The relationship between the soleus H-reflex amplitude and vibratory inhibition in controls and spastic subjects. II. Computer model

A computer model is presented that describes soleus H-reflex recruitment as a function of electric stimulus intensity. The model consists of two coupled non-linear transfer functions. The first transfer function describes the activation of muscle spindle (Ia) afferent terminals as a function of the electric stimulus intensity; whereas the second describes the activation of a number of motoneurons as a function of the number of active Ia afferent terminals. The effect of change in these transfer functions on the H-reflex recruitment curve is simulated. In spastic patients, a higher average maximal H-response amplitude is observed in combination with a decreased H-reflex threshold. Vibration of the Achilles tendon reduces the H-reflex amplitude, presumably by reducing the excitatory afferent input. Vibratory inhibition is diminished in spasticity. In the model, the afferent-motoneuron transfer function was modified to represent the possible alterations occurring in spasticity. The simulations show that vibratory suppression of the H-reflex is determined only in part by the inhibition level of the afferent input. With a constant level of presynaptic inhibition, the suppression of reflexes of different sizes may vary. A lowering of the motoneuron activation thresholds in spastic patients will directly contribute to a decrease of vibratory inhibition in spasticity.     

Simulation system of spinal cord motor nuclei and associated nerves and muscles,in a Web-based architecture

A Web-based simulation system of the spinal cord circuitry responsible for muscle control is described. The simulator employs two-compartment motoneuron models for S, FR and FF types, with synaptic inputs acting through conductance variations. Four motoneuron pools with their associated interneurons are represented in the simulator, with the possibility of inclusion of more than 2,000 neurons and 2,000,000 synapses. Each motoneuron action potential is followed, after a conduction delay, by a motor unit potential and a motor unit twitch. The sums of all motor unit potentials and twitches result in the electromyogram (EMG), and the muscle force, respectively. Inputs to the motoneuron pool come from populations of interneurons (Ia reciprocal inhibitory interneurons, Ib interneurons, and Renshaw cells) and from stochastic point processes associated with descending tracts. To simulate human electrophysiological experiments, the simulator incorporates external nerve stimulation with orthodromic and antidromic propagation. This provides the mechanisms for reflex generation and activation of spinal neuronal circuits that modulate the activity of another motoneuron pool (e.g., by reciprocal inhibition). The generation of the H-reflex by the Ia-motoneuron pool system and its modulation by spinal cord interneurons is included in the simulation system. Studies with the simulator may include the statistics of individual motoneuron or interneuron spike trains or the collective effect of a motor nucleus on the dynamics of muscle force control. Properties associated with motor-unit recruitment, motor-unit synchronization, recurrent inhibition and reciprocal inhibition may be investigated.     

Modeling of the H-reflex facilitation during ramp and hold contractions

Healthy subjects were asked to make a voluntary ramp and hold contraction. The duration of the ramp stage was 500 ms, and the torque increment in this period was set to 15 Nm. The contraction was made from a relaxed and from a 5 Nm background torque situation. Hoffmann (H-) reflexes were elicited during the voluntary contraction, mostly with 100 ms intervals. These experiments showed an increase (facilitation) in the H-reflex before the torque or the EMG started to increase. This facilitation of the H-reflex remained during all the stages of the voluntary movement and declined to normal levels again only at the very end of the hold phase, which lasted for one second. This specific pattern of facilitation during a voluntary contraction was modeled using a modeling language, that is specifically designed to calculate neuronal systems with a high degree of reality (Ekeberg et al., 1991). Our model consisted of a motoneuron pool with 200 neurons connected to an EMG-model of the human soleus muscle and an extra group of higher-level neurons for controlling the amount of decrease of presynaptic inhibition. The model was used to simulate the observed modulation of the H-reflex with both a presynaptic and a postsynaptic mechanism. Simulations showed that a continuous change in the descending control signals is needed to make the model based on postsynaptic mechanism fit with the experimental data, whereas no extra control from the CNS over the excitatory drive to the motoneuron pool is needed when the decrease of presynaptic inhibition mechanism is applied.     

