Correlative microscopy of cerebellar Bergmann glial cells.

Double fluorescent labelling of rat cerebellar cortex using antibody to glial fibrillary acidic protein (GFAP) and Alexa fluor conjugates for secondary detection for confocal laser scanning microscope (CLSM), field emission scanning electron microscopy (FESEM) of Rhesus monkey cerebellar cortex, ultrathin sectioning and freeze-etching replica method for transmission electron microscopy of mouse cerebellar cortex have been examined in an attempt to obtain a new and more accurate view of three-dimensional image of Bergmann glial cells (BGC) and their topographic relations in the molecular layer. Intense immunopositive GFAP green staining was observed in the BGC and glial limiting layer. Secondary antibody conjugated with Alexa fluor 488 and Alexa fluor 668-1B4 stained in red capillary endothelial cells and microglial cells. BGC morphology revealed the existence of several cell types or subpopulations of BGC. Bergmann glial fibers, in palisade arrangement, branch and rebranch forming a complex glial network in the molecular layer. Field emission SEM and freeze-fracture SEM method show the SE-I image of high mass dense Bergmann glial cytoplasm ensheathing like a veil the Purkinje cell (PC) soma and dendritric arborization. Bergmann glial fibers appeared completely surrounding individual parallel fibers or parallel fiber bundles, terminal climbing fiber collaterals, basket and stellate cells and capillaries. Freeze-etching direct replicas showed the typical orthogonal arrangement of intramembrane particles, corresponding to the large repertoire of BGC receptors. The study reveals three-dimensional Bergmann glial cells heterogeneity and the complex network formed by Bergmann glial cells in the molecular layer.

A common oocyst surface antigen of Cryptosporidium recognized by monoclonal antibodies.

Two hybridoma clones, CMYL3 and CMYL30, were generated by immunizing Balb/c mice with excysted oocysts of Cryptosporidium muris. Both clones secreted monoclonal antibodies against an oocyst-wall antigen with apparent molecular mass of 250 kDa (called CM250) from C. muris and C. parvum. The epitope appeared to be periodate-sensitive, suggesting the involvement of a carbohydrate moiety. Immunofluorescence and confocal microscopy on purified oocysts and infected mouse tissues revealed staining confined to the oocyst wall of both Cryptosporidium species. Immunogold labeling further revealed the presence of the CM250 antigen in electron-dense vesicles and cytoplasm of developing macrogametocytes, and ultimately localized to the oocyst wall of mature oocysts. Both antibodies cross-reacted with C. serpentis oocysts but did not recognize the other enteropathogenic protozoans Giardia muris, Eimeria falciformis and E. nischulz. These antibodies may be valuable tools for the analysis of oocyst-wall formation in Cryptosporidium and characterization of the common antigen.

Hippocampal mossy fibers induce assembly and clustering of PSD95-containing postsynaptic densities independent of glutamate receptor activation.

Factors that regulate the formation, spatial patterning, and maturation of CNS synapses are poorly understood. We used organotypic hippocampal slice cultures derived from developing (P5-P7) rat to test whether synaptic activity regulates the development and organization of postsynaptic structures at mossy fiber (MF) giant synapses. Antibodies to a prominent postsynaptic density (PSD) scaffold protein, PSD95, identified large (>1 microm) and irregularly shaped PSD assemblies that codistributed with synapsin-I or metabotropic glutamate receptor 7b (mGluR7b) -immunolabeled MF terminals in area CA3. To investigate the spatial organization of synaptic PSDs on individual pyramidal cells, neurons in slice cultures were transfected with a vector encoding a GFP-PSD95 fusion protein. Confocal three-dimensional reconstructions revealed clusters of PSDs along proximal dendrites of transfected pyramidal neurons in area CA3, but not in CA1. Clusters averaged 7.6 microm in length (range, 2.2-29 microm) and contained up to 35 individual PSDs (mean, 8.3). PSD clusters failed to form when slices were cultured without MFs, indicating that MFs induce cluster assembly. Chronic blockade of N-methyl-D-apartate- and AMPA/kainate-type glutamate receptors did not disrupt MF targeting or de novo formation of PSD clusters with a normal distribution on target cells. Additionally, glutamate receptor blockers did not alter the ultrastructural development of MF giant synapses containing multiple puncta adherens-like junctions and asymmetric synaptic junctions at dendritic shaft and spine domains, respectively. The results indicate that MF axons can induce the assembly and clustering of PSD95-containing postsynaptic complexes, displaying a normal subcellular and tissue distribution, by mechanisms that are independent of ionotropic glutamate receptor activation.

