PCR driven DNA-DNA competitive hybridization: a new method for sensitive differential cloning.
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Animals↗
Biomedical subjects
S Catsicas
Publications and source records attributed to S Catsicas.
Ribosomal protein L27 is identical in chick and rat.
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Amino Acid Sequence↗
Expression of a conserved cell-type-specific protein in nerve terminals coincides with synaptogenesis.
Contact of axons with target territories results in the formation of synapses, specific junctional complexes that may represent a final stage of neuronal maturation. Synaptosomal-associated protein 25 (SNAP-25) is a component of particular nerve terminals recently identified in rodent brain. To evaluate the structure and regulation of molecular components of the synapse, we investigated the expression of SNAP-25 in the developing chicken nervous system. Analysis of SNAP-25 cDNA clones demonstrated that the chicken homologue is identical in amino acid sequence to the mouse protein. In chicken retina and neural tube, the onset of SNAP-25 mRNA and protein expression was found to correspond to the time of synaptogenesis. These results suggest that SNAP-25 plays a role in the physiology of mature nerve terminals and that its expression may be regulated by specific cell-cell interactions occurring during synapse formation.
Amino Acid Sequence↗
Limits to the dependence of developing neurons on protein synthesis in their axonal target territory.
Our basic question was whether the survival of developing neurons is critically dependent on the level of protein synthesis in the axonal target region. The experiments were carried out on the projection from the isthmo-optic nucleus (ION) to the contralateral retina in chick embryos. The ION is known to undergo almost 60% neuronal death between embryonic days (E) 12 and E17 and to be critically dependent on the retina for trophic support throughout this period and shortly afterwards. Various concentrations of the protein synthesis inhibitor cycloheximide were infused into one eye from E15 to E19. Moderate inhibition (up to about 40%) of retinal protein synthesis, which did not lead to retinal degeneration, had no detectable effects on the number of neurons, nor on the general morphology, in the ION. Only when the inhibition was as high as 50%, leading to widespread degeneration in the retina, did massive degeneration occur also in the ION. It was also shown that a single intraocular injection of cycloheximide at E15 that inhibited retinal protein synthesis by as much as 70-90% during the subsequent 24 h had little effect on the ION in embryos fixed at E19. These results indicate that although the ION neurons are critically dependent on the retina, they can resist major reductions in the level of retinal protein synthesis, which argues against the widespread belief that neuronal survival during development is regulated by the limited production of trophic molecules in the axonal target area. The data are, however, compatible with alternative hypotheses. Most plausibly, survival may be regulated by limited access to a nonlimiting supply of trophic molecules.
Animals↗
Retrograde modulation of dendritic geometry in the vertebrate brain during development.
The neurons of the chick's isthmo-optic nucleus (ION) are known to innervate the retina. We here show that removing the retinal primordia causes the ION dendritic trees to be much less polarized than normal. Our observations were made at 11 embryonic days, which is before the isthmo-optic neurons become dependent on the retina for survival. Other parameters such as neuronal size were unchanged, so the effect seems to have been specific to dendritic shape. Our interpretation is that early target removal eliminates a retrograde signal that normally enhances dendritic polarization.
Animals↗
Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control.
To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.
Animals↗
Spatiotemporal gradients of kainate-sensitivity in the developing chicken retina.
We have studied the age-dependence of the effects of kainate (KA) on the chick retina as a prelude to the accompanying paper on the effects of target-removal on the isthmo-optic nucleus. KA was injected into the eyes of chick embryos and chicks at different ages, and the retinas were fixed a few hours or several days later. The former group of retinas was scanned for pyknotic cells. The earliest age at which KA caused pyknosis was embryonic day 10 (E10), when pyknotic cells appeared in a ventrotemporal patch in the amacrine sublayer near the fundus. Over the next two days the sensitive region expanded tangentially, reaching the periphery first temporally, then nasally. Only after E12 did the KA cause pyknotic cells to occur also in the bipolar sublayer, where the sensitivity spread in the same spatiotemporal sequence as the initial wave, but two days later. Cell loss was examined in embryos that survived a week or more after the KA injection. Substantial cell depletion was found in both the inner nuclear and ganglion cell layers, but only when the injection had been made after E12. With progressively later injections, the depleted zone expanded in the same spatiotemporal sequence as described above, until at E15 the injections caused depletion throughout the entire extent of the retina. The reasons for the lack of cell depletion after KA injections made before E12 are discussed. Cell counts in the ganglion cell layer and studies of anterograde transport of intravitreally injected peroxidase along the retinofugal fibers showed that about half the ganglion cells (including the displaced ganglion cells) pass through a period of vulnerability to the KA injections, to which they subsequently become sensitive.
