High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation.

A longstanding limitation of imaging with serial block-face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block-face due to image jitter. Typically, variable-pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal-to-noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block-face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block-face ultramicrotome. This system enables the application of nitrogen gas precisely over the block-face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high-resolution block-face imaging of even the most charge prone of epoxy-embedded biological samples.

Spiers Memorial Lecture. Breeding and building molecular spies.

To circumvent the limited spatial resolution of fluorescent protein imaging, we are developing genetically encoded tags for electron microscopy (EM).

Automated microscopy system for mosaic acquisition and processing.

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide-field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast-scanning, multiphoton, confocal microscope fitted with a motorized, high-precision stage and custom-developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.

PKA, PKC, and AKAP localization in and around the neuromuscular junction.

BACKGROUND: One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIalpha in addition to the RII subunits. RESULTS: Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIalpha at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIalpha and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIalpha and RIIbeta) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCbeta appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. CONCLUSIONS: The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIalpha and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIalpha in these regions. Likewise, gravin may be an anchor of PKCbeta at the NMJ.

Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels.

We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.

Selective localization of high concentrations of F-actin in subpopulations of dendritic spines in rat central nervous system: a three-dimensional electron microscopic study.

Dendritic spines differ considerably in their size, shape, and internal organization between brain regions. We examined the actin cytoskeleton in dendritic spines in hippocampus (areas CA1, CA3, and dentate gyrus), neostriatum, and cerebellum at both light and electron microscopic levels by using a novel high-resolution photoconversion method based in the high affinity of phalloidin for filamentous (F)-actin. In all brain regions, labeling was strongest in the heads of dendritic spines, diminishing in the spine neck. The number of labeled spines varied by region. Compared with the cerebellar molecular layer and area CA3, where nearly every dendritic spine was labeled, less than half the spines were labeled in CA1, dentate gyrus, and neostriatum. Serial section reconstructions of spines in these areas indicated that phalloidin labeling was restricted to the largest and most morphologically diverse dendritic spines. The resolution of the photoconversion technique allowed us to examine the localization and organization of actin filaments in the spine. The most intense staining for actin was found in the postsynaptic density and associated with the spines internal membrane system. In mushroom-shaped spines, F-actin staining was particularly strong between the lamellae of the spine apparatus. Three-dimensional reconstruction of labeled spines by using electron tomography showed that the labeled dense material was in continuity with the postsynaptic density. These results highlight differences in the actin cytoskeleton between different spine populations and provide novel information on the organization of the actin cytoskeleton in vivo.

A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis.

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.

Aberrant development of motor axons and neuromuscular synapses in erbB2-deficient mice.

Receptor tyrosine kinase erbB2, which is activated by neuregulin, is expressed in Schwann and muscle cells in the developing neuromuscular junction (NMJ). In vitro studies have shown that neuregulin promotes the survival and migration of Schwann cells and stimulates acetylcholine receptor gene transcription in cultured muscle cells. These findings suggest an important role for erbB2 in the development of the NMJ. Here we examine erbB2-deficient mice to determine whether erbB2 is required for NMJ development in vivo. Our analysis shows that there are pre- and postsynaptic defects of developing NMJ in erbB2-deficient embryos. The presynaptic defects include defasciculation and degeneration of the motor nerves, and an absence of Schwann cells. The postsynaptic defect features an impairment of junctional folds at the neuromuscular synapse in the mutants. These results demonstrate that erbB2 is essential for in vivo development of the NMJ.

Localization of a passively transferred human recombinant monoclonal antibody to herpes simplex virus glycoprotein D to infected nerve fibers and sensory neurons in vivo.

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea--identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase--and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.

NH2-Terminal targeting motifs direct dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum.

Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase-anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. Deletion of N0(1-30) abolished mitochondrial targeting while N0(1-30)-GFP localized to mitochondria. Residues 1-30 of N0 are therefore necessary and sufficient for mitochondria targeting. Addition of the 33 residues of N1 targets D-AKAP1 to the ER and residues 1-63 fused to GFP are necessary and sufficient for ER targeting. Residues 14-33 of N1 are especially important for targeting to ER; however, residues 1-33 alone fused to GFP gave a diffuse distribution. N1(14-33) thus serves two functions: (a) it suppresses the mitochondrial-targeting motif located within residues 1-30 of N0 and (b) it exposes an ER-targeting motif that is at least partially contained within the N0(1-30) motif. This represents the first example of a differentially targeted AKAP and adds an additional level of complexity to the PKA signaling network.

Identification and localization of Ca(2+)-activated K+ channels in rat sciatic nerve.

To understand the physiology of Schwann cells and myelinated nerve, we have been engaged in identifying K+ channels in sciatic nerve and determining their subcellular localization. In the present study, we examined the slo family of Ca(2+)-activated K+ channels, a class of channel that had not previously been identified in myelinated nerve. We have determined that these channels are indeed expressed in peripheral nerve, and have cloned rat homologues of slo that are more than 95% identical to the murine slo. We found that sciatic nerve RNA contained numerous alternatively spliced variants of the slo homologue, as has been seen in other tissues. We raised a polyclonal antibody against a peptide from the carboxyl terminal of the channels. Immunocytochemistry revealed that the channel proteins are in Schwann cells and are associated with canaliculi that run along the outer surface of the cells. They are also relatively concentrated near the node of Ranvier in the Schwann cell outer membrane. This staining pattern is quite similar to what we previously reported for the voltage-dependent K+ channel Kv 1.5. We did not observe staining of axons or connective tissue in the nerve and so it seems likely that most or all of the splicing variants are located in the Schwann cells. The localization of these channels also suggests that they may participate in maintaining the resting potential of the Schwann cells during K+ buffering.

The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis.

The IkappaB kinase (IKK) complex is composed of three subunits, IKKalpha, IKKbeta, and IKKgamma (NEMO). While IKKalpha and IKKbeta are highly similar catalytic subunits, both capable of IkappaB phosphorylation in vitro, IKKgamma is a regulatory subunit. Previous biochemical and genetic analyses have indicated that despite their similar structures and in vitro kinase activities, IKKalpha and IKKbeta have distinct functions. Surprisingly, disruption of the Ikkalpha locus did not abolish activation of IKK by proinflammatory stimuli and resulted in only a small decrease in nuclear factor (NF)-kappaB activation. Now we describe the pathophysiological consequence of disruption of the Ikkbeta locus. IKKbeta-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype that is remarkably similar to that of mice deficient in both the RelA (p65) and NF-kappaB1 (p50/p105) subunits of NF-kappaB. Accordingly, IKKbeta-deficient cells are defective in activation of IKK and NF-kappaB in response to either tumor necrosis factor alpha or interleukin 1. Thus IKKbeta, but not IKKalpha, plays the major role in IKK activation and induction of NF-kappaB activity. In the absence of IKKbeta, IKKalpha is unresponsive to IKK activators.

Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase.

The oligomeric IkappaB kinase (IKK) is composed of three polypeptides: IKKalpha and IKKbeta, the catalytic subunits, and IKKgamma, a regulatory subunit. IKKalpha and IKKbeta are similar in structure and thought to have similar function-phosphorylation of the IkappaB inhibitors in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IkappaB and activation of nuclear factor kappaB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKalpha-deficient mice. IKKalpha was not required for activation of IKK and degradation of IkappaB by proinflammatory stimuli. Instead, loss of IKKalpha interfered with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes.

The distribution of ganglioside-like moieties in peripheral nerves.