Changes in H reflex and V wave following short-term endurance and strength training

This study examined the effects of 3 wk of either endurance or strength training on plasticity of the neural mechanisms involved in the soleus H reflex and V wave. Twenty-five sedentary healthy subjects were randomized into an endurance group (n = 13) or strength group (n = 12). Evoked V-wave, H-reflex, and M-wave recruitment curves, maximal voluntary contraction (MVC), and time-to-task-failure (isometric contraction at 40% MVC) of the plantar flexors were recorded before and after training. Following strength training, MVC of the plantar flexors increased by 14.4 ± 5.2% in the strength group (P < 0.001), whereas time-to-task-failure was prolonged in the endurance group (22.7 ± 17.1%; P < 0.05). The V wave-to-maximal M wave (V/M(max)) ratio increased significantly (55.1 ± 28.3%; P < 0.001) following strength training, but the maximal H wave-to-maximal M wave (H(max)/M(max)) ratio remained unchanged. Conversely, in the endurance group the V/M(max) ratio was not altered, whereas the H(max)/M(max) ratio increased by 30.8 ± 21.7% (P < 0.05). The endurance training group also displayed a reduction in the H-reflex excitability threshold while the H-reflex amplitude on the ascending limb of the recruitment curve increased. Strength training only elicited a significant decrease in H-reflex excitability threshold, while H-reflex amplitudes over the ascending limb remained unchanged. These observations indicate that the H-reflex pathway is strongly involved in the enhanced endurance resistance that occurs following endurance training. On the contrary, the improvements in MVC following strength training are likely attributed to increased descending drive and/or modulation in afferents other than Ia afferents.     

Modulation of spinal inhibitory reflexes depends on the frequency of transcutaneous electrical nerve stimulation in spastic stroke survivors

Neurophysiological studies in healthy subjects suggest that increased spinal inhibitory reflexes from the tibialis anterior (TA) muscle to the soleus (SOL) muscle might contribute to decreased spasticity. While 50?Hz is an effective frequency for transcutaneous electrical nerve stimulation (TENS) in healthy subjects, in stroke survivors, the effects of TENS on spinal reflex circuits and its appropriate frequency are not well known. We examined the effects of different frequencies of TENS on spinal inhibitory reflexes from the TA to SOL muscle in stroke survivors. Twenty chronic stroke survivors with ankle plantar flexor spasticity received 50-, 100-, or 200-Hz TENS over the deep peroneal nerve (DPN) of the affected lower limb for 30?min. Before and immediately after TENS, reciprocal Ia inhibition (RI) and presynaptic inhibition of the SOL alpha motor neuron (D1 inhibition) were assessed by adjusting the unconditioned H-reflex amplitude. Furthermore, during TENS, the time courses of spinal excitability and spinal inhibitory reflexes were assessed via the H-reflex, RI, and D1 inhibition. None of the TENS protocols affected mean RI, whereas D1 inhibition improved significantly following 200-Hz TENS. In a time-series comparison during TENS, repeated stimulation did not produce significant changes in the H-reflex, RI, or D1 inhibition regardless of frequency. These results suggest that the frequency-dependent effect of TENS on spinal reflexes only becomes apparent when RI and D1 inhibition are measured by adjusting the amplitude of the unconditioned H-reflex. However, 200-Hz TENS led to plasticity of synaptic transmission from the antagonist to spastic muscles in stroke survivors.     

Rhythmic arm swing enhances long latency facilitatory effect of transcranial magnetic stimulation on soleus motoneuron pool excitability

The purpose of this study was to investigate whether rhythmic arm swing modulates the long latency effect of transcranial magnetic stimulation (TMS) on soleus motoneuron pool excitability. Ten healthy humans rhythmically swung the left arm back and forth in a sitting position. The soleus H-reflex was evoked when the arm was in the backward swing phase. Conditioning TMS was delivered over the motor cortex 8?ms before the soleus H-reflex was evoked. The soleus H-reflex amplitude in both legs was depressed by the rhythmic arm swing. In contrast, rhythmic arm swing enhanced the facilitatory effect of conditioning TMS over the motor cortex contralateral to the arm swing side on the soleus H-reflex ipsilateral to the arm swing side. This finding indicates that rhythmic arm swing enhances some polysynaptic facilitatory pathways from the motor cortex contralateral to the arm swing side to the soleus motoneuron pool ipsilateral to the arm swing side.     

