Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins.

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.

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.

Growth inhibition, tumor maturation, and extended survival in experimental brain tumors in rats treated with phenylacetate.

Phenylacetate is a naturally occurring plasma component that suppresses the growth of tumor cells and induces differentiation in vitro. To evaluate the in vivo potential and preventive and therapeutic antitumor efficacy of sodium phenylacetate against malignant brain tumors, Fischer 344 rats (n = 50) bearing cerebral 9L gliosarcomas received phenylacetate by continuous s.c. release starting on the day of tumor inoculation (n = 10) using s.c. osmotic minipumps (550 mg/kg/day for 28 days). Rats with established brain tumors (n = 12) received continuous s.c. phenylacetate supplemented with additional daily i.p. dose (300 mg/kg). Control rats (n = 25) were treated in a similar way with saline. Rats were sacrificed during treatment for electron microscopic studies of their tumors, in vivo proliferation assays, and measurement of phenylacetate levels in the serum and cerebrospinal fluid. Treatment with phenylacetate extended survival when started on the day of tumor inoculation (P < 0.01) or 7 days after inoculation (P < 0.03) without any associated adverse effects. In the latter group, phenylacetate levels in pooled serum and cerebrospinal fluid samples after 7 days of treatment were in the therapeutic range as determined in vitro (2.45 mM in serum and 3.1 mM in cerebrospinal fluid). Electron microscopy of treated tumors demonstrated marked hypertrophy and organization of the rough endoplasmic reticulum, indicating cell differentiation, in contrast to the scant and randomly distributed endoplasmic reticulum in tumors from untreated animals. In addition, in vitro studies demonstrated dose-dependent inhibition of the rate of tumor proliferation and restoration of anchorage dependency, a marker of phenotypic reversion. Phenylacetate, used at clinically achievable concentrations, prolongs survival of rats with malignant brain tumors through induction of tumor differentiation. Its role in the treatment of brain tumors and other cancers should be explored further.

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.

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.

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.

Ultrastructure of the crayfish stretch receptor in relation to its function.

The crayfish slow-adapting abdominal stretch receptor was fixed under the relaxed or stretched condition. During this procedure action potentials of the sensory neuron were recorded by a suction electrode. The receptor organ consists of a receptor muscle and a sensory neurons with its dendrites embedded in the connective tissue zone in the receptor muscle. From the cell body of the neuron, several "primary dendrites" arise, branch successively into "dendritic branches", and finally terminate as "dendritic tips," which are cylindrical processes of fairly uniform diameter. In contrast to the primary dendrites and the dendritic branches, the dendritic tips have neither mitochondria nor sheaths and are embedded in the connective tissue zone or apposed to the receptor muscle with a gap of about 15 nm. Microtubules and smooth ER are seen in all parts of the dendrites. When the receptor is stretched and then fixed with 1.6% glutaraldehyde in 0.12 M phosphate buffer (total osmolarity of this solution is isosmotic with the physiological solution), dendritic tips became more parallel to the long axis of the receptor muscle and showed marked deformation consisting of alternate regions of swelling and shrinkage, resulting in a bead-like appearance. When fixed with 1.6% glutaraldehyde in 0.2 M phosphate buffer (total osmolarity of this solution is hyperosmotic), the dendritic tips showed less tendency toward such deformation. These results suggest that the dendritic tip membrane is susceptible to stretch and might be the region where the generator potential is produced.

Thin-section and freeze-fracture studies of crayfish stretch receptor synapses including the reciprocal inhibitory synapse.

The crayfish slow-adapting abdominal stretch receptor organ is innervated by three inhibitory and several excitatory axons. A previous study by Tisdale and Nakajima ('76) showed that under certain fixation conditions inhibitory and excitatory synapses can be distinguished on the basis of synaptic vesicle structure. Using this morphological criterion we describe six types of synapses in the receptor: (1) the inhibitory axo-dendritic synapse, (2) the excitatory neuromuscular synapse, (3) the inhibitory neuromuscular synapse, (4) the axo-axonic synapse which suggests presynaptic inhibition of the excitatory synapse, (5) the axo-axonic synapse which suggests presynaptic inhibition on the inhibitory synapse, (6) the reciprocal inhibitory axo-axonic synapse, which is a new type of synapse. The presence of these six types of synapse suggest that inhibitory and excitatory axons interact synaptically in a complicated manner, resulting in a delicate control of receptor function. In freeze fracture we have observed the presynaptic membrane structures of inhibitory and excitatory synapses. The active zone of the inhibitory synapse has ridges with loosely aggregated particles on the tops of the ridges and indentations (vesicle attachment sites) along their sides. The active zone of the excitatory neuromuscular synapse consists of bands of particle aggregates which are situated on slightly elevated membrane regions and surrounded by wide, relatively particle-free, flat membrane areas.

