Syntaxin 4 is concentrated on plasma membrane of astrocytes.

Syntaxins are a family of transmembrane proteins that participate in SNARE complexes to mediate membrane fusion events including exocytosis. Different syntaxins are thought to participate in exocytosis in different compartments of the nervous system such as the axon, the soma/dendrites or astrocytes. It is well known that exocytosis of synaptic vesicles at axonal presynaptic terminals involves syntaxin 1 but distributions of syntaxins on neuronal somal and dendritic, postsynaptic or astroglial plasma membranes are less well characterized. Here, we use pre-embedding immunogold labeling to compare the distribution of two plasma membrane-enriched syntaxins (1 and 4) in dissociated rat hippocampal cultures as well as in perfusion-fixed mouse brains. Comparison of Western blots of neuronal cultures, consisting of a mixture of hippocampal neurons and glia, with glial cultures, consisting of mostly astrocytes, shows that syntaxin 1 is enriched in neuronal cultures, whereas syntaxin 4 is enriched in glial cultures. Electron microscopy (EM)-immunogold labeling shows that syntaxin 1 is most abundant at the plasma membranes of axons and terminals, while syntaxin 4 is most abundant at astroglial plasma membranes. This differential distribution was evident even at close appositions of membranes at synapses, where syntaxin 1 was localized to the plasma membrane of the presynaptic terminal, including that at the active zone, while syntaxin 4 was localized to nearby peri-synaptic astroglial processes. These results show that syntaxin 4 is available to support exocytosis in astroglia.

Homer is concentrated at the postsynaptic density and does not redistribute after acute synaptic stimulation.

Homer is a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity, calcium signaling and neurological disorders. Here, we use pre-embedding immunogold electron microscopy to illustrate the differential localization of three Homer gene products (Homer 1, 2, and 3) in different regions of the mouse brain. In cross-sectioned PSDs, Homer occupies a layer ∼30-100nm from the postsynaptic membrane lying just beyond the dense material that defines the PSD core (∼30-nm-thick). Homer is evenly distributed within the PSD area along the lateral axis, but not at the peri-PSD locations within 60nm from the edge of the PSD, where type I-metabotropic glutamate receptors (mGluR1 and 5) are concentrated. This distribution of Homer matches that of Shank, another major PSD scaffold protein, but differs from those of other two major binding partners of Homer, type I mGluR and IP3 receptors. Many PSD proteins rapidly redistribute upon acute (2min) stimulation. To determine whether Homer distribution is affected by acute stimulation, we examined its distribution in dissociated hippocampal cultures under different conditions. Both the pattern and density of label for Homer 1, the isoform that is ubiquitous in hippocampus, remained unchanged under high K(+) depolarization (90mM for 2-5min), N-methyl-d-asparic acid (NMDA) treatment (50µM for 2min), and calcium-free conditions (EGTA at 1mM for 2min). In contrast, Shank and calcium/calmodulin-dependent kinase II (CaMKII) accumulate at the PSD upon NMDA treatment, and CaMKII is excluded from the PSD complex under low calcium conditions.

Effects of CaMKII inhibitor tatCN21 on activity-dependent redistribution of CaMKII in hippocampal neurons.

TatCN21 is a membrane permeable calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor derived from the inhibitor protein CaMKIIN. TatCN21 has been used to demonstrate the involvement of CaMKII in a variety of physiological and pathological phenomena, and it also limits excitotoxic damage in neurons. Here we use preembedding immunogold electron microscopy to examine the effect of tatCN21 on the redistribution of CaMKII in cultured hippocampal neurons. Incubation of cultures with tatCN21 (20 µM for 20 min) prior to exposure to N-methyl-d-asparic acid (NMDA) (50 µM for 2 min) inhibited both the accumulation of CaMKII at postsynaptic densities (PSDs) and CaMKII clustering in the dendrites. Under these conditions, CaMKII also formed morphologically distinct aggregates with polyribosomes near the PSD and in dendrites. Formation of these CaMKII-polyribosome aggregates requires the presence of both tatCN21 and calcium, and was augmented upon exposure to high K(+) or NMDA. CaMKII-polyribosome aggregates formed consistently with 20 µM tatCN21, but minimally or not at all with 5 µM. However, these aggregates are not induced by another CaMKII inhibitor, KN93. Formation of CaMKII-polyribosome aggregates was completely reversible within 1h after washout of tatCN21. Effects of tatCN21 were largely restricted to dendrites, with minimal effect in the soma. The effects of tatCN21 on CaMKII distribution can be used to dissect the mechanism of CaMKII involvement in cellular events.

