Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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ABSTRACT |
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Bennett, David J.,
Yunru Li, and
Merek Siu.
Plateau Potentials in Sacrocaudal Motoneurons of Chronic Spinal
Rats, Recorded In Vitro.
J. Neurophysiol. 86: 1955-1971, 2001.
Intracellular
recordings were made from sacrocaudal tail motoneurons of acute and
chronic spinal rats to examine whether plateau potentials contribute to
spasticity associated with chronic injury. The spinal cord was
transected at the S2 level, causing, over time,
exaggerated long-lasting reflexes (hyperreflexia) associated with a
general spasticity syndrome in the tail muscles of chronic spinal rats
(1-5 mo postinjury). The whole sacrocaudal spinal cord of chronic or
acute spinal rats was removed and maintained in vitro in normal
artificial cerebral spinal fluid (ACSF). Hyperreflexia in chronic
spinal rats was verified by recording the long-lasting ventral root
responses to dorsal root stimulation in vitro. The intrinsic properties
of sacrocaudal motoneurons were studied using intracellular injections
of slow triangular current ramps or graded current pulses. In
chronic spinal rats, the current injection triggered
sustained firing and an associated sustained depolarization (plateau potential; 34/35 cells; mean, 5.5 mV; duration >5
s; normal ACSF). The threshold for plateau initiation was low and usually corresponded to an acceleration in the membrane potential just
before recruitment. After recruitment and plateau activation, the
firing rate changed linearly with current during the slow ramps [63%
of cells had a linear frequency-current (F-I) relation] despite the presence of the plateau. The persistent inward
current (IPIC) producing the
plateau and sustained firing was estimated to be on average 0.8 nA as
determined by the reduction in injected current needed to stop the
sustained firing [I =
0.8 ± 0.6 (SD) nA], compared with the current needed to start firing
(I = 1.7 ± 1.5 nA; 47% reduction). In
motoneurons of acute spinal rats, plateaus were rarely seen
(3/22), although they could be made to occur with bath application of
serotonin. In motoneurons of chronic spinal rats there were
no significant changes in the mean passive input resistance,
rheobase or amplitude of the spike afterhyperpolarization (AHP) as
compared with acute spinal rats. However, there were significant
increases in AHP duration and initial firing rate at recruitment and
decreases in minimum firing rate and F-I slope. We suggest
that the higher initial firing rate resulted from the plateau
activation at recruitment and the lower F-I slope resulted from an increase in active conductance during firing, due to
IPIC. Brief dorsal root stimulation
also triggered a plateau and sustained discharge (long-lasting
reflexes; 2-5 s) in motoneurons of chronic (but not acute) spinal
rats. When the plateau was eliminated by a hyperpolarizing current
bias, the reflex response was significantly shortened (to 1 s).
Thus plateaus contributed substantially to the long-lasting reflexes in
vitro and therefore should contribute significantly to the
corresponding exaggerated reflexes and spasticity in awake chronic
spinal rats.
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INTRODUCTION |
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Following spinal cord injury, exaggerated
reflexes and muscle tone often emerge that contribute to a general
spasticity syndrome in humans (Ashby and McCrea 1987;
Kuhn and Macht 1948
; Noth 1991
; Young 1994
) and animals (Ashby and McCrea
1987
; Bennett et al. 1999a
; Heckman
1994
; Taylor et al. 1997
). A central complaint of patients with spasticity involves intense muscle contractions, lasting for many seconds, that are triggered by numerous stimuli. These
are related to various long-lasting reflexes that have been described experimentally in humans and animals with injury, including: spastic stretch reflexes (Burke et al. 1970
;
Powers et al. 1989
; Thilmann et al.
1991
), cutaneous/flexor-afferent reflexes (Bennett et
al. 1999a
; Kuhn and Macht 1948
;
Remy-Neris et al. 1999
), general oligosynaptic reflexes
(Hultborn and Malmsten 1983a
,b
; Mailis and Ashby
1990
), and radiating muscle spasms (Kuhn and Macht
1948
). Such long-lasting reflexes may have numerous causes
including loss of inhibition from descending and segmental pathways
(Cavallari and Pettersson 1989
; Heckman
1994
; Mailis and Ashby 1990
; Thompson et
al. 1998
), neuronal sprouting (Krenz and Weaver
1998
), and direct changes in intrinsic properties of spinal
neurons (Eken et al. 1989
).
The possibility that the intrinsic properties of spinal motoneurons
change with spinal cord injury is consistent with data from motor unit
firing in humans and animals after injury, including sustained poorly
modulated discharges (Gorassini et al. 1999a; Thomas and Ross 1997
), unusually low minimum firing
rates (Carp et al. 1991
; Powers and Rymer
1988
; Thomas and Ross 1997
), and generally
inefficient control of firing rate in force production (Blaschak et al. 1988
; Wiegner et al.
1993
). The objective of the present paper was thus to
investigate whether long-lasting reflexes that emerge after injury
result, in part, from changes in intrinsic excitability of motoneurons,
such that relatively uncontrolled firing can be triggered by a brief
stimulation (e.g., due to plateau potentials) (Russo and
Hounsgaard 1999
).
Although motoneurons usually fire in proportion to the net excitatory
input, their response can also be altered substantially by numerous
voltage-dependent currents intrinsic to the motoneuron membrane
(Binder et al. 1996; Rekling et al. 2000
;
Russo and Hounsgaard 1999
). For example,
voltage-dependent persistent inward currents (IPIC) can at times be activated by a
brief stimulus and regeneratively cause sustained depolarizations
(i.e., plateau potentials; abbreviated plateaus) and firing
(Bennett et al. 1998a
,b
; Hounsgaard and Kiehn 1989
; Hounsgaard et al. 1988
; Lee and
Heckman 1998a
,b
; Schwindt and Crill 1984
).
Persistent inward currents are likely present in most motoneurons but
are only manifested as plateaus when they are either directly
facilitated or uncovered by reducing opposing outward currents (e.g.,
Ca2+-dependent K+ current),
both of which may occur with extrinsically administered serotonin
(5-HT) (Hultborn and Kiehn 1992
; Russo and
Hounsgaard 1999
). The IPIC
responsible for plateaus is often mediated by L-type calcium channels
(Hounsgaard and Kiehn 1989
), although any relatively
long-lasting voltage-gated inward current could also be involved,
including persistent sodium currents (Schwindt and Crill
1995
) and ligand-gated currents from
N-methyl-D-aspartate (NMDA) receptors
(Guertin and Hounsgaard 1998
; Hochman et al. 1994
; Kiehn et al. 1996
).
The existence of plateaus in motoneurons appears to normally require
monoaminergic facilitation from the brain stem [5-HT, norepinephrine
(NE)]. That is, plateaus occur in brain-stem-intact decerebrate cats, are largely eliminated by acute spinalization, and
return with application of monoaminergic drugs (Conway et al.
1988; Hounsgaard et al. 1988
). As there are few
sources of 5-HT or NE within the spinal cord below a chronic spinal
transection (except perhaps related to the autonomic system)
(McNicholas et al. 1980
; see also Newton and
Hamill 1988
), it is unlikely that plateaus mediated by
monoamines could re-emerge with long-term complete spinal cord
transection. However, because the inward currents
(IPIC) are ubiquitous, as discussed in
the preceding text, and IPIC and
plateaus are facilitated by various other neuromodulators (acetylcholine, substance P and glutamate; via metabotropic receptors) (Russo and Hounsgaard 1999
; Russo et al.
