Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.     

Synaptic potential in the motor giant axon of the crayfish

Some electrical properties of the synapses between central giant axons (presynaptic) and the motor giant axon (postsynaptic) of the crayfish abdominal nerve cord have been investigated. Postsynaptic potential change in response to presynaptic volleys contains two components: a spike potential and a synaptic potential of very long time course. Amplitude of the synaptic potential is graded according to the number of active presynaptic axons. Conductance increase in the synaptic membrane endures over most of the period of potential change, and it is this rather than the "electrical time constant" of the membrane that in large measure determines the form of the synaptic potential. Temporal summation of synaptic potential occurs during repetitive presynaptic stimulation, and after such stimulation the rate of decay of synaptic potential is greatly slowed.     

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.     

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.     

Central nervous adaptations following 1 wk of wrist and hand immobilization.

Plastic neural changes have been documented in relation to different types of physical activity, but little is known about central nervous system plasticity accompanying reduced physical activity and immobilization. In the present study we investigated whether plastic neural changes occur in relation to 1 wk of immobilization of the nondominant wrist and hand and a corresponding period of recovery in 10 able-bodied volunteers. After immobilization, maximal voluntary contraction torque decreased and the variability of submaximal static contractions increased significantly without evidence of changes in muscle contractile properties. Hoffmann (H)-reflex amplitudes and the ratios of H-slope to M-slope increased significantly in flexor carpi radialis and abductor pollicis brevis at rest and during contraction without changes in corticospinal excitability, estimated from motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation. Corticomuscular coherence measures were derived from EEG and EMG obtained during static contractions. After immobilization, corticomuscular coherence in the 15- to 35-Hz range associated with maximum negative cumulant values at lags corresponding to MEP latencies decreased. One week after cast removal, all measurements returned to preimmobilization levels. The increased H-reflex amplitudes without changes in MEPs may suggest that presynaptic inhibition or postactivation depression of Ia afferents is reduced following immobilization. Reduced corticomuscular coherence may be caused by changes in afferent input at spinal and cortical levels or by changes in the descending drive from motor cortex. Further studies are needed to elucidate the mechanisms underlying the observed increased spinal excitability and reduced coupling between motor cortex and spinal motoneuronal activity following immobilization.     

Structural plasticity at crustacean neuromuscular synapses

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.     

Localization of the Repetitive Firing Generation Site Induced by 4-Aminopyridine at the Frog Neuromuscular Junction

In the neuromuscular junction, blockade of potassium channels can produce multiple discharges after single nerve stimulation. Multielectrode recording from the nerve trunk and myelinated and non-myelinated parts of the nerve ending demonstrated that repetitive presynaptic spikes elicited by 4-aminopyridine arise earliest within the part of the axon proximal to the motor nerve ending.     

Asymmetric activation of the primary motor cortex during observation of a mirror reflection of a hand

Mirror therapy is an effective technique for pain relief and motor function recovery. It has been demonstrated that magnetic 20-Hz activity is induced in the primary motor cortex (M1) after median nerve stimulation and that the amount of the stimulus-induced 20-Hz activity is decreased when the M1 is activated. In the present study, we investigated how the image or the mirror reflection of a hand holding a pencil modulates the stimulus-induced 20-Hz activity in the M1. Neuromagnetic brain activity was recorded from 13 healthy right-handed subjects while they were either viewing directly their hand holding a pencil or viewing a mirror reflection of their hand holding a pencil. The 20-Hz activity in the left or the right M1 was examined after the right or the left median nerve stimulation, respectively, and the suppression of the stimulus-induced 20-Hz in the M1 by viewing directly one hand holding a pencil or by viewing the mirror image of the hand holding a pencil was assumed to indicate the activation of the M1. The results indicated that the M1 innervating the dominant hand was suppressed either by viewing directly the dominant hand holding a pencil or by viewing the mirror image of the non-dominant hand holding a pencil. On the other hand, the M1 innervating the non-dominant hand was activated by viewing the mirror image of the dominant hand holding a pencil, but was not activated by viewing directly the non-dominant hand holding a pencil. The M1 innervating either the dominant or the non-dominant hand, however, was not activated by viewing the hand on the side ipsilateral to the M1 examined or the mirror image of the hand on the side contralateral to the M1 exaimined. Such activation of the M1 might induce some therapeutic effects of mirror therapy.     

