Cholinergic development in chick brain reaggregated cell cultures

Reaggregated cell cultures from dissociated 7-day-old chick embryo whole brains were prepared, and the developmental profiles of acetylcholinesterase and choline acetyltransferase, in the aggregates, determined over a 30-day period. Enzyme activities in vitro, at different times of culture, typically lie between 30 and 60% of the values obtained for embryos or chicks of the same developmental age, up to day-10 posthatching. The increase in acetylcholinesterase activity over a 24-day period of culture/incubation is fourfold in the aggregates vs. sixfold for embryos, while the choline acetyltransferase values increase, during the same period of time, 32-fold in the aggregates vs. 17-fold in vivo. Choline acetyltransferase activity seems to be more dependent on good cell-to-cell contact than acetylcholinesterase activity. On the other hand, morphological studies on the aggregates with light and electron microscopy reveal a number of structural features characteristic of well-developed nervous tissue. It is suggested that aggregate cultures of chick brain cells are an adequate model system that is especially useful in analyzing developmental phenomena requiring free tridimensional interaction.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - BW284 C51 dibromide 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide - ACh acetylcholine     

Temporal changes in embryonic nerve cell recognition: correlate with cholinergic development in aggregate cultures

Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in vivo development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development.     

Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A₁₂ collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A₁₂ activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G₄ species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G₂ and G₁ forms are present in comparable concentrations in both tissues. Besides, G₄ is the predominant globular form in the chick optic lobe after hatching, G₂ and G₁ being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered.The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.     

Cell interactions and the regulation of cholinergic enzymes during neural differentiation in vitro.

Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rembencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates.The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and 10-day-old NR and OL cells show that the interactive capacities of the cells change with development.     

Laminar Distributions of Choline Acetyltransferase and Acetylcholinesterase Activities in the Inner Plexiform Layer of Rat Retina

Choline acetyltransferase and acetylcholinesterase activities were measured in samples taken at 7-micron increments through the inner plexiform layer of rat retina. These enzyme activities were not uniformly distributed through the depth of the inner plexiform layer. Peaks of choline acetyltransferase activity occurred at about one-third and peaks of acetylcholinesterase activity at about one-fifth of the depth into the inner plexiform layer from either side. The positions of the two peaks of choline acetyltransferase activity most likely correspond to the locations of processes from cholinergic amacrine somata in the inner nuclear layer, which spread in sublamina a, and processes from cholinergic amacrine somata "displaced" in the ganglion cell layer which spread in sublamina b of the inner plexiform layer. The peaks of acetylcholinesterase activity may in addition correspond to the processes of cholinoceptive amacrine and ganglion cells. The magnitudes of choline acetyltransferase and acetylcholinesterase activities are as high as found anywhere in rat brain, emphasizing the important role of cholinergic mechanisms in visual processing through the rat inner plexiform layer.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical and cytochemical localization of acetylcholinesterase in retina and optic tectum of teleost fish.

The activity of cholinesterase and its cellular and subcellular localization were investigated in the retina and optic tectum of Eugerres plumieri and in the retina of Carassius carassius by means of radiometric, histochemical, and cytochemical procedures. In both fishes only the presence of acetylcholinesterase could be demonstrated. This study, besides confirming previous findings that acetylcholinesterase is located in the ganglion and amacrine cells of the retina as well as in the inner plexiform layer, in addition provides evidence that the enzyme is also present at the region of photoreceptor synapses between the cell bodies and apposing extensions of the horizontal cells of the same layer. The latter localization may indicate the involvement of a cholinergic mechanism at the functional contacts (transferapses) between the horizontal cells. In the optic tectum of Eugerres plumieri, histochemistry reveals fine distinguishable bands of acetylcholinesterase activity; two of the bands are quite sharply defined, whereas three others have rather a more diffuse appearance. The presence of these bands and their distribution may suggest a widespread distribution of cholinergic elements in the optic tectum.     

Activity of choline acetyltransferase and acetylcholinesterase in the goldfish optic tectum after disconnection

Activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were investigated in the goldfish optic tectum after disconnection of the optic afferents. Permanent disconnection was achieved by eye removal, and optic nerve crush produced a temporary disconnection until regeneration. There was a rapid loss in total activity per tectum for both enzymes under the two disconnection conditions. At longer intervals after optic nerve crush the levels of total activity for both enzymes returned toward control levels, as regeneration of the nerve proceeded. Total activity for both enzymes remained depressed after eye removal, however. Variable results were obtained in specific activity data, expressed per unit protein, although ther was a 10% loss in specific activity of CAT at early intervals after eye removal. The data are interpreted as consistent with the possibility that at least a fraction of the axons in the retinotectal pathway of goldfish are cholinergic, and parallel our previous observations showing similar rapid losses of nicotinic-cholinergic receptor activity in this system.     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

Levels of choline intermediates in the visual system structures and in peripheral nerve of the rat: Comparison with neural tissues of a lower vertebrate (Mustelus canis) and an invertebrate (Loligo pealei)

