Immobilization Induces Changes in Presynaptic Control of Group Ia Afferents in Healthy Humans Lundbye, 2008

Physiology in Press
Immobilization induces changes in presynaptic control of group Ia afferents in healthy

humans
Jesper Lundbye-Jensen and Jens Bo Nielsen
J. Physiol. published online Jul 3, 2008;
DOI: 10.1113/jphysiol.2008.156547
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Physiology in Press; published online on July 3, 2008 as 10.1113/jphysiol.2008.156547
Immobilization induces changes in presynaptic control of group Ia afferents in
healthy humans.
Jesper Lundbye-Jensen1,2*, Jens Bo Nielsen1,2
1
Department of Neuroscience and Pharmacology, University of Copenhagen. The
Panum Institute 24.6 Blegdamsvej 3 DK-2200 København N Denmark
2
Department of Exercise and Sport Sciences University of Copenhagen Blegdamsvej 3
Panum Institute 24.6 DK-2200 København N Denmark
*
To whom correspondence should be addressed:
Jesper Lundbye-Jensen
Department of Neuroscience and Pharmacology & Department of Exercise & Sport
Sciences
University of Copenhagen
The Panum Institute 22.3
Blegdamsvej 3,
j.lundbye@mfi.ku.dk
Phone: + 45 35 32 74 51
Fax: + 45 35 32 74 99
Running title: immobilization changes presynaptic control of group Ia afferents
1
Abstract
Neural plasticity occurs throughout adult life in response to maturation, use and disuse.
Recent studies have documented that H-reflex amplitudes increase following a period of
immobilization.
To elucidate the mechanisms contributing to the increase in H-reflex size following
immobilization we immobilized the left foot and ankle joint for 2 weeks in 12 able-
bodied subjects. Disynaptic reciprocal inhibition of soleus (SOL) motoneurones and
presynaptic control of SOL group Ia afferents was measured before and after the
immobilization as well as following 2 weeks of recovery.
Following immobilization maximal voluntary plantar- and dorsiflexion torque (MVC)
was significantly reduced and the maximal SOL H-reflex amplitude increased with no
changes in Mmax. Decreased presynaptic inhibition of the Ia afferents likely contributed
to the increase of the H-reflex size, since we observed a significant decrease in the long-
latency depression of the SOL H-reflex evoked by peroneal nerve stimulation (D2
inhibition) and an increase in the size of the monosynaptic Ia facilitation of the SOL H-
reflex evoked by femoral nerve stimulation. These two measures provide independent
evidence of changes in presynaptic inhibition of SOL Ia afferents and taken together
suggest that GABAergic presynaptic inhibition of the SOL Ia afferents is decreased
following 2 weeks of immobilization. The depression of the SOL H-reflex when evoked
at intervals shorter than 10 s (homosynaptic post-activation depression) also decreased
following immobilization, suggesting that the activity-dependent regulation of
transmitter release from the afferents was also affected by immobilization. We observed
no significant changes in disynaptic reciprocal Ia inhibition. Two weeks after cast
removal measurements returned to pre immobilization levels.
2
Together, these observations suggest that disuse causes plastic changes in spinal
interneuronal circuitries responsible for presynaptic control of sensory input to the
spinal cord. This may be of significance for the motor disabilities seen following
immobilization as well as the development of spasticity following central motor lesions.
Introduction
It is well established that motor skill training may induce use-dependent functional and
structural plasticity within the central nervous system. Experience-driven central
nervous plasticity has been extensively documented at a cortical level in relation to
motor learning (Buonomano & Merzenich, 1998; Sanes & Donoghue, 2000) and there
is growing evidence that the cortical plasticity accompanying motor skill learning is
paralleled by changes in the properties of the spinal neuronal circuitries as demonstrated
in monkey (Wolpaw et al., 1983a; Wolpaw et al., 1983b), rats (Chen & Wolpaw, 1995),
in the spinal central pattern generator properties in cats (de Leon et al., 1998; Pearson,
2000; Barriere et al., 2008) and human subjects following skill training (Perez et al.,
2005; Meunier et al., 2007; Wolpaw, 2007).
It is less well investigated how and to which extent immobilization is also accompanied
by changes in transmission in spinal neuronal circuitries. Spinal cord plasticity can
occur at numerous neuronal and synaptic sites and through a variety of mechanisms. In
humans, spinal cord plasticity has mostly been inferred from modifications in the size of
H-reflexes (Perez et al., 2005; Gruber et al., 2007; Meunier et al., 2007; Perez et al.,
2007). The H-reflex provides a gross measure of motoneuron pool excitability and
transmission across the synapses of Ia afferents and thus reflects both presynaptic
3
mechanisms, postsynaptic inhibition and excitation, intrinsic motoneuronal mechanisms
and descending inputs.
Recent studies have provided evidence that H-reflex amplitudes increase following a
period of immobilization. In the rat, (Anderson et al., 1999) reported increases in the H-
reflex gain following 3 weeks of hindlimb unloading and in human (Clark et al., 2006b)
similarly observed increased H-reflex amplitudes at rest following 4 weeks of lower
limb suspension. We have recently demonstrated an increase in the excitability of H-
reflex following a period of one week of wrist and hand immobilization (Lundbye-
Jensen & Nielsen, 2008). In this study the size of the evoked H-reflexes increased with
no significant changes in corticospinal excitability estimated from motor evoked
potentials (MEPs) evoked by transcranial magnetic stimulation (TMS). Although this
finding does not provide evidence of the specific mechanisms involved, it led to the
hypothesis that immobilization may be accompanied by changes in the amount of
presynaptic inhibition of the Ia afferents or post activation depression (homosynaptic
depression). It has previously been demonstrated that a considerable part of the H-reflex
amplitude modulation is explained by changes in presynaptic inhibition of the synapses
of Ia afferents on spinal motoneurones (Hultborn et al., 1987a; Hultborn et al., 1987b;
Meunier & Pierrot-Deseilligny, 1989; Nielsen & Kagamihara, 1993b; Faist et al., 1996).
Presumably, gating of sensory inputs to spinal motoneurones is of functional importance
in regulating the contribution of the stretch reflex circuitry to the ongoing motor activity
(Nielsen & Sinkjaer, 2002; Rudomin, 2002). One advantage of regulating this
contribution at the presynaptic level is that sensory inputs may be selectively gated,
4
while allowing effective activation of the muscles by central commands (Rudomin,
2002). Furthermore, regulation of sensory information at the presynaptic level may
permit conveyance of proprioceptive information to the cortex, which might contribute
to adjusting supraspinal motor commands (Llewellyn et al., 1990; Morita et al., 1998).
In line with this notion both GABAergic presynaptic inhibition and post activation
depression have been demonstrated to increase following skill learning (Perez et al.,
2005; Meunier et al., 2007), and it is possible that presynaptic control of sensory input
changes during a period of immobilization. If this is the case it would be consistent with
the findings of Manabe et al. (1989) who observed a disuse-induced enhancement of Ia
synaptic transmission in spinal motoneurones of the rat.
The hypothesis that immobilization induces modifications of the transmission in the
monosynaptic Ia pathway through changes in presynaptic control mechanisms was
consequently tested in the present study.
We recorded the soleus H-reflexes before and after 2 weeks of ankle joint
immobilization and following a corresponding period of recovery. Disynaptic reciprocal
inhibition was evaluated through conditioning the SOL H-reflex by stimulation of the
common peroneal nerve (CPN). To evaluate presynaptic inhibition of the soleus Ia
afferents we measured the size of the femoral nerve monosynaptic Ia facilitation of the
soleus H-reflex (Hultborn et al., 1987a; Hultborn et al., 1987b) and the long-latency
(D2) inhibition of the soleus H-reflex induced by CPN nerve stimulation (Mizuno et al.,
1971). To assess post-activation (homosynaptic) depression we used the frequency-
related depression of the Sol H reflex (Crone & Nielsen, 1989b; Hultborn et al., 1996;
Kohn et al., 1997).
5
Methods
Ethics Approval
In twelve healthy volunteers (9 male and 3 female) with an average age of 25 ± 6 years
(mean ± S.D.) the left foot and ankle joint was immobilized by application of a cast for
two weeks. No volunteers had any history of neurological disease. Subjects gave
informed written consent to the experiments which were approved by the local ethics
committee (J. No. KF 100.1969/1991). The experimental procedures conformed to the
standards set by the latest revision of the Declaration of Helsinki.
Experimental protocol
All subjects participated in 4 identical experimental sessions on 4 separate days. In
order to assess day-to-day variability, measurements were obtained on two different
days prior to immobilization (pretests). In addition, subjects were tested immediately
after cast removal following two weeks of immobilization (posttest) and once again
following two weeks of recovery after cast removal (retest).
During all experimental sessions subjects were seated in a comfortable armchair with
the head, torso and arms supported. The examined left leg was flexed in the hip (120°),
knee (160°) and ankle (110°) and the foot was fixed to a pedal with a rotational axis
located coaxially with the axis of rotation of the ankle joint. The pedal had a built-in
potentiometer and was connected to a strain gauge transducer providing information on
position and the torque applied. The pedal was fixed and the position of each individual
was recorded and reestablished in all tests.
6
Maximal Voluntary Contraction
In each test maximal voluntary isometric contraction torque (MVC) was measured for
the ankle plantar flexors and dorsi flexors respectively in order to evaluate alterations in
the maximal strength of the subjects before and after immobilization. Subjects were
instructed to perform a maximal contraction by increasing the torque to maximum
