Release and recycling of the readily releasable vesicle population in a synapse possessing no reserve population.

We previously demonstrated that the tergotrochanteral muscle (TTM) of Drosophila is innervated by unique synapses that possess a small readily releasable/recycling vesicle population (active zone population), but not the larger reserve vesicle population observed at other neuromuscular junctions in this animal. Using light and electron microscopic techniques and intracellular recording from the G1 muscle fiber of the TTM, the release and recycling characteristics of the readily releasable/recycling population were observed without any possible contribution from a reserve population. Our results indicate 1) the total number of vesicles in synapses presynaptic to the G1 fiber correlates with the total number of quanta that can be released onto this fiber; 2) the number of quanta released by a single action potential onto the G1 fiber is about one half the number of morphologically "docked" vesicles in active zones onto the G1, and this ratio decreases in a partially depleted state; 3) the recycling rate at 1-Hz stimulation, a frequency that does not cause any depression, is 0.24 recycled vesicle/active zone/s; and 4) normal-appearing spontaneous release occurs from the active zone vesicle population and, unlike synapses that possess a reserve population, the frequency of this release is reduced after high-frequency evoked activity.

Relationship of the reserve vesicle population to synaptic depression in the tergotrochanteral and dorsal longitudinal muscles of Drosophila.

We have previously demonstrated that Drosophila synapses possess two vesicle populations-a small active zone population replenished by "fast" recycling and a much larger reserve population replenished by a slower recycling mechanism that includes endosomal intermediates. In this paper, we demonstrate that the synapses onto the tergotrochanteral muscle (TTM) are very unusual in that they possess only the active zone vesicle population but not the reserve population. The depression characteristics to repetitive stimulation of the TTM were compared with those of the dorsal longitudinal muscle (DLM), the synapses of which possess both an active zone and a reserve population. It was observed that the TTM response depressed more quickly than that of the DLM. To further explore the possible contribution of the reserve population to release, using the shibire mutant, DLM synapses were experimentally constructed that possess only the active zone population, and their depression characteristics were compared with those of the same synapses possessing both populations. It was observed that responses from DLM synapses possessing only the active zone population depressed more quickly than the same synapses possessing both populations. These experiments were conducted under conditions of blocked recycling so that the difference in stimulation tolerance represents the contribution of the reserve population to release. Furthermore, the depression curve of the DLM synapses lacking a reserve population now closely approximated that of the TTM synapses. These data suggest that the reserve vesicle population of DLM synapses may contribute to transmitter release during repetitive firing at physiological frequencies (5-10 Hz).

Contribution of active zone subpopulation of vesicles to evoked and spontaneous release.

Our previous work on Drosophila synapses has suggested that two vesicle populations possessing different recycling pathways, a fast pathway emanating from the active zone and a slower pathway emanating from sites away from the active zone, exist in the terminal. The difference in recycling time between these two pathways has allowed us to create a synapse that possesses the small, active zone subpopulation without the larger, nonactive zone population. Synapses were depleted using the temperature-sensitive endocytosis mutant, shibire, which reversibly blocks vesicle recycling at the restrictive temperature. In the depleted state, both the excitatory junction potential (EJP) and spontaneous release are abolished. After shibire-induced depletion, the active zone population begins to reform within 30 s at the permissive temperature, whereas the nonactive zone population does not begin to reform until approximately 10-15 min later. Evoked release recovered at approximately the same time as the active zone population. During the time when the active zone population existed in the terminal without the nonactive zone population, enough transmitter release was available to sustain a normal evoked response for many minutes at frequencies above those produced during normal activity (flight) by this motor neuron. When only the active zone population existed in the terminal, the frequency of spontaneous release was greatly attenuated and possessed abnormal release characteristics. Spontaneous release recovered its predepletion frequency and release characteristics only after the nonactive zone population was reformed.

Omega images at the active zone may be endocytotic rather than exocytotic: implications for the vesicle hypothesis of transmitter release.