Rapid formation and remodeling of postsynaptic densities in developing dendrites.

The dynamics of postsynaptic density (PSD) formation and remodeling were investigated in live developing hippocampal tissue slices. Time lapse imaging of transfected neurons expressing GFP-tagged PSD95, a prominent PSD protein, revealed that up to 40% of PSDs in developing dendrites are structurally dynamic; they rapidly (<15 min) appear or disappear, but also grow, shrink and move within shafts and spines. New spines containing PSDs were formed by conversion of dynamic filopodia-like spine precursors in which PSDs appeared de novo, or by direct extension of spines or spine precursors carrying preformed PSDs from the shaft. PSDs are therefore highly dynamic structures that can undergo rapid structural alteration within dendrite shafts, spines and spine precursors, permitting rapid formation and remodeling of synaptic connections in developing CNS tissues.

Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices.

The dynamics of microglial cell activation was studied in freshly prepared rat brain tissue slices. Microglia became activated in the tissue slices, as evidenced by their conversion from a ramified to amoeboid form within several hours in vitro. To define better the cytoarchitectural dynamics underlying microglial activation, we performed direct three-dimensional time-lapse confocal imaging of microglial cells in live brain slices. Microglia in tissue slices were stained with a fluorescent lectin conjugate, FITC-IB(4), and stacks of confocal optical sections through the tissue were collected repeatedly at intervals of 2-5 min for several hours at a time. Morphometric analysis of cells from time-lapse sequences revealed that ramified microglia progress to amoeboid macrophages through a stereotypical sequence of steps. First, in the withdrawal stage, the existing ramified branches of activating microglia do not actively extend or engulf other cells, but instead retract back (mean rate, 0.5-1.5 microm/min) and are completely resorbed into the cell body. Second, in the motility stage, a new set of dynamic protrusions, which can exhibit cycles of rapid extension and retraction (both up to 4 microm/min), abruptly emerges. Sometimes new processes begin to emerge even before the old branches are completely withdrawn. Third, in the locomotory stage, microglia begin translocating within the tissue (up to 118 microm/h) only after the new protrusions emerge. We conclude that the rapid conversion of resting ramified microglia to active amoeboid macrophages is accomplished not by converting quiescent branches to dynamic ones, but rather by replacing existing branches with an entirely new set of highly motile protrusions. This suggests that the ramified branches of resting microglia are normally incapable of rapid morphological dynamics necessary for activated microglial function. More generally, our time-lapse observations identify changes in the dynamic behavior of activating microglia and thereby help define distinct temporal and functional stages of activation for further investigation.

Polymerizing microtubules activate site-directed F-actin assembly in nerve growth cones.

We identify an actin-based protrusive structure in growth cones termed "intrapodium." Unlike filopodia, intrapodia are initiated exclusively within lamellipodia and elongate in a continuous (nonsaltatory) manner parallel to the plane of the dorsal plasma membrane causing a ridge-like protrusion. Intrapodia resemble the actin-rich structures induced by intracellular pathogens (e.g., Listeria) or by extracellular beads. Cytochalasin B inhibits intrapodial elongation and removal of cytochalasin B produced a burst of intrapodial activity. Electron microscopic studies revealed that lamellipodial intrapodia contain both short and long actin filaments oriented with their barbed ends toward the membrane surface or advancing end. Our data suggest an interaction between microtubule endings and intrapodia formation. Disruption of microtubules by acute nocodazole treatment decreased intrapodia frequency, and washout of nocodazole or addition of the microtubule-stabilizing drug Taxol caused a burst of intrapodia formation. Furthermore, individual microtubule ends were found near intrapodia initiation sites. Thus, microtubule ends or associated structures may regulate these actin-dependent structures. We propose that intrapodia are the consequence of an early step in a cascade of events that leads to the development of F-actin-associated plasma membrane specializations.