1997
), plateaus may still play a role in chronic injury.
Indeed, the similarity of the long-lasting reflexes in the chronic
spinal cat to the tonic stretch reflex in the decerebrate cat led
Hultborn's group to propose that both are mediated by plateaus on
motoneurons (Eken et al. 1989
; Nielsen and
Hultborn 1993
). Their preliminary evidence, from motoneurons recorded in two chronic spinal cats, supports this hypothesis.
In the present study, we have made intracellular recordings from
motoneurons of chronic spinal rats to test for the presence of
plateaus. The spinal cord transection was made at the sacral level,
which, within a month, leads to pronounced spasticity in the tail
muscles while sparing bladder and locomotor function (Bennett et
al. 1999a). In these chronic spinal rats, there was often
sustained tail motor unit firing, suggestive of the involvement of
plateaus (Bennett et al. 2001
). This preparation has the
unique advantage that the affected adult sacrocaudal spinal cord is
small enough to survive when acutely explanted and studied in vitro (whole adult cord) (Bennett et al. 1999b
; Long et
al. 1988
; Bennett and Li, in preparation). Ventral root
recordings were used to verify that the spastic reflex behavior
persisted in vitro (Bennett et al. 1999b
), and
intracellular recordings were made from identified motoneurons. As
anticipated, we found that with chronic spinal cord injury, the
motoneurons recovered their ability to exhibit plateau behavior. These
plateaus could be triggered by dorsal root stimulation, and they
markedly prolonged reflex responses, thus playing a major role in the
long-lasting spastic reflexes seen after chronic spinal cord injury.
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METHODS |
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Intracellular recordings were made from sacrocaudal motoneurons
of adult female Sprague-Dawley rats (age: 1.5-7 mo; 200-800 g)
following sacral spinal cord injury. The spinal cord was transected at
the S2 sacral spinal level either acutely
(acute spinal condition; n = 29 cells,
n = 14 rats) or 1 mo prior to the experiment
(chronic spinal, 1-5 mo post lesion, n = 35 cells, n = 14 rats), as described in Bennett et
al. (1999a)
. In the latter group, only rats with clear
spasticity symptoms were used (rating 3-5 in Table 1 of Bennett
et al. 1999a
). Recordings were made while the whole sacrocaudal spinal cord was maintained in vitro (Bennett et al.
1999b
; Bennett and Li, in preparation). All procedures were
approved by a local animal-welfare committee.
Surgery
The in vitro whole sacrocaudal adult rat preparation has been
described previously (Bennett et al. 1999b; Long
et al. 1988
; Bennett and Li, in preparation) and is only
briefly summarized here. Normal and chronic spinal rats were deeply
anesthetized with urethan (0.18 mg/100 g; maximum of 0.45 mg per rat
for rats 250 g), and the spinal cord caudal to the
T12 vertebrae was transferred to a dissection
dish containing oxygenated modified artificial cerebral spinal fluid
(mACSF) at room temperature (20-21°C). In the dissection dish, a
transection was made at the upper S2 level with
fine iridectomy scissors, just rostral to the original transection site
in the chronic spinal rats. In normal rats, this
S2 transection was also made and served to
provide an acute spinal lesion. Following a 1-h resting period in
mACSF, the cord was transferred to a recording chamber, where it was
submerged in normal ACSF flowing at 6 ml/min and maintained at 25°C.
The cord was supported on a nappy paper mesh and secured by passing
insect pins through lateral vasculature and connective tissue and into
a silicone elastomer (Sylgard) base below the nappy paper.
Solutions
The normal ACSF had the following composition (in mM): 122 NaCl,
24 NaHCO3, 3 KCl, 2.5 CaCl2, 1 MgSO4, and 12 glucose in distilled water, bubbled with 95%
O2-5% CO2 and pH 7.4. mACSF was used during dissection and recovery to prevent excitotoxic
injury. Initially mACSF consisted of ACSF with NaCl replaced by sucrose
at equal osmolarity [0 NaCl, 213.6 mM sucrose (295 mOsm)]
(Aghajanian and Rasmussen 1989). Later we noticed that
sucrose tended to toughen the pia, making subsequent intracellular
penetrations more difficult (e.g., dimpling occurred). We thus changed
to a mACSF based on kynurenic acid, a broad spectrum antagonist of
glutamate transmission. This mACSF composition was (in mM) 118 NaCl, 24 NaHCO3, 3 KCl, 1.5 CaCl2,
1.3 MgSO4, 25 glucose, 1.4 NaH2PO4, 5 MgCl2, and 1 kynurenic acid (McQuiston and
Madison 1999
). Regardless of the mACSF used, recordings were
made in the same normal ACSF, and qualitatively similar results were
obtained, although in general we noticed that the cords prepared with
kynurenic-based mACSF were healthier, with larger reflexes and more
pronounced plateaus. 5-HT (10-100 µM) was at times added to the ACSF
in some acute spinal preparations after the main recordings in normal ACSF.
Intracellular and root recording
The long ventral and dorsal roots (at least sacral
S3, S4, and caudal
Ca1) were mounted on silver-chloride wires
supported above the recording chamber and covered in grease. Brief
dorsal root stimulations were used to evoke long-lasting reflexes in spastic rats, and also to confirm the viability of the preparation (Bennett et al. 1999b). Reflex responses were recorded
in the ventral roots and intracellularly on the motoneurons. Ventral roots were also stimulated to antidromically activate motoneurons for identification.
All segments of the in vitro sacrocaudal spinal cord could produce
ventral root reflexes and thus had viable motoneurons (see also:
Bennett and Li, in preparation). However, we focused on motoneurons in
the caudal Ca1 and sacral
S4 regions because this is the smallest, and
presumably best oxygenated, portion of the cord in vitro, ventral root
reflexes were largest and remained viable for the longest periods (>5
h and 18 h), and motoneurons in this region innervate only the tail
muscles as opposed to bladder and pelvic regions served by higher
sacral segments (Bennett et al. 1999a
; Steers
1994
).
Sharp intracellular electrodes were made from thick-walled glass
capillary tubing (Warner, GC150F-10, 1.5 mm OD) with a Narishige puller
(PE-2), filled with 2 M potassium acetate and bevelled with a rotary
grinder (Sutter, BV-10) to give a final resistance of 50-100 M. An
Axoclamp2b intracellular amplifier (Axon Instruments) was used, either
in bridge or discontinuous current-clamp modes (DCC; 4-5 kHz
switching; all figures are from DCC recordings), with capacitance
maximally compensated. The electrode was advanced with a stepper-motor
micromanipulator (660, Kopf) while observing the electrode resistance
changes and antidromic ventral root field potentials. Final cell
penetration was achieved either by passing high-frequency current
(buzz) or making a fast step with a piezoelectric element (WPI) mounted
on the tip of the Kopf manipulator. On penetration, antidromic spike
properties were measured. Only cells with >55 mV resting potential and
>60 mV spike amplitude were accepted for analysis. For approximate
classification, the injected current required to initiate firing during
a slow current ramp (0.5 nA/s; see following text; recruitment current)
was computed. All cells with a recruitment current less than the mean
(1.75 nA, mean of all acute and chronic cells) were considered low
recruitment threshold cells and the remainder high threshold. The
intracellular current and membrane potential was low-pass filtered at 6 kHz and sampled at 16 kHz with an Axoscope system (Axon Instruments).