A Ca2+-dependent synaptic vesicle-recycling pathway emanating from the plasma membrane adjacent to the dense body at the active zone has been demonstrated by blocking pinch-off of recycling membrane by using the Drosophila mutant, shibire. Exposure of wild-type Drosophila synapses to low Ca2+/high Mg2+ saline is shown here to block this active zone recycling pathway at the stage in which invaginations of the plasma membrane develop adjacent to the dense body. These observations, in combination with our previous demonstration that exposure to high Ca2+ causes "docked" vesicles to accumulate in the identical location where active zone endocytosis occurs, suggest the possibility that a vesicle-recycling pathway emanating from the active zone may exist that is stimulated by exposure to elevated Ca2+, thereby causing an increase in vesicle recycling, and is suppressed by exposure to low Ca2+ saline, thereby blocking newly forming vesicles at the invagination stage. The presence of a Ca2+-dependent endocytotic pathway at the active zone opens up the following possibilities: (i) electron microscopic omega-shaped images (and their equivalent, freeze fracture dimples) observed at the active zone adjacent to the dense body could represent endocytotic images (newly forming vesicles) rather than exocytotic images; (ii) vesicles observed attached to the plasma membrane adjacent to the dense body could represent newly formed vesicles rather than vesicles "docked" for release of transmitter.

Synaptic vesicles have two distinct recycling pathways.

In this paper, evidence is presented that two distinct synaptic vesicle recycling pathways exist within a single terminal. One pathway emanates from the active zone, has a fast time course, involves no intermediate structures, and is blocked by exposure to high Mg2+/low Ca2+ saline, while the second pathway emanates at sites away from the active zone, has a slower time course, involves an endosomal intermediate, and is not sensitive to high Mg2+/low Ca2+. To visualize these two recycling pathways, the temperature-sensitive Drosophila mutant, shibire, in which vesicle recycling is normal at 19 degrees C but is blocked at 29 degrees C, was used. With exposure to 29 degrees C, complete vesicle depletion occurs as exocytosis proceeds while endocytosis is blocked. When the temperature is lowered to 26 degrees C, vesicle recycling membrane begins to accumulate as invaginations of the plasmalemma, but pinch-off is blocked. Under these experimental conditions, it was possible to distinguish the two separate pathways by electron microscopic analysis. These two pathways were further characterized by observing the normal recycling process at the permissive temperature, 19 degrees C. It is suggested that the function of these two recycling pathways might be to produce two distinct vesicle populations: the active zone and nonactive zone populations. The possibility that these two populations have different release characteristics and functions is discussed.

Calcium-induced translocation of synaptic vesicles to the active site.

The effect of increasing [Ca2+]o on the positioning of synaptic vesicles relative to the active site in resting coxal neuromuscular junctions of Drosophila was investigated. In normal saline (1.8 mM Ca2+) only a very small percentage of sites possess a vesicle docked under the dense body plate close to the plasma membrane in a readily releasable position. However, after exposure to elevated Ca2+ salines (3.6, 9, 18 mM), an increase in the number of active sites possessing docked vesicles was observed. Also, an increase in the average number of docked vesicles/site was seen. Intracellular recordings from coxal muscle fibers in normal saline and elevated Ca2+ salines were made, and it was observed that exposure to elevated Ca2+ saline caused an increase in miniature excitatory junction potential (mejp) frequency and in multiquantal and clustered mejps. Thus, when the number of active sites possessing docked vesicles increases, the frequency of spontaneous release also increases. Furthermore, when the number of docked vesicles/site increases, the number of multiquantal mejps increases. The data suggest that Ca2+ may be involved in vesicle translocation to the active site, and that the concentration of Ca2+ in the terminal may regulate the number of active sites that possess readily releasable vesicles. The effects of increasing the number of docked vesicles on spontaneous release characteristics are discussed.

Transformational process of the endosomal compartment in nephrocytes of Drosophila melanogaster.

This study investigates by electron microscopy the transformational process of the endosomal compartment of the Drosophila nephrocyte, the garland cell, which occurs during endocytotic processing of internalized material. The endosomal compartment of the garland cell consists of a prominent tubular/vacuolar complex in the cortical cytoplasm. When internalization of coated pits is blocked at 29 degrees C using the endocytosis mutant, shibire(ts), the tubules gradually disappear after 7 min at 29 degrees C. By 12 min at 29 degrees C, the vacuoles also disappear. Thus, the endosomal compartment appears to constantly undergo a transformational process that necessitates continuous replenishment by coated vesicles. The data suggest that the tubular component of the endosomal compartment gradually transforms into vacuoles by the expansion of the tubular membrane. The vacuoles then transform by invaginating into themselves, creating flattened cisternae. The electron-lucent substance in the lumina of the vacuoles appears to be extruded into the cytoplasm through the invaginating membrane. No shuttle vehicles such as vesicles or tubules could be identified that might have been involved in the transporting of endocytosed materials and membrane from the endosomal compartment to lysosomes or back to the plasma membrane.

Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster.

This study demonstrates that endocytosis in the oocyte of Drosophila melanogaster is reversibly blocked at the stage of pit formation by the temperature-sensitive, single-gene mutant, shibirts1. Uptake of horeradish peroxidase conjugated with wheat-germ agglutinin was observed to be normal in mutant oocytes at 19 degrees C, but was blocked at 29 degrees C. After 10 min at 29 degrees C, there was a build-up of coated pits along invaginations of the plasma membrane. Also, the endosomal compartment consisting of tubules, bulbs, and small yolk spheres, disappeared. Lowering the temperature to 19 degrees C after 10 min at 29 degrees C released a synchronized wave of endocytosis into a cytoplasm cleared of uptake-related organelles. By observing this synchronized wave after exposure to 19 degrees C for varying durations, we determined that endocytosis proceeds as follows: coated pits/vesicles----tubules----small yolk spheres----mature yolk spheres. The observations suggest that these organelles transform one into another within this sequence.

Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval.

The temperature-sensitive mutant of Drosophila, shibire(ts-1), which is normal at 19 degrees C, but in which endocytosis is reversibly blocked at 29 degrees C, was used to deplete synapses of vesicles by inducing transmitter release while membrane retrieval was blocked. When the synapse was kept at 29 degrees C for 8 min, complete vesicle depletion occurred. However, no compensatory increase in the terminal plasma membrane, either as invaginations or evaginations, was observed. Also, no internalized membranous compartment, such as cisternae or coated vesicles, appeared. No invaginations or out-pocketings were seen along the axon between release sites, and no evidence for elongation of the whole axon was found. Thus, the vesicle membrane compartment became unobservable as a result of transmitter release. Depleted synapses were observed by electron microscopy at various times after lowering the temperature, so that the process of synaptic vesicle reformation could be observed. In the first 2-3 min at 19 degrees C, gradually enlarging uncoated invaginations of the plasma membrane were observed. Between 5-10 min at 19 degrees C, these invaginations pinched off to form large cisternae. Newly formed synaptic vesicles were observed associated with these cisternae by an electron-dense material. Between 10-20 min at 19 degrees C, the number of synaptic vesicles increased, while the size of the cisternae decreased. Within 30 min, the full complement of vesicles had reappeared. No involvement of the coated vesicle pathway in synaptic vesicle reformation was observed. The data suggest that synaptic vesicle membrane is dissembled at the time of transmitter release and then is reassembled at sites along the plasma membrane and internalized in the form of large cisternae, from which new vesicles are formed.

Membrane pinch-off and reinsertion observed in living cells of Drosophila.

The garland cell of Drosophila is a nephrocyte which takes up waste products from the haemolymph. Endocytosis is thought to occur by the pinch-off of coated vesicles from deep invaginations of the plasma membrane called labyrinthine channels. Electron microscopic studies show that the length of these channels is variable, depending on the relative rates of membrane pinch-off and reinsertion (recycling). Thus, in wild-type garland cells, if the temperature is raised from 19 degrees C to 30 degrees C, the channels shorten, because at high temperature the pinch-off rate exceeds the reinsertion rate. On the other hand, in garland cells of the temperature-sensitive, single-gene mutant shibirets1 (shi), in which endocytosis is reversibly blocked at the pinch-off stage at 30 degrees C, the labyrinthine channels elongate considerably, as membrane insertion proceeds while pinch-off is blocked. The rates of membrane pinch-off and insertion were quantitated in living garland cells by observing the changes in the capacitance of the whole cell membrane which occur as a result of changes in the total area of the plasma membrane. In wild-type cells, the capacitance gradually decreased as the temperature was raised to 30 degrees C, reflecting the shortening of the channels. In shi cells, the capacitance decreased between 19 degrees C and 26 degrees C but then began to increase at higher temperatures as the blockage of endocytosis caused by the shi gene took effect, causing the channels to elongate. The observations suggest that in shi cells the surface area of the cell more than doubles in 12 min by channel elongation. Estimates of the amount of membrane which is pinched off and reinserted were made.

The relationship between the number of synaptic vesicles and the amount of transmitter released.