Rhythmic arm swing enhances long latency facilitatory effect of transcranial magnetic stimulation on soleus motoneuron pool excitability

The purpose of this study was to investigate whether rhythmic arm swing modulates the long latency effect of transcranial magnetic stimulation (TMS) on soleus motoneuron pool excitability. Ten healthy humans rhythmically swung the left arm back and forth in a sitting position. The soleus H-reflex was evoked when the arm was in the backward swing phase. Conditioning TMS was delivered over the motor cortex 8 ms before the soleus H-reflex was evoked. The soleus H-reflex amplitude in both legs was depressed by the rhythmic arm swing. In contrast, rhythmic arm swing enhanced the facilitatory effect of conditioning TMS over the motor cortex contralateral to the arm swing side on the soleus H-reflex ipsilateral to the arm swing side. This finding indicates that rhythmic arm swing enhances some polysynaptic facilitatory pathways from the motor cortex contralateral to the arm swing side to the soleus motoneuron pool ipsilateral to the arm swing side.     

Somatosensory graviception inhibits the soleus H-reflex in standing man - a parabolic flight experiment-

The purpose of this study was to investigate how gravity level affects the excitability of the soleus muscle (SOL) motoneuron pool to la afferent input while erect posture is maintained in humans. Three healthy male subjects participated in an experiment whereby three different gravity conditions (micro gravity (MG), normal gravity (NG), and hyper gravity) were imposed using a parabolic flight procedure. The SOL H-reflex was evoked every 2 seconds while the subjects kept an erect posture. The background electromyographic activity (BGA) of the SOL was almost absent during MG. The SOL H-reflex amplitude was significantly larger during MG than during NG. These results suggest that the somatosensory systems detecting a load at the lower limbs and/or vertebral column play a role in reducing the excitability of the SOL motoneuron pool to la afferent inputs by presynaptic inhibition.     

Inhibition of inspiratory upper airway motoneuron activity by phasic volume feedback

The effects of phasic volume feedback on efferent hypoglossal, recurrent laryngeal and phrenic nerve activity were studied in decerebrate, paralyzed intubated cats ventilated with a phrenic-driven servo-respirator. The gain of the respirator was altered for single inspirations, and the resulting changes in neural activities were quantified by comparison with respective neural activities without phasic volume feedback. The volume thresholds for suppression of hypoglossal and recurrent laryngeal activities were time independent. Above these two thresholds and extending over a substantial range, volume feedback caused graded inhibition of upper airway motoneuron outputs. At any particular time during inspiration the relationships between hypoglossal or recurrent laryngeal inhibition and volume were concave to the volume axis. Rate of airflow appeared to exert an effect on upper airway motoneuron activity independent of volume. These results indicate that for hypoglossal and recurrent laryngeal efferent activity 1) volume feedback can cause a sustained graded inhibition throughout inspiration; 2) the volume thresholds are time independent; and 3) partial inhibition decreases susceptibility to additional inhibition. These actions of volume feedback on upper airway motoneuron output differ from those on phrenic efferent discharge and show that phasic vagal volume feedback has a marked and differential effect on upper airway motoneuron activity. The vagus, in this preparation, appears to play a critical role in the regulation of upper airway motoneuron activity and therefore maintenance of upper airway patency.     

Neural adaptation to resistance training: changes in evoked V-wave and H-reflex responses.

Combined V-wave and Hoffmann (H) reflex measurements were performed during maximal muscle contraction to examine the neural adaptation mechanisms induced by resistance training. The H-reflex can be used to assess the excitability of spinal alpha-motoneurons, while also reflecting transmission efficiency (i.e., presynaptic inhibition) in Ia afferent synapses. Furthermore, the V-wave reflects the overall magnitude of efferent motor output from the alpha-motoneuron pool because of activation from descending central pathways. Fourteen male subjects participated in 14 wk of resistance training that involved heavy weight-lifting exercises for the muscles of the leg. Evoked V-wave, H-reflex, and maximal M-wave (M(max)) responses were recorded before and after training in the soleus muscle during maximal isometric ramp contractions. Maximal isometric, concentric, and eccentric muscle strength was measured by use of isokinetic dynamometry. V-wave amplitude increased approximately 50% with training (P < 0.01) from 3.19 +/- 0.43 to 4.86 +/- 0.43 mV, or from 0.308 +/- 0.048 to 0.478 +/- 0.034 when expressed relative to M(max) (+/- SE). H-reflex amplitude increased approximately 20% (P < 0.05) from 5.37 +/- 0.41 to 6.24 +/- 0.49 mV, or from 0.514 +/- 0.032 to 0.609 +/- 0.025 when normalized to M(max). In contrast, resting H-reflex amplitude remained unchanged with training (0.503 +/- 0.059 vs. 0.499 +/- 0.063). Likewise, no change occurred in M(max) (10.78 +/- 0.86 vs. 10.21 +/- 0.66 mV). Maximal muscle strength increased 23-30% (P < 0.05). In conclusion, increases in evoked V-wave and H-reflex responses were observed during maximal muscle contraction after resistance training. Collectively, the present data suggest that the increase in motoneuronal output induced by resistance training may comprise both supraspinal and spinal adaptation mechanisms (i.e., increased central motor drive, elevated motoneuron excitability, reduced presynaptic inhibition).     

