Porocytosis: a new approach to synaptic function.

We propose a new approach to address the question of how a single quantum of neurotransmitter is secreted from a presynaptic terminal whose clustered secretory vesicles are locally bathed in high levels of calcium ions [Proceedings of the Symposium on Bioelectrogenesis (1961) 297-309; The Physiology of Synapses (1964) Chapters 1, 4, 5, 6; How the Self Controls its Brain (1994) Chapters 1, 4, 5, 6; Science 256 (1992) 677-679]. This hypothesis, which we term 'porocytosis', posits that the post-synaptic quantal response results from transmitter secreted through an array of docked vesicle/secretory pore complexes. The transient increase in calcium ions, which results from the voltage activated calcium channels, stimulates the array of secretory pores to simultaneously flicker open to pulse transmitter. Porocytosis is consistent with the quantal nature of presynaptic secretion and transmission, and with available biochemical, morphological and physiological evidence. It explains the frequency dependency of quantal size as a function of the secretion process. It permits a signature amount of transmitter release for different frequencies allowing a given synapse to be employed in different behavioral responses. The porocytosis hypothesis permits fidelity of secretion and the seemingly apposed characteristic of synaptic plasticity. The dynamics inherent in an array insure a constant quantal size as a function of the number of units within the array. In this hypothesis, plasticity is a consequence of concurrent pre- and post-synaptic changes due to a change in array size. Changes in the number of docked vesicle-secretory pore complexes composing the array can explain facilitation, depletion, graded excitation-secretion and long term plasticity.

KCl stimulation and ultrastructural responses of tannic acid-stained cholinergic synaptic terminals.

The quantal-vesicular hypothesis equates miniature end-plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense-core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid-elasmobranch saline, KCl stimulation produced MEPPs at 40/s/microm(2)of terminal surface for several minutes with gradual reduction to spontaneous levels by 25-30 min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800 MEPPs/microm(3)of terminals having densities of 567 vesicles/microm(3). No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.

Transmitter quantal size in Torpedo electrocytes is determined by frequency of release.

Miniature end-plate potentials (MEPPs) were focally recorded from the cytoplasmic surface of electrocytes in isolated columns of the Torpedo electric organ. Double electrode studies showed that the junctional area was restricted to 12 micron2. MEPP frequencies ranging from 1/min to 400/s were controlled with electrode advancement against the cytoplasmic surface. Stable membrane potentials and noise levels indicated constant intracellular, focal recording conditions. Focal MEPPs are only 1-3 mV and MEPP amplitudes smoothly decreased with an increase in MEPP frequency which demonstrates a process that meters quantal size at moment of release. Thus, release if not from a prepackaged store. MEPP interval analyses showed that events are weakly interactive at low frequencies and periodic at higher frequencies. The interdependency of MEPP amplitudes and intervals indicates that the mechanism of release controls both rate and quantal size. We propose that the amplitude and frequency dependencies of MEPPs at the Torpedo nerve-electrocyte junction are best described by a membrane channel (e.g., mediatophore, Israël and Dunant, Neurochem. Int. 28 (1996) 1-9) that meters transmitter from a presynaptic store.

The unitary evoked potential at the frog nerve-muscle junction results from synchronous gating of fusion pores at docked vesicles.

Exocytosis of a single vesicle has been proposed as the mechanism which determines quantal size by releasing a prepackaged and standard amount of acetylcholine. As first described by del Castillo and Katz (1954) the endplate potential is composed of 100 unitary events and the small variance suggests a binomial release from 100 "discrete patches of membrane". However, exocytosis of 100 vesicles selected randomly from 5000 docked vesicles would yield a variance that is 7 times greater than observed values. We propose that the presynaptic ridge with its compliment of docked vesicles functions as the "discrete patch of membrane" such that arrays of calcium activated fusion pores meter transmitter to form the unit of release. A model based on the synchronous flicker of a large number of fusion pores produces the small variance of both miniature end plate potentials and unitary end plate potentials. Release from a single locus (fusion pore) would generate the sub-MEPP. This model permits vesicle trafficking and vesicular content depletion during tetanic stimulation and explains the frequency dependency of MEPP amplitudes and changes in sub-MEPP to bell-MEPP class ratios.