The relationship between the number of synaptic vesicles and the amount of transmitter released from identified synapses was investigated in the dorsal longitudinal flight muscle (DLM) of the temperature-sensitive endocytosis mutant of Drosophila melanogaster, shibirets-1(shi). In the shi fly at 29 degrees C, vesicle recyling is blocked, but transmitter release proceeds normally. Thus, by inducing transmitter release at 29 degrees C, shi synapses gradually become depleted of synaptic vesicles. In this way it was possible to regulate the number of vesicles in a synapse. Intracellular recordings were made from individual fibers of the DLM in shi flies after various periods at 29 degrees C while stimulating at 0.5 Hz. The amplitude of the evoked excitatory junction potential (ejp), gradually decreased with longer exposure and was brought to various levels. The fiber was then rapidly fixed for electron microscopy. The number of vesicles per synapse was compared with the amplitude of the ejp at the time of fixation. It was observed that the smaller the ejp amplitudes became, the fewer vesicles were in the synapses. Also, as the ejp amplitude decreased, an increased number of synapses contained no vesicles. It is concluded that synaptic vesicles are directly involved in the release process.

Spontaneous release of multiquantal miniature excitatory junction potentials induced by a Drosophila mutant.

1. Intracellular recordings were made from muscle fibre No. 6 of the dorsal longitudinal flight muscle (DLM) of Drosophila melanogaster in both wild-type flies and the temperature-sensitive paralytic mutant, shibirets-1 (shi). 2. Continuous recordings of the miniature excitatory junction potentials (MEJPs) in this fibre were made as the temperature was changed from 19 to 29 degrees C, and back to 19 degrees C. In shi flies, synapses become depleted of vesicles at 29 degrees C due to a temperature-dependent blockage in the recycling process, while transmitter release proceeds normally. When the temperature is lowered to 19 degrees C, recycling is allowed to proceed and recovery of the full complement of synaptic vesicles gradually occurs in about 20 min. 3. It was observed that the MEJP amplitude distribution in shi flies was unimodal at 19 degrees C prior to heating (as was wild-type), but during recovery from 8 min exposure to 29 degrees C became multimodal, with peaks at roughly integral multiples of the original peak prior to heating. This effect was never seen in wild-type flies. 4. Also, during recovery, the MEJP did not occur randomly, but rather occurred in a clustered fashion. 5. It is concluded that during recovery from depletion in shi neuromuscular junctions, a condition exists which causes the synchronization of spontaneous release, causing multiquantal MEJPs or clustering of MEJPs, depending on the degree of synchronization. 6. The possible role of Ca2+ in this phenomenon is discussed.

Morphological identification of the motor neurons innervating the dorsal longitudinal flight muscle of Drosophila melanogaster.

The dorsal longitudinal flight muscle (DLM) of Drosophila is composed of six muscle fibers (DLM1-6 from ventral to dorsal) and innervated by five motor neurons (MNs), DLM1-4 being innervated singly by MN1-4, and DLM5 and 6 being jointly innervated by MN5. This study identifies and describes the five motor neurons that innervate these six muscle fibers. Horseradish peroxidase (HRP) applied intracellularly to single identified DLM fibers resulted in the labeling of the single motor neuron innervating that muscle fiber by retrograde transsynaptic transport. This method allowed positive identification of the motor neuron innervating a particular muscle fiber, since only the innervating neuron was labeled. The axonal pathway, soma, and dendritic distribution of each labeled motor neuron was traced in the thoracic ganglion, and their relative positions were determined. The somata of MN1-4 lie in a cluster located near the lateral surface of the thoracic ganglion at the border of the pro- and mesothoracic regions, ipsilateral to the muscle fibers innervated. The somata of MN1 and 2 lie side by side in a horizontal plane with MN1 in a more anterior position. Those of MN3 and 4 lie ventrally to those of MN1 and 2 in a horizontal plane with MN3 in the more anterior position. The soma of MN5 is located contralaterally to the muscle fiber it innervates, lying in the dorsal outermost layer of the thoracic ganglion next to the midline at the border of the pro- and mesothoracic regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a presynaptic blockage of transmission in a temperature-sensitive mutant of Drosophila.

In the temperature sensitive mutant of Drosophila, shibirets1 (shi), synaptic transmission in the dorsal longitudinal flight muscles (DLM) is normal at 19 degrees C, but is diminished progressively as the temperature is raised, and is blocked at 29 degrees C. The purpose of this paper is to determine whether this defect is located presynaptically, postsynaptically, or both. It is demonstrated here that the postsynaptic sensitivity to L-glutamate, the putative transmitter for this synapse, is not decreased at 29 degrees C. Furthermore, studies conducted with genetic mosaics of this mutant show that transmission is blocked when a mutant motor neuron synapses on a wild-type muscle fiber, but is not blocked when a wild-type motor neuron synapses on a mutant muscle fiber. Thus, the shi phenotype (temperature dependent transmission block) correlates with a shi motor neuron, not with a shi muscle fiber. The data, therefore, suggest that the defect is not postsynaptic, but presynaptic.