The levels of choline intermediate endogenous pools in structures of the visual system (retina, optic nerve, lateral geniculate body, superior colliculus) and in sciatic nerve of adult (4-month-old) and young (30-day-old) rats were measured. The amounts were also obtained from retina, optic nerve, optic tectum and cranio-spinal nerves of a primitive elasmobranch, the smooth dogfish Mustelus canis, and from related nervous structures (retina, optic lobe, fin nerve, stellar nerve and stellate ganglia) of a marine invertebrate, the squid Loligo pealei. In all regions of rat nervous system, the pool size of CDP-choline was much smaller than that of free choline, whereas GroPCho was present in a relatively higher content. The pool sizes of choline intermediates in 30- and 120-day-old rats were nearly the same. In nervous system regions of the dogfish and squid, the values followed the same general trend as observed for rat. Squid nervous tissues had the lowest choline and GroPCho contents. The rat retina showed the lowest glycerophosphorylcholine phosphodiesterase activity. The chemical studies described here confirm the basic similarity in the pattern of choline intermediate pool sizes among animal species widely different in phylogenetic position. The data highly reinforce the idea that the precursor role of choline and catabolic pathways for the maintenance of the PtdCho membraneous pool seem highly conserved during evolution.     

Properties of endogenous,membrane-associated sialidase activity (N-acetylneuraminidase) of the goldfish visual system

The endogenous sialidase (N-acetylneuraminidase) activity of membranes prepared from goldfish retina and optic tectum displays characteristics similar to those reported for neural plasma membrane sialidases of other organisms. Endogenous membrane sialidase activity was found to be optimal at ph 4.0, and maximal release was obtained at 37-50 degrees C, above which temperature thermal instability of the preparations was observed. Optic nerve crush, which results in regeneration of retinal ganglion cell axons, did not result in significant changes in measured endogenous membrane sialidase activity in either the retina or the optic tectum. Enzymatic hydrolysis of membrane sialoglycolipid (ganglioside) accounted for about 70% of the total sialic acid released. Ganglioside GM1 accumulated as the major lipid product in both retina and tectum, indicating that the inner sialosylgalactosyl linkage in the ganglio oligosaccharide series was resistant to hydrolysis by the endogenous enzyme.     

Composition and labeling by [3H]glycerol and [3H]serine of phospholipids of the chicken retina and optic tectum

The content and fatty acid composition of phospholipids and the in vivo labeling of lipids by [3H]glycerol and [3H]serine was studied in the retina and the optic tectum of young chickens. The tectum had a higher content of phospholipids and a significantly lower ratio of choline (CGP) to ethanolamine (EGP) glycerophospholipids than the retina. Lipids of the chicken optic system were characterized by a high proportion of polyenoic fatty acids of the n-6 series compared to other species. Intravitreally injected [3H]glycerol was incorporated into all glycerol-containing lipids of the retina, especially in CGP and EGP. Most of the label from [3H]serine was found in serine glycerophospholipids (SGP). The time-dependent distribution of both precursors among retinal lipids was consistent with de novo synthesis as well as metabolic interconversions of lipids. Thus, [3H] from serine also appeared in EGP and CGP, indicating the presence and activity of SGP decarboxylase and EGP-n-methyl transferase. Lipids labeled with both precursors in retina were subsequently found in the tectum, via axoplasmic transport. Even though different lipid classes were labelled by each precursor the proportion of lipids transported to the tectum was similar in both cases (about 1% of the label present in retina).     

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.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE ON RAT RETINA: CHANGES IN PUTATIVE TRANSMITTERS AND THEIR CORRESPONDING ENZYMES

Abstract– Subcutaneous administration of high doses of sodium glutamate to rats during their first week after birth produced an almost total loss of choline acetyltransferase, a 90% reduction in glutamate decarboxylase and 70% reductions in acetylcholinesterase and DOPA decarboxylase activities in the adult retina. In addition there was a 70% decrease in GABA and 35-55% decrease in aspartate, glutamate, glycine, alanine and glutamine. No reduction in taurine was observed. The results support the view that the enzymes are mainly localized in the interneurons of retina and that taurine is present in the photoreceptor cells.
Glutamate treatment was also followed by a small reduction in choline acetyltransferase and glutamate decarboxylase of the superior colliculus and in choline acetyltransferase of hippocampus, whereas no changes could be detected in the lateral geniculate body of the adult rat. Unilateral enucleation performed on 1-day-old animals did not alter choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase and DOPA decarboxylase activities in the superior colliculus and in the lateral geniculate body of the adult rat.     

Quantitative histochemical studies of the hypothalamus enzymes of acetylcholine metabolism.

