p21(ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells.

Initiation and elongation of neurites in PC12 cells has been shown to be stimulated by nerve growth factor (NGF). Initiation of NGF-stimulated neurites in a PC12 subclone (PC12-N09) is rapid, giving rise to short neurites that do not elongate after 1 day. To determine whether increasing activation of p21(ras) could restore neurite elongation in these cells and whether it would affect the phosphorylation of signaling proteins, the subclone PC12-N09 was transfected with constitutively active p21(ras61L) (PC12-N09ras61L) and neurite outgrowth with or without NGF was determined. Overexpression of wild-type p21(ras) (PC12-N09rasWT) did not lead to spontaneous neurite initiation but restored the ability of NGF to stimulate continuous neurite elongation. However, NGF-stimulated phosphorylation of ERK, p38, and Akt in PC12-N09rasWT cells is similar in duration to that in PC12-N09 cells, indicating that the p21(ras) signaling through ERK, p38, and Akt was not involved in the restoration of normal neurite elongation in PC12-N09 cells. These results show that p21(ras)-activated pathways other than ERK, p38, and Akt are necessary for appropriate NGF-stimulated neurite elongation in PC12 cells.

Corticotropin releasing factor induces proliferation of cerebellar astrocytes.

In the adult cerebellum, corticotropin releasing factor (CRF), that is localized in climbing fibers, mossy fibers, and a fine varicose plexus along the Purkinje cell layer, modulates the responsiveness of Purkinje cells to excitatory amino acids. During development, CRF has been detected in the primitive cerebellar anlage as early as embryonic day (E)10, and is continuously expressed throughout embryonic and postnatal cerebellar ontogeny. To investigate a possible trophic role for CRF during cerebellar development, cerebellar culture studies using E18 mouse embryos were carried out. In our culture paradigm, that used serum-free defined medium to suppress cell proliferation, CRF induced proliferation of cells in a dose-dependent manner in a range of concentrations between 0.1-10 microM. The proliferating cells were identified as astrocytes based on their expression of vimentin and GFAP. BrdU incorporation studies supported the proposed mitogenic effect of CRF on developing astrocytes. The mitogenic effects of CRF seemed to be primarily on immature astrocytes determined by their differential expression of vimentin and GFAP. Astrocytes at more advanced stages of development, as determined by the extent of process outgrowth and GFAP expression, incorporated less BrdU compared to immature astrocytes. CRF receptors were localized in astrocytes, and the proliferation of astrocytes induced by CRF was inhibited by astressin, a competitive CRF receptor antagonist. In conclusion, CRF induces proliferation of astrocytes derived from the developing cerebellum, that suggests a gliotrophic role for CRF during cerebellar development.

Specificity controls for immunocytochemical methods.

Immunocytochemistry is used for antibody localization of proteins in cells and tissues. The specificity of the results depends on two independent criteria: the specificity of the antibody and of the method used. The antibody specificity is best determined by immunoblot and or immunoprecipitation. Absorption of the antibody with a protein does not determine that the antibody would have bound to the same protein in the tissue, and therefore is not a good control for antibody specificity. The specificity of the method is best determined by both a negative control, replacing the primary antibody with serum, and a positive control, using the antibody with cells known to contain the protein. With the increasing use of immunocytochemistry, it is important to be aware of the appropriate controls needed to show specificity of the labeling. (J Histochem Cytochem 48:163-165, 2000)

Ultrasmall immunogold particles: important probes for immunocytochemistry.

In this article, we review the immunocytochemical literature with respect to a comparison between conventional colloidal gold and ultrasmall gold particles as immunoprobes. We discuss the relative advantages and disadvantages of each of these types of particles for immunocytochemical applications. We present results from our own laboratories, in which we compared these immunoprobes in selected experimental situations. In addition, we discuss our work on the use of a fluorescently labeled ultrasmall immunoprobe for correlative microscopy.

PKC activators (phorbol ester or bryostatin) stimulate outgrowth of NGF-dependent neurites in a subline of PC12 cells.