Usually the cord was placed horizontally in the recording chamber with the ventral side upward, and the intracellular electrode was advanced vertically, perpendicular to the cord, directly into the motor nucleus. The disadvantage of this approach was that dimpling of the pia during penetration could damage underlying neurons.
An alternate longitudinal approach through a transverse cut
was used in some of the animals as follows. After removal of the cord
from the animal, it was transferred to a vibratome in mACSF and a
transverse cut was made at the S4
level. Then following the usual incubation in mACSF, the remaining
S4 and caudal cord was transferred to the
recording chamber and supported on a 30° ramp with the cut face
pointing up the ramp. The cord was not submerged as usual but covered
with nappy paper superfused with ACSF through a wick near the cut face
of the cord (Long et al. 1988). The intracellular
electrode was advanced into the cut face, longitudinally to the cord,
directly into the motor nucleus near the visible interface of the white
and gray matter (thus avoiding the pia). The main disadvantage of this
approach is that the transverse cut is an additional injury and the
reflexes recorded in the remaining S4 and caudal
Ca1 roots were diminished. However, we did not
notice differences in the intracellular plateau properties of
motoneurons recorded with the perpendicular and longitudinal
approaches, and cells from both methods have been included in the analysis.
Analysis of plateaus
Intracellular current pulses and slow triangular current ramps
(0.5 nA/s standard, 0.4-3 nA/s range) were used to evoke
voltage-dependent plateaus as described previously (Bennett et
al. 1998a; Hounsgaard et al. 1988
). During
the current ramps, the IPIC producing
the plateau, and sustaining the firing, was estimated from the
difference in injected current at recruitment, compared with
de-recruitment (
I; see RESULTS). For
computing the average
I for each cell, we only used
responses from small, slow current ramps (0.5 nA/s as in Fig.
5A, unless there was a late, high-threshold plateau), optimized to avoid firing rate adaptation, as described in
RESULTS. Also, to avoid any interactions between successive
ramps (e.g., warmup) (Bennett et al. 1998b
), we
separated ramps by
10 s and removed any depolarizing current between ramps.
The size of the plateau was estimated from the afterpotential seen
during current ramps (V in Fig. 5A, see
RESULTS). This was quantified by subtracting the potential
at the end of firing (just before the last spike) from the potential at
a matched current during the ascending ramp before the
plateau and firing started. Because the potential can rise no higher
than the firing level (ignoring the spike), this afterpotential
measure,
V, underestimates the plateaus size that might
be seen if spiking were not present. In cells that stopped firing
early, and at higher currents than at recruitment (i.e., without
plateau; acute spinal), the afterpotential was still computed but by
comparing the potential just before recruitment to that at a matched
current level after de-recruitment (see RESULTS). Some
cells were recorded in bridge mode (not shown) instead of the usual DCC
mode, and so the potential changes were corrupted by electrode
rectification. However, by comparing the potential at matched currents
on the ascending and descending current ramps, this electrode
rectification problem was usually avoided (with a few exceptions, where
rectification was sufficiently bad that we did not compute the
afterpotential, thus missing the points in Fig. 4C).
To directly test the role of voltage-dependent plateaus on spastic
reflexes, we also studied the amplitude and duration of excitatory
postsynaptic potentials (EPSPs) evoked by dorsal root stimulation while
changing the background depolarization of the cell with intracellular
current bias (Bennett et al. 1998a).
The intracellular records were analyzed with Axoscope (Axon Instruments), Sigmaplot (Jandel Scientific) and a custom Linux-based program (G. R. Detillieux, Winnipeg). In the text and figures means ± SDs are shown. Statistical differences were assessed with a Student's t-test at the 95% confidence level (P < 0.05).
Histology
The spinal cords of additional rats (6) were sectioned and
stained to examine the size and anatomy of the sacrocaudal cord. The
animals were anesthetized and perfused with 4% paraformaldehyde as in
Bennett et al. (1999a). The spinal cord was removed,
serially dehydrated in ethanol, imbedded in paraffin for 7 days, and
then sectioned at 10 µm on a microtome. Tissue was stained with
silver nitrate (Bielschowsky method) and cresyl violet
(Kiernan 1990
).
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RESULTS |
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Anatomy of the sacrocaudal ventral horn and motoneuron properties recorded in vitro
The small diameter of the sacrocaudal spinal cord (Fig.
1, A and C) was a
major factor that enabled it to survive whole (unsliced) in vitro when
it was acutely isolated from normal or chronic spinal adult rats
because oxygen and nutrients only diffuse ~300 µm into tissue
(Nicholson and Hounsgaard 1983). When intracellular
recordings were made by passing the microelectrode into the cord
directly through the ventral surface (perpendicular approach; see
METHODS), motoneurons were encountered at between 50 and
150 µm from the surface (>50 µm at Ca1;
>100 µm at S4). Despite the small size of the
sacrocaudal cord, the sacrocaudal motoneuron cell bodies were not
significantly smaller than motoneurons in the lumbar enlargement (Fig.
1, B and D; average sacrocaudal motoneuron
diameter: 35 ± 6 µm, n = 6 rats) and had
similar basic electrical properties (Table
1).
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In acute and chronic spinal rats, we recorded from motoneurons
with a moderately wide range of input resistance
(Rin; 9-25 M), presumably relating
to cell size (Binder et al. 1996
). We found that
Rin covaried with other cell
properties, including afterhyperpolarization (AHP) duration,
recruitment threshold, and rheobase (as with other motoneurons)
(Binder et al. 1996
). Despite the marked differences in
the ability of cells to maintain plateaus in acute and chronic spinal
rats (see following text), only slight (non-significant) differences
were seen between these two populations in
Rin, AHP amplitude, and rheobase
(Table 1), consistent with previous studies in spinal animals
(Baker and Chandler 1987a
; Cope et al.
1986
; Gustafsson et al. 1982
; Hochman and
McCrea 1994
). There was a significant increase in AHP duration in chronic spinal rat motoneurons (Table 1).
Motoneurons in acute spinal rats lack plateaus
We studied the firing behavior during intracellular current injection in 22 motoneurons from the acutely isolated sacrocaudal cord of normal rats (acute spinal condition) in normal ACSF. In most of these cells, there was no evidence of plateau activation (19/22). A brief current pulse did not trigger a sustained depolarization (Fig. 2E). Usually cells responded proportionally during slow triangular ramp current injections with the membrane potential and firing rate increasing linearly during the upward portion of the ramp and decreasing symmetrically during the downward portion of the ramp (see triangular reference lines drawn below potential in Fig. 2A and the overlapping frequency-current plots during the upward and downward ramps in Fig. 2C). Firing usually stopped at about the same current as where it started (Fig. 2, A and C). Overall, 59% of cells responded linearly like in Fig. 2A, and we have classified these cells as type 1 cells (i.e., linear firing and no plateau).
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In another 36% of acute spinal rat motoneurons, firing slowed
substantially on the downward portion of the slow current ramps (0.5 nA/s standard speed; Fig. 3A),
and the frequency-current plots showed a clockwise shape and an early
de-recruitment, characteristic of cells with late firing rate
adaptation (Fig. 3C) (Kernell and Monster
1982). We refer to these cells as type 2 cells (rate
adapting). Larger (or faster) ramps increased the incidence of rate
adaptation, likely due to the higher firing rates achieved (not shown)
(Kernell and Monster 1982
).
