Effect of hindlimb unloading on motor activity in adult rats: impact of prenatal stress.

Environmental changes that occur in daily life or, in particular, in situations like actual or simulated microgravity require neuronal adaptation of sensory and motor functions. Such conditions can exert long-lasting disturbances on an individual's adaptive ability. Additionally, prenatal stress also leads to behavioral and physiological abnormalities in adulthood. Therefore, the aims of the present study were (a) to evaluate in adult rats the behavioral motor adaptation that follows 14 days of exposure to simulated microgravity (hindlimb unloading) and (b) to determine whether restraint prenatal stress influences this motor adaptation. For this purpose, the authors assessed rats' motor reactivity to novelty, their skilled walking on a ladder, and their swimming performance. Results showed that unloading severely impaired motor activity and skilled walking. By contrast, it had no effect on swimming performance. Moreover, results demonstrated for the first time that restraint prenatal stress exacerbates the effects of unloading. These results are consistent with the role of a steady prenatal environment in allowing an adequate development and maturation of sensorimotor systems to generate adapted responses to environmental challenges during adulthood.

Soleus motoneuron excitability after rat hindlimb unloading using histology and a new electrophysiological approach to record a neurographic analogue of the H-reflex.

Hindlimb unloading (HU) induces neuromuscular disturbances in slow postural muscles such as the soleus. Among these perturbations, a reorganization of soleus motor units and decreases in peripheral afferent feedback have been reported and could change the alpha-motoneuron excitability. The purpose of this study was, therefore, to examine the distribution of soleus motoneuron soma sizes and the neurographic analogue of the H-reflex (NAH) induced by the soleus afferent excitation after 14 HU days. With this aim in view, we have elaborated a new electrophysiological method, discounting antidromic motor potentials, in order to record a neurographic analogue of the H-reflex, induced by soleus nerve stimulation at the L5 ventral root level instead of the electromyographic H-reflex response. Our results showed that, from a morphological point of view, the distribution of motoneuron soma sizes was modified and was shifted towards smaller sizes after HU (median 1065 microm2) in comparison with the control group (median 1314 microm2). The electrophysiological part of this study showed that thresholds and the maximal intensity of the NAH were respectively reduced by 30.5% and 27% after HU. No significant difference was obtained for the latency and the maximal amplitude of the NAH. Modifications of motoneuron excitability could be involved in the reflex adaptations observed after HU. However, changes in the presynaptic inhibition levels after HU cannot be discounted.

Variations in amino acid neurotransmitters in the rat ventral spinal cord after hindlimb unloading.

We have measured by HPLC the neurotransmitter content in L(4) and L(5) spinal segmental levels in CONT rats, after 7 (HU7) and after 14 days (HU14) of hindlimb unloading. These segments are known to contain the hindlimb muscle motoneurons. The main result is the increase of two neuroexcitators (glutamate and aspartate) and two neuroinhibitors (glycine and GABA) at the L(5) spinal segmental level in HU7 group. Our data indicated that the neurotransmitter changes are restricted to spinal segmental level containing motoneurons from muscles which are strongly modified by HU condition.

Physiologically adaptive changes of the L5 afferent neurogram and of the rat soleus EMG activity during 14 days of hindlimb unloading and recovery.

The hindlimb unloading rat model (HU, Morey's model) is usually used to mimic and study neuromuscular changes that develop during spaceflights. This Earth-based model of microgravity induces a muscular atrophy of the slow postural muscle of hindlimbs, such as the soleus, a loss of strength, modifications of contraction kinetics, changes in histochemical and electrophoretical profiles and modifications of the tonic EMG activity. It has been suggested in the literature that some of these neuromuscular effects were due to a reduction of afferent feedback during HU. However, no direct data have confirmed this hypothesis. The aim of this study was to clearly establish if changes of the L5 afferent neurogram are closely related to the soleus EMG activity during and after 14 days of HU. Immediately after HU, the EMG activity of the soleus muscle disappeared and was associated with a decrease in the afferent neurogram. The soleus electromyographic and afferent activities remained lower than the pre-suspension levels until the sixth day of HU and were recovered between the sixth and the ninth day. On the twelfth and fourteenth days, they were increased beyond the pre-suspension levels. During the first recovery day, these activities were significantly higher than those on the fourteenth HU day and returned to the pre-suspension levels between the third and sixth recovery days. To conclude, our study directly demonstrates that the HU conditions cannot be considered as a functional deafferentation, as suggested in the literature, but only as a reduction of afferent information at the beginning of the HU period.

