Division of Neurophysiology, Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark
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ABSTRACT |
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Kjaerulff, Ole and
Ole Kiehn.
5-HT Modulation of Multiple Inward Rectifiers in Motoneurons in
Intact Preparations of the Neonatal Rat Spinal Cord.
J. Neurophysiol. 85: 580-593, 2001.
This study
introduces novel aspects of inward rectification in neonatal rat spinal
motoneurons (MNs) and its modulation by serotonin (5-HT). Whole cell
tight-seal recordings were made from MNs in an isolated lumbar spinal
cord preparation from rats 1-2 days of age. In voltage clamp,
hyperpolarizing step commands were generated from holding potentials of
50 to
40 mV. Discordant with previous reports involving slice
preparations, fast inward rectification was commonly expressed and in
44% of the MNs co-existed with a slow inward rectification related to
activation of Ih. The fast inward
rectification is likely caused by an
IKir. Thus it appeared around
EK and was sensitive to low
concentrations (100-300 µM) of Ba2+ but not to
ZD 7288, which blocked Ih. Both
IKir and
Ih were inhibited by
Cs2+ (0.3-1.5 mM). Extracellular addition of
5-HT (10 µM) reduced the instantaneous conductance, most strongly at
membrane potentials above EK. Low
[Ba2+] prevented the 5-HT-induced
instantaneous conductance reduction below, but not that above,
EK. This suggests that 5-HT inhibits IKir, but also other instantaneous
conductances. The biophysical parameters of
Ih were evaluated before and under
5-HT. The maximal Ih conductance,
Gmax, was 12 nS, much higher than
observed in slice preparations. Gmax
was unaffected by 5-HT. In contrast, 5-HT caused a 7-mV depolarizing
shift in the activation curve of Ih.
Double-exponential fits were generally needed to describe Ih activation. The fast and slow time
constants obtained by these fits differed by an order of magnitude.
Both time constants were accelerated by 5-HT, the slow time constant to
the largest extent. We conclude that spinal neonatal MNs possess
multiple forms of inward rectification.
Ih may be carried by two spatially
segregated channel populations, which differ in kinetics and
sensitivity to 5-HT. 5-HT increases MN excitability in several ways,
including inhibition of a barium-insensitive leak conductance,
inhibition of IKir, and enhancement of
Ih. The quantitative characterization of these effects should be useful for further studies seeking to
understand how neuromodulation prepares vertebrate MNs for concerted
behaviors such as locomotor activity.
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INTRODUCTION |
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The discharge pattern of
motoneurons (MNs) in the spinal cord depends on the characteristic of
each of a number of ionic currents selectively channeled through
membrane proteins (Kiehn 1991b; Rekling et al.
2000
; Schwindt and Crill 1984
). A subset
of currents preferably flow at hyperpolarized membrane potentials and
are therefore known as inward rectifiers (Hille 1992
).
Dependent on the speed of current activation, inward rectification (IR)
is seen as a downward bend in either the instantaneous or the
steady-state current-voltage (I-V) relationship, or both.
The presence of one particular type of inward rectifier causes a slow
sag toward the original membrane potential when the cell is
hyperpolarized from the resting membrane potential. In spinal cord MNs,
Ito and Oshima (1965)
originally described this slowly
developing inward rectification in the cat. Later studies in a slice
preparation of the neonatal rat spinal cord suggested that the
underlying current is a slow, noninactivating inward cation current
with a reversal potential more positive than rest (Takahashi
1990a
). This current corresponds to the
hyperpolarization-activated inward mixed cation current, or
Ih, found in many vertebrate and
invertebrate neurons (Pape 1996
). The slow activation of
Ih explains the time dependency of the
sag. Moreover, since Ih also
deactivates slowly when the hyperpolarization is released, this current
contributes to the formation of a long-lasting rebound excitation.
Another form of inward rectification is found in various types of
excitable cells including neurons. This "instantaneous" current,
IKir, is carried by potassium ions
(Constanti and Galvan 1983
; Katz 1949
;
Nichols and Lopatin 1997
; Standen and Stanfield
1979
; Travagli and Gillis 1994
; Williams
et al. 1997
; Yamoah et al. 1998
).
IKir, as well as
Ih, typically contribute to the
resting membrane potential. This is one important reason why inward
rectifiers play essential roles for the excitability and response
properties of nerve cells. Moreover, the contribution of both
IKir and
Ih to the resting membrane potential
makes these currents prime targets for neuromodulators. One such
neuromodulator is serotonin (5-HT), which is often used to induce
locomotion in the isolated spinal cord of the neonatal rat
(Cazalets et al. 1992
; Kiehn and Kjaerulff
1996
; Kjaerulff and Kiehn 1996
,
1997
). 5-HT strongly excites spinal MNs in the rat
(Berger and Takahashi 1990
; Kiehn 1991a
;
Takahashi and Berger 1990
; Wang and Dun
1990
). A part of this excitation has been ascribed to an
enhancement of Ih (Takahashi
and Berger 1990
). However, the exact mechanism behind the
enhancement has not been determined.
We have started to investigate the contribution of inward rectification
to the discharge pattern in neonatal rat lumbar MNs during
transmitter-induced rhythmic locomotor activity (Kiehn et al.