SynGAP moves out of the core of the postsynaptic density upon depolarization.

SynGAP is a Ras GTPase activating protein present at the postsynaptic density (PSD) in quantities matching those of the core scaffold protein PSD-95. SynGAP is reported to inhibit synaptic accumulation of AMPA receptors. Here, we characterize by immunogold electron microscopy the distribution of SynGAP at the PSD under basal and depolarizing conditions in rat hippocampal neuronal cultures. The PSD core, extending up to 40 nm from the postsynaptic membrane, typically shows label for SynGAP, while half of the synapses exhibit additional labeling in a zone 40-120 nm from the postsynaptic membrane. Upon depolarization with high K(+), labeling for SynGAP significantly decreases at the core of the PSD and concomitantly increases at the 40-120 nm zone. Under the same depolarization conditions, label for PSD-95, the presumed binding partner of SynGAP, does not change its localization at the PSD. Depolarization-induced redistribution of SynGAP is reversible and also occurs upon application of N-methyl-d-aspartic acid (NMDA). Activity-induced movement of SynGAP could vacate sites in the PSD core allowing other elements to bind to these sites, such as transmembrane AMPA receptor regulatory proteins (TARPs), and simultaneously facilitate access of SynGAP to CaMKII and Ras, elements of a regulatory cascade.

Rapid turnover of spinules at synaptic terminals.

Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7-day-old rats, 10-14 days in culture) were exposed to high K+ for 0.5-5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with approximately 1 min treatment at 37 degrees C, decreased with prolonged treatment, and disappeared after 1-2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 degrees C. N-methyl-D-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but to a lesser extent than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4 degrees C, extremely rare in slices allowed to recover at 28 degrees C, but frequent after high K(+) depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.

Inhibition of phosphatase activity facilitates the formation and maintenance of NMDA-induced calcium/calmodulin-dependent protein kinase II clusters in hippocampal neurons.

The majority of hippocampal neurons in dissociated cultures and in intact brain exhibit clustering of calcium/calmodulin-dependent protein kinase II (CaMKII) into spherical structures with an average diameter of 110 nm when subjected to conditions that mimic ischemia and excitotoxicity [Neuroscience 106 (2001) 69]. Because clustering of CaMKII would reduce its effective concentration within the neuron, it may represent a cellular strategy to prevent excessive CaMKII-mediated phosphorylation during episodes of Ca2+ overload. Here we employ a relatively mild excitatory stimulus to promote sub-maximal clustering for the purpose of studying the conditions for the formation and disappearance of CaMKII clusters. Treatment with 30 microM N-methyl-D-aspartic acid (NMDA) for 2 min produced CaMKII clustering in approximately 15% of dissociated hippocampal neurons in culture, as observed by pre-embedding immunogold electron microscopy. These CaMKII clusters could be labeled with antibodies specific to the phospho form (Thr286) of CaMKII, suggesting that at least some of the CaMKII molecules in clusters are autophosphorylated. To test whether phosphorylation is involved in the formation and maintenance of CaMKII clusters, the phosphatase inhibitors calyculin A (5 nM) or okadaic acid (1 microM) were included in the incubation medium. With inhibitors more neurons exhibited CaMKII clusters in response to 2 min NMDA treatment. Furthermore, 5 min after the removal of NMDA and Ca2+, CaMKII clusters remained and could still be labeled with the phospho-specific antibody. In contrast, in the absence of phosphatase inhibitors, no clusters were detected 5 min after the removal of NMDA and Ca2+ from the medium. These results suggest that phosphatases type 1 and/or 2A regulate the formation and disappearance of CaMKII clusters.

Calcium/calmodulin-dependent protein kinase II clusters in adult rat hippocampal slices.

We have previously reported the formation of calcium/calmodulin-dependent protein kinase II (CaMKII) clusters approximately 110 nm in diameter in hippocampal neurons in culture and in the intact adult brain, under conditions that simulate ischemic stress and increase [Ca(2+)](i) [Dosemeci et al. (2000) J. Neurosci. 20, 3076-3084; Tao-Cheng et al. (2001) Neuroscience 106, 69-78]. These observations suggest that ischemia-like conditions that prevail during the dissection of brain tissue for the preparation of hippocampal slices could lead to the formation of CaMKII clusters. We now show by pre-embedding immuno-electron microscopy that, indeed, CaMKII clusters are present in the CA1 pyramidal neurons in hippocampal slices from adult rats fixed immediately after dissection, and that the number of CaMKII clusters increases with the delay time between decapitation and fixation. Moreover, CaMKII clusters are typically localized near the endoplasmic reticulum. When acute slices are allowed to recover in oxygenated medium for 2 h, CaMKII clusters mostly disappear, indicating that clustering is reversible. Also, the postsynaptic density, another site for CaMKII accumulation under excitatory conditions, becomes thinner upon recovery. Treatment of recovered slices with high potassium for 90 s causes the re-appearance of CaMKII clusters in nearly all CA1 pyramidal cells examined. On the other hand, when dissociated hippocampal neurons in primary culture are exposed to the same depolarizing conditions, only approximately 25% of neurons exhibit CaMKII clusters, indicating a difference in the susceptibility of the neurons in culture and in acute slices to excitatory stimuli. Altogether these observations indicate that the effect of CaMKII clustering should be considered when interpreting experimental results obtained with hippocampal slices.

