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Restless legs syndrome (RLS) is a disabling sensorimotor disorder associated with sleep disturbances and excessive sleepiness during the day.1 Periodic leg movements during sleep (PLMs) are found in 80% of RLS patients.1 Research studies addressing the functional anatomy of RLS have shown derangement of supraspinal inhibitory systems2 and impaired spinal cord circuitry regulation in RLS patients with associated PLMs.3, 4
Ib interneurons are involved in agonist-antagonist reciprocal modulation and exert some influences on the spinal locomotor rhythm generator.5 These interneurons receive afferents inputs from Golgi tendon organs, other segmental afferents, i.e., group Ia and cutaneous, and from descending systems.6 Ib pathways have been found deranged in pathological states involving spinal circuitry motor control.7–9
To further explore spinal cord excitability changes in primary RLS, we evaluated the soleus H reflex threshold, latency, amplitude, Hmax/Mmax ratio, and the modulation of group I nonreciprocal inhibition according to the method of Pierrot-Deseilligny and colleagues.10
SUBLECTS AND METHODS
Seven subjects with primary RLS and 10 healthy age-matched volunteers were enrolled in the study. All subjects gave informed consent for the experimental procedure. Patients fulfilled International RLS study group diagnostic criteria1 and were medication free at the neurophysiological evaluation. All had a history of PLMs reported by bed partners. International Restless Legs Syndrome Study Group restless legs syndrome rating scale for severity (IRLSSGRS) was administered on the morning of the study.11 The local department ethical committee gave approval for the study.
All experiment described herein were performed in the morning in a silent constant temperature room. Subjects were comfortably seated in a purpose-built armchair for the reflex activity study with the examined leg fixed, the knee flexed at 120° and the ankle at 100°. All experiments were performed on the right leg. A strict methodology was applied to evoke the H reflex.12 For testing Ib inhibition, we followed the procedures described by Pierrot-Deseilligny et al.10 A constant current stimulator (GRASS S88) delivered single rectangular 1 ms duration stimuli to the posterior tibial nerve in the popliteal fossa through a ball electrode (Simon's electrode) to evoke test H reflexes. Threshold was defined as the minimal stimulation intensity at which a reflex response with the characteristic of H reflex was detectable. Latency and peak to peak amplitude of conditioned and unconditioned H responses were automatically determined. The ratio of maximal H reflex (Hmax) to the maximal direct motor response (Mmax) was calculated from 10 measurements. For group Ib nonreciprocal inhibition evaluation test stimulus intensity was adjusted so that the reflex amplitude corresponded to 15% (10–20%) of the soleus maximal direct motor response (M response) to ensure a comparable sensitivity to inhibition and facilitation.13 The test response was conditioned by a 0.5 ms electrical shock applied through a second stimulator positioned over the gastrocnemius medialis (GM) nerve, 6 to 10 cm distal to the electrodes stimulating the posterior tibial nerve. Intensity was adjusted to 95% of motor threshold for the GM nerve. Subjects were frequently reminded not to change the posture of neck, arms and shoulders. Stimulation was repeated if electromyographic activity was recorded.
H reflexes were conditioned by stimuli delivered to the GM nerve at 2–4–5–6–8–10 ms before stimuli eliciting H reflex. For each interstimulus interval, paired and unpaired stimulations were randomly delivered with an interval at least of 15 seconds. Electrical soleus reflex activities were recorded through Ag-AgCl surface electrodes placed over the belly of the soleus muscle. A ground electrode was placed over the calf between recording and stimulating electrodes. After amplification and filtering (band pass 20 Hz – 10 kHZ) the signals were digitized with a sampling frequency of 10 kHz (BIOPAC) and stored on a PC with a dedicated software package (LIGHT) for subsequent analysis.
Ten unconditioned and 10 conditioned responses were obtained for each interstimulus interval. The average of 10 conditioned responses was compared to the average of 10 unconditioned responses. Changes in the amplitude of H reflex were expressed as percentages of its unconditioned values and plotted in terms of delays separating conditioning and test stimulation.
The mean values of threshold, latency, and Hmax/Mmax ratio in the normal group (N) were compared with the values obtained in patients (RLS) using one way ANOVA. Changes in conditioned H-reflex mean amplitude at each interstimulus interval were used as the primary outcome. We verified the nonsignificance of Box's test of equality of covariance matrices and Mauchly's test of Sphericity. A repeated measures analysis of variance between percentage of inhibition of control subjects and RLS patients was made (SPSS 15.0 for windows) with the inner-subject factor interstimulus interval and the inter-subject factor group. Post hoc testing was done using the least significant differences (LSD) method following correction for multiple tests. For correlations we used the Pearson test. Differences were considered significant if P < 0.05.