within a few seconds and to exert maximal torque for 2 s, while maintaining the
standardized position. Verbal encouragement and visual feedback of the torque exerted
were provided. Typically four or five successive trials were performed until the peak
torque did not increase any further. The peak torque recorded in either of the trials was
taken as the MVC. After completion of the strength tests electrophysiological testing
procedures involving electrical stimulation of the peripheral nerves were performed.
Stimulation and recording
EMG recording
Electromyographic (EMG) activity was recorded from the soleus (SOL) and tibialis
anterior (TA) muscles using a bipolar Silver/silver chloride (Ag/AgCl) surface electrode
configuration (0.5 cm diameter of electrodes; Blue Sensor, Ambu Inc., Ølstykke,
Denmark) with an inter electrode distance of 2 cm. The amplified EMG signals were
filtered (band-pass, 25 Hz to 1 kHz), sampled at 2 kHz, and stored on a PC for off-line
analysis (CED 1401+ with Signal & Spike 2.5 software, Cambridge Electronic Design
Ltd., Cambridge, UK). In addition all reflex responses were quantified, averaged and
related to Mmax and/or unconditioned response amplitudes online in a custom-build
software (Winflex).
7
Soleus H-reflexes
SOL H-reflexes were evoked by stimulating the tibial nerve through a
monopolar electrode (1 ms rectangular pulse) in the popliteal fossa (constant-current
stimulator model DS7A, Digitimer, UK). The indifferent electrode was placed proximal
to patella. The reflex response was measured as peak-to-peak amplitude of the non-
rectified EMG reflex response. The interstimulus interval was 4 seconds. The stimulus
intensity was randomly increased in steps of 0.05 mA, starting below H-reflex threshold
(HT) and increasing up to supramaximal intensity to measure the maximal compound
motor response (Mmax). SOL H-reflex recruitment curves were generated by averaging 5
responses at all obtained stimulus intensities. In a recent study we found Hmax to be
increased following immobilization and special care was taken to identify H max
(Lundbye-Jensen & Nielsen, 2008). Stimulus intensities ranged from 0-100 mA. In
order to enable comparison between reflex responses obtained in different sessions H-
reflex amplitudes were normalized to the corresponding Mmax and stimulation intensities
were normalized to Mthreshold in pretest1. The sensitivity of the H-reflex to facilitatory or
inhibitory conditioning effects has been shown to depend crucially on its size (Crone et
al., 1990). During measurements of the effect of a conditioning stimulus, the size of the
SOL control reflex was therefore maintained 20-25% of M max. Due to low Hmax
amplitudes a SOL control H-reflex of 15% of Mmax was used in 2 subjects in order to
allow both inhibitory and facilitatory effects.
Disynaptic Reciprocal Inhibition
In order to assess the amount of disynaptic reciprocal inhibition the SOL H-reflex was
8
conditioned by previous stimulation of the common peroneal nerve (CPN) at rest. At
conditioning test intervals of 2-3 ms (Crone et al., 1987) CPN stimulation elicits a
depression of the SOL H-reflex, which is in all likelihood mediated by the disynaptic
reciprocal Ia inhibitory pathway (Crone et al., 1987). The CPN was stimulated (1 ms
rectangular pulse) through bipolar surface electrodes (Blue Sensor, Ambu Inc.,
Denmark. Diameter 0.5 cm) placed 1-3 cm distal to the neck of the fibula. Electrodes
were placed in a position to evoke a threshold motor response in the TA muscle without
activation of a motor response in the peroneal muscles. The specificity of this
stimulation was checked several times during each experiment. The stimulus strength
was expressed in multiples of the motor response threshold (1.1xMT) in the TA muscle.
This stimulation intensity was chosen because it elicited a small TA M-response, which
could be monitored throughout the measurements and thereby ensure that the effect of
the conditioning stimulus was comparable (Petersen et al., 1998). Higher stimulation
intensities were avoided to minimize influence from other pathways than the disynaptic
Ia reciprocal pathway to the measured inhibition (Petersen et al., 1998). Also, a
stimulation intensity of 1.1 x MT is submaximal for activation of all inhibitory
interneurones and thus permits both facilitatory and inhibitory effects on transmission in
the pathway to be demonstrated (Petersen et al., 1998). In each experiment a time
course of the effect of the CPN stimulation was recorded for every subject with cond.-
test intervals ranging from 1-6 ms. In a random sequence 10 reflex responses were
obtained at each conditioning test interval with an inter stimulus interval (ISI) of 4s.
Responses were averaged and expressed relative to the obtained unconditioned SOL H-
reflex amplitude.
9
Presynaptic inhibition of the Ia afferent terminals
In order to assess the role of presynaptic inhibition of the Ia afferent terminals we
applied two different protocols involving conditioning of the SOL H-reflex: femoral
nerve (FN) facilitation and “D2”-inhibition. Changes in heteronymous Ia facilitation
and D2 inhibition reflect changes in different Ia pathways to SOL motoneurons,
heteronymous and homonymous respectively. Previous studies have however
documented that presynaptic inhibition of heteromymous and homonymous Ia pathways
to SOL motoneurons is similarly controlled by descending and/or afferent input (Katz et
al., 1988; Meunier & Pierrot-Deseilligny, 1989; Faist et al., 1996), suggesting that it is
mediated by the same or similar primary afferent depolarization (PAD) interneurones
(Hultborn et al., 1987a; Hultborn et al., 1987b). It is however advantageous to obtain
both measures in order to interpret the observed changes.
Heteronymous facilitation
The SOL H-reflex was conditioned by stimulating the femoral nerve (FN). This method
was used to assess the ongoing presynaptic inhibition exerted on the Ia afferents
mediating a monosynaptic conditioning excitatory volley (Hultborn et al., 1987a). In
man, there are heteronymous, monosynaptic excitatory projections from Ia fibers in the
FN nerve to SOL motoneurons and from Ia afferents in the PT nerve on quadriceps
motoneurons (Hultborn et al., 1987a; Meunier et al., 1993). The FN was stimulated
through a monopolar ball electrode placed over the femoral triangle. The indifferent
10
electrode was placed just below the gluteus maximus muscle. The intensity for
stimulating the FN was 1.5 x MT in the quadriceps muscle. In each experiment a time
course of the effect of the FN facilitation was recorded for every subject (Hultborn et
al., 1987a; Hultborn et al., 1987b) with cond.-test intervals ranging from -8 to -3 ms (fig
5). The negative cond.-test intervals designate that the conditioning stimulation was
applied after the test stimulation. In a random sequence, 10 responses were obtained for
each cond.-test interval and the average response amplitudes were related to the
unconditioned reflex amplitude. The onset of facilitation was considered to be the
earliest interval at which the conditioned reflex was 10% larger than the test reflex.
Following this procedure, a more detailed time course with steps of 0.2 ms for the 1 ms
prior to the onset of facilitation was obtained. Using this procedure FN facilitation was
quantified for the optimal cond.-test interval for each subject.
As demonstrated by (Hultborn et al., 1987a; Hultborn et al., 1987b), the short-latency
facilitation of the SOL H-reflex produced by stimulation of femoral nerve Ia afferents
can be ascribed to a monosynaptic heteronymous Ia excitatory postsynaptic potential
(EPSP). Hultborn et al. (1987a) provided experimental evidence that during its first 0.5
ms this heteronymous Ia facilitation is only mediated through a monosynaptic pathway
and not contaminated by input from oligo- or polysynaptic pathways (Pierrot-
Deseilligny et al., 1981; Pierrot-Deseilligny, 1996). This is why in the present study the
onset of the facilitation was examined in details in the individual subjects with
conditioning test intervals in steps of 0.2 ms to carefully measure the amount of
facilitation within the initial 0.5 ms after this onset. Provided that stimulations were
elicited at these appropriate conditioning test intervals this early heteronymous Ia
11
facilitation depends only on the size of the conditioning monosynaptic Ia EPSP.
Changes in the size of this early facilitation can therefore be used to assess ongoing
presynaptic inhibition of Ia terminals to SOL motoneurons (Hultborn et al., 1987a;
Hultborn et al., 1987b). Accordingly, a constant conditioning stimulation of the femoral
nerve, which in this study was adjusted to 1.5xMT should elicit an EPSP of a constant
size in the motoneurons, and thus a constant reflex facilitation, unless presynaptic
inhibition of Ia afferents mediating the conditioning Ia volley has changed: The smaller
this ongoing presynaptic inhibition the larger the reflex facilitation.
D2 inhibition
Using CPN conditioning of the SOL H-reflex at longer cond.-test intervals than those
applied when investigating disynaptic reciprocal inhibition, CPN stimulation elicits a
pronounced depression of the SOL H-reflex reaching a maximum for cond.-test
intervals around 15-20 ms. At cond.-test intervals around 60 ms a second, more
pronounced, depression occurs. Although it is thought of as one long depression
separated by a period of facilitation, the early and late depressions are referred to as D1
and D2 respectively (Mizuno et al., 1971). Previous findings have suggested that both
D1 and D2 are in all likelihood influenced/caused by presynaptic inhibition of the
terminal of Ia afferents on SOL motoneurons. Considering D2, there is no inhibition in
the EMG corresponding to the D2 time-course as it would have been in the case of
postsynaptic inhibition (Iles & Roberts, 1986). In this study we looked at D2 inhibition
primarily because pilot experiments demonstrated D2 to have a better reproducibility
than D1 between experimental sessions. Secondly, postsynaptic inhibition may last 20-
30 ms (Baldissera et al., 1981) and may therefore interfere with D1.
12
In the present experiment D2 inhibition was assessed by conditioning the SOL H-reflex
by CPN stimulation at cond.-test intervals of 40, 50, 60, 70, 90, 100 ms. In a random
sequence, 10 responses were obtained for each cond.-test interval and the average
