Functional ultrasound imaging of the human spinal cord.

Utilizing the first in-human functional ultrasound imaging (fUSI) of the spinal cord, we demonstrate the integration of spinal functional responses to electrical stimulation. We record and characterize the hemodynamic responses of the spinal cord to a neuromodulatory intervention commonly used for treating pain and increasingly used for the restoration of sensorimotor and autonomic function. We found that the hemodynamic response to stimulation reflects a spatiotemporal modulation of the spinal cord circuitry not previously recognized. Our analytical capability offers a mechanism to assess blood flow changes with a new level of spatial and temporal precision in vivo and demonstrates that fUSI can decode the functional state of spinal networks in a single trial, which is of fundamental importance for developing real-time closed-loop neuromodulation systems. This work is a critical step toward developing a vital technique to study spinal cord function and effects of clinical neuromodulation.

And yet it moves: Recovery of volitional control after spinal cord injury.

Preclinical and clinical neurophysiological and neurorehabilitation research has generated rather surprising levels of recovery of volitional sensory-motor function in persons with chronic motor paralysis following a spinal cord injury. The key factor in this recovery is largely activity-dependent plasticity of spinal and supraspinal networks. This key factor can be triggered by neuromodulation of these networks with electrical and pharmacological interventions. This review addresses some of the systems-level physiological mechanisms that might explain the effects of electrical modulation and how repetitive training facilitates the recovery of volitional motor control. In particular, we substantiate the hypotheses that: (1) in the majority of spinal lesions, a critical number and type of neurons in the region of the injury survive, but cannot conduct action potentials, and thus are electrically non-responsive; (2) these neuronal networks within the lesioned area can be neuromodulated to a transformed state of electrical competency; (3) these two factors enable the potential for extensive activity-dependent reorganization of neuronal networks in the spinal cord and brain, and (4) propriospinal networks play a critical role in driving this activity-dependent reorganization after injury. Real-time proprioceptive input to spinal networks provides the template for reorganization of spinal networks that play a leading role in the level of coordination of motor pools required to perform a given functional task. Repetitive exposure of multi-segmental sensory-motor networks to the dynamics of task-specific sensory input as occurs with repetitive training can functionally reshape spinal and supraspinal connectivity thus re-enabling one to perform complex motor tasks, even years post injury.

Effects of spinal cord stimulation on motor functions in children with cerebral palsy.

Is it possible to regulate the functional properties of abnormally developed spinal neuronal locomotor networks using transcutaneous spinal cord stimulation? This question has been studied in twenty-eight participants (∼9 yrs) with spastic cerebral palsy, and mainly Gross Motor Function Classification System for Cerebral Palsy level III. The participants were randomly assigned to two groups. The experimental group received transcutaneous spinal cord stimulation at two spinal levels (over T11 and L1 spinous processes), combined with locomotor treadmill training, whereas the participants of the control group received locomotor treadmill training only. After spinal cord stimulation in the experimental group we found an incremental increase in knee torque whereas in the control group this effect was absent. The amplitude of hip motion increased in both groups. A decrease of co-activation of hip and muscles of the lower extremities was observed in the experimental group while in the control group co-activation decreased only in hip muscles. The results support the idea that locomotor function can be improved significantly with the combination of training and transcutaneous spinal cord stimulation than with training alone.

SENSORIMOTOR REGULATION OF MOVEMENTS: NOVEL STRATEGIES FOR THE RECOVERY OF MOBILITY.

A series of observations have provided important insight into properties of the spinal as well as supraspinal circuitries that control posture and movement. We have demonstrated that spinal rats can regain full weight-bearing standing and stepping over a range of speeds and directions with the aid of electrically enabling motor control (eEmc), pharmacological modulation (fEmc), and training. Also, we have reported that voluntary control movements of individual joints and limbs can be regained after complete paralysis in humans. However, the ability to generate significant levels of voluntary weight-bearing stepping with or without epidural spinal cord stimulation remains limited. Herein we introduce a novel method of painless transcutaneous electrical enabling motor control (pcEmc) and sensory enabling motor control (sEmc) strategy to neuromodulate the physiological state of the spinal cord. We have found that a combination of a novel non-invasive transcutaneous spinal cord stimulation and sensory-motor stimulation of leg mechanoreceptors can modulate the spinal locomotor circuitry to that enables voluntary rhuthmic locomotor movements.

