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Hemicerebellectomy impairs the modulation of cutaneomuscular reflexes by the motor cortex following repetitive somatosensory stimulation
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Hemicerebellectomy impairs the modulation of cutaneomuscular reflexes by the motor cortex following repetitive somatosensory stimulation Nordeyn Oulad Ben Taib a , Mario Manto b,⁎ a
Service de Neurochirurgie, Hôpital Erasme-ULB, Bruxelles, Belgium Laboratoire de Neurologie Expérimentale, Hôpital Erasme-ULB, Bruxelles, Belgium
b
A R T I C LE I N FO
AB S T R A C T
Article history:
We examined the cutaneomuscular reflex of the plantaris muscle of rats in response to
Accepted 16 March 2006
cutaneous stimulation in isolation and in conjunction with subthreshold high-frequency
Available online 25 April 2006
trains of stimuli applied on the motor cortex, prior to and following repetitive peripheral stimulation. The cutaneomuscular reflex was also investigated under the same paradigm
Keywords:
following hemicerebellectomy. The enhancement of cutaneomuscular responses
Cerebellum
associated with subthreshold high-frequency trains of stimulation following repetitive
Sensory input
peripheral stimulation was prevented by hemicerebellectomy. Our results suggest that
Sciatic nerve
the pathways passing through the cerebellum are involved in the calibration of
Cutaneomuscular response
cutaneomuscular responses. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Cortical network functions require a maintenance and a fine tuning of the excitability of the motor cortex (Kaelin-Lang et al., 2002; Knash et al., 2003; Luft et al., 2002, 2005). It has been shown previously that a prolonged period of peripheral nerve stimulation can induce a lasting increase of corticomotoneuronal responses both in rodents and in human (Knash et al., 2003; Luft et al., 2002, 2005; Manto et al., 2006). It is assumed that the somatosensory input adjusts the excitability of motor pathways by influencing the motor cortex (Kaelin-Lang et al., 2002; Luft et al., 2002). Enhanced excitability indicates that the sensorimotor system is sensitized and could therefore learn quicker. Increasing the excitability of the motor cortex might have beneficial effects on motor training procedures or could improve the performance of novel motor tasks (Kaelin-Lang et al., 2002; Knash et al., 2003).
In an attempt to understand how the cerebellum interacts with the motor cortex and shapes motor commands, we recently tested the hypothesis that the cerebellum could be involved in the adaptative changes of the intensity of the corticomotor responses after a session of somatosensory stimulation. First, we showed that cerebellar nuclei contributed to the sensory modulation (Luft et al., 2005; Manto et al., 2006; Nixon, 2003; Oulad Ben Taib et al., 2004, 2005a). Second, we investigated the effects of hemicerebellectomy, which induces major motor deficits in rats (Oulad Ben Taib et al., 2005b; Tarnecki, 2003). Both disruption of cerebellar nuclei and hemicerebellectomy weakened the adaptation of the motor cortex to repetitive trains of peripheral stimulation (Luft et al., 2005). Taken together, these results underline that the cerebellum is part of the circuitry regulating the excitability of the motor cortex in a situation of steady somatosensory stimulation at the level of the peripheral nervous system.
⁎ Corresponding author. Neurologie-ULB, Fonds National de la Recherche Scientifique (FNRS), 808, Route de Lennik, 1070 Bruxelles, Belgium. Fax: +32 2 555 39 42. E-mail address: [email protected] (M. Manto). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.052
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Understanding how sensory information is used to shape motor commands has direct experimental and clinical applications. Recent studies suggest that the sensorimotor system has a modular organization, each reflex module performing sensorimotor transformations as a function of ‘efficacy’ of the muscle (Schouenborg, 2003). Error signals detected in the module are encoded by the cerebellum via spino–olivo–cerebellar pathways (Schouenborg, 2003), allowing a tuning of the sensorimotor transformation. According to this modular concept, (a) the circuitry is self-organizing and the weight distribution of the cutaneous input adjusts the synaptic organization of the module, (b) conditions that would enhance the efficacy of some muscles could reinforce the power of a given module during motor tasks. Repetitive peripheral stimulation meets the requirements to represent one of these conditions, enhancing the responses in the muscle depending on which peripheral nerve is stimulated (Luft et al., 2005). Moreover, cutaneomuscular responses could be very good candidates to understand the circuitry in reflex modules because their intensity is strongly correlated with background activity of muscles (Zehr and Chua, 2000). The pathways that normally pass through the cerebellum could influence important features of cutaneomuscular reflexes (Bloedel and Bracha, 1995; Timmann et al., 1996). Therefore, we conducted the following experiments: we checked whether cutaneomuscular reflexes were affected by repetitive peripheral stimulation and we tested the effects of trains of stimulation in the motor cortex on the intensity of cutaneomuscular reflexes. We also analyzed the effects of hemicerebellectomy on cutaneomuscular reflexes, and we tested the hypothesis that hemicerebellectomy influenced the adaptation of cutaneomuscular reflexes to peripheral repetitive stimulation.
2.
