Mechanisms of the release of anterogradely transported neurotrophin-3 from axon terminals

Neurotrophins have profound effects on synaptic function and structure. They can be derived from presynaptic, as well as postsynaptic, sites. To date, it has not been possible to measure the release of neurotrophins from axon terminals in intact tissue. We implemented a novel, extremely sensitive assay for the release and transfer of anterogradely transported neurotrophin-3 (NT-3) from a presynaptic to a postsynaptic location that uses synaptosomal fractionation after introduction of radiolabeled NT-3 into the retinotectal projection of chick embryos. Release of the anterogradely transported NT-3 in intact tissue was assessed by measuring the amount remaining in synaptosomal preparations after treatment of whole tecta with pharmacological agents. Use of this assay reveals that release of NT-3 from axon terminals is increased by depolarization, calcium influx via N-type calcium channels, and cAMP analogs, and release is most profoundly increased by excitation with kainic acid or mobilization of calcium from intracellular stores. NT-3 release depends on extracellular sodium, CaM kinase II activity, and requires intact microtubules and microfilaments. Dantrolene inhibits the high potassium-induced release of NT-3, indicating that release of calcium from intracellular stores is required. Tetanus toxin also inhibits NT-3 release, suggesting that intact synaptobrevin or synaptobrevin-like molecules are required for exocytosis. Ultrastructural autoradiography and immunolabel indicate that NT-3 is packaged in presumptive large dense-core vesicles. These data show that release of NT-3 from axon terminals depends on multiple regulatory proteins and ions, including the mobilization of local calcium. The data provide insight in the mechanisms of anterograde neurotrophins as synaptic modulators

Calibration of the stereological estimation of the number of myelinated axons in the rat sciatic nerve: a multicenter study.

Several sources of variability can affect stereological estimates. Here we measured the impact of potential sources of variability on numerical stereological estimates of myelinated axons in the adult rat sciatic nerve. Besides biological variation, parameters tested included two variations of stereological methods (unbiased counting frame versus 2D-disector), two sampling schemes (few large versus frequent small sampling boxes), and workstations with varying degrees of sophistication. All estimates were validated against exhaustive counts of the same nerve cross sections to obtain calibrated true numbers of myelinated axons (gold standard). In addition, we quantified errors in particle identification by comparing light microscopic and electron microscopic images of selected consecutive sections. Biological variation was 15.6%. There was no significant difference between the two stereological approaches or workstations used, but sampling schemes with few large samples yielded larger differences (20.7%±3.7% SEM) of estimates from true values, while frequent small samples showed significantly smaller differences (12.7%±1.9% SEM). Particle identification was accurate in 94% of cases (range: 89–98%). The most common identification error was due to profiles of Schwann cell nuclei mimicking profiles of small myelinated nerve fibers. We recommend sampling frequent small rather than few large areas, and conclude that workstations with basic stereological equipment are sufficient to obtain accurate estimates. Electron microscopic verification showed that particle misidentification had a surprisingly variable and large impact of up to 11%, corresponding to 2/3 of the biological variation (15.6%). Thus, errors in particle identification require further attention, and we provide a simple nerve fiber recognition test to assist investigators with self-testing and training

Quantifying tumour-infiltrating lymphocyte subsets : a practical immuno-histochemical method

Background: Efficient histological quantification of tumour-infiltrating T and B lymphocyte (TIL) subsets in archival tissues would greatly facilitate investigations of the role of TIL in human cancer biology. We sought to develop such a method. Methods: Ten ×40 digital images of 4 μ sections of 16 ductal invasive breast carcinomas immunostained for CD3, CD4, CD8, and CD20 were acquired (a total of 640 images). The number of pixels in each image matching a partition of Lab colour space corresponding to immunostained cells were counted using the ‘Color range’ and ‘Histogram’ tools in Adobe Photoshop 7. These pixel counts were converted to cell counts per mm2 using a calibration factor derived from one, two, three or all 10 images of each case/antibody combination. Results: Variations in the number of labelled pixels per immunostained cell made individual calibration for each case/antibody combination necessary. Calibration based on two fields containing the most labelled pixels gave a cell count minimally higher (+ 5.3%) than the count based on 10-field calibration, with 95% confidence limits − 14.7 to + 25.3%. As TIL density could vary up to 100-fold between cases, this accuracy and precision are acceptable. Conclusion: The methodology described offers sufficient accuracy, precision and efficiency to quantify the density of TIL sub-populations in breast cancer using commonly available software, and could be adapted to batch processing of image files

