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Hindlimb unloading affects cortical motor maps and decreases corticospinal excitability
Experimental Neurology 237 (2012) 211–217
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Hindlimb unloading affects cortical motor maps and decreases corticospinal excitability Cécile Langlet a, Bruno Bastide b, c, Marie-Hélène Canu b, c,⁎ a b c
Laboratoire d'Automatique humaine et de Sciences Comportementales, Campus Bridoux, Rue du général Delestraint, Université Lorraine, F-57000 Metz, France Université Lille Nord de France, F-59000 Lille, France Laboratoire «Activité Physique, Muscle et Santé», EA 4488, IFR 114, Université Lille 1, Sciences et Technologies, F-59650 Villeneuve d'Ascq, France
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Article history: Received 17 October 2011 Revised 18 June 2012 Accepted 20 June 2012 Available online 27 June 2012 Keywords: Motor cortex Excitability Somatotopy Disuse Bed-rest
a b s t r a c t A chronic reduction in neuromuscular activity through prolonged body immobilization of humans results in muscle atrophy and weakness as well as motor tasks performance impairment, which is correlated to a change in corticospinal excitability. In rats, hindlimb unloading (HU) is commonly used to mimic the effects of confinement to bed in patients. Several studies have reported changes in the representation of the somatosensory cortex in rodents submitted to HU or sensorimotor restriction by casting: remapping and enlargement of receptive fields, changes in the response of layer 4 neurons to peripheral stimulation. However, we have no data about motor cortical maps in rats submitted to a period of motor restriction during adulthood. Therefore, the objectives of the present study were twofold: to determine, in control rats and in rats submitted to a 14-day period of HU, the size and organization of hindlimb representation in the M1 cortex and to evaluate the overall excitability of M1 cortex by determining the stimulation thresholds. HU led to a dramatic decrease in the hindlimb representation on the M1 cortex (− 61%, p b 0.01). In addition, current thresholds for eliciting a movement were increased. The toes were less strongly affected by HU than other joint. Our main conclusion is that HU dramatically affects the organization and functioning of cortical motor maps and decreases corticospinal excitability. © 2012 Elsevier Inc. All rights reserved.
Introduction A chronic reduction in neuromuscular activity through prolonged body immobilization in humans results in motor tasks performance impairment. In particular, during prolonged bed-rest or lower limb immobilization by casting, posture and gait are strongly affected (Viguier et al., 2009; for review: Fortney et al., 1996), and the performance on a test of functional mobility (walking task with proprioceptive challenge) is altered (Roberts et al., 2010). Impaired movement arises from a combination of factors, including muscle atrophy and weakness, and/or neuroplastic changes. According to Clark et al. (2006), neural factors explain 48% of the variability in muscle strength loss which occurs as a result of 4 weeks of casting, whereas muscular factors explain only 39% of variability. In addition, the nervous factors that accompany behavioral alterations after casting are primarily of supraspinal origin (deficit in central activation) (Clark et al., 2006; Moisello et al., 2008). Abbreviations: HU, hindlimb unloading; M1, primary motor cortex; TMS, transcranial magnetic stimulation; C, control; EMG, electromyographic. ⁎ Corresponding author at: Laboratoire «Activité Physique, Muscle et Santé», EA 4488, IFR 114, Université Lille 1, Sciences et Technologies, Bâtiment SN4, F-59655 Villeneuve d'Ascq cedex, France. Fax: +33 3 20 43 68 88. E-mail addresses: [email protected] (C. Langlet), [email protected] (B. Bastide), [email protected] (M-H. Canu). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.06.018
In human, the development of transcranial magnetic stimulation (TMS) allows a painless examination of the motor cortex. However, studies are rare, and results are sometimes conflicting, and depend on the type of disuse (bed-rest, casting…), duration, and which member (upper or lower limb) is affected. For instance, casting of the lower limb during 16 weeks induces a decrease of the cortical representation of the immobilized limb (Liepert et al., 1995), whereas Zanette et al. (1997) reported no change in motor cortical map following a 4-week casting of the upper limb. Casting of the lower limb in healthy subjects (Roberts et al., 2007) or of the upper limb for traumatic fractures (Zanette et al., 2004) is associated with increased corticospinal excitability, whereas a decrease in excitability is reported immediately following bed rest (Roberts et al., 2010). In rats, hindlimb unloading (HU) is used to mimic the effects of confinement to bed in patients or of microgravity on astronauts, since this model reproduces the chronic weightless bearing and reduction in hindlimb movement (Morey-Holton and Globus, 2002). It is a valuable model, which allows to gain new insights into the underlying mechanisms of motor adaptation that occurs in response to bed-rest. A 14-day period of HU induces abnormalities in the postural and locomotor tasks (Canu and Garnier, 2009; Canu et al., 2007). This functional impairment might be the result of an alteration in the functioning of the somatosensory pathway (Canu et al., 2003; Dupont et al., 2003; Langlet et al., 2001; Ren et al.,
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2012). It has also been shown recently that intrinsic properties of pyramidal neurons within foot representation on the motor cortex were changed towards a decreased excitability (Canu et al., 2010). However, we have no data on the functional organization of motor cortex in rats submitted to HU. Therefore, the objectives of the present study were twofold: to determine the size and organization of hindlimb representation in the M1 cortex and to evaluate the overall excitability of M1 cortex by determining the stimulation thresholds. Our main conclusion is that HU dramatically affects the organization and functioning of cortical motor maps and decreases corticospinal excitability. Experimental procedures Ethics statement All procedures described below were approved by both the Agricultural and Forest Ministry and the National Education Ministry (veterinary service of health and animal protection, authorization 59‐00999). All efforts were made to minimize suffering. Animals and treatment Adult male Wistar rats (280–320 g) were divided into two groups: C (control, n = 8), and HU (Hindlimb Unloading for 14 days, n = 8). Both groups were housed in the same facilities, under temperature and light controlled conditions (23 °C, 12-h light/12-h dark cycle). Control rats were group housed (4 per cage). Hindlimb unloading was obtained using the model adapted from Morey-Holton and Globus (2002). The tail of each rat was cleaned, dried, and wrapped in antiallergenic adhesive plaster. This cast was secured to an overhead swivel that permitted 360° rotation. The rats were unloaded by the tail at a ~ 30° head-down angle in order to avoid a contact of the hindlimbs with the ground, whereas they were allowed to walk freely on their forelimbs. Animals had ad libitum access to food and water, and could have social interactions with their neighbors. Electrophysiological procedure The rats were anesthetized with ketamine hydrochloride (70 mg/ kg, i.p.), xylazine (5 mg/kg, i.p.) and acepromazine (0.2 mg/kg, i.p.). Supplementary doses of ketamine (20 mg/kg, i.p.) and acepromazine (0.02 mg/kg, i.p.) were delivered as needed to suppress hindlimb withdrawal reflex. The body temperature was maintained at approximately 37 °C with a thermostatically controlled heating pad. The rat was placed in a stereotaxic apparatus. Lidocaine 2% was applied on ear bars and scalp incision line. Electromyographic (EMG) recording 9 rats (4 C and 5 HU) were implanted for EMG recording. Bipolar electrodes were made from two insulated multistranded stainless steel wires (seven strands, 50 μm gauge, AM System, USA). The recording surface was obtained by mechanically removing the insulation for a length of 0.5 mm. A pair of electrode wires was inserted into the midbelly of triceps surae and tibialis anterior muscles of the left hindlimb and secured by means of sutures (Monosof, Tyco, France). Electrodes were linked to a differential amplifier (Model 1700, AM System, USA). The raw EMG signal was amplified (×1000, band pass 300 Hz–10 kHz), full-wave rectified and digitized at 2000 Hz. EMG analysis was performed with Spike2 software (Cambridge Electronic Design, UK). Microstimulation mapping The cortex was exposed by making a craniotomy that extended from 3 mm anterior to 3 mm posterior to bregma, and from 1 to 5 mm lateral from midline. A small puncture was made in the