A Unilateral Cervical Spinal Cord Contusion Injury Model in Non-Human Primates (Macaca mulatta).

The development of a non-human primate (NHP) model of spinal cord injury (SCI) based on mechanical and computational modeling is described. We scaled up from a rodent model to a larger primate model using a highly controllable, friction-free, electronically-driven actuator to generate unilateral C6-C7 spinal cord injuries. Graded contusion lesions with varying degrees of functional recovery, depending upon pre-set impact parameters, were produced in nine NHPs. Protocols and pre-operative magnetic resonance imaging (MRI) were used to optimize the predictability of outcomes by matching impact protocols to the size of each animal's spinal canal, cord, and cerebrospinal fluid space. Post-operative MRI confirmed lesion placement and provided information on lesion volume and spread for comparison with histological measures. We evaluated the relationships between impact parameters, lesion measures, and behavioral outcomes, and confirmed that these relationships were consistent with our previous studies in the rat. In addition to providing multiple univariate outcome measures, we also developed an integrated outcome metric describing the multivariate cervical SCI syndrome. Impacts at the higher ranges of peak force produced highly lateralized and enduring deficits in multiple measures of forelimb and hand function, while lower energy impacts produced early weakness followed by substantial recovery but enduring deficits in fine digital control (e.g., pincer grasp). This model provides a clinically relevant system in which to evaluate the safety and, potentially, the efficacy of candidate translational therapies.

Integrating multiple sensory systems to modulate neural networks controlling posture.

In this study we investigated the ability of sensory input to produce tonic responses in hindlimb muscles to facilitate standing in adult spinal rats and tested two hypotheses: 1) whether the spinal neural networks below a complete spinal cord transection can produce tonic reactions by activating different sensory inputs and 2) whether facilitation of tonic and rhythmic responses via activation of afferents and with spinal cord stimulation could engage similar neuronal mechanisms. We used a dynamically controlled platform to generate vibration during weight bearing, epidural stimulation (at spinal cord level S1), and/or tail pinching to determine the postural control responses that can be generated by the lumbosacral spinal cord. We observed that a combination of platform displacement, epidural stimulation, and tail pinching produces a cumulative effect that progressively enhances tonic responses in the hindlimbs. Tonic responses produced by epidural stimulation alone during standing were represented mainly by monosynaptic responses, whereas the combination of epidural stimulation and tail pinching during standing or epidural stimulation during stepping on a treadmill facilitated bilaterally both monosynaptic and polysynaptic responses. The results demonstrate that tonic muscle activity after complete spinal cord injury can be facilitated by activation of specific combinations of afferent inputs associated with load-bearing proprioception and cutaneous input in the presence of epidural stimulation and indicate that whether activation of tonic or rhythmic responses is generated depends on the specific combinations of sources and types of afferents activated in the hindlimb muscles.

[Neuronal control of posture and locomotion in decerebrated and spinalized animals].

We have found that the brainstem-spinal cord circuitry of decerebrated cats actively maintain the equilibrium during standing, walking and imposed mechanical perturbations similar to that observed in intact animals. The corrective hindlimb motor responses during standing included redistribution of the extensor activity ipsilateral and contralateral to perturbation. The postural corrections in walking cats were due to considerable modification of EMG pattern in the limbs as well as changing of the swing-stance phases of the step cycle and ground reaction forces depending of perturbation side. Thus the basic mechanisms for balance control of decerebrated animals in these two forms of motor behavior are different. Balance-related adjustments relied entirely on the integration of somatosensory information arising from the moving hindquarters because of the suppression of vestibular, visual, and head-neck-trunk sensory input. We propose that the somatosensory input from the hindquarters in concert with the lumbosacral spinal circuitry can control the dynamics of the hindquarters sufficient to sustain balance. We found that, after isolation from the brainstem or forebrain, lumbosacral circuits receiving tonic epidural electrical stimulation can effectively control equilibrium during standing and stepping. Detailed analyses of the relationships among muscle activity, trunk kinematics, and limb kinetics indicate that spinal motor systems utilize a combination of feedback and feedforward strategies to maintain dynamic equilibrium during walking. The unexpected ability of spinal circuitries to exert efficient postural control in the presence of epidural electrical stimulation in decerebrated and spinal cats have significant implications for the potential of humans with a severe spinal cord injury to regain a significant level of functional standing and walking capacities.

