able to generate a measurable fraction of the apamin sensitive current (~20%).
Examples of outward currents recorded before or after adding 50 µM carbachol are shown in Fig. 7, A and B. On average carbachol depressed the outward current by 28 ± 3% (n = 6), a phenomenon associated with the generation of an inward current (40 ± 20 pA). Analysis of tail currents (Fig. 7C) indicated that carbachol blocked the slow component (by 80 ± 10%) strongly and the fast one (by 35 ± 9%) weakly (n = 6). Nevertheless, 50 µM carbachol in the presence of 100 nM apamin could still reduce the steady-state outward current by 27 ± 1% (n = 3 cells) as shown in the example in Fig. 7, D and E. In apamin solution the monoexponentially decaying (20 ± 5 ms) tail current was also depressed in amplitude by 20 ± 10% (Fig. 7F).
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DISCUSSION |
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The major novel findings of the present study were that, by using patch clamp recording from rat hypoglossal motoneurons, it was possible to quantify macroscopically the outward current apparently responsible for the mAHP and to demonstrate its modulation by muscarinic receptor activation. In addition, the present patch clamp experiments provided new evidence for the role of the mAHP in spike firing and its regulation by muscarinic receptors.
mAHP
Single or repeated action potentials were followed by a series of
depolarizing and hyperpolarizing afterpotentials as exemplified in Fig. 1. The fAHP, mAHP, and fADP were present in all HMs. A few
cells possessed the sAHP (see also Nishimura et al.
1989; Viana et al. 1993b
) or the sADP, which has
also been observed in facial motoneurons (Nishimura et al.
1989
).
The HM mAHP was qualitatively similar to the one recorded with sharp
electrodes from brain stem (Chandler et al. 1994;
Mosfeldt Laursen and Rekling 1989
; Nishimura et
al. 1989
; Sah and McLachlan 1992
; Viana
et al. 1993b
) or spinal (Walton and Fulton 1986
;
Zhang and Krnjevic 1987
) motoneurons. This similarity
demonstrates that whole-cell patch clamping allowed us to measure this
response without introducing artifacts inherent to the recording
technique. The mAHP was completely blocked by
Ca2+-free solution or apamin, and it reversed at
membrane potential near the predicted
EK+. All these observations indicate
that mAHP was a Ca2+-dependent
K+ conductance. Its sensitivity to apamin, a very
selective blocker of SK channels (for review see Sah
1996
), suggests that the Ca2+-dependent
K+ conductance responsible for this phenomenon
was probably mediated by SK channels and turned on by
Ca2+ entry after one (or more) AP. In fact, the
lack of effect of apamin on resting potential or leak conductance shows
that such a Ca2+-dependent
K+ conductance was inactive at resting levels of
intracellular Ca2+.
Apamin sensitive current
The outward current selectively inhibited by apamin may be termed
IAHP (see Sah 1996). On
HMs this represented about one third of the total outward current
induced by membrane depolarization and deactivated slowly. For 1-s-long
membrane depolarization to +20 mV the cord conductance of
IAHP (under apparently steady-state conditions) was 7 ± 2 nS. The corresponding conductance value for
the slow K+ current
(Islow; values taken from Fig.
2A in Lape and Nistri 1999
) was 14 nS,
whereas the fast transient current
(Ifast) was strongly inactivated at
this time point. Thus during sustained depolarization
Islow generated a membrane shunt
considerably larger than IAHP. Note
that in addition to these K+ currents there was a
residual, unidentified outward current sensitive to muscarinic agents
as discussed below. Because the largest component of the total outward
steady current was apparently made up of Islow (which deactivated with a faster
time course; Lape and Nistri 1999
), it was difficult to
study IAHP in isolation. For this
reason kinetic parameters pertaining to
IAHP were obtained by analyzing the
current (and especially its slow tail) obtained after subtraction. The
I-V relation of IAHP
indicated an apparent activation threshold at about
40 mV; its
nonlinear voltage dependence in the
40/
10 mV range might have
reflected the strong voltage dependence of Ca2+
conductance activation (Hille 1992
). The time course of
IAHP development was biexponential.
