Dynamics of presynaptic endosomes produced during transmitter release.

The movements of the presynaptic endocytotic structures produced during tetanic stimulation at 10 Hz were examined morphometrically and cytochemically in the cat superior cervical ganglion in vivo. The longitudinal profiles of the axon terminal and preterminal area were subdivided into five zones, I-V. Zone I, the area adjacent to the active zone, was assigned a hemicircle with a diameter equivalent to the active zone width (2R). Zones II-IV were defined by subdividing successively the presynaptic and preterminal areas within hemicircles with diameters equivalent to three-, five-, and sevenfold of 2R, respectively. Zone V was composed of the rest of the preterminal profile. The endocytotic structures, macropinocytotic endosomes and coated vesicles, observed in each zone were morphometrically analyzed with the time course of stimulation. The lateral surface of zone II was shown to be the main site for internalization of the terminal surface membrane during transmitter release. A large amount of the plasmalemma of zone II was rapidly retrieved by macropinocytosis to produce early endosomes at an increased rate of about three times that at rest. The population of coated vesicles, few in number at rest, increased to two- to threefold in zones II-V following the stimulation. Cytochemical examinations showed the incorporation of HRP into synaptic vesicles, endosomes and multivesicular bodies. An antibody against synaptophysin labeled the presynaptic endosomes, multivesicular bodies, coated vesicles as well as synaptic vesicles. The results have suggested that a considerable part of these endosomes was transported retrogradely at an increasing rate from zone II to zone V via zones III and IV. On the other hand, synaptophysin was observed to be distributed on the tubular protrusions of the presynaptic endosomes, suggesting the segregation and recycling of a part of synaptic vesicle proteins from the early endosomes in the nerve endings of the cat superior cervical ganglion.

Rapid recovery of structure and function of the cholinergic synapses in the cat superior cervical ganglion in vivo following stimulation-induced exhaustion.

Cat superior cervical ganglia (SCG) were tetanically stimulated in vivo at 30-100 Hz until neural transmission was exhausted, and then were allowed to rest and recover. Changes in their cholinergic synapses were examined electrophysiologically and morphologically during the time of tetanic stimulation and during recovery. For morphometric analysis the presynaptic terminal was subdivided into two areas: an area directly over the active zone, termed zone-I, (bounded by a hemicircle with a diameter equivalent to the active zone length), and the remaining preterminal area, termed zone-II. In control ganglia before stimulation synaptic vesicle density in zone-I (SVD-I) averaged 90 microns-2 and the number of vesicles actually attached to the active zone (SVA) averaged about 2.5 per single profile of nerve terminal. Upon stimulation, the postganglionic potential immediately began to decline in amplitude and disappeared after 1 min of stimulation. Simultaneously, SVD-I declined to less than 35 microns-2 and SVA declined to less than 1 per section. Thereafter, stimulation was terminated and the ganglion was allowed to rest. Recovery of the postganglionic potential was monitored by stimulation at 1 Hz. The postganglionic potential reached control levels after only 1 min of rest. Likewise, the structural parameters, SVD-I and SVA, also rapidly recovered, reaching control levels after only 30 sec of rest, slightly faster than the postganglionic potential. This illustrates that stimulation-induced fatigue of transmitter output and depletion of synaptic vesicles recover to the control level at a high rate in synapses of the cat SCG with a normal supply of blood. In fact, morphological recovery may be slightly faster than electrophysiological recovery. Mechanisms of vesicle formation and migration to the presynaptic area are discussed in light of these observations.

Synaptic vesicle increase correlated to potentiation of transmission at the synapse of the cat superior cervical ganglion in vivo.

