Motor units of juvenile rat lumbrical muscles and fibre type compositions of the glycogen-depleted component.

1. Fourth deep lumbrical muscles were dissected out, with their nerve supply, from juvenile rats aged 8-15 days (a period corresponding to maximal rate of decline of polyneuronal innervation), and aged 28, 29 and 30 days (when developmental synapse elimination is complete). Preparations were superfused with rat Ringer solution at 25 degrees C. 2. Isometric twitches and tetani were recorded from the whole muscles and from a single motor unit in the muscle. Unit isolation was by partial section of the sural or lateral plantar nerves. The axon of a single unit occurred naturally in the sural nerve in some cases. 3. Fibres in single units were depleted of glycogen by repetitive stimulation, and studied histologically in frozen midbelly sections of the muscle, stained for glycogen with periodic acid-Schiff's reagent (PAS). Most fibre counts were based on transmittance measurements made with an image analysis system. Contralateral muscles were unstimulated and acted as controls. 4. Motor unit sizes were estimated from tetanic tensions and from muscle fibre cross-sectional area measurements. Comparison of the two methods indicated that in most units glycogen depletion was not complete. This effect was maximal at 8 and 10 days postnatally. It is suggested that this is due to weak neuromuscular transmission at synapses in the process of natural elimination during development. 5. Other sections (serial and semi-serial) were immunostained with a polyclonal antibody raised against slow myosin. Fibres staining for the antibody (slow; S-fibres) contribute about 12-9% of muscle fibres depending on age. In some muscles, fibre types were determined by myosin ATPase staining following alkali pre-incubation. Fast fibres (F-fibres) contained no slow myosin. 6. Some units had no S-fibres (i.e. they were homogeneous), and many units had a small proportion of S-fibres, though less than in the whole muscle (i.e. they were heterogeneous but composition was biased in favour of F-fibres). 7. One unit from a 10-day-old rat contained more S- than F-fibres. Many of the F-fibres were small. It is proposed that this was a developing IIC/IIA unit, a type known to occur in adults. 8. It is concluded that mismatched connections in developing motor units possibly become weak early (by 8 days postnatally) in the process of synapse elimination (which is complete by 20 days postnatally), but that the time course of actual withdrawal cannot be followed by the technique of glycogen depletion.

Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43.

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Comparison of FM1-43 staining patterns and electrophysiological measures of transmitter release at the frog neuromuscular junction.

Frog motor nerve terminals were stained with the activity-dependent dye FM1-43, which appears to stain recycled synaptic vesicles. Superficial end plates which could be visualized in their entirety were imaged and end plate potentials (EPPs) evoked by low frequency nerve stimulation were recorded from the muscle fibers which were innervated by the imaged terminals. Curare was present to block muscle contractions. The amplitude of the EPPs correlated reasonably well with the number of fluorescent spots counted in the terminals (r = 0.68). Each fluorescent spot probably represents a cluster of synaptic vesicles localized at an active zone. Several other morphological and electrophysiological values were measured and calculated. The results are consistent with the ideas that FM1-43 stains recycled synaptic vesicles, and that the number of vesicle clusters in a terminal is a good predictor of synaptic efficacy.

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.

The importance of competition between motoneurones in developing rat muscle; effects of partial denervation at birth.

1. The number of motor units in developing fourth deep lumbrical muscles was reduced by unilateral partial denervation of the muscle at birth, by cutting the lateral plantar nerve. A minority of the motor axons arrive via the sural nerve, and were thus not cut. Those muscles that contained one motor unit (one-unit muscles) after partial denervation developed in the absence of competition between motoneurones. Muscles with two motor units had little competition. A few four-unit muscles were studied for comparison. 2. Isometric twitch and tetanic tensions of single motor units were recorded in vitro at 60 days of age in response to stimulation of the sural nerve. On average, units in partially denervated muscles generated more tension than normal units. The isometric tension characteristics of the units in the one-unit and two-unit muscles were different from the normal units (e.g. slower contracting and more fatiguable). The units of four-unit muscles had properties similar to those of normal muscles. 3. Fibres of an individual unit were identified by glycogen depletion and S (slow) fibres were identified in cross-section that bound a polyclonal antibody to slow type I myosin. Those fibres that did not bind the antibody were designated F fibres. The units of one-unit muscles had the same total number of fibres and fibre type composition (both S and F fibres in the same unit) as estimated from previous work to exist at birth. The units of two-unit muscles contained the same total number of fibres, but apparently fewer S fibres, though this may have been as a result of incomplete glycogen depletion of some fibres. 4. It is concluded that competition between motoneurone terminals is necessary for the withdrawal of mismatched connections on muscle fibres present at birth; or, alternatively, that such withdrawal cannot take place if it would result in denervation of the muscle fibre.