Nerve growth factor (NGF) stimulation of PC12 cells activates signaling pathways leading to new protein expression and growth of neurites. In wild type PC12 cells, incubation with phorbol ester (PMA) will activate protein kinase C (PKC) leading to the expression of many proteins necessary for neurite outgrowth, but this activation of PKC alone will not stimulate growth of long neurites. Here, we show in the subline of PC12-N09, which lacks NGF-stimulated growth of long neurites, that a brief incubation with PKC activators, PMA or bryostatin 1 (bryostatin), before NGF incubation, stimulates the growth of long neurites. However, incubation in the reverse order is ineffective. A short incubation with PMA or bryostatin followed by NGF induced tyrosine phosphorylation of MAP kinase (MAPK), which is of the same duration as that induced by NGF alone. Thus, PMA preincubation did not increase the length NGF activation of MAPK. Twenty-four hr after incubation with PMA or bryostatin, PKC isoforms were downregulated but PKC isoforms delta-, and epsilon- were still present. In these cells chronically treated with either PMA or bryostatin to downregulate PKC, NGF incubation preceded by PMA preincubation still led to long neurite outgrowth. These results suggest that a PMA or bryostatin incubation followed by NGF activates PKC isoforms delta-, and epsilon-leading to outgrowth of long neurites, and that the PMA signaling is independent of the MAPK pathway.

Gangliosides inhibit growth factor-stimulated neurite outgrowth in SH-SY5Y human neuroblastoma cells.

Exogenously added gangliosides are known to promote neurite outgrowth in a variety of cell types, including some neuroblastoma cell lines. To study neuritogenesis in SH-SY5Y human neuroblastoma we serum starved the cells for 24 hr and exposed them to gangliosides (GM1, GM3, or GT1b), platelet-derived growth factor (PDGF), insulin, nerve growth factor (NGF), insulin-like growth factor I (IGF-I), or combinations of these for 3 days. We measured four parameters of neurite outgrowth using image analysis. PDGF induced neurite outgrowth in SH-SY5Y and GM1 inhibited this. Both phenomena were dose-dependent with neurites/cell and neurite length being below controls with 100 microM GM1, and percent of neurite-bearing cells being below controls with 25, 50, and 100 microM GM1. Similar but more inhibitory results were obtained with GM3 and GT1b. Insulin and IGF-I induced a neuritogenic response that was less potent than that of PDGF and was also inhibited by gangliosides. NGF had no effect on neurite outgrowth but gangliosides were still inhibitory even in cells not treated with growth factors. From this we conclude that gangliosides inhibit spontaneous and trophic factor-induced neurite outgrowth in SH-SY5Y cells. For GM1 and GT1b, but not GM3, this probably involves inhibition of trophic factor receptor function.

Neuronal thermosensitivity and survival of rat hypothalamic slices in recording chambers.

Several studies have examined the activity of neurons in hypothalamic tissue slices. The present experiments studied relationships between neuronal activity (firing rate and thermosensitivity) and tissue survival as a function of time and slice thickness. Rat hypothalamic tissue slices were sectioned at different thicknesses (350, 450, and 600 microm) and maintained in an oxygenated interface chamber which was perfused with artificial cerebrospinal fluid (ACSF). Electron and light microscopy were used to examine tissue morphology at different depths from the slice surfaces, and extracellular recordings were used to measure each cell's spontaneous activity and response to changes in temperature. Tissue damage was most evident at tissue layers nearest the gas-exposed surface. At 9 h in the chamber, 350 microm thick slices showed subtle changes in morphology with little difference between the gas-exposed and ACSF-exposed surfaces. In the 450 and 600 microm thick slices, tissue degeneration became more evident with increased damage at the gas-exposed surface. This damage extended fully into the tissue of the 600 microm section. There were no differences in firing rate or thermosensitivity between 350 and 450 microm slices; but in 600 microm slices, there were fewer spontaneously active neurons, although these neurons had a higher mean thermosensitivity. Based on the incidence of spontaneous activity and morphological integrity, the results suggest that electrophysiological experiments using 350 microm slices are preferable to experiments using thicker slices.

