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.

Activity induced changes in the distribution of Shanks at hippocampal synapses.

Dendritic spines contain a family of abundant scaffolding proteins known as Shanks, but little is known about how their distributions might change during synaptic activity. Here, pre-embedding immunogold electron microscopy is used to localize Shanks in synapses from cultured hippocampal neurons. We find that Shanks are preferentially located at postsynaptic densities (PSDs) as well as in a filamentous network near the PSD, extending up to 120 nm from the postsynaptic membrane. Application of sub-type specific antibodies shows that Shank2 is typically concentrated at and near PSDs while Shank1 is, in addition, distributed throughout the spine head. Depolarization with high K+ for 2 min causes transient, reversible translocation of Shanks towards the PSD that is dependent on extracellular Ca2+. The amount of activity-induced redistribution and subsequent recovery is pronounced for Shank1 but less so for Shank2. Thus, Shank1 appears to be a dynamic element within the spine, whose translocation could be involved in activity-induced, transient structural changes, while Shank2 appears to be a more stable element positioned at the interface of the PSD with the spine cytoplasm.

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.

Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate.

Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.

Activity-related redistribution of presynaptic proteins at the active zone.

Immunogold labeling distributions of seven presynaptic proteins were quantitatively analyzed under control conditions and after high K+ depolarization in excitatory synapses from dissociated rat hippocampal cultures. Three parallel zones in presynaptic terminals were sampled: zones I and II, each about one synaptic vesicle wide extending from the active zone; and zone III, containing a distal pool of vesicles up to 200 nm from the presynaptic membrane. The distributions of SV2 and synaptophysin, two synaptic vesicle integral membrane proteins, generally followed the distribution of synaptic vesicles, which were typically evenly distributed under control conditions and had a notable depletion in zone III after stimulation. Labels of synapsin I and synuclein, two synaptic vesicle-associated proteins, were similar to each other; both were particularly sparse in zone I under control conditions but showed a prominent enrichment toward the active zone, after stimulation. Labels of Bassoon, Piccolo and RIM 1, three active zone proteins, had very different distribution profiles from one another under control conditions. Bassoon was enriched in zone II, Piccolo and RIM 1 in zone I. After stimulation, Bassoon and Piccolo remained relatively unchanged, but RIM 1 redistributed with a significant decrease in zone I, and increases in zones II and III. These results demonstrate that Bassoon and Piccolo are stable components of the active zone while RIM 1, synapsin I and synuclein undergo dynamic redistribution with 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.

Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis.

We present evidence that regulation of dense-core secretory granule biogenesis and hormone secretion in endocrine cells is dependent on chromogranin A (CGA). Downregulation of CGA expression in a neuroendocrine cell line, PC12, by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected prohormone, and reduction of secretory granule proteins. Transfection of bovine CGA into a CGA-deficient PC12 clone rescued the regulated secretory phenotype. Stable transfection of CGA into a CGA-deficient pituitary cell line, 6T3, lacking a regulated secretory pathway, restored regulated secretion. Overexpression of CGA induced dense-core granules, immunoreactive for CGA, in nonendocrine fibroblast CV-1 cells. We conclude that CGA is an "on/off" switch that alone is sufficient to drive dense-core secretory granule biogenesis and hormone sequestration in endocrine cells.

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.

Snap-25 is polarized to axons and abundant along the axolemma: an immunogold study of intact neurons.

SNAP-25, synaptosomal associated protein of 25 kDa, is reported to be a t-SNARE (target receptor associated with the presynaptic plasma membrane) involved in the docking and fusion of synaptic vesicles. We present here the first ultrastructural localization of SNAP-25 in intact neurons by pre-embedding EM immunocytochemistry in rat brains, hippocampal slice cultures, and PC12 cells. In differentiated neurons, SNAP-25 labeling was clearly membrane-associated. The labeling was most prominent in the plasma membrane of axons and excluded from the plasma membranes of soma and dendrites. Furthermore, SNAP-25 did not appear to be restricted to the synaptic junctions. SNAP-25 labeling was seen in the cytoplasm of the soma and large dendrites, mostly associated with the Golgi complexes. There were also some SNAP-25 labeled tubulo-vesicular structures in the cytoplasm of the soma and the axons, but rarely in the smaller dendrites. In PC12 cells, after 5-10 minutes of high potassium (75 mM) stimulation in the presence of HRP, SNAP-25 labeling appeared, additionally, on HRP-filled early endosomes. After a longer (20-30 minutes) HRP incubation, most of the later stage endosomes and lysosomes were loaded with HRP but they were negative for SNAP-25. These results suggest that SNAP-25 is sorted out of these late endosomal compartments, and that the bulk of the SNAP-25 protein is probably recycled back to the axolemma from the early endosomes. In contrast, in those samples which were incubated with HRP for longer periods, there were still some SNAP-25-positive vesicular structures which were HRP-negative. These structures most likely represent anterograde vesicles that carry newly synthesized SNAP-25 from the soma to the axolemma by axonal transport. SNAP-25 appears to be sorted at the Golgi complex to reach the axolemma specifically. Its widespread distribution all along the axolemma does not support the view of SNAP-25 as a t-SNARE limited for synaptic exocytosis.

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.

Differential polarization of serotonin transporters in axons versus soma-dendrites: an immunogold electron microscopy study.