Confocal imaging of microglial cell dynamics in hippocampal slice cultures.

Methods are described for imaging the cellular dynamics of microglia in live mammalian brain slice cultures. Brain slices prepared from developing rat hippocampus are cultured for up to 2 weeks by the roller tube or static filter culture technique, stained with one or more fluorescent dyes, and imaged by scanning laser confocal microscopy. One of several cell type-specific or nonspecific fluorescent dyes can be used independently or in combination to label cells in live brain tissues. The fluorescently conjugated plant isolectin GSA-IB4 is useful for identifying microglia and for following their structure, movement, and proliferation. Live and dead neurons and glia can be distinguished using membrane-permeant and -impermeant fluorescent nucleic acid dyes. Nonspecific fluorescent lipids such as DiIC18 can be used as a vital stain to label populations of endocytic and phagocytic cells. Using multichannel confocal imaging, tissue slices that are single-, double-, or triple-labeled can be imaged in the living state in two or three spatial dimensions as well as in time. This provides a means for investigating the cell-cell interaction and dynamic behavior of microglia and other cell types in live brain tissues cultured under various physiological conditions.

Hormone replacement therapy and other potential treatments for dementias.

The past decade has seen a substantial increase in the number of individuals affected by dementia. Dementia places a tremendous personal and economic burden on millions of patients and caregivers annually. Consequently, many scientists have been searching for a treatment for dementia to avoid the imminent public health crisis that will occur if this trend continues. Primary and secondary prevention studies, as well as animal research, demonstrate the potential for hormone replacement therapy (HRT) as an efficacious treatment for dementia. Recently, the Women's Health Initiative-Memory Study began the first randomized, longterm clinical trial to test the hypothesized role of HRT at the onset and in the progression of dementia in women. Researchers also are investigating the potential of other treatments for dementias, such as nonsteroidal anti-inflammatory drugs and free radical scavengers.

The dynamics of dendritic structure in developing hippocampal slices.

Time-lapse fluorescence confocal microscopy was used to directly visualize the formation and dynamics of postsynaptic target structures (i.e., dendritic branches and spines) on pyramidal neurons within developing tissue slices. Within a 2 week period of time, pyramidal neurons in cultured slices derived from early postnatal rat (postnatal days 2-7) developed complex dendritic arbors bearing numerous postsynaptic spines. At early stages (1-2 d in vitro), many fine filopodial protrusions on dendrite shafts rapidly extended (maximum rate approximately 2.5 microM/minute) and retracted (median filopodial lifetime, 10 min), but some filopodia transformed into growth cones and nascent dendrite branches. As dendritic arbors matured, the population of fleeting lateral filopodia was replaced by spine-like structures having a low rate of turnover. This developmental progression involved a transitional stage in which dendrites were dominated by persistent (up to 22 hr) but dynamic spiny protrusions (i.e., protospines) that showed substantial changes in length and shape on a timescale of minutes. These observations reveal a highly dynamic state of postsynaptic target structures that may actively contribute to the formation and plasticity of synaptic connections during CNS development.

Mossy fiber growth and synaptogenesis in rat hippocampal slices in vitro.

