Multielectrode spiral cuff for ordered and reversed activation of nerve fibres

In this paper we present the modelling, design, and experimental testing of a nerve cuff multielectrode system for selective activation of fibres in superficial peripheral nerve trunk regions which is capable of activating fibres in physiological order. The multielectrode system consists of 45 platinum electrodes embedded within a self-curling spiral silicone sheet organized in fifteen longitudinal groups consisting of three electrodes spaced equidistally around the circumference of the cuff. Electrodes in the centre band acted as stimulating cathodes while the two electrodes of the same group in the two outer bands were connected together and corresponded to the position of a particular cathode, serving as anodes to block the nascent action potentials by membrane hyperpolarization. The interpolar distance was 6 mm on both sides, resulting in a total cuff length of about 20 mm. The cuff was constructed with a diameter to fit the size of the dog sciatic nerve. Preliminary animal testing of the nerve cuff was performed on the sciatic nerve of a Bigley female dog. In the 45-electrode stimulation system, biphasic cathodic first pulses with quasitrapezoidal-shaped cathodic and square anodic parts were delivered through the particular group of tripolar electrodes to effect both selective stimulation of motor axons within the gastrocnemius muscle fascicle, and differential block by membrane hyperpolarization. The test was repeated using rectangular cathodic first biphasic current pulses delivered monopolarly on the central electrode of the same group while connected anodes were replaced by a common anode situated elsewhere in the surrounding tissue. In both experiments an isometric torque in the ankle joint elicited by the gastrocnemius muscle was measured and compared. It was shown that tripolar activation with quasitrapezoidal stimulation pulses elicited an isometric torque with a peak value of 0.83 Nm in 65 ms after onset of delivering stimulating pulses in comparison to the monopolar activation with rectangular biphasic pulses where the peak of the same value was observed in 45 ms after onset. Thus, the multipolar cuff stimulating monopolarly provided an effective means of activating motor axons selectively within the gastrocnemius muscle fascicle, while more physiological recruitment of the muscle fibres was evident when stimulating tripolarly.     

Importance of satellite cells in the strength recovery after eccentric contraction-induced muscle injury

The purpose of this study was to determine if the elimination of satellite cell proliferation using gamma-irradiation would inhibit normal force recovery after eccentric contraction-induced muscle injury. Adult female ICR mice were implanted with a stimulating nerve cuff on the common peroneal nerve and assigned to one of four groups: 1) irradiation- and eccentric contraction-induced injury, 2) eccentric contraction-induced injury only, 3) irradiation only, and 4) no intervention. Anterior crural muscles were irradiated with a dose of 2,500 rad and injured with 150 in vivo maximal eccentric contractions. Maximal isometric torque was determined weekly through 35 days postinjury. Immediately after injury, maximal isometric torque was reduced by approximately 50% and had returned to normal by 28 days postinjury in the nonirradiated injured mice. However, torque production of irradiated injured animals did not recover fully and was 25% less than that of injured nonirradiated mice 35 days postinjury. These data suggest that satellite cell proliferation is required for approximately half of the force recovery after eccentric contraction-induced injury.     

Neuromuscular organization of feline anterior sartorius: II. Intramuscular length changes and complex length-tension relationships during stimulation of individual nerve branches.

The feline anterior sartorius is a long strap-like muscle composed of short muscle fibers. Nerve branches that enter this muscle contain the axons of motor units whose constituent muscle fibers are distributed asymmetrically within the muscle. In the present study, twitch and tetanic isometric contractions were evoked by stimulating individual nerve branches while muscle force was recorded and intramuscular length changes were monitored optically by the movement of reflective markers on the muscle. Contractions elicited by stimulating the parent nerve produced little change in the positions of the surface markers. Contractions elicited by stimulating the proximally or distally directed nerve branches caused the muscle to shorten at the end closest to the nerve branch and lengthen at the opposite end. Some muscles were supplied by a centrally directed nerve branch whose stimulation produced variable effects: in some cases a portion of the muscle shortened whereas the rest lengthened, but in other cases, the positions of the surface markers showed little change. The intramuscular length changes produced by stimulating single nerve branches were greater during isometric contractions at short whole-muscle lengths than at long whole-muscle lengths. The twitch and tetanic length-tension relationships obtained by stimulating the individual nerve branches were not congruent with the length-tension relationship produced when the parent nerve was stimulated. At short whole-muscle lengths, stimulation of a single nerve branch generated only a small fraction of the force that could be generated by the muscle when the parent nerve was stimulated. As whole-muscle length increased, an increased fraction of total muscle force could be generated by stimulating a single nerve branch. The results suggest that a complex relationship between passive and active elements contributes to the total muscle force and depends on the distribution of active and passive muscle units throughout the muscle.     