Dynamic responses of presynaptic terminal membrane pools following KCl and sucrose stimulation.

The cholinergic presynaptic terminals of Torpedo electric organ have been examined morphometrically following stimulation by KCI and sucrose. The objective was to confirm correlations predicted by the vesicle hypothesis between miniature end-plate potentials (MEPPs) and morphometric changes in terminal ultrastructure. Both secretegogues generated high frequencies of MEPPs and also distinctive though differing ultrastructural changes. The synaptic vesicles show classes of 68 and 90 nm diameters and both store acetylcholine (ACh). KCl stimulation depleted the 90 nm class first whereas sucrose reversed the order of depletion. Very few instances of actual vesicle fusion were seen. Dose-response correlations between vesicle density and secretegogue strength (mM) and duration were higher with sucrose. Both secretegogues produced declines in vesicle numbers and densities and yielded multimodal distributions of large vesicles with an average 160 nm mean diameter. No meaningful correlations were detected between numbers of MEPPs and vesicles and little evidence was found to indicate that vesicles were fusing to terminal plasma membrane in numbers approximating MEPP release. Linear regression analysis was used to quantitatively examine relationships between the vesicle membrane pool and other pools of the putative exo/endocytotic pathway. Correlation coefficients between vesicle and terminal plasma membrane pools were non-significant and of positive sign, indicating independent, similar responses. Non-significant, negative coefficients were obtained when vacuole and 160 nm vesicle membrane values were included. These tests further argue against claims that vesicles are actively fusing with the plasma membrane. These conflicting findings for both secretegogues preclude meaningful correlations between vesicle changes and numbers of MEPPs generated and again emphasize the difficulty of validating the vesicle hypothesis by ultrastructural means. On the other hand, the study shows that vesicular, vacuolar and terminal membrane pools are dynamically changing during transmitter release, presumably interacting with cytosolic membrane constituents. A dynamical release process therefore has been proposed to account for the two classes of MEPPs, the rapid changes in class ratio and the mutable characteristics of the bell-MEPP that presently challenge the quantal-vesicular claims of prepackaged, immutable, exocytotically released packets of transmitter. This model features a state for each MEPP class with class and size determined at moment of release. For example, a single flicker of a channel would generate the sub-MEPP (defined subunit of an MEPP) and 7-20 flickering channels would generate the bell-MEPP.

Dynamics of ethanol-induced transmitter packet release in the frog neuromuscular junction.

Intervals of spontaneous miniature endplate potentials (MEPPs) are usually thought to follow random kinetics because number vs. interval size plots can fit a Poisson distribution such that the largest number of intervals are found in the smallest interval bin. Even though several studies report that MEPPs have a tendency to clump together, an alternative dynamic has not gained acceptance. We found that spontaneous MEPP interval plots with commonly used bin sizes (10 ms or larger) from muscle fibers of diameters usually studied (20 microns) also skew to the smallest interval bin. In order to study very small junctions which do not generate enough intervals for a detailed analysis of high resolving power (small bin width) at normal frequencies of 0.05 MEPPs/s we increased MEPP frequencies with ethanol to obtain large samples. Ethanol accentuated the normal clustering of MEPPs and number vs. interval plots of 0.5 ms show periodic peaks at 400 Hz. A periodogram of power vs. frequency also showed a preferred frequency of 400 Hz. The first peak indicates a refractory period and the 400 Hz oscillation demonstrates that MEPPs are not independent. A release channel with a rhythmical nature is discussed as a possible molecular process which generates clusters of MEPPs.

Hypertonic treatment reversibly increases the ratio of giant skew-miniature endplate potentials to bell-miniature endplate potentials.