cisterna magna to reduce edema. The dura mater was removed, and the cortex was covered with 37° artificial cerebrospinal fluid. Monophasic cathodal pulses were delivered with platinum-iridium microelectrodes (tip diameter: 1–2 μm, impedance: 1 MΩ at 1 kHz, parylene insulated, PTM23B10, WPI, UK). The anodal electrode was a screw implanted in the frontal bone. Electrodes were positioned at a depth of approximately 1800 μm below cortical surface, and then cautiously adjusted in order to evoke movement at the lowest threshold, corresponding to a localization within the layer V (Young et al., 2011). Electrode penetrations were placed in a grid-like fashion at 250 μm intervals. We never made two successive stimulations at closed locations. Stimulation consisted of 100 ms trains of 0.2 ms pulses (333 Hz) delivered at interval of 1 s from an electrically isolated, constant current stimulator (A300 Pulsemaster stimulator with A360 isolation unit, WPI, UK). Animals were maintained in a prone position, with hindlimbs unsupported. Hindlimbs were regularly mobilized (flexion and extension in a stepping-like movement). In addition, the threshold was first established with the limb approximately halfway between flexion and extension. Since changes in threshold could occur with respect to the limb position, the position was changed to from complete flexion to complete extension to search for the lowest threshold. The experimenter maintained firmly the different joints to verify that distal movement was not due to a movement of a more proximal joint. At each site, the stimulation was initiated at the lowest intensity (5 μA) and was progressively increased until a movement was evoked. Identification of joint movements was performed by visual inspection and palpation. At each penetration site, the minimal threshold required to elicit a movement of any joint of the hindlimb was recorded. This minimal current intensity was defined as “Absolute threshold”. Then, current intensity was gradually increased until a movement of a specific hindlimb joint (hip, knee, ankle or toes) was detected. This second intensity was considered as “Relative threshold”. Penetration sites that failed to elicit a movement of the hindlimb at any current intensity, up to 80 μA, were defined as unresponsive. If a movement of another joint (forelimb, vibrissae…) was obtained at current intensity corresponding to the absolute threshold, it was considered as an unresponsive site for hindlimb. Furthermore, as the anesthesia level varied during the experiment, we checked regularly in some already recorded penetration sites for changes in the current intensity. In addition, we tested the same point just before and several minutes after a new injection of anesthetic, and suspended the manipulation until we recovered the same threshold value. The cortical representation of the hindpaw was reconstructed by drawing boundaries to enclose the sites eliciting hindlimb movement or movement of a given joint. We considered that the area reconstruction was complete when it was surrounded by unresponsive sites (or sites responding to another body part). The area was determined by using Photoshop software. Statistical analyses Results are presented as mean ± SEM. Normality was tested with Kolmogorov–Smirnov test. Multi-group results were compared using Statistical Analysis Systems General Linear Model procedures and were subjected to two-way ANOVA to determine significant main effects and interactions. The Bonferroni test was used for post-hoc comparison. A t-test was performed when appropriate. A p-value of less than 0.05 was chosen as the significance level for all statistical analyses. A p-value of less than 0.1 was reported as a tendency. Results A total of 972 penetrations were made (535 in C rats and 437 in HU ones). In C rats, 60% of penetrations gave responses with current
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under 80 μA. Movements were evoked in the hindlimb (40%) or in another body part (20%). In HU rats, the proportion of responsive sites was only 46% (hindlimb: 29%, another body part: 17%) (Fig. 1). The ANOVA performed with Group (C vs. HU) as independent variable and Response (proportion of hindlimb, another body part, or unresponsive sites) as dependent variable revealed a Group × Response interaction (F = 6.07, p b 0.05). Unresponsive sites were more numerous in HU rats (post-hoc test: p b 0.01), whereas the proportion of sites eliciting a movement of hindlimb was reduced (post-hoc test: p b 0.05). Movements elicited at absolute threshold concerned one single joint. In C as in HU rats, we observed flexions of ankle, knee and hip, except for toes where extensions were reported. Hip adductions were also reported, and in one single case, an ankle extension. When the current intensity was increased, several joints were active, but the movement (i.e. flexion vs. extension) kept unchanged. Effect of HU on the M1 hindlimb map area In C rats, the M1 hindlimb map extended from A+0.75 to A− 2.00 with respect to Bregma, and from L1.50 to L4.00. The probability to encounter a responsive to HL site was the highest (>50%) at the following coordinates: A + 0.25 to − 1.50 and L 2.00 to 3.75. The hindlimb cortical area was surrounded by cortical regions with no response, or with forelimb representation in the rostral border, vibrissae in the caudal border, extending laterally and medially, and tail in the medial caudal border. The same organization was observed in HU rats, although the map was smaller. The mean anteroposterior extent was 1.75 ± 0.14 mm in C rats and only 1.08 ± 0.12 mm in HU ones (t-test: p b 0.01), and the mediolateral extent was respectively 1.87 ± 0.18 mm and 1.25 ± 0.14 mm (t-test: p b 0.05). The shrinkage from C to HU map was homogenous in all directions (− 0.25 mm in the caudal, rostral, lateral and medial directions). The area whose stimulation elicited hindlimb movements was 2.98 mm 2 at absolute threshold. It was dramatically reduced after 14 days of HU (− 61%, p b 0.01) (Fig. 2A). The same organization was observed in HU rats. We also measured the total representation of the different joints obtained with supra-threshold currents (i.e. relative threshold) (Fig. 2B). In C rats, a proximo-distal gradient is shown, the most proximal joint (hip) being more represented than the most distal one (toes). HU profoundly affected the organization of the hindlimb area. The two-way ANOVA performed with Group (C vs. HU) and Joint (toes, ankle, knee, hip) as independent variables, and Area as dependent variable, revealed a Joint effect (F = 8.15, p b 0.001). In addition, it confirmed the results obtained with absolute threshold currents (see above), namely that the area was decreased in HU rats (Group effect: F = 12.26, p b 0.01). There was also a Group× Joint interaction (F = 6.09, p b 0.01). The representational areas of the hip, knee and
