Journal of Neurocytology最新文献

Individual neurons dissected from immunohistochemically stained paraffin sections of the developing rat geniculate (VIIth cranial) ganglion were assayed for their content of mRNA of the neurotrophin receptor genes, p75 , trkA , trkB and trkC. Fetal and postnatal rats, from the 13th embryonic day (E13) until the 20th postnatal day (P20), were used. Single cells were subjected to RNA amplification, followed by treatment with reverse transcriptase and DNA amplification by the polymerase chain reaction (PCR). The identity of the PCR products was verified by subcloning and sequencing. A total of 227 neurons were examined, of which 212 (93%) gave a PCR signal for at least one neurotrophin receptor. We found: (1) Approximately half of the neurons expressed more than one receptor. (2) A truncated version of trkB , possessing the ligand-binding region but lacking the tyrosine kinase domain, occurred quite frequently, often in combination with the full-length trkB, with trkA or both. (3) The pattern of staining for trkB-like immunoreactivity was usually predictive that either its full length or truncated mRNA would be present. This was not the case for trkC-like immunoreactivity. Western blots on E15 brain tissue showed no band for full-length trkC ( approximately 150 kDa), suggesting the antibody may have been immunoreactive with a truncated ( approximately 120 kDa) but not a full-length version of the trkC receptor. (4) The pattern of neurotrophin receptor gene expression changed during development. (5) p75 expression occurred infrequently--in only 7 of the 212 neurons that gave a signal for any receptor.

The vanilloid receptor VR1 (TRPV1) is a temperature- and capsaicin-sensitive cation channel expressed by a class of primary afferents involved in nociception. To confirm the hypothesis that VR1-positive primary afferents are glutamatergic and contact spinal neurons that express the main classes of ionotropic glutamate receptors, we performed multiple immunofluorescent staining for VR1 and the glutamate transporter VGLUT2 (a specific marker for glutamatergic transmission) or AMPA and NMDA receptor subunits. VR1-positive cells in the dorsal root ganglion and boutons of their central afferent fibers in the dorsal horn expressed VGLUT2, and the latter contacted AMPA- or NMDA receptor-positive perikarya. Based on our previous observations of preferential targeting of VR1-positive primary afferents to spinal neurons that express the neurokinin receptor NK1 (Hwang et al., 2003), we further quantified the frequency of termination of VR1-positive afferents onto NK1-positive neurons co-expressing glutamate receptors. A larger fraction of NK1/NMDA receptors-positive than NK1/AMPA receptors-positive sites were contacted by VR1-positive boutons. We conclude that VR1-positive primary afferents in the rat use glutamate as neurotransmitter and contact postsynaptic sites that co-express NK1 and ionotropic glutamate receptors.

A variety of data suggest that noradrenaline and acetylcholine may interact in the basal forebrain, however no morphological studies have addressed whether indeed cholinergic neurons express adrenergic receptors. We have investigated the presence of alpha-adrenergic receptor subtype alpha2A-AR in cholinergic neurons of the basal forebrain. Cholinergic neurons were identified with an antibody against choline acetyltransferase and the receptor with a polyclonal antibody raised against a 47 amino acid fragment of the third intracellular loop of the alpha2A-AR. For double labeling at the light microscopic level the Ni-DAB/DAB technique was used, and for electron microscopy an immunoperoxidase/immunogold method was applied. We detected the alpha2A-AR protein in cholinergic as well as in non-cholinergic neurons. Almost half of all cholinergic neurons contained this adrenergic receptor. Double-labeled neurons were distributed throughout the rostro-caudal extent of the basal forebrain cholinergic continuum, including the medial septum, vertical and horizontal diagonal band nuclei, pallidal regions, substantia innominata and the internal capsule. Non-cholinergic neurons that expressed the alpha2A-AR outnumbered cholinergic/alpha2A-AR neurons by several factors. Electron microscopy confirmed the presence of alpha2A-AR in cholinergic neurons in the medial septum, vertical and horizontal diagonal band nuclei. Gold particles (10 nm) indicative of alpha2A-AR were diffusely distributed in the cytoplasm and accumulated in cytoplasmic areas near the Golgi complex and cysterns of the endoplasmic reticulum and were associated with the cellular membranes at synaptic and non-synaptic locations. Since many of the alpha2A-AR+/non-cholinergic neurons we detected are likely to be GABAergic cells, our data support the hypothesis that noradrenaline may act via basal forebrain cholinergic and non-cholinergic neurons to influence cortical activity.

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.

Labeling central nervous tissue from mature animals with antibodies to NG2 chondroitin sulfate proteoglycan reveals the existence of large numbers of NG2 positive cells, at least some of which are oligodendroglial progenitors. It is generally agreed that these cells differ from the classically defined neuroglia, since they are antigenetically different from astrocytes, oligodendrocytes, or microglial cells. Although the NG2 positive cells have been well characterized in light microscopic preparations, examination of the labeled cells by electron microscopy have not led to general agreement about their morphological features. The basic reason for this is that it is difficult to obtain good preservation of the fine structure of NG2 labeled neurons. Since these NG2 positive cells are abundant in the central nervous system, it was decided to examine routinely prepared tissue from the brains of mature monkeys and rats by electron microscopy to determine if there is a neuroglial cell type whose presence has been overlooked. It soon became evident that there is a fourth type of neuroglial cell. These cells have pale, irregular shaped nuclei with a thin rim of heterochromatin beneath the nuclear envelope, and they have pale cytoplasm. Superficially they resemble astrocytes, which is the probable reason why the presence of this fourth type of neuroglial cell has been largely overlooked. However, the fourth type of neuroglial cell, here referred to as a ss neuroglial cell, has no intermediate filaments in its cytoplasm, the mitochondria are thinner than those of astrocytes, centrioles are frequently encountered in their cytoplasm, and when they are adjacent to capillaries they are always separated from the basal membrane by an astrocytic processes.