2000). A complete understanding of how inward rectification contributes to the discharge patterns in neonatal rat lumbar MNs requires knowledge of the biophysical parameters of the underlying currents. Furthermore, earlier studies have generally been performed on
MNs located superficially in thin slice preparations (e.g., Takahashi 1990a
). Despite the importance of the
information obtained under such circumstances, it has hardly been
possible to avoid elimination of part of the dendritic arbor. This
might lead to important qualitative and quantitative deviations from
the membrane current characteristic in the more intact MNs located in
whole spinal cords used for locomotor studies.
Our study shows that, in addition to the slowly developing inward
rectification caused by activation of
Ih, most neonatal rat MNs possess an
instantaneous rectifier with kinetics and pharmacology similar to
IKir. We show that 5-HT
enhances Ih by causing a depolarizing shift in the activation curve. In contrast, the fast inward rectifier IKir is inhibited by 5-HT. In
addition, 5-HT appears to inhibit other instantaneous currents,
possibly "leak" currents, as well. These effects of 5-HT enhance
resting MN excitability and cause a theoretical enhancement of rebound
firing and phase-transition during rhythmic motor discharges.
Preliminary results from these experiments have been presented in
abstract form (Kjaerulff and Kiehn 1998).
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METHODS |
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Dissection
The procedure for isolating the spinal cord has been described
in detail earlier (Kjaerulff and Kiehn 1996,
1997
) and is only partially summarized here. Neonatal
rat pups (1-2 days old) were anesthetized with ether and quickly
decapitated. The spinal cord was isolated and a piece of cord
comprising approximately the Th12-S1 segments was placed in a recording
chamber perfused with standard Krebs solution containing (in mM) 128 NaCl, 4.69 KCl, 25 NaHCO3, 1.18 KH2PO4, 1.25 MgSO4, 2.52 CaCl2, and 22 glucose, aerated with 5% CO2 in
O2. The composition of this extracellular solution is identical to that used in our previous locomotor studies (e.g., Kiehn and Kjaerulff 1996
; Kjaerulff and
Kiehn 1997
) (see INTRODUCTION) and was selected
also for the present study to be able to directly relate the findings
on the inward rectifier currents to the results of the locomotor experiments.
Recordings
Recordings from MNs were made with patch electrodes pulled from
thick-walled borosilicate glass (OD: 1.5 mm; ID: 1.0 mm, Clark Electromedical Instruments, Pangbourne, England) to a final resistance of 4-7 M. Recordings were made in the whole cell configuration (Hamill et al. 1981
) using an Axoclamp 2B amplifier
(Axon Instruments, Foster City, CA) in the continuous single-electrode
voltage-clamp (cSEVC) mode. With maximal phase-advance, it was usually
possible to increase the clamp gain to 100 nA/mV (maximal gain).
However, the gain was generally kept slightly lower (~90 nA/mV) to
prevent fatal oscillations of the clamp circuit induced by changes in the access resistance. pCLAMP (Axon Instruments) was used for data
acquisition and off-line current measurements. Current signals were
low-pass filtered at 1-2 kHz and digitized at 2.667 kHz.
In most experiments the spinal cord was pinned down with the ventral
side up and the electrode lowered blindly into the ventrolateral region
of the L4 (occasionally L5)
segment through a slit cut in the white matter. In a few cases the
ventral part of the cord was isolated (Kjaerulff and Kiehn
1996), pinned down dorsal side up, and the electrode lowered
into the motor column via the dorsal cut surface. The pipette electrode
solution contained (in mM) 128 potassium gluconate, 4 NaCl, 1 glucose,
10 HEPES, 0.0001 CaCl2, 4 ATP-Mg, and 0.3 GTP-Li.
The pH was adjusted to 7.3 with KOH. Recordings were made at room temperature.
Correction of the voltage
In general, the access resistance,
Ra, was within 10-30 M. Since the
current could reach amplitudes of 1 nA or more, the predicted voltage
drop over Ra was not negligible. To
compensate for this error, we monitored
Ra pertaining to individual voltage steps and corrected the membrane potential off-line using the relation
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(1) |
JUNCTION POTENTIAL.
Voltages were not corrected for the liquid junction potential
(Neher 1992), which was within the range 6-8 mV
(pipette interior negative) with the intracellular and various
extracellular solutions used in the present experiments. Correcting
potentials would lead to more hyperpolarized values than those reported
in the text.
Database
Recordings from 138 MNs were included in this study. Cells were
identified as MNs when ventral root stimulation through a suction
electrode evoked an antidromic action potential. For a MN to be
included in the database, the holding current corresponding to a
holding potential of 50 mV had to be more positive than 0 pA.
Pharmacological compounds
Serotonin creatine sulfate (5-HT, 10 µM), tetrodotoxin (TTX,
0.2-0.3 µM), tetraethylamminium-chloride (TEA 20-30 mM), and 4-aminopyridine (4-AP, 2-4 mM) were from Sigma (St. Louis, MO). The
5-HT concentration chosen is at the plateau of the dose-response curve
for activation of the previously described 5-HT-induced inward
current, I5-HT, in neonatal rat MNs
(Takahashi and Berger 1990).
D,L-2-amino-5-phosphonovaleric acid (APV, 20 µM) and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) were from RBI
(Natick, MA) and
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrdinium chloride (ZD 7288, 100 µM) from Tocris Cookson (Bristol, UK). In experiments involving the extracellular use of
BaCl2 (100-300 µM),
KH2PO4 was omitted and
MgSO4 replaced with an equimolar concentration of
MgCl2.