Wide-pulse-width, high-frequency neuromuscular stimulation: implications for functional electrical stimulation.

Electrical stimulation (1-ms pulses, 100 Hz) produces more torque than expected from motor axon activation (extra contractions). This experiment investigates the most effective method of delivering this stimulation for neuromuscular electrical stimulation. Surface stimulation (1-ms pulses; 20 Hz for 2 s, 100 Hz for 2 s, 20 Hz for 3 s) was delivered to triceps surae and wrist flexors (muscle stimulation) and to median and tibial nerves (nerve stimulation) at two intensities. Contractions were evaluated for amplitude, consistency, and stability. Surface electromyograph was collected to assess how H-reflexes and M-waves contribute. In the triceps surae, muscle stimulation produced the largest absolute contractions (23% maximal voluntary contraction), evoked the largest extra contractions as torque increased by 412% after the 100-Hz stimulation, and was more consistent and stable compared with tibial nerve stimulation. Absolute and extra contraction amplitude, consistency, and stability of evoked wrist flexor torques were similar between stimulation types: torques reached 11% maximal voluntary contraction, and extra contractions increased torque by 161%. Extra contractions were 10 times larger in plantar flexors compared with wrist flexors with muscle stimulation but were similar with nerve stimulation. For triceps surae, H reflexes were 3.4 times larger than M waves during nerve stimulation, yet M waves were 15 times larger than H reflexes during muscle stimulation. M waves in the wrist flexors were larger than H reflexes during nerve (8.5 times) and muscle (18.5 times) stimulation. This is an initial step toward utilizing extra contractions for neuromuscular electrical stimulation and the first to demonstrate their presence in the wrist flexors.     

Effects of eccentric exercise training on cortical bone and muscle strength in the estrogen-deficient mouse.

The purpose of this study was to determine whether eccentrically biased exercise training could attenuate changes in muscle and bone function associated with estrogen deficiency in the mouse model. Four groups of ICR mice were used: control (Con), sham ovariectomized (Sham), ovariectomized (OVX), and ovariectomized + high-force resistance training (OVX+Train). All groups except Con were implanted with a nerve cuff surrounding the peroneal nerve to stimulate the left ankle dorsiflexors. Training consisted of 30 stimulated eccentric contractions of the left ankle dorsiflexors at approximately 150% of peak isometric torque every third day for 8 wk. After the training period, groups were not significantly different with regard to peak torque or muscle size. However, the tibial midshaft of the trained leg in the OVX+Train mice exhibited greater stiffness (+15%) than that in the untrained OVX mice, which could not be explained by changes in cross-sectional geometry of the tibia. Scaling of bone mechanical properties to muscle strength were not altered by ovariectomy or training. These data indicate that eccentric exercise training in adult mice can significantly increase bone stiffness, despite the absence of ovarian hormones.     

The mechanics of agonistic muscles

IntroductionIn this study, we tested two assumptions that have been made in experimental studies on muscle mechanics: (i) that the torque-angle properties are similar among agonistic muscles crossing a joint, and (ii) that the sum of the torque capacity of individual muscles adds up to the torque capacity of the agonist group.MethodsMaximum isometric torque measurements were made using a specifically designed animal knee extension dynamometer for the intact rabbit quadriceps muscles (n = 10) for knee angles between 60 and 120°. The nerve branches of the vastus lateralis (VL), vastus medialis (VM) and rectus femoris (RF) muscles were carefully dissected, and a custom made nerve cuff electrode was implanted on each branch. Knee extensor torques were measured for four maximal activation conditions at each knee angle: VL activation, VM activation, RF activation, and activation of all three muscles together.ResultsWith the exception of VL, the torque-angle relationships of the individual muscles did not have the shape of the torque-angle relationship obtained when all muscles were activated simultaneously. Furthermore, the maximum torque capacity obtained by adding the individual torque capacities of VL, VM and RF was approximately 20% higher than the torques produced when the three muscles were activated simultaneously.DiscussionThese results bring into question our understanding of in-vivo muscle contraction and challenge assumptions that are sometimes made in human and animal muscle force analyses.     