Freeze-fracture study of the crayfish stretch receptor.

The crayfish slow-adapting stretch receptor was fixed under relaxed or stretched conditions (twice the relaxed length) and then processed for freeze-fracture study. The sensory neuron membrane had evenly distributed intramembrane particles mostly on its P face. The density of these particles was higher in the cell body than in the dendritic tips, which are the terminal portions of the dendrites. The dendritic tips were cylindrical under the relaxed condition and showed deformations with stretch stimuli. When they were fixed under the stretched condition with 1.6% glutaraldehyde in 0.12 M phosphate buffer (the total osmolarity of this fixative is isosmotic with the physiological solution), the dendritic tips showed regional swelling and shrinkage. The intramembrane particle density of the swollen parts decreased and there were particle-free patches of membrane, whereas the particle density of the shrunken parts increased. On the other hand when the receptor was fixed with 1.6% glutaraldehyde in 0.2 M phosphate buffer (the total osmolarity is hyperosmotic but buffer osmolarity is isosmotic), the diameter of the dendritic tips became smaller, and their membrane particle densities were almost the same as that under the relaxed condition. The sheath cells covering the sensory neuron were characterized by their sheet-like profiles, gap junctions, and crater-like protrusions. The receptor muscle membrane had longitudinal foldings, occasional invaginations, peripheral couplings, string-shaped particle aggregates, and band-shaped particle aggregates.

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.

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.

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.

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.

Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.

We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single organelle.

Developmental induction of glutaminase in primary cultures of cerebellar granule cells.

Glutaminase mRNA levels increased over 3-fold relative to total RNA, poly(A)+ RNA, and beta-actin mRNA in neonatal rat cerebellar granule cells as the cells differentiated between days 3 and 8 in culture. In contrast, mRNA levels of another glutamate cycle enzyme, glutamine synthetase, remained constant. Glutaminase protein levels increased per cell more than 2-fold between days 3 and 8, and at least 3-fold by day 10 in these cells. The total amount of glutamate per cell increased about 40% during this period. Glutaminase induction paralleled the development of Ca2+-dependent glutamate release, and the formation of neurites, synaptic vesicles, and synapses. The induction of glutaminase in developing granule cells is consistent with a special role for glutaminase in the synthesis of neurotransmitter glutamate.

Nodal and paranodal membrane structure in complementary freeze-fracture replicas of amphibian peripheral nerves.

Complementary freeze-fracture replicas of frog peripheral nerves have revealed new details of membrane structures at the node of Ranvier and paranodal axon-Schwann cell junction. At the node both E and P fracture faces of the axolemma have high particle concentrations (approximately 1350/sq. micron and 1600/sq. micron respectively) and these particles do not overlap when tracings from the respective fracture faces are superimposed. A high proportion of the E face particles are large (> 9.5 nm) and cast long shadows while the proportion of large particles in the P face is much lower. In the paranodal region the diagonal pattern of parallel rows in the junctional axolemma always has the same orientation within a given fracture face. In the E face, the parallel rows form a positive (+ 30 degrees) angle to the groove below and in the P face, a negative (-30 degrees) angle to the ridge above. This implies that the diagonal pattern derives from asymmetric subunits that are able to associate along only one axis and are unable to 'flip over' with respect to the junctional membranes.

Development of nodal and paranodal membrane specializations in amphibian peripheral nerves.