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ankle were strongly affected (−65%, −73%, −72% respectively; post-hoc test: p b 0.001), whereas toe representation kept unchanged (−28%; ns). Thus, proximo-distal gradient observed in C rats disappeared in HU rats, all joints being almost equally represented. Effect of HU on the M1 hindlimb thresholds HU induced an increase in both absolute and relative thresholds (Fig. 3). Absolute threshold was increased from 16% (knee, p = 0.06) to 50% (ankle, p b 0.01). The decreased excitability was limited to the hindlimb area and was not an effect of HU on the whole motor cortex, since the current which elicited the movement of the forelimb was strictly unchanged in HU rats. The increase in relative threshold extended from 12% for the knee (p b 0.05) to 19% for the hip (p b 0.01). It should be noted that the increase was not significant for the toes (+15%, p = 0.07). Electromyography In 4 C and 5 HU rats, the electromyographic activity of ankle flexor (tibialis anterior) and extensor (gastrocnemius) muscles was recorded. Fig. 4 presents the threshold for the two groups (C and HU) and for different methods of determination (visual inspection and/or palpation vs. EMG). The threshold for electromyographic activation of flexor muscle was very close to the threshold determined visually (apparition of movement). By contrast, the value was higher for the extensor muscle. The two-way ANOVA reveals an effect of the method of determination (F = 6.76, p b 0.05). As indicated previously, the threshold was increased in HU rats (+ 27.6% and + 29.6% in flexor and extensor muscles, respectively) (Group effect: F = 30.99, p b 0.001). The response latency and duration have been measured on the flexor muscle. The analysis was not performed on the extensor muscle since the threshold was higher (Fig. 5). At threshold, a burst of activity was evoked approximately 95 ms after the onset of stimulation (C: 93.5 ± 4.9 ms; HU: 95.5 ± 5.1 ms; t-test: p = 0.89), and ended 25 ms after the offset of stimulation (C: 25.5 ± 4.4 ms; HU: 24.3 ± 2.4 ms; t-test: p = 0.88). No difference was reported between C and HU rats. When stimulus intensity was increased, the latency was reduced and the burst duration was increased (Fig. 5). Discussion The two main findings of the present work are first that HU led to a dramatic decrease in the hindlimb representation on the M1 cortex; second, current thresholds for eliciting a movement were increased. In addition, these effects were not identical according to the joint considered: the toes were less affected than the other joints. Methodological issues
Fig. 1. Proportion of sites eliciting a movement of hindlimb or of another body part (forelimb, trunk, tail, vibrissae), or of unresponsive sites (unresp.), in C rats (white bars) and HU rats (black bars). Data are means ± S.E.M. * and **: pb 0.05 and p b 0.01 respectively, with respect to C value.
Only a few papers have quantified the area of hindlimb representation in rats, and the variability between studies is particularly striking. VandenBerg et al. (2002) have reported values of ~ 0.5 mm 2 in Fischer-344 rats and ~ 1 mm 2 in Long-Evans rats. In the latter strain, Neafsey et al. (1986) reported an area of 7 mm 2. Value was 3.9 mm 2 in Sprague–Dawley rats (Delcour et al., 2011). Finally, we found that, in Wistar rats, the hindlimb representation extended on 2.98 mm 2 at relative threshold. These discrepancies might be explained in part by strain differences (VandenBerg et al., 2002). It is also well known that the level of anesthesia influences the apparition of movement, and thus the map extent and threshold values (Potez and Larkum, 2008; Tandon et al., 2008; Young et al., 2011; Zandieh et al., 2003). Comparison between studies is thus awkward. To avoid any bias due to variation in anesthetic state within the
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Fig. 2. Cortical maps obtained by electrophysiological mapping procedure. A. Representative individual maps in C (left) and HU (right) rats. Black dots indicate electrode penetrations in layer V of the M1 area. The map was reconstructed by drawing boundaries to enclose the sites eliciting movement of a given joint at absolute threshold. Note the strong reduction in hip and ankle representation. Values are stereotaxic coordinates in mm, with respect to bregma. B. Cortical surface area occupied by hindlimb movements in C (white bars) and HU rats (black bars). Maps were constructed with (relative) suprathreshold currents. Data are means ± S.E.M. ***: p b 0.001 with respect to C value.
present work, the level was carefully controlled and the same point was checked regularly. Our maps were probably slightly over-estimated since the maximum stimulation current used was 80 μA, instead of 60 μA in most studies. We chose this value as high current limit because in HU rats, due to the higher threshold and the smaller hindlimb representation area (in particular for the ankle), maps were difficult to build with lower currents. Large stimulation currents might activate neurons with a decreased excitability; they could also spread through a volume of tissue. Larger currents have also a repercussion on threshold mean values, which are higher in the present study than in others. However, when values in the range 60–80 μA were excluded, our mean threshold values were 15% decreased and were then very similar to those reported by Delcour et al. (2011). The latency for EMG activation was very long (~ 50 ms at twice the threshold), and was unchanged by HU. Maybe the 300 Hz high pass cut-off frequency could have produced a slight attenuation of signal, and could have affected the identification of EMG onset, since maximum EMG power in rats is ~ 300 Hz (Li et al., 2011). The high value could also be due to the anesthetic state. Liang et al. (1993) and Brus-Ramer et al. (2009) found latencies near 10 ms for the forelimb in the rat. Their anesthetic level was obtained by ketamine alone,
whereas we used a mixture of ketamine and acepromazine, which is known to induce a muscle relaxation, and thus could have impacted motor threshold and response latency. The hindlimb representation is decreased by hindlimb unloading Many studies have shown that when a limb disappears or is disconnected from the supraspinal structures, for instance following an amputation (Cohen et al., 1991a; Donoghue and Sanes, 1988; Pascual-Leone et al., 1996), nerve section (Sanes et al., 1990; Toldi et al., 1996), or spinal cord injury (Cohen et al., 1991b; Martinez et al., 2010), its cortical representation is profoundly altered. Our work shows that a non-traumatic situation also has heavy consequences on the cortical motor map, since the hindlimb representation area was decreased by 61%. The size of the decrease of hindlimb area in motor maps following HU (− 61%) is surprising when compared to the modest reduction in tactile representation of the hindpaw (− 15%) (Dupont et al., 2011) following 14 days of HU. Studies that evaluated in the same time both tactile and motor maps in response to a change in activity are very scarce. After training to a reaching task, the overall size of the somatosensory forepaw map was not altered, in spite of a strong increase (×1.6) of the forepaw
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Fig. 3. Absolute and relative threshold (in μA) in C rats (white bars) and HU rats (black bars), for the different joints of the hindlimb. Data are means ± S.E.M. *, **, #: p b 0.05, p b 0.01 and p b 0.1 with respect to C value.