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.

Changes in rat soleus muscle phenotype consecutive to a growth in hypergravity followed by normogravity.

It has been demonstrated that a long-term stay in hypergravity (HG: 2G) modified the phenotype and the contractile properties of rat soleus muscle. The ability of this muscle to contract was drastically reduced, which is a sign of anticipated aging. Consequently, our aim was to determine whether rats conceived, born, and reared in hypergravity showed adaptative capacities in normogravity (NG: 1G). This study was performed on rats divided into two series: the first was reared in HG until 100 days and was submitted to normogravity until 115 to 220 postnatal days (HG-NG rats); the second was made up of age paired groups reared in normogravity (NG rats). The contractile, morphological, and phenotypical properties of soleus muscle were studied. Our results showed that the NG rats were characterized by coexpressions of slow and fast myosin, respectively, 76.5 and 23.5% at 115 days. During their postnatal maturation, the fast isoform was gradually replaced by slow myosin. At 220 days, the relative proportions were respectively 91.05% and 8.95%. From 115 to 220 days, the HG-NG rats expressed 100% of slow myosin isoform and they presented a slower contractile behavior compared with their age-matched groups; at 115 days, the whole muscle contraction time was increased by 35%, and by 15%, at 220 days. Our study underlined the importance of gravity in the muscular development and suggested the existence of critical periods in muscle phenotype installation.

Enhancement of hybrid-fiber types in rat soleus muscle after clenbuterol administration during hindlimb unloading.

Our objective was to determine the effects of a clenbuterol (CB) treatment orally administered (2 mg per kg) to rats submitted to 14 days of hindlimb unloading (HU). The morphological and the contractile properties as well as the myosin heavy chain isoforms contained in each fiber type were determined in whole soleus muscles. As classically described after HU, a decrease in muscle wet weight and in body mass associated with a loss of muscular force, an evolution of the contractile parameters towards those of a fast muscle type, and the emergence of fast myosin heavy chain isoforms were observed. The CB treatment in the HU rats helped reduce the decrease in 1) muscle and body weights, 2) force and 3) the proportion of slow fibers, without preventing the emergence of fast myosin isoforms. Clenbuterol induced a complex remodelling of the muscle typing promoting the combination of both slow and fast myosin isoforms within one fiber. To conclude, our data demonstrate that CB administration partially counteracts the effects produced by HU, and they allow us to anticipate advances in the treatment of muscular atrophy.

A 14-day period of hindpaw sensory deprivation enhances the responsiveness of rat cortical neurons.

Hypodynamia-hypokinesia (HH) is a model of hindpaw sensory deprivation. It is obtained by unloading of the hindquarters during 14 days. In this situation, the feet are not in contact with the ground and as a consequence, the cutaneous receptors are not activated; the sensory input to the primary somatosensory cortex (SmI) is thus reduced. In a previous study, we have shown that HH induced a cortical reorganisation of the hindlimb representation. The understanding of the mechanisms involved in cortical map plasticity requires a close examination of the changes in response properties of cortical neurons during HH. The aim of the present study was thus to study the characteristics of neurons recorded from granular and infragranular layers in hindlimb representation of SmI. A total of 289 cortical neurons were recorded (158 from control rats and 131 from HH rats) in pentobarbital-anaesthetized rats. Cutaneous threshold, cutaneous receptive fields, spontaneous activity (discharge rate and instantaneous frequency) and activity evoked by air-jet stimulation (response latency and duration, amplitude) were analysed. The present study suggests that activity-dependent changes occur in the cortex. The duration of the spike waveform presented two populations of spikes: thin-spike cells (<1 ms, supposed to be inhibitory interneurons) and regular cells (>1 ms). Thin-spike cells were less frequently encountered in HH than in control rats. The analysis of regular cells revealed that after HH (1) spontaneous activity was unchanged and (2) cortical somatosensory neurons were more responsive: the cutaneous threshold was reduced and the response magnitude increased. Taken together, these results suggest a down-regulation of GABAergic function.