Hippocampal slices from early postnatal rat were used to study mossy fiber (MF) growth and synaptogenesis. The ability of MFs to form new giant synapses within isolated tissue slices was established by a series of experiments involving synapsin I immunohistochemistry, electron microscopy, and whole-cell recordings. When hippocampal slices from immature rats were cultured for up to 2 weeks, the distribution of giant MF terminals was similar to that found in vivo. Using a lesioning procedure, we determined that MFs in slices extend and form appropriate synaptic connections with normal target CA3 pyramidal cells. MF terminals were dispersed more widely than normal within the CA3 pyramidal layer after a lesion, but electron microscopy indicated that synaptic junctions were still primarily associated with pyramidal cell dendrites and not the somata. Establishment of functional synaptic input in vitro was confirmed by whole-cell recordings of MF-driven excitatory postsynaptic currents (50 pA to 1 nA) in pyramidal cells. The results establish for the first time that an MF projection with appropriate and functional synaptic connections can be formed de novo and not just maintained in excised hippocampal slices. The cellular dynamics underlying MF growth and synaptogenesis were examined directly by time-lapse confocal imaging of fibers selectively stained with a fluorescent membrane dye (Dil or DiO). MFs growing deep within isolated tissue slices were tipped by small (5-10 microns), active growth cones that advanced at variable rates (5-25 microns/hr). Furthermore, dynamic filopodial structures were seen at small varicosities along the length of developing MFs, which may identify nascent en passant synaptic contacts. The hippocampal slice preparations are shown to support normal development of MF connections and allow for direct visualization of the cellular dynamics of synapse formation in a mammalian CNS tissue environment.

Spontaneous Ca2+ transients in developing hippocampal pyramidal cells.

To determine the spatiotemporal pattern of hippocampal pyramidal cell activity during development, we examined cytosolic Ca2+ dynamics in tissue slices derived from early postnatal rats. After a brief (12-60 h) culture period, slices were stained with a calcium-sensitive dye, Fluo-3. Fluorescence imaging of the Fluo-3-stained slices with a scanning laser confocal microscope afforded simultaneous observation of many cells at high spatial resolution. Time-lapse imaging revealed spontaneous Ca2+ transients in the somata dendrites of many pyramidal cells in areas CA1 and CA3. For the most part, Ca2+ activity in neighboring pyramidal cells appeared to be uncorrelated, although we occasionally observed synchronous Ca2+ transients in adjacent cells. The transients were blocked by both tetrodotoxin (1 microM) and a mixture of the glutamate receptor antagonists, APV (50 microM) + CNQX (10 microM). Thus, spontaneous Ca2+ transients appear to be a consequence of activity-dependent release of glutamate acting postsynaptically through ionotropic glutamate receptors. Although gamma-aminobutyric acid (GABA) is thought to be an excitatory neurotransmitter during hippocampal development (Cherubini et al., 1991, Trends Neurosci. 14 (12):515-519), the spontaneous Ca2+ transients were not blocked by the GABAA receptor antagonist picrotoxin (100 microM). Furthermore, application of GABA (50 microM) abolished the spontaneous Ca2+ events, possibly via GABAB receptor-mediated inhibition of postsynaptic cells. The present results join other recent observations suggesting that isolated neural tissues support spontaneous activity, although the patterns and mechanisms of the activity reported here appear to differ from those of previous studies. Differences in the patterns of spontaneous activity during development may contribute to variations in the functional organization of different regions of CNS tissue.

Vacuole dynamics in growth cones: correlated EM and video observations.