Statistical analysis
Commercially available software (Statistica; StatSoft, Tulsa,
OK) was used to test for differences between the amplitude of the
difference current, Iss Iin, before, during, and after
application of 5-HT in the same MNs. Data were transformed by taking
the square root of the absolute current amplitudes prior to
significance testing (Altman 1991
) to conform with the
assumptions underlying the statistical model used, i.e., repeated
measures ANOVA. This model was also used to evaluate changes in holding
current induced by 5-HT in normal medium. Duncans multiple range test
was employed for post hoc comparisons.
INSTANTANEOUS I-V RELATIONSHIP. We used a statistical criterion to help decide whether rectification was present in the instantaneous I-V relationship. If the I-V relationship was judged by eye to be linear, this was accepted without further analysis. However, if the I-V curve appeared to bend around a given voltage, Vbend, linear regression was used to determine Gdep and Ghyp, i.e., the average slope conductance in the voltage range more depolarized and hyperpolarized, respectively, than Vbend (see Figs. 3A and 5, A and B, for indications of Vbend, Gdep, and Ghyp). The 97.5% confidence interval for Gdep was then compared with the 97.5% confidence interval for Ghyp. Fast rectification was set to be present if the two confidence intervals did not overlap. The bend in the instantaneous I-V curve was generally sharp enough to permit the identification of Vbend with an uncertainty below ~10 mV.
Pharmacologically induced changes in Gdep, Ghyp, and in fast rectification were analyzed using paired t-tests. Since the distributions of the instantaneous conductance data were positively skewed, data were transformed by taking the logarithm before testing (Altman 1991 ![]() |
RESULTS |
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Generally, experiments were done in the presence of TTX to block
the fast Na+ current,
INa (Takahashi 1990b).
TTX also blocks synaptically mediated indirect effects of 5-HT on MNs
caused by spike activation of presynaptic interneurons (Wang and
Dun 1990
). In most experiments, the glutamate receptor
antagonists APV and CNQX were also added to reduce spontaneous synaptic noise.
Current responses in normal medium
LINEAR.
In some MNs, when hyperpolarizing voltage steps from a holding
potential of 50 mV were generated, the size of the instantaneous current response, Iinst, was
proportional to the size of the voltage step (Fig.
1, A and B). In
normal medium (with or without TTX/APV/CNQX), such a linear
instantaneous I-V relationship was observed in 30 of 72 MNs
(41.7%).
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FAST INWARD RECTIFICATION. In a larger fraction of the MNs, however, the Iinst elicited by large hyperpolarizing voltage steps was disproportionally larger than Iinst evoked by smaller hyperpolarizing steps. Thus these MNs showed fast IR (Fig. 1, C and D). Typically, the membrane potential at which the increase in conductance occurred was rather sharply defined, producing an inflection point on the instantaneous I-V relationship at 79.8 ± 1.1 mV (mean ± SE; Vbend, see METHODS). A comparison of the 97.5% confidence interval of the average instantaneous slope conductance at voltages above Vbend, termed Gdep, with that of the conductance, Ghyp, below Vbend confirmed the presence of fast IR in a high number of MNs (35 of 72 MNs, 48.6%).
OUTWARD RECTIFICATION.
In relatively few MNs, the Iinst
elicited by large hyperpolarizing voltage steps was disproportionally
smaller than the Iinst evoked by smaller hyperpolarizing steps (7 of 72 MNs, 9.7%; not shown). Thus fast outward rectification (OR) was also observed, but
only in a minority of the MNs. The average
Vbend for fast OR was 83.3 ± 2.8 mV.
SLOWLY DEVELOPING INWARD RECTIFICATION, Ih. An inwardly increasing current usually followed the instantaneous current jump. The rate of inward increase was fast initially but leveled off with time (inward relaxation). The steady-state current, Iss, was reached only after several seconds (e.g., Fig. 1, A and C). The activation rate and the amplitude of this slowly developing inward relaxation increased with increasing hyperpolarization. The amplitude increase lead to inward rectification in the steady-state I-V relationship (steady-state IR; Fig. 1, B and D).
The slowly developing inward rectification shares voltage and time dependency with the hyperpolarization-activated cation current, Ih, previously described by Takahashi (1990a)Multiple forms of inward rectification caused by distinct conductances co-exist in MNs
PERSISTENCE OF FAST INWARD RECTIFICATION IN ZD 7288.
In roughly half of the MNs, Ih
appeared to be the only form of inward rectifying current present,
since these cells showed no fast IR (33 of 72 MNs, 45.8%; Fig.
1B). However, fast IR co-existed with the steady-state IR
indicative of Ih in a substantial
number of MNs (32 of 72 MNs, 44.4%; Fig. 1, C and
D). In contrast to Ih, fast
IR has not previously been reported to be present in spinal rat MNs. It
therefore became important to determine whether the two forms of inward
rectification are mediated by different conductances, or whether they
are different expressions of the same conductance, e.g.,
Ih. For this purpose, we employed ZD
7288, an established blocker of Ih
(BoSmith et al. 1993; Harris and Constanti
1995
; Hughes et al. 1998
; Khakh and
Henderson 1998
; Williams et al. 1997
). Figure
2A shows a MN, in which both
fast IR and the slowly developing IR indicative of
Ih was present in control conditions.
ZD 7288 reduced both the instantaneous and the steady-state conductance
(Fig. 2B). Moreover, ZD 7288 abolished the slow inward
relaxation. In contrast, fast IR persisted (Fig. 2B; Fig.