Functional anatomy of the vagal innervation of the cervical trachea of the dog.

The canine cervical trachea has been used for numerous studies regarding the neural control of tracheal smooth muscle. The purpose of the present study was to determine whether there is lateral dominance by either the left or right vagal innervation of the canine cervical trachea. In anesthetized dogs, pressure in the cuff of the endotracheal tube was used as an index of smooth muscle tone in the trachea. After establishment of tracheal tone, as indicated by increased cuff pressure, either the right or left vagus nerve was sectioned followed by section of the contralateral vagus. Sectioning the right vagus first resulted in total loss of tone in the cervical trachea, whereas sectioning the left vagus first produced either a partial or no decrease in tracheal tone. After bilateral section of the vagi, cuff pressure was recorded during electrical stimulation of the rostral end of the right or left vagus. At the maximum current strength used, stimulation of the left vagus produced tracheal constriction that averaged 28.5% of the response to stimulation of the right vagus (9.0 +/- 1.8 and 31.6 +/- 2.5 mmHg, respectively). In conclusion, the musculature of cervical trachea in the dog appears to be predominantly controlled by vagal efferents in the right vagus nerve.     

Turning on the central contribution to contractions evoked by neuromuscular electrical stimulation.

Neuromuscular electrical stimulation can generate contractions through peripheral and central mechanisms. Direct activation of motor axons (peripheral mechanism) recruits motor units in an unnatural order, with fatigable muscle fibers often activated early in contractions. The activation of sensory axons can produce contractions through a central mechanism, providing excitatory synaptic input to spinal neurons that recruit motor units in the natural order. Presently, we quantified the effect of stimulation frequency (10-100 Hz), duration (0.25-2 s of high-frequency bursts, or 20 s of constant-frequency stimulation), and intensity [1-5% maximal voluntary contraction (MVC) torque generated by a brief 100-Hz train] on the torque generated centrally. Electrical stimulation (1-ms pulses) was delivered over the triceps surae in eight subjects, and plantar flexion torque was recorded. Stimulation frequency, duration, and intensity all influenced the magnitude of the central contribution to torque. Central torque did not develop at frequencies < or = 20 Hz, and it was maximal at frequencies > or = 80 Hz. Increasing the duration of high-frequency stimulation increased the central contribution to torque, as central torque developed over 11 s. Central torque was greatest at a relatively low contraction intensity. The largest amount of central torque was produced by a 20-s, 100-Hz train (10.7 +/- 5.5 %MVC) and by repeated 2-s bursts of 80- or 100-Hz stimulation (9.2 +/- 4.8 and 10.2 +/- 8.1% MVC, respectively). Therefore, central torque was maximized by applying high-frequency, long-duration stimulation while avoiding antidromic block by stimulating at a relatively low intensity. If, as hypothesized, the central mechanism primarily activates fatigue-resistant muscle fibers, generating muscle contractions through this pathway may improve rehabilitation applications.     

A nerve clamp electrode design for indirect stimulation of skeletal muscle

A nerve clamp electrode was developed to indirectly stimulate skeletal muscle innervated by α motor neurons as an alternative to conventional electrodes. The stimulating electrode device consists of a spring coil-activated nerve clamp mounted inside a 1-mL syringe barrel. Supramaximal pulses were generated by a Grass stimulator and delivered to the nerve segment via the nerve clamp electrode. The salient feature of the electrode is its ability to produce muscle contractions indirectly through stimulation of the attached nerve. Indirect muscle stimulation is critical for studying the paralytic actions of presynaptic-acting toxins such as botulinum neurotoxins (BoNT), a potent inhibitor of acetylcholine (ACh) release from α motor neurons. This device enables stimulation of muscle contraction indirectly as opposed to contraction from direct muscle stimulation. The electrode is able to stimulate indirect muscle contraction when tested on ex vivo preparations from rodent phrenic nerve-hemidiaphragm muscle in similar fashion to conventional electrodes. In addition, the electrode stimulated external intercostal nerve-muscle preparations. This was confirmed after applying BoNT serotype A, a potent inhibitor of ACh release, to induce muscle paralysis. Alternative methods, including suction and bipolar loop electrodes, were unsuccessful in stimulating indirect muscle contraction. Therefore, this novel electrode is useful for physiological assessment of nerve agents and presynaptic actions of toxins that cause muscle paralysis. This electrode is useful for stimulating nerve-muscle preparations for which the length of nerve is a concern.     

