Developmental characteristics of dendritic spines in the dentate gyrus of Fmr1 knockout mice.

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The neuroanatomical phenotype of adult FXS patients, as well as adult Fmr1 knockout (KO) mice, includes elevated dendritic spine density and a spine morphology profile in neocortex that resembles younger individuals. Developmental studies in mouse neocortex have revealed a dynamic phenotype that varies with age, especially during the period of synaptic pruning. Here we investigated the hippocampal dentate gyrus to determine if the FXS spine phenotype is similarly tied to periods of maturation and pruning in this brain region. We used high-voltage electron microscopy to characterize Golgi-stained spines along granule cell dendrites in Fmr1 KO and wildtype (WT) mouse dentate gyrus at postnatal days 15, 21, 30, and 60. In contrast to neocortex, dendritic spine density was higher in Fmr1 KO mice across development. Interestingly, neither genotype showed specific phases of synaptogenesis or pruning, potentially explaining the phenotypic differences from neocortex. Similarly, although the KO mice showed a more immature morphological phenotype overall than WT (higher proportion of thin headed spines, lower proportion of mushroom and stubby spines), both genotypes showed gradual development, rather than impairments during specific phases of maturation. Finally, spine length showed a complex developmental pattern that differs from other brain regions examined, suggesting dynamic regulation by FMRP and other brain region-specific proteins. These findings shed new light on FMRP's role in development and highlight the need for new techniques to further understand the mechanisms by which FMRP affects synaptic maturation.

Differential regulation of Purkinje cell dendritic spines in rolling mouse Nagoya (tg/tg), P/Q type calcium channel (α1(A)/Ca(v)2.1) mutant.

Voltage dependent calcium channels (VDCC) participate in regulation of neuronal Ca(2+). The Rolling mouse Nagoya (Cacna1a(tg-rol)) is a spontaneous P/Q type VDCC mutant, which has been suggested as an animal model for some human neurological diseases such as autosomal dominant cerebellar ataxia (SCA6), familial hemiplegic migraine and episodic ataxia type-2. Morphology of Purkinje cell (PC) dendritic spine is suggested to be regulated by signal molecules such as Ca(2+) and by interactions with afferent inputs. The amplitude of excitatory postsynaptic current was decreased in parallel fiber (PF) to PC synapses, whereas apparently increased in climbing fiber (CF) to PC synapses in rolling mice Nagoya. We have studied synaptic morphology changes in cerebella of this mutant strain. We previously found altered synapses between PF varicosity and PC dendritic spines. To study dendritic spine plasticity of PC in the condition of insufficient P/Q type VDCC function, we used high voltage electron microscopy (HVEM). We measured the density and length of PC dendritic spines at tertiary braches. We observed statistically a significant decrease in spine density as well as shorter spine length in rolling mice compared to wild type mice at tertiary dendritic braches. In proximal PC dendrites, however, there were more numerous dendritic spines in rolling mice Nagoya. The differential regulation of rolling PC spines at tertiary and proximal dendrites in rolling mice Nagoya suggests that two major excitatory afferent systems may be regulated reciprocally in the cerebellum of rolling mouse Nagoya.

High-voltage electron microscopy reveals direct synaptic inputs from a spinal gastrin-releasing peptide system to neurons of the spinal nucleus of bulbocavernosus.

The spinal nucleus of bulbocavernosus (SNB) is a sexually dimorphic motor nucleus located in the anterior horn of the fifth and sixth lumbar segments of the spinal cord that plays a significant role in male sexual function. We recently found that a sexually dimorphic expression of gastrin-releasing peptide (GRP) in the lumbar spinal cord regulates male copulatory reflexes. Although it is reported that these systems are both profoundly regulated by circulating androgen levels in male rats, no direct evidence has been reported regarding GRP synaptic inputs onto SNB motoneurons. The aim of the current study was to determine the axodendritic synaptic inputs of spinal GRP neurons to SNB motoneurons. Immunoelectron microscopy, combined with a retrograde tracing technique using high-voltage electron microscopy (HVEM), provided a three-dimensional visualization of synaptic contacts from the GRP system in the lumbar spinal cord onto SNB motoneurons. HVEM analysis clearly demonstrated that GRP-immunoreactive axon terminals directly contact dendrites that extend into the dorsal gray commissure from the SNB. These HVEM findings provide an ultrastructural basis for understanding how the spinal GRP system regulates male sexual behavior.