111
Results
2.1. Results in control group and in rats 48 h after operation Fig. 1 shows the full-wave rectified cutaneomuscular responses in a control rat and in a hemicerebellectomized rat. In the control rat, high-frequency stimulation of the right motor cortex did not modify the intensity of the cutaneomuscular response in the basal condition. The enhancement of the intensity of the cutaneomuscular response appeared following peripheral repetitive stimulation. This enhancement was absent in the hemicerebellectomized rat. Fig. 2 illustrates the values obtained in both groups. In the control group, statistical analysis confirmed an increase of the intensity of the integrated cutaneomuscular responses when trains of subthresholds stimuli were applied to the motor cortex after peripheral repetitive stimulation (P = 0.01 as compared to baseline). In the hemicerebellectomy group, there was no difference between baseline cutaneomuscular responses and following peripheral repetitive stimulation (P > 0.20). Values of integrated cutaneomuscular responses were significantly higher in the control group than in the hemicerebellectomy group following peripheral repetitive stimulation (inter-group effect; P = 0.009).
2.2.
Other control experiments
The intensity of the cutaneomuscular response in right plantaris muscle remained similar before and after peripheral repetitive stimulation of left sciatic nerve, both in control rats (P = 0.19) and in rats with left hemicerebellectomy (P = 0.27).
Fig. 1 – Cutaneomuscular responses in a control rat (top traces) and in a rat with left hemicerebellectomy (bottom traces), before (left and middle left) and after peripheral repetitive stimulation (middle right and right). From left to right: (A) cutaneous stimulation; (B) cutaneous stimulation combined with high-frequency subthreshold stimulation of the motor cortex; (C) cutaneous stimulation; (D) cutaneous stimulation combined with high-frequency subthreshold stimulation of the motor cortex. Cutaneomuscular responses recorded in left plantaris muscle. Responses are rectified and averaged from 10 successive trials.
112
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Fig. 2 – Cutaneomuscular responses in control rats (gray bars) and in hemicerebellectomized rats (white bars). Conditions 1 and 2: before peripheral repetitive stimulation of the sciatic nerve; conditions 3 and 4: after peripheral repetitive stimulation of the sciatic nerve. Conditions 1 and 3: cutaneous stimulation; conditions 2 and 4: cutaneous stimulation combined with high-frequency stimulation of the right motor cortex at a subthreshold level. Values are mean ± SD. Values are expressed in ms μV. *P < 0.05.
Stimulation of left motor cortex was associated with an enhancement of the cutaneomuscular response of the right plantaris muscle following peripheral repetitive stimulation of right sciatic nerve, both in the control group (P = 0.015) and in left hemicerebellectomized rats (P = 0.01).
3.
Discussion
The findings of this study are (1) the enhancement of the cutaneomuscular responses when subthresholds stimuli are applied in the contralateral motor cortex following a period of peripheral repetitive stimulation and (2) the absence of such a tuning following ipsilateral hemicerebellectomy. The organization of the motor cortex is greatly dependent on the balance between excitatory and inhibitory influences over the network of cortical connections (Luft et al., 2005). The cerebellar output exerts an excitatory effect on the contralateral motor cortex via the cerebello–thalamo–cortical pathway. This tract is the most probable candidate for providing the input for gating the information flow (Luft et al., 2005; Molinari et al., 2002). Cerebellar outputs are guided to the primary motor cortex via the ventrolateral thalamic group which projects mainly to layers IV and V (Sanes and Donoghue, 2000). Through this channel, inputs can modulate the efficacy of the interconnections among cortical neurons, adjusting the circuitry of the motor cortex (Luft et al., 2005; Sanes and Donoghue, 2000). Cutaneous reflexes change according to the behavioral context such that they function in an appropriate fashion to
assist motion (Zehr and Chua, 2000). Cutaneomuscular reflexes require the integrity of the dorsal columns, the sensorimotor cortex and the corticospinal tract (Gibbs et al., 1995). Cutaneous stimuli activate complex pathways that include spinal cord tracts (Floeter et al., 1998). The effects of the cerebellum on these pathways have not been elucidated, and many questions remain unsolved regarding the interaction between peripheral feedback and the function of these reflexes (Zehr and Chua, 2000). We previously observed a decreased excitability of the spinal cord in hemicerebellectomized rats, with depressed H-reflex recruitment and decreased excitability of the anterior horn, as described in rodents and in human (Drozdowski, 1995; Fox and Hitchcock, 1982; Oulad Ben Taib et al., 2005a,b). The efferences of cerebellar nuclei modify the excitability of segmental motoneurons via ascending and descending pathways (Bantli and Bloedel, 1975). Experimental data support the existence of an excitatory cerebello–thalamo–corticospinal pathway which affects the excitability of motoneurons since cooling of the motor cortex removes an excitatory component from the intracellularly recorded response evoked in lumbar motoneurons by dentate stimulation (Bantli and Bloedel, 1975). In addition, descending pathways from the brainstem, such as the rubrospinal tract, provide a part of the substrate by which the cerebellum participates in the control of muscle tension associated with limb movements. One of the important loops subserving the two-way communication between the sensory and motor systems is the spino–cerebello–rubro–spinal projection system (Tarnecki, 2003). This loop is linked to the musculature by the rubral projection to spinal motoneurons. Rubrospinal neurons receive major input from cerebellar nuclei (Courville, 1966) and show intense responses to sensory stimuli. Moreover, behavioral data indicate that the cerebello– rubral system is involved in learning and memory (Rosenfield and Moore, 1983; Tarnecki, 2003). Among cerebellar nuclei, the interpositus nucleus, which receives inputs from both the spinal cord and the sensorimotor cortex and which issues outputs to both the premotor neurons