The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.

Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organelles. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed the distribution of glutamic acid decarboxylase (GAD), a synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), in the cerebellar nuclei of the mouse. Pre-embedding immunocytochemistry was carried out on brain sections fixed with high concentrations of glutaraldehyde and sodium metabisulfite. After incubations with a monoclonal antibody to GAD and a 1.4-nm NanoGold-labeled secondary antibody, sections were silver-enhanced with N-propyl gallate as a reducing agent and MES as a new buffer system. In the cerebellar nuclei, GAD label was specifically localized in axon terminals over clusters of synaptic vesicles. These terminals formed axosomatic and axodendritic contacts. The majority of GAD-labeled terminals had cytological characteristics indicating their origin from Purkinje cells, which are known to contain GAD.

An analysis of GABAergic afferents to basket cell bodies in the cat's cerebellum.

An antibody to glutamic acid decarboxylase (GAD) was used to identify GABAergic terminals around the somata of basket cells in the cat's cerebellar cortex. Two sources for GAD immunoreactive terminals were identified based on size and cytological characteristics including the recurrent collaterals derived from Purkinje cells and the axons of stellate cells. The majority of the unlabeled terminals likely arise from parallel fibers. The present analysis indicates that GAD-positive terminals form 13% of the synaptic contacts on basket cells. The remaining 87% are unlabeled. Thus, GAD-positive terminals represent a small proportion of the synaptic input to basket cell bodies as compared to afferent endings that likely mediate excitation.

Nerve growth factor stimulates GAP-43 expression in PC12 cell clones independently of neurite outgrowth.

Expression of the growth associated protein GAP-43 (B-50, F1, neuromodulin) increases with the onset of neuronal development as seen by the growth of axons. To investigate the relationship of the signaling events leading to GAP-43 expression and neurite outgrowth, we examined PC12 clones with different phenotypes. Three clones, PC12-N09, PC12-N15, and PC12-N21, responded to NGF with increased expression of GAP-43, but only two clones, PC12-N15 and PC12-N21, responded with growth of neurites. Similar increases in expression of GAP-43 were obtained when these clones were exposed to the phorbol ester PMA. Thus, NGF and PMA induced GAP-43 expression in PC12-N09 cells in the absence of neurite outgrowth. In contrast, all three clones, were able to respond to forskolin (FOR) by initiation of long neurites which had synaptophysin in the growth cones, but showed only low levels of GAP-43. Combined stimulation of PC12-N09 cells with FOR and PMA both initiated neurites and increased expression of GAP-43 as seen in normal PC12 cells. These results show that PC12-N09 cells, in response to either NGF or PMA, can express GAP-43, but without neurite outgrowth, and that all the PC12 clones were also able to respond to FOR with increased neurite outgrowth in the presence of low levels of GAP-43. The dissociation of GAP-43 expression and growth of neurites observed in PC12-N09 cells suggests that signaling mechanisms can independently regulate GAP-43 expression and neurite outgrowth during neuronal differentiation.

Neuronotypic differentiation results in reduced levels and altered distribution of synaptophysin in PC12 cells.

Several synaptic vesicle proteins including synaptophysin and p65/synaptotagmin are expressed by the pheochromocytoma cell line PC12. Stimulation of these cells with nerve growth factor for 7 days induces morphologic neuronotypic differentiation, but the levels of synaptophysin are markedly reduced. Stimulation with cyclic AMP analogs also produces neuronotypic differentiation of PC12 cells, and the degree of morphologic differentiation induced by these agents parallels their ability to effect reduction in synaptophysin levels. By contrast, levels of p65/synaptotagmin are increased following neuronotypic differentiation. The contrasting effects of neuronotypic differentiation on levels of synaptophysin and p65/synaptotagmin indicate potential differences in the regulation of these proteins in PC12 cells. Immunocytochemical labeling of undifferentiated PC12 cells reveals concentrations of synaptophysin in the perinuclear region. After neuronotypic differentiation, there is reduction in perinuclear labeling and concentration of label in swellings along PC12 cell processes. At the ultrastructural level, synaptophysin labeling is found on similar organelles in both undifferentiated and nerve growth factor-stimulated PC12 cells. Although the highest labeling densities were seen on small clear vesicles, specific labeling was also seen on dense core vesicles. The presence of synaptophysin on both small clear vesicles and dense core vesicles indicates potential functional similarities in these vesicle types. The changes in the levels and immunocytochemical distribution of synaptophysin after neuronotypic differentiation suggest possible functional heterogeneity among morphologically similar populations of small clear vesicles.