Three-dimensional structure of the cytoskeleton in Trichomonas vaginalis revealed new features.

The flagellated protozoan Trichomonas vaginalis has been widely studied owing to its medical significance and unique structure. The complicated three-dimensional (3D) structure of the cellular components of T. vaginalis was reconstructed from serial sections to enable observation of the spatial features of the whole cell. Electron tomography was used to examine the detailed structure of the cellular organelles. Tomographic reconstruction showed the mastigont system and the parabasal filament of T. vaginalis in detail. The last thin filament (Pf3) was located close to the adjacent filament, and the two filaments appeared to be vertically parallel in the cross-sectional view. It is likely that Pf3 cannot be distinguished from the adjacent filament in 2D images obtained from transmission electron microscopy. Our 3D reconstruction of T. vaginalis revealed the presence of an additional striated fiber, and 3D reconstruction by electron tomography showed twisting of the split parabasal filament.

Cytokinesis of neuroepithelial cells can divide their basal process before anaphase.

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time-lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal-to-apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F-actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP-anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal-to-apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M-phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.

Morphological changes in dendritic spines of Purkinje cells associated with motor learning.

Experience-dependent changes of spine structure and number may contribute to long-term memory storage. Although several studies demonstrated structural spine plasticity following associative learning, there is limited evidence associating motor learning with alteration of spine morphology. Here, we investigated this issue in the cerebellar Purkinje cells using high voltage electron microscopy (HVEM). Adult rats were trained in an obstacle course, demanding significant motor coordination to complete. Control animals either traversed an obstacle-free runway or remained sedentary. Quantitative analysis of spine morphology showed that the density and length of dendritic spines along the distal dendrites of Purkinje cells were significantly increased in the rats that learned complex motor skills compared to active or inactive controls. Classification of spines into shape categories indicated that the increased spine density and length after motor learning was mainly attributable to an increase in thin spines. These findings suggest that motor learning induces structural spine plasticity in the cerebellar Purkinje neurons, which may play a crucial role in acquiring complex motor skills.

Three-dimensional, computer-tomographic analysis of membrane proteins (TrkA, caveolin, clathrin) in PC12 cells.

Signaling of nerve growth factor (NGF) and its receptor (TrkA) promotes neuronal differentiation, synapse formation and survival. It has been known that the complex of NGF and TrkA is internalized into the cytoplasm and transported for further signal transduction, but the ultrastructural information of this process is virtually unknown. In order to clarify the relationship between the internalization of TrkA and the membrane-associated proteins (caveolin and clathrin), the localization and three-dimensional structures of those proteins were examined with computer tomography of high voltage electron microscopy in PC12 cells. TrkA immunoreactivity was found only at definite areas in the plasma membrane, as ring and cluster structures. Its 3D image indicated that those cluster structures contained small pits, which did not appear to be typical caveolae in size and shape. 3D images of clathrin and caveolin-1 immunoreactivities indicated that the formation of those small pits was associated with clathrin, but not with caveolin-1. Caveolin-1 immunoreactivity was found as a mesh-like structure just beneath the plasma membrane. These results suggest that clathrin rather than caveolin is mainly involved in the process of TrkA internalization, at least in differentiated PC12 cells.

Ultrastructural localization of the neurotrophin receptor (TrkA) in cultured rat pheochromocytoma PC12 Cells: three-dimensional image analysis by high voltage electron microscopy.

Nerve growth factor (NGF) is a well-known neurotrophic factor and the NGF signaling through the receptor, TrkA, plays important roles in regulating neuronal differentiation and survival. A recent study has demonstrated that the TrkAs expressed in undifferentiated PC12 cells were associated with caveolae, which were invaginated small pits on the plasma membrane. Caveolae are frequently seen in many cell types such as endothelial cells, fibroblasts and hepatocytes, but few in neurons. In the present study, we performed immunocytochemistry of TrkA in differentiated PC12 cells and analyzed the ultrastructural localization of TrkA by conventional electron microscopy and high-voltage electron microscopic (HVEM) tomography. The TrkA immunoreactivities were mainly associated with the cytoplasmic vesicles (10-30 nm in diameter) and a part of the plasma membrane. The HVEM tomography showed that the TrkA immunoreactivities were often assembled into ring-like structures (400-800 nm in diameter) near the plasma membrane, unlike typical flask-shaped invaginations of caveolae (50-100 nm in diameter). These results suggest that TrkA are not localized in the caveolae, at least in differentiated PC12 cells, but other invaginations are involved in a novel process of internalization of ligand-bound TrkA.