and the motor cortex, seems to have a major contribution in the modulation of the activity of the motor cortex when peripheral stimuli are applied (Luft et al., 2005; Oulad Ben Taib et al., 2004). Neurons in interpositus nuclei are especially involved in somesthetic reflex behaviors, with their discharges being tuned in relation to sensory feedback. The interpositus neurons integrate signals principally from the motor cortex, somatosensory cortex, premotor areas and spinal cord (Bosco and Poppele, 2003; Luft et al., 2005; Oulad Ben Taib et al., 2004). This convergence suggests a role in updating skilled movements. We aim to perform additional experiments such as cutting the superior or middle cerebellar peduncles or generating lesions in cerebellar cortex to increase our understanding of the results. Indeed, cerebellar cortex ablation removes the inhibitory effect of Purkinje cells on cerebellar nuclei. This causes a considerable increase in the background firing and eliminates the pauses in discharges occurring in responses generated by somatosensory stimuli. The choice of the pattern of stimulation is one of the factors influencing strongly the interaction between peripheral somatosensory stimulation and adaptation in the motor cortex (Luft et al., 2005). In human, some specific patterns of
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stimulation influence the excitability of spinal circuits, unlike others. The strength of reciprocal inhibition between ankle flexor and extensor muscles has been assessed before and after peroneal nerve stimulation at motor threshold intensity (Perez et al., 2003). Short-latency reciprocal inhibition from ankle flexor to extensor muscles was measured by conditioning the soleus H-reflex with stimulation of the common peroneal nerve. A short-term plasticity within inhibitory circuits in the spinal cord was disclosed. The pattern of sensory input appears to be a critical factor for inducing changes in the spinal circuit in humans. These integrative spinal mechanisms might be necessary to establish the compatibility with signals which converge in the cerebellum from spinal sensory and motor cortical areas (Bosco and Poppele, 2003). Somatosensory input is implicated in motor learning and in the recovery of function following a cerebral lesion and gives therapeutic benefits in functional recovery following vascular lesions (Johansson et al., 1993; Luft et al., 2005). Repetitive stimulation is currently used in human to enhance reorganization of sensorimotor representations (Johansson et al., 1993). The motor pathways sensitized by peripheral input could increase their learning capacities (Ziemann et al., 2001). It is assumed that the increased excitability represents an early electrophysiological adjustment of the sensorimotor pathways (Luft et al., 2005). In a second stage, this functional change is transformed into a structural plasticity. According to the sensory prediction hypothesis (Nixon, 2003), the cerebellum is necessary to make predictions based on past sensory events and is critical to implement such predictions in the sensorimotor system. The cerebellum is involved in learning to anticipate sensory events (Nixon, 2003). It operates with an outstanding degree of temporal precision for the formation of expectations arising from movements and the interactions with the environment. This precision forms the signature of skilled motor acts (Nixon, 2003). A basic function of cerebellar circuitry could be the online monitoring of sensory inputs (Luft et al., 2005; Manto et al., 2006; Oulad Ben Taib et al., 2004). By permitting the motor cortex to influence the intensity of cutaneomuscular responses in a context of repetitive sensory information, the pathways running through the cerebellum prior to ascending or descending to other nervous system structures facilitate the dynamic adaptation of the brain to consistent environmental changes.
4.
Experimental procedures
4.1.
Description of the procedures
Studies were performed following approval of the institutional animal care committee of the Free University of Brussels. The facilities housing animals are inspected on a regular basis and meet the current national regulations of Belgium. The laboratory has received the agreement to perform surgery in animals (Number LA1230492, Ministère des Classes Moyennes et de l'Agriculture, Belgium). Adequate food, water, ventilation and space are provided. Procedures to minimize discomfort were used during the experiments. Surgical procedures were
113
conducted by a neurosurgeon familiar with aseptic techniques (to minimize risks of infection), and animals were under close supervision on a daily basis. Postoperative monitoring and care were provided. Details of the procedures were given elsewhere (Oulad Ben Taib et al., 2004, 2005a,b). Male Wistar rats (weight: 250 to 300 g; left hemicerebellar ablation: n = 5) were anesthetized using an intraperitoneal administration of chloral hydrate (400 mg/kg ip). In the 5 rats, a caudal craniotomy was performed over one half of the cerebellum (Florenzano et al., 2002; Oulad Ben Taib et al., 2005b). The dura was exposed, incised and the left hemicerebellum was removed. Subsequently, the overlying flaps of skin were opposed and sutured. Animals were allowed to recover from anesthesia and surgical stress and had free access to water and food. At the end of the experiments, brains were dissected to evaluate the extent of the lesion after administration of an overdose of chloral hydrate ip. Histological verification of the lesion was performed. Complete ablation of the left cerebellar hemisphere and deep nuclei was carried out in all animals. There was no histological evidence of brainstem injury. We found no evidence of brain infection. Forty-eight hours after surgery, we investigated the responses evoked in the left plantaris muscle following stimulation of the left plantar skin before (basal condition) and after repetitive electrical stimulation of the left sciatic nerve. We selected this timing because the severity of deficits observed both in human and in rodents is greatest early after ablation of the cerebellum. A second group of 7 rats was used as the control group. Chloral hydrate was administered continuously using the ip route (CMA micropump, CMA, Sweden). Anesthesia depth was adjusted for absence of abdominal contractions in response to tail pinch.
