CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN CULTURED BRAIN CELLS FROM CHICK EMBRYOS

—Dissociated cells from brains of 7-day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4-6 days. Total DNA and protein increased five-fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40-fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three-fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l -Thyroxine, cyclic AMP (adenosine-3′,5′-monophosphate) or theophylline promoted increased levels of both enzymes by 30-200 per cent. l -Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10^-3m -5-flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10^-3m -morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.     

Factors regulating development of an embryonic mouse sympathetic ganglion.

Embryonic development of the mouse superior cervical ganglion (SCG) is defined in vivo and in vitro using morphologic, morphometric, and biochemical approaches. Catecholamine fluorescence was present in the SCG on Day 14 of gestation and underwent characteristic changes in distribution among neurons between this time and adulthood. During prenatal ontogeny, choline acetyltransferase (ChAc) activity increased 2-fold, while tyrosine hydroxylase (T-OH) activity rose 30-fold and total protein increased 4-fold. Ganglionic explants from 14-day embryos extended neurites and exhibited specific biochemical development in medium without added nerve growth factor (NGF). However, the addition of NGF further stimulated neuronal development: Ganglia exhibited significant increases in ChAc and T-OH activities and in total protein compared to controls grown in medium without added NGF. The presence of target submandibular gland radically altered development of T-OH activity in cultured sympathetic ganglia. By 5 days in culture, ganglia grown with target tissue, even in the presence of anti-NGF, exhibited a 10- to 15-fold increase in T-OH activity compared to zero-time controls, and a 2-fold increase over ganglia grown alone or with nontarget tissue. Ganglia grown with target salivary glands showed a correspondingly greater elaboration and directionality of nerve fiber outgrowth, even in the presence of anti-NGF.     

Acetylcholine and its metabolic enzymes in developing antennae of the moth, Manduca sexta.

Antennae of the moth, Manduca sexta, are thickly populated with sensory neurons, which send axons through antennal nerves to the brain. These neurons arise by cell divisions and differentiate synchronously during the 18 days of metamorphosis from pupa to adult. Biochemical studies support the hypothesis that antennal neurons use acetylcholine (ACh) as a neurotransmitter: (1) Antennae incubated with [¹⁴C]choline synthesize and store [¹⁴C]ACh; several other transmitter candidates do not accumulate detectably when appropriate radioactive precursors are supplied; (2) antennae and antennal nerves contain endogenous ACh; and (3) extracts of mature antennae contain choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) with properties similar to those reported for the enzymes from other arthropods. Levels of ACh, ChAc, and AChE begin to increase in antennae soon after the sensory neurons are “born.” Levels rise exponentially for over a week as the neurons differentiate and then reach a plateau, at about the time the neurons reach morphological maturity, that is maintained into adulthood. In contrast, levels of carnitine acetyltransferase, cholinesterase, and soluble protein, presumably not confined to nervous tissue, change little during metamorphosis. Levels of ACh, ChAc, and AChE rise in an intracranial segment of antennal nerve at about the same time as in the antenna, indicating that axons can transport neurotransmitter machinery at an early stage in their development.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry

Under certain culture conditions, neonatal rat superior cervical ganglion neurons display not only a number of expected adrenergic characteristics but, paradoxically, also certain cholinergic functions such as the development of hexamethonium-sensitive synaptic contacts and accumulation of choline acetyltransferase (ChAc). The purpose of this study was to determine whether the entire population of cultured neurons was aquiring cholinergic capabilities, or whether this phenomenon was restricted to a subpopulation. After 1--6 and 8 wk in culture, neurons were fixed in KMnO4 after incubation in norepinephrine and prepared for electron microscopy analysis of synaptic vesicle content to determine whether vesicles were dense cored or clear. ChAc, acetylcholinesterase (AChE), and DOPA-decarboxylase (DDC) activities were assayed in sister cultures. In the period from 1 to 8 wk in culture, the average ChAc activity per neuron increased 1,100-fold, and the DDC and AChE activities increased 20- and 30-fold, respectively. After 1 wk in culture, 48 of 50 synaptic boutons contained predominantly dense-cored vesicles, but by 8 wk the synaptic vesicle population was predominantly of the clear type. At intermediate times, the vesicle population in many boutons was mixed. The morphology of the synaptic contacts on neuronal surfaces was that characteristic of autonomic systems, with no definite clustering of the vesicles adjacent to the area of contact. Increased vesicle size correlated with increasing age in culture and the presence of a dense core. Considering these data along with available physiological studies, we conclude that these cultures contain one population of neurons that is initially adrenergic. Over time, under conditions of this culture system, this population develops cholinergic mechanisms. That a neuron may, at a given time, express both cholinergic and adrenergic mechanisms is suggested by the approximately equal numbers of clear and dense-cored vesicles in the boutons found at the intermediate times.     