Tri-dimensional morphometric analysis of astrocytic processes with high voltage electron microscopy of thick Golgi preparations.

A characteristic feature of the astrocytic processes is to assume the form of shin sheets or lamellate coverings of other brain constituents. We analyzed the extensive and finely divided processes of the protoplasmic astrocyte in the molecular layer of the rat dentate gyrus by means of computer electron tomography and stereo-photogrammetry using tilted high voltage electron microscope images of thick Golgi preparations. The surface area and volume of the astrocytic processes were measured and the surface/volume ratios were estimated. The surface/volume ratios of astrocytic processes in the neuropile ranged from 18.9 to 33.0 per microm, and the mean value was 26.2+/-5.0 per microm. The values were roughly comparable to those previously reported for the microdomain of Bergmann glia cell terminal processes in the rat cerebellum, which were estimated from reconstructions using thin serial section electron microscope images. The large surface to volume ratio of the astrocytic processes in the neuropile resulted from the lamellar nature of the processes interposed between other cellular elements, and may reflect the functional activities of the astrocyte. The results suggest the usefulness of the electron tomography and stereo-photogrammetry for three-dimensional morphometrical analysis of the astrocytic processes, although both techniques can be expected to be refined further in order to provide more precise measurements of these complicated processes.

Bidirectional alterations in cerebellar synaptic transmission of tottering and rolling Ca2+ channel mutant mice.

Hereditary ataxic mice, tottering (tg) and rolling Nagoya (tg(rol)), carry mutations in the P/Q-type Ca(2+) channel alpha(1A) subunit gene. The positions of the mutations and the neurological phenotypes are known, but the mechanisms of how the mutations cause the symptoms and how the different mutations lead to various onset and severity have remained unsolved. Here we compared fundamental properties of excitatory synaptic transmission in the cerebellum and roles of Ca(2+) channel subtypes therein among wild-type control, tg, and tg(rol) mice. The amplitude of EPSC of the parallel fiber-Purkinje cell (PF-PC) synapses was considerably reduced in ataxic tg(rol). Although the amplitude of the parallel fiber-mediated EPSC was only mildly decreased in young non-ataxic tg mice, it was drastically diminished in adult ataxic tg mice of postnatal day 28-35, showing a good correlation between the impairment of the PF-PC synaptic transmission and manifestation of ataxia. In contrast, the EPSC amplitude of the climbing fiber-Purkinje cell (CF-PC) synapses was preserved in tg, and it was even increased in tg(rol), which was associated with altered properties of the postsynaptic glutamate receptors. The climbing fiber-mediated EPSC was more dependent on other Ca(2+) channel subtypes in mutant mice, suggesting that such compensatory mechanisms contribute to maintaining the CF-PC synaptic transmission virtually intact. The results indicate that different mutations of the P/Q-type Ca(2+) channel not only cause the primary effect of different severity but also lead to diverse additional secondary effects, resulting in disruption of well balanced neural networks.

Measurements of electron beam damage for organic crystals in a high voltage electron microscope with image plates.

The critical dose for extinction of the diffraction pattern from behenic acid monolayer crystals increased with increasing accelerating voltages. The mean values at 114, 500 and 1000kV were 0.6, 1.8 and 2.2 electrons/A(2), respectively. The critical dose at 500-1000kV is three or more times as large as that at 114kV. Considering this with the recent measured value of the detective quantum efficiency of the image plate, 0.5 at 500kV and 0.4 at 1000kV, 1.5 times as much information can be collected from a crystal at 500-1000kV as at 114kV. Therefore, the combined use of high voltage electron microscopy and image plate detectors offers a significant improvement over conventional electron microscopy approaches for the study of radiation sensitive organic molecule crystals.