response amplitudes were related to the unconditioned reflex amplitude.
FN facilitation and D2 inhibition provide independent information about presynaptic
inhibition and help us to exclude changes in recruitment gain in the soleus motoneurons
as a cause of changes in the H-reflex size (Nielsen & Kagamihara, 1993b, a). D2 (and
D1) inhibition reflects the level of presynaptic inhibition evoked by peripheral nerve
stimulation and FN facilitation reflects the level of ongoing presynaptic inhibition of FN
Ia afferents (which is paralleled by SOL Ia afferents).
Post activation depression
Another presynaptic mechanism, which could be affected by immobilization, is post
activation (or homosynaptic) depression originally described by (Curtis & Eccles,
1960). Post activation depression reflects a reduced Ia afferent transmitter release
probability due to previous activation (Crone & Nielsen, 1989a; Nielsen et al., 1993;
Nielsen et al., 1995; Aymard et al., 2000) and it is therefore only observed when the Ia
afferents have been previously activated by a conditioning stimulus of the same
afferents (e.g. vibration, tendon tap, electrical nerve stimulation; (Crone & Nielsen,
1989a). The depression of the monosynaptic Ia EPSP results primarily from
mechanisms operating within the presynaptic terminals, which depends on the history of
activation of the synapse (Lev-Tov & Pinco, 1992; Pinco & Lev-Tov, 1993). Usually,
13
post activation depression is observed for more than 10 s following a preceding
activation (conditioning stimulus), compared with only 300-400 ms for ‘classic’
presynaptic inhibition (Hultborn et al., 1996). Unlike GABAergic presynaptic inhibition
post activation depression is also not widely distributed among the afferent fibres
(Hultborn et al., 1996).
[Insert fig 1 around here]
In the present study post activation depression was induced by electrical activation of
the soleus Ia afferents and evaluated by frequency related changes in the H-reflex. Ten
SOL H-reflexes (15-25% of Mmax) were evoked with an inter stimulus interval of 10s.
After this 10 SOL H-reflexes were evoked with an identical stimulus intensity and an
inter stimulus interval of 1s. Since the first reflex in this series was not influenced by
post activation depression it was excluded and the remaining 9 responses were
averaged. Post activation depression was quantified as the difference between the
average reflex amplitudes obtained at 10s and 1s inter stimulus interval. That is, the
depression of the SOL H-reflex elicited by shortening the interstimulus interval from
10s to 1s.
Contractile Properties
In order to test changes in muscular contractile properties supra-maximal electrical
stimulation was delivered to the PTN and CPN to elicit maximal twitch contractions.
Stimulation consisted of a 1 ms rectangular pulse delivered by a constant-current
stimulator (model DS7A, Digitimer, UK). The elicited twitch torque was registered and
14
peak torque, time to peak torque and half relaxation time was calculated during offline
analysis.
Immobilization
At the end of the second pretest a custom-molded circular ankle cast (X-lite®, Camp
Scandinavia) was configured for each subject. The subject´s left ankle and foot was
covered by a stockinette and foam padding and the cast was positioned around the
ankle, foot and toes, maintaining the ankle joint in a neutral position restricting ankle
joint movements. Subjects were not able to remove the cast themselves, ensuring that
joint immobilization was effective for the full period. Subjects were instructed to walk
with crutches and to avoid ground contact with the immobilized foot throughout the
immobilization period. After 2 weeks of immobilization the cast was removed by the
experimenter immediately before beginning of the posttest.
Data Analysis and Statistics
Reflex measurements
The mean and standard error of the mean were calculated for all parameters and
conditions.
Considering SOL H-reflexes all stimulation intensities were normalized to Mthreshold in
pretest 1 and all response amplitudes were normalized to the corresponding Mmax. Hmax
was quantified from the H-reflex recruitment curve.
One-way repeated measures ANOVA test was used to determine the effect of
immobilization on MVC, twitch torque, time to peak, half relaxation time, Mmax,
15
Hmax/Mmax ratios, disynaptic reciprocal inhibition, femoral nerve facilitation, D2
inhibition and post activation depression with time of measurements as a factor. Tukey
post hoc test was performed on significant comparisons. All values are reported as
mean±sd unless stated otherwise. In all tests, statistical significance was assumed if the
P < 0.05.
Results
Following immobilization maximal voluntary plantar and dorsi flexion torque
decreased significantly (fig. 2). Plantar flexion torque decreased ~15% from 69.1±21.2
Nm in pretest 1 and 69.3±19.8 Nm in pretest 2 to 59±20 Nm immediately after cast
removal (p < 0.001 and p < 0.001). After two weeks of recovery plantar flexion MVC
returned to baseline level 69.9±20.5 Nm (p < 0.001 compared to post immobilization, p
= 0.42 and p = 0.46 compared to pretest 1 and 2 respectively). For dorsi flexion MVC
torque also decreased significantly ~23% from 44.1±9.2 Nm in pretest1 and 43.7±11
Nm in pretest 2 to 33.6±8.1 Nm immediately after cast removal (p < 0.001). After two
weeks of recovery MVC torque returned to 40.4±11.5 Nm (p = 0.004 compared to post
immobilization, p = 0.753 and 0.65 compared to pretest 1 and 2 respectively).
Following supra-maximal PTN stimulation three parameters were quantified from the
elicited twitch contraction in order to assess changes in contractile properties. These
three parameters were twitch contraction torque (TT), contraction time (CT) (time to
peak torque) and half-relaxation time (HRT). TT was 5.34±2.83 and 5.24±2.83 Nm in
16
pretest 1 & 2. Following immobilization TT increased significantly to 8.29±4.6 Nm
(p=0.03 and p=0.048 respectively). After two weeks of recovery TT decreased to
6.72±2.83 Nm (p=0.084 compared to post immobilization, p=0.994 and 0.994 compared
to pretest 1 and 2 respectively). There were no significant changes in the CT from
before immobilization (116.5±21 ms and 111.4±17 ms) to cast removal (114.7±19 ms)
or following two weeks of recovery (106.1±9 ms) (p=0.206). There was however a
tendency towards a change in half relaxation time between tests. Before immobilization
HRT was 83±21 ms and 86±23 ms. Following immobilization HRT was prolonged to
101.6±16 ms (p=0.067) After 2 weeks of recovery HRT decreased to 93.7±20 ms.
The peak-to-peak amplitude of the SOL maximal compound action potential (Mmax) did
not change significantly following immobilization (p=0.28). The group average
Mmax±s.d. was 33.5±12.9 mV and 31.9±12.5 mV in pretests, 30.9±14 mV following
immobilization and 31.6±9.1 mV after 2 weeks of recovery.
H-reflexes
SOL H-reflex amplitudes measured at rest increased significantly following
immobilization (fig 3). We recently demonstrated how Hmax/Mmax ratios and H-reflex
recruitment curve properties changed significantly following immobilization. Because
of this, and in order to avoid an excessive amount of stimulations we focused on
determining the Hmax/Mmax ratio in the present study (Lundbye-Jensen & Nielsen, 2008).
The SOL Hmax/M max ratio increased significantly from 0.566±0.21 and 0.553±0.2 in
pretests to 0.644±0.23 after immobilization (p=0.03 & p=0.021). Following recovery
the Hmax / Mmax ratio returned to pre immobilization levels 0.565±0.24 (p=0.032
17
compared to post immobilization, p=0.999 and p=0.988 compared to pretest 1 and 2
respectively). There were no significant changes in the H-reflex latency from 31.7±2.7
and 31.8±3.1 ms in pretests to 32.3±2.9 ms following immobilization. In the retest the
H-reflex latency was 32.2±3.3 ms (p=0.2).
Disynaptic Reciprocal Inhibition
Figure 4 illustrates the time-course of the inhibition of the SOL H-reflex at rest
following single stimuli at 1.1 x M-threshold to the common peroneal nerve expressed
relative to the unconditioned H-reflex size. The inhibition was apparent when the
conditioning CPN stimulation preceded the PTN test stimulus by 2 ms (88.7±11%;
90±7.2%; 90.9±8.7%; 90.9±13%), 3 ms (88.2±13%; 86.9 ±9.1%; 92.4±10.3%;
84.8±14%) and 4 ms (89.1±10.9%; 89.2±14.6%; 91±14.4%; 85.6±9.2%) in the pre-,
post- and retests, respectively (Figure 4). No significant differences in disynaptic
reciprocal inhibition were observed between the pre-, post- and retest data at any of the
cond.-test intervals (p = 0.39-0.94).
Femoral Nerve Facilitation
Figure 5 illustrates the early facilitation of the SOL H-reflex evoked by stimulation of
the femoral nerve stimulation at 1,5 x M-threshold for quadriceps expressed relative to
18
the unconditioned H-reflex size. The facilitation of the SOL H-reflex induced by
femoral nerve stimulation had an onset at a cond.-test interval around –5.5 ms (negative
conditioning–test intervals designate that the control stimulus preceded the conditioning
stimulus). At a cond.-test interval of -6 ms the average facilitation was 110.6±18.7%;
111±19%; 120.3±21%; 101.6±18% in pre- post and retests respectively with a
significantly increased facilitation following immobilization (p=0.037). At a cond.-test
interval of -7 ms the facilitation was 108.2±9.9% and 108.2±11.7% in pretest 1 and 2. It
increased significantly to 119.5±13.8% following immobilization (p=0.01) and
decreased to 109.3±9% after two weeks of recovery (p=0.089 compared to post and
p=0.916 compared to pretests)
In order to ensure that the facilitation was caused solely by transmission in the Ia
monosynaptic pathway from the quadriceps to SOL motoneurones, an individually
optimal conditioning–test interval was applied with 0.2ms accuracy to evaluate the size
of the early facilitation before and after immobilization and recovery (Hultborn et al.,
1987a) (bottom fig.5). The average size of the FN facilitation from all subjects at this
individual interval was 112.2±4.6% and 115±5.2% in the pretests (size of the
conditioned reflex expressed as a percentage of the control reflex). After immobilization
the facilitation increased to 128.2±5.2% (p=0.013). After 2 weeks of recovery the
facilitation returned to baseline values at baseline 113.4±3.1% (p=0.033 compared to
post, p=0.995 and p=0.989 compared to pretest 1 and 2 respectively.
19
D2 Inhibition