2C, compare
and
). In current clamp (not shown; see Kiehn et al. 2000
) ZD 7288 removed the sag response,
again indicating an efficient blockade of
Ih. As in voltage clamp, however, fast IR persisted. Similar results were obtained in a total of five MNs (2 in voltage clamp, 3 in current clamp). The fact that ZD 7288 blocked
Ih but not fast IR suggests that the
fast IR is caused by a different inward rectifier than
Ih.
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Identity of the fast inward rectifying current
Several of our observations suggest that at least part of the fast
IR in spinal neonatal rat MNs is caused by an
IKir, i.e., an inward
("anomalous") rectifying current carried by potassium ions
(Constanti and Galvan 1983; Hagiwara et al.
1976
; Katz 1949
; Nichols and Lopatin
1997
; Standen and Stanfield 1979
;
Travagli and Gillis 1994
). First,
IKir is generally associated with fast or "instantaneous" IR. Second, fast IR in the rat MNs occurred around a Vbend of
80 mV on average;
this voltage is close to the K+ equilibrium
potential (EK, predicted to be
79 mV
with the intra- and extracellular solutions used). This similarity
suggests that fast IR, like IKir, is
largely K+ dependent. Third, we sometimes
observed an apparent inactivation during strong hyperpolarizing steps
(Fig. 4). This behavior of IKir has
been reported previously in a number of cell types (Biermans et
al. 1987
; Constanti and Galvan 1983
;
Hagiwara et al. 1976
; Silver and DeCoursey
1990
; Yamoah et al. 1998
) and has generally been
attributed to an external voltage- and time-dependent block by
Na+ ions rather than true inactivation of the
IKir channels themselves.
EFFECTS OF DIVALENT CATIONS ON FAST IR.
If the fast IR in spinal neonatal rat MNs is caused by
IKir, it should be sensitive to
Cs2+ concentrations in the millimolar range
(Benson and Levitan 1983; Constanti and Galvan
1983
; Hagiwara et al. 1976
; Standen and
Stanfield 1979
; Travagli and Gillis 1994
).
Indeed, we found that fast IR was reduced or blocked by
CsCl2 (0.3-1.5 mM, n = 6 MNs;
not shown).
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REVERSAL POTENTIAL FOR
IBa2+.
The reversal potential for the current blocked by
Ba2+, IBa2+,
was determined as the intersection between the instantaneous
I-V curves obtained in the same cells in control and during
Ba2+ application (Fig. 3). The reversal potential
for IBa2+ was 75.0 ± 3.4 mV
(n = 5); this is close to the potassium equilibrium potential (i.e.,
79 mV). IBa2+ was
generally of small, albeit not negligible, amplitude at membrane
potentials depolarized to its reversal potential.
OUTWARD CURRENT RELAXATION BLOCKED BY Ba2+.
An outward current relaxation was sometimes observed in
response to large hyperpolarizing voltage steps (i.e., beyond
approximately 80 mV; Fig. 4A). This outward relaxation,
generally preceded by a brief inward relaxation, was slow, and
typically did not reach steady state before the voltage step was
terminated, typically after 3 s. Since
Ih or its related currents do not
inactivate (DiFrancesco 1985
; Pape 1996
),
the outward relaxation in response to extreme hyperpolarizing steps
cannot be caused by Ih inactivation. However, it may be caused by the "inactivation" of
IKir (Biermans et al.
1987
; Yamoah et al. 1998
). In accordance with
this notion, addition of the IKir
antagonist Ba2+ (300 µM, Fig. 4B)
profoundly altered the response by abolishing the outward relaxation.
This effect was reversible on washout of Ba2+
(Fig. 4C).
BLOCKING IKir ENHANCES Ih. The inactivation of IKir can efficiently mask Ih. This important point is illustrated in Fig. 4. Before Ba2+, the inward relaxation characteristic of Ih was inconspicuous during voltage steps of small/medium amplitude (Fig. 4A). Furthermore, during stronger hyperpolarizations, it was largely obscured by the persistent outward relaxation that we ascribe to the inactivation of IKir. These observations provide the false impression that Ih was largely absent in this MN. However, this was not the case, since an inward relaxation with a voltage dependency of amplitude and activation rate characteristic of Ih was observed after the Ba2+ block of IKir (Fig. 4B). This and similar findings in other MNs points out that to study the behavior of Ih in detail, it is necessary to first eliminate IKir.
We conclude that in neonatal rat spinal motor neurons a fast inward rectifier very likely to be a potassium-dependent IKir often co-exists with the slow mixed cation-dependent inward rectifier Ih.5-HT influences the instantaneous I-V relation
The discovery of IKir in neonatal
rat spinal MN prompted an investigation of the possible influence of
5-HT on IKir and other instantaneous
currents. By separately analyzing the effect of 5-HT on the
"depolarized" and the "hyperpolarized" instantaneous conductances (Gdep and
Ghyp, respectively; see
METHODS), we found that 5-HT reduces the instantaneous
conductance in a voltage-dependent manner. 5-HT reduced
Gdep from 21.4 ± 3.0 nS
(control) to 15.8 ± 2.1 nS (5-HT). This reduction was significant
(P < 0.00001, n = 24 MNs, paired
t-test; Fig. 5A)
and partially reversible, since Gdep
rose again on washout of 5-HT (16.7 ± 4.4 nS, 5-HT; 19.8 ± 5.1 nS, wash, n = 9, P < 0.05). In the
same MNs, 5-HT also significantly reduced
Ghyp, from 28.2 ± 4.6 nS in
control to 24.4 ± 3.9 nS in 5-HT (P < 0.005, Fig. 5A). This effect is probably normally reversible,
although we were not able to demonstrate this in the present
experiments (Ghyp = 24.8 ± 8.6 nS, 5-HT; 23.5 ± 7.3 nS, P > 0.5). Thus 5-HT
reduced the instantaneous conductance over the entire voltage range
tested. Interestingly, this reduction was significantly stronger in the
depolarized voltage range than in the hyperpolarized range (reduction
in Gdep = 5.7 ± 1.3 nS vs.