The two groups N and RLS did not differ for the H reflex mean threshold (N = 12.6 ± 3.1 mA vs. RLS 13.2 ± 3.8 mA; ns), latency (N = 35.1 ± 0.1 ms vs. RLS = 35.3 ± 0.5 ms; ns) or the Hmax/Mmax ratio (N = 0.38 vs. RLS = 0.41; ns).
Mean changes in amplitude of the conditioned H reflex expressed as percentage of control values in N and RLS groups are shown in Table 1 with standard error of the mean (SEM). Figure 1 (filled triangles) illustrates soleus H reflex amplitude reduction in control subjects. Amplitude was reduced from 4 to 10 ms of interstimulus interval with a maximum reduction at 6 ms (79.6% of control value). Figure 1 (empty circles) illustrates the mean ± SEM results obtained after various delays in the RLS group. The curve reveals facilitation from 2 to 10 ms of interstimulus interval with an amplitude peak at 4 ms (135.3% of basal value). Box's test of equality of covariance matrices (P = 0.2) and Mauchly's test of Sphericity (P = 0.1) were not statistically significant. The repeated measure analysis of variance demonstrated a significant difference between patients and control group (F = 9.53 P = 0.007). According to post hoc testing of the different interstimulus intervals, the mean amplitude differences between RLS patients and controls were statistically significant at 4 ms (P = 0.043), 5 ms (P = 0.007) and 6 ms (P = 0.001), indicating the replacement of short latency autogenic inhibition in RLS patients with a facilitation (Table 1). Clinical scores did not correlate with the area under the curve between the recovery curve of the control subjects and the RLS patients (R = 0.411; P = 0.359). Figure 2 shows autogenic inhibition in one RLS patient and in one control subject at different interstimuli intervals.
Table 1. Soleus conditioned H reflex amplitude changes in RLS and normal subjects at each interstimulus intervals
Patients (n = 7)
Controls (n = 10)
P = 0.043.
P = 0.007.
P = 0.001 (LSD test) in comparison between patients and controls.
ISI, inter-stimulus interval; SEM, standard error of the mean.
Electrophysiological investigations have show abnormalities in RLS motor excitability pathways, with or without PLMs.3, 4 Entezari-Taher et al.2 found a normal peripheral silent period with a shortened central silent period, and suggested a supraspinal impairment of inhibitory system in RLS. Bara-Jimenez et al.3 evaluated state dependent changes in spinal cord flexor reflex (FRs) excitability of 10 RLS patients. On the basis of electrophysiological and polysomnographic findings, it was suggested that PMLs in RLS and FRs share a common spinal mechanism and that PMLs may result from enhanced spinal cord excitability in RLS patients. Video-polysomnographic and electromyographic recordings of the PMLs motor pattern distribution during sleep in idiopathic RLS patients concluded for an enhanced spinal cord excitability not spreading along pyramidal pathways.14, 15 In addition, a different motor phenomenon, i.e., propriospinal myoclonus, detected in RLS16 further suggests spinal hyperexcitability. The origin of the enhanced spinal cord excitability may be sought in primary dysfunction of spinal effectors, changes in interneuronal circuitry at spinal level itself, modified influences from descending pathways, or a combination of these three possibilities.4
Enhanced RLS and PLMs described in patients with spinal cord lesions have been considered to favor the hyperexcitability of motoneurones,17 but the Hmax/Mmax ratio and the threshold level of motoneurones pool excitability in RLS patients have been found unmodified4, 18 (present data).
Rijsman et al.4 demonstrated an abnormal late facilitation (about 200 ms of interstimulus interval) and late inhibition (between 300 and 500 ms of interstimulus interval) in the RLS H-reflex recovery curve, confirming earlier observations,19 and a reduced H reflex vibratory inhibition. They accounted for these findings hypothesizing changes in inhibition or facilitation through descending spinal tracts, or changes in inter neural circuitry at spinal level itself.4
Our findings disclosed a significantly less active spinal autogenic inhibition in therapy free RLS patients. The decrease in Ib inhibition observed in RLS patients may be a consequence of different mechanisms acting on Ib inhibition.