Inference of motor unit recruitment order in voluntary and electrically elicited contractions

The relationship between surface myoelectric signal parameters and the level of voluntary or electrically elicited contractions was studied in 32 experiments on the tibialis anterior muscle of 22 healthy human subjects. Contractions were performed at 20 and 80% of the maximum voluntary contraction torque. Two levels of stimulation current were used, yielding, respectively, a maximum M wave and an M wave approximately 30% of the maximum. A four-bar electrode probe was used to detect single- and double-differential signals from which mean and median frequency of the power spectrum and average muscle fiber conduction velocity were estimated. Measurements obtained from voluntary contractions showed a positive correlation between contraction levels and both conduction velocity and spectral parameters. Conduction velocity increased by 21.2 +/- 10.9% when voluntary contraction level increased from 20 to 80% of the maximal value. Spectral parameters increased by similar amounts. Tetanic electrical stimulation was applied to a muscle motor point for 20 s via surface electrodes. Rectangular current pulses with 0.1-ms width and frequencies of 20, 25, 30, 35, and 40 Hz were used. Four types of behavior were observed with increasing stimulation level: 1) the two spectral parameters and conduction velocity both increased with stimulation in 15 experiments, 2) the two spectral parameters decreased and conduction velocity increased in 8 experiments, 3) the two spectral parameters and conduction velocity both decreased in 6 experiments, and 4) the two spectral parameters increased and conduction velocity decreased in 3 experiments. Conduction velocity increased with increasing stimulation current in 72% of the experiments, indicating a recruitment order similar to that of voluntary contractions, although it decreased in the other 28% of the cases, indicating a reverse order of recruitment. Contrary to what is observed in direct stimulation of nerves, motor units are not in general recruited in reverse order of size during electrical stimulation of a muscle motor point. This discrepancy may be the result of geometric factors or a lack of correlation between axonal branch diameter and the diameter of the parent motoneuron axon. Changes of conduction velocity and spectral parameters in opposite directions may be the result of the combined effect of the motor unit recruitment order and of the different tissue filtering function associated with the geometric location of the recruited motor units within the muscle.     

Form and classification of motor endings in mammalian muscle spindles

The presynaptic features of 234 motor endings supplied to cat hindlimb muscle spindles have been studied in teased, silver preparations, and the postsynaptic features of a further 27 endings have been studied in serial, 1 micron thick, transverse sections. In the presynaptic study motor endings received by the three types of intrafusal muscle fibre were compared with the endings supplied to spindles by the various functional categories of motor axon. Three forms of motor ending were found that had significantly different presynaptic features. These forms correspond closely to those previously identified in the literature as p1 (beta), p2 (dynamic gamma) and trail (static gamma). The results of the postsynaptic study showed that the degree of indentation of the intrafusal muscle fibres by motor axon terminals increases with greater distance from the primary ending, irrespective of muscle-fibre type. We conclude that the postsynaptic form of intrafusal motor endings is determined by distance from primary ending and muscle-fibre type. It is not determined by type of motor axon, and cannot be correlated with presynaptic form so as to produce a unified classification of intrafusal motor endings.     

Coordinated motor neuron axon growth and neuromuscular synaptogenesis are promoted by CPG15 in vivo

We have used in vivo time-lapse two-photon imaging of single motor neuron axons labeled with GFP combined with labeling of presynaptic vesicle clusters and postsynaptic acetylcholine receptors in Xenopus laevis tadpoles to determine the dynamic rearrangement of individual axon branches and synaptogenesis during motor axon arbor development. Control GFP-labeled axons are highly dynamic during the period when axon arbors are elaborating. Axon branches emerge from sites of synaptic vesicle clusters. These data indicate that motor neuron axon elaboration and synaptogenesis are concurrent and iterative. We tested the role of Candidate Plasticity Gene 15 (CPG15, also known as Neuritin), an activity-regulated gene that is expressed in the developing motor neurons in this process. CPG15 expression enhances the development of motor neuron axon arbors by promoting neuromuscular synaptogenesis and by increasing the addition of new axon branches.     

Ultrastructural changes in somatosensory cortex of albino rats during space flight

For the first time, mammalian brain has been studied during space flight aboard NASA orbital laboratory Spacelab-2. The main ultrastructural differences in the somatosensory cortex of the brain fixed under microgravity conditions and after landing include an increased number of degenerating presynaptic axon terminals after landing. Apparently, this is due to a sharp increase in afferent impulsation in the cortex during and after landing.     

