Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and “housekeeping” enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways. Thus, GABAergic differentiation in developing retina is sensitive to cell signaling mediated in part by cognin.     

The cell recognition molecule, cognin, mediates choline acetyltransferase activity in embryonic chick retina.

Cell signaling and cell-cell interactions play an important role in neuronal differentiation in the embryonic CNS. Previous work (Hausman, R.E., Vivek Sagar, G.D. and Shah, B.H., Dev. Brain Res., 59 (1991) 31-37) had shown that cholinergic differentiation in the embryonic chick retina depends on insulin and neuron-neuron interactions. Here, we pursued the molecular nature of that dependence on cell interactions. The embryonic chick retina is known to contain several cell adhesion or recognition molecules. We asked if retina cognin, a 50 kDa cell surface-associated protein, played a role in controlling cholinergic differentiation in the developing chick retina. As previously, cholinergic differentiation was measured by two markers: choline acetyltransferase (ChAT) activity and high-affinity choline uptake. We used polyclonal antibody to cognin to determine if blocking cognin-mediated cell interactions would affect the normal embryonic increases in these cholinergic markers. We demonstrated a 40% inhibition of the normal developmental appearance of ChAT activity in retina neuronal cultures from early development, but no effect in cultures from more differentiated retina. The inhibition was selective for retina, since it was not seen in neural tissues like cerebrum and cerebellum that also express ChAT. In contrast to the effect of insulin, choline uptake was not affected by treatment with cognin antibody. Antibodies to two other cell recognition molecules present in the retina (Ng-CAM and N-cadherin) did not block the normal developmental appearance of ChAT. These results suggest that cognin-mediated interactions play a unique role in the control of one aspect of cholinergic differentiation in the developing chick retina.     

Evidence for plasticity in neurotransmitter expression in neuronal cultures derived from 3-day-old chick embryo

We have previously reported the developmental profiles of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) bio- and immunocytochemically, assessing GABAergic and cholinergic neuronal phenotypes respectively, in neuroblast-enriched cultures from 3-day-old chick embryo, plated on poly-L-lysine. We have also reported that collagen as culture substrate inhibits neuronal aggregation and neuritic fasciculation in this culture system. In this study we assessed the same parameters for cultures on collagen. In addition, we evaluated the effects of nerve growth factors (NGF) on cholinergic and GABAergic expression on neurons plated either on polylysine or collagen. We found that non-neuronal cells and NGF prolonged the survival of cholinergic and GABAergic neuronal populations and that both markedly stimulated GABAergic expression. In contrast, cholinergic expression was only enhanced by NGF. Immunostaining for GABA and ChAT reflected the biochemical findings. Glutamine synthetase and cyclic nucleotide phosphohydrolase, used as markers for astrocytes and oligodendrocytes respectively, showed very low activity in both substrata and were not related to GAD or ChAT peak activities. Our findings suggest that humoral factors and cell-cell contacts markedly influence neuronal phenotypic expression in culture. Moreover, it appears that during early neuronal differentiation GABAergic neurons are more responsive to microenvironmental regulation compared to cholinergic neurons.     

Cloning of chicken choline acetyltransferase and its expression in early embryonic retina

The enzyme choline acetyltransferase [EC 2.3.1.6] (ChAT) synthesizes the neurotransmitter acetylcholine that plays a key morphogenic role in vertebrate retina development. As the embryonic avian retina is particularly useful for morphogenetic studies, we cloned the complete coding region of chicken ChAT cDNA. At the deduced amino acid level, chicken ChAT is approximately 76% identical to mammalian ChAT proteins. We also report here the cloning of the complete 5' end of the complex cholinergic locus. This locus contains both the ChAT gene and the nested intronless gene for the vesicular acetylcholine transporter (VAChT). The genomic organization of the 5' end of the chicken cholinergic locus is similar to that reported in other vertebrate species. A 5.7 kb mRNA corresponding to the ChAT message was detected in both embryonic retina and post-hatch brain. An analysis of the ChAT mRNA in embryonic chick retina shows that the message can be detected by E6 and its level increased during early retinal development. Vertebrate ChAT mRNAs can contain one or more of three non-coding exons, M, N or R and by RT-PCR we demonstrate, at least, a chicken ChAT mRNA containing exon M.     