Time-dependent effects of neuromuscular electrical stimulation on changes in spinal excitability are dependent on stimulation frequency: A preliminary study in healthy adults

Neuromuscular electrical stimulation (NMES) can be used as treatment for spasticity. The present study examined differences in time-dependent effects of NMES depending on stimulation frequency. Forty healthy subjects were separated into four groups (no-stim, NMES of 50, 100, and 200?Hz). The un-conditioned H-reflex amplitude and the H-reflex conditioning-test paradigm were used to measure the effectiveness on monosynaptic Ia excitation of motoneurons in the soleus (SOL) muscle, disynaptic reciprocal Ia inhibition from tibialis anterior (TA) to SOL, and presynaptic inhibition of SOL Ia afferents. Each trial consisted of a 30-min period of NMES applied to the deep peroneal nerve followed by a 30-min period with no stimulation to measure prolonged effects. Measurements were performed periodically. Stimulation applied at all frequencies produced a significant reduction in monosynaptic Ia excitation of motoneurons in the SOL muscle, however, only stimulation with 50?Hz showed prolonged reduction after NMES. NMES frequency did not affect the amount of disynaptic reciprocal Ia inhibition and presynaptic inhibition of Ia afferents. The results show a frequency-dependent effect of NMES on the monosynaptic Ia excitation of motoneurons. This result has implications for selecting the optimal NMES frequency for treatment in patients with spasticity.     

Influences of neck vibration on lower limb extensor muscles in man

The effects of neck vibration were studied in ten healthy subjects standing in unrestrained or restrained conditions. An involuntary slow and gradual displacement of the body axis associated with a subjective experience of forward tilt occurred during vibration of the dorsal surface of the neck. This effect was associated with a decreased tonic contration of the antigravitary lower limb musculature. The amplitude of the H-reflex from the soleus muscle, recorded at progressively increasing time interval after the onset of the vibratory stimulus, showed complex interactions: in particular, in unrestrained conditions an early inhibitory phase occurred at about 100 msec, followed by a short-lasting facilitatory phase between 150-300 msec and by a late long-lasting excitatory component which started 500-600 msec after the onset of vibratory stimulus. In restrained subjects, the late excitatory phase disappeared and was substituted by a delayed depression. In this instance, the short-lasting facilitatory phase appeared to be superimposed on a background of inhibition. The effects produced by the neck input on the H-reflex were attenuated during vibration of the dorsal muscolature at L4-L5. The mechanisms involved in the cervical control of posture and reflex movements following neck vibration are discussed.     

Post-tetanic potentiation of reciprocal Ia inhibition in human lower limb.