Fatigue from incompletely fused tetanic contractions in skeletal muscle in situ

The purpose of this study was to investigate the contractile response of skeletal muscle in situ when stimulation results in an unfused tetanic contraction. The left gastrocnemius-plantaris muscle group of anesthetized (pentobarbital sodium) dogs (n = 16) was connected to an isometric lever and stimulated indirectly for 30 min. During 10-Hz stimulation, total tension (the peak of each oscillation in tension) increased during the first 2 min of stimulation (staircase), then decreased during the remaining 28 min of stimulation. Since relaxation was incomplete at this rate of stimulation, the developed tension, the difference between peak tension and the lowest tension between successive contractions, did not follow the same pattern of staircase and fatigue as the peak tension did. Developed tension (delta T) decreased during the staircase response then increased from 2 to 10 min before finally decreasing again during the last 20 min, ending at 56 +/- 15 (mean +/- SE) % of the initial (first contraction) delta T. At 2 min of 10-Hz contractions, half-relaxation time (1/2 RT) was too long to measure (insufficient relaxation between contractions), but later, 1/2 RT decreased from greater than 65 ms to less than 40 ms. Increased 1/2 RT has been associated with reduced energy availability. If an increased 1/2 RT is an indication of insufficient energy, then it can be concluded that fatigue continued in spite of a recovery of energy supplies. This suggests a possible dissociation of fatigue and energy availability.     

Muscle plasticity: comparison of a 30-Hz burst with 10-Hz continuous stimulation

The changes in the contractile properties induced by a 30-Hz phasic stimulation paradigm were measured and compared with the changes induced by a 10-Hz continuous stimulation paradigm. The study was performed on the tibialis anterior muscles of cats with one paradigm applied to one hindlimb muscle and the other to the contralateral limb. Both hindlimb muscles received the same number of stimuli in a day, making the average stimulation frequency 10 Hz. Two periods of daily stimulation were studied, 8 and 24 h/day. Muscles stimulated at 30 Hz produced greater overall tetanic tension and, during a prolonged stimulation test, exerted a greater mean tension than muscles stimulated at 10 Hz (50 and 32% increase for animals stimulated for 8 and 24 h/day, respectively). Muscle mass was least reduced and fewer pathological abnormalities were observed in the muscles stimulated at 30 Hz. There were no apparent differences in the histochemistry or biochemistry between muscles stimulated at 10 and 30 Hz, which could account for these differences in muscle properties. These results indicate the 30-Hz paradigm may be better suited than 10 Hz continuous stimulation for applications requiring sustained muscle tension such as correction of scoliosis or muscle conditioning for motor prostheses.     

Proliferation and relocation of developing lobster neuromuscular synapses

The development of multiterminal innervation from a single identifiable excitatory motoneuron to the lobster distal accessory flexor muscle (DAFM) was studied by serial section electron microscopy. The number, size, and location of neuromuscular synapses and presynaptic dense bars within the peripheral branching pattern of the axon was determined in cross sections of the DAFM in 1st (24-hr-old)-, 4th (2-week-old)-, and 12th (1-year-old)-stage lobsters. The mean size of synapses remains fairly constant in these three stages but synaptic density, i.e., the number of synapses per unit length of fiber, increased more than 20-fold between the 1st and 4th stages and more than 5-fold between the 4th and 12th stages. Synaptic surface area per fiber length showed a parallel increase. Consequently there is a proliferation of synapses along the length of individual muscle fibers during primary development. Furthermore from the 1st stage where only a few fibers are innervated, synapses proliferate to many more fibers in the 4th and to all fibers in the 12th stage. The neuromuscular synapses are distributed in different proportions within the axonal branching pattern in the three stages. Based on the number and size of synapses and presynaptic dense bars, the main axon and primary branches provide almost equal amounts of innervation in the 1st stage. With further branching in the 4th stage, the main axon accounts for only 20–25% of the innervation; the primary branches for 45% and other finer branches the remainder. By the 12th-stage synapses are found only on branches other than the main axon and its primary offshoots. There is therefore a shift in innervation from the main axon to the primary branches and then to the finer branches during primary development. This shift in innervation involves the formation of new synaptic terminals and the restructuring of existing ones into axonal areas. In this way the multiterminal innervation arising from an identifiable motoneuron is remodeled.     

Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode

Measurements of resting potential and action potential in presynaptic branches of the excitatory motor axon to the crayfish opener muscle were made with intracellular microelectrodes during application of serotonin (10(-9)-10(-3) M). A 5-min exposure to 10(-6) M serotonin produced enhancement of excitatory junction potentials (EJPs) lasting about 1 h. The membrane potential of the presynaptic terminal was depolarized by about 5 mV; the depolarization subsided within 1/2 h. Concomitant reduction in amplitude of the presynaptic action potential, not accompanied by spike broadening, was observed. The presynaptic depolarization, and the enhancement of EJPs, were dependent on the presence of extracellular sodium but not extracellular calcium. A possible mechanism for serotonin's effect involves initial entry of sodium into the nerve terminal, with consequent increased availability of intracellular calcium. The subsequent long-lasting phase of EJP enhancement may result from an additional effect on the metabolism of the nerve terminal.     