Cholinergic amacrine cells are not required for the progression and atropine-mediated suppression of form-deprivation myopia

Muscarinic cholinergic pathways have been implicated in the visual control of ocular growth. However, the source(s) of acetylcholine and the tissue(s) which regulate ocular growth via muscarinic acetylcholine receptors (mAChRs) remain unknown. We sought to determine whether retinal sources of acetylcholine and mAChRs contribute to visually guided ocular growth in the chick. Cholinergic amacrine cells were ablated by intraocular injections of either ethylcholine mustard aziridinium ion (ECMA; a selective cholinotoxin) or quisqualic acid (QA; an excitotoxin that destroys many amacrine cells, including those that release acetylcholine). Disruption of cholinergic pathways was assessed immunocytochemically with antibodies to the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and three different isoforms of mAChR, and by biochemical assay for ChAT activity. ECMA (25 nmol) destroyed two of the four subtypes of cholinergic amacrine cells and attenuated retinal ChAT activity, but left retinal mAChR-immunoreactivity intact. QA (200 nmol) destroyed the majority of all four subtypes of cholinergic amacrine cells, and ablated most mAChR-immunoreactivity and ChAT activity in the retina. ECMA and QA had no apparent effect on mAChRs or cholinergic fibres in the choroid, only marginally reduced choroidal ChAT activity, and had little effect on ChAT activity in the anterior segment. Toxin-treated eyes remained emmetropic and responded to form-deprivation by growing excessively and becoming myopic. Furthermore, daily intravitreal injection of 40 μg atropine for 6 days into form-deprived toxin-treated eyes completely prevented ocular elongation and myopia. We conclude that neither cholinergic amacrine cells nor mAChRs in the retina are required for visual regulation of ocular growth, and that atropine may exert its growth-suppressing influence by acting upon extraretinal mAChRs, possibly in the choroid, retinal pigmented epithelium, or sclera.     

Distribution of cholinergic neurons in the chick spinal cord during embryonic development. Comparison of ChAT immunocytochemistry with AChE histochemistry.

The location of cholinergic neurons was studied during the development of the chick embryo spinal cord. A comparison between choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry was performed. ChAT-positive neurons could be detected only from embryonic day 9 (E9) onwards by the FITC technique and from E12 onwards by the PAP technique. These neurons were located mainly in the medial and lateral motor columns in the ventral horn of the gray matter and some of them were observed in the intermediate region of the spinal cord. AChE-containing cell bodies were much more numerous than the ChAT immunoreactive ones and were distributed in the ventral horn of the gray matter, the intermediate gray region and mostly off the apical part of the dorsal horn. ChAT should provide a reliable and specific marker for cholinergic neurons.     

Muscle-derived factors reverse the cholinotoxic effects of ethanol during early neuroembryogenesis in the chick embryo.

The interaction between muscle-derived factors and ethanol on cholinergic neuronal expression was studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase (ChAT) as cholinergic neuronal marker. Ethanol (10 mg/50 microliters) and limb muscle extract (130 micrograms protein/50 microliters) (LME) were administered in ovo either alone or concomitantly at embryonic days 1-3 (E1-E3); or ethanol was given E1-E3 and followed by LME at E4-E7. All groups were sacrificed at embryonic day 8 (E8) and ChAT activity was assayed in homogenates of whole brain and of spinal cord. As previously reported, ethanol at E1-3 produced a 30% decrease in brain ChAT activity and 35% in spinal cord. Concomitant administration of ethanol and LME at embryonic days E1-E3 eliminated the decrease in choline acetyltransferase activity produced by ethanol in the brain, but not in the spinal cord. On the other hand, administration of LME at embryonic days E4-E7 to embryos pretreated with ethanol at embryonic days E1-E3, raised ChAT activity to control level in the spinal cord, but only partially restored ChAT activity in the brain. In view of the alleged neurotrophic effects of muscle-derived factors on neuronal survival and neuronal growth, we interpret these findings to suggest that LME in addition to its ability to decrease natural neuronal death, may prevent death resulting from neurotoxicity.     