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Motoneurons of spastic chronic spinal rats have plateaus
Following the S2 sacral spinal cord
transection, exaggerated long-lasting reflexes associated with a
general spasticity syndrome developed in the tail musculature within a
month, as in Bennett et al. (1999a). Motoneurons
(n = 35) were recorded from the isolated sacrocaudal
cord of these spastic rats
1 mo after injury (chronic spinal;
recordings in normal ACSF). Prior to each experiment, the hyperreflexia
was verified in vitro by recording the associated long-lasting ventral
root reflexes (Bennett et al. 1999b
; Bennett and Li, in preparation).
When a brief intracellular current pulse was injected into motoneurons
of chronic spinal rats, a sustained depolarization (afterpotential) and
afterdischarge was produced (Fig. 2F; i.e., plateau
potential), lasting many seconds, and not seen in acute spinal
rats (Fig. 2E). Symmetrical triangular current ramps also triggered a sustained afterdischarge and afterpotential
(V) due to the plateau activation and a very
asymmetrical response in relation to the current (Figs. 2B
and 3B; 34/35 cells). That is, following recruitment of
firing at a particular current on an upward current ramp (e.g., left
vertical dashed line in Fig. 2B, 0.4 nA), de-recruitment
only occurred when the current was reduced to a substantially lower
level (right vertical dashed line;
I =
1 nA). Thus
firing stopped much later than expected (referred to as
self-sustained firing; also see Fig. 3B)
(Bennett et al. 1998a
). The interpretation of this
self-sustained firing is that the inward current
IPIC, associated with the plateau, was
activated during the ascending current ramp, and then
IPIC effectively provided a
depolarizing bias current, allowing the injected current to be
substantially reduced before firing stopped (approximately:
I =
IPIC; see
following text). The reduction in current at de-recruitment compared
with recruitment,
I, thus provides a measure of the inward current IPIC that helped to
sustain the firing.
Note that the term "plateau" can be somewhat misleading during
current ramps because the depolarizing inward current
IPIC combines with the injected
current to produce the final response, and the potential is
not locked at a fixed level (see details in Bennett
et al. 1998a).
Quantification of plateau and comparison in acute and chronic spinal rats
As mentioned in the preceding text, in acute spinal rats
there was little tendency for plateaus, and de-recruitment occurred at
or above the current for recruitment, as summarized in Fig. 4A for all motoneurons
(I 0;
I not significantly different from 0, Fig. 4B). In three acute spinal rat motoneurons,
there was a drop in injected current at de-recruitment
(
I < 0), indicating self-sustained firing and
plateaus (3/22 cells). In contrast, in most chronic spinal motoneurons
(34/35) the current dropped substantially at de-recruitment compared
with recruitment (i.e.,
I was significantly less than 0;
IPIC =
I =
0.8 ± 0.6 nA). The average estimated IPIC is
summarized for acute and chronic spinal rats in Fig. 4B,
which indicates a very significant (1.0 nA) increase in
IPIC with chronic injury. Note that in
chronic spinal rats both neurons with a low (<1.75 nA) and high
(>1.75 nA) recruitment threshold had plateaus, as indicated by
I. Further, there was no significant difference between
the plateaus (
I) in low- and high-threshold neurons
(
I =
0.7 ± 0.5 nA compared with
1.1 ± 0.9 nA), and there was considerable scatter in
I (regression value r = 0.2 in Fig. 4A).
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The depolarization produced by the plateau was most easily seen at the
end of firing, and we have referred to this as the afterpotential
(V in Figs. 2B and 3B; see
METHODS). In chronic spinal rats, the afterpotential, and
thus plateau estimate, was 5-10 mV (5.1 ± 6.0 mV) and was
significantly greater than in acute spinal rats (Fig. 4D).
In acute spinal rats the afterpotential was not significantly different
from zero.
In 12 motoneurons recorded in acute spinal rats, the ability of 5-HT to
facilitate plateaus was studied (as in Hounsgaard and Kiehn
1989) (5 cells studied before and after 5-HT and 7 with 5-HT
only). We used a concentration between 10 and 100 µM, which produced
sustained activation of the ventral roots in response to dorsal root
stimulation (not shown). The 5-HT usually depolarized cells, reduced
the AHP and lowered their firing threshold current (by 2.2 nA, on
average). Subsequent ramp current injections showed plateaus, although
the estimated plateau current (IPIC =
I; Fig. 4B) and afterpotential
(
V; Fig. 4D) were not as large as in chronic
spinal rats.
Characteristics of plateaus in chronic spinal rats
LOW-THRESHOLD PLATEAUS, INITIATED BEFORE RECRUITMENT (TYPE 3 CELLS).
In the majority of chronic spinal rat motoneurons (63%, classified as
type 3 firing behavior), the plateau activation started before or simultaneous to recruitment during the current ramp, and
firing rate acceleration was either not seen (Fig. 3D) or only seen in the first few spikes (Figs.
5, A and B, left
arrows; and Fig. 6D). In these
cells, during the slow current ramp, the membrane potential increased
linearly until it was within ~5 mV of the firing threshold, after
which there was a gradual acceleration in the depolarization (lasting
~0.5-1.0 s; double arrow in Fig. 5A). This acceleration
marked the onset of the plateau because the current could be reduced at
any time afterward and there was still a sustained depolarization and
firing (self-sustained firing). The most dramatic examples of this
occurred with small current ramps, where the upward current just
activated the plateau (at acceleration in potential, Fig.
5C, arrow), and then immediately the downward current ramp
started just prior to recruitment. In this case, the plateau
continued to depolarize the cell and produce sustained firing even when
the current was reduced by 1 nA [i.e., 1 nA sustained
IPIC; in acute spinal rats a
comparable small current ramp did not evoke any firing or a plateau
(not shown)].
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FIRING RATE ADAPTATION (TYPE 2) AND EFFECT OF RAMP SPEED AND
AMPLITUDE.
In 17% of motoneurons of chronic spinal rats, there was firing rate
adaption (slowing of firing; type 2 cells), with clockwise hysteresis in the frequency-current plots, and in these cells, the
self-sustained firing was weaker than average (i.e., plateau, I, not as pronounced). As in acute spinal rats, this
slowing of firing was accentuated with large or fast ramps. Indeed even cells with a clear plateau and little firing rate adaptation during slow ramps showed some slowing of firing, with less afterdischarge (
I) and less afterpotential (
V) during
faster or larger ramps (Fig. 5, B compared with
A).
LATE-ACTIVATED PLATEAUS (TYPE 4).
In some motoneurons (20%; as in Figs. 6E and 2B;
classified as type 4 cells), although firing initially
increased linearly with current, there was a late acceleration in
firing, which was assumed to mark the main activation of
IPIC and the plateau (Fig. 2B, double arrows), as has been described in cat motoneurons
(Bennett et al. 1998a; Hounsgaard et al.
1988
). In these cells, there was a corresponding
counterclockwise hysteresis loop in the F-I plot (Figs.
6E and 2D) and a marked drop in injected current
at de-recruitment compared with recruitment (
I < 0, i.e., large sustained IPIC). All cells
with this late plateau activation after recruitment were low
recruitment threshold, presumably small, cells (7/35 cells; 0.12- to
2.5-nA recruitment thresholds).
