How is the cytoplasmic calcium concentration controlled in nerve terminals?

1. The ability of intraterminal organelles to sequester calcium and buffer the cytoplasmic free Ca2+ concentration ([Ca2+]i) has been investigated in isolated mammalian presynaptic nerve terminals (synaptosomes). A combination of biochemical and morphological methods has been used. 2. When the plasmalemma of synaptosomes is disrupted by osmotic shock or saponin, Ca from the medium can be sequestered by two types of intraterminal organelles in the presence of ATP. 2. Typical mitochondrial poisons (e.g., oligomycin, azide and 2,4-dinitrophenol) block the Ca uptake into one type of organelle (mitochondria); the second type of organelle, which has a higher affinity for Ca (half-saturation congruent to 0.35 microM Ca2+) is spared by the mitochondrial poisons. 4. When the "leaky" synaptosomes are incubated in media containing oxalate, and then fixed and prepared for electron microscopy, electron-dense deposits are observed in the intraterminal mitochondria and smooth endoplasmic reticulum (SER). Mitochondrial poisons block the formation of the deposits in the mitochondria, but spare the SER. 5. X-ray microprobe analysis demonstrates that these deposits contain Ca. 6. Experiments with the Ca-sensitive metallochromic indicator, arsenazo III, demonstrate that the intraterminal organelles in the "leaky" synaptosomes can buffer Ca2+ in the medium to below 5 X 10(-7) M. With small (physiological) Ca loads, the Ca2+ is effectively buffered (to < 5 X 10(-7) M) even in the presence of mitochondrial poisons. 7. The data indicate that the SER in presynaptic terminals may play an important role in helping to buffer the Ca that normally enters during neuronal activity.

Probing for calcium at presynaptic nerve terminals.

The nonmitochondrial ATP-dependent calcium sequestration site within "pinched-off" presynaptic nerve terminals (synaptosomes) was localized by morphological techniques. The terminals contain mitochondria, smooth endoplasmic reticulum (SER), synaptic vesicles, and occasional coated vesicles. Three-dimensional reconstructions of serial sections of synaptosomes reveal that the SER consists, in part, of flattened sacs or cisterns often situated adjacent to mitochondria. Synaptosomes with leaky plasma membranes (induced by saponin treatment) were incubated in physiological salt solutions containing Ca, ATP, and oxalate to promote Ca sequestration. After incubation in these solutions, synaptosomes contained electron-dense deposits, presumably calcium oxalate, localized within mitochondria, SER cisterns, and vesicular profiles. Electron probe microanalyses of these electron-dense deposits confirmed the presence of calcium. When mitochondrial poisons were included in the incubation media, electron-dense deposits were still observed in the SER; however, under these conditions, mitochondria very rarely contained such deposits. When A23187 or EGTA was included in the incubation solutions, electron-dense deposits rarely were observed in any organelles within the terminals. When oxalate was omitted from the incubation media, no electron-dense deposits were found in the synaptosomes. These results show that the nerve terminal SER is capable of sequestering Ca. The data are consistent with biochemical and physiological evidence that the SER plays a significant role in intraterminal Ca buffering during neuronal activity.

Localization of calcium in presynaptic nerve terminals. An ultrastructural and electron microprobe analysis.

Ultrastructural techniques and electron probe microanalysis were used to determine whether or not the smooth endoplasmic reticulum (SER) within presynaptic nerve terminals is a Ca-sequestering site. The three-dimensional structure of the SER was determined from serial sections of synaptosomes. The SER consists of flattened cisterns that may branch and are frequently juxtaposed to mitochondria. To investigate intraterminal Ca sequestration, synaptosomes were treated with saponin to disrupt the plasmalemmal permeability barrier. When these synaptosomes were incubated in solutions containing Ca, ATP, and oxalate, electrondense Ca oxalate deposits were found in intraterminal mitochondria, SER cisterns, and large vesicular profiles. Saponin-treated synaptosomes that were incubated in the presence of mitochondrial poisons contained electron-dense deposits within SER cisterns and large vesicular profiles, but very rarely in mitochondria. Similar deposits were observed within saponin-treated synaptosomes that were not post-fixed with OSO4, and within saponin-treated synaptosomes that were prepared for analysis by freeze-substitution. Electron-probe microanalyses of these deposits confirmed the presence of large concentrations of Ca. When oxalate was omitted from the incubation solutions, no electron-dense deposits were present in saponin-treated synaptosomes. In other control experiments, either the Ca ionophore A23187 or the Ca chelator EGTA was added to the incubation media; electron-dense deposits were very rarely observed within the intraterminal organelles of these saponin-treated synaptosomes. The data indicate that presynaptic nerve terminal SER is indeed a Ca-sequestering organelle.

Fine structural studies of synaptogenesis in the superficial layers of the chick optic tectum.

Synaptogenesis in the superficial layers of the rostral pole of the optic tectum has been studied in the chick from embryonic day six (E6) to seven days post-hatching. Symmetrical membrane densities of puncta adhaerentia are observed prior to the detection of synapses and throughout development. Immature synaptic contacts are observed by E7. These early synapses are primarily axodendritic; however, somatodendritic, dendrodendritic, axosomatic and axoglial synapses are also observed. The majority of these synapses have asymmetrical membrane densities and the presynaptic terminals contain clear, spherical, synaptic vesicles. Synaptic terminals containing pleomorphic vesicles and making symmetrical synaptic contacts are not commonly observed until the third week of embryonic development, and may represent the onset of inhibitory function within the tectum. Comparison of the number of synapses per unit area in control versus experimental tecta, after unilateral eye enucleations of E3, indicates that the presynaptic terminals of some synapses present at E8 are of retinal origin. It is suggested that the development of retinotectal synapses follows a rostrocaudal gradient in the tectum and corresponds to the intrinsic tectal pattern of cytoarchitectonic differentiation.

A freeze-fracture study of synaptic junction development in the superficial layers of the chick optic tectum.

Synaptogenesis in the superficial layers of the rostral pole of the chick optic tectum has been studied using freeze-fracture techniques. The developmental sequence of intramembrane organization at synaptic junctions involves the accumulation and assembly of intramembrane particles into aggregates characteristic of the mature junctions. By embryonic day seven, areas of loosely-arranged clusters of medium-sized particles are observed on the cytoplasmic membrane leaflets (P-faces) of developing neurites. These clusters are characteristic of the intramembrane organization at presynaptic active zones. At later stages, small pits, characteristic of vesicle fusion sites, are observed interspersed among such P-face particle clusters. Complementary intramembrane specializations are also present on the external leaflets (E-faces) of presynaptic membranes at the active zones. Small solitary aggregates of large-sized particles on the E-faces of neurite plasma membranes are also seen at early embryonic stages. As development progresses, these aggregates increase in size and packing density and occupy large oval domains in postsynaptic membranes. These intramembrane specializations may represent the postsynaptic active zones of asymmetric synapses. Another type of intramembrane specialization, observed during the third week of incubation, is characterized by aggregates of small- and medium-sized particles on the P-face of postsynaptic membranes and is often seen directly apposed to the E-face of a presynaptic terminal. This type of intramembrane specialization may represent the postsynaptic active zone region at symmetrical synaptic contacts.