An update on the Golgi staining technique improving cerebellar cell type specificity.

The detailed morphological characterization of single cells was a major breakthrough in neuroscience during the turn of the twentieth century, enabling Ramon y Cajal to postulate the neuron doctrine. Even after 150 years, single cell analysis is an intriguing goal, newly motivated by the finding that autism might be caused by intricate and discreet changes in cerebellar morphology. Besides new single labelling technologies, the Golgi staining technique is still in use due to its whole cell labelling characteristics, its superior contrast performance over other methods and its apparent randomness of staining cells within a whole tissue block. However, the specificity and whole cell labelling of Golgi staining are also disputed controversially, and the method still has a poor reputation for being time consuming and needing high expenditures. We demonstrate here, how a classical Golgi technique can be adapted for staining different cerebellar cell types using a time-saving and efficient protocol, enabling the identification of the detailed morphological characteristics of single cells.

Antigen-presenting cell marker expression and phagocytotic activity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) cells are the main cellular constituents of the periodontium, maintain the integrity of the connective tissue, and impact pathology in periodontitis. The aim of this study was to analyze whether PDL cells recognize foreign particles and participate in the immune response to periodontal pathogens. METHODS: Expression of surface proteins characteristic of antigen-presenting cells (APCs) (major histocompatibility complex [MHC] class II, CD40, CD80, CD86) was analyzed in PDL cells after challenge with the cytokines interleukin (IL)-1ß, IL-17A, and interferon-gamma (IFN-γ) or with heat-killed Aggregatibacter actinomycetemcomitans using real-time PCR and flow cytometry. Confocal laser scanning microscopy, transmitted light microscopy, flow cytometry, and time-lapse microscopy were applied to analyze their phagocytotic capacity of collagen (carboxylate-modified microspheres), non-periodontal (Escherichia coli) and periodontal (Aggregatibacter actinomycetemcomitans) pathogens. Furthermore, it was examined whether cytokine activation of PDL cells affects the phagocytosis of collagen or bacteria. RESULTS: PDL cells upregulated MHC class II after cytokine stimulation on transcriptional level, whereas co-stimulatory molecules characteristic of professional APCs were not induced. Analyses on protein level revealed that MHC class II was not constitutively expressed in all PDL cell lines used. PDL cells phagocytosed both collagen and bacteria via acidic vesicles, suggesting the formation of phagosomes. Phagocytosis could be partially inhibited by inhibitors of phagocytosis, i.e., dynasore and wortmannin. Pre-incubation with cytokines did not further enhance the phagocytosis rate of collagen or bacteria. CONCLUSIONS: These results suggest that PDL cells do not only represent bystanders in periodontal infections, but display non-professional APC characteristics, suggesting possible participation in immune reactions of the oral cavity.

Merlin modulates process outgrowth and synaptogenesis in the cerebellum.

Neurofibromatosis type 2 (NF2) patients are prone to develop glial-derived tumors in the peripheral and central nervous system (CNS). The Nf2 gene product -Merlin is not only expressed in glia, but also in neurons of the CNS, where its function still remains elusive. Here, we show that cerebellar Purkinje cells (PCs) of isoform-specific Merlin-deficient mice were innervated by smaller vGluT2-positive clusters at presynaptic terminals than those of wild-type mice. This was paralleled by a reduction in frequency and amplitude of miniature excitatory postsynaptic currents (mEPSC). On the contrary, in conditional transgenic mice in which Merlin expression was specifically ablated in PCs (L7Cre;Nf2fl/fl), we found enlarged vGluT2-positive clusters in their presynaptic buttons together with increased amplitudes of miniature postsynaptic currents. The presynaptic terminals of these PCs innervating neurons of the deep cerebellar nuclei were also enlarged. When exploring mice with Merlin-deficient granule cells (GCs) (Math1Cre;Nf2fl/fl), we found cerebellar extracts to contain higher amounts of vGluT1 present in parallel fiber terminals. In parallel, mEPSC frequency was increased in Math1Cre;Nf2fl/fl mice. On the contrary, VGluT2 clusters in cerebellar glomeruli composed of NF2-deficient presynaptic Mossy fiber terminals and NF2-deficient postsynaptic GC were reduced in size as shown for isoform-specific knockout mice. These changes in Math1Cre;Nf2fl/fl-deficient mice were paralleled by an increased activation of Rac1-Cofilin signaling which is known to impact on cytoskeletal reorganization and synapse formation. Consistent with the observed synaptic alterations in these transgenic mice, we observed altered ultrasonic vocalization, which is known to rely on proper cerebellar function. No gross morphological changes or motor coordination deficits were observed in any of these transgenic mice. We therefore conclude that Merlin does not regulate overall cerebellar development, but impacts on pre- and post-synaptic terminal organization.