movement representation (Coq et al., 2009). Taken together, it appears that the motor cortical maps seem to be more reactive to disuse or overuse than somatosensory ones. Threshold values are increased in unloaded rats Our data provided evidence for a decreased excitability of the corticospinal system. In humans, the excitability of the motor cortex is also modulated during immobilization, although studies are conflicting (Facchini et al., 2002; Kaneko et al., 2003; Liepert et al., 1995; Roberts et al., 2010; Zanette et al., 1997, 2004). The threshold measured herein was 1.4 greater for the ankle in HU rats, and 1.2 greater for the other joints. These values are similar to those reported by Martinez et al. (2010), who found a 1.4 fold increase in threshold after cervical spinal cord hemisection. Once again, this result underlines that a sensorimotor restriction can lead to changes comparable to those obtained after a traumatic section. A decreased excitability of corticospinal neurons was expected in the light of other studies on motor cortex. However, this result is
Fig. 4. Absolute threshold (in μA) in C rats (white bars) and HU rats (black bars), for the ankle. Threshold was determined by visual inspection (detection of ankle movement) or on EMG (activity detectable on flexor or extensor muscles). Data are means ± S.E.M. *, ** and ***: p b 0.05, 0.01 and 0.001 with respect to C value. #: p b 0.05 with respect to visual threshold.
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Fig. 5. A: Electromyographic activity of ankle flexor and extensor muscles, at flexor threshold (A1), or extensor threshold (2× flexor threshold, A2). The horizontal bar indicates the application of stimulating current, arrows indicate the latency. B: mean latency of flexor burst onset and offset, at flexor threshold (above) and at 2× flexor threshold (below), in C (white bars) and HU rats (hatched bars), The gray square represents the stimulus duration.
surprising in view of studies performed in the somatosensory cortex. In fact, extracellular recordings performed in the somatosensory cortex of HU rats have provided evidence for a higher activation of cortical neurons to electrical stimulation of the sciatic nerve (Langlet et al., 2001). In addition, studies have shown a reduction of threshold and an increase in cell activity in response to low tactile stimulation (Dupont et al., 2003). Taken together, these data indicate that excitability of the somatosensory cortex of HU rats was increased. Thus, as mentioned above, motor and sensory cortex react very differently to disuse. Toes are less affected than other joints by hindlimb unloading The reorganization of M1 cortex by HU concerns primarily the representation of proximal joints, whereas the toe representation is less affected. In C rats, the area occupied by the toes on the motor cortex is far smaller than other joints: supra-threshold currents can evoke movements of the toes from only 33% of the total area of hindlimb (against 75% for the hip). After HU, the proximo-distal gradient disappeared. Contrary to our data, after spinal cord hemisection, Martinez et al. (2010) reported an increase in the relative area representing the proximal forelimb whereas distal movements were drastically under-represented. The type and degree of reorganization should be considered with respect to the function of the limb: representation of distal parts of the forelimb is more affected by disuse since this limb is implied in fine manipulation; on the contrary, representation of proximal parts of the hindlimb is more concerned by disuse since hindlimb is functionally implied in the stabilization of the body during posture and gait. If remodeling of motor cortical maps is directly correlated to motor performance (see below), the animals should develop difficulties to produce efficient and harmonious movements, due to the fact that all joints are not affected in the same degree by HU. As a matter
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of fact, a kinematic study of hindlimb motion during locomotion has shown that intralimb coordination was profoundly affected in HU rats (Canu and Garnier, 2009). Mechanisms of motor plasticity Despite some attempts to determine the origin of plasticity which occurs in adults in response to disuse, mechanisms are not fully elucidated and are probably the combination of several factors (see Sanes and Donoghue, 2000 for review). Recently, in our model of unloading, an electrophysiological study performed in vitro has demonstrated that the basic electrophysiological characteristics of “regular spiking” neurons, identified as spiny pyramidal neurons and located in the hindlimb representation, were changed towards a decreased excitability (Canu et al., 2010). This change in input/output coupling could account for the increased threshold in HU rats. Dopamine is also known to modulate activity of cortical neurons, although data are limited (Luft and Schwartz, 2009 for review). Hosp et al. (2009) have shown that an acute injection of D2-receptor antagonist in the motor forelimb area of rats affected motor cortical excitability and somatotopy: the size of the forelimb representation was reduced by 68% and threshold increased by 37%. These values are quite similar to those observed in the present study (−61% and +40%, respectively); they suggest that a poor activation of D2-receptors within the deep cortical layers in response to the low dopamine level in HU rats might play a role in motor plastic mechanisms. As a matter of fact, we have shown that the overall level of dopamine and its metabolites was decreased in the hindlimb area of the sensorimotor cortex of HU rats (Canu et al., unpublished data). A change in the balance between excitation and inhibition, towards a decreased excitation of cortical neurons or an increase in inhibitory drive, could also explain the increase in threshold values. To sustain this hypothesis, a higher GABAergic inhibition by diazepam treatment results in significantly smaller forelimb movement areas and higher mean movement threshold (Young et al., 2011). However, a previous study has demonstrated that the level of GABA was decreased in brain tissue sampled from the hindlimb area cortex of HU rats (Canu et al., 2006), in particular within the layer IV (D'Amelio et al., 1996). Taken together, these data suggest a release from inhibition, which affects essentially the sensory processing, but not the motor command. Activity dependent plasticity is known to induce morphological changes within the motor cortex. A recent study performed by our team has shown an increase (~10%) in the density of spines of pyramidal neurons in HU rats (unpublished results). However, a great proportion of new spines are filopodia-like protrusions, which are immature shapes lacking synapses. This data suggests that a reorganization in the synaptic connectivity could occur after a period of HU. Functional implications and conclusion To conclude, the present study demonstrates that HU dramatically affects the organization and functioning of cortical motor maps and decreases corticospinal excitability. The cortical remodeling and the increase of the thresholds may participate to the alteration of the motor performance reported in rats submitted to HU (Canu and Garnier, 2009; Canu et al., 2007). In humans, studies have reported a deterioration of dexterity, a reduced muscle strength and changes in limb kinematics following 12 h (Moisello et al., 2008), 72 h (Weibull et al., 2011) or 1 week (Lundbye-Jensen and Nielsen, 2008) of arm immobilization. The change in motor performance was more likely attributed to a change in functional properties of the central nervous system (“voluntary deficit”) and/or to inter-hemispheric plasticity rather than to pure muscular deficits. Yet, most rehabilitation techniques for patients suffering from deconditioning after bed rest or inactivity are essentially muscle strengthening exercise programs (electrostimulation, resistive