Figure 6 illustrates the D2 inhibition which could be observed at conditioning test
intervals of 70, 80, 90 and 100 ms. At 80 ms cond.-test interval the conditioned SOL H-
reflex was 69.8±22.2% and 72.7±28% of the unconditioned reflex amplitude before
immobilization and 84.5±22.9% (p=0.156) following immobilization. After recovery
the conditioned response returned to pre immobilization level of 70.6±36%. At 90 ms
cond.-test interval the conditioned reflex was depressed to 58.9±21.5%, and
59.7±25.7% before immobilization. This inhibition decreased following immobilization
to 72.6±17.2% (p=0.08) but returned to pre immobilization level (59.6±35%) following
2 weeks of recovery.
At 100 ms cond.-test interval the conditioned H-reflex was 52.9±19.8% and
51.7±25.7% of the unconditioned H-reflex before immobilization. Following
immobilization the response was significantly larger 71.1±26.9% (p=0.024 and
p=0.015). After two weeks of recovery the conditioned response returned to pre
immobilization level of 54.4±34% (p=0.063 compared to post immobilization, p=0.996
and p=0.998 compared to pretests).
Post Activation Depression
20
Figure 7 illustrates the depression of the SOL H-reflex observed when decreasing inter
stimulus intervals from 10s to 1s. At 10s inter stimulus interval the SOL H-reflex
amplitude was 27.7±7% and 26.9±7.6% of Mmax in pretest 1 and 2, 26.8±7% of Mmax
after immobilization and 27.7±8.2% of M max in the retest. There was no significant
difference in this parameter between tests (p=0.935).
Before immobilization the depression of the SOL H-reflex amplitude was 61.7±5% in
pretest 1 and 59.6±6.6% in pretest 2. Following immobilization the amount of post
activation depression decreased significantly to 43.7± 9.4% (p=0.001 and p=0.005
compared to pretests). After 2 weeks of recovery post activation depression increased to
pre immobilization level of 58.6±11.8% (p=0.008 compared to posttest, p=0.874 and
p=0.995 compared to pretests).
Discussion
The primary purpose of the present study was to elucidate adaptations in spinal neuronal
circuitries in relation to immobilization. The main behavioral finding was that 2 weeks
of ankle joint immobilization leads to significant decreases of maximal voluntary
strength. This was accompanied by an increase in the SOL H-reflex excitability in the
immobilized leg as has previously been reported (Duchateau, 1995; Anderson et al.,
1999; Clark et al., 2006b; Lundbye-Jensen & Nielsen, 2008). Based on the applied
conditioning-test protocols of the SOL H-reflex it appears that the observed increase of
H-reflex excitability at least in part relates to changes in presynaptic control of group Ia
21
afferents following immobilization whereas disynaptic reciprocal inhibition was
unaffected by the immobilization.
MVC and twitch contractions
In the present study, immobilization led to a 14% reduction of plantar flexion MVC and
a 23% reduction of dorsiflexion MVC. These changes are in agreement with the
findings in previous studies reporting 17% and 19% decreases in triceps surae MVC
following 2 weeks (Gondin et al., 2004) and 3 weeks (Davies et al., 1987) of
immobilization respectively. Depending on the duration of the immobilization period,
the observed decrease in voluntary strength has been attributed partly to changes in
neural activation and partly to changes in muscle contractile properties (White et al.,
1984; Davies et al., 1987; Duchateau & Hainaut, 1987; Duchateau, 1995; Seki et al.,
2001a, b; Gondin et al., 2004; Clark et al., 2006a; Clark et al., 2006b). Several studies
have found decreases in voluntary activation following immobilization (Vandenborne et
al., 1998; Kawakami et al., 2001; Gondin et al., 2004). Seki et al. (2001b) reported a
restriction of motoneurone firing to lower rates following immobilization and reduced
maximal motoneuronal firing rates following only one week of immobilization (Seki et
al., 2001b; Seki et al., 2007). Judged from the plantar flexor twitch measures used in the
present study, it is not unlikely that changes in muscle contractile properties may have
contributed to the reduced voluntary strength observed following immobilization. The
finding of increased twitch contraction torque (TT) and the tendency toward prolonged
half relaxation time (HRT) following immobilization is indeed in agreement with
previous studies on short-term immobilization (White et al., 1984; Davies et al., 1987;
Seki et al., 2001a; Gondin et al., 2004). The trend towards increased HRT has
22
previously been suggested to relate to reduction in the reuptake of Ca 2+ by the
sarcoplasmatic reticulum following immobilization (Thom et al., 2001). Although
immobilization was accompanied by changes in twitch contraction parameters in the
present study, the exact origin of these alterations remains to be determined.
Interestingly, immobilization was accompanied by a larger reduction of the MVC for
dorsiflexion compared to plantar flexion. One possible reason for this could be
differences in muscle characteristics, fibre type distribution and myosin heavy chain
(MHC) expression demonstrating different sensitivities to inactivity. Secondly, ankle
joint immobilization, while restricting TA and SOL contractions, does not completely
immobilize the biarticular gastrocnemius muscle. Although twitch contraction measures
changed for triceps surae as a whole, the fact that contraction of gastrocnemius was
possible during the immobilization may have counteracted loss of plantar flexion MVC
following immobilization. In line with this, (Gondin et al., 2004) found SOL, but not
gastrocnemius EMG activity to be significantly reduced following ankle joint
immobilization.
Increased Hmax/Mmax ratio
In the present study we observed an increase of the Hmax/Mmax ratio at rest following
immobilization. It has previously been reported that the Hmax/Mmax ratio is increased
following a period of immobilization in man (Duchateau, 1995; Clark et al., 2006b;
Lundbye-Jensen & Nielsen, 2008) and hindlimb suspension in the rat (Anderson et al.,
1999) and that the gain (Hslope/Mslope ratio) of the H-reflex is increased (Lundbye-Jensen
& Nielsen, 2008).
23
Modulation of the H-reflex amplitude can result from a number of factors, including
changes in GABAergic presynaptic inhibition of the Ia afferents, variation in the amount
of Ia neurotransmitter release, and changes in the excitability of the motoneurons due to
changes in either the membrane potential arising from excitatory or inhibitory input
(postsynaptic) or the intrinsic properties of the neurons. Cormery et al. (2005) recently
observed changes in the intrinsic properties of rat motoneurones following 2 weeks of
hindlimb unloading. This was signified by increased rheobase, decreased spike
amplitudes and deceased membrane time constants. Some properties of ‘slow’
motoneurones resembled those of ‘fast’ motoneurones following hindlimb unloading
(Cormery et al., 2005). This finding indicates that the motoneurones are not more
excitable or can be more easily activated. However, a selective change in the properties
of the ‘slow’ motoneurones means that the changes observed in H-reflex following
immobilization in the present study could be due to changes in the input-output relation
or ‘recruitment gain’ of the motoneurone pool following immobilization (Kernell &
Hultborn, 1990; Hultborn et al., 2004).
In a recent study we observed increased H-reflex amplitudes following one week of
wrist immobilization with no significant changes in corticospinal excitability estimated
from TMS measures (Lundbye-Jensen & Nielsen, 2008). Although many mechanisms
may potentially be involved in this phenomenon, we hypothesized based on this finding
that presynaptic control of Ia afferent input to the spinal cord could change (decrease)
following a period of immobilization. If so, this would be in agreement with Manabe et
al. (1979) who observed a disuse induced enhancement of monosynaptic Ia afferent
24
transmission (EPSPs) to rat spinal motoneurones following conduction block with
tetrodotoxin and nerve section (Manabe et al., 1989).
Presynaptic inhibition of Ia afferents
It has previously been demonstrated that part of the H-reflex modulation during
voluntary movement may be explained by changes in presynaptic inhibition of the
synapses of Ia afferents on spinal motoneurones (Hultborn et al., 1987a; Hultborn et al.,
1987b; Meunier & Pierrot-Deseilligny, 1989; Nielsen & Kagamihara, 1993b; Faist et
al., 1996; Perez et al., 2005). Presumably, gating of sensory input to spinal
motoneurones is of functional importance in regulating the contribution of the stretch
reflex circuitry to the ongoing motor activity (Rudomin & Schmidt, 1999; Nielsen &
Sinkjaer, 2002). One advantage of regulating this contribution at the presynaptic level is
that sensory inputs may be selectively gated while allowing effective activation of the
muscles by central commands (Rudomin, 2002). Furthermore regulation of sensory
information at the presynaptic level may permit conveyance of proprioceptive
information to the cortex which might contribute to adjusting supraspinal motor
commands (Llewellyn et al., 1990; Morita et al., 1998). There is good evidence from
previous studies that changes in presynaptic inhibition of the synapses between sensory
afferents and motoneurons is fundamental in the adaptation of the reflex circuitry during
motor learning (Kandel et al., 2000; Wolpaw, 2007). Recently, Perez et al. (2005)
demonstrated that presynaptic inhibition of Ia afferents from SOL was increased
following a period of visuomotor learning. However, it has not to our knowledge been
investigated whether presynaptic inhibition changes following a period of
immobilization.
25
Presynaptic inhibition consists of a GABA-mediated depolarization of the terminals of
primary afferents, which results in a decreased transmitter release (Schmidt, 1971;
Rudomin, 1990). In animal experiments, the most direct demonstration of presynaptic
inhibition of Ia afferents - see (Eccles, 1964) is the depression of the monosynaptic
EPSP evoked in motoneurones by a constant stimulation of Ia afferents without a
change in the motoneurone membrane potential and conductance (Frank & Fuortes,
1957). In human studies, information about presynaptic inhibition can only be obtained
indirectly.
In the present study we observed significant increases in the heteronymous facilitation