Activation and inhibition of the micturition reflex by penile afferents in the cat

Coordination of the urinary bladder and the external urethral sphincter is controlled by descending projections from the pons and is also subject to modulation by segmental afferents. We quantified the effects on the micturition reflex of sensory inputs from genital afferents traveling in the penile component of the somatic pudendal nerve by electrical stimulation of the dorsal nerve of the penis (DNP) in alpha-chloralose anesthetized male cats. Depending on the frequency of stimulation (range, 1-40 Hz), activation of penile afferents either inhibited contractions of the bladder and promoted urine storage or activated the bladder and produced micturition. Stimulation of the DNP at 5-10 Hz inhibited distension-evoked contractions and increased the maximum bladder capacity before incontinence. Conversely, stimulation at 33 and 40 Hz augmented distension-evoked contractions. When the bladder was filled above a threshold volume (70% of the volume necessary for distension-evoked contractions), stimulation at 20-40 Hz activated de novo the micturition reflex and elicited detrusor contractions that increased voiding efficiency compared with distension-evoked voiding. Electrical stimulation of the DNP with a cuff electrode or percutaneous wire electrode produced similar results. The ability to evoke detrusor contractions by activation of the DNP was preserved following acute spinal cord transection. These results demonstrate a clear role of genital afferents in modulating the micturition reflex and suggest the DNP as a potential target for functional restoration of bladder control using electrical stimulation.     

Selective recording of electroneurograms from the left vagus nerve of a dog during stimulation of cardiovascular or respiratory systems

Selective electroneurograms (ENGs) from superficial regions of the left vagus nerve of a dog were recorded with a 33-electrode spiral cuff (cuff) implanted on the nerve at the neck in an adult Beagle dog. The electrodes in the cuff were arranged in thirteen groups of three electrodes (GTE 1-13). To identify the relative positions of the particular nerve regions that innervated the heart and lungs, stimulating pulses (2 mA, 200 micros, 20 Hz) were individually delivered to all thirteen GTEs. It was shown that by delivering stimulating pulses to GTEs 4 and 9, heart rate, blood pressure and respiratory rate were modulated. Precisely, only when the stimuli were delivered to GTE 9, the heart rate began to fall and only when the stimuli were delivered to GTE 4 the rate of breathing decreased. To test the selectivity of recording the above-defined groups GTEs 4 and 9 and randomly chosen GTEs 1 and 7 were simultaneously used as recording GTEs while cardio-vascular or respiratory systems were stimulated by carotid artery compression, epinephrine injection and non-invasive, positive end-pressure ventilation. Results showed that stimulations elicited site-specific changes in ENG power spectra recorded from the superficial regions of the vagus nerve. Power spectrum of the ENG recorded with GTE 9, contained frequencies belonging to the neural activity elicited by compression of the carotid artery and injection of epinephrine. The power spectrum of the ENG recorded with GTE 4, contained frequencies belonging to the neural activity elicited by non-invasive, positive end-expiratory pressure ventilation. We concluded that the multi-electrode nerve cuff enables selective stimulation and recording of nerve activity from internal organs.     

Procedures for rat in situ skeletal muscle contractile properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation. To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37°C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship. With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.     

Limb movements generated by stimulating muscle,nerve and spinal cord

We have compared the movements generated by stimulation of muscle, nerve, spinal roots and spinal cord in anesthetized, decerebrate and spinalized cats. Each method produced a full range of movements of the cat's hind limb in the sagittal plane against a spring load, except for stimulation of the roots. Stimulation of the dorsal roots produced movements that were mainly up and forward, whereas stimulation of the ventral roots produced complementary movements (down and backward). Results from stimulation in the intermediate areas of the spinal cord were compared to predictions of the "movement primitives" hypothesis. We could not confirm that the directions were independent of stimulus amplitude or the state of descending inputs. Pros and cons of stimulating at some sites were provisionally considered for the reliable control of limb movements with functional electrical stimulation (FES) in clinical conditions.     