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Hindlimb unloading affects cortical motor maps and decreases corticospinal excitability Cécile Langlet a, Bruno Bastide b, c, Marie-Hélène Canu b, c,⁎ a b c
Laboratoire d'Automatique humaine et de Sciences Comportementales, Campus Bridoux, Rue du général Delestraint, Université Lorraine, F-57000 Metz, France Université Lille Nord de France, F-59000 Lille, France Laboratoire «Activité Physique, Muscle et Santé», EA 4488, IFR 114, Université Lille 1, Sciences et Technologies, F-59650 Villeneuve d'Ascq, France
a r t i c l e
i n f o
Article history: Received 17 October 2011 Revised 18 June 2012 Accepted 20 June 2012 Available online 27 June 2012 Keywords: Motor cortex Excitability Somatotopy Disuse Bed-rest
a b s t r a c t A chronic reduction in neuromuscular activity through prolonged body immobilization of humans results in muscle atrophy and weakness as well as motor tasks performance impairment, which is correlated to a change in corticospinal excitability. In rats, hindlimb unloading (HU) is commonly used to mimic the effects of confinement to bed in patients. Several studies have reported changes in the representation of the somatosensory cortex in rodents submitted to HU or sensorimotor restriction by casting: remapping and enlargement of receptive fields, changes in the response of layer 4 neurons to peripheral stimulation. However, we have no data about motor cortical maps in rats submitted to a period of motor restriction during adulthood. Therefore, the objectives of the present study were twofold: to determine, in control rats and in rats submitted to a 14-day period of HU, the size and organization of hindlimb representation in the M1 cortex and to evaluate the overall excitability of M1 cortex by determining the stimulation thresholds. HU led to a dramatic decrease in the hindlimb representation on the M1 cortex (− 61%, p b 0.01). In addition, current thresholds for eliciting a movement were increased. The toes were less strongly affected by HU than other joint. Our main conclusion is that HU dramatically affects the organization and functioning of cortical motor maps and decreases corticospinal excitability. © 2012 Elsevier Inc. All rights reserved.
Introduction A chronic reduction in neuromuscular activity through prolonged body immobilization in humans results in motor tasks performance impairment. In particular, during prolonged bed-rest or lower limb immobilization by casting, posture and gait are strongly affected (Viguier et al., 2009; for review: Fortney et al., 1996), and the performance on a test of functional mobility (walking task with proprioceptive challenge) is altered (Roberts et al., 2010). Impaired movement arises from a combination of factors, including muscle atrophy and weakness, and/or neuroplastic changes. According to Clark et al. (2006), neural factors explain 48% of the variability in muscle strength loss which occurs as a result of 4 weeks of casting, whereas muscular factors explain only 39% of variability. In addition, the nervous factors that accompany behavioral alterations after casting are primarily of supraspinal origin (deficit in central activation) (Clark et al., 2006; Moisello et al., 2008). Abbreviations: HU, hindlimb unloading; M1, primary motor cortex; TMS, transcranial magnetic stimulation; C, control; EMG, electromyographic. ⁎ Corresponding author at: Laboratoire «Activité Physique, Muscle et Santé», EA 4488, IFR 114, Université Lille 1, Sciences et Technologies, Bâtiment SN4, F-59655 Villeneuve d'Ascq cedex, France. Fax: +33 3 20 43 68 88. E-mail addresses: [email protected] (C. Langlet), [email protected] (B. Bastide), [email protected] (M-H. Canu). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.06.018
In human, the development of transcranial magnetic stimulation (TMS) allows a painless examination of the motor cortex. However, studies are rare, and results are sometimes conflicting, and depend on the type of disuse (bed-rest, casting…), duration, and which member (upper or lower limb) is affected. For instance, casting of the lower limb during 16 weeks induces a decrease of the cortical representation of the immobilized limb (Liepert et al., 1995), whereas Zanette et al. (1997) reported no change in motor cortical map following a 4-week casting of the upper limb. Casting of the lower limb in healthy subjects (Roberts et al., 2007) or of the upper limb for traumatic fractures (Zanette et al., 2004) is associated with increased corticospinal excitability, whereas a decrease in excitability is reported immediately following bed rest (Roberts et al., 2010). In rats, hindlimb unloading (HU) is used to mimic the effects of confinement to bed in patients or of microgravity on astronauts, since this model reproduces the chronic weightless bearing and reduction in hindlimb movement (Morey-Holton and Globus, 2002). It is a valuable model, which allows to gain new insights into the underlying mechanisms of motor adaptation that occurs in response to bed-rest. A 14-day period of HU induces abnormalities in the postural and locomotor tasks (Canu and Garnier, 2009; Canu et al., 2007). This functional impairment might be the result of an alteration in the functioning of the somatosensory pathway (Canu et al., 2003; Dupont et al., 2003; Langlet et al., 2001; Ren et al.,
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2012). It has also been shown recently that intrinsic properties of pyramidal neurons within foot representation on the motor cortex were changed towards a decreased excitability (Canu et al., 2010). However, we have no data on the functional organization of motor cortex in rats submitted to HU. Therefore, the objectives of the present study were twofold: to determine the size and organization of hindlimb representation in the M1 cortex and to evaluate the overall excitability of M1 cortex by determining the stimulation thresholds. Our main conclusion is that HU dramatically affects the organization and functioning of cortical motor maps and decreases corticospinal excitability. Experimental procedures Ethics statement All procedures described below were approved by both the Agricultural and Forest Ministry and the National Education Ministry (veterinary service of health and animal protection, authorization 59‐00999). All efforts were made to minimize suffering. Animals and treatment Adult male Wistar rats (280–320 g) were divided into two groups: C (control, n = 8), and HU (Hindlimb Unloading for 14 days, n = 8). Both groups were housed in the same facilities, under temperature and light controlled conditions (23 °C, 12-h light/12-h dark cycle). Control rats were group housed (4 per cage). Hindlimb unloading was obtained using the model adapted from Morey-Holton and Globus (2002). The tail of each rat was cleaned, dried, and wrapped in antiallergenic adhesive plaster. This cast was secured to an overhead swivel that permitted 360° rotation. The rats were unloaded by the tail at a ~ 30° head-down angle in order to avoid a contact of the hindlimbs with the ground, whereas they were allowed to walk freely on their forelimbs. Animals had ad libitum access to food and water, and could have social interactions with their neighbors. Electrophysiological procedure The rats were anesthetized with ketamine hydrochloride (70 mg/ kg, i.p.), xylazine (5 mg/kg, i.p.) and acepromazine (0.2 mg/kg, i.p.). Supplementary doses of ketamine (20 mg/kg, i.p.) and acepromazine (0.02 mg/kg, i.p.) were delivered as needed to suppress hindlimb withdrawal reflex. The body temperature was maintained at approximately 37 °C with a thermostatically controlled heating pad. The rat was placed in a stereotaxic apparatus. Lidocaine 2% was applied on ear bars and scalp incision line. Electromyographic (EMG) recording 9 rats (4 C and 5 HU) were implanted for EMG recording. Bipolar electrodes were made from two insulated multistranded stainless steel wires (seven strands, 50 μm gauge, AM System, USA). The recording surface was obtained by mechanically removing the insulation for a length of 0.5 mm. A pair of electrode wires was inserted into the midbelly of triceps surae and tibialis anterior muscles of the left hindlimb and secured by means of sutures (Monosof, Tyco, France). Electrodes were linked to a differential amplifier (Model 1700, AM System, USA). The raw EMG signal was amplified (×1000, band pass 300 Hz–10 kHz), full-wave rectified and digitized at 2000 Hz. EMG analysis was performed with Spike2 software (Cambridge Electronic Design, UK). Microstimulation mapping The cortex was exposed by making a craniotomy that extended from 3 mm anterior to 3 mm posterior to bregma, and from 1 to 5 mm lateral from midline. A small puncture was made in the