Animals↗
Abrupt loss of dependence of retinopetal neurons on their target cells, as shown by intraocular injections of kainate in chick embryos.
We have examined the capacity of neurons in the chick isthmo-optic nucleus (ION) to survive when their target neurons in the contralateral retinal are destroyed by intraocular injections of kainate (KA) at different stages in development. The retinal vulnerability to KA builds up progressively from embryonic day 10 (E10) until a plateau is reached at E15 (see accompanying paper); and the effects on the ION increase in parallel, almost all the ION neurons being rapidly lost after the E15 injections. KA injection before E15 lesioned only part of the retina and caused degeneration only in the topographically corresponding region of the ION. Near the end of the natural cell death period in the ION (E17), this initial dependence on the target cells is rapidly lost. Already at E16 the injections kill less ION neurons, and by E19 they kill none of them. The ION neurons have become completely insensitive to the KA injections and appear normal more than 4 months later, although axotomy (by eye removal) at a similar age would by then have killed them. The ectopic ION neurons, scattered outside the ION but projecting to the retina, are never affected by KA injections at any age.
Animals↗
Major role for neuronal death during brain development: refinement of topographical connections.
The precision of the topographic projection from the isthmo-optic nucleus (ION) to the retina has been examined in chicken embryos and chicks by the retrograde transport of a fluorescent carbocyanine dye from restricted retinal sites. At all ages, the labeled neurons are most numerous in the topographically appropriate part of the ION, but in younger embryos up to 49% of them are found outside this region. The distribution of these "aberrantly" projecting neurons is variable, but they generally occur throughout the entire ION. They all die during the ION's period of neuronal death, accounting for most of the 60% cell loss that then occurs. We therefore suggest that a major role of neuronal death during brain development is to reduce the imprecision of neuronal connections.
Animals↗
A combination of Golgi impregnation and fluorescent retrograde labeling.
Central nervous system structures containing neurons labeled by the fluorescent tracers Fast blue (FB), Diamidino yellow dihydrochloride (DY), Rhodamine B isothiocyanate (RITC) and Rhodamine-labeled latex microspheres (RLM) were processed with the Golgi method. The goal was to improve the visualization of the fluorescent labeled neurons and to allow their ultrastructural examination. While the fluorescence of FB and RITC is greatly attenuated by the Golgi method, RLM and DY are still visible in Golgi-impregnated neurons. However, it is usually necessary to remove the silver precipitate by gold-toning.
Amidines↗
Age-dependent accumulation of advanced glycosylation end products in human neurons.
Glucose can react nonenzymatically with free amino groups on proteins and form advanced glycosylation end-products (AGEPs), that have been previously isolated and characterised in aging human connective tissues. In this study, we used immunocytochemistry to examine the distribution of AGEPs in the aging human brain. Our findings show that the pyramidal neurons selectively accumulate AGEP-containing vesicles in an age-dependent manner. In addition, our results demonstrate that AGEPs accumulate in the same type of neuron that degenerates in Alzheimer's disease.
Adolescent↗
A centrifugally controlled circuit in the avian retina and its possible role in visual attention switching.
The isthmo-optic nucleus (ION) is the main source of efferents to the retina in birds. Isthmo-optic neurons project in topographical order on amacrine cells in the ventral parts of the retina, and a subclass of these known as proprioretinal neurons project onto the dorsal retina. We propose that, through the intermediary of the amacrine target cells, activity in the isthmo-optic pathway excites ganglion cells locally in the ventral retina but inhibits those in dorsal regions. This circuit would thereby mediate centrifugally controlled switches in attention between the dorsal retina, involved in feeding, and the more ventral parts, involved in scanning for predators. This hypothesis accounts for a wide range of disparate data from behavior, comparative anatomy, endocrinology, hodology, and neurophysiology.
Animals↗
Long-distance intraretinal connections in birds.
Electrophysiological experiments have shown in both birds and mammals that remote parts of the retina, several millimetres apart, interact at the retinal level. The anatomical basis of this is poorly understood, although in mammals some cells in the ganglion cell layer have axons that terminate in the inner plexiform layer several millimetres from the cell body. In birds, the longest previously reported intraretinal connections were from amacrine cells, extending only a few hundred microns. But we here describe very long connections that span almost the entire extent of the retina in chicks and chick embryos. The parent cell bodies are in the inner nuclear layer of the ventral half of the retina, and they project in topographical order onto the dorsal half. They do not project to the brain. They may be involved in selective switching of attention between the upper and lower parts of the visual field, at an unprecedentedly early stage of visual processing.
Animals↗