Motor units of the fourth deep lumbrical muscle of the adult rat: isometric contractions and fibre type compositions.

1. Isometric twitch and tetanic tensions were recorded from whole muscles and single motor units in fourth deep lumbrical muscles isolated from young adult (60 days) rats. Muscles were superfused with oxygenated Ringer solution at 25 degrees C except where stated otherwise. 2. It was confirmed that the muscle is supplied most commonly by eleven motor axons, nine via the lateral plantar nerve (LPN), and two via the sural nerve (SN). Motor units whose axons were isolated from either LPN or SN were studied. There was no difference in mean motor unit size. 3. In their unfused tetani most units showed 'sag' and some 'no sag', with no segregation between LPN and SN. 'No sag' units were always small (unit tetanic tension less than 8% whole-muscle tetanic tension), tended to be relatively slowly contracting and relaxing during an isometric twitch, and tended to have relatively low twitch:tetanus ratios. Units showing sag ranged from large to small. 4. In some motor units muscle fibres were depleted of their glycogen by repetitive stimulation at 30 degrees C in glucose-free Ringer solution, and the muscle and its unstimulated control frozen and sectioned. Neighbouring sections were stained for glycogen and for binding of two myosin-specific antibodies, one specific for slow myosin and the other for type IIA myosin. Myosin ATPase and succinic dehydrogenase histochemistry were also carried out in some muscles. 5. Serial reconstructions showed that all or virtually all extrafusal fibres in the muscle were present in a midbelly section, and that the myosin type of individual fibres did not change significantly along their length. Spindle profiles were seen frequently and in two muscles eight and twelve spindles were identified. 6. Of twenty-six motor units examined twenty contained almost exclusively muscle fibres of the recently described type IIX. All these units showed sag in their isometric tetani. 7. Six units each contained 50% or more of slow myosin-containing fibres (IIC and a few type I). The remaining fibres in these units were IIA. All these units were therefore of mixed fibre composition, and are discussed as IIC/IIA units. In whole muscles slow-myosin-containing fibres were generally distributed evenly (non-randomly) throughout the muscle cross-section. 8. Whole muscles contained on average 970 fibres (S.D. +/- 70) of which 82 (+/- 9) were slow-myosin-containing. A few muscles from older rats (3-24 months) contained very few such fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Competitive mechanisms underlying synapse elimination in the lumbrical muscle of the rat.

The process of neuromuscular synapse elimination has been studied in the fourth deep lumbrical (4DL) muscle of the rat, a preparation which offers technical advantages for some types of experimental work. Studies have been performed both during development and in adult denervated muscles undergoing reinnervation. Results indicate that synapse elimination is dependent upon competition between motoneurons. Cellular mechanisms underlying this competition have also been explored. Both neuromuscular activity and muscle fiber type recognition appear to play a role, but positional cues appear unimportant in this small muscle.

Inhibitory interactions between motoneurone terminals in neonatal rat lumbrical muscle.