of the SOL H-reflex evoked by femoral nerve stimulation (Hultborn et al., 1987a) and a
reduction in the amount of D2 inhibition. These two measures provide independent
evidence of changes in presynaptic inhibition of SOL Ia afferents and taken together
with the observation of unchanged disynaptic reciprocal inhibition these findings
strongly suggest that presynaptic GABAergic inhibition of the SOL Ia afferents is
reduced following 2 weeks of ankle joint immobilization.
The early facilitation of the SOL H-reflex evoked by femoral nerve stimulation provides
indirect information about the heteronymous Ia EPSPs evoked in the motoneuron pool
(Hultborn et al. 1987a). It is essential for the method that the investigated facilitation of
the SOL H-reflex evoked by FN stimulation is monosynaptic in origin, as a change in
facilitation may otherwise be caused by postsynaptic changes at an interneuronal level.,
meaning that changes in facilitation of the test reflex cannot be ascribed unequivocally
to changes in the size of the conditioning Ia EPSP. This is why in the present study the
onset of facilitation was examined in detail with cond.-test intervals in steps of 0.2 ms.
26
Considering the homonymous D2 inhibition, PAD interneurons mediating presynaptic
inhibition of the test reflex’ Ia afferent volley are activated by a conditioning group I
volley. Although the D1 and D2 inhibition (Mizuno et al. 1979) is probably
contaminated by other effects (Meunier & Pierrot-Deseilligny, 1998) the size of these
depressions can be assessed to estimate the excitability of PAD interneuron’s mediating
presynaptic inhibition. Following immobilization D2 inhibition decreased significantly
which also indicates that presynaptic inhibition is decreased. Decreased inhibition could
result from decreased excitability of PAD interneurones or occlusion following
activation by another excitatory input (Faist et al., 1996). However, in the lower limb
Iles (1996) using very weak stimuli has shown that cortically evoked decrease in CPN-
induced inhibition is not due to occlusion in PAD pathways. Furthermore, in the present
experiments, the decreased D2 inhibition was associated with increased heteronymous
facilitation, which cannot be due to an occlusion in PAD pathways (see Hultborn et al.
1987b).
As previously mentioned, it may be argued, that an increased heteronymous Ia
facilitation could be observed as a consequence of skewed input to or effects within the
motoneuron pool. This could potentially affect the recruitment gain by increasing the
slope of the input-output relation of the H-reflex (Kernell & Hultborn, 1990; Nielsen &
Kagamihara, 1993b). This would be in agreement with the findings of (Cormery et al.,
2005). However, if recruitment gain had been changed by a skewed distribution of
effects within the motoneuron pool, this would be expected to enhance the effect of all
synaptic input to the motoneurons thereby increasing D1 and D2 inhibition. In effect,
the larger the recruitment gain the larger should be the D1 and D2 inhibition. The
present finding of increased heteronymous Ia facilitation and decreased D2 inhibition
27
following immobilization is in other words not consistent with a change in the
recruitment gain. In conclusion, the observed changes are most likely caused by changes
in presynaptic inhibition.
Post activation depression
Post activation depression also decreased following immobilization. Post activation
depression of the elicited reflex is caused by a frequency-dependent decrease in the
probability of transmitter release from previously activated synapses. This depression of
synaptic efficiency following previous synaptic activation of Ia afferents has been well
characterized in both animal (Curtis & Eccles, 1960; Hultborn et al., 1996) and human
studies (Crone & Nielsen, 1989a; Hultborn et al., 1996). We suggest that the long term
changes we observe in the frequency-related depression of the SOL H-reflex following
immobilization reflect plastic changes at the level of the synapse between Ia afferents
and SOL motoneurones causing an increase in the efficacy of the synapse. The
molecular mechanisms underlying post activation depression are not yet fully
understood and subsequently we can only speculate about the possible mechanisms
contributing to the modifications in the amount of transmitter released following
immobilization.
It has however recently been reported by Meunier et al. (2007) that post activation
depression is subject to use-dependent plasticity. They reported that post activation
depression increased immediately following and one day after a single bout of skillful
bicycle training. Contrary to the GABAergic presynaptic inhibition there is so far no
evidence for a descending control of post activation depression, but some effects of for
instance monoaminergic systems cannot be excluded. It is also not unlikely that
28
structural modifications of the synapses between Ia afferents and SOL motoneurones
have occurred during the 2 weeks of immobilization.
Post activation depression is decreased in spastic patients (Faist et al., 1994; Nielsen et
al., 1995) and (Hultborn & Nielsen, 1998) previously pointed out that the decreased
post activation depression in patients with cerebral or spinal lesions may (at least in
part) be secondary to the disuse of motoneurones and Ia afferents accompanying the
primary disorder. This notion is emphasized by the results of the present study and
further supported by the findings of (Gallego et al., 1979) that in the cat prolonged
disuse of the sensory fibers causes an increase in synaptic efficacy (EPSPs) of primary
afferents in triceps surae motoneurones. Conversely, Meunier et al. (2007)
demonstrated that motor training increased post activation depression.
As already mentioned post activation depression differs from the classical presynaptic
inhibition transmitted through GABAergic axo-axonal synapses (see review by Hultborn
& Nielsen). Even though both mechanisms contribute to the gating of sensory input to
the central nervous system through modulation of synaptic transmitter release, the two
mechanisms have distinct characteristics. Nevertheless, it is possible that an interaction
between GABAergic presynaptic inhibition and post activation depression contributes
to the observed changes following immobilization. It has previously been suggested
from animal studies (Enriquez-Denton et al., 2002) that reduced presynaptic inhibition
also results in reduced post activation depression and vice versa.
29
It is not possible from our data to make any conclusions regarding the exact
mechanism(s) of the reduced presynaptic inhibition following immobilization, but some
speculations may be made. There is good evidence from cat experiments that primary
afferent synapses on ascending neurons and motoneurones are inhibited by different
populations of interneurones (Jankowska & Padel, 1984). Indeed, the network
responsible for controlling presynaptic inhibition of primary afferents seems in general
to be organized to ensure selective control of afferent input to specific populations of
both motoneurones and ascending neurons (Rudomin & Schmidt, 1999; Rudomin,
2002) and it is not impossible that changes in sensory input to the spinal interneurones
which convey the presynaptic inhibition leads to plastic changes during a period of
immobilization. According to afferent input and the functional requirements to the
system, the CNS may increase or decrease the level of presynaptic inhibition of Ia
afferents and thereby the central gain of the monosynaptic stretch reflex (Capaday &
Stein, 1986; Hultborn et al., 1987a; Nielsen & Kagamihara, 1993b).
Based on the findings of the present study it seems plausible that the decreased
presynaptic inhibition occurred in response to the decreased motor activity
accompanying limb immobilization. The changes in presynaptic inhibition may be a
direct consequence of a reduced voluntary motor activity. It is however also possible
that immobilization is accompanied by decreased proprioceptive input to the central
nervous system. Reducing the amount of presynaptic inhibition of Ia afferents may in
this way be a means of the central nervous system to increase the gain of the actual
incoming afferent input. Since stimulation of low threshold cutaneous
mechanoreceptors by light brushing of both distal dorsal and plantar surfaces of the foot
30
has also been demonstrated to decrease presynaptic inhibition (Iles, 1996) it may also be
possible that the changes observed following immobilization occur in response to
wearing the ankle cast. Measurements were however obtained after removal of the cast.
Functional and Clinical perspectives
Although the present study involved limb immobilization in able-bodied subjects, the
findings may also be of clinical relevance. This is especially the case in relation to
neurological disorders leading to physical inactivity. It is noteworthy that the findings of
increased H-reflexes, decreased GABAergic presynaptic inhibition and decreased post
activation depression following immobilization to some extent matches the findings of
previous studies in spastic patients (Nielsen et al., 1993; Faist et al., 1994; Nielsen et
al., 1995; Grey et al., 2008) and it is worth considering the effects of reduced physical
activity in itself. As mentioned previously, it is possible that the decreased presynaptic
inhibition and post activation depression observed in patients with cerebral or spinal
lesions may at least in part be a consequence of the disuse of motoneurones and Ia
afferents.
Sensory feedback mechanisms through spinal reflex circuits help to ensure that the muscle
activity is optimally adjusted to the immediate environment during movement. From a
functional perspective it is interesting how changes in the functional properties of the
central nervous system following a period of immobilization relate to functional motor
control and further how subsequent training or rehabilitation affects these changes.
31
Acknowledgements
This work was supported by grants from The Danish Health Science Research Council,
The Danish Ministry of Culture, The Novo Nordisk Foundation, The Carlsberg
Foundation, The Elsass Foundation and the Danish Society of Multiple Sclerosis
References
Anderson J, Almeida-Silveira MI & Perot C. (1999). Reflex and muscular adaptations