This latter property probably reflects the multifactorial process
underlying IAHP generation and might have been due to phased recruitment of SK channels by increasingly larger amounts of intracellular Ca2+ diffusing
over a wide cytoplasmic area.
It is noteworthy that membrane depolarization as short as 1 ms could
elicit a measurable IAHP. It is
suggested that even very short voltage changes (as brief as a single
AP, normally lasting 1-3 ms) can trigger Ca2+
entry sufficient to turn on an adequate number of SK channels to
generate an mAHP. Similar observations have been obtained with ganglion
neurons (Lancaster and Pennefather 1987).
It is useful to assess the relative contribution of
Ifast,
Islow, and
IAHP to a single action potential: by
assuming a spike lasting 3 ms the calculated cord conductances are 4 nS
(from Fig. 3C of Lape and Nistri 1999), 1 nS
(from Fig. 2A of Lape and Nistri 1999
), and 3 nS (present study; see Fig. 6E), respectively. Even if
calculations based on responses to a fast step pulse cannot fully take
into account the dynamic changes in spike shape, it appears that the
main K+ conductance activated during a single
spike was Ifast. The role of
IAHP would probably be more
conspicuous after the spike itself because
IAHP activation continued because of
its dependence on delayed Ca2+ entrance. This
property makes IAHP the most suitable
mechanism to generate the mAHP as
Ifast and
Islow deactivate with a more rapid
time course (Lape and Nistri 1999
).
It is interesting that IAHP
deactivation (observed as monoexponential decay time constant of the
apamin-sensitive tail current) showed no voltage dependence, thus
suggesting that the membrane conductance underlying
IAHP was probably voltage independent. IAHP with similar kinetic properties
was described in sympathetic neurons (Cassell and McLachlan
1987; Goh and Pennefather 1987
), vagal
motorneurons (Sah and McLachlan 1992
), trigeminal
motoneurons (Chandler et al. 1994
), and cortical neurons
(Schwindt et al. 1992
). The present data will help
reconstructing HM firing behavior with computer-generated modeling
based on experimentally acquired data.
Effects of muscarinic receptor activity on mAHP
The mAHP of HMs is a target for neuromodulation by 5-HT or
norepinephrine (Bayliss et al. 1995; Parkis et
al. 1995
). Both substances do not act directly on the mAHP
underlying conductance but operate indirectly by either inhibiting
Ca2+ currents in the case of 5-HT (Bayliss
et al. 1995
) or reducing leak conductance (and activating an
inward current) in the case of norepinephrine (Parkis et al.
1995
). The present study shows that the mAHP (and
IAHP) is also a target for muscarinic
receptor activity. Carbachol or muscarine reduced mAHP amplitude
without changing its rise and decay times, suggesting that muscarinic receptors apparently led to inhibition of a fraction of
IAHP channels. It should be pointed
out that previous studies on cortical neurons have reported that
muscarinic receptors usually block the sAHP (Schwindt et al.
1988
, 1992
) while sparing the mAHP. On the other hand, as in
the case of HMs, the mAHP of hippocampal neurons is reduced by
carbachol (Fiszman et al. 1991
; Storm
1989
; Williamson and Alger 1990
; Zhang
and McBain 1995
). It is however clear that in hippocampal cells
several conductances participate in the generation of mAHP
(Storm 1989
) and that one of them, the so called
IM, may be the main target for the
blocking action by carbachol on the mAHP (Halliwell and Adams
1982
). It seems unlikely that on HMs IM was responsible for generating the mAHP
because IM is not
Ca2+ dependent and has slow activation kinetics
(Adams et al. 1982
, Brown and Selyanko
1985
) which preclude its turning on by a single AP. The present
study thus indicates the mAHP as a novel site of action for muscarinic
receptors of hypoglossal motoneurons. Furthermore, atropine enhanced
the mAHP amplitude, suggesting that under the recording conditions of
the slice preparation there was background release of acetylcholine
sufficient for partial inhibition of the mAHP.