4.2. Preliminary assessment of M responses and corticomuscular responses In preliminary experiments (Oulad Ben Taib et al., 2005a,b), we assessed in each rat the direct motor responses (M responses) and the corticomotor responses. M responses were studied in the left plantaris muscle using a technique adapted from Gozariu et al. (1998). Electrical stimulation of the left tibial nerve was performed using needle electrodes inserted subcutaneously at the ankle, behind the medial malleolus. Electrical stimuli consisted of single square wave shocks of 0.5 ms duration. EMG recordings were made from the ipsilateral plantaris muscle through a pair of needle electrodes inserted in the distal third of the sole (filters: 30 Hz–1.5 kHz). We determined the corticomotor threshold which was defined as the lowest intensity eliciting at least 5 out of 10 evoked responses with an amplitude >20 μV. Stimuli (rectangular pulses, duration of a pulse: 1 ms) were applied via screws which were fixed on the skull at the level of the right motor cortex (Paxinos and Watson, 1986). The sigmoid feature of the recruitment curve was checked in each rat. The peak-to-peak amplitude of corticomuscular responses (10 responses, before and after repetitive peripheral stimulation) was measured for a stimulus intensity of 130% of motor threshold. Filter settings were 30 Hz–1.5 kHz (NeuroMax 4, Xltek, Canada). These preliminary tests are required given the possibility of abnormal corticomotor responses in rats.
114 4.3.
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Cutaneomuscular responses
For cutaneomuscular responses, needles were implanted in the left plantar skin using a microscope. Duration of stimuli was 0.1 ms. Intensities of stimulation was 2.5 mA. Cutaneomuscular responses had a typical onset latency of 11.5 to 13 ms and a duration of 5–7 ms. Intensities of cutaneomotor responses of the left plantaris muscle were studied. EMG responses were rectified and averaged. We analyzed the integral under the averaged EMG trace. Areas were averaged for 10 successive trials in each rat and in each condition. We consistently averaged the same number of responses (Harrison et al., 2000).
4.4. Description of the stimulation parameters: peripheral stimulation of the left sciatic nerve and trains of stimulation of the right motor cortex Regarding peripheral repetitive stimulation, the left sciatic nerve was surgically exposed for bipolar stimulation (Oulad Ben Taib et al., 2004). Duration of stimulation was 1 h, as previously reported (Oulad Ben Taib et al., 2005a). Trains of stimulation were delivered at a rate of 10 Hz (a train being composed of 5 stimuli of a 1 ms duration; A310–A365 stimulator—World Precision Instruments, UK). Stimulus intensity was adjusted to produce constant somatosensoryevoked potentials (SEP) in EEG (Luft et al., 2002). For stimulation of the motor cortex, we first studied whether the duration of trains had an influence in our experiments using trains of 2–3, 4–6, 7–9, or 10–12 stimuli, because the duration of trains of stimuli has been shown to exert inhibiting or facilitating activities in the motor cortex (Arai et al., 2005; Modugno et al., 2001). We found no effect or a slight effect of trains of 2 or 3 stimuli on the intensity of cutaneomuscular responses following repetitive peripheral stimulation (no change or slight increase in the intensity of cutaneomuscular responses in the condition of motor cortex stimulation following repetitive peripheral stimulation: mean values of 98.76% and 109.63% of baseline responses in 3 rats, respectively, for trains of 2 and 3 successive stimuli). By contrast, we observed that trains made of 4 to 6 consecutive stimuli did increase the intensity of cutaneomuscular responses following repetitive peripheral stimulation (mean increase of 126.40%, 139.57% and 134.48% of baseline values, respectively, for trains of 4, 5 and 6 consecutive stimuli). This increase was also observed for trains of 7, 8 or 9 stimuli but at a lesser extent (mean increase of 124.29%, 117.74% and 113.96% of baseline values, respectively, for trains of 7, 8 or 9 stimuli), while no effect was found with trains of 10, 11 or 12 stimuli (mean increase of 102.15%, 99.03% and 103.62% of baseline values, respectively, for trains of 10, 11 or 12 stimuli). Therefore, we selected trains of stimuli made of 5 rectangular pulses (duration of a pulse: 600 μs, frequency of stimulation 1000 Hz) and were applied at the level of the right motor cortex. We selected a very high frequency of stimulation on the basis of studies underlining that high-frequency repetitive stimulation of the motor cortex increases corticospinal excitability (Quartarone et al., 2005). The timing between the cutaneous stimulation and the train of stimuli in motor cortex was calculated as follows: the latency of the corticomotor
response was subtracted from the latency of the onset of the cutaneomuscular responses. Trains of cortical stimulation were delivered 3 to 4 ms after skin stimulation. The intensity of trains of stimulation was set at 90% of motor threshold (Quartarone et al., 2005). In both groups (control rats; rats with left hemicerebellectomy), studies were performed in the basal condition and were repeated 1 h later after repetitive stimulation of the sciatic nerve.
4.5. Other control experiments performed 48 h after surgery—assessment of the enhancement of the cutaneomuscular response in left plantaris muscle and contralateral effects In 5 of the control rats and in 5 hemicerebellectomized rats investigated 48 h after operation, we checked the enhancement of the cutaneomuscular response in right plantaris muscle following repetitive stimulation of left sciatic nerve. Moreover, in 4 control rats and in 4 hemicerebellectomized rats, we also analyzed the effects of left hemicerebellectomy on the modulation of the cutaneomuscular response on the right plantaris muscle (stimulation of left motor cortex; repetitive stimulation of the right sciatic nerve).
4.6.
Statistical analysis
We compared the intensities of cutaneomuscular responses in the basal condition and following peripheral repetitive stimulation in the 2 groups (control rats, rats with left hemicerebellectomy) using the analysis of variance (SigmaStat, Jandel, Germany). Multiple comparisons were applied. For control experiments, the Student's t test was applied to compare intensities of cutaneomuscular responses in each group of rats before and after peripheral repetitive stimulation. P values lower than 0.05 were considered as statistically significant.