in rat soleus muscle after hindlimb suspension. J ExpBiol 202, 2701-2707.
Aymard C, Katz R, Lafitte C, Lo E, Penicaud A, Pradat-Diehl P & Raoul S. (2000).

Presynaptic inhibition and homosynaptic depression: a comparison between
lower and upper limbs in normal human subjects and patients with hemiplegia.
Brain 123 ( Pt 8), 1688-1702.
Baldissera F, Hultborn H & Illert M. (1981). Integration in spinal neuronal systems. In

Handbook of Physiology, section 1, The Nervous System, ed. Brooks VB, pp.
509-595. American Physiological Society, Bethesda, MD, USA.
Barriere G, Leblond H, Provencher J & Rossignol S. (2008). Prominent role of the

spinal central pattern generator in the recovery of locomotion after partial spinal
cord injuries. J Neurosci 28, 3976-3987.
Buonomano DV & Merzenich MM. (1998). Cortical plasticity: from synapses to maps.
AnnuRevNeurosci 21, 149-186.
Capaday C & Stein RB. (1986). Amplitude modulation of the soleus H-reflex in the
human during walking and standing. J Neurosci 6, 1308-1313.
Chen XY & Wolpaw JR. (1995). Operant conditioning of H-reflex in freely moving
rats. Journal of neurophysiology 73, 411-415.
Clark BC, Fernhall B & Ploutz-Snyder LL. (2006a). Adaptations in human

neuromuscular function following prolonged unweighting: I. Skeletal muscle
contractile properties and applied ischemia efficacy. J ApplPhysiol 101, 256-
263.
Clark BC, Manini TM, Bolanowski SJ & Ploutz-Snyder LL. (2006b). Adaptations in
human neuromuscular function following prolonged unweighting: II.
Neurological properties and motor imagery efficacy. J ApplPhysiol 101, 264-
272.
32
Cormery B, Beaumont E, Csukly K & Gardiner P. (2005). Hindlimb unweighting for 2
weeks alters physiological properties of rat hindlimb motoneurones. J Physiol
568, 841-850.
Crone C, Hultborn H, Jespersen B & Nielsen J. (1987). Reciprocal Ia inhibition between

ankle flexors and extensors in man. J Physiol 389, 163-185.
Crone C, Hultborn H, Mazieres L, Morin C, Nielsen J & Pierrot-Deseilligny E. (1990).

Sensitivity of monosynaptic test reflexes to facilitation and inhibition as a
function of the test reflex size: a study in man and the cat. ExpBrain Res 81, 35-
45.
Crone C & Nielsen J. (1989a). Methodological implications of the post activation

depression of the soleus H-reflex in man. ExpBrain Res 78, 28-32.
Crone C & Nielsen J. (1989b). Spinal mechanisms in man contributing to reciprocal

inhibition during voluntary dorsiflexion of the foot. J Physiol 416, 255-272.
Curtis DR & Eccles JC. (1960). Synaptic action during and after repetitive stimulation.
J Physiol 150, 374-398.
Davies CT, Rutherford IC & Thomas DO. (1987). Electrically evoked contractions of
the triceps surae during and following 21 days of voluntary leg immobilization.
European journal of applied physiology and occupational physiology 56, 306-
312.
de Leon RD, Hodgson JA, Roy RR & Edgerton VR. (1998). Locomotor capacity
attributable to step training versus spontaneous recovery after spinalization in
adult cats. Journal of neurophysiology 79, 1329-1340.
Duchateau J. (1995). Bed rest induces neural and contractile adaptations in triceps
surae. Med SciSports Exerc 27, 1581-1589.
Duchateau J & Hainaut K. (1987). Electrical and mechanical changes in immobilized

human muscle. J ApplPhysiol 62, 2168-2173.
Eccles JC. (1964). Presynaptic Inhibition in the Spinal Cord. Progress in brain research
12, 65-91.
Enriquez-Denton M, Morita H, Christensen LO, Petersen N, Sinkjaer T & Nielsen JB.

(2002). Interaction between peripheral afferent activity and presynaptic
inhibition of ia afferents in the cat. J Neurophysiol 88, 1664-1674.
Faist M, Dietz V & Pierrot-Deseilligny E. (1996). Modulation, probably presynaptic in

origin, of monosynaptic Ia excitation during human gait. Experimental brain
research Experimentelle Hirnforschung 109, 441-449.
33
Faist M, Mazevet D, Dietz V & Pierrot-Deseilligny E. (1994). A quantitative
assessment of presynaptic inhibition of Ia afferents in spastics. Differences in
hemiplegics and paraplegics. Brain 117 ( Pt 6), 1449-1455.
Frank K & Fuortes M. (1957). Presynaptic and postsynaptic inhibition of monosynaptic

reflexes. Federation Proceeding, 39-40.
Gallego R, Kuno M, Nunez R & Snider WD. (1979). Disuse enhances synaptic efficacy
in spinal mononeurones. J Physiol 291, 191-205.
Gondin J, Guette M, Maffiuletti NA & Martin A. (2004). Neural activation of the

triceps surae is impaired following 2 weeks of immobilization. EurJ ApplPhysiol
93, 359-365.
Grey MJ, Klinge K, Crone C, Lorentzen J, Biering-Sorensen F, Ravnborg M & Nielsen