Critical periods to ethanol exposure during early neuroembryogenesis in the chick embryo: cholinergic neurons

The acute and chronic effects of ethanol on cholinergic neuronal expression were studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase (ChAT) activity as a cholinergic marker. Ethanol administered to embryos in ovo on day 1 (E1) produced a 30% decrease in ChAT activity, while ethanol administration on day 3 elicited no significant change. Similar effects were produced by ethanol on ChAT activity in the spinal cord. The decrease in ChAT activity in both brain and spinal cord was not accompanied by a significant change in protein content. Of significance were our findings with chronic ethanol treatment: in embryos treated from E1 to E5 and sacrificed at E6, ChAT activity was decreased. In contrast, in embryos treated similarly but sacrificed at E8 ChAT activity was increased. These findings establish that the critical period of cholinergic neuronal sensitivity to ethanol is confined to E. Moreover, the increase in ChAT activity observed after chronic ethanol treatment indicates that the developing neurons have the capability to adapt to ethanol. This apparent adaptation results in overcompensation, as reflected by the increase in ChAT activity. Whether this overcompensation is at the expanse of another neuronal population remains to be investigated.     

Selective persistent reduction in choline acetyltransferase activity in basal forebrain of the rat after thyroid deficiency during early life

The effect of thyroid deficiency on the activity of choline acetyltransferase (ChAT; the marker for cholinergic neurons) was studied in different parts of the rat brain at ages 5, 10, 15 and 25 days, and at day 130 following 102 days of rehabilitation. During normal development, the activity of ChAT increased in the cerebral cortex, hippocampus and basal forebrain, and decreased in the cerebellum. Neonatal thyroid deficiency resulted in a marked retardation of the developmental patterns of the enzyme activity. In the hippocampus the effect diminished with age even during the period of thyroid hormone deprivation, while in the cerebral cortex and cerebellum the enzyme activity was restored to normal only after rehabilitation. In contrast, ChAT activity in the basal forebrain remained persistently reduced in comparison with controls. The results indicate that neonatal thyroid deficiency causes selective irreversible biological damage to subcortical cholinergic neurons.     

β-Bungarotoxin-induced cell-death of neurons in chick retina

The cytotoxicity of β-bungarotoxin (β-BTX), a snake venom neurotoxin with phospholipase A₂ activity, for chick neurons was investigated using organ and monolayer cultures of retina. β-BTX led to a marked reduction in the total activities of choline acetyltransferase and glutamate decar☐ylase of retina cultures at concentrations as low as 100 pM. The total activity of lactate dehydrogenase was, however, much less affected by β-BTX. Also, the total activity of tyrosine hydroxylase of organ-cultured retina decreased only at 30–50 fold higher concentrations of the toxin. The total activity of the glial marker glutamine synthetase was not changed by β-BTX. In contrast to this selectivity for neurons displayed by β-BTX, non-neurotoxic phospholipases A₂ from bee venom and porcine pancreas led to a simultaneous loss of both neuronal and glial marker enzymes.

Light and electron microscopy of organ-cultured retina showed that only cells in the ganglion cell layer and the inner third of the amacrine cell layer degenerated after incubation with β-BTX. In the toxin-sensitive cells, the Golgi apparatus and the endoplasmatic reticulum appeared the first subcellular structures to be affected.

It is concluded that β-BTX preferentially recognizes and/or destroys cholinergic and GABAergic cells in the amacrine and ganglion cell layers of the developing chick retina. This toxin may thus be a useful probe to investigate cell surface properties of cholinergic and GABAergic neurons in the chick central nervous system.     