Our data however indicate that, in addition to
IAHP, other K+
currents of HMs were modulated by muscarinic receptors. In fact, in the
presence of apamin when IAHP should
have been completely blocked, carbachol could still reduce a component
of the sustained outward current. The multiple sites of carbachol
action on HMs were confirmed in experiments under current clamp
conditions, as a reduction in mAHP and membrane depolarization could be
differentially antagonized by apamin. Although block of a variety of
K+ conductances contributes to the
carbachol-evoked depolarization (Benardo and Prince
1982; Storm 1990
; Womble and Moises
1992
), it is possible that enhancement of a
Ca2+-dependent nonspecific cationic current is
also a factor leading to membrane depolarization (Colino and
Halliwell 1993
). Unlike the case of CA1 hippocampal cells
(Figenschou et al. 1996
), the present study did not
observe any change in action potential duration or threshold in the
presence of carbachol.
Repetitive firing
Adult HMs in brain stem slices show three distinct phases of spike
frequency adaptation (Sawczuk et al. 1995, 1997
). In
most cases HMs of neonatal rats display fast spike adaptation or, in a
minority of cases, firing acceleration (Viana et al. 1993b
, 1995
). The present study found no evidence for spike frequency acceleration, whereas fast adaptation was the most common response (a
minority of cells had a regular firing pattern). The differences might
be due to postnatal developmental changes (as the present results were
obtained from younger rat cells that often show fast adaptation;
Viana et al. 1995
) or to the recording conditions (sharp
versus patch electrodes; blind recording versus visually identified
motoneurons; large current pulses versus weaker ones). In the present
investigation the crucial role of the mAHP in firing behavior became
immediately apparent after the mAHP was blocked by apamin, carbachol,
or Ca2+-free solution. Strong firing adaptation
was readily manifested as a result. Previous studies have shown the
importance of the mAHP in controlling repetitive firing in different
neurons (Baldissera and Gustafsson 1974
; Chandler
et al. 1994
; Kernel and Sjoholm 1973
;
Nishimura et al. 1989
; Powers et al.
1999
; Storm 1990
; Viana et al.
1993b
). Whenever the mAHP was present to hyperpolarize the
membrane potential of HMs, the duration of membrane potential sojourns
at depolarized level became inadequate for full activation or
inactivation of various voltage-dependent conductances (for instance
compare AP duration lasting ~3 ms with activation time constants of
8 ± 3 ms for Ifast and 18 ± 3 ms for Islow; Lape and
Nistri 1999
). These kinetic properties thus prevented the onset
of the adaptation process. The present results therefore demonstrate
that the mAHP of neonatal HMs had the fundamental property of
maintaining a slow firing frequency (with a relatively regular pattern)
but, at the same time, it could not be the principal component for
spike adapting properties.
The fact that strong spike frequency adaptation appeared when the mAHP
was fully suppressed by apamin raises the question of the relative
contribution by different conductances to the control of repetitive
firing. In the presence of apamin (either alone or plus carbachol which
should have also blocked IM or leak conductance as discussed earlier), fast adaptation probably developed because of the kinetic properties of
Ifast. Slow adaptation (which developed over a matter of hundreds of milliseconds) presumably relied
on Islow especially as the baseline
membrane potential, elevated during the spike train, should have
facilitated persistent Islow
activation. Modulation of mAHP by neuromodulators like muscarine, 5-HT
(Bayliss et al. 1995), or norepinephrine (Parkis
et al. 1995
) suggests that HM firing could change during
different behavioral states associated with various degrees of activity
of cholinergic, serotoninergic, or noradrenergic pathways. These
transmitters might thus act via dynamic alterations in the role of
various K+ conductances to the total outward current.
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ACKNOWLEDGMENTS |
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This work was supported by grants from Instituto Nazionale Fisica Della Materia and Ministero dell' Università e della Ricerca Scientifica e Tecnologica (MURST) (co-finanziamento ricerca).
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FOOTNOTES |
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Address reprint requests to R. Lape.
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 3 December 1999; accepted in final form 10 February 2000.
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REFERENCES |
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