1. Evoked synaptic potentials and currents were recorded in neonatal rat fourth deep lumbrical muscle during the period of polyneuronal innervation. Signs of inhibitory interactions between converging mononeurone terminals were detected. 2. Muscle fibres innervated by axons from the lateral plantar nerve (LPN) and from the sural nerve (SN) were studied. In unblocked preparations the muscle contracted, and electrode tips were mounted on flexible wires to prevent loss of impalements. 3. In voltage recordings from unblocked preparations, paired two-shock stimulation of one nerve revealed synaptic depression: the second response was smaller than the first. When the two stimuli were delivered to different nerves (SN and LPN), the second response was smaller than its own control. 4. In voltage clamped, unblocked preparations, a similar result was obtained. Conditioning stimulation of one nerve (SN, for example) inhibited the response to test stimulation of the other nerve (LPN). The inhibition was greater with larger conditioning responses, was maximal when the conditioning and test stimuli were approximately superimposed, and decayed with a time course of several tens of milliseconds. Several tests showed that the end-plate was well clamped: the observed inhibition could not be explained by voltage escape at the end-plate. 5. The inhibition was not constant during the tail of the test end-plate current (EPC). Instead, it declined during the EPC tail, suggesting that the mechanism of inhibition was active, though diminishing, throughout the time course of the test EPC. 6. The amount of inhibition was not noticeably affected by altering the muscle membrane potential (two cells studied). 7. Treatment with curare or alpha-bungarotoxin to block most ACh receptors reduced the inhibition. In about half of the fibres studied, no inhibition was evident: in the others, up to 50% inhibition was observed. The average inhibition for all receptor-blocked fibres was about 15%. 8. In six alpha-bungarotoxin-treated cells, multiple conditioning stimuli were delivered. In most cases, the amount of inhibition increased with increasing numbers of conditioning stimuli. 9. Several possible mechanisms of inhibition are discussed, including reduction of current through ACh channels during the test response owing to alterations in synaptic cleft ion concentrations produced by the conditioning response, presynaptic inhibition of transmitter release, and postsynaptic receptor saturation.

Adult rat lumbrical muscle fibres are not polyneuronally innervated.

The existence of polyneuronal innervation in adult rat skeletal muscle reported by Taxt (1983) has been reinvestigated. The extent of polyneuronal innervation was measured by intracellular recording in cut muscle fibre preparations. Only fibres in which spontaneously occurring miniature end-plate potentials were visible were tested for the presence of multiple inputs. Only one in sixty-nine fibres penetrated was found to have more than one axonal input. High-threshold, low-amplitude end-plate potentials were sought, but none was found. Thus we were unable to find any evidence for extensive polyneuronal innervation persisting in rats of 40 days of age.

Rat muscle during post-natal development: evidence in favour of no interconversion between fast- and slow-twitch fibres.

1. It was confirmed that in the fourth deep lumbrical muscle of the rat the number of muscle fibres at birth is about half that in the adult. 2. The average number of slow-myosin-containing (S) fibres (as determined by specific antibody binding) remains constant from birth to adulthood. Therefore it is likely that all the muscle fibres generated post-natally are type F (i.e. slow-myosin-free). 3. A comparison in the electron microscope between transverse mid-belly sections from new-born (day 0) and 4 day muscles showed many muscle fibres or myotubes to be intimately associated with other fibres and cells of other types in the new-born, but to be much less closely grouped at 4 days. 4. A full cell count was obtained from electron microscopy of a mid-belly section of a lumbrical muscle at birth. 5. Cross-sectional area measurements in the light microscope at 3-5 days and in the adult showed that at 3-5 days on average the S fibres have a greater cross-sectional area than the F fibres. This is reversed in the adult where the S fibres are the smaller. At 3-5 days the range of cross-sectional areas of F fibres is much wider than for S fibres. Some F fibres are among the largest fibres in the muscle. 6. It is argued from the data that the motor units of adult muscle, which are homogeneous with respect to muscle-fibre types, are produced by selective withdrawal of neonatal motor-unit contacts during developmental synapse elimination.

Motor units in a skeletal muscle of neonatal rat: mechanical properties and weak neuromuscular transmission.

1. Isometric twitch and tetanic tensions were recorded from whole muscles and single motor units in isolated fourth deep lumbrical muscles from neonatal rats (most at 3-5 days old) and from older rats of various ages. 2. Whole-muscle time to peak contraction reduced from about 120 ms at birth to about 20-25 ms at 20 days and older. 3. The number of motor units in the muscle was constant with age (eleven on average) and there was no significant branching of motor axons below the common peroneal nerve branching point in the thigh. 4. In the 3-5 days age range, mean twitch:tetanus ratio for whole muscles was 0.299 and for single units was 0.177. As a consequence, mean motor unit size (as a percentage of whole-muscle tension) was greater for tetani (29.7%) than for twitches (19.9%). This was not the case in muscles from animals 22 days or older. Evidence is given that the cause of this is low junctional efficacy in some neuromuscular junctions in neonatal muscle. Intracellular recordings supported this view. 5. The relationships of motor-unit size to the contraction time, to the ratio of contraction time:half-relaxation time, and to fatigue index are given. There was no indication of clear segregation of motor units into more than one population, but it is concluded that small motor units are more likely to contain a higher proportion of slowly contracting, fatigue-resistant fibres than large units. 6. The level of overlap by axons in the lateral plantar nerve onto muscle fibres in a single sural nerve motor unit was greater in tetani than in twitches. The results indicate that the distribution of weak and strong inputs was not random, but that there was a tendency for one strong input to accompany a number of weak inputs (on average about two) on each muscle fibre. 7. Intracellular recording indicates that about 12% of fibres at 3-5 days may be electrically coupled.