Changes in the pattern, number and size of synaptic vesicles during transmitter release were examined in the synapses of the cat superior cervical ganglion (SCG) in vivo in relation to alteration in the amplitude of postganglionic compound action potential (PGP). Stimulation of the preganglionic nerve fibers at 10 Hz caused an increase in the mean amplitude of PGP. It became augmented by approximately 30% compared to control 10-30 sec after starting the stimulation, and then gradually declined to reach a plateau after 4-6 min. This level, about 20% higher in value than control, was sustained until the end of 30 min of stimulation. The nerve terminal was divided into two areas to examine topographically the numerical changes in synaptic vesicles (SVs): zone I on the presynaptic membrane encircled with a diameter equivalent to the active zone length, and zone II occupying the remaining area outside zone I. The synaptic vesicle density in zone I (vesicle number/microns 2) was 96.9 +/- 4.8 (mean +/- S.E.M.) in the unstimulated control ganglia and 128.8 +/- 9.4 (mean +/- S.E.M.) in the ganglia stimulated for 10-30 sec, which was 30% higher in value than control. Then, it decreased slightly reaching a plateau, 20% higher in value than control. The diameter distribution of the SVs showed that their diameters in zone I (56.6 nm mean) were larger than those (51.6 nm mean) in zone II, and that prolonged stimulation induced smaller vesicles in both areas. The results showed that the increase in SV number in zone I correlated well with the elevation of PGP.(ABSTRACT TRUNCATED AT 250 WORDS)

Intact inhibition of the stretch reflex during general tetanus.

Tetanus toxin at lethal doses (2-10 x 10(3) mouse minimal lethal doses per kg body weight, mMLD/kg) was injected i.v. into 10 cats under pentobarbital anaesthesia. After the appearance of the first sign of generalized tetanus the animal was anaesthetized by a mixture of urethane and chloralose. Experiments were performed several hr thereafter when the toxin action was anticipated to be optimal. The stretch reflexes were elicited manually, by the contraction of the antagonistic muscles or by a stretch device. In toxin treated animals the spontaneous electromyographic activity was inhibited by strong stretching of the tested muscle or by that of the antagonistic muscle. The stretch reflex of the extensor muscle elicited by a contraction of the flexor muscle was inhibited by electrical stimulation of the flexor afferent fibres. The stretch reflex elicited by a stretch device as well as the electrically elicited monosynaptic reflex were inhibited by conditioning stimulation of the antagonistic nerve. The inhibition curves were almost the same as those of healthy animals. It is concluded that the spinal inhibitions, such as antagonistic group Ia, autogenic group Ib, groups II and III and the presynaptic inhibitions, were kept intact in severe general tetanus.

Saltatory conduction of peripheral nerve impulse in clioquinol-treated rats.

By using a new method, unidimensional latency-topography, which shows the saltatory conduction pattern of an impulse along peripheral nerve fibers, the internodal length, internodal conduction time and conduction velocity were determined from the L5 ventral and/or dorsal root filaments of clioquinol-treated rats (CTR). The saltatory conduction pattern was preserved in most of the CTR fibers tested, but was not seen in some fibers. A positive correlation was seen between the conduction velocity and the internodal length in the nerve fibers of both the normal rats and CTR. Although there was no difference in the internodal length between normal rats and CTR, conduction velocities determined in CTR fibers were lower than those in normal rat fibers. Myelin length was calculated from the saltatory conduction pattern in the topography to represent the functional length of the saltatory conduction. The functional myelin length of the CTR fiber was shorter than that of normal rats. Shortening of the functional myelin length in CTR is due to the widening of the Ranvier node, which corresponds to the exposure of the Ranvier node, i.e. demyelination. It was concluded that the decrease in conduction velocity in CTR fibers was due to exposure which caused delayed excitation at the Ranvier nodes.

Saltatory conduction revealed by unidimensional latency-topography of peripheral nerve impulse.

