The effects of chronic stimulation on the morphology of the frog neuromuscular junction

Abstract

A quantitative study was made of the effects of 24 h continuous stimulation on the morphology of the frog neuromuscular junction. The synaptic vesicle concentration in the nerve endings of frog sartorius muscles stimulated in vitro for 24 h at 2 Hz was the same as that in controls stimulated for only 0.3 h at 2 Hz. The control preparations were either freshly dissected or maintained at rest in vitro for 23 h prior to stimulation. Chronically stimulated terminals differed from their controls only in having more cisternae and fewer dense cored vesicles. Varying the lengths of the nerves to both chronically stimulated an in vitro control muscles had little effect on the morphology of the nerve endings. Continuous recording of muscle twitch tension demonstrated that neurotransmission was effective throughout the 24 h period of stimulation. Additional evidence that nerve failure or degeneration was not a factor in the results came from a second set of control and chronically stimulated preparations that were tetanized at 30 Hz for 0.3 h before fixation. Changes attributable to rapid stimulation were evident in 87 to 100% of their nerve terminals. Although the distribution of membrane among various membrane organelles differed from one treatment group to another, the total amount of measurable membrane in the nerve terminals was the same in all of the treatment groups; that is, the total amount of membrane was not altered by maintenance in vitro, chronic stimulation at 2 Hz, rapid stimulation at 30 Hz, reduced nerve length, or any tested combination of these treatments. This conservation of total membrane suggests that membrane exchange between axon and nerve terminal occurs at a relatively slow rate which is unaffected by synaptic activity, and that the local mechanism for recycling synaptic vesicle membrane in frog neuromuscular junctions is more autonomous and durable than has been suspected.

Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin

“…Our study is perhaps more comparable to an earlier study, which describes 3 million quanta being released over 24 hours using a 2 Hz nerve-evoked simulation paradigm (Lynch, 1982). This type of stimulation would produce a higher rate of release (700-800 quanta/second), which was shown to induce significant depletion of synaptic vesicles (Lynch, 1982). To the best of our knowledge, our study establishes that the machinery for synaptic-vesicle recycling at the frog NMJ can cope with the release of over 100 but less than 700 vesicles/second.…”

“…Assuming that each stimulation results in 380 quanta being released (Van der Kloot and Molgo, 1994), the total number of quanta released is 684,000. Our study is perhaps more comparable to an earlier study, which describes 3 million quanta being released over 24 hours using a 2 Hz nerve-evoked simulation paradigm (Lynch, 1982). This type of stimulation would produce a higher rate of release (700-800 quanta/second), which was shown to induce significant depletion of synaptic vesicles (Lynch, 1982).…”

“…These compartments were not detected upon short-term high-rate stimulation; smaller oblongshaped cisternae were mostly observed in this case (Richards et al, 2000). Similarly, low-rate chronic stimulation (24 hours at 2 Hz) has been shown to increase the number of these small cisternae at the frog NMJ (Lynch, 1982). One could speculate that the large endosomal structures highlighted in our study might result from the accumulation of plasma membrane derived from the hot-spots detected on the plasma membrane after 5 minutes exposure to GLTx (Fig.…”

Veratridine‐stimulated central synapses in culture: A quantitative ultrastructural analysis

“…The source of this excess membrane might well be the synaptic vesicle compartment, thus suggesting a membrane incorporation process in association with veratridine stimulation. The total membrane, however, remained unchanged after stimulation ( Table 2), which supports the view that no net loss of membrane occurs during synapse activity (Heuser and Reese, 1973;Zimmermann, 1979;Lynch, 1982), although net loss of presynaptic membrane has also been reported following electrical stimulation (Tremblay and Philippe, 1981 The frequency occurrence of vacuolar/cisternal profiles increased (Table l), reflecting either swelling of these organelles or vesicle membrane recycling, and therefore contributing to the preservation of the total membrane. The frequency of coated vesicles, however, did not increase significantly after veratridine stimulation.…”

Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.

“…However, when terminals are stimulated under conditions that interfere with vesicle recycling, then the number of quanta secreted approximately equals the number of vesicles lost and it approaches the number of vesicles stored in control terminals. Agents or procedures that stimulate secretion and permit extensive recycling include electric stimulation of the nerve (Ceccarelli et al 1972(Ceccarelli et al , 1973Heuser & Reese, 1973; Rose, Pappas & Kriebel, 1978;Lynch, 1982;Koenig et al 1983), La3+ (041-2 mm) applied at room temperature (Heuser & Miledi, 1971;Segal et al 1985) or at 3-5 0C (Heuser & Miledi, 1971;Florey & Kriebel, 1983;Dekhuijzen et al 1989), and low doses of black widow spider venom (BWSV), or a-latrotoxin (az-LTX), applied at room temperature in solutions with normal levels of Ca2` (Ceccarelli & Hurlbut, 1980;Fesce et al 1986a;Valtorta, Jahn, Fesce, Greengard & Ceccarelli, 1988). Agents or conditions that stimulate secretion and interfere with recycling include electric stimulation of motor nerves in a Drosophila mutant with defective vesicle recycling (Koenig et al 1983(Koenig et al , 1989, solutions with 0-05-041 mM-ouabain (Haimann et al 1985), K+-rich, ClP-deficient solutions (Gennaro, Nastuk, & Rutherford, 1978;Ceccarelli, Molenaar, Oen, Polak, Torri-Tarelli & van Kempen, 1989), high doses of a-LTX applied at 1-3 0C , and low doses of BWSV or a-LTX applied at room temperature in Ca2+-free solutions with 4 mmMg2+ (Ceccarelli & Hurlbut, 1980;Fesce et al 1986a;Valtorta et al 1988).…”