The non-selective innervation of muscle fibres and mixed composition of motor units in a muscle of neonatal rat.

1. Motor-unit size was measured by tension recording in neonatal (3-5 day) rat skeletal muscle (fourth deep lumbrical muscle). Each unit was then depleted of glycogen and its fibres studied in mid-belly frozen sections, by staining for glycogen (periodic acid-Schiff reagent and antibody labelling for slow myosin. The contralateral muscle acted as control, and further controls for the method are described. 2. All the motor units contained both slow-myosin-containing (S; antibody-positive) and slow-myosin-free (F; antibody-negative) fibres. 3. The proportion of each unit that was made up of S fibres was compared with the whole muscle. Of the twelve units studied seven were not selectively innervated, four may have been selectively innervated in favour of F fibres, and one was selectively innervated in favour of S fibres. The last unit was much smaller than the others. 4. Fibre cross-sectional areas were measured in units and in the whole muscles. Mean cross-sectional areas for individual F fibres in all the motor units were smaller than in the corresponding whole muscles (ratio 0.71), implying that small fibres have higher levels of polyneuronal innervation than larger ones (each small fibre occurring in more overlapping units than each larger fibre). There was no such difference in S fibres (ratio 0.96). 5. Motor-unit sizes (as a percentage of whole muscle) were smaller when obtained from summed fibre cross-sectional areas than from fibre counts (this follows from 4, above). Comparisons with unit sizes from tension recording are discussed. 6. Controls show that there is little, if any, non-specific fatigue of muscle fibres that are not part of the unit subjected to glycogen depletion. 7. Evidence is given that muscle fibre conduction block occurs during the depletion regime, leading to less glycogen depletion towards the ends of the muscle fibres than in the end-plate zone.

The effect of selective, chronic stimulation on motor unit size in developing rat muscle.

One of the two peripheral nerves which innervate rat lumbrical muscle was stimulated chronically in vivo during the postnatal period of synapse elimination to determine whether the differential stimulation would affect the outcome of the elimination process. Rats were anesthetized for about 4 hr a day for 5 to 6 consecutive days, during which time the sural nerve (or, in other animals, the lateral plantar nerve) was electrically stimulated. Each animal received about 10(6) stimuli. After the last stimulation period, the sizes of motor units in both nerves were estimated from motor unit tension recorded in vitro. We found that, on average, sural motor units were larger than others in animals which had received sural nerve stimulation and smaller than others in animals which had received lateral planter nerve stimulation. These results are consistent with the hypothesis that more active nerve terminals possess a relative advantage in competing for occupancy of the endplate.

The effects of deafferentation and spinal cord transection on synapse elimination in developing rat muscles.

The proportion of muscle fibres innervated by more than one motoneurone (polyneuronal innervation) was measured in the fourth deep lumbrical muscle of young rats following either unilateral deafferentation or spinal cord isolation (cord transection combined with bilateral deafferentation). 2. Unilateral deafferentation at 7 days of age did not affect the subsequent time course of synapse elimination in the developing muscle. 3. Spinal cord isolation at 7 days led to a prolongation of the time course of synapse elimination in the muscle, levels of polyneuronal innervation approaching zero beyond about 30 days (about 20 days in normal animals). The raised levels of polyneuronal innervation in these animals were not associated with significant terminal sprouting at end-plates, but were associated with multiple axonal inputs to end-plates as is found in younger normal animals. This was shown by zinc iodide and osmium staining. Also some muscle fibres had two and sometimes three separate end-plates. 4. Spinal cord isolation in rats 19 or more days old resulted in a reappearance of multiple innervation. This was largely due to ultraterminal sprouting. 5. It is concluded that unilateral deafferentation alone causes no change in the time course of synapse elimination. The multiple innervation caused by total deafferentation and spinal transection is attributed to the lack of activity of these muscles.

