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Electron microscopic tomography is proving useful to examine the organization of protein molecules in large molecular machines at critical neural sites, such as synapses. Methods are still in a state of flux, and many steps exist between a neuron and a molecular model of one of its synapses. Here, we show our approach for doing tomography on synaptic structure, hoping thereby to shed light generally on the emerging field of EM tomography.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIalpha translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIalpha in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha accumulation at synapses is a memory trace of past synaptic activity.

Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We determined the positions of calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95 within the three-dimensional structure of isolated PSDs using immunogold labeling, rotary shadowing, and electron microscopic tomography. The results show that all PSDs contain a central mesh immediately underlying the postsynaptic membrane. Label for PSD-95 is found on both the cytoplasmic and cleft sides of this mesh, averaging 12 nm from the cleft side. All PSDs label for PSD-95. The properties of CaMKII labeling are quite different. Label is virtually absent on the cleft sides of PSDs, but can be heavy on the cytoplasmic side at a mean distance of 25 nm from the cleft. In tomograms, CaMKII holoenzymes can be visualized directly, appearing as labeled, tower-like structures reflecting the 20 nm diameter of the holoenzyme. These towers protrude from the cytoplasmic side of the central mesh. There appears to be a local organization of CaMKII, as judged by fact that the nearest-neighbor distances are nearly invariant over a wide range of labeling density for CaMKII. The average density of CaMKII holoenzymes is highly variable, ranging from zero to values approaching a tightly packed state. This variability is significantly higher than that for PSD-95 and is consistent with an information storage role for CaMKII.

Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca2+/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of ∼100 nm and, in distinction to postsynaptic densities, do not immunolabel for PSD-95. These structures were purified to near homogeneity by extraction with the detergentN-lauryl sarcosinate. Biochemical analysis revealed that CaMKII accounts for virtually all of the protein in the purified preparation, suggesting that spherical structures are clusters of self-associated CaMKII. Exposure of cultured hippocampal neurons to a mitochondrial uncoupler in glucose-free medium promotes the formation of numerous CaMKII-immunopositive structures identical in size and shape to the CaMKII clusters observed in subcellular fractions. Clustering of CaMKII would reduce its kinase function by preventing its access to fixed substrates. On the other hand, clustering would not affect the ability of the large cellular pool of CaMKII to act as a calmodulin sink, as demonstrated by the Ca2+-dependent binding of gold-conjugated calmodulin to CaMKII clusters. We propose that the observed clustering of CaMKII into spherical structures is a protective mechanism preventing excessive protein phosphorylation upon loss of Ca2+homeostasis, without compromising calmodulin regulation.

Synaptic activity-dependent changes in the spatio-temporal distribution of calcium ions regulate important neuronal functions such as dendritic integration and synaptic plasticity, but the processes that terminate the free Ca2+transients associated with these changes remain unclear. We have characterized at the electron microscopic level the intracellular compartments involved in buffering free Ca2+transients in dendritic cytoplasm of CA3 neurons by measuring the larger changes in the concentrations of total Ca that persist for several minutes after neuronal activity. Quantitative energy-dispersive x-ray microanalysis of cryosections from hippocampal slice cultures rapidly frozen 3 min after afferent synaptic activity identified a subset of dendritic endoplasmic reticulum (ER) as a high-capacity Ca2+buffer. Calcium sequestration by cisterns of this subset of ER was graded, reversible, and dependent on a thapsigargin-sensitive Ca2+-ATPase. Sequestration was so robust that after repetitive high-frequency stimulation the Ca content of responsive ER cisterns increased as much as 20-fold. These results demonstrate that a subpopulation of ER is the major dendritic Ca sequestration compartment in the minutes after neuronal activity.

Previous work suggests that organelles contacting microtubules in axons are in fast transport. Here, we examine the distribution of organelles contacting microtubules in growing axons and growth cones from chick optic tectum. Five axon segments, each 10 microns long, and 4 entire growth cones were reconstructed from serial electron micrographs of quick-frozen, freeze-substituted chick optic tectum. Organelles contacting microtubules in axons are evenly distributed along all microtubules. Smaller organelles, presumably in anterograde transport, are enclosed in fascicles of microtubules, while larger organelles in retrograde transport lie outside the fascicles. In contrast, organelles contacting microtubules are prevalent only in the most proximal parts of the growth cone, before the microtubule fascicles splay out more distally. The distance between noncontacting organelles and microtubules also becomes progressively greater, reaching a maximum in the mid- and more distal region of the growth cone. Contacts with microtubules of both the smaller, presumably anterogradely transported organelles, as well as the larger, presumably retrogradely transported organelles, abruptly become less frequent in the proximal midregion of the growth cone. It is therefore of possible significance in stopping and starting microtubule-based organelle transport that microtubules change from a straight to an undulating configuration in the midregion of the growth cone. The decrease in organelle binding to microtubules at the demarcations between the straight and undulating microtubule segments may depend on proteins or other local factors as well as the splaying out of the microtubule bundles.

The freeze-fracture technique was used to study differences in membrane structure which could explain differences in the number of quanta released from axon terminals on twitch and tonic muscle fibers in Anolis intercostal muscles. The protoplasmic leaflets of axon terminals facing lizard twitch muscle fibers have intramembrane particle specializations characterized by two parallel linear particle arrays each composed of two particle rows which lie perpendicular to the axis of shallow ridges in the axolemma. During K+ depolarization, vesicles open between the arrays, confirming that these structures are the active zones for synaptic vesicle opening. Active zones at axon terminals on tonic fibers are defined by one linear particle array composed of two parallel particle rows oriented along the axis of a shallow presynaptic ridge; vesicles open beside these arrays. Thus, there are more particles near active zone vesicles in terminals on twitch fibers. Even though terminals on twitch and tonic muscle fibers seem to have similar numbers of synaptic vesicles associated with their active zones, a presynaptic action potential is reported to release at least 10 times more quanta from terminals on twitch fibers. We postulate that the differences in quantal output are related to the observed differences in the number of active zone particles flanking synaptic vesicles at the active zone. Indeed, the correlation between the distribution of these particles and the level of transmitter release provides additional support for the idea that they are the calcium channels which couple transmitter release to the action potential.

Interactions between synaptic vesicles and the presynaptic membrane which accompany transmitter release were examined at excitatory, cholinergic synapses in bullfrog sympathetic ganglia. Ganglia were fixed at rest or during electrical stimulation of the preganglionic axons and then were either thin-sectioned or freeze-fractured. Release of transmitter for brief periods is accompanied by selective depletion of four-fifths of the synaptic vesicles aligned at active zones, an overall loss of half of the synaptic vesicles in the terminals, and synaptic vesicle openings at active zones. These findings are consistent with the hypotheses that synaptic vesicles which are ready to be released are aligned at active zones and that these vesicles fuse with and add their membrane to the presynaptic membrane as they release transmitter. Larger vesicles with dense cores also contact and open onto the presynaptic membrane at the active zone, appearing to release their contents by exocytosis. The arrangement of intramembrane particles at fractured postsynaptic specializations resembles that at other excitatory, cholinergic synapses.