The neuronal growth cone is a major site of surface membrane dynamics associated with uptake and release of materials, motility, and axon extension. Although intracellular membrane organelles are thought to mediate surface membrane addition and retrieval at the growth cone, membrane events are fleeting and therefore difficult to study directly. In an effort to capture transient interactions between intracellular membrane organelles and the plasmalemma at the growth cone, embryonic rat sympathetic neuron cultures were prepared for whole-mount electron microscopy (EM) by rapid freezing and freeze substitution. We identified a set of vacuole-like organelles (> or = 150 nm in diameter) that appeared to interact directly with the plasmalemma. In stereo-pair EM images the bounding membrane of some of these vacuoles had an orifice at sites where the organelle was adjoining the plasmalemma, suggesting that the organelle and surface membranes were confluent. Since this population of organelles could be labeled with cationized ferritin or HRP when added to living cultures just prior to freezing or chemical fixation, they were probably derived from the plasmalemma. Combined light microscopy and EM of individual growth cones showed that these same vacuoles had a conspicuous reverse shadowcast appearance in differential interference contrast images. Thus, we used real-time video microscopy to follow these organelles in living growth cones. Many of these vacuoles spontaneously appeared, remained visible for several minutes, and then disappeared. Reverse shadowcast vacuoles were formed at various sites throughout the growth cone, including surface membrane ruffles at the leading edge [P (peripheral)-domain] as well as quiescent and retracting regions at the growth cone base [C (central)-domain]. Vacuoles in the P-domain moved centripetally and rarely grew in size. In contrast, those in the C-domain exhibited Brownian-like movements and sometimes appeared to increase in size, raising the possibility that new membrane may be added to these organelles. Vacuoles within both the P- and C-domains shrank before rapidly disappearing, but rarely vesiculated, suggesting that they had fused with the plasmalemma. The results indicate that vacuoles are a highly dynamic population of organelles that directly communicate with the plasma membrane at the growth cone; they provide a major route of surface membrane uptake and may also play a role in membrane recycling.

Confocal imaging of mossy fiber growth in live hippocampal slices.

Acutely isolated slices of developing rat hippocampus have been used to study axon growth and synapse formation. Mossy fibers, which are the axons of dentate granule cells, were labeled in living brain slices by injection of a fluorescent membrane dye (DiI or DiO) into the dentate gyrus. Time-lapse observations were made in area CA3 at a time when mossy fibers are normally growing in and forming en passant synapses with pyramidal neurons. Single scan images were collected at 1-2 min intervals over a period of several hours using a scanning laser confocal microscope. At the tips of growing mossy fibers were highly motile growth cones with several filopodia and small lamellae. Labeled fibers typically extended at rates up to 15 microns/h, but occasionally individual axons abruptly stopped elongating and the leading growth cone became quiescent. In addition, dynamic filopodia-like structures were found to be associated with axonal varicosities proximal to the leading growth cone. We are currently pursuing methods to determine whether these motile activities correlate with synapse formation.

Diverse migratory pathways in the developing cerebral cortex.

During early development of the mammalian cerebral cortex, young neurons migrate outward from the site of their final mitosis in the ventricular zone into the cortical plate, where they form the adult cortex. Time-lapse confocal microscopy was used to observe directly the dynamic behaviors of migrating cells in living slices of developing cortex. The majority of cells migrated along a radial pathway, consistent with the view that cortical neurons migrate along radial glial fibers. A fraction of cells, however, turned within the intermediate zone and migrated orthogonal to the radial fibers. This orthogonal migration may contribute to the tangential dispersion of clonally related cortical neurons.

Structure and organization of membrane organelles along distal microtubule segments in growth cones.

Advance and stabilization of organelle-rich cytoplasm within the neuronal growth cone is coupled to axon elongation (Goldberg and Burmeister, 1986; Aletta and Greene, 1988), and this involves forward movement of organelles from the growth cone base along distinct tracks toward the leading edge. Membrane-bound organelles that advance first within the growth cone often make transient excursions toward the leading edge, and at the light microscope level these leading organelles appear to colocalize with distal microtubule (MT) segments (Dailey and Bridgman, 1989). We have used electron microscopy (EM) to identify the membranous organelles adjacent to distal MT segments, and to examine their structural interactions with MTs. In both glutaraldehyde-fixed and rapid frozen whole-mount growth cones, attenuated endoplasmic reticulum (ER)-like membrane elements were the most common organelle type located adjacent to distal MT segments. These ER-like membrane elements coursed roughly parallel to MTs and frequently terminated within an electron-dense bulb at the MT tip. Blind-ended membrane tubes, dense-core vesicles, clear vesicles, and vacuoles were also found adjacent to distal MT segments. Quantitative analyses of organelle-MT associations suggest that elements of the ER-like membrane system may frequently advance ahead of other membrane-bound organelles. Freeze-etch EM revealed crossbridging structures between MTs and membranous organelles, which is consistent with the idea that advance of leading membrane organelles into the growth cone periphery is mediated by microtubule-based motor transport mechanisms. The results suggest that distal microtubule segments serve as transport elements for advance of membrane organelles into more peripheral growth cone regions, and together MTs and ER-like membrane organelles may initiate the conversion of dynamic F-actin-rich cytoplasm to more stable organelle-rich cytoplasm (i.e., axoplasm).