Inhibition of phosphatase activity prolongs NMDA-induced modification of the postsynaptic density.

NMDA-induced modification of postsynaptic densities (PSDs) was studied by immunoelectron microscopy. Treatment of cultured hippocampal neurons with NMDA for 2 min promotes a 2.3 fold thickening of the PSD and a 4 fold increase in PSD-associated CaMKII immunolabel. These changes are reversed 5 min after the removal of NMDA and Ca2+ from the medium. In addition, following NMDA treatment, PSDs exhibit a 7.5 fold increase in labeling with an antibody specific to the (Thr286) phospho-form of CaMKII, indicating that CaMKII translocated to the PSD is phosphorylated. When the phosphatase inhibitors, calyculin A or okadaic acid, are included in the medium, the NMDA-induced thickening of the PSD as well as the increase in PSD-associated CaMKII immunolabeling are largely maintained (75% and 88% of the peak values respectively) at 5 min after removal of NMDA and Ca2+ from the medium. These results imply that NMDA receptors can mediate activity-induced changes in the PSD and that phosphatases of type 1 and/or 2A are involved in the reversal of these changes.

Sustained elevation of calcium induces Ca(2+)/calmodulin-dependent protein kinase II clusters in hippocampal neurons.

Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-D-aspartate (250 microM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca(2+). Indeed, intracellular Ca(2+) monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca(2+). Moreover, CaMKII does not cluster when the rise in intracellular Ca(2+) is prevented by the omission of extracellular Ca(2+) during CCCP treatment, confirming that clustering is Ca(2+)-dependent. A lag period of 1-2 min between the onset of high intracellular Ca(2+) levels and the formation of CaMKII clusters suggests that a sustained increase in Ca(2+) level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca(2+) overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.

Glutamate-induced transient modification of the postsynaptic density.

Depolarization of rat hippocampal neurons with a high concentration of external potassium induces a thickening of postsynaptic densities (PSDs) within 1.5-3 min. After high-potassium treatment, PSDs thicken 2.1-fold in cultured neurons and 1.4-fold in hippocampal slices compared with their respective controls. Thin-section immunoelectron microscopy of hippocampal cultures indicates that at least part of the observed thickening of PSDs can be accounted for by an accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) on their cytoplasmic faces. Indeed, PSD-associated gold label for CaMKII increases 5-fold after depolarization with potassium. The effects of high-potassium treatment on the composition and structure of the PSDs are mimicked by direct application of glutamate. In cultures, glutamate-induced thickening of PSDs and the accumulation of CaMKII on PSDs are reversed within 5 min of removal of glutamate and Ca(2+) from the extracellular medium. These results suggest that PSDs are dynamic structures whose thickness and composition are subject to rapid and transient changes during synaptic activity.

Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium.

BACKGROUND: Many bacteria swim by rotating helical flagellar filaments. Waterbury et al. discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces. The hypothesis that Synechococcus might swim using traveling surface waves prompted this investigation. RESULTS: Using quick-freeze electron microscopy, we have identified a crystalline surface layer that encloses the outer membrane of the motile strain Synechococcus sp. WH8113, the components of which are arranged in a rhomboid lattice. Spicules emerge in profusion from the layer and extend up to 150 nm into the surrounding fluid. These spicules also send extensions inwards to the inner cell membrane where motility is powered by an ion-motive force. CONCLUSION: The envelope structure of Synechococcus sp. WH8113 provides new constraints on its motile mechanism. The spicules are well positioned to transduce energy at the cell membrane into mechanical work at the cell surface. One model is that an unidentified motor embedded in the cell membrane utilizes the spicules as oars to generate a traveling wave external to the surface layer in the manner of ciliated eukaryotes.

Activation of calpain may alter the postsynaptic density structure and modulate anchoring of NMDA receptors.