The purpose of this study was to investigate how reciprocal Ia inhibition is changed during muscle fatigue of lower limb muscle, induced with a voluntary contraction or height frequency electrical stimulation. Reciprocal Ia inhibition from ankle flexors to extensors has been investigated in 12 healthy subjects. Hoffmann reflex (H-reflex) in the soleus muscle was used to monitor changes in the amount of reciprocal Ia inhibition from common peroneal nerve as demonstrated during voluntary dorsi or planterflexion and 50 Hz electrical stimulation induced dorsi or planterflexion. The test soleus H-reflex was kept at 20-25% of maximum directly evoked motor response (M response) and the strength of the conditioning common peroneal nerve stimulation was kept at 1.0 x motor threshold. At rest, weak la inhibition was demonstrated in 12 subjects, maximal inhibition from the common peroneal nerve was 28.8%. During voluntary dorsiflexion and 50 Hz electrical stimulation induced dorsiflexion, there absolute amounts of inhibition increased as compared to at rest, and decreased or disappeared during voluntary planterflexion and 50 Hz electrical stimulation induced planterflexion as compared to at rest. During voluntary or electrical stimulation induced agonist muscle fatigue, the inhibition of the soleus H-reflex from the common peroneal nerve was greater during voluntary dorsiflexion (maximal, 11.1%) and 50 Hz (maximal, 6.7%) electrical stimulation induced dorsiflexion than at rest. The inhibition was decreased or disappeared during voluntary planterflexion 50 Hz electrical stimulation induced planterflexion. It was concluded that the results were considered to support the hypothesis that alpha-motoneurones and la inhibitory intemeurones link to antagonist motoneurones in reciprocal inhibition. The diminished reciprocal Ia inhibition of voluntary contraction during muscle fatigue as compared to electrical stimulation, is discussed in relation to its possible contribution to ankle stability.     

Presynaptic inhibition in humans: a comparison between normal and spastic patients.

During the last 40 years, several studies in man have been devoted to the pathophysiological mechanisms underlying spasticity. Spasticity is characterised by a velocity dependent increase in muscle tone. Many spinal pathways control stretch reflex excitability and a malfunction in any one of them could theoretically produce the exaggeration of the stretch reflex. Delwaide showed that the vibration-induced inhibition of Ia fibres is reduced in spastic patients. However, the relation between a decrease in presynaptic Ia inhibition and the pathophysiology of spasticity has been recently questioned since it was argued that homosynaptic depression (or post-activation depression) also contributes to the vibratory-induced depression of monosynaptic reflexes. This paper is thus devoted to a review of the methods recently developed to study selectively presynaptic Ia inhibition in man and to a reevaluation of the relations between modifications in presynaptic Ia inhibition and spasticity in hemiplegic and spinal spastic patients.     

Nonlinear systems analysis of computer models of repetitive firing

The inhibitory influences of recurrent inhibition and afterhyperpolarization are studied theoretically insofar as they affect the density of the interspike interval and the frequency transfer characteristic. The methods employed involve exact results for excitation with decay and constant threshold, computer simulations for decaying thresholds representing afterhyperpolarization, and the diffusion approximation for excitation with inhibition and a constant threshold. Afterhyperpolarization tends to preserve the linearity of the frequency transfer characteristic and the lognormality of the interspike time. Recurrent inhibition which grows linearly with frequency of excitation can lead to frequency limiting. Some forms of nonlinear recurrent inhibition may lead to a filter type effect whereby the neuron responds significantly only over certain ranges of input intensity. A simple network model is analysed which exhibits recurrent inhibitory frequency growing linearly with frequency of excitation. An estimate of 10 to 50 is made for the number of Renshaw cells which connect with a spinal motoneuron. The frequency limiting of motoneurons is discussed and the stabilizing influence attributed to Renshaw cells is questioned. It is postulated that Renshaw recurrent inhibition is of functional significance at low levels of excitatory drive to motoneurons and that its effect is diminished by reciprocal inhibition at high excitatory input frequencies.     

Effects of whole body vibration on motor unit recruitment and threshold

Whole body vibration (WBV) has been suggested to elicit reflex muscle contractions but this has never been verified. We recorded from 32 single motor units (MU) in the vastus lateralis of 7 healthy subjects (34 ± 15.4 yr) during five 1-min bouts of WBV (30 Hz, 3 mm peak to peak), and the vibration waveform was also recorded. Recruitment thresholds were recorded from 38 MUs before and after WBV. The phase angle distribution of all MUs during WBV was nonuniform (P < 0.001) and displayed a prominent peak phase angle of firing. There was a strong linear relationship (r = -0.68, P < 0.001) between the change in recruitment threshold after WBV and average recruitment threshold; the lowest threshold MUs increased recruitment threshold (P = 0.008) while reductions were observed in the higher threshold units (P = 0.031). We investigated one possible cause of changed thresholds. Presynaptic inhibition in the soleus was measured in 8 healthy subjects (29 ± 4.6 yr). A total of 30 H-reflexes (stimulation intensity 30% Mmax) were recorded before and after WBV: 15 conditioned by prior stimulation (60 ms) of the antagonist and 15 unconditioned. There were no significant changes in the relationship between the conditioned and unconditioned responses. The consistent phase angle at which each MU fired during WBV indicates the presence of reflex muscle activity similar to the tonic vibration reflex. The varying response in high- and low-threshold MUs may be due to the different contributions of the mono- and polysynaptic pathways but not presynaptic inhibition.     