Miniature endplate potentials were recorded from single frog muscle fibers before, during and after treatment with hypertonic saline (200-500 mM NaCl or Na gluconate added to frog saline). Miniature endplate potential amplitude distributions were plotted from small muscle fibers so that the modes and ratios of the skew-miniature endplate potential to bell-miniature endplate potential classes could be defined. Muscle fibers were voltage clamped with two electrodes to determine the input resistance before, during and after treatment. Input resistance increased from two to 100 times during treatment and rapidly fell towards control values (no more than 30% greater) when preparations were returned to normal frog saline. Short duration treatments with 200-300 mM hypertonic salines immediately increased frequencies (100-fold) of both skew-miniature endplate potential and bell-miniature endplate potential classes. Preparations when returned to normal frog saline after a few minutes of treatment showed control miniature endplate potential distributions within minutes. One to two hour treatments left only the skew-miniature endplate potential class and with hour-long recovery periods bell-miniature endplate potentials reappeared and ratios of skew-miniature endplate potential to bell-miniature endplate potential classes returned to control values. Treatment with 500 mM NaCl added to frog saline immediately increased the percentage of skew-miniature endplate potentials (from 2 to 50%) with little or no increase in overall miniature endplate potential frequencies. The mode of the skew-miniature endplate potential class was unchanged after hypertonic treatment, whereas that of the bell-miniature end plate potential class either remained about the same size or decreased depending on the duration of treatment. The number and percentage of giant-miniature endplate potentials belonging to the skew-miniature endplate potential class increased as a function of the duration of 200-300 mM hypertonic saline treatments. Most giant-miniature endplate potentials had a slow rising phase with a foot and/or breaks demonstrating a composite structure. Sequentially recorded giant-miniature endplate potentials had similar initial slopes indicating either repetitive releases from single sites or releases from cooperative sites. After hypertonic treatment the bell-miniature endplate potential size was never more than that expected with the increase (under 30%) in input resistance. The results presented here are completely different from those of Yu and Van der Kloot [(1991) J. Physiol. 433, 677-704] who reported that the bell-miniature endplate potential amplitude was increased two- to four-fold after hypertonic treatment. The wide range of results in the ratio of skew-miniature endplate potential to bell-miniature endplate potential classes is discussed in regards to the quantal hypothesis which is based on a single class of immutable amounts of transmitter; and, a hypothesis based on a dynamical process that meters transmitter in subunit amounts to control miniature endplate potential size and class during release.

Detached, purified nerve terminals from skate electric organ for biochemical and physiological studies.

Electric organs of skate (Raja species) dissociate to form populations of individual electrocytes when incubated in saline solutions containing collagenase. The rate of dissociation was highly temperature dependent, with an apparent Q10 of > 6 in the range of 6 degrees-26 degrees C. The number of electrocytes per organ was relatively constant and independent of electric organ size, whereas mean cell diameters increased with organ size. The activities of two cholinergic marker enzymes, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in extracts of whole fresh organs were much less than those reported for the electric ray Torpedo, suggesting a lower volume of terminals in the organ. Electrocytes prepared from collagenase-treated organs had good resting potentials and generated postsynaptic evoked potentials. Spontaneous and electrode pressure-evoked miniature endplate potentials (MEPPs) were readily recorded from isolated electrocytes. Incubation periods of more than 4 days in collagenase at 6 degrees C produced electrocytes with good resting potentials and very low MEPP frequencies, indicating denervation. Detachment of terminals and decreased MEPP frequencies were concurrent. The time course of denervation was followed with the appearance of ChAT and AChE activities in a small particulate fraction derived from washed electrocytes. Peak activities of both enzymes were seen at 4 days of incubation at 16 degrees C, but after 20 h at 16 degrees C. Electrocytes from 4-day, 6 degrees C incubations showed detached, mitochondria-rich nerve terminals and dissociated Schwann cells. In unfixed preparations examined with Nomarski optics, isolated nerve terminals were recognized and distinguished from nucleated Schwann cells. Electron micrographs show that isolated terminals were similar to attached terminals just before they dissociated. The MEPP frequencies and evoked potentials were normal at terminals just before dissociation. We conclude that the transmitter release process was normal in detached terminals and in terminals free of Schwann cells.

Dynamic responses of presynaptic terminal membrane pools to electrical stimulation.

The anatomical tenets of the quantal-vesicular hypothesis of neurotransmission are a 1:1 ratio between numbers of releasable quanta and vesicles, a reciprocal response between vesicle and terminal membrane pools and constancy of the total membrane pool. We have used electrical stimulation and morphometry to study these relationships in the cholinergic presynaptic terminals of Torpedo electric organ. Our results show that during neurotransmission changes in vesicle numbers do not correlate with quantal release, vesicle and terminal membranes do not change in reciprocal fashion and total nerve terminal membrane does not remain constant. We conclude that these vesicular tenets of quantal release are not verifiable at the Torpedo electric organ junction.