Properties of motor units in a small foot muscle of Xenopus laevis.

1. Extensor digitorum longus IV muscle (EDL IV) of Xenopus laevis was isolated together with its nerve and the 8th and 9th spinal roots. Motor units were functionally isolated and their properties investigated. 2. The motor unit isometric twitch and tetanic tensions, contraction time and axon conduction time and distance were measured. Axon conduction velocity and twitch:tetanus ratio were calculated. 3. The distributions of motor unit properties were continuous and showed no tendency to separate into types. 4. Axon conduction velocity was directly related to unit tetanic tension (expressed as a percentage of whole muscle tetanic tension and to twitch:tetanus ratio and inversely related to contraction time. Unit tetanic tension was directly related to twitch:tetanus ratio and inversely related to contraction time. Contraction time was inversely related to twitch: tetanus ratio. Possible explanations for these relationships are discussed. 5. The units of EDL IV show a greater resistance to fatigue than fast units of other amphibian muscles. There was no separation of units into fatiguable and fatigue resistant types.

Polyneural innervation: mechanical properties of overlapping motor units in a small foot muscle of Xenopus laevis.

1. Extensor digitorum longus IV of Xenopus laevis was isolated together with its nerve and the 8th and 9th spinal roots. Motor units were functionally isolated, by splitting and stimulating ventral root filaments. 2. Motor units were stimulated separately and in pairs. Between many pairs, overlap of motor innervation was found (evidence of polyneuronal innervation of muscle fibres). Evidence for overlap was a deficit in the tension developed when the two units were stimulated together compared with the sum of the tetanic tensions developed when the two units were stimulated separately. This provided a measure of the extent of overlap. 3. Polyneuronal innervation was confirmed by intracellular recording from muscle fibres during stimulation of each motor axon. 4. For each motor unit, isometric twitch and tetanic tensions, twitch contraction time and the motor axon conduction time and distance were measured and axon conduction velocity and twitch:tetanus ratio were calculated. 5. The properties of the pairs of motor units found to overlap are reported and marked disparities between the properties of some units in overlapping pairs demonstrated. 6. Frequency distributions for the properties of motor units shown to overlap with others were not significantly different from those for total unit samples., Therefore polyneuronal innervation of muscle fibres occurs throughout the motor unit population of this muscle. 7. In several motor units the isometric twitches of several individual muscle fibres were recorded, each fibre being stimulated by intracellular current injection. It was found that contraction times of single fibres differed by up to 36% between muscle fibres in the same unit and that twitch tension varied by up to 400%.

Electrical responses of muscle fibres in a small foot muscle of Xenopus laevis.

1. Extensor digitorum longus IV (EDL IV) of Xenopus laevis was isolated together with its nerve and the 8th and 9th spinal roots. Motor units were functionally isolated.2. Glass micro-electrodes were used to penetrate the muscle fibres and to record their electrical responses to stimulation of single motor axons, or the total motor supply to the muscle.3. Three types of electrical response were recorded from muscle fibres: an action potential in response to a single nerve stimulus (type 1), an action potential following repetitive nerve stimulation but not in response to single shocks (type 2) and end-plate potentials which summed to a plateau of depolarization during repetitive nerve stimulation without producing action potentials (type 3).4. In general muscle fibres giving type 1 responses belonged to fast motor units with high twitch: tetanus ratios; those giving type 3 responses, to small units with low twitch: tetanus ratios; and those giving type 2 responses, to units with intermediate properties.5. In many cases the several muscle fibres innervated by a single axon gave different responses to stimulation of that axon and a single muscle fibre might give different responses to stimulation of two single axons. Thus it was not possible to classify motor units or muscle fibres by electrical responses.6. Stimulation of the total motor supply to the muscle favoured the production of type 1 responses so that the proportion of muscle fibres giving type 1 responses was higher when the muscle nerve was stimulated than when single motor axons were stimulated. The proportion of fibres giving type 2 responses was lower, and the proportion of fibres giving type 3 responses was similar, in both cases. The implications of this are discussed.7. Low neuromuscular junctional efficacy is suggested as an explanation for fluctuating twitch tensions and low twitch: tetanus ratios in many motor units in this muscle, and may also affect the activity patterns imposed on the muscle fibres by the motor axons which supply them.

