Journal of Neurocytology最新文献

Cultured hippocampal slices prepared from apolipoprotein E (apoE)-deficient mice were exposed to an inhibitor of cathepsins B and L and then processed for an ultrastructural analysis of neuronal features for pyramidal cell bodies. Electron microscopy showed that the nuclei of pyramidal cells from treated hippocampal slices were more eccentrically located than those from untreated slices. In addition, increased numbers of vesicles were associated with the Golgi complex while microtubules were less frequent in the proximal dendrites. Consistent with previous studies in rats, treated apoE-deficient slices had increased numbers of lysosomes and multivesicular bodies. Finally, there were reductions in the number of synapses around the cell body, a finding similar to that found in the brains from Alzheimer's disease patients. These results provide ultrastructural data indicating that partial lysosomal dysfunction in apoE-deficient brains rapidly induces characteristic features of the aged human brain.

Primary sensory information from neurons innervating whisker follicles on one side of a rat's face is relayed primarily through two subnuclei of the brainstem trigeminal complex to the contralateral thalamus. The present experiments were undertaken to separate the contribution of the principal trigeminal nucleus (PrV) from that of the spinal trigeminal nucleus (SpV) to whisker evoked responses in the ventral posterior medial (VPM) nucleus in the adult rat thalamus. Extracellular single-unit responses of VPM neurons to controlled stimulation of the contralateral whiskers under urethane anesthesia were quantified in terms of receptive field size, modal latency, response probability and response magnitude. The SpV contribution to VPM cell responses was isolated by making kainic acid lesions of the PrV. The PrV contribution was ascertained by cutting the trigeminothalamic axons arising from SpV just before they cross the midline. After destruction of the PrV, the SpV pathway alone produced large receptive fields (mean: 9.04 whiskers) and long latency (mean: 11.07 ms) responses from VPM neurons. In contrast, PrV input alone (SpV disconnected) generated small receptive fields (mean: 1.06 whiskers) and shorter latency (mean: 6.74 ms) responses. With both pathways intact the average receptive field size was 2.4 whiskers and peak (modal) response latency was 7.33 ms. The responses with both pathways intact were significantly different from either pathway operating in isolation. Response probability and magnitude followed the same trend. We conclude that normal responses of individual VPM neurons represent the integration of input activity transmitted through both PrV and SpV pathways.

Abundant evidence indicates important functional differences between the two cerebral hemispheres of humans, although the cellular basis of these differences is unknown. A recent hypothesis proposes that these functional differences depend on differences between sides in the "repertoire" of axonal conduction delays for cortico-cortical axons. In morphological terms this corresponds to differences in caliber, or proportion, of myelinated versus unmyelinated axons. Several behavioural studies have indicated that cerebral asymmetry occurs in rodents, in which rigorous morphological analysis is possible. The hypothesis was therefore tested for the first time in adult male Wistar rats, using transmission electron microscopy and stereological methods. Subcortical white matter was compared between left and right sides in three regions (frontal, parietal, and occipital). The average caliber and numerical density of unmyelinated and myelinated axons was compared between sides and between regions. All data were corrected for shrinkage. No significant differences between sides were found in the average caliber of either type of axon in any region. The numerical density of either type of axon also yielded no significant differences between sides in any region. Significant differences were evident between regions in both caliber and numerical density of the two axonal types, and these quantitative data are reported. The proportion of unmyelinated axons in the lateral white matter was also higher than in previous studies of hemispheric white matter that studied the corpus callosum. The present study provides no evidence supporting the hypothesis that functional hemispheric specialization is due to differences in axonal number, caliber or type.

Brain derived neurotrophic factor (BDNF) when added to explant cultures of both embryonic and adult retinal ganglion cell (RGC) axons exerted a marked effect on their growth cone size and complexity and also on the intensity of GAP-43, beta-III tubulin and F-actin immunoreaction product in their axons. GAP-43 was distributed in axons, lamellipodia, and filopodia whereas beta-III tubulin was distributed along the length of developing and adult regenerating axons and also in the C-domain of their growth cones. BDNF-treated developing RGC growth cones were larger and displayed increased numbers of GAP-43 and microtubule-containing branches. Although filopodia and lamellipodia were lost from both developing and adult RGC growth cones following trkB-IgG treatment, the intensity of the immunoreaction product of all these molecules was reduced and trkB-IgGs had no effect on the axonal distribution of betas-III tubulin and GAP-43. BDNF-treated growth cones also displayed increased numbers of F-actin containing filopodia and axonal protrusions. This study demonstrates, for the first time, that trkB-IgG treatment causes the loss of F-actin in the P-domain of growth cone tips in developing and regenerating RGC axons. Although microtubules and F-actin domains normally remained distinct in cultured growth cones, beta-III tubulin and F-actin overlapped within the growth cone C-domain, and within axonal protrusions of adult RGC axons, under higher concentrations of BDNF. The collapse of RGC growth cones appeared to correlate with the loss of F-actin. In vitro, trkB signalling may therefore be involved in the maintenance and stabilisation of RGC axons, by influencing F-actin polymerisation, stabilisation and distribution.