GM1 ganglioside has been implicated as a target of immune attack in some diseases of the peripheral nervous system. Anti-GM1 ganglioside antibodies are associated with certain acquired immune-mediated neuropathies. It is not clear how anti-GM1 antibodies cause nerve dysfunction and injury; however, sodium and/or potassium ion channel dysfunction at the node of Ranvier has been implicated. To gain insight into the pathogenesis of these neuropathies, we examined the distribution of GM1 ganglioside and Gal(beta1-3)GalNAc moieties in nerve fibres and their relationship to voltage-gated sodium and potassium (Kv1.1, 1.5) channels at the nodes of Ranvier in peripheral nerves from human, rat and dystrophic mice. Gal(beta1-3)GalNAc moieties were localized via the binding of cholera toxin and peanut agglutinin. As a control for the specificity of these findings, we compared the distribution of GM1 moieties to that of the ganglioside GT1b. Our study provides definitive evidence for the presence of Gal(beta1-3)GalNAc bearing moieties on the axolemmal surface of mature myelinated fibres and on Schwann cells. Gal(beta1-3)GalNAc binding sites did not have an obligatory co-localization with voltage-gated sodium channels or the potassium ion channels Kv1.1 and Kv1.5 and are thus not likely carried by these ion channels. In contrast with Gal(beta1-3)GalNAc, GT1b-like moieties are restricted to the axolemma.

The perinucleolar compartment and transcription.

The perinucleolar compartment (PNC) is a unique nuclear structure localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III and two hnRNP proteins have been localized in the PNC (Ghetti, A., S. Piñol-Roma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181- 1193; Timchenko, L.T., J.W. Miller, N.A. Timchenko, D.R. DeVore, K.V. Datar, L. Lin, R. Roberts, C.T. Caskey, and M.S. Swanson. 1996. Nucleic Acids Res. 24: 4407-4414; Huang, S., T. Deerinck, M.H. Ellisman, and D.L. Spector. 1997. J. Cell Biol. 137:965-974). In this report, we show that the PNC incorporates Br-UTP and FITC-conjugated CTP within 5 min of pulse labeling. Selective inhibition of RNA polymerase I does not appreciably affect the nucleotide incorporation in the PNC. Inhibition of all RNA polymerases by actinomycin D blocks the incorporation completely, suggesting that Br-UTP incorporation in the PNC is due to transcription by RNA polymerases II and/or III. Treatment of cells with an RNA polymerase II and III inhibitor induces a significant reorganization of the PNC. In addition, double labeling experiments showed that poly(A) RNA and some of the factors required for pre-mRNA processing were localized in the PNC in addition to being distributed in their previously characterized nucleoplasmic domains. Fluorescence recovery after photobleaching (FRAP) analysis revealed a rapid turnover of polypyrimidine tract binding protein within the PNC, demonstrating the dynamic nature of the structure. Together, these findings suggest that the PNC is a functional compartment involved in RNA metabolism in the cell nucleus.

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Electron tomography of mitochondria from brown adipocytes reveals crista junctions.

Electron microscope tomography was used to examine the membrane topology of brown adipose tissue (BAT) mitochondria prepared by cryofixation or chemical fixation techniques. These mitochondria contain an uncoupling protein which results in the conversion of energy from electron transport into heat. The three-dimensional reconstructions of BAT mitochondria provided a view of the inner mitochondrial membrane different in important features from descriptions found in the literature. The work reported here provides new insight into BAT mitochondria architecture by identifying crista junctions, including multiple junctions connecting a crista to the same side of the inner boundary membrane, in a class of mitochondria that have no tubular cristae, but only lamellar cristae. Crista junctions were defined previously as the tubular membranes of relatively uniform diameter that connect a crista membrane with the inner boundary membrane. We have also found that the cristae architecture of cryofixed mitochondria, including crista junctions, is similar to that found in chemically fixed mitochondria, suggesting that this architecture is not a fixation artifact. The stacks of lamellar cristae extended through more of the BAT mitochondrial volume than did the cristae we observed in neuronal mitochondria. Hence, the inner membrane surface area was larger in the former. In chemically fixed mitochondria, contact sites were easily visualized because the outer and inner boundary membranes were separated by an 8 nm space. However, in cryofixed mitochondria almost all the outer membrane was observed to be in close contact with the inner boundary membrane.

Myosin I is associated with zymogen granule membranes in the rat pancreatic acinar cell.