Neuronal responses of the reticular and anterior ventral thalamic nuclei to stimulation of the thalamic ventrolateral nucleus and motor cortex

Responses of 137 neurons of the rostral pole of the reticular and anterior ventral thalamic nuclei to electrical stimulation of the ventrolateral nucleus and motor cortex were studied in 17 cats immobilized with D-tubocurarine. The number of neurons responding antidromically to stimulation of the ventrolateral nucleus was 10.5% of all cells tested (latent period of response 0.7–3.0 msec), whereas to stimulation of the motor cortex it was 11.0% (latent period of response 0.4–4.0 msec). Neurons with a dividing axon, one branch of which terminated in the thalamic ventrolateral nuclei, the other in the motor cortex, were found. Orthodromic excitation was observed in 78.9% of neurons tested during stimulation of the ventrolateral nucleus and in 52.5% of neurons during stimulation of the motor cortex. Altogether 55.6% of cells responded to stimulation of the ventrolateral nucleus with a discharge of 3 to 20 action potentials with a frequency of 130–350 Hz. Similar discharges in response to stimulation of the motor cortex were observed in 30.5% of neurons tested. An inhibitory response was recorded in only 6.8% of cells. Convergence of influences from the thalamic ventrolateral nucleus and motor cortex was observed in 55.7% of neurons. The corticofugal influence of the motor cortex on responses arising in these cells to testing stimulation of the ventrolateral nucleus could be either inhibitory or facilitatory.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 5, pp. 460–468, September–October, 1978.     

Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens

The postganglionic axons of sympathetic neurons innervating the mouse vas deferens were stimulated transmurally in vitro by passing square pulses between two platinum electrodes. The ultrastructural appearance of the adrenergic nerve terminals was compared to samples fixed immediately after 30 min of stimulation and in samples allowed to recover for 2 h before fixation. The contralateral vasa deferentia served as controls, and these were incubated in Krebs solution for the same period as stimulated muscles. For each of four experiments, the mean number of large and small dense-core vesicles per square micrometer was calculated, as were the mean area and perimeter of the axon varicosities in each group. It was found that the number of small vesicles per square micrometer decreased by 60% during the stimulation period, but returned almost to control levels 2 h later. Large vesicles did not change in number during the stimulation or recovery periods. The proportion of vesicles containing cores was also determined for each group and found to decline just after stimulation in the small vesicle population, but to remain constant in the large vesicle population. The core depletion was partly reversed after 2 h. The vesicle recovery process was studied by use of the extracellular tracer horseradish peroxidase (HRP). When HRP was present in the extracellular space during stimulation, large numbers of vesicles contained the marker after recovery from stimulation. Thus, it is proposed that adrenergic axon varicosities recycle vesicle membrane through the plasma membrane in a manner similar to that already described for cholinergic nerve terminals.     

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is ～4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.     

Cooperative Ca2+ removal from presynaptic terminals of the spiny lobster neuromuscular junction

Stimulation-induced changes in presynaptic free calcium concentration ([Ca2+]i) were examined by fluorescent imaging at the spiny lobster excitor motor nerve terminals. The Ca2+ removal process in the terminal was analyzed based on a single compartment model, under the assumption that the Ca2+ removal rate from the terminal cytoplasm is proportional to nth power of [Ca2+]i. During 100 nerve stimuli at 10-100 Hz, [Ca2+]i reached a plateau that increased in a less-than-linear way with stimulation frequency, and the power index, n, was about 2. In the decay time course after stimulation, n changed with the number of stimuli from about 1.4 after 10 stimuli to about 2 after 100 stimuli. With the change of n from 1.4 to 2, the rate became larger at high [Ca2+]i (>1.5 microM), but was smaller at low [Ca2+]i (<1 microM). These results suggest that a cooperative Ca2+ removal mechanism of n = 2, such as mitochondria, may play an important role in the terminal. This view is supported by the gradual increase in the [Ca2+]i plateau during long-term stimulation at 20-50 Hz for 60 s and by the existence of a very slow [Ca2+]i recovery process after this stimulation, both of which may be due to accumulation of Ca2+ in the organelle.