Peripheral nerves from the hind legs of frog tadpoles were examined in order to ascertain the pattern of development of nodal and paranodal specializations in myelinated fibers. In thin sections the earliest detectable node-related specializations resemble "intermediate" junctions between axons and Schwann cell processes. These occur in individually ensheathed axons near the edges of the sheath segments and could represent early nodal or paranodal components or transient structures. The characteristic nodal "undercoating" is indistinct and highly variable in thickness in immature fibers and its density is lower in developing nodes than in adult nodes. Corresponding freeze-fracture replicas of developing axons demonstrate aggregates of nodal E face particles whose concentration is lower than that in the adult. Such aggregates usually occur immediately adjacent to Schwann cell indentations, even though early in development the latter may not exhibit the paracrystalline pattern seen in the adult paranodal axolemma. On rare occasions, node-like particle aggregates and presumptive nodal undercoatings have been observed without recognizable paranodal junctions or indentations nearby. However, neither specialization has been found in axons not individually ensheathed by Schwann cells. Paranodal Schwann cell loops are widely separated and irregularly arranged in the developing nodes, and the paranodal regions flanking a node usually mature asymmetrically. Differentiated paranodal junctions appear early in axons ensheathed by only a few loose Schwann cell lamellae. However, such junctions are not formed by all paranodal loops; they consistently appear first in the loops close to the node and only later in those further removed. No junctional specialization has been observed in either the axolemma or the Schwann cell membrane without the close association of the other.

Axolemmal differentiation in myelinated fibers of rat peripheral nerves.

In developing rat peripheral fibers, nodal specialization appears early, prior to myelin compaction, and is first detected as a junction between the axon and the overhanging Schwann cell process characterized by a uniformly wide (approximately 18 nm) intercellular gap containing a patchy dense substance and a cytoplasmic undercoating subjacent to the axolemma. The gap width is rather consistent but the axolemmal undercoating is more variable and lower in density than that found at more mature nodes of Ranvier, and it is also highly variable in length, ranging from 0.5 to 3 micron. The outermost Schwann cell layer is usually prominent with a large volume of cytoplasm and many organelles. In freeze-fracture replicas, modal specializations are characterized by accumulations of large (approximately 10 nm) particles in the axolemma, especially the E face, but immature nodes generally have a lower particle concentration than mature nodes. No node-like particle aggregates have been found in axons not intimately associated with Schwann cells. Mature paranodal axon-Schwann cell junctions are usually formed first by the loops closest to the node and are characterized by a 2-3 nm gap between the apposed membranes, periodic intercellular densities (transverse bands) in the gap and cisternae flattened against the junctional Schwann cell membrane. The loops further removed from the node display a wider gap containing irregularly spaced or diffuse intercellular densities, or none. Mature junctions appear relatively late in the rat, and it is not unusual to find developing nodes with several Schwann cell loops present that do not indent the axolemma significantly and are not associated with the paracrystalline pattern characteristic of the mature junctional axolemma. In such instances, the nodal particle aggregates do not have sharply circumscribed boundaries. The majority of the developing nodes are asymmetric with one paranodal segment more mature than the other.

Extranodal particle accumulations in the axolemma of myelinated frog optic axons.

Optic nerves of adult frogs were freeze-fractured with the proximal to distal orientation and distances from retina monitored throughout the process. E face particle accumulations are commonly found (approximately 90% of all examples) in the juxtaparanodal portion of the internode (JPI) immediately adjacent to the paranodal junction. The concentration of these particles is usually highest (200-700/micron 2) immediately adjacent to the last strip of the paranodal junction and then decreases over approximately 1-4 micron to the background level (approximately 100/micron 2) of the more remote portions of the internode. Accumulations with high particle concentrations generally extend further into the internode than those with low concentrations. JPI particle accumulations occur with equal frequency in proximal and distal JPIs, and no apparent difference was seen between optic nerve segments adjacent to or distant from the retina. The majority of the JPI particles are large (10 nm or more in diameter), and they resemble the large nodal particles in size and shape. Particle size analysis in different areas of the internode shows that the concentration of small particles does not change significantly along the internode (including the JPI), but the concentration of large particles is significantly higher in the immediate JPI (140-600/micron 2) than in internodal regions (30-55/micron 2). Thus, the high particle concentration at the JPI region is mainly due to the accumulation of large particles. Such accumulations also occur frequently in irregularly shaped 'lakes' between paranodal junctional strips. Here too the particles are primarily large, and the accumulations occur equally in segments adjacent to or distant from the retina and in both proximal and distal paranodal regions. Heminodes occur in all segments of the frog optic nerve. Most of these lack typical nodal specializations.