Nerve stimulation with a multi-contact cuff electrode: validation of model predictions

The recruitment characteristics of muscle selective nerve stimulation by a multi-contact nerve cuff electrode, as predicted by computer modeling, have been investigated in acute experiments on rabbits. A nerve cuff containing five or six dot electrodes was placed around the sciatic nerve in five rabbits. M-waves were recorded with wire electrodes from the lateral gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles. The muscle recruitment performances of three contact configurations (monopole, transverse bipole, transverse tripole) were compared. The selectivity was quantified by the recruitment of two muscles (one extensor and one flexor) in response to a particular stimulus. The results showed that only in a few cases, transverse bi- and tripolar stimulation provided a better selectivity than monopolar stimulation. Neither of the two extensors, nor of the two flexors could be stimulated separately. In accordance with the results of the modeling studies, bi- and tripolar stimulation required higher stimulus currents than monopolar stimulation, whereas maximum recruitment and slopes of recruitment curves were lower. The rabbit sciatic nerve appears to be a less suitable preparation for reproducible selectivity experiments, due to the variability in the number and size of the fascicles and their position in this nerve.     

Functional evaluation of nerve-skeletal muscle constructs engineered in vitro

Summary Previously, we have engineered three-dimensional (3-D) skeletal muscle constructs that generate force and display a myosin heavy-chain (MHC) composition of fetal muscle. The purpose of this study was to evaluate the functional characteristics of 3-D skeletal muscle constructs cocultured with fetal nerve explants. We hypothesized that coculture of muscle constructs with neural cells would produce constructs with increased force and adult MHC isoforms. Following introduction of embryonic spinal cord explants to a layer of confluent muscle cells, the neural tissue integrated with the cultured muscle cells to form 3-D muscle constructs with extensions. Immunohistochemical labeling indicated that the extensions were neural tissue and that the junctions between the nerve extensions and the muscle constructs contained clusters of acetylcholine receptors. Compared to muscles cultured without nerve explants, constructs formed from nerve-muscle coculture showed spontaneous contractions with an increase in frequency and force. Upon field stimulation, both twitch (2-fold) and tetanus (1.7-fold) were greater in the nerve-muscle coculture system. Contractions could be elicited by electrically stimulating the neural extensions, although smaller forces are produced than with field stimulation. Severing the extension eliminated the response to electrical stimulation, excluding field stimulation, as a contributing factor. Nervemuscle constructs showed a tendency to have higher contents of adult and lower contents of fetal MHC isoforms, but the differences were not significant. In conclusion, we have successfully engineered a 3-D nerve-muscle construct that displays functional neuromuscular junctions and can be electrically stimulated to contract via the neural extensions projecting from the construct.     

Reinnervation of adult muscle in organ culture restores tetrodotoxin sensitivity in the absence of electrical activity.

Strips of denervated adult mouse diaphragm muscle maintained in organ culture were reinnervated by nerve processes growing out from explants of embryonic mouse spinal cord. In vivo, following denervation, the action potential loses its sensitivity to tetrodotoxin; this sensitivity is regained upon reinnervation. Similarly, action potentials in cultured muscle fibres were insensitive to tetrodotoxin, and sensitivity was restored in muscle fibres that became reinnervated in vitro. Tetrodotoxin sensitivity was also restored in cultured muscle fibres reinnervated in the continuous presence of d-tubocurarine, but it was not induced by 4 days of direct electrical stimulation of noninnervated muscles. We conclude that developing nerve terminals can exert a trophic action on adult muscle fibres that is independent of electrical activity in the muscle.     

Glenohumeral stability in simulated rotator cuff tears

Rotator cuff tears disrupt the force balance in the shoulder and the glenohumeral joint in particular, resulting in compromised arm elevation torques. The trade-off between glenohumeral torque and glenohumeral stability is not yet understood. We hypothesize that compensation of lost abduction torque will lead to a superior redirection of the reaction force vector onto the glenoid surface, which will require additional muscle forces to maintain glenohumeral stability. Muscle forces in a single arm position for five combinations of simulated cuff tears were estimated by inverse dynamic simulation (Delft Shoulder and Elbow Model) and compared with muscle forces in the non-injured condition. Each cuff tear condition was simulated both without and with an active modeling constraint for glenohumeral stability, which was defined as the condition in which the glenohumeral reaction force intersects the glenoid surface. For the simulated position an isolated tear of the supraspinatus only increased the effort of the other muscles with 8%, and did not introduce instability. For massive cuff tears beyond the supraspinatus, instability became a prominent factor: the deltoids were not able to fully compensate lost net abduction torque without introducing destabilizing forces; unfavorable abductor muscles (i.e. in the simulated position the subscapularis and the biceps longum) remain to compensate the necessary abduction torque; the teres minor appeared to be of vital importance to maintain glenohumeral stability. Adverse adductor muscle co-contraction is essential to preserve glenohumeral stability.     