reduction in Ghyp =
3.9 ± 2.0 nS; n = 24; P < 0.05). Stated equivalently, 5-HT enhanced the inward bend in the instantaneous I-V relationship. These data suggest that the fast IR is
under neuromodulatory control in spinal MNs.
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EFFECTS OF 5-HT ON INSTANTANEOUS CURRENTS IN BARIUM. It has been argued above that low concentrations of barium provide a relatively selective blockade of the putative IKir. In an attempt to extract the effect of 5-HT on IKir from effects on other instantaneous currents, we repeated the experiments described in the previous paragraph, but this time with barium present both before and during the addition of 5-HT, to permanently block IKir. In Ba2+ (100-300 µM), 5-HT still reduced Gdep (28.3 ± 0.8 nS, Ba2+ alone; 24.3 ± 0.9 nS, Ba2+ plus 5-HT, n = 17, P < 0.001, Fig. 5B). In contrast, the 5-HT-induced reduction of Ghyp observed in normal medium was not seen in Ba2+ (21.9 ± 0.6 nS, Ba2+ alone; 23.5 ± 0.7 nS, Ba2+ and 5-HT, P > 0.10, Fig. 5B).
DEDUCED EFFECTS OF 5-HT ON IKir AND OTHER INSTANTANEOUS CURRENTS. Comparing the results of 5-HT application on the instantaneous currents in normal medium and in barium leads to the following conclusions. Since barium, in concentrations favoring the antagonism of IKir, prevented the 5-HT-dependent reduction in Ghyp, we suggest that the Ghyp reduction stems from 5-HT diminishing IKir. In contrast, the 5-HT-dependent reduction of Gdep observed in normal medium is unlikely to strongly involve IKir, since this effect was not prevented by the IKir antagonist barium. Rather, 5-HT appears to reduce other instantaneous conductances in addition to IKir (see DISCUSSION).
Reversal potential of Ih
In the following we discuss the effects of 5-HT on
Ih. To permit the evaluation of these
effects in terms of conductance rather than current alone (see
below), we determined Eh, the
reversal potential of Ih. We used the
method previously employed by others (Bayliss et al.
1994; Mayer and Westbrook 1983
; Takahashi
1990a
), where the intercept of two instantaneous I-V
curves evoked from two different holding potentials is used to estimate
Ih (not shown) (see Fig.
6 in Takahashi 1990a
for
an illustration of a similar protocol). The holding potentials were
kept below the threshold for depolarization-induced currents.
Eh was determined in 125 experiments
(50 MNs) under various pharmacological conditions. Due to the presence
of some clear outliers, the median rather than the mean was chosen for
calculations (see below) of the Ih conductance. The median Eh was
33
mV, well in agreement with previous measurements in the same MNs
(Takahashi 1990a
).
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5-HT effects on Ih in barium
ENHANCEMENT OF THE Ih
AMPLITUDE.
The fact that Ba2+ blocks
IKir (Figs. 3 and 4) allowed us to
isolate the effect of 5-HT on Ih.
Ih was elicited by hyperpolarizing voltage steps
before and after the addition of 5-HT, in the constant presence of
200-300 µM Ba2+ to eliminate
IKir. Measurements in 5-HT were taken
as soon as possible after control measurements, to minimize the impact
of time-dependent rundown (see later). In the example MN in Fig. 6, the
access resistance, Ra, associated with
the recording was low, so that voltage steps of the same nominal
amplitude were similar even after correcting the membrane potential for
the voltage drop over Ra (see
METHODS). In this MN, therefore, the current responses to
voltage steps of the same order, elicited before and during 5-HT, could
be compared directly (Fig. 6A). Responses were identical
down to 66 mV. Beginning at
72 mV, however, the Ih amplitude was larger in 5-HT than
in control. This difference (in steady state) increased with further
hyperpolarization, peaked at
90 mV, and then gradually declined again
to reach zero at
107 mV. The I-V curve in Fig.
6B summarizes the 5-HT enhancement of the steady-state
Ih, calculated as the difference
between the steady-state current, Iss,
and the instantaneous current, Iin. The average amplitude measured at
80 mV was 144 ± 36 pA in
barium alone, and 252 ± 62 pA in barium plus 5-HT
(P < 0.001, n = 16 MNs, Fig.
6C). Thus the enhancement of the
Ih amplitude in the intermediate
voltage range was a general finding in the barium-containing solution.
5-HT ENHANCEMENT OF V1/2.