cisterna magna to reduce edema. The dura mater was removed, and the cortex was covered with 37° artificial cerebrospinal fluid. Monophasic cathodal pulses were delivered with platinum-iridium microelectrodes (tip diameter: 1–2 μm, impedance: 1 MΩ at 1 kHz, parylene insulated, PTM23B10, WPI, UK). The anodal electrode was a screw implanted in the frontal bone. Electrodes were positioned at a depth of approximately 1800 μm below cortical surface, and then cautiously adjusted in order to evoke movement at the lowest threshold, corresponding to a localization within the layer V (Young et al., 2011). Electrode penetrations were placed in a grid-like fashion at 250 μm intervals. We never made two successive stimulations at closed locations. Stimulation consisted of 100 ms trains of 0.2 ms pulses (333 Hz) delivered at interval of 1 s from an electrically isolated, constant current stimulator (A300 Pulsemaster stimulator with A360 isolation unit, WPI, UK). Animals were maintained in a prone position, with hindlimbs unsupported. Hindlimbs were regularly mobilized (flexion and extension in a stepping-like movement). In addition, the threshold was first established with the limb approximately halfway between flexion and extension. Since changes in threshold could occur with respect to the limb position, the position was changed to from complete flexion to complete extension to search for the lowest threshold. The experimenter maintained firmly the different joints to verify that distal movement was not due to a movement of a more proximal joint. At each site, the stimulation was initiated at the lowest intensity (5 μA) and was progressively increased until a movement was evoked. Identification of joint movements was performed by visual inspection and palpation. At each penetration site, the minimal threshold required to elicit a movement of any joint of the hindlimb was recorded. This minimal current intensity was defined as “Absolute threshold”. Then, current intensity was gradually increased until a movement of a specific hindlimb joint (hip, knee, ankle or toes) was detected. This second intensity was considered as “Relative threshold”. Penetration sites that failed to elicit a movement of the hindlimb at any current intensity, up to 80 μA, were defined as unresponsive. If a movement of another joint (forelimb, vibrissae…) was obtained at current intensity corresponding to the absolute threshold, it was considered as an unresponsive site for hindlimb. Furthermore, as the anesthesia level varied during the experiment, we checked regularly in some already recorded penetration sites for changes in the current intensity. In addition, we tested the same point just before and several minutes after a new injection of anesthetic, and suspended the manipulation until we recovered the same threshold value. The cortical representation of the hindpaw was reconstructed by drawing boundaries to enclose the sites eliciting hindlimb movement or movement of a given joint. We considered that the area reconstruction was complete when it was surrounded by unresponsive sites (or sites responding to another body part). The area was determined by using Photoshop software. Statistical analyses Results are presented as mean ± SEM. Normality was tested with Kolmogorov–Smirnov test. Multi-group results were compared using Statistical Analysis Systems General Linear Model procedures and were subjected to two-way ANOVA to determine significant main effects and interactions. The Bonferroni test was used for post-hoc comparison. A t-test was performed when appropriate. A p-value of less than 0.05 was chosen as the significance level for all statistical analyses. A p-value of less than 0.1 was reported as a tendency. Results A total of 972 penetrations were made (535 in C rats and 437 in HU ones). In C rats, 60% of penetrations gave responses with current
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under 80 μA. Movements were evoked in the hindlimb (40%) or in another body part (20%). In HU rats, the proportion of responsive sites was only 46% (hindlimb: 29%, another body part: 17%) (Fig. 1). The ANOVA performed with Group (C vs. HU) as independent variable and Response (proportion of hindlimb, another body part, or unresponsive sites) as dependent variable revealed a Group × Response interaction (F = 6.07, p b 0.05). Unresponsive sites were more numerous in HU rats (post-hoc test: p b 0.01), whereas the proportion of sites eliciting a movement of hindlimb was reduced (post-hoc test: p b 0.05). Movements elicited at absolute threshold concerned one single joint. In C as in HU rats, we observed flexions of ankle, knee and hip, except for toes where extensions were reported. Hip adductions were also reported, and in one single case, an ankle extension. When the current intensity was increased, several joints were active, but the movement (i.e. flexion vs. extension) kept unchanged. Effect of HU on the M1 hindlimb map area In C rats, the M1 hindlimb map extended from A+0.75 to A− 2.00 with respect to Bregma, and from L1.50 to L4.00. The probability to encounter a responsive to HL site was the highest (>50%) at the following coordinates: A + 0.25 to − 1.50 and L 2.00 to 3.75. The hindlimb cortical area was surrounded by cortical regions with no response, or with forelimb representation in the rostral border, vibrissae in the caudal border, extending laterally and medially, and tail in the medial caudal border. The same organization was observed in HU rats, although the map was smaller. The mean anteroposterior extent was 1.75 ± 0.14 mm in C rats and only 1.08 ± 0.12 mm in HU ones (t-test: p b 0.01), and the mediolateral extent was respectively 1.87 ± 0.18 mm and 1.25 ± 0.14 mm (t-test: p b 0.05). The shrinkage from C to HU map was homogenous in all directions (− 0.25 mm in the caudal, rostral, lateral and medial directions). The area whose stimulation elicited hindlimb movements was 2.98 mm 2 at absolute threshold. It was dramatically reduced after 14 days of HU (− 61%, p b 0.01) (Fig. 2A). The same organization was observed in HU rats. We also measured the total representation of the different joints obtained with supra-threshold currents (i.e. relative threshold) (Fig. 2B). In C rats, a proximo-distal gradient is shown, the most proximal joint (hip) being more represented than the most distal one (toes). HU profoundly affected the organization of the hindlimb area. The two-way ANOVA performed with Group (C vs. HU) and Joint (toes, ankle, knee, hip) as independent variables, and Area as dependent variable, revealed a Joint effect (F = 8.15, p b 0.001). In addition, it confirmed the results obtained with absolute threshold currents (see above), namely that the area was decreased in HU rats (Group effect: F = 12.26, p b 0.01). There was also a Group× Joint interaction (F = 6.09, p b 0.01). The representational areas of the hip, knee and
