Journal of Neurocytology最新文献

The present study aimed to elucidate the distribution of betaine/gamma-aminobutyric acid (GABA) transporter-1 (BGT-1) in the normal monkey cerebral neocortex and hippocampus by immunoperoxidase and Immunogold labelling. BGT-1 was observed in pyramidal neurons in the cerebral neocortex and the CA fields of the hippocampus. Large numbers of small diameter dendrites or dendritic spines were observed in the neuropil. These made asymmetrical synaptic contacts with unlabelled axon terminals containing small round vesicles, characteristic of glutamatergic terminals. BGT-1 label was observed in an extra-perisynaptic region, away from the post-synaptic density. Immunoreactivity was not observed in portions of dendrites that formed symmetrical synapses, axon terminals, or glial cells. The distribution of BGT-1 on dendritic spines, rather than at GABAergic axon terminals, suggests that the transporter is unlikely to play a major role in terminating the action of GABA at a synapse. Instead, the osmolyte betaine is more likely to be the physiological substrate of BGT-1 in the brain, and the presence of the transporter in pyramidal neurons suggests that these neurons utilize betaine to maintain osmolarity.

Membrane damage has been postulated as a critical factor in mediating axonal degeneration in brain and spinal cord trauma. Despite compelling evidence of membrane disruption as a result of physical insults in both in vivo and in vitro studies, the dynamics of such damage over the time post injury in in vivo studies has not been well documented. Using a well-characterized in vivo guinea pig spinal cord compression model and horseradish peroxidase exclusion assay, we have documented significant membrane disruption at 1 hr, 3 days, and 7 days following injury. Furthermore, the membrane damage was found to spread laterally 10 mm beyond the center of original compression site in both rostral and caudal directions. A second-degree polynomial fit of the measured data predicts a bilateral spread of approximately 20-21 mm of membrane disruption from the epicenter of injury over a period of about 20 days. Thus, this study shows that membrane damage exists days, and possibly weeks, after spinal cord trauma in live guinea pigs. This provides the evidence necessary to investigate the role of membrane damage in triggering axonal deterioration in the future. Furthermore, this study has also revealed a long therapeutical window for membrane repair and functional enhancement following traumatic injury in the central nervous system.

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x(L) in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7-14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3-7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10-14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.

For quantitative studies of rat dorsal root ganglion (DRG) in experimental models stereological principles offer a number of different techniques. The application, however, requires knowledge of the anatomy and cytology of the ganglion, considerations of sampling and choosing between the many estimators available. For number and volume estimates in thick glycolmethacrylate sections the optical fractionator and the vertical planar rotator technique in most cases provide sufficient efficiency and are simple to use. Classification of the neurons in the DRG into A- and B-cells is of value in experimental conditions where the two cell types can react differently. Studies on development and subclassification of neuronal DRG cells will gain from application of stereological methods, also. Until now the methods have mainly been applied in studies of axotomy and in a few intoxication models where the time course of cell loss and changes in perikarya volume are important parameters. Further quantitative studies providing better understanding of distribution and expression of neuropeptides, cytokines, apoptotic molecules etc. will provide insight for future therapeutic approaches in neurodegenerative disorders. The more demanding staining techniques require less restrictive embedding media, but unbiased principles are available for almost all the stereological techniques applied to tissue only deformed after sectioning.

In the present study, an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure was used to map and quantify the pathways involved in the trafficking and subcellular targeting of gephyrin in identified abducens motoneurons. Gephyrin is a scaffolding protein, which plays a crucial role in the clustering of the GABA(A) and glycine receptors to the cytoskeleton. TEMCI associated several accurate tools: (i) nanogold immunodetection of gephyrin in motoneurons identified on the basis of their immunoreactivity to Choline Acetyl Transferase, (ii) low magnification color scale coding of gephyrin densities on series of ultrathin sections of motoneurons, which gave a map of the cytoplasmic distribution of the protein, (iii) statistical analysis of the subcellular distribution of the immunolabeling. The color map of gephyrin densities in the cell bodies reflected the distribution of inhibitory synapses over the membrane. The TEMCI analysis of motoneurons with various patterns of synaptic covering made it possible to visualize for the first time the cytoplasmic transport pathway of gephyrin towards its target at synaptic contact. A high magnification quantitative analysis, including the study of 109 inhibitory synapses, showed that most gephyrin-associated immunogold particles (67%) were located in the subsynaptic regions facing the active zones, and the second most densely occupied regions were the perisynaptic regions (19.5% of immunogold particles). A consistent proportion of the gephyrin (11.5%), significantly higher than densities present in the rest of the cytoplasm (2%), was detected in the extrasynaptic submembrane region.

Atomic Force Microscopy (AFM) has been used to image the morphology of developing neurons and their processes. Additionally, AFM can physically interact with the cell under investigation in numerous ways. Here we use the AFM to both three-dimensionally image the neuron and to inflict a nano/micro-puncture to its membrane. Thus, the same instrument used as a tool to precisely penetrate/cut the membrane at the nanoscale level is employed to image the morphological responses to damage. These first high resolution AFM images of living chick dorsal root ganglion cells and cells of sympathetic ganglion and their growing processes provide confirmation of familiar morphologies. The increased resolution of the AFM revealed these structures to be significantly more complex and variable than anticipated. Moreover we describe novel, dynamic, and unreported architectures, particularly large dorsally projecting ridges, spines, and ribbons of cytoplasm that appear and disappear on the order of minutes. In addition, minute (ca. 100 nm) hair-like extensions of membrane along the walls of nerve processes that also shift in shape and density, appearing and disappearing over periods of minutes were seen. We also provide "real time" images of the death of the neuron cell body after nano/micro scale damage to its membrane. These somas excreted their degraded cytoplasm, revealed as an enlarging pool beneath and around the cell. Conversely, identical injury, even repeated perforations and nanoslices, to the neurite's membrane do not lead to demise of the process. This experimental study not only provides unreported neurobiology and neurotrauma, but also emphasizes the unique versatility of AFM as an instrument that can (1) physically manipulate cells, (2) provide precise quantitative measurements of distance, surface area and volume at the nanoscale if required, (3) derive physiologically significant data such as membrane pressure and compliance, and (4) during the same period of study--provide unexcelled imaging of living samples.