The ventral or dorsal root of the rat was placed on 12 electrodes arranged side by side at 0.4 mm intervals. Impulse conduction along the fiber was displayed using unidimensional latency-topography, which corresponds to the relation between latency and electrode distance. The relation revealed step-like displacement, which implies saltatory conduction of the impulse. Since the distance between the plateaux correspond to internodal length, the conduction velocity could be calculated from the length and the time differences between plateaux. It was found from 36 observations that : (i) mean internodal length was 0.92 +/- 0.13 mm (range 0.70--1.25 mm); (ii) mean conduction velocity was 76.1 +/- 20.7 m/s; and (iii) the correlation coefficient of conduction velocity and internodal length was statistically significant at r = 0.38 (P less than 0.025), so the longer the internodal length, the higher the conduction velocity.

The effect of digital nerve stimulation on recruitment order of motor units in the first deep lumbrical muscle of the cat.

The normal recruitment order of EMG spikes of the first deep lumbrical muscle of the cat's hindpaw, usually seen during cortical stimulation, pad pinch and weak plantar nerve stimulation, was temporarily reversed after stimulation of the medial digital nerve of the foot at 50 Hz for 2 min, and normal order was recovered in 2 to 10 min. The longer the period of stimulation of the medial digital nerve was, the longer the time for recovery. Alteration of order is repeatable and reversible after an interval of more than 15 min. After prolonged medial digital nerve stimulation, EMG responses to pad pinch and plantar nerve stimulation were facilitated. Combination of some stimuli (e.g. cortical stimulation and plantar nerve stimulation, pad pinch and plantar nerve stimulation, plantar nerve and medial digital nerve stimulation) also produced reversal of recruitment order during the period of stimulation. A functionally single motor nerve fiber to the first deep lumbrical muscle was isolated from the motor nerve axons in L7 and S1 of the spinal cord of cats, and physiological properties of pairs of motor units whose recruitment order was temporarily altered by plantar nerve stimulation after prolonged stimulation of the medial digital nerve of a foot were examined. Motor units with large action potentials were more facilitated than motor units with small action potentials after prolonged stimulation of the medial digital nerve. The former motor units showed fast contraction time, large twitch tension, low resistance to fatigue and presence of sag-behavior. The latter motor units showed slow contraction time, small twitch tension and high resistance to fatigue. High threshold motor units with middle-sized surface EMG records were recruited after motor units with large potentials had been recruited; motor units with middle-size action potentials were beta motor axons branching to both intrafusal and extrafusal muscle fibers. The beta motor axons also showed fast conduction velocity and the presence of sag-behavior.

Participation of mono- and polysynaptic transmission during tonic activation of the stretch reflex arcs.

Through intracellular observations of the cat spinal motoneuron both mono- and polysynaptic reflex arcs were shown to participate in repetitive activation of stretch reflex. Experiments were performed on 25 cats anesthetized by intraperitoneal (i.p.) injection of 3 cc/kg of a mixture of 1% chloralose and 10% urethane. Driving of the motoneuronal discharges accomplished by electric stimulation (16-100 Hz) of the gastrocnemius lateralis and medialis nerves. Repeated electric stimulation of muscle nerves elicited monosynaptic "vibratory" EPSPs and a polysynaptic "augmenting" EPSP in the motoneuron. The firing of the motoneuron occurred when a temporal summation of the "vibratory" EPSPs was sufficiently obtained. The discharge frequency of motoneuron, MNf, was expressed as a product of electrostimulus frequency, Sf, and reciprocal of an integer, n, i.e. MNf=(1/n)Sf. An increase in the stimulus intensity resulted in a dimimution of the vibratory EPSP and a remarkable increase of n. This was assumed to be due to an inhibitory process produced by the Group II fibers. The minimum value of integer n was always a determinant factor for the maximum value of MNf which was referred to as the "preferred" frequency of the motoneuron. Long-lasting electric stimulation of muscle nerves was followed by a more remarkable recruitment of the augmenting EPSP which made the motoneuron attain its critical threshold of firing. Motoneuronal spikes elicited by such a large augmenting EPSP were not locked to the Ia impulses. Unlocked spikes were observed only in a later stage of electric stimulation of Ia fibers.