JB. (2008). Post-activation depression of Soleus stretch reflexes in healthy and
spastic humans. Experimental brain research Experimentelle Hirnforschung
185, 189-197.
Gruber M, Taube W, Gollhofer A, Beck S, Amtage F & Schubert M. (2007). Training-

specific adaptations of H- and stretch reflexes in human soleus muscle. J
MotBehav 39, 68-78.
Hultborn H, Brownstone RB, Toth TI & Gossard JP. (2004). Key mechanisms for
setting the input-output gain across the motoneuron pool. Progress in brain
research 143, 77-95.
Hultborn H, Illert M, Nielsen J, Paul A, Ballegaard M & Wiese H. (1996). On the

mechanism of the post-activation depression of the H-reflex in human subjects.
ExpBrain Res 108, 450-462.
Hultborn H, Meunier S, Morin C & Pierrot-Deseilligny E. (1987a). Assessing changes

in presynaptic inhibition of I a fibres: a study in man and the cat. J Physiol 389,
729-756.
Hultborn H, Meunier S, Pierrot-Deseilligny E & Shindo M. (1987b). Changes in

presynaptic inhibition of Ia fibres at the onset of voluntary contraction in man. J
Physiol 389, 757-772.
Hultborn H & Nielsen JB. (1998). Modulation of transmitter release from Ia afferents
by their preceding activity - a 'postactivation depression'. In Presynaptic
inhibition & Neural Control, ed. Rudomin PM, L., pp. 178-191. Oxford
University Press, New York.
Iles JF. (1996). Evidence for cutaneous and corticospinal modulation of presynaptic
inhibition of Ia afferents from the human lower limb. J Physiol 491 ( Pt 1), 197-
207.
34
Iles JF & Roberts RC. (1986). Presynaptic inhibition of monosynaptic reflexes in the
lower limbs of subjects with upper motoneuron disease. Journal of neurology,
neurosurgery, and psychiatry 49, 937-944.
Jankowska E & Padel Y. (1984). On the origin of presynaptic depolarization of group I

muscle afferents in Clarke's column in the cat. Brain research 295, 195-201.
Kandel ER, H. SJ & Jessell TM. (2000). Principles of Neural Science McGraw-Hill
Fourth Edition.
Katz R, Meunier S & Pierrot-Deseilligny E. (1988). Changes in presynaptic inhibition

of Ia fibres in man while standing. Brain 111 ( Pt 2), 417-437.
Kawakami Y, Akima H, Kubo K, Muraoka Y, Hasegawa H, Kouzaki M, Imai M,

Suzuki Y, Gunji A, Kanehisa H & Fukunaga T. (2001). Changes in muscle size,
architecture, and neural activation after 20 days of bed rest with and without
resistance exercise. EurJ ApplPhysiol 84, 7-12.
Kernell D & Hultborn H. (1990). Synaptic effects on recruitment gain: a mechanism of

importance for the input-output relations of motoneurone pools? Brain research
507, 176-179.
Kohn AF, Floeter MK & Hallett M. (1997). Presynaptic inhibition compared with
homosynaptic depression as an explanation for soleus H-reflex depression in
humans. Experimental brain research Experimentelle Hirnforschung 116, 375-
380.
Lev-Tov A & Pinco M. (1992). In vitro studies of prolonged synaptic depression in the
neonatal rat spinal cord. J Physiol 447, 149-169.
Llewellyn M, Yang JF & Prochazka A. (1990). Human H-reflexes are smaller in

difficult beam walking than in normal treadmill walking. Experimental brain
Lundbye-Jensen J & Nielsen JB. (2008). Central nervous adaptations following one
week of wrist and hand immobilization. J Appl Physiol.
Manabe T, Kaneko S & Kuno M. (1989). Disuse-induced enhancement of Ia synaptic

transmission in spinal motoneurons of the rat. J Neurosci 9, 2455-2461.
Meunier S, Kwon J, Russmann H, Ravindran S, Mazzocchio R & Cohen L. (2007).

Spinal use-dependent plasticity of synaptic transmission in humans after a single
cycling session. J Physiol 579, 375-388.
Meunier S & Pierrot-Deseilligny E. (1989). Gating of the afferent volley of the

monosynaptic stretch reflex during movement in man. J Physiol 419, 753-763.
35
Meunier S & Pierrot-Deseilligny E. (1998). Cortical control of presynaptic inhibition of
Ia afferents in humans. Experimental brain research Experimentelle
Hirnforschung 119, 415-426.
Meunier S, Pierrot-Deseilligny E & Simonetta M. (1993). Pattern of monosynaptic

heteronymous Ia connections in the human lower limb. Experimental brain
Mizuno Y, Tanaka R & Yanagisawa N. (1971). Reciprocal group I inhibition on triceps

surae motoneurons in man. Journal of neurophysiology 34, 1010-1017.
Morita H, Petersen N & Nielsen J. (1998). Gating of somatosensory evoked potentials

during voluntary movement of the lower limb in man. ExpBrain Res 120, 143-
152.
Nielsen J & Kagamihara Y. (1993a). Differential projection of the sural nerve to early
and late recruited human tibialis anterior motor units: change of recruitment
gain. Acta Physiol Scand 147, 385-401.
Nielsen J & Kagamihara Y. (1993b). The regulation of presynaptic inhibition during co-
contraction of antagonistic muscles in man. J Physiol 464, 575-593.
Nielsen J, Petersen N, Ballegaard M, Biering-Sorensen F & Kiehn O. (1993). H-reflexes

are less depressed following muscle stretch in spastic spinal cord injured patients
than in healthy subjects. ExpBrain Res 97, 173-176.
Nielsen J, Petersen N & Crone C. (1995). Changes in transmission across synapses of Ia

afferents in spastic patients. Brain 118 ( Pt 4), 995-1004.
Nielsen JB & Sinkjaer T. (2002). Afferent feedback in the control of human gait. J
ElectromyogrKinesiol 12, 213-217.
Pearson KG. (2000). Plasticity of neuronal networks in the spinal cord: modifications in
response to altered sensory input. Progress in brain research 128, 61-70.
Perez MA, Lundbye-Jensen J & Nielsen JB. (2007). Task-specific depression of the
soleus H-reflex after co-contraction training of antagonistic ankle muscles.
Journal of neurophysiology.
Perez MA, Lungholt BK & Nielsen JB. (2005). Presynaptic control of group Ia afferents
in relation to acquisition of a visuo-motor skill in healthy humans. J Physiol 568,
343-354.
Petersen N, Morita H & Nielsen J. (1998). Evaluation of reciprocal inhibition of the

soleus H-reflex during tonic plantar flexion in man. J NeurosciMethods 84, 1-8.
36
Pierrot-Deseilligny E. (1996). Transmission of the cortical command for human
voluntary movement through cervical propriospinal premotoneurons. Progress
in neurobiology 48, 489-517.
Pierrot-Deseilligny E, Morin C, Bergego C & Tankov N. (1981). Pattern of group I

fibre projections from ankle flexor and extensor muscles in man. Experimental
brain research Experimentelle Hirnforschung 42, 337-350.
Pinco M & Lev-Tov A. (1993). Modulation of monosynaptic excitation in the neonatal

rat spinal cord. Journal of neurophysiology 70, 1151-1158.
Rudomin P. (1990). Presynaptic inhibition of muscle spindle and tendon organ afferents
in the mammalian spinal cord. Trends in neurosciences 13, 499-505.
Rudomin P. (2002). Central control of information transmission through the intraspinal

arborizations of sensory fibers examined 100 years after Ramon y Cajal.
Progress in brain research 136, 409-421.
Rudomin P & Schmidt RF. (1999). Presynaptic inhibition in the vertebrate spinal cord
revisited. ExpBrain Res 129, 1-37.
Sanes JN & Donoghue JP. (2000). Plasticity and primary motor cortex.
AnnuRevNeurosci 23, 393-415.
Schmidt RF. (1971). Presynaptic inhibition in the vertebrate central nervous system.
Ergebnisse der Physiologie, biologischen Chemie und experimentellen
Pharmakologie 63, 20-101.
Seki K, Kizuka T & Yamada H. (2007). Reduction in maximal firing rate of

motoneurons after 1-week immobilization of finger muscle in human subjects. J
Electromyogr Kinesiol 17, 113-120.
Seki K, Taniguchi Y & Narusawa M. (2001a). Alterations in contractile properties of

human skeletal muscle induced by joint immobilization. J Physiol 530, 521-532.
Seki K, Taniguchi Y & Narusawa M. (2001b). Effects of joint immobilization on firing

rate modulation of human motor units. J Physiol 530, 507-519.
Thom JM, Thompson MW, Ruell PA, Bryant GJ, Fonda JS, Harmer AR, De JX &
Hunter SK. (2001). Effect of 10-day cast immobilization on sarcoplasmic
reticulum calcium regulation in humans. Acta Physiol Scand 172, 141-147.
Vandenborne K, Elliott MA, Walter GA, Abdus S, Okereke E, Shaffer M, Tahernia D