BACKGROUND & AIMS: The mechanisms whereby intracellular messengers mediate zymogen granule transport and exocytosis in the pancreatic acinar cell are not well defined. Electron microscopy has shown a periluminal network of actin in the acinar cell, suggesting a role for actin and myosin in the transport process. The possible involvement of two types of myosin in the secretory process was investigated, and their distribution in acinar cells was determined. METHODS: Antibodies specific to myosin I or to myosin II were used for immunocytochemistry and Western blot analysis. Ultrastructural studies were also performed. RESULTS: Western blot analysis showed that myosin I and myosin II were present in total pancreatic homogenate but that only myosin I was present on isolated zymogen granules and their membranes. By immunocytochemistry, myosin I was shown in the apical aspect of acinar cells colocalized with glycoprotein 2, a marker for zymogen granules, and actin. By immunocytochemistry, myosin I was also localized on isolated zymogen granules. CONCLUSIONS: The immunolocalization of myosin I to zymogen granule membranes and its close association with periluminal actin suggest that myosin I plays a direct role in the process of transport and exocytosis of zymogen granules in the pancreatic acinar cell.

Subcellular localization of the K+ channel subunit Kv3.1b in selected rat CNS neurons.

Voltage-gated potassium channels constitute the largest group of heteromeric ion channels discovered to date. Over 20 genes have been isolated, encoding different channel subunit proteins which form functional tetrameric K+ channels. We have analyzed the subcellular localization of subunit Kv3.1b, a member of the Kv3 (Shaw-like) subfamily, in rat brain at the light and electron microscopic level, using immunocytochemical detection. Detailed localization was carried out in specific neurons of the neocortex, hippocampus and cerebellum. The identity of Kv3.1b-positive neurons was established using double labeling with markers for specific neuronal populations. In the neocortex, the Kv3.1b subunit was expressed in most parvalbumin-containing bipolar, basket or chandelier cells, and in some bipolar or double bouquet neurons containing calbindin. In the hippocampus, Kv3.1b was expressed in many parvalbumin-containing basket cells, as well as in calbindin-positive neurons in the stratum oriens, and in a small number of interneurons that did not stain for either parvalbumin or calbindin. Kv3.1b protein was not present in pyramidal cells in the neocortex and the hippocampus, but these cells were outlined by labeled presynaptic terminals from interneuron axons that surround the postsynaptic cell. In the cerebellar cortex, granule cells were the only population expressing the channel protein. Careful examination of individual granule cells revealed a non-uniform distribution of Kv3.1 staining on the somata: circular bands of labeling were present in the vicinity of the axon hillock. In cortical and hippocampal interneurons, as well as in cerebellar granule cells, the Kv3.1b subunit was present in somatic and unmyelinated axonal membranes and adjacent cytoplasm, as well as in the most proximal portion of dendritic processes, but not throughout most of the dendrite. Labeling was also seen in the terminals of labeled axons, but not at a higher concentration than in other parts of the axon. The distribution in the cells analyzed supports a role in action potential transmission by regulating action potential duration.

Clustering of voltage-sensitive sodium channels on axons is independent of direct Schwann cell contact in the dystrophic mouse.

The distribution of voltage-sensitive sodium channels on axons in the dorsal and ventral spinal roots of the dystrophic mouse 129/ReJ-Lama2dy was determined via immunocytochemistry. In these nerves there are regions in which Schwann cells fail to proliferate and myelinate axons in a normal manner, leaving bundles of closely packed large-diameter amyelinated axons. We have identified discrete and focal concentrations of sodium channel immunoreactivity on these axons by both confocal immunofluorescence and immunoelectron microscopy, using a peptide-derived polyclonal antibody. In addition, simultaneous labeling with an antibody recognizing neuronal-specific ankyrinG revealed a distinct colocalization with the sodium channels on both normal and amyelinated axons. The presence of patches of sodium channels along with their anchoring protein on amyelinated axons in the absence of intervening Schwann cells demonstrates that axons can form and maintain independently these initial aggregations. This confirms that direct contact between Schwann cell and axon is not required for the formation of sodium channel patches of nodal dimensions and density. Furthermore, this strongly suggests that local transfer of sodium channels from Schwann cells to axons is not required for this process.