Synaptic damage and loss are factors that affect the degree of dementia experienced in Alzheimer disease (AD) patients. Multicolor DiOlistic labeling of the hippocampus has been undertaken which allows the full dendritic arbor of targeted neurons to be imaged. Using this labeling technique the neuronal morphology of two transgenic mouse lines (J20 and APP/PS1) expressing mutant forms of the Amyloid Precursor Protein (APP), at various ages, have been visualized and compared to Wild Type (WT) littermate controls. Swollen bulbous dystrophic neurites with loss of spines were apparent in the transgenic animals. Upon quantification, statistically significant reductions in the number of spines and total dendrite area was observed in both transgenic mouse lines at 11 months of age. Similar morphological abnormalities were seen in human AD hippocampal tissue both qualitatively and quantitatively. Immunohistochemistry and DiOlistic labeling was combined so that Abeta plaques were imaged in relation to the dendritic trees. No preferential localization of these abnormal dystrophic neurites was seen in regions with plaques. DiI labeled reative astrocytes were often apparent in close proximity to A beta plaques.

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ss-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.

The hard palate of rodents is a mucous membrane covered by a keratinized epithelium that typically contains Merkel cell (MC)-neurite complexes. MCs have engendered considerable research activity because of their involvement in mechanoreception and possibly also Merkel cell carcinomas. MCs derive from the neural crest, differentiate under control of peripheral nerve factors, are enriched in large dense core vesicles, and secrete neuropeptides and other neuroactive molecules. Upon stimulation, MC-neurite complexes produce slowly adapting type I responses. Here we emphasize that the murine hard palate is a highly differentiated sensory region, as shown by intravital staining with a styryl dye and immunocytochemistry with antibodies to vesicular glutamate transporters (VGLUTs). The entire palate contained densities of sensory endings and MC-neurite complexes, that nearly paralleled in abundance the vibrissal pads. MCs were differentially distributed in the murine palate; clusters of MCs were most abundant in the antemolar and intermolar rugae, while individual MCs were particularly enriched in the rugae at the mid-portion of the palate and in the postrugal field. VGLUT1, VGLUT2 and VGLUT3 were expressed in MCs throughout, although immunostained MCs were most frequently encountered in intermolar than antemolar rugae. The same transporters were also present in corpuscular endings at the summit of the rugae and in intraepithelial free nerve endings throughout the palate. VGLUTs presumably load glutamate into large dense core vesicles in MCs and into small clear vesicles in corpuscular and free nerve endings. The data suggest that glutamate release, or co-release, is likely to represent an important functional aspect of palatine Merkel cells and neighboring corpuscular and free nerve endings.

Filamentous aggregates of the protein tau are a prominent feature of Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). However, the extent to which the molecular structure of the tau in these aggregates is similar or differs between these diseases is unclear. We approached this question by examining these disorders with a panel of antibodies that represent different structural, conformational, and cleavage-specific tau epitopes. Although each of these antibodies reveals AD pathology, they resolved into three classes with respect to PSP and CBD: AD2 and Tau-46.1 stained the most tau pathology in all cases; Tau-1, 2, 5, and 12 exhibited variable reactivity; and Tau-66 and MN423 did not reveal any tau pathology. In addition, hippocampal neurofibrillary tangles in these cases showed a predominantly PSP/CBD-like, rather than AD-like, staining pattern. These results indicate that the patterns of the tau epitopes represented by this panel that reside in the pathological aggregates of PSP and CBD are similar to each other but distinct from that of AD.

A previous study using the 2'3'cyclic nucleotide 3'phosphodiesterase (CNPase), an oligodendroglial marker that also stain ensheathed fibers, showed a decrease in the number of immunoreactive fibers and a change in the pattern of CNPase immunoreactivity (CNPase+) in hypothyroid animals. CNPase+ fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. As CNPase, in adult animals, can be found only in regions in which oligodendrocyte cytoplasm remains as internal, external and paranodal loops, it was suggested that the reduction of thyroid hormone levels leads to a failure in myelin compaction. Previous data showed a higher frequency of some abnormalities in myelin sheath as multiple cytoplasmic loops and redundant myelin profiles in mutant animals that present a failure in myelin compaction. The increase in the frequency of these abnormalities (multiple internal and external loops and redundant myelin) indicates a failure in the interrelations between the axons and the oligodendroglial processes. To verify if the thyroid hormone deficiency during CNS development disturbs these interrelations, we evaluated the frequency of the morphological abnormalities (multiple internal and external loops and redundant myelin) in myelin sheath of corpus callosum (cc) in experimental hypothyroidism. Randomic fields were kept by electron microscopy and the analysis of the frequency of morphological abnormalities showed a significant difference in hypothyroid animals at 60-day-old (PND60), with no significant differences at 90-day-old (PND90) animals. The frequency of multiple internal loops is higher in hypothyroid animals at PND60 that indicates a disturbance in the wrapping by the oligodendroglial process. These findings showed that thyroid hormone might modulate the axon-oligodendroglial relationships that are important for the adequate temporal sequence of events that occur during myelinogenesis, with possible consequences on myelin compaction.