From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum.

The analysis of patterned gene expression has been an important tool for dissecting the molecular and developmental bases of functional compartmentalization in the mammalian cerebellum. In particular, sagittally-oriented cellular aggregates arranged along the mediolateral axis are the patterning element most commonly invoked to illustrate cerebellar compartmentalization, and these are revealed both by patterns of afferent projection and by a number of classical biochemical markers that are distributed in a pattern of'zebra stripes'. Compartmentation along both the mediolateral and rostrocaudal axes might be linked mechanistically to segmentation in the fruit fly, since early cerebellar development is especially dependent upon the expression of mammalian homologs of Drosophila segmentation genes. In addition, as has been demonstrated in the retinotectal system, some of these genes are likely to control positional information required for the sagittal organization of cerebellar afferent projections. However, in contrast to these global or macro zones, the cerebellum is also compartmentalized at the subcellular or micro level. This can be visualized by differential patterns of mRNA distribution within the sole cerebellar efferent system, the Purkinje cell, defining within such cells a number of distinct subcellular domains or 'postal zones'. The global versus subcellular levels of cerebellar compartmentalization are related since they both appear to be linked to patterns of afferent innervation.A major goal of cerebellar research will be to unravel the true nature of such a relationship, and its relevance to function and behavior.

Mobilization of iron from cellular ferritin by ascorbic acid in neuroblastoma SK-N-SH cells: an EPR study.

The mobilization of iron from intracellular ferritin by ascorbic acid has been analysed in situ by electron paramagnetic resonance (EPR) spectroscopy. EPR enables a distinction between ferritins and other Fe(3+)-binding cellular components. The ordered iron core of ferritin gives rise to a resonance signal which can be observed only at temperatures above 50 K. In the present study we clearly demonstrate that ascorbic acid is capable of mobilizing iron from ferritin in the cellular system by reduction of the ferric ion core in neuroblastoma SK-N-SH cells. This mechanism may open new ways in the therapy of this hardly curable tumor in stage IV, especially in combination with some cytostatic drugs.

Nitric oxide synthase expression reveals compartments of cerebellar granule cells and suggests a role for mossy fibers in their development.

The developmental expression and cellular distribution of nitric oxide synthase was investigated in the murine cerebellum and in cerebellar neurons developing under controlled in vitro conditions. Cerebellar granule cells expressed nitric oxide synthase only after migration to the internal granule cell layer. Initially, the nascent internal granule cell layer throughout the cerebellum stained uniformly for nitric oxide synthase, but during the second postnatal week, a pattern emerged consisting of clusters of heavily stained granule cells separated by areas of unstained granule cells. This pattern persisted into adulthood. There was a close temporal correlation between innervation of the granule cell layer by mossy fibers and the emergence of granule cell compartments as defined by levels of nitric oxide synthase expression. Granule cells in dissociated cultures derived from cerebellar anlagen prior to mossy fiber innervation also express nitric oxide synthase. The time-course of nitric oxide expression was independent of electrical activity of the neuronal network forming in vitro. However, suppression of spontaneous electrical activity resulted in enhanced nitric oxide synthase expression. These findings indicate that granule cell precursors are endowed with an intrinsic program which regulates nitric oxide synthase induction and which is executed independently of correct positional cues. The data also suggest that electrical activity of ingrowing mossy fibers down regulates nitric oxide synthase expression and plays an important role in the generation of granule cell compartments. These compartments may contribute to the functional organization of the cerebellar cortex.

The differentiation of cerebellar interneurons is independent of their mitotic history.

A narrow time window centered around the terminal mitosis of their precursors has been recognized to be critical for the determination and/or realization of the developmental fate of a variety of neuronal phenotypes. In contrast, individual cell lineages in the cerebellum get separated early during embryonic development, and at least precursors for granule neurons have been found to be specified while still proliferating. We utilized primary dissociated cultures to address the issue of whether the faithful development of cerebellar granule cells and basket/stellate cells is dependent on their mitotic history and on the completion of a fixed number of cell cycles. Neuroblasts derived from embryonic cerebellar anlagen and transferred into primary dissociated cultures stopped proliferating as assessed by a loss of expression of the cell proliferation marker, Ki-67, and a failure to incorporate 5-bromo-2'-deoxyuridine. Although these cells had been forced to leave the proliferating cell pool prematurely, they developed into granule neurons or basket/stellate cells as judged by their distinct pattern of expression of specific molecular markers and the acquisition of a typical morphology. This included the cell intrinsic capacity of granule neurons to position their afferent synapses specifically to their dendrites. Thus, the competence of cerebellar interneurons to differentiate appropriately is independent of the precise timing of their final mitosis; however, their sensitivity towards extrinsic developmental signals appears to vary in a cell cycle-dependent manner, as suggested by the failure to survive of those cells that were in S-phase at the time of cultivation.

