Small-field, binocular neurons in the superficial layers of the frog optic tectum

Binocular neurons with receptive fields about 5 degrees across were recorded just beneath the pia. Most of them responded to dark stimuli in the lower half of their receptive field and to light stimuli above. There was almost no vertical disparity between the left and right fields and the modal value of the horizontal disparity of the population of cells was 1.7 degrees. Because frogs do not verge their eyes it is possible to calculate at what distance the receptive fields through the two eyes are superimposed. This calculation suggests that the neurons are tuned to detect features in the external world about 50 cm away. This is too far for the neurons to be involved in the frog's everyday distance vision. It is more likely that they are concerned with assessing the vertical position of a horizontal surface.     

The contribution of axoplasmic flow in optic nerve and protein synthesis within the optic tectum to synaptic membrane proteins of the chick optic tectum

In the 5-day-old chick, radioactive leucine was incorporated into proteins of synaptosomal and subsynaptosomal fractions both by fast axoplasmic flow and synthesis within the optic tectum. The distribution of radioactivity in subsynaptosomal fractions suggested that both pathways contribute to the protein constituents of each fraction. The relative contributions to each fraction were similar except for the supernatant proteins, for which fast axoplasmic flow contributed less than the synthesis within the optic tectum. The qualitative contribution of fast flow and synthesis within the optic tectum to the synaptic membrane fraction was distinctive. Fast axoplasmic flow preferentially labelled the high molecular weight proteins, whereas synthesis within the optic tectum labelled a larger percentage of smaller molecular weight proteins.     

Vasculogenesis in the chick embryo optic tectum

The initial formation and further development of the intraneural blood vessel network in the tectum opticum of the chick from the 4th to the 14th incubation day have been analyzed and some quantitative data morphometrically recorded. Vessels have been filled by intracardial injection of India ink in vivo. As inferred from our previous investigations on the vasculogenesis of several districts of the central and peripheral nervous system in the chick embryo, also in the developing optic tectum growth and distribution pattern of the vessels seem to unfold step by step under the local influence of earlier occurring morpho-histogenetic processes of the corresponding neural substratum.     

Cholinergic development in chick optic tectum and retina reaggregated cell cultures

The developmental profiles of acetylcholinesterase and choline acetyltransferase in chick optic tectum and retina cell aggregates, over a 30-day period, have been determined and compared with the corresponding developmental curves obtained in vivo. Both acetylcholinesterase and choline acetyltransferase activities in retina cell aggregates and the acetylcholinesterase activity in optic tectum cell aggregates usually lie between 40 and 90% of the values measured in vivo for the same cell (tissue) type and developmental age. However, the choline acetyltransferase activity in tectum aggregates increases only during the first 7 days of culture, and then decreases to reach a low value of 8% of that measured in vivo, by day 24. This fact, which is associated with widespread degeneration and cell death, could be attributed to the condition of natural deafferentiation occurring in a tectum cell aggregate. A parallel has been drawn between this behavior of a tectum cell aggregate and the effect of early embryonic eye removal on the development of the contralateral optic tectum in vivo. Thus, the tectum may have a biphasic pattern of development, with an autonomous period of growth of about 2 wk, followed by an afference-dependent phase, while the retina behaves, from a cholinergic point of view, as a relatively self-sufficient structure.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - ACh acetylcholine - BW284 C51 dibromide 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide     

GFAP-immunoreactive perivascular glia in the chick optic tectum.

Immunocytochemical staining of the glial fibrillary acidic protein (GFAP) was utilized to characterize the processes of the astrocytes enveloping the vessel wall in the central nervous system. The study was carried out in the mesencephalic lobes of 18 and 20 incubation-day chick embryos and of 20 day chickens. A perivascular GFAP positivity was mainly detectable in the vessel portions running within the tectum white layers, while it was scarce, or absent, in the grey ones. The perivascular GFAP negativity in the tectum cellular layers was not considered result of the absence of astrocytic endfeet since our previous electronmicroscopical studies evidenced an almost complete perivascular astrocytic ring throughout the tectum layers at hatching time. Present data rather suggest that the expression of the GFAP-made intermediate filaments in developing astrocytes might be controlled by the surrounding microenvironment.     