Acknowledgment Mario Manto is supported by the Fonds National de la Recherche Scientifique-Belgium.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Hemicerebellectomy impairs the modulation of cutaneomuscular reflexes by the motor cortex following repetitive somatosensory stimulation Nordeyn Oulad Ben Taib a , Mario Manto b,⁎ a
Service de Neurochirurgie, Hôpital Erasme-ULB, Bruxelles, Belgium Laboratoire de Neurologie Expérimentale, Hôpital Erasme-ULB, Bruxelles, Belgium
b
A R T I C LE I N FO
AB S T R A C T
Article history:
We examined the cutaneomuscular reflex of the plantaris muscle of rats in response to
Accepted 16 March 2006
cutaneous stimulation in isolation and in conjunction with subthreshold high-frequency
Available online 25 April 2006
trains of stimuli applied on the motor cortex, prior to and following repetitive peripheral stimulation. The cutaneomuscular reflex was also investigated under the same paradigm
Keywords:
following hemicerebellectomy. The enhancement of cutaneomuscular responses
Cerebellum
associated with subthreshold high-frequency trains of stimulation following repetitive
Sensory input
peripheral stimulation was prevented by hemicerebellectomy. Our results suggest that
Sciatic nerve
the pathways passing through the cerebellum are involved in the calibration of
Cutaneomuscular response
cutaneomuscular responses. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Cortical network functions require a maintenance and a fine tuning of the excitability of the motor cortex (Kaelin-Lang et al., 2002; Knash et al., 2003; Luft et al., 2002, 2005). It has been shown previously that a prolonged period of peripheral nerve stimulation can induce a lasting increase of corticomotoneuronal responses both in rodents and in human (Knash et al., 2003; Luft et al., 2002, 2005; Manto et al., 2006). It is assumed that the somatosensory input adjusts the excitability of motor pathways by influencing the motor cortex (Kaelin-Lang et al., 2002; Luft et al., 2002). Enhanced excitability indicates that the sensorimotor system is sensitized and could therefore learn quicker. Increasing the excitability of the motor cortex might have beneficial effects on motor training procedures or could improve the performance of novel motor tasks (Kaelin-Lang et al., 2002; Knash et al., 2003).
In an attempt to understand how the cerebellum interacts with the motor cortex and shapes motor commands, we recently tested the hypothesis that the cerebellum could be involved in the adaptative changes of the intensity of the corticomotor responses after a session of somatosensory stimulation. First, we showed that cerebellar nuclei contributed to the sensory modulation (Luft et al., 2005; Manto et al., 2006; Nixon, 2003; Oulad Ben Taib et al., 2004, 2005a). Second, we investigated the effects of hemicerebellectomy, which induces major motor deficits in rats (Oulad Ben Taib et al., 2005b; Tarnecki, 2003). Both disruption of cerebellar nuclei and hemicerebellectomy weakened the adaptation of the motor cortex to repetitive trains of peripheral stimulation (Luft et al., 2005). Taken together, these results underline that the cerebellum is part of the circuitry regulating the excitability of the motor cortex in a situation of steady somatosensory stimulation at the level of the peripheral nervous system.
⁎ Corresponding author. Neurologie-ULB, Fonds National de la Recherche Scientifique (FNRS), 808, Route de Lennik, 1070 Bruxelles, Belgium. Fax: +32 2 555 39 42. E-mail address: [email protected] (M. Manto). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.052
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Understanding how sensory information is used to shape motor commands has direct experimental and clinical applications. Recent studies suggest that the sensorimotor system has a modular organization, each reflex module performing sensorimotor transformations as a function of ‘efficacy’ of the muscle (Schouenborg, 2003). Error signals detected in the module are encoded by the cerebellum via spino–olivo–cerebellar pathways (Schouenborg, 2003), allowing a tuning of the sensorimotor transformation. According to this modular concept, (a) the circuitry is self-organizing and the weight distribution of the cutaneous input adjusts the synaptic organization of the module, (b) conditions that would enhance the efficacy of some muscles could reinforce the power of a given module during motor tasks. Repetitive peripheral stimulation meets the requirements to represent one of these conditions, enhancing the responses in the muscle depending on which peripheral nerve is stimulated (Luft et al., 2005). Moreover, cutaneomuscular responses could be very good candidates to understand the circuitry in reflex modules because their intensity is strongly correlated with background activity of muscles (Zehr and Chua, 2000). The pathways that normally pass through the cerebellum could influence important features of cutaneomuscular reflexes (Bloedel and Bracha, 1995; Timmann et al., 1996). Therefore, we conducted the following experiments: we checked whether cutaneomuscular reflexes were affected by repetitive peripheral stimulation and we tested the effects of trains of stimulation in the motor cortex on the intensity of cutaneomuscular reflexes. We also analyzed the effects of hemicerebellectomy on cutaneomuscular reflexes, and we tested the hypothesis that hemicerebellectomy influenced the adaptation of cutaneomuscular reflexes to peripheral repetitive stimulation.
2.
111
Results
2.1. Results in control group and in rats 48 h after operation Fig. 1 shows the full-wave rectified cutaneomuscular responses in a control rat and in a hemicerebellectomized rat. In the control rat, high-frequency stimulation of the right motor cortex did not modify the intensity of the cutaneomuscular response in the basal condition. The enhancement of the intensity of the cutaneomuscular response appeared following peripheral repetitive stimulation. This enhancement was absent in the hemicerebellectomized rat. Fig. 2 illustrates the values obtained in both groups. In the control group, statistical analysis confirmed an increase of the intensity of the integrated cutaneomuscular responses when trains of subthresholds stimuli were applied to the motor cortex after peripheral repetitive stimulation (P = 0.01 as compared to baseline). In the hemicerebellectomy group, there was no difference between baseline cutaneomuscular responses and following peripheral repetitive stimulation (P > 0.20). Values of integrated cutaneomuscular responses were significantly higher in the control group than in the hemicerebellectomy group following peripheral repetitive stimulation (inter-group effect; P = 0.009).