PARTIAL DEACTIVATION OF PLATEAU DURING FIRING?
In cells with a clear late acceleration in firing (type 4), which we
have previously taken to mark the main activation of the plateau (Figs.
2B and 6E) (Bennett et al. 1998a),
we were surprised to find that self-sustained firing (plateaus) could be evoked even when the current ramp was kept below the
point where the frequency acceleration occurred [below main plateau threshold; compare Fig. 6, E and F, responses
from the same cell; contrast to Fig. 2 of Bennett et al. (1998b)
].
However, on closer inspection, we found that there was also an
acceleration in potential just prior to recruitment (Fig.
2B, single arrow), and this was likely associated with an
early plateau activation. Thus possibly there were two distinct plateau
activations, one at recruitment and a second one later. We, however,
favor an alternate interpretation based on two observations:
1) during the current ramps, the accumulated effect of the
AHPs tended to hyperpolarize the membrane following recruitment (i.e.,
mean potential between spikes; e.g., Fig. 5B) compared with
the potential just before recruitment. 2) Following the
firing produced by a current pulse, there was, at times, a very slow
hyperpolarization (sAHP), and pause in firing, before the
afterdischarge (plateau) continued [Fig. 8G described
later; see similar effects in Figs. 7 and 8 of Russo and
Hounsgaard (1996)
]. Thus while the plateau may be partly
activated before recruitment, with moderate firing rates the
accumulated hyperpolarization from AHPs may have prevented further
activation or even caused partial deactivation of the plateau. Only
when the cell was further depolarized by the increasing current ramp
was the plateau fully activated (late acceleration, at high firing rate).
VERY SLOW FIRING.
When the current was reduced during a plateau and firing slowed,
surprisingly long intervals often occurred between spikes (1 s),
often many times the AHP duration and the related theoretical maximum
interval (Figs. 5A and 8H) (Kernell
1999
). In these cases, the plateau was just at threshold to
deactivate, and thus perhaps each AHP transiently deactivated the
plateau, and the plateau was then only slowly reactivated to produce a
subsequent spike at a long interval. Indeed this slow rise before each
spike was similar to the slow rise in potential when the
plateau was first activated just before recruitment (Fig. 5,
A and B). This phenomenon was not transient
because slow firing could continue for many seconds when pulses were
used to evoke plateaus (Fig. 8H, described later). The
possibility of slow firing generated by voltage-dependent inward
currents with slow kinetics near firing threshold has been discussed by
others (e.g., Carp et al. 1991
; Hodgkin
1948
; Kernell 1999
).
Summary of firing behavior in acute and chronic spinal rat motoneurons
F-I TYPES. The distribution of cells between the four types of firing behaviors described in the preceding text (types 1-4) is summarized in Fig. 7A; the majority of the acute and chronic spinal cells behaved linearly, as type 1 and 3 cells, respectively. To summarize the type definitions: type 1 cells had a linear F-I relation, with overlapping frequency points for the ascending and descending current ramps, but no self-sustained firing (no plateau, all from acute spinal rats, Fig. 6A; see also Fig. 2C). Type 2 cells showed firing rate adaptation and usually no plateau (Fig. 6B; see also Fig. 3C). Some cells in chronic spinal rats were of this type, and they had only weak self-sustained firing with rate adaptation countering the plateau. Type 3 cells had linear characteristics as in type 1 but also showed self-sustained firing (plateau). Remarkably the firing rate remained on the linear F-I regression line even when the current was brought well below the recruitment current on the descending ramp (Figs. 6, C and D, and 5A). Some of Type 3 cells started firing directly on the F-I linear regression line (thin line in Fig. 6C), and we assume that the plateau was fully activated at recruitment. Others included in this type had a few accelerating spikes just after recruitment below the linear regression for the F-I relation (Fig. 6D), which we supposed indicated the early activation of the plateau, continuing for a second after recruitment (see preceding text). Type 4 cells had a late frequency acceleration, a few seconds after recruitment, followed by self-sustained firing (high-threshold plateau; Figs. 6E and 2D). As mentioned previously, type 4 cells behaved linearly, as with type 3 cells, when the current was kept below the level for a late frequency acceleration (Fig. 6F). With the exception of type 4 cells, which were purely small, low recruitment-threshold cells, the other three types included both cells of low and high recruitment threshold (see preceding text).
|
INITIAL AND FINAL RATES.
In previous motor unit experiments, motoneurons with presumed plateaus
were found to have significantly higher firing rates at recruitment,
compared with at de-recruitment (Gorassini et al. 1998,
2001a
), and this was thought to be due to an early plateau activation at recruitment that boosted the initial firing rate above
the minimum rate. In chronic spinal rats with plateaus, this was also
found to be the case, with recruitment at ~8 Hz, and de-recruitment
at half that value (significant difference; Fig. 7B). In
contrast, the firing rates at recruitment and de-recruitment were not
significantly different in motoneurons of acute spinal rats (~8 Hz;
Fig. 7B). The firing rate achieved at de-recruitment (minimum rate) in chronic spinal rats was significantly
lower than in acute spinal rats, and, at times, as low as 1 Hz (see preceding text).
LOWER F-I SLOPE AND SLOWER STEADY FIRING IN CHRONIC SPINAL RATS. Overall, motoneurons of chronic spinal rats fired at lower rates than in acute spinal rats not just lower minimum rates. To further quantify this, we have fit a linear regression to the F-I ramp responses. The first instantaneous firing rate point that fell on the F-I regression line after recruitment was measured and referred to as the initial steady firing rate. This measure is not necessarily the instantaneous rate at recruitment because there were often overshoots (early adaptation) or undershoots in firing before the rate was modulated linearly with current (Fig. 6D). The initial steady firing rate was found to be significantly lower in chronic spinal rats than in acute spinal rats (Fig. 7C).
The slope of the F-I linear regressions was also significantly lower in chronic spinal rats (Fig. 7D). This lower slope may be explained by an increased conductance provided by IPIC in chronic spinal rats (see DISCUSSION). Thus while the plateau may have enabled recruitment at twice the minimum rate, its associated conductance change may have made it more difficult to produce further increases in rate (i.e., lower F-I gain).Voltage dependence of plateaus evoked with brief pulses
Although a brief current pulse in some chronic spinal cells could
be readily used to evoke a plateau from rest (Fig. 2F), in
others, a plateau could only be evoked by a pulse when there was an
appropriate steady depolarizing bias current. We found that for a given
cell, the parameters for producing a plateau from a pulse could be
estimated from the ramp response as follows: first, the plateau
threshold current was estimated, which was usually at the acceleration
in potential just prior to firing (1.5 nA in Fig.
8, A and B, see
arrow). Second, the plateau current was estimated
(IPIC = I, which is
0.6 nA in Fig. 8B). Finally, a bias current was chosen that
when added to the plateau current, exceeded the threshold current for
plateau activation (e.g., bias +0.6 > 1.5 nA), thus allowing the
plateau to remain activated after the pulse. By varying the bias level,
we have been able to verify this recipe for plateau activation, as
shown for a typical cell in Fig. 8, C-H. With no bias
current (Fig. 8, C-E), a pulse could not produce a
sustained afterdischarge, regardless of the pulse height, presumably
because the plateau current, IPIC, was only 0.6 nA, compared with the plateau threshold of 1.5 nA. [There was, however, evidence that the plateau was activated during the pulse
because a delayed onset in firing and slow rise in potential could be
seen when the plateau threshold current was reached (Fig. 8D, arrow; see details in Fig.