High percentage of skew-distributed miniature endplate currents in old mice.

Muscle fibers from diaphragms of old (14 to 24 months) and young adult (1 to 2 months) inbred (strain C57BL/6) mice were voltage clamped at -140 mV with two microelectrodes near the neuromuscular junction. Miniature endplate currents (MEPCs) were digitized so that peak amplitudes and rise times could be determined. MEPC amplitude distributions from old mice varied greatly between fibers from the same diaphragm, and the mean MEPC amplitude (2.1 +/- 0.83 nA, mean +/- SD) was smaller than in young mice (5.2 +/- 0.59 nA). In old mice, some (50%) amplitude distributions were bell shaped, composed of mainly bell-MEPCs with a 2- to 5-nA mode, whereas others (30%) were skewed with a 0.5- to 2-nA mode, and some (20%) showed two peaks, representing both skew- and bell-MEPC classes. MEPC rise-time distributions from old mice varied between fibers, although they all had similar modes. Some (30%) were bell shaped (similar to those in young mice) with a mode between 0.5 and 1 ms (coefficient of variation, 40%), but most distributions were skewed. Endplates with smaller mean MEPC amplitudes showed a longer mean rise time, and for a given junction, MEPC amplitudes were correlated positively to the corresponding rise times. This observation, together with analyses of the rising phases, indicates that MEPCs with long rise times were not generated at remote sites. We discuss our results with regard to the hypothesis of a dynamic formation of transmitter packets, and we attribute long rise time, skew-MEPCs to a prolonged release process. During aging, the state of release that generates the skew-MEPC class appears more dominant than the state generating the bell-MEPC class.

Effects of calcium on the dynamic process of transmitter release which generates either skew- or bell-MEPPS.

Miniature endplate potential (MEPP) amplitudes, MEPP frequencies and ratios of skew:bell-MEPPs were determined as well as synaptic vesicle diameters and densities at the mouse diaphragm neuromuscular endplate during exposure to elevated calcium concentrations. Additions of external Ca2+ had variable effects on MEPP frequencies and percentages of skew-MEPPs, regardless of concentrations used (1-25 mM). Nevertheless, changes in MEPP amplitudes were most sensitive (4-fold decrease) to low value increases of Ca2+. Changes in MEPP frequencies produced by an increase in Ca2+ were very sensitive to initial frequencies as well as the initial calcium concentration. An increase in Ca2+ usually increased MEPP frequency (providing skew-MEPPs were measured). Changes in the percentage of skew-MEPPs were extremely variable (4-90%) and these changes depended on initial frequencies, initial skew- to bell-MEPP ratios and age of the mouse. With a change in Ca2+ concentration, synaptic vesicle diameters and densities remained constant during changes in MEPP frequencies and large changes in the skew:bell-MEPP ratios; and, vesicle numbers were sometimes slightly increased. Because of the wide range in MEPP frequencies and amplitudes, this study demonstrates that the effect of various treatments should be evaluated on identified endplates and that analyses of randomly selected endplates must consider the large variability between endplates. These results show that the skew-MEPP class must not be ignored in studies of spontaneous MEPP release, and that initial frequencies and age of the mouse are also important in evaluating changes in skew-MEPP to bell-MEPP ratios. The rapid changes in skew- to bell-MEPP classes indicate that MEPP class and size are determined at the moment of release by the state of the release process as proposed by Kriebel et al. (1990). Because changes in calcium concentration can immediately alter the ratio of skew- to bell-MEPPs we conclude that the release process has two states to generate the two classes of MEPPs, and that the release process is very sensitive to conditions so that states are easily changed. We propose that the release process meters transmitter in subunit amounts to form both classes of MEPPS and that the calcium ions modulate the process.

Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction.

Fatt and Katz (Nature 166:597-598, 1950; J Physiol 117:109-128, 1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol 124:560-573, 1954). Quantal packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): "Microphysiologie comparée des éléments excitables" 67:245-258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between MEPPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal-vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15:167-178, 1990). The drop formation process (Shaw, "The Dripping Faucet as a Model Chaotic System," Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew- to bell-MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter "packets" are formed at the moment of release and that transmitter supply to the process that forms packets is continuous.