Airway compression secondary to left atrial enlargement and increased pulmonary artery pressure.

Although congenital cardiac defects are infrequently considered a cause of major airway compression in neonates and infants, patients with left-sided cardiac enlargement can develop compression of the left mainstem bronchus. This is a consequence of the intimate relationship of the trachea and left mainstem bronchus to the left atrium, left pulmonary veins and left pulmonary artery. If the mean pulmonary arterial pressure, mean left atrial pressure and carinal angle are increased, the likelihood of major airway compression is high.

Dynamics of the endoplasmic reticulum and other membranous organelles in growth cones of cultured neurons.

The fluorescent lipophilic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] was used to examine the distribution of membrane-bound organelles in growth cones of cultured rat sympathetic neurons. Within chemically fixed growth cones, intense DiOC6(3) fluorescence was localized predominately to the base or central region of growth cones. However, in most growth cones several thin DiOC6(3)-fluorescent processes radiated from the base into the periphery, and double fluorescence imaging of single growth cones indicated that these processes were highly colocalized (approximately 79%) with microtubules. The distribution of DiOC6(3) fluorescence in living growth cones was examined using low light-level fluorescence video microscopy. We observed thin fluorescent processes within the periphery of growth cones to undergo length excursions (extension/retraction) and to change orientation (move laterally). During growth cone advance, processes became progressively thicker and were gradually engulfed by the advancing fluorescent mass. When growth cones were viewed with video-enhanced differential interference contrast microscopy, the position of the fluorescent processes correlated with thickened extensions of central-type cytoplasm through which vesiclelike organelle transport often occurred. These observations indicate several features concerning the organization and movement of membranous organelles (MOs) in growth cones: (1) MOs are highly compartmentalized, the majority being localized to the growth cone base; (2) MOs advance into the periphery along distinct pathways probably associated with microtubules; (3) one or more thin continuous MOs, which most likely represent a thin tubular component of the endoplasmic reticulum, generally precedes advance of vesiclelike MOs along individual transport pathways; and (4) transport pathways with their associated MOs are spatially and temporally dynamic.

The organization of myosin and actin in rapid frozen nerve growth cones.

Rapid freezing and freeze substitution were used in conjunction with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin in growth cones of cultured rat superior cervical ganglion neurons. The general cytoplasmic organization was determined by whole mount EM; tight microfilament bundles formed the core of filopodia while a dense meshwork formed the underlying structure of lamellipodia. Although the central microtubule and organelle-rich region of the growth cone had fewer microfilaments, dense foci and bundles of microfilaments were usually observed. Anti-actin immunofluorescence and rhodamine phalloidin staining of f-actin both showed intense staining of filopodia and lamellipodia. In addition, staining of bundles and foci were observed in central regions suggesting that the majority of the microfilaments seen by whole mount EM are actin filaments. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern. Although less intense, anti-myosin staining was also seen in peripheral regions; it was most prominent at the border with the central region, in portions of lamellipodia undergoing ruffling, and in spots along the shaft and at the base of filopodia. Immunoelectron microscopy of myosin using postembedment labeling with colloidal gold showed a similar distribution to that seen by immunofluorescence. Label was scattered throughout the growth cone, but present as distinct aggregates in the peripheral region mainly along the border with the central region. Less frequently, aggregates were also seen centrally and along the shaft and at the base of filopodia. This distribution is consistent with myosins involvement in the production of tension and movements of growth cone filopodia and lamellipodia that occur during active neurite elongation.