Glutamate-like immunoreactivity in chick cerebellum and optic tectum

Glutamate was coupled via glutaraldehyde to bovine serum albumin. The conjugate was used for raising specific anti-glutamate antibodies. The purified antibody was used for immunostaining of chick cerebellum and optic tectum. Staining was intense in the molecular layer and in cell bodies of the granule cell layer. In the optic tectum a diffuse staining was detected in the superficial layers of stratum griseum fibrosum superficiale and in cell bodies especially in the layers a and e. Large cell bodies located in the stratum griseum centrale were also stained.     

Membrane alterations during chick neural retina development: A freeze-fracture study

As part of a study of cell surface differentiation during chick retina development, a freeze-fracture study of neural retinas from 5 to 10 day embryonic chicks was undertaken. Three classes of changes have been detected. (1) As cells differentiate and become recognizable by their position within the tissue, they acquire characteristic numbers of intramembrane particles in the surfaces in each layer. (2) Small gap junctions appear between cells at the outer limiting membrane of the 5 day retina. At 6 days, they are larger, more numerous and are also found in deeper layers of the tissue. By the seventh day, the size and number of the junctions is greatly reduced; they are not visible after the tenth day. (3) The characteristic lack of particles in the outer limiting membrane of the mature retina appears at the ninth day of incubation, at the time that presumptive photoreceptors extend through the outer limiting membrane. Tight junctions between cells were not observed during this study.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

Layer by layer analysis of synaptic organization in the optic tectum of the prog

The distribution of axo-axonal and axo-dendritic synapses, nerve endings, and bodies of neurons by depth in the optic tectum ofRana temporaria L. was investigated under normal conditions and 6–9, 60, and 134 days after removal of the contralateral eye. Counting was carried out on long oriented sections examined in the electron microscope. In outer plexiform layer 9 the density of synapses was greatest near the surface of the tectum and decreased in the direction away from it; no inner sublayers with differing density of synapses could be distinguished. In the outer zone of layer 9 (to a depth of about 30 ) many axo-axonal synpases were discovered. Endings of myelinated optic fibers of large diameter ("dark" terminal degeneration) were widely distributed in the same layer. The density of axo-dendritic synapses in deep plexiform layer 5 was similar to that in layer 9. Many nerve endings containing granular vesicles as well as pale synaptic vesicles were found in layer 5 and neighboring zones.A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 11, No. 2, pp. 130–136, March–April, 1979.     

Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion

The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(-) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(-) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (approximately 50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(-) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (-) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 M) was abolished by 8-phenyltheophylline (15 uM). Glutamate and the glutamatergic agonists kainate or trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 M, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 M 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (rs)-alpha-methyl-4-carboxyphenylglycine [(rs)M-CPG], a metabotropic, antagonist, while 1 mMl-2-amino-3-phosphonopropionate (l-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 M and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.Abbreviations ADO adenosine - DNQX 6,7-dihydroxy-2,3-dinitro-quinoxaline - KA kainate - l-AP3 l-2-amino-3-phosphonopropionate - mGluRs metabotropic glutamate receptors - P-THEO 8-phenyltheophylline - (rs)M-CPG (rs)-alpha-methyl-4-carboxyphenyl-glycine - trans-ACPD trans-d,l-1-aminocyclopentane-1,3-dicarboxyho acid     

Ganglioside patterns and cholera toxin-peroxidase labeling of aggregating cells from the chick optic tectum

The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. G_D and G_D1b were the predominant gangliosides early in development, while G_D1a and several other multisialogan gliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin-peroxidase binding site G_M1 showed conjugate binding correlates with increasing levels of G_M1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of G_M1 in the synaptic cleft region.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.