Ascorbic-acid-mediated iron release from cellular ferritin and its relation to the formation of DNA strand breaks in neuroblastoma cells.

Ascorbic acid at pharmacologically attainable concentrations effectively inhibited the growth of the catecholamine-positive neuroblastoma cell line SK-N-SH; it inhibited LS cells to a smaller extent and catecholamine-negative SK-N-LO cell growth least effectively. In all three cell lines high concentrations of H2O2 were found. Since ascorbic acid was shown to release iron from ferritin in vitro and to keep it in the reduced state, we suggested that it acted as a pro-oxidant in ferritin-rich neuroblastoma cells in the presence of H2O2 and Fe2+ (Fenton reaction), implying iron release from cellular ferritin. We show here that iron could be mobilized from cellular ferritin by 1 mM ascorbic acid in iron-59-preloaded SK-N-SH and LS cells, but not in SK-N-LO cells. In agreement with these results, DNA strand break formation by ascorbate was only observed in SK-N-SH and LS cells. In SK-N-LO cells, DNA strand breaks could be induced by a combination of 1 mM ascorbic acid and 100 microM H2O2. Since cell-damaging effects caused by chemotherapy further facilitate iron release from ferritin, we conclude that ascorbate could be a powerful enhancer of some cytostatic drugs in neuroblastoma therapy.

Software implementation of statistical methods for the analysis of structure and patterns in neuroanatomical objects.

Neuroanatomical research has greatly benefited from the availability of a large number of cell-specific and region-specific molecular markers. In fact, the analysis of spatial patterns of gene expression in individual cells or patterns within cell populations often provides an inroad into understanding the functional significance of distinct structures. However, it can be difficult to discern whether the arrangement of different morphologically or biochemically defined structures represents a defined pattern. To address this issue, we adapted a series of established statistical procedures for the analysis of uni- and bivariate point patterns in histological specimens. We implemented these statistical procedures in an easy-to-use computer program. The methods are scale independent and easy to expand for various applications. The utility of this approach is demonstrated with examples from tissue sections and cultured cells at the light and electron microscopical levels.

Uptake and cytotoxicity of ascorbic acid and dehydroascorbic acid in neuroblastoma (SK-N-SH) and neuroectodermal (SK-N-LO) cells.

Ascorbic acid (AA) was found to be cytotoxic to neuroblastoma cells in vitro and in vivo. In the present, study we investigated whether the reduced--(AA) or oxidized form (dehydroascorbic acid, DhAA) and its rapidly formed metabolites were the main cytotoxic agents. In neuroblastoma SK-N-SH cells, AA was found to be more cytotoxic than DhAA, although considerably higher amounts of [14C]DhAA than of [14C]AA were incorporated. In contrast, SK-N-LO cells derived from neuroectodermal tissue in fact showed a similar uptake but were much less injured by both substances. We observed that uptake of [14C]AA and [14C]DhAA was impaired in the presence of dithiothreitol and glutathione. Once inside the cell, [14C]DhAA was partially reduced to [14C]AA. From these data we conclude first that at least part of AA is oxidized prior to its uptake, and second that the reduced form of AA and perhaps ascorbyl radicals but not DhAA or its metabolites are the most important forms in mediating cytotoxic reactions in neuroblastoma cells. Furthermore, the results strengthen the previous assumption that AA acts as a pro-oxidant in neuroblastoma cells and supports its use in the treatment of neuroblastoma, especially in combination with existing chemotherapeutics.

Ascorbic acid induces lipid peroxidation on neuroectodermal SK-N-LO cells with high endogenous ferritin content and loaded with MAb-ferritin immunoconjugates.

Neuroblasma-and other malignant cells often contain elevated amounts of iron-rich ferritin and H2O2 and may therefore be a potential target for pro-oxidative effects of ascorbic acid (AA), generating cytotoxic products e.g. by lipid peroxidation (LPO). The influence of H2O2 and iron, either in its free form or bound to ferritin, on AA induced LPO was first investigated using erythrocyte ghosts as a model system. Results of these experiments showed that AA induced LPO not only in the presence of free available iron but also in the presence of ferritin. Similarly, AA induced significant LPO in neuroectodermal SK-N-LO cells with elevated intracellular ferritin levels. These LPO promoting effects of ferritin in the presence of AA on SK-N-LO cells could also be observed using ferritin-immunoconjugates: for this purpose, ferritin was bound to human monoclonal antibodies (MAb-ferritin) recognizing ganglioside GD2 which is present in large quantities on cell surfaces of SK-N-LO and many neuroblastoma cells. We conclude that the pro-oxidative effects of AA could be exploited in the treatment of ferritin rich neuroblastoma in combination with chemotherapy or with MAb-ferritin immunoconjugates.