Ankle morphology amplifies calcaneus movement relative to triceps surae muscle shortening.

The present study investigated the mechanical role of the dorsoventral curvature of the Achilles tendon in the conversion of the shortening of the plantarflexor muscles into ankle joint rotation. Dynamic, sagittal-plane magnetic resonance spin-tagged images of the ankle joint were acquired in six healthy subjects during both passive and active plantarflexion movements driven by a magnetic resonance compatible servomotor-controlled foot-pedal device. Several points on these images were tracked to determine the 1) path and deformation of the Achilles tendon, 2) ankle's center of rotation, and 3) tendon moment arms. The degree of mechanical amplification of joint movement was calculated as the ratio of the displacements of the calcaneus and myotendinous junction. In plantarflexion, significant deflection of the Achilles tendon was evident in both the passive (165.7 ± 7.4°; 180° representing a straight tendon) and active trials (166.9 ± 8.8°). This bend in the dorsoventral direction acts to move the Achilles tendon closer to the ankle's center of rotation, resulting in an ∼5% reduction of moment arm length. Over the entire range of movement, the overall displacement of the calcaneus exceeded the displacement of the myotendinous junction by ∼37%, with the mechanical gains being smaller in dorsi- and larger in plantarflexed joint positions. This is the first study to assess noninvasively and in vivo using MRI the curvature of the Achilles tendon during both passive and active plantarflexion movements. The dorsoventral tendon curvature amplifies the shortening of the plantarflexor muscles, resulting in a greater displacement of the tendon's insertion into the calcaneus compared with its origin.

Growth hormone plus resistance exercise attenuate structural changes in rat myotendinous junctions resulting from chronic unloading.

Myotendinous junctions (MTJs) are specialized sites on the muscle surface where forces generated by myofibrils are transmitted across the sarcolemma to the extracellular matrix. At the ultrastructural level, the interface between the sarcolemma and extracellular matrix is highly folded and interdigitated at these junctions. In this study, the effect of exercise and growth hormone (GH) treatments on the changes in MTJ structure that occur during muscle unloading, has been analyzed. Twenty hypophysectomized rats were assigned randomly to one of five groups: ambulatory control, hindlimb unloaded, hindlimb unloaded plus exercise (3 daily bouts of 10 climbs up a ladder with 50% body wt attached to the tail), hindlimb unloaded plus GH (2 daily injections of 1 mg/kg body wt, i.p.), and hindlimb unloaded plus exercise plus GH. MTJs of the plantaris muscle were analyzed by electron microscopy and the contact between muscle and tendon was evaluated using an IL/B ratio, where B is the base and IL is the interface length of MTJ's digit-like processes. After 10 days of unloading, the mean IL/B ratio was significantly lower in unloaded (3.92), unloaded plus exercise (4.18), and unloaded plus GH (5.25) groups than in the ambulatory control (6.39) group. On the opposite, the mean IL/B ratio in the group treated with both exercise and GH (7.3) was similar to control. These findings indicate that the interaction between exercise and GH treatments attenuates the changes in MTJ structure that result from chronic unloading and thus can be used as a countermeasure to these adaptations.

[Transcutaneous electrical stimulation of the spinal cord: non-invasive tool for activation of locomotor circuitry in human].

A new tool for locomotor circuitry activation in the non-injured human by transcutaneous electrical spinal cord stimulation (tSCS) has been described. We show that continuous tSCS over T11-T12 vertebrae at 5-40 Hz induced involuntary locomotor-like stepping movements in subjects with their legs in a gravity-independent position. The increase of frequency of tSCS from 5 to 30 Hz augmented the amplitude of evoked stepping movements. The duration of cycle period did not depend on frequency of tSCS. During tSCS the hip, knee and ankle joints were involved in the stepping performance. It has been suggested that tSCS activates the locomotor circuitry through the dorsal roots. It appears that tSCS can be used as a non-invasive method in rehabilitation of spinal pathology.

A new age for rehabilitation.