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ankle were strongly affected (−65%, −73%, −72% respectively; post-hoc test: p b 0.001), whereas toe representation kept unchanged (−28%; ns). Thus, proximo-distal gradient observed in C rats disappeared in HU rats, all joints being almost equally represented. Effect of HU on the M1 hindlimb thresholds HU induced an increase in both absolute and relative thresholds (Fig. 3). Absolute threshold was increased from 16% (knee, p = 0.06) to 50% (ankle, p b 0.01). The decreased excitability was limited to the hindlimb area and was not an effect of HU on the whole motor cortex, since the current which elicited the movement of the forelimb was strictly unchanged in HU rats. The increase in relative threshold extended from 12% for the knee (p b 0.05) to 19% for the hip (p b 0.01). It should be noted that the increase was not significant for the toes (+15%, p = 0.07). Electromyography In 4 C and 5 HU rats, the electromyographic activity of ankle flexor (tibialis anterior) and extensor (gastrocnemius) muscles was recorded. Fig. 4 presents the threshold for the two groups (C and HU) and for different methods of determination (visual inspection and/or palpation vs. EMG). The threshold for electromyographic activation of flexor muscle was very close to the threshold determined visually (apparition of movement). By contrast, the value was higher for the extensor muscle. The two-way ANOVA reveals an effect of the method of determination (F = 6.76, p b 0.05). As indicated previously, the threshold was increased in HU rats (+ 27.6% and + 29.6% in flexor and extensor muscles, respectively) (Group effect: F = 30.99, p b 0.001). The response latency and duration have been measured on the flexor muscle. The analysis was not performed on the extensor muscle since the threshold was higher (Fig. 5). At threshold, a burst of activity was evoked approximately 95 ms after the onset of stimulation (C: 93.5 ± 4.9 ms; HU: 95.5 ± 5.1 ms; t-test: p = 0.89), and ended 25 ms after the offset of stimulation (C: 25.5 ± 4.4 ms; HU: 24.3 ± 2.4 ms; t-test: p = 0.88). No difference was reported between C and HU rats. When stimulus intensity was increased, the latency was reduced and the burst duration was increased (Fig. 5). Discussion The two main findings of the present work are first that HU led to a dramatic decrease in the hindlimb representation on the M1 cortex; second, current thresholds for eliciting a movement were increased. In addition, these effects were not identical according to the joint considered: the toes were less affected than the other joints. Methodological issues
Fig. 1. Proportion of sites eliciting a movement of hindlimb or of another body part (forelimb, trunk, tail, vibrissae), or of unresponsive sites (unresp.), in C rats (white bars) and HU rats (black bars). Data are means ± S.E.M. * and **: pb 0.05 and p b 0.01 respectively, with respect to C value.
Only a few papers have quantified the area of hindlimb representation in rats, and the variability between studies is particularly striking. VandenBerg et al. (2002) have reported values of ~ 0.5 mm 2 in Fischer-344 rats and ~ 1 mm 2 in Long-Evans rats. In the latter strain, Neafsey et al. (1986) reported an area of 7 mm 2. Value was 3.9 mm 2 in Sprague–Dawley rats (Delcour et al., 2011). Finally, we found that, in Wistar rats, the hindlimb representation extended on 2.98 mm 2 at relative threshold. These discrepancies might be explained in part by strain differences (VandenBerg et al., 2002). It is also well known that the level of anesthesia influences the apparition of movement, and thus the map extent and threshold values (Potez and Larkum, 2008; Tandon et al., 2008; Young et al., 2011; Zandieh et al., 2003). Comparison between studies is thus awkward. To avoid any bias due to variation in anesthetic state within the
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Fig. 2. Cortical maps obtained by electrophysiological mapping procedure. A. Representative individual maps in C (left) and HU (right) rats. Black dots indicate electrode penetrations in layer V of the M1 area. The map was reconstructed by drawing boundaries to enclose the sites eliciting movement of a given joint at absolute threshold. Note the strong reduction in hip and ankle representation. Values are stereotaxic coordinates in mm, with respect to bregma. B. Cortical surface area occupied by hindlimb movements in C (white bars) and HU rats (black bars). Maps were constructed with (relative) suprathreshold currents. Data are means ± S.E.M. ***: p b 0.001 with respect to C value.
present work, the level was carefully controlled and the same point was checked regularly. Our maps were probably slightly over-estimated since the maximum stimulation current used was 80 μA, instead of 60 μA in most studies. We chose this value as high current limit because in HU rats, due to the higher threshold and the smaller hindlimb representation area (in particular for the ankle), maps were difficult to build with lower currents. Large stimulation currents might activate neurons with a decreased excitability; they could also spread through a volume of tissue. Larger currents have also a repercussion on threshold mean values, which are higher in the present study than in others. However, when values in the range 60–80 μA were excluded, our mean threshold values were 15% decreased and were then very similar to those reported by Delcour et al. (2011). The latency for EMG activation was very long (~ 50 ms at twice the threshold), and was unchanged by HU. Maybe the 300 Hz high pass cut-off frequency could have produced a slight attenuation of signal, and could have affected the identification of EMG onset, since maximum EMG power in rats is ~ 300 Hz (Li et al., 2011). The high value could also be due to the anesthetic state. Liang et al. (1993) and Brus-Ramer et al. (2009) found latencies near 10 ms for the forelimb in the rat. Their anesthetic level was obtained by ketamine alone,
whereas we used a mixture of ketamine and acepromazine, which is known to induce a muscle relaxation, and thus could have impacted motor threshold and response latency. The hindlimb representation is decreased by hindlimb unloading Many studies have shown that when a limb disappears or is disconnected from the supraspinal structures, for instance following an amputation (Cohen et al., 1991a; Donoghue and Sanes, 1988; Pascual-Leone et al., 1996), nerve section (Sanes et al., 1990; Toldi et al., 1996), or spinal cord injury (Cohen et al., 1991b; Martinez et al., 2010), its cortical representation is profoundly altered. Our work shows that a non-traumatic situation also has heavy consequences on the cortical motor map, since the hindlimb representation area was decreased by 61%. The size of the decrease of hindlimb area in motor maps following HU (− 61%) is surprising when compared to the modest reduction in tactile representation of the hindpaw (− 15%) (Dupont et al., 2011) following 14 days of HU. Studies that evaluated in the same time both tactile and motor maps in response to a change in activity are very scarce. After training to a reaching task, the overall size of the somatosensory forepaw map was not altered, in spite of a strong increase (×1.6) of the forepaw
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Fig. 3. Absolute and relative threshold (in μA) in C rats (white bars) and HU rats (black bars), for the different joints of the hindlimb. Data are means ± S.E.M. *, **, #: p b 0.05, p b 0.01 and p b 0.1 with respect to C value.