Synaptophysin has a selective distribution in early endosomes of PC12 cells.

We have studied the endocytic pathway in PC12 cells and localized synaptophysin to a subpopulation of early endosomes. Endocytosis was examined by electron microscopy using horseradish peroxidase as an endocytic tracer. Immediately following brief incubations with horseradish peroxidase, label was seen in small vesicles and tubules which appeared to be part of the tubular early endosomal network. A large vacuolar structure, containing a rim of horseradish peroxidase reaction product and an electron-lucent central region, was also labelled at the earliest time point examined. Within 5 min after the horseradish peroxidase pulse, reaction product was seen in multivesicular bodies. After prolonged chase periods, horseradish peroxidase label was lost from the early endosomal structures and accumulated in large dense vesicles containing lamellar stacks of membranes. The observed pattern of horseradish peroxidase distribution is consistent with delivery of the tracer into tubular and vacuolar early endosomes with subsequent movement of horseradish peroxidase out of these compartments and into lysosomes. Examination of synaptophysin distribution by EM immunocytochemistry following incubations with horseradish peroxidase revealed selective immunogold labelling of early endosomal structures. Notably, small vesicular and tubular profiles were frequently double-labelled while vacuolar early endosomes were only rarely labelled for synaptophysin. Immunocytochemical labelling was not observed in multivesicular bodies or large dense vesicles with lamellar stacks. Results of experiments in which endosomal structures were immunoprecipitated with antibody to synaptophysin were consistent with the immunocytochemical findings. Maximal recovery of endocytosed horseradish peroxidase activity was seen immediately following the horseradish peroxidase pulse, and a significant decrease was seen after brief chase periods. These results indicate the presence of synaptophysin in vesicles and tubules of the early endosomal compartment.

Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.

Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.

In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.

GAP-43 distribution is correlated with development of growth cones and presynaptic terminals.

GAP-43 (F1, B-50, pp46) has been associated with neuronal development and regeneration, but precise localization within neurons is not known. Pre-embedding electron microscopic immunocytochemistry using silver-enhanced 1 nm gold particles was used to localize GAP-43 label in cell cultures of cerebellar neurons. In the plasma membranes of early cultures, high levels of GAP-43 were seen in all parts of the neuron. In older cultures, consistent with previous reports, the first loss of GAP-43 label was seen in the soma and then the axon. Growth cones had high levels of GAP-43 label on the plasma membrane, with increased distribution over unattached relative to attached filopodia. The amount of GAP-43 seen over the plasma membrane of forming presynaptic terminals is lower than over growth cones, indicating a possible correlation between the presence of GAP-43 and the stage of presynaptic terminal development. Intracellular GAP-43 in axons and growth cones was highest in membranes of smooth cisternae. The levels of GAP-43 in smooth cisternae in axons fell by seven days in culture while the levels of GAP-43 in smooth cisternae of growth cones fell at 14 days. When mini-explant cerebellar cultures were examined with light microscopic immunocytochemistry, GAP-43 label of plasma membrane was highest at the periphery of the radial axonal outgrowth, suggesting that addition of GAP-43 to the plasma membrane can occur in the distal axon or at the growth cone.

Transitional elements with characteristics of both growth cones and presynaptic terminals observed in cell cultures of cerebellar neurons.