9).] When the bias current was increased
to 0.7 nA (in Fig. 8F), a pulse evoked a sustained
depolarization that outlasted the pulse (afterpotential; Fig.
8F, right arrow). This potential was slowly decrementing,
suggesting that the bias current was set just below the plateau
threshold (0.7 + 0.6 < 1.5 nA). A larger bias current (0.9 nA)
produced more robust plateau activations (0.9 + 0.6 = 1.5 nA),
with a greater afterpotential and afterdischarge (Fig. 8G).
|
|
Interestingly, following the pulse, a hyperpolarization and pause in
firing often occurred (sAHP; Fig. 8, G and H),
and this was followed by an acceleration in firing as the plateau
continued. Probably this occurred because the accumulated effect of
AHPs (sAHP) during the pulse produced a partial deactivation of the plateau, which in this case was only reversed once the pulse ended (see
DISCUSSION). A related observation is that the plateau
caused a greater afterdischarge when it was evoked by a lower amplitude pulse (compare Fig. 8, F and H). Thus smaller
pulses, which produced less firing during the pulse, caused less
plateau inactivation and a greater afterdischarge, presumably by
producing less sAHP (as in Fig. 6 of Russo and Hounsgaard
1996).
Slow and fast plateau activation
The plateau activation speed increased systematically with the depolarizing pulse amplitude (Fig. 9, bias current fixed at 0.75 nA). With a minimum pulse size (0.34 nA in Fig. 9A), the membrane potential only very slowly depolarized as the plateau was being activated, and firing was substantially delayed (4 s in this case). Larger pulses produced faster plateau activation and less delay in recruitment (the latter summarized in Fig. 9C, solid circles; note that the time scale is faster in Fig. 10B, compared with 9A). With the largest pulses, the plateau was activated simultaneously with recruitment (Figs. 9B, bottom, and 8G). In these cases, the presence of the plateau had to be verified by looking for a discharge after the pulse (not shown in fast time scale in Fig. 9, but see Fig. 8G).
|
In some cells, there was a delayed acceleration in firing associated with the plateau activation during the pulse (mentioned in the preceding text in relation to Fig. 2F), and the delay for this acceleration also depended on the pulse amplitude. To quantify this, we have computed the time to reach steady-state firing during pulses of different amplitude (Fig. 9B, small arrows). For larger amplitude pulses, the firing increased to its steady state value faster, as summarized in Fig. 9C. The cell shown in Fig. 9 had its plateau primarily activated before recruitment and then had about a 1.5-Hz increase in rate that resulted from the further plateau activation during firing (Fig. 9D).
Voltage-dependent long-lasting reflexes
Because our ultimate goal was to relate the presence of plateaus
to the exaggerated long-lasting reflexes seen with spasticity, we have
examined the reflex activation of the motoneurons by dorsal root
stimulation. In acute spinal rats, a brief dorsal root stimulation only
triggers a brief ventral root response (Bennett et al.
1999a,b
). In contrast, in chronic spinal rats, a brief dorsal
root stimulation triggers a sustained response in the ventral roots
with a duration similar to tail reflexes in awake spastic rats, of ~2
s (stimuli 1.5-10 times threshold) (Bennett et al.
1999a
,b
). Corresponding long-lasting reflex responses were seen
during intracellular recording in chronic spinal rat motoneurons in
response to the brief dorsal root stimulation, with an EPSP duration
ranging from 2 to 5 s at rest (see Fig. 10B; no
intracellular current bias, 2-s duration). The duration of this EPSP
was always substantially reduced (to ~1 s in Fig. 10C) by
hyperpolarizing the motoneuron with a steady bias current to eliminate
any effect of the plateau. Conversely, a depolarizing current bias
increased the duration of the reflex responses (EPSP; to 6 s in
Fig. 10A) as might be expected from Fig. 8. The duration of
the dorsal root stimulus could be as short as a single shock yet still
evoke the long-lasting voltage-dependent reflexes as just described
(not shown; see also ventral root reflexes) (Bennett et al.
1999b
; Bennett and Li, unpublished data). This voltage-dependent behavior suggests that plateaus were triggered by the
dorsal root stimulation and played a major role in amplifying and
prolonging the reflex responses in chronic spinal rats. However, the
plateau was not the only contributor to the sustained reflexes because
when the plateau was eliminated by hyperpolarization, the stimulus
still evoked a depolarization that outlasted the stimulus by ~1 s
(Fig. 10C).
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DISCUSSION |
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The results demonstrate that plateau potentials are prominent in
sacrocaudal motoneurons of chronic spinal rats with spasticity. Further, the plateaus contribute substantially to the exaggerated long-lasting reflexes, prolonging the synaptic input by many seconds, and thus playing an important role in spasticity (Bennett et al. 1999a). The plateaus occurred spontaneously when recorded in
normal ACSF in vitro, without exogenous neuromodulators added to the ACSF, and thus these motoneurons become somewhat like deep dorsal horn
neurons that normally exhibit plateaus and related oscillations spontaneously (Jiang et al. 1995
; Morrisset and
Nagy 1999
; Russo and Hounsgaard 1996
). In
contrast, plateau behavior was not usually seen in motoneurons of acute
spinal rats although exogenously applied 5-HT could enable plateaus.
Because previous studies have provided evidence for plateaus in
hindlimb/leg motoneurons of intact rats (Eken et al.
1989; Gorassini et al. 1999a
), decerebrate cats
(Hounsgaard et al. 1988
), and awake humans
(Gorassini et al. 1998
, 2001a
), it is reasonable to
assume that plateaus were also present in sacrocaudal tail motoneurons
of intact rats prior to injury. In addition, the finding that 5-HT
facilitates plateaus in these sacrocaudal cells after acute injury is
consistent with this assumption because such monoamines are thought to
be a major facilitator of plateaus in normal animals (Eken et
al. 1989
). We can therefore conclude that motoneuron excitability provided by plateau behavior is acutely removed by spinal
cord injury and is recovered in chronic spinal rats that develop
spasticity, thus verifying the hypothesis and results of Eken et
al. (1989)
and Nielsen and Hultborn (1993)
, and
the more recent inferences of plateau behavior from motor unit
recordings in awake spinal-cord-injured rats (Bennett et al.
2001
) and humans (Gorassini et al. 1999b
).
We have found a remarkably high incidence of plateaus in motoneurons of
chronic spinal rats (from intracellular and motor unit recordings)
(Bennett et al. 2001). Nielsen and Hultborn
(1993)
found a lower incidence of plateaus in motoneurons of
chronic spinal cats; however, we suggest that this was partly because of the criterion of hysteresis in firing that they used to identify plateaus. We found that, while most cells have plateaus, the plateaus are activated just before recruitment (low threshold), and thus clear
counterclockwise hysteresis loops (F-I plot) with a late frequency acceleration are infrequent (type 4; 20%), compared with
linear frequency plots with self-sustained firing and no open
counterclockwise hysteresis loop (type 3; 63%).