Elevation of calcium during sustained synaptic activity may lead to the activation of the postsynaptic calcium-dependent protease calpain and thus could alter the integrity and localization of endogenous proteins. The distribution of anchoring proteins for neuroreceptors is an important determinant of the efficacy of neuronal transmission. Many of these anchoring proteins are concentrated within the postsynaptic density (PSD). In the present study, we examined the effects of calpain II on isolated PSDs using biochemical and electron microscopic techniques. Biochemical analysis reveals that PSD-95, a clustering molecule which anchors NMDA receptors by interaction with their NR2 subunits, as well as the NR2 subunits themselves, are cleaved by calpain. On the other hand, under conditions where all the PSD-95 protein is cleaved, actin and alpha-actinin-a protein thought to anchor NMDA receptors to actin filaments-remain intact. For analysis by electron microscopy, PSDs were adsorbed on glass, immunogold-labeled with an antibody to PSD-95, slam frozen, freeze dried, and rotary shadowed. Electron micrographs of replicas indicate that PSDs are disc-shaped and are composed of a lattice-like structure which labels with PSD-95 immunogold. After calpain treatment, PSDs adsorbed on glass become thinner overall and the lattice becomes fragmented. Altogether, these results suggest that calpain activity could produce changes in the organization of the PSD and, by cleaving PSD-95 associated with the PSD lattice, could modify the anchoring of NMDA receptors.

Retrograde axonal transport of herpes simplex virus: evidence for a single mechanism and a role for tegument.

Herpes simplex virus type I (HSV) typically enters peripheral nerve terminals and then travels back along the nerve to reach the neuronal cell body, where it replicates or enters latency. To monitor axoplasmic transport of HSV, we used the giant axon of the squid, Loligo pealei, a well known system for the study of axoplasmic transport. To deliver HSV into the axoplasm, viral particles stripped of their envelopes by detergent were injected into the giant axon, thereby bypassing the infective process. Labeling the viral tegument protein, VP16, with green fluorescent protein allowed viral particles moving inside the axon to be imaged by confocal microscopy. Viral particles moved 2.2 +/- 0.26 micrometer/sec in the retrograde direction, a rate comparable to that of the transport of endogenous organelles and of virus in mammalian neurons in culture. Electron microscopy confirmed that 96% of motile (stripped) viral particles had lost their envelope but retained tegument, and Western blot analysis revealed that these particles had retained protein from capsid but not envelope. We conclude that (i) HSV recruits the squid retrograde transport machinery; (ii) viral tegument and capsid but not envelope are sufficient for this recruitment; and (iii) the giant axon of the squid provides a unique system to dissect the viral components required for transport and to identify the cellular transport mechanisms they recruit.

A novel particulate form of Ca(2+)/calmodulin-dependent [correction of Ca(2+)/CaMKII-dependent] protein kinase II in neurons.

Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of approximately 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 detergent N-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 Ca(2+)-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 Ca(2+) homeostasis, without compromising calmodulin regulation.

Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation.

Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (approximately 10-130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores, a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA, lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pull membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing.

Association of actin filaments with axonal microtubule tracts.

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.

Localization of the linker domain of Ca2+/calmodulin-dependent protein kinase II.

Electron micrographs of rotary shadowed replicas of alpha-Ca2+/calmodulin-dependent protein kinase II reveal a flower-shaped multimeric molecule with a central particle surrounded by 8-10 smaller peripheral particles. Peripheral particles are attached to the central particle by thin arms or "linkers." Movement of peripheral particles to contact each other for autophosphorylation is likely to involve these linkers. It has generally been accepted that the segment 317-328 of the alpha-subunit constitutes the linker domain. In the present study we test this assumption by generating a mutant lacking the proposed sequence. The mutant has biochemical and morphological properties similar to those of the wild type, and a thin linker is occasionally observed in replicas from either type. The results indicate that the deleted sequence does not correspond to the linker domain. This conclusion, combined with observations from two recent studies which identify the C-terminal domain involved in oligomerization, narrows down the location of the linker domain within the sequence 330-354.

Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons.

Transient changes in the intracellular concentration of free calcium ([Ca2+])i) act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand- or Ca2+(-)gated release from intracellular stores. Characterizing the sources and spatio-temporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of fluorescent indicators sensitive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendritic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative information on the amount and location of intracellular total, i.e., free plus bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity state of the neuron. When optical measurements of [Ca2+]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively general picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here results from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and termination of [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx.

Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon.

A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm/day, whereas neurofilament was moved at 1.1 mm/day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to approximately 20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 micrometer(2)/s compared with only 0.43 micrometer(2)/s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole/cold-sensitive transport mechanism.