2.2.
Other control experiments
The intensity of the cutaneomuscular response in right plantaris muscle remained similar before and after peripheral repetitive stimulation of left sciatic nerve, both in control rats (P = 0.19) and in rats with left hemicerebellectomy (P = 0.27).
Fig. 1 – Cutaneomuscular responses in a control rat (top traces) and in a rat with left hemicerebellectomy (bottom traces), before (left and middle left) and after peripheral repetitive stimulation (middle right and right). From left to right: (A) cutaneous stimulation; (B) cutaneous stimulation combined with high-frequency subthreshold stimulation of the motor cortex; (C) cutaneous stimulation; (D) cutaneous stimulation combined with high-frequency subthreshold stimulation of the motor cortex. Cutaneomuscular responses recorded in left plantaris muscle. Responses are rectified and averaged from 10 successive trials.
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Fig. 2 – Cutaneomuscular responses in control rats (gray bars) and in hemicerebellectomized rats (white bars). Conditions 1 and 2: before peripheral repetitive stimulation of the sciatic nerve; conditions 3 and 4: after peripheral repetitive stimulation of the sciatic nerve. Conditions 1 and 3: cutaneous stimulation; conditions 2 and 4: cutaneous stimulation combined with high-frequency stimulation of the right motor cortex at a subthreshold level. Values are mean ± SD. Values are expressed in ms μV. *P < 0.05.
Stimulation of left motor cortex was associated with an enhancement of the cutaneomuscular response of the right plantaris muscle following peripheral repetitive stimulation of right sciatic nerve, both in the control group (P = 0.015) and in left hemicerebellectomized rats (P = 0.01).
3.
Discussion
The findings of this study are (1) the enhancement of the cutaneomuscular responses when subthresholds stimuli are applied in the contralateral motor cortex following a period of peripheral repetitive stimulation and (2) the absence of such a tuning following ipsilateral hemicerebellectomy. The organization of the motor cortex is greatly dependent on the balance between excitatory and inhibitory influences over the network of cortical connections (Luft et al., 2005). The cerebellar output exerts an excitatory effect on the contralateral motor cortex via the cerebello–thalamo–cortical pathway. This tract is the most probable candidate for providing the input for gating the information flow (Luft et al., 2005; Molinari et al., 2002). Cerebellar outputs are guided to the primary motor cortex via the ventrolateral thalamic group which projects mainly to layers IV and V (Sanes and Donoghue, 2000). Through this channel, inputs can modulate the efficacy of the interconnections among cortical neurons, adjusting the circuitry of the motor cortex (Luft et al., 2005; Sanes and Donoghue, 2000). Cutaneous reflexes change according to the behavioral context such that they function in an appropriate fashion to
assist motion (Zehr and Chua, 2000). Cutaneomuscular reflexes require the integrity of the dorsal columns, the sensorimotor cortex and the corticospinal tract (Gibbs et al., 1995). Cutaneous stimuli activate complex pathways that include spinal cord tracts (Floeter et al., 1998). The effects of the cerebellum on these pathways have not been elucidated, and many questions remain unsolved regarding the interaction between peripheral feedback and the function of these reflexes (Zehr and Chua, 2000). We previously observed a decreased excitability of the spinal cord in hemicerebellectomized rats, with depressed H-reflex recruitment and decreased excitability of the anterior horn, as described in rodents and in human (Drozdowski, 1995; Fox and Hitchcock, 1982; Oulad Ben Taib et al., 2005a,b). The efferences of cerebellar nuclei modify the excitability of segmental motoneurons via ascending and descending pathways (Bantli and Bloedel, 1975). Experimental data support the existence of an excitatory cerebello–thalamo–corticospinal pathway which affects the excitability of motoneurons since cooling of the motor cortex removes an excitatory component from the intracellularly recorded response evoked in lumbar motoneurons by dentate stimulation (Bantli and Bloedel, 1975). In addition, descending pathways from the brainstem, such as the rubrospinal tract, provide a part of the substrate by which the cerebellum participates in the control of muscle tension associated with limb movements. One of the important loops subserving the two-way communication between the sensory and motor systems is the spino–cerebello–rubro–spinal projection system (Tarnecki, 2003). This loop is linked to the musculature by the rubral projection to spinal motoneurons. Rubrospinal neurons receive major input from cerebellar nuclei (Courville, 1966) and show intense responses to sensory stimuli. Moreover, behavioral data indicate that the cerebello– rubral system is involved in learning and memory (Rosenfield and Moore, 1983; Tarnecki, 2003). Among cerebellar nuclei, the interpositus nucleus, which receives inputs from both the spinal cord and the sensorimotor cortex and which issues outputs to both the premotor neurons and the motor cortex, seems to have a major contribution in the modulation of the activity of the motor cortex when peripheral stimuli are applied (Luft et al., 2005; Oulad Ben Taib et al., 2004). Neurons in interpositus nuclei are especially involved in somesthetic reflex behaviors, with their discharges being tuned in relation to sensory feedback. The interpositus neurons integrate signals principally from the motor cortex, somatosensory cortex, premotor areas and spinal cord (Bosco and Poppele, 2003; Luft et al., 2005; Oulad Ben Taib et al., 2004). This convergence suggests a role in updating skilled movements. We aim to perform additional experiments such as cutting the superior or middle cerebellar peduncles or generating lesions in cerebellar cortex to increase our understanding of the results. Indeed, cerebellar cortex ablation removes the inhibitory effect of Purkinje cells on cerebellar nuclei. This causes a considerable increase in the background firing and eliminates the pauses in discharges occurring in responses generated by somatosensory stimuli. The choice of the pattern of stimulation is one of the factors influencing strongly the interaction between peripheral somatosensory stimulation and adaptation in the motor cortex (Luft et al., 2005). In human, some specific patterns of