The emergence of plateaus with chronic injury is very significant from
a functional point of view because the normal descending inhibitory
control is lacking, and uncontrolled long-lasting contractions may be
triggered by brief stimuli (e.g., spasms and hypertonus) (Bennett et al. 1999a). The threshold, amplitude, and
duration of the plateau is therefore important to quantify functionally as discussed in the following text. We suggest that spasticity associated with injury is not so much a condition related to motoneuron overexcitability (see discussion of plateau amplitude in the following text) but instead to a recovery of relatively normal excitability and
plateau behavior without the normal inhibitory control to turn off plateaus and associated sustained firing.
Possible mechanisms for emergence of plateau in chronic spinal rats
The cause of plateaus after chronic spinal cord transection is
unknown. The spinal cord has essentially no endogenous neurons that
release monoamines (NE, 5-HT; only 1 5-HT spinal neuron per rat)
(Newton and Hamill 1988), and peripherally derived
monoamines [from sympathetic terminal sprouts (McNicholas et
al. 1980
); or other hormones] could not play a role in
producing plateaus in the explanted in vitro spinal cord
preparation that we have used. However, as mentioned in the
INTRODUCTION, persistent inward currents are likely present
in many neurons, and a number of neuromodulators, outside of
monoamines, can enable them to dominate sufficiently over outward
currents to enable plateaus (Russo and Hounsgaard 1999
).
Indeed, even in acute spinal animals a few motoneurons have plateaus
(3/22 in our case; 1/20 in Hounsgaard et al. 1988
), suggesting a latent endogenous capability for plateaus, perhaps controlled intrinsically or by interneuronal or afferent inputs (via
substance P and glutamate) (Russo and Hounsgaard 1999
;
Russo et al. 1997
).
Plateaus may have emerged as a result of unmasking the persistent
inward currents by reducing voltage- and calcium-gated outward K+ currents, many of which normally participate
in the AHP (Russo and Hounsgaard 1999). For example,
5-HT-mediated plateaus are associated with a reduction in the AHP
(Hounsgaard and Kiehn 1989
; Hultborn and Kiehn
1992
). However, the AHP amplitude (or duration) in chronic
spinal rats was not significantly smaller than in acute spinal rats, suggesting that the plateaus that emerged in chronic spinal rats were not facilitated by a reduction in these
AHP-related K+ currents (in contrast to how 5-HT
works). Further, because there was a plateau and a large
AHP, the accumulated hyperpolarization from the AHPs in chronic spinal
rats produced peculiar effects not seen in motoneurons of plateaus
mediated by 5-HT (Hounsgaard and Kiehn 1989
;
Hounsgaard et al. 1988
), such as partial plateau deactivation during firing. Similar plateau deactivation has been reported following high-frequency firing in plateau-generating turtle
dorsal horn neurons and was associated with a large slow AHP (sAHP)
that slowed or stopped repetitive firing (Russo and Hounsgaard
1996
). Also, we found that, with low levels of injected current, the plateau was partially deactivated by each large AHP and
then only slowly reactivated, enabling a further spike and AHP, etc,
thus explaining the very slow steady firing rates seen in chronic
spinal rats (1-4 Hz; see Fig. 8H) (see also similar discussions in Carp et al. 1991
; Hodgkin
1948
; Kernell 1999
).
Alternatively, the plateaus may have emerged after chronic injury
because of a direct facilitation of the persistent inward current
(IPIC) by metabotropic actions (e.g.,
mGluR1 or muscarine receptors) (Svirskis and Hounsgaard
1998) or even a permanent up-regulation of the associated
channels or channel subunits (Ma et al. 1997
). One
prediction of a direct increase in
IPIC would be that the conductance
should be increased in chronic spinal rats with plateaus compared with
acute spinal rats, whereas if IPIC was
simply unmasked by reducing the opposing outward currents, the opposite
might occur (Kernell 1999
). Although we did not directly measure the conductance during the plateau (i.e., during firing), we
did find that the F-I slope was lower in chronic spinal rats than in acute spinal rats. This finding is consistent with a greater conductance resulting from IPIC in
chronic spinal rats (i.e., more current needed to increase firing),
especially considering that the basic cell properties in acute and
chronic spinal rats were otherwise similar (similar passive
Rin, rheobase, AHP amplitude; Table
1). Also, an increase in F-I slope has been associated with
a decrease in the AHP and associated conductances in the presence of
neuromodulators (Berger et al. 1992
; see Fig. 1 of Kernell 1999
).
The inward currents involved in the plateau after chronic injury remain
uncertain and could, in principle, include persistent calcium (L-type),
persistent sodium, NMDA and ICAN
currents (Russo and Hounsgaard 1999). Considering the
plateau timing and broad activation range seen in chronic spinal rats,
we suggest the involvement of L-type calcium channels, as in plateaus
of other motoneurons and dorsal horn neurons in turtle and young rat
(Morisset and Nagy 1999
; Russo and Hounsgaard
1999
). In particular, the very slow plateau onset during small
current pulses (at threshold; Fig. 9A) is a characteristic
property of plateaus mediated by L-type calcium channels of motoneurons
(Hounsgaard et al. 1989
; Svirskis and Hounsgaard
1997
).
Low plateau threshold
Functionally, one important characteristic of plateaus is the
threshold for activation. In decerebrate cats, the threshold varies
widely depending on the cell type (Lee and Heckman
1998a) and mode of activation (intracellular vs. synaptic,
somatic vs. dendritic) (Bennett et al. 1998a
,b
; see also
turtle motoneurons, Svirskis and Hounsgaard 1997
, 1998
).
That is, with intracellular current ramps, some cat motoneurons have
plateaus activated near the recruitment level, but the majority have
plateaus activated well after recruitment (at high firing rates). With
synaptic activation, the threshold for plateau activation is lowered in
cat motoneurons (Bennett et al. 1998a
). In contrast, in
chronic spinal rats the majority of motoneurons have plateaus that are
initiated below the firing level, even during intracellular current
injections (Figs. 5 and 7A).
Low plateau thresholds are normally characteristic only of slow, early
recruited motoneurons in the ("normal") decerebrate cat (Lee
and Heckman 1998a). Thus the reason why all motoneuron types
have a low threshold after chronic injury is unclear, especially considering that the tail muscles have both fast and slow twitch muscles (Steg 1964
). Interestingly, chronic spinal rats
additionally have slower AHPs (Table 1), lower firing rates (Fig. 7)
and longer muscle twitches (Stephens et al. 1999
) than
acute spinal rats, consistent with the whole motor unit behaving more
slowly (see also Powers and Rymer 1988
). This occurs
even though the recruitment threshold (Fig. 4A) and input
resistance (Table 1) are similar to that of acute spinal rats (as in
Baker and Chandler 1987a
; Hochman and McCrea
1994
).