Effects of opiates on the growth of neuron-enriched cultures from chick embryonic brain

Neuron-enriched cultures derived from 6-day-old chick embryo cerebral hemispheres were treated with morphine or methadone, 10(-5) M or 10(-6) M, on days 4-6 or 6-8 in culture and were evaluated morphologically and biochemically at day 9 using phase contrast microscopy and choline acetyltransferase activity (ChAT) as a cholinergic marker. The treatment of the cultures with morphine markedly affected their growth pattern; specifically, we observed an increased number of flat cells presumptively glia, and aggregates sided by flat cells and devoid of thick bundles of neuritic processes that normally characterize neuron-enriched cultures. These morphologic changes were reflected in a drastic decrease of ChAT activity in cultures treated from day 4 to day 6 but not from 6 to 8. In contrast to morphine, exposure to 10(-6) M methadone from day 4 to day 6 resulted in reduced ChAT activity but the growth pattern of the cultures remained morphologically intact. We suggest that morphine exerts a general neurotoxic effect whereas methadone may affect some specific cholinergic function.     

Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic

Published evidence that ganglion cells in the retinae of nonmammalian species are cholinergic is strong but indirect. In this paper we report results of attempts to demonstrate choline acetyltransferase immunoreactivity in ganglion cells of goldfish retina using two different antisera against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme. We obtained ChAT-immunoreactive staining of amacrine and displaced amacrine cells in the retina and type XIV cells in the tectum, but we obtained no direct immunocytochemical evidence that ganglion cells in the goldfish retina are cholinergic. Thus, ganglion cells identified by retrograde transport of propidium iodide were never ChAT-immunoreactive. Intraocular injections of colchicine did not result in the appearance of a population of ChAT-immunoreactive neurons in the ganglion cell layer. ChAT-immunoreactive axons were not observed in intact, ligated, or transected optic nerves. And finally, the ChAT immunoreactivity of cells and fibers in the optic tectum was unaffected by deafferentation. These experiments provide no positive evidence that any ganglion cells in goldfish retina contain the acetylcholine-synthesizing enzyme, ChAT. While it is possible that our method is too insensitive to detect the enzyme in ganglion cell somata or too specific to recognize the form of ChAT present in these cells, the fact that we can stain putatively cholinergic retinal amacrine cells and tectal neurons makes these alternative explanations improbable. We conclude that it is unlikely that any of the ganglion cells in the retina are cholinergic and that alternative explanations should be sought for previously published results that suggest that they are.     

Muscle-derived factors enhance cholinergic neuronal expression in the chick embryo--II. In culture studies

The effects of muscle-derived factors on the cholinergic and GABAergic neuronal phenotypes were studied in cultures derived from 3-day-old whole chick embryo (E3WE) consisting of proliferating neuroblasts and 8-day-old chick embryo cerebral hemispheres (E8CH) consisting of differentiated neurons. The effects of limb muscle extract (LME) were examined either when added to the culture medium or to the polylysine coating substratum cultures. We also compared the effects of LME derived from 8- to 15-day-old chick embryos. We found that LME added to the medium not only increased choline acetyltransferase activity, a marker for cholinergic neurons throughout the development of neurons in E3WE or E8CH culture, but also delayed the decline in the activity observed in untreated cultures. The marked increase in choline acetyltransferase activity in E8CH cultures grown on substratum-bound LME as compared to those grown in medium-containing LME, suggests that LME factors may be adhesion-promoting substances stimulating neuronal growth. These findings provide evidence that muscle-derived factors may be important in early cholinergic phenotypic expression and support our previous in ovo studies indicating that target-derived factors in limb muscle extract have general cholinotrophic effect.     