Focal, extracellular recording of slow miniature junctional potentials at the mouse neuromuscular junction.

Miniature endplate potentials (MEPPs) with slow rising phase can be attributed either to burst of transmitter releases or to distortion of conduction from remote releasing sites. The spontaneous activity of neuromuscular junctions recorded extracellularly at mouse diaphragms using sharp electrodes was analyzed to test these two hypotheses. The miniature junctional potentials (MEJPs) frequencies observed intracellularly as compared to MEPP frequency measured intracellularly in controls indicate that most events recorded extracellularly are induced by the presence of the electrode. All types of MEPPs (bell-MEPPs, skew-MEPPs, slow-, and giant MEPPs) previously described with intracellular recording methods (Vautrin and Kriebel, Neuroscience 41:71-88, 1991) were observed extracellularly and showed similar characteristics. This means that the presynaptic and postsynaptic zones that generate these synaptic events are restricted within areas of a few micrometers squared of synaptic contact. Long rise times of extracellularly recorded synaptic spontaneous events may be explained by multiple transmitter releases at intervals shorter than the rise time of individual events, which postsynaptic responses fuse into a single peak.

Characteristics of slow-miniature endplate currents show a subunit composition.

The normal neuromuscular junction shows two classes of spontaneous miniature endplate potentials. These classes are based on a discontinuity in the profile of miniature endplate potential amplitude distributions. The amplitude of one class of miniature endplate potentials from a bell-shaped amplitude distribution and the remaining miniature endplate potentials compose a population which forms a left-hand skew distribution with a mode 1/7 to 1/10 that of the bell-miniature endplate potentials [Kriebel M. E. and Gross C. E. (1974) J. gen. Physiol, 64, 85-103]. Some skew-miniature endplate potentials have a slow time-to-peak and show breaks on the rising phase. Most treatments that alter the miniature endplate potential frequency change the ratio of skew-miniature endplate potentials/bell-miniature endplate potentials [Kriebel M. E. et al. (1976) J. Physiol. 262, 553-581]. The time characteristics of miniature endplate currents were readily altered in the isolated frog and mouse neuromuscular junctions with several agents known to increase the percentage of slow-miniature endplate potentials (heat, botulinum toxin, 4-aminoquinoline and increases in bath osmolarity). The slow-miniature endplate potential amplitudes were a continuum of amplitudes from skew- to giant miniature endplate potentials. The rising phases of miniature endplate potentials were a continuum from smooth to many with breaks and offsets. In a series of sequentially recorded slow-miniature endplate currents, many had congruent rising phases of constant slope regardless of amplitude or of time-to-peak. The rising phases of congruent slow-miniature endplate currents which showed a change in slope deviated at similar amplitudes. The least value of the slope of a slow-miniature endplate current was that of the sub-miniature endplate current; and, miniature endplate currents with overall lower slope values showed a wave pattern and/or irregular breaks which suggests summation of sequentially delayed sub-miniature endplate currents. Plots of the amplitude vs time-to-peak of miniature endplate currents from identified junctions demonstrated that the normal percentage of slow-miniature endplate currents was greatly increased with the treatments used here and that the time-to-peak of giant miniature endplate currents usually was longer than that of normally occurring bell-miniature endplate currents. Giant miniature endplate currents with short time-to-peak values are probably from two miniature endplate currents occurring, by chance, almost simultaneously. During and/or after treatments, miniature endplate currents formed clusters of similar size miniature endplate currents, not randomly distributed in time, which graded from distinct miniature endplate currents to giant miniature endplate currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Transmitter release: prepackaging and random mechanism or dynamic and deterministic process.