& Esterhai JL. (1998). Longitudinal study of skeletal muscle adaptations during
immobilization and rehabilitation. Muscle Nerve 21, 1006-1012.
37
White MJ, Davies CT & Brooksby P. (1984). The effects of short-term voluntary
immobilization on the contractile properties of the human triceps surae. QJ
ExpPhysiol 69, 685-691.
Wolpaw JR. (2007). Spinal cord plasticity in acquisition and maintenance of motor
skills. Acta physiologica (Oxford, England) 189, 155-169.
Wolpaw JR, Braitman DJ & Seegal RF. (1983a). Adaptive plasticity in primate spinal
stretch reflex: initial development. Journal of neurophysiology 50, 1296-1311.
Wolpaw JR, Kieffer VA, Seegal RF, Braitman DJ & Sanders MG. (1983b). Adaptive
plasticity in the spinal stretch reflex. Brain research 267, 196-200.
LEGENDS
Fig. 1 Spinal mechanisms. During the experiments EMG was obtained from SOL and
TA muscles. Peripheral nerve electrical stimulation was delivered to the posterior tibial
nerve (PTN), the common peroneal nerve (CPN) and the femoral nerve (FN) in order to
investigate disynaptic reciprocal inhibition, ‘classical’ presynaptic inhibition and post
activation depression.
Fig. 2 Maximal Voluntary Contraction Torque (MVC) was measured during ankle
joint plantar flexion and dorsi flexion. Measures of maximal isometric muscle strength
(MVC) before immobilization (gray bars), after immobilization (black bars) and after
one week of recovery (white bars). The figure illustrates group mean peak torque (Nm)
± SD for plantar flexion and dorsiflexion. * denotes p 0.05, *** denotes p<0.001
Fig. 3 Hoffmann reflex and M-wave recruitment curves & Hmax/Mmax ratios
Hoffmann reflexes were elicited in SOL before (gray), after (black) and 2 weeks after
38
immobilization (white). The upper figure illustrates Hoffmann reflex (circles) and M-
wave (squares) recruitment curves for a representative subject. The abscissa represents
the stimulation intensity normalized to the M-wave threshold in pretest 1, the ordinate
illustrates response amplitude normalized to the corresponding Mmax. The lower figure
illustrates group average Hmax/Mmax ratios (± SD) before (pre1 and pre2), after (cast
removal) and 2 weeks after (retest) immobilization. * denotes p 0.05.
Fig. 4: Measurement of disynaptic reciprocal inhibition from ankle dorsiflexors to
ankle plantar flexors by the H-reflex technique at rest. The time course (at rest) of
the effect of conditioning peroneal nerve stimulation (single 1ms) stimuli applied to the
peroneal nerve 2-3 cm distal to the caput fibulae at 1.1 x MT) on the size of the soleus
test H-reflex. (This was evoked by single 1 ms stimuli applied to the tibial nerve in the
popliteal fossa. Size of the unconditioned soleus H-reflex 20–25% of Mmax.) The
abscissa shows the interval in ms between the conditioning stimulus and the test
stimulus. The ordinate shows the size of the conditioned test reflex expressed in
percentage of the test reflex. The time course was obtained before immobilization
(pretests 1, light grey and 2, dark grey) after immobilization (posttest, black) and 2
weeks after cast removal (retest, white). The short-latency, presumed disynaptic,
reciprocal inhibition was seen at conditioning-test (CT) intervals between 2 and 4 ms. It
was expressed as the size of the conditioned H-reflex as a percentage of the control H-
reflex size. The figure illustrates group average ±SD.
Fig. 5 Presynaptic inhibition estimated from SOL H-reflex facilitation by
conditioning stimulation of the femoral nerve (FN). The soleus H reflex was
39
facilitated by a heteronymous Ia volley from quadriceps and the amount of reflex
facilitation was used to estimate the size of the conditioning I a excitatory post-synaptic
potential (e.p.s.p). The upper part of fig.5 illustrates the time course (at rest) of the
effect of conditioning femoral nerve (FN) stimulation (single 1ms) stimuli applied to the
FN at the femoral triangle at 1.5 x MT) on the size of the soleus test H-reflex. Size of
the unconditioned soleus H-reflex 20–25% of Mmax. Conditioning test intervals were -8
to -3 ms. The abscissa shows the interval in ms between the conditioning stimulus and
the test stimulus. The ordinate shows the size of the conditioned test reflex expressed in
percentage of the test reflex. The time course was obtained before immobilization
weeks after cast removal (retest, white). Based on the onset of facilitation a detailed
time course in steps of 0.2 ms was obtained for each subjects in each test. The bottom of
fig.5 illustrates this facilitation at best individual conditioning test intervals before, after
and 2 weeks after immobilization. All depicted values in fig. 5 are group average ± SD.
* denotes p 0.05.
Fig. 6 D2 inhibition. Time course of the long-latency depression of the SOL H-reflex
(D2 inhibition) evoked at 40-100 ms conditioning–test interval by conditioning peroneal
nerve stimulation (single 1ms) stimuli applied to the peroneal nerve 2-3 cm distal to the
caput fibulae at 1.1 x MT). The abscissa shows the interval in ms between the
conditioning stimulus and the test stimulus. The ordinate shows the size of the
conditioned test reflex expressed in percentage of the test reflex.The time course was
obtained before immobilization (pretests 1, light grey and 2, dark grey) after
40
immobilization (posttest, black) and 2 weeks after cast removal (retest, white). All
depicted values in fig. 6 are group average ± SD. * denotes p 0.05.
Fig. 7 Post activation depression. The figure illustrates the frequency-related
depression of the SOL H-reflex at interstimulus intervals of 1s relative to 10s. In A) the
ordinate shows the average amplitude of the SOL H-reflex obtained at 1s ISI in
percentage of the average SOL H-reflex amplitude obtained at 10s ISI. In B) the
ordinate shows the amount of the frequency related (post activation) depression from
the control reflex obtained at 10s ISI. The data were obtained before immobilization
weeks after cast removal (retest, white). All depicted values in fig. 6 are group average
± SD. * denotes p 0.05.
41
42
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Immobilization induces changes in presynaptic control of group Ia afferents in healthy
humans
Jesper Lundbye-Jensen and Jens Bo Nielsen
J. Physiol. published online Jul 3, 2008;
DOI: 10.1113/jphysiol.2008.156547
This information is current as of August 4, 2008
Updated Information including high-resolution figures, can be found at:

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Immobilization induces changes in presynaptic control of group Ia afferents in healthy

Jesper Lundbye-Jensen and Jens Bo Nielsen

J. Physiol. published online Jul 3, 2008;

This information is current as of August 4, 2008

The latest version of this article is at:

Downloaded from jp.physoc.org by Marco Narici on August 4, 2008

Immobilization induces changes in presynaptic control of group Ia afferents in

Jesper Lundbye-Jensen1,2*, Jens Bo Nielsen1,2

Panum Institute 24.6 Blegdamsvej 3 DK-2200 København N Denmark

Panum Institute 24.6 DK-2200 København N Denmark

Department of Neuroscience and Pharmacology & Department of Exercise & Sport

The Panum Institute 22.3

Running title: immobilization changes presynaptic control of group Ia afferents

To elucidate the mechanisms contributing to the increase in H-reflex size following

bodied subjects. Disynaptic reciprocal inhibition of soleus (SOL) motoneurones and

immobilization as well as following 2 weeks of recovery.

Following immobilization maximal voluntary plantar- and dorsiflexion torque (MVC)

changes in Mmax. Decreased presynaptic inhibition of the Ia afferents likely contributed

evidence of changes in presynaptic inhibition of SOL Ia afferents and taken together

suggest that GABAergic presynaptic inhibition of the SOL Ia afferents is decreased

at intervals shorter than 10 s (homosynaptic post-activation depression) also decreased

following immobilization, suggesting that the activity-dependent regulation of

no significant changes in disynaptic reciprocal Ia inhibition. Two weeks after cast

removal measurements returned to pre immobilization levels.

interneuronal circuitries responsible for presynaptic control of sensory input to the

immobilization as well as the development of spasticity following central motor lesions.

structural plasticity within the central nervous system. Experience-driven central

nervous plasticity has been extensively documented at a cortical level in relation to

paralleled by changes in the properties of the spinal neuronal circuitries as demonstrated

2005; Meunier et al., 2007; Wolpaw, 2007).

by changes in transmission in spinal neuronal circuitries. Spinal cord plasticity can

and descending inputs.

similarly observed increased H-reflex amplitudes at rest following 4 weeks of lower

limb suspension. We have recently demonstrated an increase in the excitability of H-

no significant changes in corticospinal excitability estimated from motor evoked

potentials (MEPs) evoked by transcranial magnetic stimulation (TMS). Although this

hypothesis that immobilization may be accompanied by changes in the amount of

presynaptic inhibition of the Ia afferents or post activation depression (homosynaptic

amplitude modulation is explained by changes in presynaptic inhibition of the synapses

of Ia afferents on spinal motoneurones (Hultborn et al., 1987a; Hultborn et al., 1987b;

Presumably, gating of sensory inputs to spinal motoneurones is of functional importance

2002). Furthermore, regulation of sensory information at the presynaptic level may

permit conveyance of proprioceptive information to the cortex, which might contribute

the findings of Manabe et al. (1989) who observed a disuse-induced enhancement of Ia

synaptic transmission in spinal motoneurones of the rat.

The hypothesis that immobilization induces modifications of the transmission in the

monosynaptic Ia pathway through changes in presynaptic control mechanisms was

consequently tested in the present study.

immobilization and following a corresponding period of recovery. Disynaptic reciprocal

common peroneal nerve (CPN). To evaluate presynaptic inhibition of the soleus Ia

1971). To assess post-activation (homosynaptic) depression we used the frequency-

Kohn et al., 1997).

committee (J. No. KF 100.1969/1991). The experimental procedures conformed to the

standards set by the latest revision of the Declaration of Helsinki.

All subjects participated in 4 identical experimental sessions on 4 separate days. In

order to assess day-to-day variability, measurements were obtained on two different

days prior to immobilization (pretests). In addition, subjects were tested immediately