Plateau amplitude and IPIC
How large the plateaus are in chronic spinal rats in relation to
those in normals or brain-stem-intact animals is another important
functional question. In chronic spinal rats, the plateau is ~5-10 mV
(Figs. 4 and 5). Further, our estimates of the sustained current
supplied by the plateau (IPIC) ranged
from ~0.5 to 1.5 nA, with an average of ~0.81 nA, which is
substantial in relation to the current required to recruit the cell to
steady firing (1.7 nA). That is, the plateaus provide about half the
average current needed to recruit a motoneuron and maintain moderate
firing rates (0.81/1.7). With 5-HT administration, the plateau effects
are smaller (Fig. 4, B and D), suggesting that
plateaus in the chronic spinal rat may be at least as large as in the
normal brain-stem-intact rat. Motoneurons in brain-stem-intact
decerebrate cats have very similar plateau amplitudes to the chronic
spinal rat (~8 mV; Bennett et al. 1998a; see also:
Hounsgaard et al. 1988
; Lee and Heckman 1998a
,b
), and the sustained
IPIC is on average 6 nA (sustained peak in Lee and Heckman 1998b
), which is again about
half the average current to recruit the motoneurons (14 nA; cat
motoneurons require 10 times more current to activate, compared with
rat and turtle) (Lee and Heckman 1998a
,b
). Note,
however, that the plateau can be further augmented in cats with
additional monoaminergic agonists
(IPIC doubled with methoxamine)
(Lee and Heckman 1998b
; see also Svirskis
and Hounsgaard 1998
). Finally, in humans the plateau has a
similar effect as in decerebrate cats, with the plateau again providing
half the estimated input to maintain moderate firing rates
(Gorassini et al. 1998
, 2001a
). Thus the sustained depolarization and effective IPIC
current provided by the plateau in chronic spinal rats is comparable to
that predicted in intact animals and humans.
Linear firing rate profiles with slow, low-amplitude current ramps
Considering the presence of plateau behavior in motoneurons,
it is remarkable that the firing rate profiles were usually linear with
the plateau prolonging the firing even though the firing rate remained
on the F-I regression line (type 3 neurons, Fig. 6C). We suggest that this linearity occurred because the
plateau was mostly activated at or before recruitment and thus did not markedly affect the linearity of firing afterward, other than to
provide a depolarizing bias that brought the cell to a relatively high
rate (possibly optimal rate), compared with its minimum rate. In some
cells, there was evidence that the plateau was being further activated
(or deactivated) during firing, and this produced some nonlinearities
in firing (type 4 neurons). However, we have primarily studied the
plateaus with intracellular current injection, which should raise the
plateau threshold in comparison to the threshold seen with synaptic
activation (see preceding text) (Bennett et al. 1998a)
and exaggerate the firing rate nonlinearities. Importantly, the
relative linearity of firing profiles implies that, even in motoneurons
with plateaus, the firing rate profile should closely reflect the
input to the motoneurons (Figs. 5A and 6,
C and D), and this profile can be used to study
the input-output properties of other higher threshold motoneurons (with
motor unit recordings in the awake rat) (Bennett et al.
2001
; Gorassini et al. 2001a
,b
).
The ramp profiles that we used were slow and small in amplitude, thus
optimized to clearly see the sustained plateau. This avoided high
firing rates that produced firing rate adaptation (Kernell and
Monster 1982) and non-steady-state dynamics of the cells (ramp
speed-related) and thus favored linear firing profiles. Nonlinear
behaviors can be seen with faster and larger inputs where higher firing
rates are achieved. Thus the stimulus parameters are very important to
consider in designing experiments to study plateaus, especially when
studying motor unit firing (Bennett et al. 2001
). This
is not to say that plateaus are not present with fast, large-amplitude
inputs: only that they are harder to study. Finally, because more
firing rate adaptation occurred in motoneurons without plateaus (acute
spinal rats; Figs. 6B and 7A), it is possible
that the plateaus themselves may have countered firing rate adaptation
(in chronic spinal rats). Thus the presence of
IPIC may determine the degree of
firing rate adaption and associated nonlinear firing, with the most
firing rate adaptation occurring in pentobarbital anesthetized animals
where IPIC is blocked (Kernell and Monster 1982
), less in the acute spinal unanesthetized
case, and the least in the chronic spinal case where
IPIC is enhanced (see Lee and
Heckman 1998a
,b
).
Role of plateaus in spasticity
When the dorsal roots were briefly stimulated in chronic spinal
rats, a long-lasting reflex was seen in the motoneurons; this reflex is
the counterpart of the long-lasting reflex seen in ventral roots and in
the tail muscles during spastic behavior (Bennett et al.
1999a). The reflex was markedly reduced in duration by hyperpolarization (Fig. 10C), indicating that intrinsic
voltage-dependent properties of the motoneurons contribute
substantially to these spastic reflexes (i.e., plateaus amplify and
prolong the reflexes). Further, spasticity in humans has been
associated with tonic or poorly modulated motor unit discharge
(Gorassini et al. 1999a
; Thomas and Ross
1997
), abnormally low firing rates (Powers and Rymer
1988
; Thomas and Ross 1997
), and impaired rate
modulation (Heckman 1994
; Wiegner et al.
1993
); these findings are each consistent with the emergence of
a plateau, as discussed in the preceding text (i.e., increased
IPIC and associated conductance,
without lower AHP). The finding that the long-lasting reflexes
associated with spasticity are mediated in large part by plateaus
throws new light on the antispastic action of baclofen, which has
recently been shown to inhibit L-type calcium currents and plateaus
(Russo et al. 1998
; Svirskis and Hounsgaard
1998
).
Our results also indicate that the exaggerated reflexes following
spinal cord injury are in part produced by a relatively protracted
synaptic input (EPSP lasting ~1 s in Fig. 10C) (see also
Baker and Chandler 1987b). A single low-threshold shock
to the dorsal roots is enough to evoke this long EPSP, even at
hyperpolarized levels. Considering that plateaus are slow activating,
we suggest that this long EPSP serves to prolong the effect of a brief
afferent stimulation sufficiently to trigger a plateau, which in turn
produces many seconds of firing. A similar long EPSP is seen in
motoneurons during flexor-reflex-afferent (FRA) stimulation in
DOPA-treated acute spinal cats, which is likewise prolonged by plateau
potentials intrinsic to the motoneurons (Conway et al.
1988
).
As we have described in the preceding text, the plateau potential
amplitude after chronic injury recovers to a level comparable to that
estimated in normal intact animals, so the fact that we do
not see spastic-like long-lasting reflexes in intact animals and humans likely involves major differences in inhibitory, as well as
excitatory, control of motoneurons in intact and spinal states.
Moderately long-lasting stimulation (1-s muscle vibration) can trigger
self-sustained motor unit firing in normal humans (plateau)
(Gorassini et al. 1998, 2001a
), but this firing can be
inhibited easily by descending inhibition (e.g., reduction in
volitional effort). This descending inhibition is lacking following complete spinal cord injury.
In summary, following chronic injury, three factors combine in the production of spasticity: moderately long-lasting synaptic events emerge in response to brief, low-threshold afferent stimuli, plateau behavior is recovered and prolongs these synaptic events by many seconds, and the normal inhibitory control is lacking, enabling firing to continue and ultimately contribute to protracted muscle spasms and hypertonus associated with spasticity.
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ACKNOWLEDGMENTS |
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We thank L. Sanelli and Y. Han for expert technical assistance and P. Harvey, F. Giddes, and M. Gorassini for comments on the manuscript.
Funding was provided by the Medical Research Council, the National Sciences and Engineering Research Council, and Canadian Foundation for Innovation of Canada, and the Alberta Heritage Foundation for Medical Research.
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FOOTNOTES |
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Address for reprint requests: D. J. Bennett, 513 HMRC, Div. of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2S2, Canada (E-mail: david.bennett{at}ualberta.ca).
Received 25 September 2000; accepted in final form 3 May 2001.
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