The estimated Eh permitted us to
convert steady-state Ih-V
curves (Fig. 6B) to Gh
activation curves (Fig. 7A),
using the equation
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5-HT effects in normal medium
To test whether the 5-HT enhancement of
Ih is a robust phenomenon present
under standard conditions and not only under circumstances aimed at
pharmacologically isolating Ih,
experiments similar to those described in the previous section were
performed in standard Krebs solution, i.e., without barium. These data
include measurements taken during wash-off of 5-HT. As in barium, 5-HT
also enhanced the difference current,
Iss - Iin under standard conditions. The effect of 5-HT was transient, such that after prolonged exposure (10-15 min) to 5-HT the difference current
Iss - Iin again began to decline, despite
the continuous presence of the drug (not shown). Furthermore, the
difference current was typically smaller in wash than in control. We
attribute these effects to rundown of
Ih (Takahashi and Berger
1990), a current known in these and other cells to be modulated
by diffusible second messengers (Hille 1992
;
Larkman et al. 1995
; Pape 1996
).
Iss - Iin at
80 mV was determined
1) before 5-HT (Fig.
8A, Control), 2)
between 5 and 10 min following the application of 5-HT (Early 5-HT),
and 3) either more than 15 min after adding 5-HT, or in
washout of 5-HT (Late 5-HT/wash). The pooling of data obtained late in
5-HT with those obtained in wash was justified by the observation that
there was no substantial differences between measurements in these two
conditions, presumably as a result of rundown. The mean of
(Iss - Iin) showed a 19.0% average increase
in 5-HT compared with control. This is close to significance at the 5%
level (P = 0.07, n = 14). A 31.4%
reduction in Iss - Iin compared with control was seen in
the wash/late 5-HT group (P < 0.001), reflecting the
rundown effect.
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Results obtained in normal medium should be interpreted with caution due to the presence of IKir (Fig. 4). However, even in normal medium Ih often appeared to form the main component of the difference current, Iss - Iin, despite the contribution from IKir. Therefore these results confirm that the 5-HT enhancement of Ih also takes place in standard conditions. Furthermore, in view of the observed rundown, it seems likely that our results may well underestimate the degree of the 5-HT enhancement occurring under physiological circumstances.
Enhanced rundown of Ih in a poly-channel antagonist solution
In some experiments, the standard Krebs solution was
replaced with a zero- or low (10%) Ca2+ solution
with an elevated [Mg2+] to which was added TEA
(20 mM) and 4-AP (2 mM). Such a "poly-channel antagonist" solution
is conventionally used to pharmacologically isolate
Ih from voltage-gated
Ca2+ currents, the delayed rectifier
(IK), and the A current
(IA) (Takahashi 1990b).
Ih was measured at
80 mV in 13 MNs
(Fig. 8B), under the same conditions as in the experiments
made in standard Krebs solution. Despite the application of 5-HT, a
progressive decline of the Ih
amplitude was typically observed in the poly-channel antagonist
solution. On average, the Ih amplitude
in 5-HT was reduced to 56% compared with control (P < 0.05, Fig. 8B) and after wash only 40% of the control
Ih remained (P < 0.001). This observation is in sharp contrast to the findings in
Ba2+ (Fig. 6C) and in normal medium
(Fig. 8A), where 5-HT enhanced Ih. These data suggest that the
poly-channel antagonist solution enhances
Ih rundown. The underlying mechanism
is currently unknown.
5-HT accelerates Ih activation
The results presented in this section all originate from the
experiments done in the Ba2+-containing Krebs
solution. Current activation in response to a family of hyperpolarizing
voltage steps was fitted to a single-exponential function of the form
![]() |
(4) |
![]() |
(5) |
The goodness-of-fit of single- and double-exponential functions
did not differ substantially for voltage steps of low amplitude. However, when the membrane potential was stepped to more hyperpolarized levels than approximately 80 mV, double exponentials clearly fitted
the current responses better than single exponentials. The fast and
slow time constants,
fast and
slow, were extracted from the
double-exponential fits (Fig. 9,
A and B) and plotted against the membrane
potential, V (Fig. 9, C and D).
V was taken as the average between
Vin and
Vss (see METHODS).
|
In 6 of 16 MNs (37.5%), there was no obvious relationship
between V and fast. This
was also the case for small-amplitude steps in the remaining 10 MNs
(62.5%, not shown). However, for larger steps,
fast systematically decreased with
increasing hyperpolarization (Fig. 9C). The relationship
between V and
fast was
well described by the hyperbolic function
![]() |
(6) |
![]() |
(7) |
The dependence of slow on
V also largely (10 of 16 MNs) obeyed Eq. 6,
although the datapoints were more scattered than those related to
fast. Again, 5-HT did not change
a (
0.022 ± 0.004 s
1
mV
1,
Ba2+;
0.026 ± 0.003 s
1
mV
1,
Ba2+ and 5-HT; n = 10, P > 0.2, paired t-test) but increased
b significantly (
1.5 ± 0.3 s
1,
Ba2+;
1.7 ± 0.3 s
1,
Ba2+ and 5-HT, P < 0.00001, n = 10). Thus like
fast,
slow was decreased by 5-HT. These
data show that activation of Ih is
accelerated by 5-HT, which also appears from direct inspection of the
raw data (Fig. 9A, see also Fig. 6A). Figure 9,
E and F, shows the overall effect of 5-HT on
fast and
slow, respectively. When comparing
the two time constants, 5-HT exerted its strongest effect on
slow. This was most obvious in the
hyperpolarized region.
We were not able to determine with certainty the time constants in the
voltage region more depolarized than approximately 70 mV. This was
partly due to weak activation of Ih
but was also related to the fact that the fitted parameters were
contaminated by the decay of the capacitive currents related to
generation of the voltage steps.