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stimulation influence the excitability of spinal circuits, unlike others. The strength of reciprocal inhibition between ankle flexor and extensor muscles has been assessed before and after peroneal nerve stimulation at motor threshold intensity (Perez et al., 2003). Short-latency reciprocal inhibition from ankle flexor to extensor muscles was measured by conditioning the soleus H-reflex with stimulation of the common peroneal nerve. A short-term plasticity within inhibitory circuits in the spinal cord was disclosed. The pattern of sensory input appears to be a critical factor for inducing changes in the spinal circuit in humans. These integrative spinal mechanisms might be necessary to establish the compatibility with signals which converge in the cerebellum from spinal sensory and motor cortical areas (Bosco and Poppele, 2003). Somatosensory input is implicated in motor learning and in the recovery of function following a cerebral lesion and gives therapeutic benefits in functional recovery following vascular lesions (Johansson et al., 1993; Luft et al., 2005). Repetitive stimulation is currently used in human to enhance reorganization of sensorimotor representations (Johansson et al., 1993). The motor pathways sensitized by peripheral input could increase their learning capacities (Ziemann et al., 2001). It is assumed that the increased excitability represents an early electrophysiological adjustment of the sensorimotor pathways (Luft et al., 2005). In a second stage, this functional change is transformed into a structural plasticity. According to the sensory prediction hypothesis (Nixon, 2003), the cerebellum is necessary to make predictions based on past sensory events and is critical to implement such predictions in the sensorimotor system. The cerebellum is involved in learning to anticipate sensory events (Nixon, 2003). It operates with an outstanding degree of temporal precision for the formation of expectations arising from movements and the interactions with the environment. This precision forms the signature of skilled motor acts (Nixon, 2003). A basic function of cerebellar circuitry could be the online monitoring of sensory inputs (Luft et al., 2005; Manto et al., 2006; Oulad Ben Taib et al., 2004). By permitting the motor cortex to influence the intensity of cutaneomuscular responses in a context of repetitive sensory information, the pathways running through the cerebellum prior to ascending or descending to other nervous system structures facilitate the dynamic adaptation of the brain to consistent environmental changes.
4.
Experimental procedures
4.1.
Description of the procedures
Studies were performed following approval of the institutional animal care committee of the Free University of Brussels. The facilities housing animals are inspected on a regular basis and meet the current national regulations of Belgium. The laboratory has received the agreement to perform surgery in animals (Number LA1230492, Ministère des Classes Moyennes et de l'Agriculture, Belgium). Adequate food, water, ventilation and space are provided. Procedures to minimize discomfort were used during the experiments. Surgical procedures were
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conducted by a neurosurgeon familiar with aseptic techniques (to minimize risks of infection), and animals were under close supervision on a daily basis. Postoperative monitoring and care were provided. Details of the procedures were given elsewhere (Oulad Ben Taib et al., 2004, 2005a,b). Male Wistar rats (weight: 250 to 300 g; left hemicerebellar ablation: n = 5) were anesthetized using an intraperitoneal administration of chloral hydrate (400 mg/kg ip). In the 5 rats, a caudal craniotomy was performed over one half of the cerebellum (Florenzano et al., 2002; Oulad Ben Taib et al., 2005b). The dura was exposed, incised and the left hemicerebellum was removed. Subsequently, the overlying flaps of skin were opposed and sutured. Animals were allowed to recover from anesthesia and surgical stress and had free access to water and food. At the end of the experiments, brains were dissected to evaluate the extent of the lesion after administration of an overdose of chloral hydrate ip. Histological verification of the lesion was performed. Complete ablation of the left cerebellar hemisphere and deep nuclei was carried out in all animals. There was no histological evidence of brainstem injury. We found no evidence of brain infection. Forty-eight hours after surgery, we investigated the responses evoked in the left plantaris muscle following stimulation of the left plantar skin before (basal condition) and after repetitive electrical stimulation of the left sciatic nerve. We selected this timing because the severity of deficits observed both in human and in rodents is greatest early after ablation of the cerebellum. A second group of 7 rats was used as the control group. Chloral hydrate was administered continuously using the ip route (CMA micropump, CMA, Sweden). Anesthesia depth was adjusted for absence of abdominal contractions in response to tail pinch.