Eye Movements, Strabismus, Amblyopia, and Neuro-Ophthalmology Differences in Gene Expression between Strabismic and Normal Human Extraocular Muscles

PURPOSE. Strabismic extraocular muscles (EOMs) differ from normal EOMs in structural and functional properties, but the gene expression profile of these two types of EOM has not been examined. Differences in gene expression may inform about causes and effects of the strabismic condition in humans. METHODS. EOM samples were obtained during corrective surgery from patients with horizontal strabismus and from deceased organ donors with normal EOMs. Microarrays and quantitative PCR identified significantly up-and down-regulated genes in EOM samples. Analysis was performed on probe sets with more than 3-fold differential expression between normal and strabismic samples, with an adjusted P value of 0.05. RESULTS. Microarray analysis showed that 604 genes in these samples had significantly different expression. Expression predominantly was upregulated in genes involved in extracellular matrix structure, and down-regulated in genes related to contractility. Expression of genes associated with signaling, calcium handling, mitochondria function and biogenesis, and energy homeostasis also was significantly different between normal and strabismic EOM. Skeletal muscle PCR array identified 22 (25%) of 87 muscle-specific genes that were significantly down-regulated in strabismic EOMs; none was significantly upregulated. CONCLUSIONS. Differences in gene expression between strabismic and normal human EOMs point to a relevant contribution of the peripheral oculomotor system to the strabismic condition. Decreases in expression of contractility genes and increases of extracellular matrix-associated genes indicate imbalances in EOM structure. We conclude that gene regulation of proteins fundamental to contractile mechanics and extracellular matrix structure is involved in pathogenesis and/or consequences of strabismus, suggesting potential novel therapeutic targets. (Invest Ophthalmol Vis Sci. 2012;53:5168-5177

One at a time, live tracking of NGF axonal transport using quantum dots

Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50–150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body

The peripheral distribution and central projections of the sensory rami of the facial nerve in goldfish, Carassius auratus

Taste buds in goldfish and other cyprinids are found not only within the oropharyngeal cavity but also scattered over the external body surface. The external taste buds are innervated by branches of the facial nerve that terminate centrally in an enlargement of the medulla termed the facial lobe. The peripheral distribution and areas of innervation of the rami of the facial sensory nerve were determined by using a modification of the Sihler technique and by examination of a Bodian-stained head series. The central projections of individual rami of the facial sensory nerve were traced by means of the horseradish peroxidase (HRP) technique. Fibers of the facial sensory nerve distribute over the head and trunk via nine rami. The supraorbital ramus distributes fibers to taste buds above the eye. The palatine, maxillary, and mandibular rami innervate taste buds of the rostral palate, upper lip, and lower lip, respectively. The three rami of the hyomandibular trunk innervate taste buds on the operculum, branchiostegal rays, and in the lower cheek region. A facial recurrent ramus was also found that distributes fibers to taste buds on the trunk and pectoral fin via two rami, the lateral recurrent ramus and pectoral recurrent ramus. The facial sensory rami map somatotopically on the facial lobe. Overall, the projections follow an anteroposterior orientation with the long axis of the body tilted slightly ventrally. The lips and rostral palate make up a disproportionately large portion of the map, taking up nearly the entire ventral extent of the lobe. The trunk and pectoral fin regions map broadly across the dorsal portion of the lobe. Further, projections to the nucleus of the descending trigeminal tract were observed with labeling of the supraorbital, maxillary, and mandibular rami, and the rami of the hyomandibular trunk. Projections to the facial motor nucleus were also observed with labeling of maxillary and mandibular rami, perhaps indicating a monosynaptic reflex arc. These projections have not been reported in previous studies on the teleostean facial taste system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50035/1/902590306_ftp.pd