5-HT effect on the holding current
At a holding potential of 50 mV, the holding current in normal
medium (including TTX/CNQX/APV) was 80 ± 23 pA (n = 12 MNs). Five minutes after adding 5-HT, the holding current measured
in the same MNs displayed a strong inward shift, reaching
51 ± 27 pA (P < 0.0005, data not shown). This level did not
change significantly during the next 5 min (5 min vs. 10 min in 5-HT,
P > 0.3). We made the qualitative observation,
however, that the holding current began to shift back in the outward
direction after ~15-20 min in 5-HT. The transient effect of 5-HT on
the holding current matches the clear but fleeting 5-HT enhancement of
the Ih amplitude described in a
previous section.
An inward shift in holding current was also induced by 5-HT in
the experiments done in the constant presence of
Ba2+ (see above). The holding potential was 40
mV, while the holding current was 440 ± 47 pA in
Ba2+ and 239 ± 46 pA in
Ba2+ plus 5-HT (P < 0.00005, n = 14 MNs, paired t-test). An inward shift
in holding current was observed in a total of 26 MNs.
Both enhancing Ih (which has a reversal potential more positive than the holding potential used in the present experiments) and inhibiting IKir and potassium-dependent leak currents (with a reversal potential more negative than the holding potential) leads to an increase in inward current. Therefore our consistent observation of an inward shift in the holding current is in accordance with our conclusions earlier in the paper that 5-HT enhances Ih and inhibits potassium-dependent currents including IKir. The frequent occurrence of the 5-HT-induced inward current suggests that this feature is important for normal motor function in the neonatal rat.
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DISCUSSION |
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Takahashi (1990a) reported the presence of a slowly
activating, mixed
Na+/K+-dependent inward
current in neonatal rat spinal MNs. While he preferred to denote this
current IIR, we have used the term
Ih, which is now a more common name
for this class of currents in neurons (Pape 1996
). The
Ih in MNs of the neonatal rat is
enhanced by 5-HT (Larkman and Kelly 1997
;
Takahashi and Berger 1990
). Some aspects of this
neuromodulatory effect, especially the pharmacology, have been closely
studied (Larkman and Kelly 1997
; Larkman et al.
1995
; Takahashi and Berger 1990
). However, a
detailed quantitative investigation of how 5-HT affects the biophysical
parameters of Ih has been missing in
mammalian MNs. Furthermore, previous studies on neonatal rat MNs have
often involved recordings from MNs in thin slices. The MNs recorded
from were situated superficially in the slices and were mechanically
cleared to allow visual inspection of the cells (Larkman and
Kelly 1998
; Takahashi and Berger 1990
). It seems
likely that this procedure may have lead to a partial pruning of the
dendritic tree, which could alter the quantitative features of whole
cell currents. In contrast, here we present the results of whole cell
tight-seal voltage-clamp recordings from MNs in the intact spinal cord.
Too strong emphasis on the quantitative aspects of our data should be
avoided due to the imperfections of the whole cell voltage-clamp
technique, including the absence of ideal space clamp and the rundown
of the second-messenger-dependent currents such as
Ih. Nevertheless, our results provide
a necessary framework for future investigations, e.g., of
5-HT-mediated control of MN behavior during integrated motor tasks in
intact preparations, including locomotor activity (Kiehn et al.
2000
).
INSTANTANEOUS INWARD RECTIFICATION. During experiments aimed at quantifying the effects of 5-HT on the time-dependent inward rectifier, Ih, we discovered that fast inward rectification in neonatal rat spinal MNs is quite common and often coexists with Ih. We treat this finding first, partly because it is novel and of independent interest, and partly because fast inward rectification complicated the analysis of neuromodulation of Ih, as discussed below.
We found that half (49%) of the spinal MNs in the neonatal rat display fast IR. Several observations support the idea that this property is due to a potassium-dependent current belonging to the IKir class (Constanti and Galvan 19835-HT modulation of the instantaneous conductance
We found that 5-HT reduced the instantaneous conductance of spinal
MNs in conjunction with a negative (depolarizing) shift in the holding
current at 50 mV. This effect may be decomposed into suppression of
at least two currents, which dominate at different voltage ranges. Low
concentrations of Ba2+, which were sufficient to
block IKir, specifically prevented the
5-HT-dependent conductance reduction at hyperpolarized potentials (below approximately
80 mV; Fig. 5); this 5-HT effect may therefore be explained at least partly by blockade of
IKir. A previous demonstration of such
a mechanism was made in nucelus accumbens neurons, where an
instantaneous inward rectifying K+ conductance is
reduced by 5-HT (North and Uchimura 1989
). Also, 5-HT
reduces K+ currents both in adult (Larkman
and Kelly 1992
) and neonatal (Larkman and Kelly
1998
) rat facial MNs; one of these currents has been suggested
to be an IKir mainly on
pharmacological grounds.
In the depolarized voltage range (above approximately 80 mV),
Ba2+ did not prevent the 5-HT-induced reduction
in the instantaneous conductance in our recordings. Therefore a
different current than IKir must be
involved in this phenomenon. An interesting possibility is a background
K+ current present in the neonatal rat spinal MNs
(Fisher and Nistri 1993
). This current is
pharmacologically clearly different from IKir, since it is insensitive to
Cs2+ (2 mM), and, although reduced in high (1.5 mM) [Ba2+], it is not affected by
Ba2+ in low concentration (200 µM). The
background current is inhibited by the neuropeptides TRH and substance
P. Co-existence of these transmitters with 5-HT in fibers innervating
spinal motor nuclei has been described (Arvidsson et al.