A simple assay for determination of iron release from ferritin in neuroblastoma cells.

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.

[Autonomic innervation of the female pelvis. Anatomic basis]. / Autonome Innervation des weiblichen Beckens. Anatomische Grundlagen.

The pelvic plexus with its sympathetic (hypogastric nerves, sacral sympathetic trunk) and parasympathetic (pelvic splanchnic nerves) sources provides and distributes the autonomic nervous supply for the pelvic organs. This homogeneous autonomic nerve plate is located within and deeply to the peritoneal fold (recto-uterine in the female pelvis) lying medially to the internal iliac vessels. Autonomic nerve fibres emerge from the pelvic plexus and travel to the anterolateral aspect of the rectum and the inferolateral and posterolateral aspects of the bladder and female genital tract. In addition, pelvic organs are innervated by nerves accompanying the vessels and the ureter which penetrate the pelvic plexus. Knowledge of the topography of the pelvic plexus indicates the most common sites for iatrogenic lesions and should help us in avoiding postoperative autonomic dysfunction.

Cellular distribution of metastasis suppressor 1 and the shape of cell bodies are temporarily altered in Engrailed-2 overexpressing cerebellar Purkinje cells.

Metastasis suppressor 1 (MTSS1, BEG4, MIM) is well described for its function as a metastasis suppressor gene and is expressed in a variety of tissues. However, only little is known about its expression in the central nervous system (CNS), and functions within the CNS have not been addressed so far. Here, we show that MTSS1 was expressed in postmitotic neurons of the cerebellar cortex. Within Purkinje cells, higher amounts of MTSS1 were temporarily localized in the axonal somatic compartment than in the dendritic compartment. In L7En-2 transgenic mice, in which the segment-polarity gene and regulator of neuronal maturation Engrailed-2 is overexpressed specifically in cerebellar Purkinje cells, MTSS1 was homogenously distributed within Purkinje cell somata throughout development. In parallel to the altered distribution of MTSS1 in L7En-2 Purkinje cells, L7En-2 Purkinje cell somata were distorted and in some cells invaginations of the plasma membrane were observed. These invaginations were only found in L7En-2 neurons, and displayed multiple synapses which could not be seen at the smooth surface of wildtype Purkinje cell somata. Current knowledge about MTSS1 function in vitro and the correlation between MTSS1 localization and the occurrence of membrane alterations in L7En-2 Purkinje cells described here suggest that MTSS1 might be involved in shaping neuronal membranes in vivo.

The developmental expression of neuronal nitric oxide synthase in cerebellar granule cells is sensitive to GABA and neurotrophins.

In the adult cerebellar cortex, the differential expression of neuronal nitric oxide synthase (nNOS) can be used to discern discrete subsets of granule neurons. These subsets originate concurrently with the afferent innervation of granule neurons. Using primary cultures established from murine cerebella at different development stages, we analyzed the effects of the neurotransmitter GABA and the neurotrophins BDNF and NT-3 on nNOS expression in developing granule cells. We show a biophasic effect of GABA on nNOS expression, which correlates with the ability of this transmitter to increase intracellular calcium levels. BDNF and NT-3 are shown to increase nNOS levels in cultures derived from postnatal, but not in those from embryonic cerebella. Together with previous findings [Baader and Schilling; J Neurosci 1996; 16:1440-1449], these data suggest a scenario in which afferent innervation and growth factors cooperate to differentially affect nNOS expression at discrete developmental stages of cerebellar granule cells.

Glutamate receptors mediate dynamic regulation of nitric oxide synthase expression in cerebellar granule cells.