Dopamine (DA) is an important neuromodulator in the visual system. The release of DA in the retina largely depends on environmental lighting conditions. Most previous studies have assessed the effect of illumination on retinal DA or its metabolites using homogenates or in vitro preparations. This study was designed to investigate the effect of transitions between lighting conditions--from dark to steady or flickering light and vice versa--on retinal DA release in zebrafish using in vivo microdialysis. The transition from dark to flickering light increased DA release, whereas the transition from flickering light to dark decreased it. This latter effect depended on time of day within the light period, e.g., it was strongest in the late afternoon. When using steady light, none of these effects were seen. Our study also demonstrates that in vivo microdialysis can successfully be applied to the investigation of retinal DA release in zebrafish.

We present an MRI-based anatomic analysis of a series of 9 human brains, representing lobar, semilobar and alobar forms of holoprosencephaly. The analysis of these variable forms of the malformation is based upon a topologic systematics established in a prior analysis of a homogeneous set of semilobar malformations. This systematics has the dual advantage that it serves both as a uniform reference for qualitative description and as a quantitative descriptive base for mathematical correlations between parameters of topology and of growth and development. Within this systematics, the prosencephalic midline is divided from caudal to rostral into diencephalic (DD-right and left, subthalamus through suprachiasmatic junction with telencephalon), telencephalic (TT-right and left, suprachiasmatic border of telencephalon midline to hippocampal commissure) and diencephalic-telencephalic (DT-right and left-hippocampal commissure through temporal limb of choroid fissure) segments. The topologic abnormality of the initial semilobar series was expressed in an orderly rostral to caudal gradient along the TT segment. In each malformation, normal midline topology began with a small posterior corpus callosum. Although the topologic anomaly in the present series invariably also involved the TT segment, this involvement was not continuous and was variably associated with anomalies of the DD in 6 and unilaterally of the DT in 1 brain. In the present as well as with the earlier series of HPE malformations but not in "normative brains," total telencephalic growth is strongly correlated with the length of the midline telencephalic segment. We propose that this system of analysis will be sensitive to the developmental stage and locus of expression of genetic and non-genetic determinants of the formal origin of HPE. For all of the present series, karyotype anlyses were normal. Mutations in the Shh and Zic2 genes were excluded in 2 cases.

Cerebellar climbing fibers have a unique relationship with the dendritic tree of cerebellar Purkinje cells and have been proposed as a key input in establishing long-term plastic changes in the cerebellar cortex. Although both glutamate and aspartate and a number of neuropeptides have been implicated as climbing fiber-released neurotransmitters/neuromodulators, the in vivo release of these substances during climbing fiber stimulation remains to be demonstrated. In the present study, climbing fibers were activated with harmaline and rats or mice were implanted with a microdialysis probe or a microperfusion probe, respectively, to measure amino acid or peptide release. Additional rats were euthanized at various timepoints post-harmaline injection and Fos immunocytochemistry was used to visualize the activation pattern of the inferior olive, cerebellar cortex and deep nuclei over time. Fos expression was first detected in the inferior olive at 15 min post-harmaline injection followed by expression in the deep cerebellar nuclei (30 min) and then in the cerebellar cortex (1 h). Between 2 and 6 h Purkinje cells expressing Fos were found in variable numbers in both the vermal and paravermal regions and there was a distinct parasagittal-banding pattern in the vermal region. Of several amino acids measured following harmaline administration only glutamate and aspartate levels increased significantly in the first dialysate sample compared to preharmaline levels and their release was blocked by prior lesion of the inferior olive. Citrulline also increased following climbing fiber stimulation, but this occurred in the second and third dialysate samples and may reflect nitric oxide production. Four peptides were examined in cerebellar microperfusates following climbing fiber stimulation. Only corticotropin releasing factor (CRF), calcitonin gene related peptide (CGRP) and bradykinin were significantly increased compared to pre-harmaline levels. These results suggest that glutamate, aspartate, CRF and CGRP are released from climbing fibers during activation of the olivocerebellar system.

In this study, we investigated the distribution and developmental expression of the GABA(B) receptor subunits, GABA(B1) and GABA(B2), in the main and accessory olfactory bulbs of the rat. Antibodies raised against these subunits strongly labelled the glomerular layer, suggesting that olfactory and vomeronasal nerve fibers express functional GABA(B) receptors. Using postembedding immunogold cytochemistry, we found that GABA(B) receptors can be present at both extrasynaptic and presynaptic sites of olfactory nerve terminals, and in the latter case they are preferentially associated with the peripheral part of the synaptic specialization. Olfactory nerve fibers expressed GABA(B1) and GABA(B2) at early developmental stages, suggesting that GABA(B) receptors may play a role in olfactory development. Output and local neurons of the main and accessory olfactory bulbs were also labelled for GABA(B1) and GABA(B2), although the subcellular distribution patterns of the two subunits were not completely overlapping. These results indicate that presynaptically located GABA(B) receptors modulate neurotransmitter release from olfactory and vomeronasal nerve fibers and that, in addition to this presynaptic role, GABA(B) receptors may regulate neuronal excitability in infraglomerular circuits.