As growth cones interact with targets, they become presynaptic terminals by losing growth cone characteristics and acquiring presynaptic characteristics. Results presented here show that transitional elements can be identified in cell cultures of rat cerebellum, which have some characteristics of both growth cones and presynaptic terminals. During the first week in culture, slender growth cones have fine filopodia. Subsequently, many growth cones in contact with the polylysine substrate spontaneously enlarge and become non-motile. In transitional elements, the synaptic vesicle protein p65 extends into the peripheral domain and in some cases, extends into filopodia. Many of these transitional elements have active filopodia but show no movement over the substrate for periods of up to nine days. These transitional elements have lost the actin-rich peripheral domain of the growth cone but retain actin labelling in the filopodia. With electron microscopy, transitional elements were seen to contain accumulations of synaptic vesicles at the site of contact with the substrate. Electron microscopic immunocytochemistry showed these synaptic vesicles labelled for p65 with silver-developed gold particles. Thus, transitional elements have characteristics of both growth cones and presynaptic terminals, suggesting that they may also have functional attributes of both growth cones and presynaptic elements.

Redistribution of GAP-43 during growth cone development in vitro; immunocytochemical studies.

The growth-associated protein GAP-43 (B-50, F1, pp46), has been found in elongating axons during development and regeneration, and has also been associated with synaptic plasticity in mature neurons. We have examined the loss of GAP-43 labelling from cerebellar granule cells with immunocytochemical localization of a polyclonal antibody to GAP-43. One day after plating, the plasma membrane of cell bodies, neurites and growth cones were all labelled with anti-GAP-43. By 10 days, most of the cell body labelling was lost, and by 20 days the neuritic and growth cone labelling was greatly reduced. Beginning at six days, anti-GAP-43 labelling of growth cones, which was initially uniform, became clustered. When growth cones were double-labelled with antibodies to GAP-43 and the synaptic vesicle protein, p65, inverse changes in the distribution of label was observed. While growth cone labelling with anti-p65 increased from three to 20 days in culture, GAP-43 label began to be lost from some growth cones by six days and showed continuing decline through 20 days. For individual growth cones, the loss of GAP-43 appeared to parallel the accumulation of p65, and first growth cones to lose GAP-43 appeared to be the first to accumulate p65 label. When cultures were grown on a substrate of basement membrane material, the time frames of neuritic outgrowth, loss of GAP-43 labelling, and increase in p65 labelling were all accelerated. At five days, labelling for GAP-43 was weak and labelling for p65 was strong, in a pattern comparable to that seen in older cultures on a polylysine substrate. These results suggest several conclusions concerning the expression and loss of GAP-43 in cultured cerebellar granule neurons. First, GAP-43 label is initially distributed in all parts of these cells. With increasing time in culture the label is first lost from cell bodies and later from neurites and growth cones. Second, the loss of GAP-43 label from growth cones is correlated with the appearance of the synaptic vesicle protein p65. Finally, in vitro developmental changes in the loss of GAP-43 can be altered by changing the growth substrate.

A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.

The availability of 1-nm gold particles permits the use of a particulate label with standard pre-embedding electron microscopic immunocytochemical techniques. We have employed these particles to localize a synaptic vesicle protein, p65, and a growth-associated protein, GAP-43, in neuron cell cultures. To be detected by standard transmission electron microscopy, these ultra-small gold particles must be enlarged. We have applied a commercially available silver development kit (IntenseM), the method of Danscher, and a neutral pH development procedure which we developed to effect this enlargement. Although IntenseM permits development with good preservation of morphology, it is limited by lack of reproducibility and by variability of final particle size. The method of Danscher provides well-controlled and reproducible enlargement, but is limited with respect to preservation of ultrastructural details. The neutral pH development procedure reproducibly enlarges gold particles with superior preservation of morphology. The use of this development procedure in conjunction with 1-nm gold probes should permit precise ultrastructural localization of a variety of intracellular antigens.

Highly basic 30- and 32-kilodalton proteins associated with synapse formation on polylysine-coated beads in enriched neuronal cell cultures.

Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.