In this review we will describe newly developed techniques that are being used to recover levels of motor function after a severe spinal cord injury that have not been observed previously. These new approaches include pharmacological neuromodulation and/or epidural stimulation of the spinal cord circuitries in combination with motor training. By combining the increased levels of excitability of the interneuronal spinal circuitries using these interventions and the ability of the spinal circuitries to interpret and respond appropriately to ongoing complex ensembles of sensory input, the peripheral sensory system can become an effective source for the control of motor function. Similar types of neuromodulation have been shown to enable the brain to regain functional connectivity with the spinal cord circuitries below a clinically complete spinal cord lesion. In fact, some level of voluntary control of movement has been observed in subjects with complete paralysis in the presence of epidural stimulation. The biological mechanisms thought to underlie the recovery of motor function after a severe spinal cord injury are based on decades of research on a wide range of animal models. Fortunately the extensive conservation of neural mechanisms of motor control has provided a window for gaining considerable insight into the mechanisms of recovery of motor function in humans.

Selectively reshaping a muscle phenotype: functional overload of cat plantaris.

INTRODUCTION: Functional overload (FO) of the fast plantaris muscle was studied in treadmill-exercised (FO-Ex) or sedentary (FO-Sed) adult cats. METHODS: Mechanical, phenotype, and kinematics analyses were performed. RESULTS: Plantigrade vs. normal digitigrade posture was observed early post-FO. Relative plantaris mass was greater in FO-Sed (10%) and FO-Ex (60%) cats than in controls 12 weeks post-FO. Specific tension was similar across groups, indicating functional hypertrophy. Fiber size was greater, percent slow fibers higher, percent IIa myosin heavy chain (MHC) higher, and IIx MHC lower in FO-Ex than controls. Twitch and half-relaxation times were longer, and the frequency-tension curve shifted toward that observed in slow muscles. Electromyography (EMG) and tendon force amplitudes during stepping were larger, and the yield (lengthening) phase occurred at a longer muscle length before compared with after FO. DISCUSSION: Reshaping the plantaris phenotype was highly dependent on the overload stimulus, indicating that electrical stimulation paradigms used during rehabilitation should be performed with the muscles under "loaded" conditions.

Functional recovery of stepping in rats after a complete neonatal spinal cord transection is not due to regrowth across the lesion site.

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.

BDNF-exercise interactions in the recovery of symmetrical stepping after a cervical hemisection in rats.

Clinical evidence indicates that motor training facilitates functional recovery after a spinal cord injury (SCI). Brain-derived neurotrophic factor (BDNF) is a powerful synaptic facilitator and likely plays a key role in motor and sensory functions. Spinal cord hemisection decreases the levels of BDNF below the injury site, and exercise can counteract this decrease [Ying Z, Roy RR, Edgerton VR, Gomez-Pinilla F (2005) Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury. Exp Neurol 193:411-419]. It is not clear, however, whether the exercise-induced increases in BDNF play a role in mediating the recovery of locomotion after a SCI. We performed a lateral cervical ( approximately C4) hemisection in adult rats. Seven days after hemisection, the BDNF inhibitor trkB IgG was injected into the cervical spinal cord below the lesion ( approximately C5-C6). Half of the rats were exposed to voluntary running wheels for 14 days. Locomotor ability was assessed by determining the symmetry between the contralateral (unaffected) vs. the ipsilateral (affected) forelimb at the most optimum treadmill speed for each rat. Sedentary and exercised rats with BDNF inhibition showed a higher level of asymmetry during the treadmill locomotion test than rats not treated with the BDNF inhibitor. In hemisected rats, exercise normalized the levels of molecules important for synaptic function, such as cyclic AMP response element binding protein (CREB) and synapsin I, in the ipsilateral cervical enlargement, whereas the BDNF blocker lessened these exercise-associated effects. The results indicate that BDNF levels play an important role in shaping the synaptic plasticity and in defining the level of recovery of locomotor performance after a SCI.

BDNF and learning: Evidence that instrumental training promotes learning within the spinal cord by up-regulating BDNF expression.