The effect of subthreshold prepulses on the recruitment order in a nerve trunk analyzed in a simple and a realistic volume conductor model

 The influence of subthreshold depolarizing prepulses on the threshold current-to-distance and the threshold current-to-diameter relationship of myelinated nerve fibers has been investigated. A nerve fiber model was used in combination with both a simple, homogeneous volume conductor model with a point source and a realistic, inhomogeneous volume conductor model of a monofascicular nerve trunk surrounded by a cuff electrode. The models predict that a subthreshold depolarizing prepulse will desensitize Ranvier nodes of fibers in the vicinity of the cathode and thus cause an increase in the threshold current of a subsequent pulse to activate these fibers. If the increase in threshold current of the excited node is large enough, the excitation will be accompanied by a strong hyperpolarization of adjacent nodes, preventing the propagation of action potentials in these fibers. As fibers close to the electrode are more desensitized by prepulses than more distant ones, it is possible to stimulate distant fibers without stimulating such fibers close to the electrode. Moreover, as larger fibers are more desensitized than smaller ones, smaller fibers have lower threshold currents than larger fibers up to a certain distance from the electrode. The realistic model has provided an additional condition for the application of this method to invert nerve fiber recruitment, i.e., real or virtual anodes should be close to the cathode. When using a cuff electrode for this purpose, in the case of monopolar stimulation the cuff length (determining the position of the virtual anodes) should not exceed twice the internodal length of the fibers to be blocked. Similarly, the distance between cathode and anodes should not exceed the internodal length of these fibers when stimulation is to be applied tripolarly. Received: 15 May 2000 / Accepted in revised form: 9 February 2001     

Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle

Skeletal muscle contraction depends on the release of Ca(2+) from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca(2+) concentration ([Ca(2+)](SR)), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca(2+)](SR) and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca(2+)](SR) decreases by approximately 50 microM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon beta-adrenergic stimulation, leading to an increased efficacy of the Ca(2+) release/reuptake cycle during motor nerve stimulation.     

Motor unit recruitment when neuromuscular electrical stimulation is applied over a nerve trunk compared with a muscle belly: triceps surae

Neuromuscular electrical stimulation (NMES) can be delivered over a nerve trunk or muscle belly and can generate contractions by activating motor (peripheral pathway) and sensory (central pathway) axons. In the present experiments, we compared the peripheral and central contributions to plantar flexion contractions evoked by stimulation over the tibial nerve vs. the triceps surae muscles. Generating contractions through central pathways follows Henneman's size principle, whereby low-threshold motor units are activated first, and this may have advantages for rehabilitation. Statistical analyses were performed on data from trials in which NMES was delivered to evoke 10-30% maximum voluntary torque 2-3 s into the stimulation (Time(1)). Two patterns of stimulation were delivered: 1) 20 Hz for 8 s; and 2) 20-100-20 Hz for 3-2-3 s. Torque and soleus electromyography were quantified at the beginning (Time(1)) and end (Time(2); 6-7 s into the stimulation) of each stimulation train. H reflexes (central pathway) and M waves (peripheral pathway) were quantified. Motor unit activity that was not time-locked to each stimulation pulse as an M wave or H reflex ("asynchronous" activity) was also quantified as a second measure of central recruitment. Torque was not different for stimulation over the nerve or the muscle. In contrast, M waves were approximately five to six times smaller, and H reflexes were approximately two to three times larger during NMES over the nerve vs. the muscle. Asynchronous activity increased by 50% over time, regardless of the stimulation location or pattern, and was largest during NMES over the muscle belly. Compared with NMES over the triceps surae muscles, NMES over the tibial nerve produced contractions with a relatively greater central contribution, and this may help reduce muscle atrophy and fatigue when NMES is used for rehabilitation.