Expression of myosin heavy chain isoforms along intrafusal fibers of rat soleus muscle spindles after 14 days of hindlimb unloading.

Morphological, contractile, histochemical, and electrophoretical characteristics of slow postural muscles are altered after hindlimb unloading (HU). However, very few data on intrafusal fibers (IFs) are available. Our aim was to determine the effects of 14 days of hindlimb unloading on the morphological and immunohistochemical characteristics of IF in rat soleus muscle. Thirty-three control and 32 unloaded spindles were analyzed. The number and distribution of muscle spindles did not appear to be affected after unloading. There was no significant difference in number, cross-sectional area, and histochemical properties of IF between the two groups. However, after unloading, a significant decrease in slow type 1 MHC isoform and a slight increase in slow-tonic MHC expression were observed in the B and C regions of the bag1 fibers. The alpha-cardiac MHC expression was significantly decreased along the entire length of the bag2 fibers and in the B and C regions of the bag1 fibers. In 12 muscle spindles, the chain fibers expressed the slow type 1 and alpha-cardiac MHC isoforms over a short distance of the A region, although these isoforms are not normally expressed. The most striking finding of the study was the relative resistance of muscle spindles to perturbation induced by HU.

Contractile properties and myosin expression in rats born and reared in hypergravity.

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.

Fictive motor activity in rat after 14 days of hindlimb unloading.

The goal of the present study was to examine the effects of chronic hindlimb unloading on fictive motor patterns which can be developed in hindlimb nerves of adult rats. The animals were divided into two groups. The first group was submitted to hindlimb unloading for 2 weeks by tail suspension. The second group served as controls. After this initial phase, the animals of both groups were acutely decorticated, paralysed and electroneurographic efferent activity was recorded from hindlimb muscle nerves under conditions of "fictive locomotion" in order to evaluate variations in central locomotor command. Fictive rhythmic motor episodes were either spontaneous or evoked by electrical stimulation of the mesencephalic locomotor region. Only the second ones were recognised as locomotor-like activities. The motor pattern was not fundamentally affected by unloading except that, after the unloading period, extensor muscle nerves were significantly more frequently activated and their burst durations were increased compared to activity in control animals, despite the fact that the phasic sensory afferent inputs were suppressed. This suggests that unloading induces plastic modifications of the central networks of neurons implicated in the locomotor command. The origin of this extensor hyperactivity is discussed. It is proposed that it could be the consequence of either changes in motoneuronal properties or of an increase in afferent input to motoneurones.

Time course of recovery of the somatosensory map following hindpaw sensory deprivation in the rat.

Hindlimb sensory deprivation is known to induce a decrease in the cortical representation of hindpaw, and an increase in the size of the cutaneous receptive fields. The aim of the present study was to determine (i) the time-course of recovery when the rat retrieves a normal use of its limbs after a 14-day period of sensory disruption and (ii) whether a 1-day period of sensory deprivation is sufficient to induce a plasticity. Our results indicate that the remodelling of the cortical map was not observed after 1 day of sensory deprivation. On the other hand, the recovery was achieved after 6 h. These findings suggest that a procedure reducing sensory function resulted in reversible changes in the somatosensory cortex. The recovery was more rapid than the induction of plasticity.

Hypodynamia--hypokinesia induced variations in expression of fos protein in structures related to somatosensory system in the rat.