We have previously shown that the spinal cord is capable of learning a sensorimotor task in the absence of supraspinal input. Given the action of brain-derived neurotrophic factor (BDNF) on hippocampal learning, the current studies examined the role of BDNF in spinal learning. BDNF is a strong synaptic facilitator and, in association with other molecular signals (e.g. cAMP-response element binding protein (CREB), calcium/calmodulin activated protein kinase II (CaMKII) and synapsin I), important for learning. Spinally transected rats given shock to one hind leg when the leg extended beyond a selected threshold exhibited a progressive increase in flexion duration that minimized shock exposure, a simple form of instrumental learning. Instrumental learning resulted in elevated mRNA levels of BDNF, CaMKII, CREB, and synapsin I in the lumbar spinal cord region. The increases in BDNF, CREB, and CaMKII were proportional to the learning performance. Prior work has shown that instrumental training facilitates learning when subjects are tested on the contralateral leg with a higher response criterion. Pretreatment with the BDNF inhibitor TrkB-IgG blocked this facilitatory effect, as did the CaMKII inhibitor AIP. Intrathecal administration of BDNF facilitated learning when subjects were tested with a high response criterion. The findings indicate that instrumental training enables learning and elevates BDNF mRNA levels within the lumbar spinal cord. BDNF is both necessary, and sufficient, to produce the enabling effect.

Differential modulation of myosin heavy chain phenotype in an inactive extensor and flexor muscle of adult rats.

The effects of chronic neuromuscular inactivity on the phenotype and size of muscle fibres in a fast ankle extensor (medial gastrocnemius, MG) and a fast ankle flexor (tibialis anterior, TA) muscle of the rat hindlimb were determined. Inactivity was produced by spinal cord isolation (SI), i.e. complete spinal cord transections at a mid-thoracic and high sacral level and bilateral deafferentation between the transection sites. After 90 days of SI, the MG and TA muscle weights were 53 and 45% lower than in age-matched controls. Overall mean fibre sizes in the deep (close to the bone) and superficial (away from the bone) regions were approximately 60 and 65% smaller in the MG and approximately 40 and 50% smaller in the TA of SI than control rats, respectively. The myosin heavy chain (MHC) composition shifted towards the faster isoforms after SI: the MG showed an increase in both types IIx (20%) and IIb (23%), whereas the TA showed a marked increase in type IIx (94%) and a decrease in type IIb (18%) MHC. Both muscles in SI rats showed no type IIa and only one MG muscle had approximately 5% type I MHC. These results show that prolonged inactivity has a stronger effect on a fast extensor compared with a fast flexor in the rat hindlimb. The larger decrease in mass and fibre size in the MG than the TA most probably reflects the larger impact of chronic inactivity on the normally more highly recruited extensor than flexor muscle. The primary shift to type IIb MHC in the MG and type IIx MHC in the TA indicate a different default mode for an inactive extensor vs. flexor muscle, and may reflect differing activity-independent neural influences, i.e. neurotrophic factors, on muscle fibre phenotype in extensors vs. flexors.

Is spinal cord isolation a good model of muscle disuse?

The patterns of normal daily activity that are required to maintain normal skeletal muscle properties remain unknown. The present study was designed to determine whether spinal cord isolation can be used as a reliable experimental model of neuromuscular inactivity, that is, as a baseline for the absence of activity. Electromyograms (EMGs) were recorded from selected hindlimb muscles of unanesthetized rats over 24-hour periods before and 7, 30, 60, and 90 days after surgical isolation of the lumbar spinal cord. Our data indicate that some rat slow muscle fibers pre-surgery were activated for less than 3 hours per day. Spinal cord isolation (SI) reduced the mean daily integrated EMG (IEMG) and daily EMG duration in the primary slow extensor muscle (soleus) to <1% of control, and in the primary fast extensor muscles [medial gastrocnemius (MG) and vastus lateralis (VL)] to <2% of control. These parameters were decreased to <8% and 3% of control, respectively, in a primary fast flexor muscle, the tibialis anterior (TA). From 30 to 90 days post-SI, the mean amplitudes of the spontaneous EMG bursts were relatively normal in the soleus, increased approximately 2-fold in the MG and VL, and increased approximately 4-fold in the TA. Some evidence of the normal antagonistic flexor-extensor relationship was apparent in the brief periods of recorded activity post-SI. These results indicate that SI eliminates nearly all of the normal EMG activity in the hindlimb muscles in the presence of relatively normal muscle innervation and functional intraspinal neural circuitry.