The quantitative histochemical distribution of acetylcholinesterase and choline acetyltransferase activity has been measured in individual hypothalamic nuclei and median eminence, as well as in entire hypothalamic sections by a mapping technique. There was an 18-fold range of nuclear choline acetyltransferase activity with highest activities in the lateral preoptic nucleus and median eminence. There was a nine-fold range of nuclear acetylcholinesterase activity with highest activities in the lateral preoptic and magnocellular nuclei and lowest activity in the median eminence. The substantial gradients of choline acetyltransferase activity found in the hypothalamus indicate the importance of using a technique that provides an objective, permanent record of contiguous sample locations thereby allowing detailed analysis of tissue areas using, but not dependent on, anatomical boundaries.     

TOPOGRAPHICAL AND SUBCELLULAR DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE IN PIGEON OPTIC TECTUM

Abstract— The distribution of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in different layers of the pigeon optic tectum and in some nuclei of the optic lobe have been investigated. About 40% of GAD and 25% of ChAT were found in the superficial part of tectum, but negligible activity was found in the stratum opticum. The highest GAD activity was found in layers 3-7 (according to the nomenclature of C ajal , 1911) with a peak in layer 4. ChAT activity peaked in layers 3, 5. 8 and 10/11. Its distribution correlated well with the staining pattern of AChE, particularly in the superficial part of the tectum. The distribution of ChAT and GAD did not change significantly 4 weeks after enucleation. ChAT and GAD activities were high in the nucleus isthmi, pars parvocellularis (Ipc). The activity of GAD was also high in the nucleus intercollicularis (ICo), the other nuclei showed less activity of both enzymes.     

Glial cells in coculture can increase the acetylcholinesterase activity in human brain endothelial cells.

The elements of the cholinergic system (acetylcholinesterase and choline acetyltransferase) and butyrylcholinesterase were studied in human cortical capillary samples, brain-derived endothelial cell cultures and glial cell cultures. It was shown that the elements of the cholinergic system are present in the microvessels, but the choline acetyltransferase activity may be due to contamination with cholinergic nerve terminals since no choline acetyltransferase could be demonstrated in endothelial cell cultures. The present results revealed that the activity of acetylcholinesterase is reduced in the cortical endothelial cell cultures after longer culture times, while butyrylcholinesterase activity is not altered. In a system where endothelial cells were cocultured with embryonic human brain astroglial cells for 12 days in vitro, the acetylcholinesterase activity was increased 2-fold. These results support a glial influence on the enzyme activity of the cerebral endothelium.     

Reciprocal Regulation of Ornithine Decarboxylase and Choline Kinase Activities by Their Respective Reaction Products in the Developing Rat Cerebellar Cortex

Changes in the activity of choline kinase were measured in the cerebellum during development. Early transient increase was found in the enzyme activity just prior to and during birth. This period of increase did not coincide with the periods of transient elevation in ornithine decarboxylase and choline acetyltransferase previously observed in the developing cerebellum. The effects of the naturally occurring polyamines (putrescine, spermidine, and spermine) on choline kinase and choline acetyltransferase activities, and of phosphorylcholine (the product of the reaction catalyzed by choline kinase) on ornithine decarboxylase and choline acetyltransferase activities, were also examined. Choline acetyltransferase activity was not influenced by either polyamines or phosphorylcholine. However, choline kinase activity from 7-day-old, but not from adult, cerebellum was increased 25% in the presence of 4 mM spermine. In contrast, low spermidine concentrations (less than 2 mM) inhibited choline kinase activity selectively in 7-day-old cerebellum. Ornithine decarboxylase activity from 7-day-old cerebellum was inhibited in a concentration-dependent manner by phosphorylcholine. The present data together with other previous reports suggest that: (a) polyamines may play a role in choline utilization during development via their regulation of choline kinase activity, on the one hand, and of acetylcholinesterase activity on the other; and (b) during development, a reciprocal regulation of choline kinase and ornithine decarboxylase activities by their respective reaction products may exist, whereby choline kinase activity is regulated in a complex manner by polyamines and, in turn, ornithine decarboxylase is inhibited by phosphorylcholine.     

Comparative enzymatic development in chick embryonic brain areas

The activities of glutamic acid decarboxylase (GAD), choline acetylase, dopa decarboxylase, and tyrosine hydroxylase were measured by radioactive assays and of acetylcholinesterase by a colorimetric procedure on homogenates of the tectum, forebrain, and cerebellum of the chick from the third embryonic day to 3 weeks post-hatch. GAD showed a rapid development beginning about day 9 and peaking at or before hatching: there were generally similar levels in all 3 areas during development although in the oldest chicks the tectum had significantly higher GAD levels than the forebrain, the cerebellar levels being intermediate. The other enzymes all showed a somewhat later development with sharp increases beginning on or after day 11 and peak levels being reached only after hatching. The different brain regions also showed much greater disparity in levels of these other enzymes than found for GAD. The tectum contained the greatest concentrations of choline acetylase and acetylcholinesterase, and the forebrain had the most tyrosine hydroxylase and dopa decarboxylase. The data may be useful for correlation with morphological developmental studies.     

Differential expression of calretinin in the developing and regenerating zebrafish visual system

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.