Reversible control of synaptic transmission in a single gene mutant of Drosophila melanogaster.

Synaptic transmission of the single gene mutant, shibirets1 (shi), of Drosophila melanogaster is reversibly blocked by elevated temperature. The presynaptic mechanism of transmission was studied in the neuromuscular junction of the dorsal longitudinal flight muscle of this mutant. It was observed that when the temperature was raised to 29 degrees C in shi flies, the amplitude of the excitatory junction potential (EJP) greatly diminished, the frequency of spontaneously released miniature excitatory junction potentials (MEJP's) was greatly reduced, and almost complete vesicle depletion was observed. These conditions were reversible if the temperature was lowered to 19 degrees C. These data suggest that the block in transmission is a result of vesicle depletion. It is suggested that depletion occurs not as a result of excessive release of transmitter but rather as a result of a block in the recycling of vesicles, which causes depletion as exocytosis (transmitter release) proceeds normally.

Organization of identified axons innervating the dorsal longitudinal flight muscle of Drosophila melanogaster.

Axons innervating the dorsal longitudinal flight muscle of Drosophila were investigated with physiological, light microscopic and electron microscopic techniques. Five motor axons innervate the six muscle fibres which compose a dorsal longitudinal flight muscle. All five axons, designated 1-5, are identified physiologically and morphologically. Axons 1-4 separately innervate muscle fibres 1-4, while axon 5 innervates both muscle fibres 5 and 6, making five motor units. The branching pattern of the nerve and the organization of these axons within the nerve is very consistent from fly to fly, making identification of every axon possible in both the nerve trunk and finer branches. Morphologically, axons 1-2 and 3-4 make particular pairs with long shared pathways. Furthermore, the axons are unusually closely associated with each other within the nerve.

Flight pattern induced by temperature in a single-gene mutant of Drosophila melanogaster.

Simultaneous intracellular recordings were made from the six dorsal longitudinal flight muscle (DLM) fibers in the temperature-sensitive mutant of Drosophila melanogaster, shibirets1 (shi). The DLM firing pattern induced by temperature in shi was compared with the DLM pattern of a wild-type fly in stationary flight. The firing pattern, induced at temperatures between about 26 and 27 degrees C, was very similar to that of the wild-type pattern. At higher temperatures, the pattern became abnormal, characterized by many synchronous firings among the fibers and occasional bursting.

Interspike interval relationship among flight muscle fibres in Drosophila.

Simultaneous intracellular recordings were made from the 10 motor units (12 fibres) comprising the bilateral pair of dorsal longitudinal flight muscles in Drosophila melanogaster while in stationary flight. The neural input which commonly drives these units was characterized by observing the influence which this input has on the interspike intervals of the various units. It was observed that the intervals of these units (both ipsilateral and contralateral), when considered collectively (that is, as a series of successively occurring intervals without regard for which unit represents which interval), fluctuate in a serially correlated manner. These interval fluctuations collectively define a fluctuation of complex waveform. The characteristics of this waveform suggest that two (or more) oscillating inputs are involved in commonly driving these units. In addition, a coupling in frequency and timing was observed between certain pairs of ipsilateral units, as well as between the units of one side relative to those of the other side. This coupling suggests that the neural pathway leading from the oscillating driving source might diverge, first to left and right sides, and then at a more peripheral level into three separate pathways, one leading to units 1 and 2, another to units 3 and 4, and a third to unit 5/6.

Neural interactions controlling timing of flight muscle activity in Drosophila.

Simultaneous intracellular recordings were made from the six ipsilateral dorsal longitudinal muscle fibres of Drosophila in stationary flight. The influence of the firing of one motor unit upon the firing of another was analysed by observing the relationship between the interspike interval of a unit and the relative firing times of the other motor units within that interval. The analysis suggests that the influence is insignificant except when one unit would have fired soon after another. Then, a neural interaction occurs that can cause a unit to fire either earlier or later, depending on its firing relationship with the other units. Thus, the observation that no DLM fibre fires soon after another is the result of both a delaying effect and an effect which causes a cell to fire earlier than it normally would have fired.