Properties of twitch motor units in snake costocutaneous muscle.

1. Single motor units of twitch muscle fibres were studied in isolated nerve-muscle preparations of m. costocutanei inferiores from grass and garter snakes. Preparations were superfused with Ringer solution at a controlled temperature of 22 degrees C.2. The peak and time-to-peak tension (contraction time) were measured for isometric twitches of forty-seven whole muscles and 83 motor units. The sample of motor units was drawn from an estimated total population of 213-355 twitch units. Peak tetanic tensions were also measured. The measurements were made at muscle lengths at which the twitch tension was maximal, and this length was not always the same for whole muscle and unit twitches. In fifty-nine cases 1 and in twelve cases 2 motor units were isolated from each muscle.3. Whole muscle contraction times ranged from 22-61 msec (mean +/- S.D. = 40.3 +/- 9.8 msec) and those for units from 18-92 msec (mean +/- S.D. = 46.9 +/- 15.9 msec). The wide range for whole muscles is discussed.4. The percentage of the whole muscle tetanic tension contributed by each unit (unit size) was calculated. Contraction time was inversely related to unit size.5. Twitch-tetanus ratios were calculated and found not to be related to unit contraction time.6. The conduction velocities of axons innervating 23 motor units were calculated from latency measurements at two points along the length of the nerve. They ranged from 1.9 to 10.4 m/sec. Axon conduction velocity was inversely related to unit contraction time, and directly related to unit size.

Post-tetanic potentiation of twitch motor units in snake costocutaneous muscle.

1. Isometric twitch responses of single motor units in snake costocutaneous muscle have been recorded before and after conditioning tetanic stimulation. 2. Most units showed post-tetanic potentiation of twitch tension, associated with increased maximal rate of rise of twitch tension, and in some cases also associated with prolongation of twitch contraction time. A few units showed a short phase of actual depression of the post-tetanic twich responses, followed by potentiation. 3. The time course and magnitude of post-tetanic changes of twitch tension and maximal rate of rise of tension could be described by the sum of three processes which are assumed to be maximal close to the end of the conditioning tetanus: (i) a rapidly declining potentiation (called here early potentiation, which lasted less than 2 sec) which may have a purely mechanical origin; (ii) a much slower-declining potentiation (called here prolonged potentiation, which lasted up to 15 min); and (iii) a process which lasted up to 60 sec during which twitch potentiation was reduced. The latter process (called here depressed potentiation) was usually apparent as a marked trough in the plots of twitch amplitude versus time after the conditioning tetanus, and occassionally was evident as an actual transient depression of twitch amplitude after the tetanus compared with that before. 4. The effects of the prolonged potentiation and the depressed potentiation on the maximal effect of each process close to the end of the tetanus were extracted by fitting single exponential equations to different portions of the data, using a computer program. 5. Twitch potentiation associated with increased maximal rate of rise of tension seemed to be a separate phenomenon to that associated with prolongation of contraction time, seen when conditioning tetani of higher frequency and numbers of stimuli were employed. The depressed potentiation of twitch tension tended to be partly masked in cases where contraction time was prolonged, but this did not affect the depressed potentiation of maximal rate of rise of tension. 6. The post-tetanic potentiation shown by a unit was related to the contraction time of the unit, in addition to the well known relationship to the initial twitch-tetanus ratio. The depressed potentiation may correlat more closely with the initial twitch-tetanus ratio than with the unit contraction time. 7. The magnitude of maximal depressed potentiation shown by a unit may be directly correlated to that of maximal prolonged potentiation. 8. The time constant of decay for prolonged potentiation of twitch tension tended to be related inversely to unit contraction time and directly to unit size and the maximal value of prolonged potentiation oftwitch tension. The time constant of decay for prolonged potentiation of maximal rate of rise of tension tended to be related to unit size and initial twitch-tetanus ratio, and the time constant of decay for depressed potentiation of rate of rise of tension tended to be related to unit size. 9...