movement representation (Coq et al., 2009). Taken together, it appears that the motor cortical maps seem to be more reactive to disuse or overuse than somatosensory ones. Threshold values are increased in unloaded rats Our data provided evidence for a decreased excitability of the corticospinal system. In humans, the excitability of the motor cortex is also modulated during immobilization, although studies are conflicting (Facchini et al., 2002; Kaneko et al., 2003; Liepert et al., 1995; Roberts et al., 2010; Zanette et al., 1997, 2004). The threshold measured herein was 1.4 greater for the ankle in HU rats, and 1.2 greater for the other joints. These values are similar to those reported by Martinez et al. (2010), who found a 1.4 fold increase in threshold after cervical spinal cord hemisection. Once again, this result underlines that a sensorimotor restriction can lead to changes comparable to those obtained after a traumatic section. A decreased excitability of corticospinal neurons was expected in the light of other studies on motor cortex. However, this result is
Fig. 4. Absolute threshold (in μA) in C rats (white bars) and HU rats (black bars), for the ankle. Threshold was determined by visual inspection (detection of ankle movement) or on EMG (activity detectable on flexor or extensor muscles). Data are means ± S.E.M. *, ** and ***: p b 0.05, 0.01 and 0.001 with respect to C value. #: p b 0.05 with respect to visual threshold.
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Fig. 5. A: Electromyographic activity of ankle flexor and extensor muscles, at flexor threshold (A1), or extensor threshold (2× flexor threshold, A2). The horizontal bar indicates the application of stimulating current, arrows indicate the latency. B: mean latency of flexor burst onset and offset, at flexor threshold (above) and at 2× flexor threshold (below), in C (white bars) and HU rats (hatched bars), The gray square represents the stimulus duration.
surprising in view of studies performed in the somatosensory cortex. In fact, extracellular recordings performed in the somatosensory cortex of HU rats have provided evidence for a higher activation of cortical neurons to electrical stimulation of the sciatic nerve (Langlet et al., 2001). In addition, studies have shown a reduction of threshold and an increase in cell activity in response to low tactile stimulation (Dupont et al., 2003). Taken together, these data indicate that excitability of the somatosensory cortex of HU rats was increased. Thus, as mentioned above, motor and sensory cortex react very differently to disuse. Toes are less affected than other joints by hindlimb unloading The reorganization of M1 cortex by HU concerns primarily the representation of proximal joints, whereas the toe representation is less affected. In C rats, the area occupied by the toes on the motor cortex is far smaller than other joints: supra-threshold currents can evoke movements of the toes from only 33% of the total area of hindlimb (against 75% for the hip). After HU, the proximo-distal gradient disappeared. Contrary to our data, after spinal cord hemisection, Martinez et al. (2010) reported an increase in the relative area representing the proximal forelimb whereas distal movements were drastically under-represented. The type and degree of reorganization should be considered with respect to the function of the limb: representation of distal parts of the forelimb is more affected by disuse since this limb is implied in fine manipulation; on the contrary, representation of proximal parts of the hindlimb is more concerned by disuse since hindlimb is functionally implied in the stabilization of the body during posture and gait. If remodeling of motor cortical maps is directly correlated to motor performance (see below), the animals should develop difficulties to produce efficient and harmonious movements, due to the fact that all joints are not affected in the same degree by HU. As a matter
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of fact, a kinematic study of hindlimb motion during locomotion has shown that intralimb coordination was profoundly affected in HU rats (Canu and Garnier, 2009). Mechanisms of motor plasticity Despite some attempts to determine the origin of plasticity which occurs in adults in response to disuse, mechanisms are not fully elucidated and are probably the combination of several factors (see Sanes and Donoghue, 2000 for review). Recently, in our model of unloading, an electrophysiological study performed in vitro has demonstrated that the basic electrophysiological characteristics of “regular spiking” neurons, identified as spiny pyramidal neurons and located in the hindlimb representation, were changed towards a decreased excitability (Canu et al., 2010). This change in input/output coupling could account for the increased threshold in HU rats. Dopamine is also known to modulate activity of cortical neurons, although data are limited (Luft and Schwartz, 2009 for review). Hosp et al. (2009) have shown that an acute injection of D2-receptor antagonist in the motor forelimb area of rats affected motor cortical excitability and somatotopy: the size of the forelimb representation was reduced by 68% and threshold increased by 37%. These values are quite similar to those observed in the present study (−61% and +40%, respectively); they suggest that a poor activation of D2-receptors within the deep cortical layers in response to the low dopamine level in HU rats might play a role in motor plastic mechanisms. As a matter of fact, we have shown that the overall level of dopamine and its metabolites was decreased in the hindlimb area of the sensorimotor cortex of HU rats (Canu et al., unpublished data). A change in the balance between excitation and inhibition, towards a decreased excitation of cortical neurons or an increase in inhibitory drive, could also explain the increase in threshold values. To sustain this hypothesis, a higher GABAergic inhibition by diazepam treatment results in significantly smaller forelimb movement areas and higher mean movement threshold (Young et al., 2011). However, a previous study has demonstrated that the level of GABA was decreased in brain tissue sampled from the hindlimb area cortex of HU rats (Canu et al., 2006), in particular within the layer IV (D'Amelio et al., 1996). Taken together, these data suggest a release from inhibition, which affects essentially the sensory processing, but not the motor command. Activity dependent plasticity is known to induce morphological changes within the motor cortex. A recent study performed by our team has shown an increase (~10%) in the density of spines of pyramidal neurons in HU rats (unpublished results). However, a great proportion of new spines are filopodia-like protrusions, which are immature shapes lacking synapses. This data suggests that a reorganization in the synaptic connectivity could occur after a period of HU. Functional implications and conclusion To conclude, the present study demonstrates that HU dramatically affects the organization and functioning of cortical motor maps and decreases corticospinal excitability. The cortical remodeling and the increase of the thresholds may participate to the alteration of the motor performance reported in rats submitted to HU (Canu and Garnier, 2009; Canu et al., 2007). In humans, studies have reported a deterioration of dexterity, a reduced muscle strength and changes in limb kinematics following 12 h (Moisello et al., 2008), 72 h (Weibull et al., 2011) or 1 week (Lundbye-Jensen and Nielsen, 2008) of arm immobilization. The change in motor performance was more likely attributed to a change in functional properties of the central nervous system (“voluntary deficit”) and/or to inter-hemispheric plasticity rather than to pure muscular deficits. Yet, most rehabilitation techniques for patients suffering from deconditioning after bed rest or inactivity are essentially muscle strengthening exercise programs (electrostimulation, resistive
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