Transgenic mice expressing mutant (P301L) human tau develop neurofibrillary tangles, amyotrophy and progressive motor disturbance. We present ultrastructural features of neuronal degeneration in this model that suggests involvement of both neurofibrillary and autophagic processes in neurodegeneration. Neurons undergoing neurofibrillary degeneration contain tau-immunoreactive, 15-20 nm-wide straight or wavy filaments with no periodic twists. Tau filaments were found in two types of affected neurons. One type resembled neurons with neurofibrillary tangles (NFT) that were filled with numerous filaments that displaced sparse cytoplasmic organelles to the periphery. Microtubules were almost completely absent. The nucleus remained centrally located, but showed lobulations due to deep infoldings. The other type resembled ballooned neurons seen in some human tauopathies. The nucleus was peripherally placed, but normal appearing. The cytoplasmic organelles were dispersed throughout the swollen perikarya, the Golgi complex was fragmented and duplicated, while mitochondria and other organelles appeared normal. Tau filaments similar to those in NFT were sparse and not tightly packed. Microtubules were also sparse. Many autophagic vacuoles were present in these cells. Heterogeneous appearing axonal swellings resembling spheroids in human tauopathies were present in gray and white matter. Unlike normal appearing axons, axonal spheroids were filled with tau-immunoreactive filaments and autophagic vacuoles, in addition to normal appearing neurofilaments and microtubules. These P301L transgenic mice exhibit many features common to human tauopathies, making them a valuable model to study the pathogenesis of these uncommon disorders.

Stereocilia side links are directly involved in the maintenance of stereociliary bundle integrity in hair cells. The structure of the stereocilia side links and morphology of the auditory hair bundle in relation to noise exposure in the chinchilla was investigated by transmission electron microscopy. The outer hair cell (OHC) stereocilia side link was suggested to consist of extracellular, juxta-membrane and thin filamentous regions. Two beaded filaments were folded at their distal ends and fastened in one globule in the center between stereocilia. An intracellular, submembraneous layer appeared to form a bridge between the actin core and the extracellular, juxta-membrane region of the side link. In normal physiological conditions, most OHC stereocilia had a regular distribution of side links, forming a 'zipper-like' lattice between stereocilium shafts. Side links of the inner hair cell (IHC) stereocilia had a similar filamentous appearance, but were observed less commonly and had decreased structural organization compared to those of the OHC stereocilia. Ultrastructural analysis of OHC and IHC stereocilia showed that a large number of the side links could survive acoustic stimulation of 114 dB SPL for 2 hrs or 123 dB SPL for 15 min, that resulted in temporarily elevated hearing thresholds in all animals. Disarray, separation, close attachment and fusion of stereocilia were more frequently observed for IHC stereocilia and OHC stereocilia that were poorly connected or that lacked side links. Most disarrayed OHC and IHC stereocilia recovered to a normal erect state with restored orientation of the side links after 14-28 days, which correlated with near-complete recovery of auditory sensitivity. However, direct attachment of plasma membranes, ruptured links, fusion and blebs were seen on some stereocilia even after 28 days and appear to be permanent.

Matrix metalloproteinases are important regulators of extracellular matrix molecules and cell-cell signaling. Antibodies to matrix metalloproteinase 3 (MMP3) recognize molecules at the frog neuromuscular junction, and MMP3 can remove agrin from synaptic basal lamina (VanSaun & Werle, 2000). To gain insight into the possible roles of MMP3 at the neuromuscular junction, detailed observations were made on the structure and function of the neuromuscular junctions in MMP3 null mutant mice. Striking differences were found in the appearance of the postsynaptic apparatus of MMP3 null mutant mice. Endplates had an increased volume of AChR stained regions within the endplate structure, leaving only small regions devoid of AChRs. Individual postsynaptic gutters were wider, containing prominent lines that represent the AChRs concentrated at the tops of the junctional folds. Electron microscopy revealed a dramatic increase in the number and size of the junctional folds, in addition to ectopically located junctional folds. Electrophysiological recordings revealed no change in quantal content or MEPP frequency, but there was an increase in MEPP rise time in a subset of endplates. No differences were observed in the rate or extent of developmental synapse elimination. In vitro cleavage experiments revealed that MMP3 directly cleaves agrin. Increased agrin immunofluorescence was observed at the neuromuscular junctions of MMP3 null mutant mice. These results provide strong evidence that MMP3 is involved in the control of synaptic structure at the neuromuscular junction and they support the hypothesis that MMP3 is involved in the regulation of agrin at the neuromuscular junction.

BDNF and NT-4 (but not NT-3 or CNTF) significantly enhanced the outgrowth of early embryonic and adult regenerating RGC axons when provided with a supportive substrate in vitro. BDNF and NT-4 treatment transiently increased RGC axon outgrowth from E15 rat retinas but not from retinas at older embryonic ages. The transient effect of BDNF and NT-4 and the inability of the neurotrophins to promote outgrowth from older embryonic retinal explants suggests a time frame of neurotrophin action and that other chemical factors (target-derived or otherwise) may be necessary for the continued maintenance of developing RGC axons. BDNF and NT-4 also enhanced the outgrowth of regenerating axons from adult retinal explants, but appeared to have a more subtle effect on axon outgrowth, in that the growth-promoting effects of BDNF and NT-4 appeared continuous throughout the incubation period. The suppression of RGC axon outgrowth from embryonic and adult retinae cultured in trkB-IgG-containing medium suggests that the response of developing and regenerating axons, to BDNF and NT-4 are likely to occur through trkB signalling.