There have been many reports describing modifications of the sensory and motor cortex following various types of disuse. Hypodynamia--hypokinesia is characterized by the absence of weight-bearing and by a decrease in motor activity. We have shown a reorganization of the cortical cartography after hypodynamia--hypokinesia. In order to give an anatomical account for this cortical plasticity, we set out to determine whether cerebral and spinal structures exhibited variations of their neuronal activation. For this purpose, immunocytochemical detection of Fos protein was performed in the rat brain and spinal cord. Following stimulation of the sciatic nerve, Fos protein was detected in the primary and secondary somatosensory cortex in control rats and in rats submitted to an episode of 14 days of hypodynamia--hypokinesia. Results showed that the stimulation of the sciatic nerve induced an increase in the number of Fos-immunoreactive neurons in all these structures. Moreover, after hypodynamia--hypokinesia, the number of Fos-immunoreactive neurons was increased in the primary and secondary somatosensory cortex and in the spinal cord. These results provide evidence for a higher activation of cortical cells after hypodynamia--hypokinesia in comparison to controls. These data support the hypothesis that hypodynamia--hypokinesia contributes to the development of functional plasticity.

Effect of the beta(2)-agonist clenbuterol on the locomotor activity of rat submitted to a 14-day period of hypodynamia-hypokinesia.

The beta(2)-adrenergic agonist clenbuterol is known for its anabolic action on normal and atrophied muscles. The aim of this work was to evaluate if chronic clenbuterol administration could prevent alterations in the locomotor activity induced by hindlimb suspension. The effects of clenbuterol were evaluated in three studies: muscle morphological characteristics, observation of locomotor movement and electromyographic activity of soleus and gastrocnemius muscles. Rats were divided into four groups: control (CON, morphological study only), hindlimb suspended (HS), clenbuterol administered (CB, 2 mg kg(-1) per day in drinking water), and hindlimb suspended+clenbuterol administered (HSCB). The soleus muscle weight was reduced in the two suspended groups (HS and HSCB) but did not change after clenbuterol treatment. By contrast, the gastrocnemius weight was not affected by suspension but was increased by clenbuterol (CB and HSCB). Some locomotor deficits were always observed in HS rats (unstable gait, ankle hyperextension, ellipsis). Clenbuterol administration did not prevent these perturbations. Cycle duration and soleus burst duration were increased in the three groups. Soleus mean EMG (burst area/duration) was decreased in HS rats, but not in the two other groups. For the gastrocnemius, burst duration was increased in CB rats, decreased in HSCB rats and unchanged in HS ones; mean EMG did not change. In conclusion, clenbuterol cannot be used as a countermeasure to reduce the alteration in locomotor performance. Moreover, our results suggest that this alteration is specifically related to changes in neuronal properties.

Effects of cutaneous receptor stimulation on muscular atrophy developed in hindlimb unloading condition.

The aim of this study was to investigate whether stimulation of the cutaneous mechanoreceptors of the rat foot sole could partially or totally prevent the soleus muscle atrophy developed after 14 days in hindlimb unloading conditions. Final experiments were achieved under deep anesthesia using pentobarbital sodium (60 mg/kg, ip injection). Atrophy was characterized by a significant decrease in muscle wet weight, fiber size, maximal twitch and tetanic tensions, contraction kinetics, and histochemical and electrophoretical changes. Our data demonstrate that the stimulation of these mechanoreceptors partially prevents the decrease in muscle weight (53%) and cross-sectional area of the soleus muscle (36%) and in all fiber types (type I: 31%; type Ic: 40%; type IIc: 49%; and type IIa: 44%) and also prevented the reductions in strength (peak twitch tension: 31%; peak tetanic tension: 25%). However, the decrease in contraction kinetics was not counteracted. Moreover, histochemical and electrophoretical changes were partially slowed. Thus our results suggest that stimulation of the sole mechanoreceptors can be used, in part, as a countermeasure to the muscular atrophy observed after a period of hindlimb unloading.

Phenotypic changes in the composition of muscular fibres in rat soleus motor units after 14 days of hindlimb unloading.