Nonreciprocal group I (Ib) inhibition is considered an example of “reflex reversal,”20 since during locomotion, even in rudimentary form, inhibition of motoneurones is suppressed and a Ib excitation emerges.5, 21 Our data, however, were obtained from subjects examined at rest, i.e., in a sitting position without any locomotor or loading activation.5 Nevertheless, PMLs may be a fragment of a locomotor pattern embedded within the spinal cord,14 so that spontaneous switching of spinal excitability toward a higher level is a conceivable explanatory mechanism of the decreased Ib inhibition observed.
An abnormal sensorimotor integration could be responsible for a derangement of spinal inputs, with a decreased Ib inhibition, as suggested by patients with neuropathy developing RLS.22, 23 No genetic or acquired causes of neuropathy characterized our patients with normal neurological evaluation, and an abnormal afferent input along Ia fibers is excluded by the normal values of latencies and amplitude of H reflex of RLS patients.24
We cannot exclude group I excitation (Ia) overwhelming transmission across the Ib inhibitory pathway. Differences in Ia afferences are not a convincing explanatory mechanism, and the normal threshold and Hmax/Mmax ratio found in RLS patients play against this hypothesis.
A diminished axo-axonal presynaptic inhibition of Ia terminals, with a possible role for postactivation depression or neurotransmitter depletion have been suggested by Rijsman et al.4 to explain reduced vibratory inhibition. However, they did not exclude a possibly modulatory effect of aminergic or dopaminergic drive acting on presynaptic gabaergic inhibition. The latter mechanism is in agreement with growing evidence in favor of an involvement of spinal dopaminergic drive in the pathophysiology of RLS.25
Taken together, the available neurophysiological findings, i.e., flexor reflexes enhancement, defective spinal interneuronal circuitry activity, lowered presynaptic Ia control, and reduction of nonreciprocal group I inhibition (or facilitation), all suggest spinal cord hyperexcitability in RLS, and are compatible with the involvement of supraspinal control.
Neurophysiological findings in RLS could depend on a modification in distinct descending pathways or on variations of the same tract. Both the corticospinal and reticulospinal tract may exert similar effects on spinal circuitry.26 Again, increased Hmax/Mmax ratios in RLS patients27 have not been subsequently confirmed4, 18 and were unchanged in the present study. In addition, temporal analysis of the recruitment pattern of PLMs in RLS ruled out constant caudal or rostral propagation,14, 15 thereby excluding a spread along corticospinal pathways. Alternatively, propriospinal pathways have been implicated as the spreading generators of abnormal involuntary muscles activity.28 Recent human and experimental studies focused on the role of the ventral/dorsal reticulospinal tract in modulation of spinal motor control.26, 29 Takakusaki et al.29 experimentally demonstrated that the nonreciprocal inhibition from the medullary reticular formation (MRF), namely the medullary inhibitory region located in the nucleus reticularis giganto cellularis (NRGC), is mediated by fibers descending in the ventral spinal cord quadrant. In view of this, they suggested that the ventral reticulospinal tract inhibits flexor reflex afferents at the level of last order interneurons and excites a group of Ib inhibitory interneurons. The NRGC also exerts a facilitatory influence on presynaptic Ia inhibition.26 A dysfunction of the ventral reticulospinal tract could induce all the electrophysiological abnormalities found in RLS with or without PMLs. In addition, MRF neuronal activity varies with sleep-awake cycle and locomotion,29 and MRF state related dysfunction is in agreement with the RLS circadian rhythm and symptoms improvement with limb movements.22 Our data demonstrated a reduced Ib inhibition in RLS patients in the morning suggesting a defective “basal” setting excitability level of NRGC. Circadian modulation of Ib inhibition in normal subjects and RLS patients are currently under investigation.
We are aware of the limits of present study, i.e., a challenge with acute or chronic dopaminergic therapy,25 however, the reduced inhibition, or facilitation, in group I nonreciprocal inhibition found in RLS can be taken as additional, albeit indirect, proof of spinal hyperexcitability with involvement of supraspinal control systems in RLS.
Further studies should compare the nonreciprocal group I inhibition pattern in RLS patients in basal condition, during different phases of the circadian rhythm, and with dopaminergic therapy.
This work was supported by MURST, RFo (ex 60%) 2005–2006 grant. Dr. Scaglione's work was supported by a research grant from Fondazione Cassa di Risparmio-Bologna, Italy. We also acknowledge Anne Collins for editing the text.