Stepwise variations in end-plate potential amplitudes that are also multiples of spontaneous miniature end-plate potentials (MEPPs) demonstrate a quantal nature of evoked transmitter release at the vertebrate neuromuscular junction. Both the number of quanta which form relatively small end-plate potentials (EPPs) and the time intervals between MEPPs were found to fit Poisson statistics. These observations suggested that the release process randomly liberates uniform quantities of transmitter. Initial studies showed that quantal size remained stable after seemingly high rates of release which was interpreted to indicate that a large store of equally sized, equally available, and independently releasable quanta are present in the nerve terminals. The observation of numerous presynaptic vesicles that contain transmitter provided a morphological basis for prepacked transmitter (i.e., quanta). However, physiological studies over the last 15 years have yielded data that are difficult to incorporate into the quantum-vesicle hypothesis. With normal conditions and during most treatments which increase the rate of release, two classes of MEPPs have been found and both show a substructure. The bell-MEPP class was characterized by Fatt and Katz and the smaller skew-MEPP class has been studied by Kriebel. The ratio of the two classes and substructure compositions of both classes are variable. Short series of MEPPs and unitary EPPs (U-EPPs) show preferred amplitudes and longer series of MEPPs and U-EPPs show stepwise variations in amplitude. Slow-MEPPs and giant MEPPs belong to the skew class and represent nearly synchronous bursts of smaller MEPPs. Transmitter packet formation, preferred amplitudes, stepwise variations in amplitudes, random-like distributions and organized bursts can be simulated by a simple deterministic system, the drop formation process, that is known for its periodic and chaotic behaviors which are determined by the single parameter of flow rate. MEPP intervals, sizes and classes, are also dependent on rates of release which demonstrate that the release process(es) is highly organized and sensitive to different conditions. We demonstrate that the processes of drop formation and release of a packet of transmitter have similar properties and that deterministic characteristics describe MEPP and U-EPP time dependencies and amplitude substructures. The data and model presented here suggest that packet size of acetylcholine may be determined at the moment of release.

Morphological, physiological and biochemical observations on skate electric organ.

The electric organs of two species of skate have been examined morphologically, physiologically and biochemically. They can be easily dissociated into innervated or denervated component electrocytes by a Torpedo Ringer's solution containing 1% collagenase. Collagenase treatment did not, however, separate the Schwann cell cover capping the synaptosomes. Isolated electrocytes generate normal MEPP frequencies and show evoked responses for two days in Torpedo Ringer's. The nerve terminals retain excitability and transmitter release properties up to the time of separation. Since isolated terminals and denervated electrocytes show normal ultrastructural characteristics for up to 12 h, the skate electric organ provides several preparations which are not attainable with Torpedo tissue. Acetylcholine (ACh) content of supernatant fractions containing the synaptosomes was comparable to that found in Torpedo (sps.). Collagenase specifically eliminates the basal lamina associated with the synaptic junctional region. Neuronal cell death and synaptic terminal degeneration were also noted in the adult organs of both species. The skate electric organ is ideally suited for the study of cholinergic development and transmission.

Reversible effect of depolarization by K-propionate on sub-miniature endplate potential to bell-miniature endplate potential ratios, on miniature endplate potential frequencies and amplitudes, and on synaptic vesicle diameters and densities in frog neuromuscular junctions.

Miniature endplate potentials were recorded from edge muscle fibers of frog sartorius muscles during high frequencies induced with K-propionate and during recovery. The identified neuromuscular junctions were studied with the electron microscope and their ultrastructure was correlated with amplitude and numbers of miniature endplate potentials generated. Miniature endplate potential amplitudes were maintained during the first 10 min of depolarization. They then decreased during the next 2-3 h until the mode was lost to the noise. Miniature endplate potential frequency was greatly increased during the first hour and there was initial depletion of vesicles. Miniature endplate potential frequencies remained high (5 x 10(5)/h) for 3 h but vesicle densities returned to nearly normal values during the second to third hour of treatment. The conspicuous infolding of the presynaptic membrane noted during the first hour of treatment suggests that recycling of vesicles is initially slower than fusion. Calculated recycling time is shorter than 25 min. During recovery after prolonged K-propionate treatment, the sub-miniature endplate potential class reappeared within minutes but about 20 min were required before it returned to control size. Subsequently, the bell-miniature endplate potentials reappeared and slowly increased in amplitude. The ultrastructure returned to a normal state. There was no change in vesicle diameters. No significant difference was found between the diameters of "touching vesicles" (vesicles touching the presynaptic membrane) and the non-touching vesicles. By comparison, lanthanum ions (1 mM) released a smaller number of quanta which did not exceed the number of vesicles present at the start of the experiment. Variations of the subunit hypothesis of the quantum of transmitter release are discussed.