Muscle-derived factors enhance cholinergic neuronal expression in the chick embryo--I. In ovo studies

The effects of muscle-derived factors were studied in the chick embryo in ovo, during early neuroembryogenesis. Limb muscle extract (LME) administration during embryonic period E1-E7 produced a significant increase in choline acetyltransferase activity of both spinal cord and brain in 8-day-old chick embryos. Similar treatment failed to induce significant change in the GABAergic phenotypes as assessed by the activity of the enzyme glutamic acid decarboxylase. Administration of limb muscle extract at either embryonic days 1-3 or 4-7 produced a significant increase in choline acetyltransferase activity in the brain, indicating that the critical period of limb muscle extract in the brain to be between embryonic days E1 and E7, a period of neuronal proliferation and differentiation in the brain. On the other hand, LME administration produced no effect on spinal cord choline acetyltransferase activity when given at embryonic days 1-3, whereas it produced a marked increase when given at embryonic days 4-7. Thus, the critical period of limb muscle extract effect in the spinal cord appears to be confined to embryonic days E4-E7, a period of neuronal differentiation and cell death in the spinal cord. These findings indicate that the cholinotrophic activity of muscle-derived factors is not limited to the muscle target tissues but have a general effect on cholinergic neurons in the CNS. Whether these cholinotrophic effects are mediated by a common factor or by different factors is still under investigation.     

Neuronal plasticity in the developing chick brain: interaction of ethanol and neuropeptides

We have examined the influence of ethanol on cholinergic and catecholaminergic neuronal expression in the chick embryonic brain using choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) activities as respective neuronal markers. Ethanol (5-20 mg/50 microliters/day), administered to embryos in ovo from day 1 to 3 of development produced a dose-dependent decrease in ChAT activity while TH activity exhibited a dose-dependent increase when embryos were sacrificed on embryonic day 8. The optimal neurotoxic dose of ethanol following this paradigm was 15 mg/day and the LD50 was 17.5 mg/day for the 3 days. Subsequently, embryos were administered ethanol (15 mg) either alone or concomitantly with growth hormone-releasing hormone (GH-RH; 100 ng/50 microliters/day). Previous studies from this laboratory have demonstrated both potent cholinotropic and catecholaminotropic effects for this neuropeptide, results confirmed in this study. Co-administration of ethanol and GH-RH resulted in a significant increase in ChAT activity as compared to both saline- and ethanol-treated controls when examined on day 8 of embryonic growth. No additive effect was observed in TH activity following co-administration of ethanol and GH-RH. The findings from this study are interpreted to mean that GH-RH represents a potent secondary signal to undifferentiated neuroblasts which may lead to a restoration of the cholinergic neuronal population following neurotoxic insult by ethanol.     

Muscle-derived factors enhance cholinergic neuronal expression in the chick embryo—II. In culture studies

The effects of muscle-derived factors on the cholinergic and GABAergic neuronal phenotypes were studied in cultures derived from 3-day-old whole chick embryo (E3WE) consisting of proliferating neuroblasts and 8-day-old chick embryo cerebral hemispheres (E8CH) consisting of differentiated neurons. The effects of limb muscle extract (LME) were examined either when added to the culture medium or to the polylysine coating substratum cultures. We also compared the effects of LME derived from 8- to 15-day-old chick embryos. We found that LME added to the medium not only increased choline acetyltransferase activity, a marker for cholinergic neurons throughout the development of neurons in E3WE or E8CH culture, but also delayed the decline in the activity observed in untreated cultures. The marked increase in choline acetyltransferase activity in E8CH cultures grown on substratum-bound LME as compared to those grown in medium-containing LME, suggests that LME factors may be adhesion-promoting substances stimulating neuronal growth. These findings provide evidence that muscle-derived factors may be important in early cholinergic phenotypic expression and support our previous in ovo studies indicating that target-derived factors in limb muscle extract have general cholinotrophic effect.     

Cholinergic and GABAergic neurons occur in both the distal and proximal turtle retina.