4.2. Preliminary assessment of M responses and corticomuscular responses In preliminary experiments (Oulad Ben Taib et al., 2005a,b), we assessed in each rat the direct motor responses (M responses) and the corticomotor responses. M responses were studied in the left plantaris muscle using a technique adapted from Gozariu et al. (1998). Electrical stimulation of the left tibial nerve was performed using needle electrodes inserted subcutaneously at the ankle, behind the medial malleolus. Electrical stimuli consisted of single square wave shocks of 0.5 ms duration. EMG recordings were made from the ipsilateral plantaris muscle through a pair of needle electrodes inserted in the distal third of the sole (filters: 30 Hz–1.5 kHz). We determined the corticomotor threshold which was defined as the lowest intensity eliciting at least 5 out of 10 evoked responses with an amplitude >20 μV. Stimuli (rectangular pulses, duration of a pulse: 1 ms) were applied via screws which were fixed on the skull at the level of the right motor cortex (Paxinos and Watson, 1986). The sigmoid feature of the recruitment curve was checked in each rat. The peak-to-peak amplitude of corticomuscular responses (10 responses, before and after repetitive peripheral stimulation) was measured for a stimulus intensity of 130% of motor threshold. Filter settings were 30 Hz–1.5 kHz (NeuroMax 4, Xltek, Canada). These preliminary tests are required given the possibility of abnormal corticomotor responses in rats.
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Cutaneomuscular responses
For cutaneomuscular responses, needles were implanted in the left plantar skin using a microscope. Duration of stimuli was 0.1 ms. Intensities of stimulation was 2.5 mA. Cutaneomuscular responses had a typical onset latency of 11.5 to 13 ms and a duration of 5–7 ms. Intensities of cutaneomotor responses of the left plantaris muscle were studied. EMG responses were rectified and averaged. We analyzed the integral under the averaged EMG trace. Areas were averaged for 10 successive trials in each rat and in each condition. We consistently averaged the same number of responses (Harrison et al., 2000).
4.4. Description of the stimulation parameters: peripheral stimulation of the left sciatic nerve and trains of stimulation of the right motor cortex Regarding peripheral repetitive stimulation, the left sciatic nerve was surgically exposed for bipolar stimulation (Oulad Ben Taib et al., 2004). Duration of stimulation was 1 h, as previously reported (Oulad Ben Taib et al., 2005a). Trains of stimulation were delivered at a rate of 10 Hz (a train being composed of 5 stimuli of a 1 ms duration; A310–A365 stimulator—World Precision Instruments, UK). Stimulus intensity was adjusted to produce constant somatosensoryevoked potentials (SEP) in EEG (Luft et al., 2002). For stimulation of the motor cortex, we first studied whether the duration of trains had an influence in our experiments using trains of 2–3, 4–6, 7–9, or 10–12 stimuli, because the duration of trains of stimuli has been shown to exert inhibiting or facilitating activities in the motor cortex (Arai et al., 2005; Modugno et al., 2001). We found no effect or a slight effect of trains of 2 or 3 stimuli on the intensity of cutaneomuscular responses following repetitive peripheral stimulation (no change or slight increase in the intensity of cutaneomuscular responses in the condition of motor cortex stimulation following repetitive peripheral stimulation: mean values of 98.76% and 109.63% of baseline responses in 3 rats, respectively, for trains of 2 and 3 successive stimuli). By contrast, we observed that trains made of 4 to 6 consecutive stimuli did increase the intensity of cutaneomuscular responses following repetitive peripheral stimulation (mean increase of 126.40%, 139.57% and 134.48% of baseline values, respectively, for trains of 4, 5 and 6 consecutive stimuli). This increase was also observed for trains of 7, 8 or 9 stimuli but at a lesser extent (mean increase of 124.29%, 117.74% and 113.96% of baseline values, respectively, for trains of 7, 8 or 9 stimuli), while no effect was found with trains of 10, 11 or 12 stimuli (mean increase of 102.15%, 99.03% and 103.62% of baseline values, respectively, for trains of 10, 11 or 12 stimuli). Therefore, we selected trains of stimuli made of 5 rectangular pulses (duration of a pulse: 600 μs, frequency of stimulation 1000 Hz) and were applied at the level of the right motor cortex. We selected a very high frequency of stimulation on the basis of studies underlining that high-frequency repetitive stimulation of the motor cortex increases corticospinal excitability (Quartarone et al., 2005). The timing between the cutaneous stimulation and the train of stimuli in motor cortex was calculated as follows: the latency of the corticomotor
response was subtracted from the latency of the onset of the cutaneomuscular responses. Trains of cortical stimulation were delivered 3 to 4 ms after skin stimulation. The intensity of trains of stimulation was set at 90% of motor threshold (Quartarone et al., 2005). In both groups (control rats; rats with left hemicerebellectomy), studies were performed in the basal condition and were repeated 1 h later after repetitive stimulation of the sciatic nerve.
4.5. Other control experiments performed 48 h after surgery—assessment of the enhancement of the cutaneomuscular response in left plantaris muscle and contralateral effects In 5 of the control rats and in 5 hemicerebellectomized rats investigated 48 h after operation, we checked the enhancement of the cutaneomuscular response in right plantaris muscle following repetitive stimulation of left sciatic nerve. Moreover, in 4 control rats and in 4 hemicerebellectomized rats, we also analyzed the effects of left hemicerebellectomy on the modulation of the cutaneomuscular response on the right plantaris muscle (stimulation of left motor cortex; repetitive stimulation of the right sciatic nerve).
4.6.
Statistical analysis
We compared the intensities of cutaneomuscular responses in the basal condition and following peripheral repetitive stimulation in the 2 groups (control rats, rats with left hemicerebellectomy) using the analysis of variance (SigmaStat, Jandel, Germany). Multiple comparisons were applied. For control experiments, the Student's t test was applied to compare intensities of cutaneomuscular responses in each group of rats before and after peripheral repetitive stimulation. P values lower than 0.05 were considered as statistically significant.
Acknowledgment Mario Manto is supported by the Fonds National de la Recherche Scientifique-Belgium.
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