1992
). Other currents may also be involved, however. In
neonatal rat facial MNs, 5-HT reduces a potassium current, which is
insensitive to antagonists of IKir, but sensitive to 4-AP (Larkman and Kelly 1998
). It
appears likely that a similar current exists in the spinal MNs. This
current could contribute to the 5-HT effect provided it is not fully
inactivated at the resting membrane potential.
The reduction of the instantaneous conductance and the outward shift in
holding current caused by ZD 7288 suggest that
Ih also contributes to the resting
conductance at potentials around 50 mV. We found that 5-HT enhances
this depolarizing contribution of Ih
to the membrane potential (see also Kiehn et al. 2000
).
Provided that IKir allows current flow at potentials more positive that EK, it will generate an outward current that decreases MN excitability. Inhibition of IKir by 5-HT in the depolarizing voltage range will therefore increase MN excitability. From the present series of experiments, it appears that there is little steady activation of IKir at potentials more positive than EK (see previous paragraph), suggesting that the 5-HT modulation of IKir in the depolarizing voltage range plays a minor role for MN firing.
Ih characteristics
We have established the biophysical parameters of
Ih in MNs in the intact cord in
detail, and in this section compare them with the parameters previously
determined in acute slices (Takahashi 1990a). Since the
co-existence of IKir with
Ih in many MNs could obscure
Ih (Fig. 4), we blocked the
IKir with low concentrations of
Ba2+. The maximal conductance,
Gmax, was determined from Boltzmann fits to Gh/V plots and
found to be 12.0 ± 1.5 nS. Takahashi (1990a)
reported a half-maximally activated chord conductance of about 0.8 nS.
Hence his Gmax value (~1.6 nS) would
be about an order of magnitude smaller than ours. A likely explanation
for this difference is that we have recorded from presumably more
intact MNs. This technical aspect would be of particular relevance if the Ih channels in neonatal rat MNs
are abundant in the distal membrane, as has been reported for
hippocampal CA1 pyramidal neurons, where the density of
Ih in the most distal regions is
roughly sevenfold higher than in the somatic region (Magee
1998
). It is also possible that our experimental approach for
an as yet undetermined reason has lead to a selection of large MNs
compared with those of previous experimenters.
We observed that for hyperpolarizing steps to approximately 80 mV or
beyond, the sum of two exponentials was necessary to obtain good fits
to Ih activation, with
fast being measured in hundreds of
milliseconds and
slow in seconds.
Double-exponential activation of Ih in
the neonatal rat MNs may indicate that two kinetically distinct
Ih channel populations are present in
these neurons (cf., Solomon and Nerbonne 1993
).
Interestingly, Takahashi (1990a)
reported that a single,
slow time constant was sufficient to describe the activation of
Ih. This apparent discrepancy can be
resolved if it is assumed that the two kinetically distinct Ih channel populations are spatially
segregated, so that the slow channels are concentrated proximally while
the fast channels mainly localize to the distal dendrites that were
presumably better conserved in our experiments (see above).
5-HT modulation of Ih
It has been reported previously that 5-HT increases a
conductance very similar to Gh in
spinal MNs (Takahashi and Berger 1990), but the
biophysical nature of this effect was not determined. We found that
this enhancement consists of a depolarizing shift in the activation
curve for Ih, amounting to 7 mV on
average. In contrast, 5-HT caused no systematic change in
Gmax. Results from other MN types have
been mixed. In adult rat facial MNs, 5-HT depolarizes V
without changing the maximum tail current amplitude of
Ih, i.e., similar to our findings
(Larkman and Kelly 1992
). On the other hand, 5-HT
increases Ih in guinea pig trigeminal MNs without shifting the (normalized) activation curve, suggesting that
this enhancement is related to an increase in
Gmax (Hsiao et al.
1997
). Apparently, the biophysical mechanism underlying the
5-HT enhancement of Ih in mammalian
MNs is heterogeneous.
In our experiments, 5-HT shortened both the fast and the slow time
constant. However, slow was clearly stronger
affected that
fast (Fig. 9, compare
E with F). In combination with the considerations
in the previous section, this observation leads us to suggest that
neonatal rat spinal rat MNs may be endowed with two kinetically,
spatially and pharmacologically distinct Ih channel populations. One of these
populations predominates in the soma and proximal dendrites, activates
slowly on hyperpolarization, and is strongly enhanced by 5-HT. The
second population, although also represented proximally, predominates
in the distal dendrites, activates quickly, and is more moderately
enhanced by 5-HT. More experiments are necessary to further
substantiate this idea.
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ACKNOWLEDGMENTS |
---|
We thank M. E. Denton and B. Johnson for participating in initial experiments in this study.
This work was supported by the NOVO Foundation and the Danish Medical Research Council.
Present address of O. Kjaerulff: The Nobel Institute for Neurophysiology, Dept. of Neuroscience, The Karolinska Institute, S-171 77 Stockholm, Sweden (E-mail: OleKjaerulff{at}neuro.ki.se).
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FOOTNOTES |
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Address for reprint requests: O. Kiehn, Section of Neurophysiology, Dept. of Medical Physiology, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen, Denmark (E-mail: O.Kiehn{at}mfi.ku.dk).
Received 19 June 2000; accepted in final form 3 October 2000.
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REFERENCES |
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