Turtle retinas were processed immunocytochemically and histochemically to detect the presence of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and glutamate decarboxylase (GAD). We observed cholinergic and gamma-aminobutyric acid (GABA)ergic neurons in the proximal retina, as expected, and in the distal retina as well. ChAT immunoreactivity in the distal retina was observed within the axons and pedicles of numerous cone photoreceptors, suggesting that a population of turtle cone photoreceptors uses ACh as a neurotransmitter. Type L2 horizontal cells were immunoreactive for GAD, and their dendrites invaginated into cone pedicles. AChE histochemistry revealed processes within the outer plexiform layer which formed a loosely organized lattice. In the proximal retina, labeling for ChAT and GAD was similar to that reported by previous investigators. Processes from ChAT-labeled amacrine cells in the inner nuclear layer formed a stratum within the distal inner plexiform layer (IPL) (at 16-21% relative IPL depth), and processes from ChAT-labeled amacrines in the ganglion cell layer formed a proximal ChAT stratum (at 55-58% relative IPL depth). In addition, six AChE-labeled bands and five GAD-labeled bands were observed within the IPL of stained retinas. Therefore, we determined that the two broadest AChE-labeled bands and the two broadest GAD-labeled bands overlapped the two labeled ChAT strata. The evidence for cholinergic and GABAergic processes in both the inner plexiform layer and the outer plexiform layer, combined with electrophysiological evidence from other investigators, raises the possibility that distal retinal neurons may be involved in the encoding of directional information.     

bFGF and heparin but not laminin are necessary factors in the mediums that affect NSCs differentiation into cholinergic neurons

OBJECTIVES: Neural stem cells (NSCs) are self-renewed, pluripotent cells that can differentiate into neurons, astrocytes and oligodendrocytes. Cholinergic neurons are an important kind of neurons that play an essential role in the treatment of Parkinsonism and epilepsy. We are interested in how different mediums affect NSCs differentiation into cholinergic neurons. METHODS: NSCs were isolated from the striatum corpora of embryonic brain in a 14-day pregnant rat. Cells were cultured in basic mediums [F12/DMEM (1:1) including 1% B27 (v/v) and 20 ng/ml EGF] but with different combinations of three supplements: bFGF (20 ng/ml), heparin (5 mug/ml) and laminin (1 mug/ml). After 7 days culturing, cells were immunized with choline acetyltransferase (ChAT), a marker enzyme of cholinergic neuron. RESULTS: We found ChAT could not be detected in the basic mediums with only one supplement. Then, we tested the combination of two out of three. We found that ChAT positive cells could only be detected in the medium with bFGF and heparin (FH). However, when we added the laminin into the FH, more ChAT positive cells appeared. DISCUSSION: This finding suggests that bFGF and heparin are essential in the mediums that affect NSCs differentiation into cholinergic neurons, and laminin is an important positive factor in this process.     

Acetyl-L-carnitine has a neuromodulatory influence on neuronal phenotypes during early embryogenesis in the chick embryo.

Studies from this laboratory and others have demonstrated that neuroblasts in early embryogenesis exhibit a high degree of plasticity with respect to neurotransmitter phenotype. The critical period within which these neuroblasts are sensitive to the effects of endogenous neurotrophins has been defined as 1-3 days of development in the chick embryo. In this study, we examined the influence of acetyl-L-carnitine (ALCAR) administered in ovo during embryonic days 1-3 (E1-E3) and sacrificed at embryonic day 8 (E8) on cholinergic and GABAergic neuronal phenotypes using as neuronal markers the activities of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), respectively. Phenotypic expression was assessed in 3 distinct anatomical regions of the embryonic brain: cerebral hemispheres (CH), optic lobes (OL), and diencephalon-midbrain-brainstem (DMBS). A single administration of ALCAR at embryonic day 1 resulted in a dose-dependent increase in ChAT activity and decrease in GAD activity in CH. ChAT activity was again increased and GAD activity decreased in CH from embryos that were administered ALCAR (100 micrograms/50 microliters/day) daily from embryonic day E1 to E3. No change was observed in either ChAT or GAD activity in OL in response to ALCAR administration during the critical period (E1-E3) at doses ranging from 10 to 500 micrograms/day. In the DMBS, the activity of ChAT exhibited a marked increase at lower doses (10 micrograms) followed by a marked decrease at higher doses (500 micrograms) of ALCAR. The decrease in ChAT activity in DMBS was again observed at an ALCAR dose of 100 micrograms/day when administered from E1 to E3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal subpopulation but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia. © 1994 Wiley-Liss, Inc.