Nitric oxide (NO) is a multifaceted messenger molecule believed to be involved in neural plasticity and development. Within the cerebellum, the NO synthesizing enzyme, NO synthase (NOS), is expressed exclusively by granule cells and stellate/basket neurons. In the adult cerebellum, levels of NOS expression can be used to define discrete clusters of granule cell populations. Differential expression of NOS by granule cells temporally coincides with the establishment of afferent innervation of granule cells. In primary cerebellar cultures that comprise a functional network of glutamatergic and GABAergic cerebellar neurons, blockade of electrical activity by tetrodotoxin induced the expression of the neuronal isoform of NOS (nNOS) in granule cells. Conversely, direct depolarization of cultured neurons with K+ completely downregulated nNOS expression. Suppression of NMDA receptor- and AMPA receptor-mediated spontaneous synaptic signaling in cultured cells resulted in a drastic upregulation of nNOS expression in granule neurons. In contrast, blockade of GABAA receptor-mediated intercellular communication did not affect nNOS expression by granule cells. Blocking N-, P-, and Q-type voltage-dependent Ca2+ channels resulted in a graded upregulation of NOS expression, whereas manipulations of the cAMP-dependent signal transduction pathway induced no changes. We conclude that nNOS expression in developing cerebellar granule cells is regulated by excitatory neurotransmission and that calcium is an important signal transduction molecule involved in this regulatory process.

Changing subcellular distribution and activity-dependent utilization of a dendritically localized mRNA in developing Purkinje cells.

In cerebellar Purkinje neurons, the degree of dendritic segregation of the Purkinje cell-specific mRNA L7/pcp-2 is correlated with their development and synaptic investment. This developmental pattern is also observed in Purkinje cells in primary dissociated culture. Short-term (12-48 h) stimulation of cultured Purkinje cells by potassium-induced depolarization or blockade of their inhibitory GABAergic input results in an increased incidence of Purkinje cells with L7/pcp-2 mRNA-positive dendrites and increased levels of L7 protein expression, the latter by a posttranscriptional mechanism. None of these treatments affected the localization of the mRNA encoding calbindin D28k nor the level of this protein in Purkinje neurons. Protracted exposure to depolarizing levels of potassium or elimination of GABAergic transmission resulted in conspicuous changes of Purkinje cell dendritic morphology. These data suggest a scenario in which activity-driven translation of subcellularly segregated mRNAs may contribute to the developmental and functional plasticity of nerve cells.

Regulation of a Purkinje cell-specific promoter by homeodomain proteins: repression by engrailed-2 vs. synergistic activation by Hoxa5 and Hoxb7.

We have previously demonstrated that a short sequence element (L7ATE) within the proximal promoter of a Purkinje cell-specific gene, pcp-2(L7), is required for the normal pattern of expression of the gene in the cerebellum of transgenic mice. The presence of a series of TAAT sequence motifs in this element suggested its interaction with homeodomain proteins. To extend these observations, degenerate oligonucleotides were used to clone by reverse-transcriptase polymerase chain reaction members of the mouse Hox gene family expressed in neonatal cerebellum but not forebrain. Two of these, HoxB7 and HoxA5, are continuously expressed from the neonatal period into adult stages in cerebellar Purkinje cells. These Hox proteins are shown to synergistically activate the L7 promoter by cotransfection assay in vitro. In contrast, another homeodomain protein that is normally expressed in Purkinje cells only during the embryonic period, En-2, has a negative effect on L7 gene expression. These data suggest a biphasic, combinatorial control mechanism for the Purkinje cell-specific expression of the pcp-2(L7) gene.

Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions.

Members of the En and Wnt gene families seem to play a key role in the early specification of the brain territory that gives rise to the cerebellum, the midhindbrain junction. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, we expressed En-2 ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity to the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures that is highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions.

Selective disruption of "late onset" sagittal banding patterns by ectopic expression of engrailed-2 in cerebellar Purkinje cells.

To explore the role of Engrailed proteins in development of the cerebellum, Engrailed-2 (En-2) was ectopically expressed in cerebellar Purkinje cells from the late embryonic stage into adulthood. The fundamental organization of Purkinje cell sagittal zones as revealed by the "early onset" markers L7-beta-gal and cadherin-8 was found to be virtually identical to that in wild type. In contrast, "late onset" sagittal banding patterns revealed by Purkinje cell markers zebrin I, zebrin II, and 9-O-acetyl GD3 Ganglioside (P-Path), and the granule cell marker NADPH-diaphorase, were disrupted. In general, although some evidence of banding was still detectable, boundaries defined by the latter markers were poorly defined, and the patterns overall took on a diffuse appearance. In parallel with the changes in late onset markers, anterograde tracing of spinocerebellar axons revealed a general diffusion of the mossy fiber projection pattern in lobule VIII and the anterior lobe. These observations suggest that at least two separate mediolateral boundary systems exist in the cerebellum, and these are differentially affected by ectopic En-2 expression. Alternatively, one boundary system exists that remains primarily intact in the mutant, but recognition of this system by a set of late developmental events is perturbed.