Characteristics of the tonic stretch reflex in spastic spinal cord and head-injured patients

Spasticity is a known sequelae of spinal cord injury and head injury. We sought to examine whether there were any significant differences in the characteristics or underlying mechanisms of spasticity in these two groups in the chronic period which may be related to the level of injury of the neuraxis. The response to vibration applied to the muscle, or the tonic vibratory reflex, has been shown to be related to the degree of spasticity, and was therefore studied along with phasic reflexes and passive movements. These studies were carried out on cooperative, stabilized patients who were otherwise healthy, 5 with head injuries, and 5 with spinal cord injuries. The patients were examined in a supine position while surface EMG recordings were made of quadriceps and triceps surae muscles bilaterally. Tendon jerk responses, passive and volitional movements, and responses to a powerful vibratory stimulator were measured. In both head injury and spinal cord injury patient groups, a large EMG response was elicited by passive maneuvers, and tendon jerks were exaggerated. The tonic vibratory response, previously shown to be dependent upon brain influence, was present in both groups. These observations suggest that similar suprasegmental mechanisms may be responsible for hypertonia in both head-injured and spinal cord-injured patients.     

Neuroprotective effect of testosterone treatment on motoneuron recruitment following the death of nearby motoneurons

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have previously shown that motoneuron death induces marked dendritic atrophy in surviving nearby motoneurons. Additionally, in quadriceps motoneurons, this atrophy is accompanied by decreases in motor nerve activity. However, treatment with testosterone partially attenuates changes in both the morphology and activation of quadriceps motoneurons. Testosterone has an even larger neuroprotective effect on the morphology of motoneurons of the spinal nucleus of the bulbocavernosus (SNB), in which testosterone treatment can completely prevent dendritic atrophy. The present experiment was performed to determine whether the greater neuroprotective effect of testosterone on SNB motoneuron morphology was accompanied by a greater neuroprotective effect on motor activation. Right side SNB motoneurons were killed by intramuscular injection of cholera toxin‐conjugated saporin in adult male Sprague‐Dawley rats. Animals were either given Silastic testosterone implants or left untreated. Four weeks later, left side SNB motor activation was assessed with peripheral nerve recording. The death of right side SNB motoneurons resulted in several changes in the electrophysiological response properties of surviving left side SNB motoneurons, including decreased background activity, increased response latency, increased activity duration, and decreased motoneuron recruitment. Treatment with exogenous testosterone attenuated the increase in activity duration and completely prevented the decrease in motoneuron recruitment. These data provide a functional correlate to the known protective effects of testosterone treatment on the morphology of these motoneurons, and further support a role for testosterone as a therapeutic agent in the injured nervous system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Internal citrate ions reduce the membrane potential for contraction threshold in mammalian skeletal muscle fibers.

An effect of internal citrate ions on excitation-contraction coupling in skeletal muscle is described. The threshold for contraction was measured in rat extensor digitorum longus, (EDL), and soleus muscle fibers using a two microelectrode voltage clamp technique with either KCl-filled or K3 citrate-filled current electrodes. Contraction thresholds were stable for many minutes with KCl current electrodes. In contrast, thresholds fell progressively towards the resting membrane potential, by as much as -15 mV over a period of 10 to 20 min of voltage-clamp with citrate current electrodes. In addition, prepulse inhibition was suppressed, subthreshold activation enhanced and steady-state inactivation shifted to more negative potentials. Fibers recovered slowly from these effects when the citrate electrode was withdrawn and replaced with a KCl electrode. The changes in contraction threshold suggest that citrate ions act on the muscle activation system at an intracellular site, since the citrate permeability of the surface membrane is probably very low. An internal citrate concentration of 5 mM was calculated to result from citrate diffusion out of the microelectrode into the recording area for 20 min. 5 mM citrate added to an artificial cell lowered the free calcium concentration from 240 to 31 microM. It is suggested that citrate modifies excitation-contraction coupling either by acting upon an anion-dependent step in activation or by reducing the free calcium and/or free magnesium concentration in the myoplasm.