Plasticity of functional connectivity in the adult spinal cord.

This paper emphasizes several characteristics of the neural control of locomotion that provide opportunities for developing strategies to maximize the recovery of postural and locomotor functions after a spinal cord injury (SCI). The major points of this paper are: (i) the circuitry that controls standing and stepping is extremely malleable and reflects a continuously varying combination of neurons that are activated when executing stereotypical movements; (ii) the connectivity between neurons is more accurately perceived as a functional rather than as an anatomical phenomenon; (iii) the functional connectivity that controls standing and stepping reflects the physiological state of a given assembly of synapses, where the probability of these synaptic events is not deterministic; (iv) rather, this probability can be modulated by other factors such as pharmacological agents, epidural stimulation and/or motor training; (v) the variability observed in the kinematics of consecutive steps reflects a fundamental feature of the neural control system and (vi) machine-learning theories elucidate the need to accommodate variability in developing strategies designed to enhance motor performance by motor training using robotic devices after an SCI.

Use of c-fos to identify activity-dependent spinal neurons after stepping in intact adult rats.

STUDY DESIGN: An investigation of c-fos activation pattern in spinal neurons of intact adult rats after acute bouts of treadmill locomotion. OBJECTIVES: To map spinal neurons that are involved in quadrupedal treadmill stepping of intact adult rats by using c-fos as a marker. SETTINGS: Los Angeles, CA, USA. METHODS: Spinal cord sections of rats that were not stepped (n = 4) were used to map the FOS-positive (+) neurons under basal conditions. The stepped group (n = 16) was placed on a treadmill to step quadrupedally for varying durations to induce c-fos activity. Spinal cord sections of thoracic and lumbar segments of Stp and Nstp rats were processed using a c-fos antibody, choline acetyl transferase and heat shock protein 27 for identifying motoneurons. RESULTS: Stepping induced a greater number of FOS+ neurons than was observed in rats that did not step on the treadmill. There was a rostrocaudal and a dorsoventral gradient of FOS labeled neurons. The number of FOS+ neurons increased with the duration of treadmill stepping. Significant increases in FOS+ neurons were in the most medial parts of laminae IV, V, and VII. FOS+ motoneurons increased with treadmill stepping, particularly in large motoneurons (> or = 700 microm2). CONCLUSION: These data suggest that FOS can be used to identify activity-dependent neuronal pathways in the spinal cord that are associated with treadmill stepping, specifically in lamina VII and in alpha motoneurons. SPONSORSHIP: NIH NS16333, NS40917, and the Christopher Reeve Paralysis Foundation (CRPF VEC 2002).

Basal and evoked levels of bioassayable growth hormone are altered by hindlimb unloading.

Bioassayable growth hormone (BGH) in rats is released in large quantities from the pituitary in response to the activation of large, proprioceptive afferent fibers from fast and mixed fiber-type hindlimb musculature. We hypothesized that hindlimb unloading (HU) of adult male rats would 1) reduce the basal levels of plasma BGH, and 2) abolish stimulus-induced BGH release. Rats were exposed to HU for 1, 4, or 8 wk. Plasma and pituitaries were collected under isoflurane anesthesia for hormone analyses. Additionally, at 4 and 8 wk, a subset of rats underwent an in situ electrical stimulation (Stim) of tibial nerve proprioceptive afferents. Basal plasma BGH levels were significantly reduced (-51 and -23%) after 1 and 8 wk of HU compared with ambulatory controls (Amb). Although Amb-Stim rats exhibited increased plasma BGH levels (88 and 143%) and decreased pituitary BGH levels (-27 and -22%) at 4 and 8 wk, respectively, stimulation in HU rats had the opposite effect, reducing plasma BGH (-25 and -33%) and increasing pituitary BGH levels (47 and 10%) relative to HU alone at 4 and 8 wk. The 22-kDa form of GH measured by immunoassay and the plasma corticosterone, T3, T4, and testosterone levels were unchanged by HU or Stim at all time points. These data suggest that BGH synthesis and release from the pituitary are sensitive both to chronically reduced neuromuscular loading and to acute changes in neuromuscular activation, independent of changes in other circulating hormones. Thus BGH may play a role in muscle, bone, and metabolic adaptations that occur in response to chronically unloaded states.