Intervention effects of Ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of Neurotrophin-4 and N-Cadherin

Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg2+ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg2+ free medium for 3 hours), iii) GLS group I (incubated with Mg2+ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg2+ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression

Development of the retinofugal projections in the embryonic and larval zebrafish ( Brachydanio rerio )

Studies of the projection from the vertebrate retina have contributed significantly to current concepts of neural development. The zebrafish has recently become a favored system for the study of development in general and neural development in particular. Although the development of both the optic nerve and the retinotectal projection of the zebrafish has been described, the retinofugal projection in its entirety has not. This paper describes it and also addresses the issue of projectional exuberance: i. e., transient projections to targets that are not innervated in the adult. The retinofugal projection of embryonic and larval zebrafish (32 hours to 7 days post-fertilization) was labeled by intraocular injection of DiI (1,1′-dioctadecyl-3,3,3′,3′, tetramethylindocarbocyanine perchlorate) and then studied in wholemounts and sections. The first optic axons crossed the chiasm at 32 hours post-fertilization and projected in a straight line to reach the tectum at about 44 hours. At 48 hours, a few optic axons deviated along either the tract of the posterior commissure or the tract of the postoptic commissure. By 72 hours (about the time of hatching) optic axons arborized in ten distinct regions, termed arborization fields. At 6–7 days post-fertilization, the same ten arborization fields (nine contralateral, one bilater) were evident. Most of the arborization fields were located in the superficial neuropil and were not associated with morphologically identifiable clusters of somata. On the basis of various landmarks, the ten arborization fields are identified as precursors of retinorecipient nuclei previously described in other adult cypriniform fishes. The development was characterized by the nearly complete absence of any transient projections. Thus, the idea that axonal outgrowth is initially exuberant and trimmed back later is not supported by these results. © 1994 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50063/1/903460410_ftp.pd

Inhibition of cell proliferation does not slow down echinoderm neural regeneration

BACKGROUND: Regeneration of the damaged central nervous system is one of the most interesting post-embryonic developmental phenomena. Two distinct cellular events have been implicated in supplying regenerative neurogenesis with cellular material – generation of new cells through cell proliferation and recruitment of already existing cells through cell migration. The relative contribution and importance of these two mechanisms is often unknown. METHODS: Here, we use the regenerating radial nerve cord (RNC) of the echinoderm Holothuria glaberrima as a model of extensive post-traumatic neurogenesis in the deuterostome central nervous system. To uncouple the effects of cell proliferation from those of cell migration, we treated regenerating animals with aphidicolin, a specific inhibitor of S-phase DNA replication. To monitor the effect of aphidicolin on DNA synthesis, we used BrdU immunocytochemistry. The specific radial glial marker ERG1 was used to label the regenerating RNC. Cell migration was tracked with vital staining with the lipophilic dye DiI. RESULTS: Aphidicolin treatment resulted in a significant 2.1-fold decrease in cell proliferation. In spite of this, the regenerating RNC in the treated animals did not differ in histological architecture, size and cell number from its counterpart in the control vehicle-treated animals. DiI labeling showed extensive cell migration in the RNC. Some cells migrated from as far as 2 mm away from the injury plane to contribute to the neural outgrowth. CONCLUSIONS: We suggest that inhibition of cell division in the regenerating RNC of H. glaberrima is compensated for by recruitment of cells, which migrate into the RNC outgrowth from deeper regions of the neuroepithelium. Neural regeneration in echinoderms is thus a highly regulative developmental phenomenon, in which the size of the cell pool can be controlled either by cell proliferation or cell migration, and the latter can neutralize perturbations in the former. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12983-017-0196-y) contains supplementary material, which is available to authorized users