In spite of the conventional belief that neurotransmitter uptake occurs at the synapses, we demonstrated previously that serotonin transporters and the high-affinity uptake of serotonin were not confined to the terminals but rather occurred throughout the axons [Zhou F. C. et al. (1998) Brain Res. 805, 241-254]. In the present study, the detailed distribution of serotonin transporters over various parts of the neuron was illustrated and analysed morphometrically using a pre-embedding immunogold method with a characterized serotonin transporter antibody at the electron microscopic level. Our findings reveal a highly polarized distribution of serotonin transporters between axons and soma-dendrites in two aspects. (1) On the plasma membrane, serotonin transporter-immunogold is extremely low on soma-dendrites and synaptic junctions, but consistently dense along the axons and perisynaptic area. (2) In contrast, serotonin transporter labeling in the cytoplasm is concentrated in soma and dendrites, particularly on the membranes of rough endoplasmic reticulum, Golgi complexes and tubulovesicular structures, but low in the axoplasm. The extensive distribution of serotonin transporter along the axolemma suggests a broad range of uptake sites beyond synaptic junctions, and is consistent with the notion that the major mode of transmission for serotonin neurons is through volume (extrasynaptic) transmission. The highly polarized distribution also indicates that the major serotonin uptake sites are on axons and not on soma-dendrites.

Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice.

Fabry disease is an X-linked metabolic disorder caused by a deficiency of alpha-galactosidase A (alpha-Gal A). The enzyme defect leads to the systemic accumulation of glycosphingolipids with alpha-galactosyl moieties consisting predominantly of globotriaosylceramide (Gb3). In patients with this disorder, glycolipid deposition in endothelial cells leads to renal failure and cardiac and cerebrovascular disease. Recently, we generated alpha-Gal A gene knockout mouse lines and described the phenotype of 10-week-old mice. In the present study, we characterize the progression of the disease with aging and explore the effects of bone marrow transplantation (BMT) on the phenotype. Histopathological analysis of alpha-Gal A -/0 mice revealed subclinical lesions in the Kupffer cells in the liver and macrophages in the skin with no gross lesions in the endothelial cells. Gb3 accumulation and pathological lesions in the affected organs increased with age. Treatment with BMT from the wild-type mice resulted in the clearance of accumulated Gb3 in the liver, spleen, and heart with concomitant elevation of alpha-Gal A activity. These findings suggest that BMT may have a potential role in the management of patients with Fabry disease.

Serotonin transporters are located on the axons beyond the synaptic junctions: anatomical and functional evidence.

The serotonin (5-HT) transporter (5-HTT) is known to play a role in depression and many 5-HT related diseases, and is the target site for drugs of abuse, such as cocaine, MDMA, and methamphetamine. The major role of the 5-HTT has long been considered to be to inactivate serotonin transmission through the elimination of serotonin at release sites. However, immunocytochemistry using an antibody against the N-terminal of the 5-HTT at the light microscopic (LM) level indicates that the 5-HTT is associated not only with 5-HT varicosities but also with axons. Electron microscopy (EM) reveals that the majority of the 5-HTTs exist on the axolemma outside the synaptic junctions. In studying whether axonal 5-HTTs are involved in the uptake of 5-HT, we found with autoradiography that [3H]citalopram bound to all major 5-HT fibers, not only in the terminal regions, but also in 5-HT axonal bundles such as the cingulum bundle and medial forebrain bundle. Furthermore, voltammetry recordings indicated that serotonin axonal bundles were actively engaged in high affinity serotonin uptake. The evidence indicates that 5-HTTs on 5-HT axons away from the synapse are likely to be functional in a manner similar to the terminal 5-HTT for serotonin uptake. It also suggests that the role of the 5-HTT may not only be for the termination of synaptic transmission, but also for the regulation of 5-HT through extrasynaptic (volume) transmission. Our findings may also impact the understanding of the sites of action of selective serotonin reuptake inhibitors and drug entry into serotonin neurons via the numerous axonal sites.

The K+ channel, Kv2.1, is apposed to astrocytic processes and is associated with inhibitory postsynaptic membranes in hippocampal and cortical principal neurons and inhibitory interneurons.

A variety of voltage-gated ion channels are expressed on principal cell dendrites and have been proposed to play a pivotal role in the regulation of dendritic excitability. Previous studies at the light microscopic level demonstrated that the K+ channel subunit Kv2.1 expression was polarized to the cell soma and dendrites of principal neurons throughout the central nervous system. Here, using double immunostaining we now show that Kv2.1 protein is similarly expressed in the majority of cortical and hippocampal parvalbumin, calbindin and somatostatin-containing inhibitory interneurons. At the electron microscopic level Kv2.1 immunoreactivity was primarily observed on the plasma membrane of the somata and proximal dendrites of both principal neurons and inhibitory interneurons; expression was low on smaller dendritic branches, and absent on axons and presynaptic terminals. Kv2.1 subunit expression was highly concentrated on the cell surface membrane immediately facing astrocytic processes. Kv2.1 expression was also concentrated in specific cytoplasmic compartments and on the subsurface cisterns underlying the plasma membrane facing astrocytes. In addition, Kv2.1 subunit immunoreactivity was associated with postsynaptic densities of a fraction of inhibitory symmetric synapses; while expression at asymmetric synapses was rare. These data demonstrate that channels formed by Kv2.1 subunits are uniquely positioned on the soma and principal dendrites of both pyramidal cells and inhibitory interneurons at sites immediately adjacent to astrocytic processes. This close apposition to astrocytes will ensure a rapid removal and limit the influence of K+ released into the extracellular space. This expression pattern suggests that channels formed by Kv2.1 are poised to provide a role in the regulation of neuronal dendritic excitability.