The main goal of this study was to identify the different fibre types of the motor units (MUs) contained in the soleus muscles from control (CONT) rats and from rats submitted to 14 days of hindlimb unloading (HU). The MU types were classified according to their contractile properties and also using glycogen depletion followed by adenosine triphosphatase (ATPase) staining. In CONT rats, the soleus muscle contained two MU types identified as slow and fast types. After HU, the MU distribution showed three populations: slow, intermediate and fast. All the MUs from HU soleus were heterogeneous in terms of fibre type composition, indicating a complex remodelling of the muscle.

Effect of microgravity on the electromyographic activity of two upperlimb muscles during a goal-directed movement and during locomotion.

It is well known that both neuromuscular and perceptual properties are affected during spaceflight. These modificaitons can therefore induce dramatic alterations in the mechanical basis of movements and locomotion disturbances. The main objectives of this study were: 1) to examine whether the nervous control of muscular activity in the upper limbs of the rhesus monkey (Macaca mulatta) was modified in a microgravity environment; and 2) to quantify the electromyographic (EMG) pattern of biceps (BI) and triceps (TRI) muscles pre-, in-, and postflight during performance of goal-directed movements and locomotion.

Influence of brief daily tendon vibration on rat soleus muscle in non-weight-bearing situation.

The purpose of this study was to investigate whether tendon vibration could prevent soleus muscle atrophy during hindlimb unloading (HU). Mechanical vibrations with a constant low amplitude (0.3 mm) were applied (192 s/day) with constant frequency (120 Hz) to the Achilles tendon of the unloaded muscle during the 14-day HU period. Significant reductions in muscle mass (-41%), fiber size, maximal twitch (-54%), and tetanic tensions (-73%) as well as changes in fiber type and electrophoretic profiles and twitch-time parameters (-31% in the contraction time and -30% in the half relaxation time) were found after 14 days of HU when compared with the control soleus. Tendon vibration applied during HU significantly attenuated, but did not prevent, 1) the loss of muscle mass (17 vs. 41%); 2) the decrease in the fiber cross-sectional area of type IIA (-28 vs. -50%) and type IIC (-29 vs. -56%) fibers; and 3) the decrease in maximal twitch (-3 vs. -54%) and maximal tetanic tensions (-29 vs. -73%) and the half relaxation time (1 vs. -30%). Changes in the contraction time and in histological and electrophoretical parameters associated with HU were not counteracted. These findings suggest that tendon vibration can be used as a paradigm to counteract the atrophic process observed after HU.

Short-term reorganization of the rat somatosensory cortex following hypodynamia-hypokinesia.

This study was performed to determine if hypodynamia-hypokinesia (HH) could induce a reorganization of the rat somatosensory cortex. The cortical hindpaw representation was determined by stimulating the limb and recording multi-unit cortical activity. The size of the cutaneous receptive fields was also measured. After 14 days of HH, the size of the cortical hindpaw representation was decreased. The proportion of small cutaneous receptive fields decreased while the large ones increased. After 7 days of HH, no change in the two studied parameters was noticed in five animals. In the other rats, a number of sites unresponsive to cutaneous stimulation or with high thresholds was observed. This study provides evidence of a plasticity of the somatosensory cortex induced by a situation that reduces both sensory and motor functions. The cortical reorganization occurs in two stages.

Short-term plasticity in primary somatosensory cortex of the rat after hindlimb suspension.

Since the last 25 years, the cortex is considered as a dynamic entity, susceptible of changes. Various types of modifications in stimuli may lead to the plasticity of the target neurons. These include immobilisation, denervation, amputation, deafferentation... In the somatosensory system, the most important changes are a substantial reorganisation of the cortical somatotopic representation, and an enlargement of the receptive fields (RF) of cortical neurons. Hindlimb suspension (HS) is characterized by the absence of weight-bearing and a reduced motor activity. In these conditions, the cutaneous receptors located on the foot sole are deactivated. Our hypothesis is that this condition of HS can produce a reorganisation of the somatosensory cortex (SmI) and a modification in the size of the cutaneous RF.