Culture of chick embryo neural retina in serum-free medium : Expression of cholinergic proteins

Dissociated retinal cells from 8-day-old chick embryos were cultivated in serum-containing and in defined serum-free media. Under the latter conditions, and using polylysine as a substrate, the proliferation of glial cells was almost completely prevented, and pure (>90%) neuronal cultures could be maintained for up to 7 days in vitro. The specific but not the total activities of choline acetyltransferase and of the nicotinic and the muscarinic acetylcholine receptor were increased under serum-free culture conditions. Autoradiographic studies with [¹²⁵I]α-bungarotoxin, a selective ligand for nicotinic cholinergic receptors, showed that serum-free culture conditions may constitute a useful tool for identifying biochemically different types of retinal neurons in tissue culture.     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

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     

DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE WITHIN THE SUBSTANTIA NIGRA AND IN OTHER BRAIN REGIONS FROM CONTROL AND PARKINSONIAN PATIENTS

—The distribution of choline acetyltransferase (ChAc, EC 2.3.1.6) and l -glutamate 1-carboxylyase (glutamate decarboxylase, GAD, EC 4.1.1.15) was studied in serial frontal slices of the substantia nigra (SN) (pars compacta, PC; pars reticulata, PR; an intermediate region, IR) as well as in other brain areas from post mortem tissue of control and Parkinsonian patients. Within the SN from control brain ChAc and GAD activities showed a distinctive distribution: ChAc activity in PC was higher than in PR and IR by 427% and 253% respectively and within PC the enzyme activity in the rostral part exceeded that in the control part by 353%. The GAD activity in PC was higher by 41% than that in PR and within PC seemed to be higher in the caudal than in the rostral part. For both enzyme activities there were no significant differences between PR and IR or within these regions. In Parkinsonian brain both ChAc and GAD activities were reduced to 15-25% of controls in all 3 regions of the SN. The distinctive distribution of ChAc and GAD activity found in the SN of control brain was abolished: no difference was observed between the 3 regions. However, within PC the ChAc activity was lower in the medial than in the rostral part. Since nigral ChAc is possibly located in interneurons, the decrease in enzyme activity may be connected with the cell loss observed in the SN of Parkinsonian brain. By contrast, nigral GAD is probably contained in terminals of strio-nigral neurons and the decrease in enzyme activity in Parkinson's disease in the absence of striatal cell loss, may reflect a change in the functional state of these GABA neurons. Among various areas of control brains ChAc activity was highest in caudate nucleus and putamen while GAD was highest in SN. caudate nucleus, putamen and cerebral cortex. In Parkinsonian brain the most severe reduction in ChAc and GAD activities was found in the SN.     

CHOLINE ACETYLTRANSFERASE (ChAc) ACTIVITY IN DEVELOPING CHICK OPTIC CENTRES AND THE EFFECTS OF MONOLATERAL REMOVAL OF RETINA AT AN EARLY EMBRYONIC STAGE AND AT HATCHING

The choline acetyltransferase (ChAc) activity was measured in the optic centres of chick embryos after early removal of the optic cup and of young chicks after monolateral extirpation of the right eyeball after hatching. The contralateral optic lobes were thus deprived of their complement of retinal fibres. The following results were obtained: in chick embryos the ChAc was slightly lower in the deafferented lobe between the 10th and the 14th day of incubation; between the 14th and the 17th day a critical fall in activity was observed leading to a significant ChAc loss of 71 per cent. In eye deprived chicks no significant change in total ChAc activity occurred during the first postoperative month; significant changes were found only in the second month. The results reached so far suggest that removal of retinal fibres does not cause short term changes in optic centre ChAc in either the embryo or the chick. ChAc contained in nerve cell bodies seems independent of synapses and its behaviour is interpreted as a reflection of metabolic disturbance of the centre.     

Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase

Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.     

Immunological properties of rat brain choline acetyltransferase.

Abstract— Four antisera active against choline acetyltransferase (ChAc) were obtained by injecting 22 rabbits with rat brain ChAc. The ChAc preparations used for immunization (specific activity from 015 to 2 μmol/min/mg of protein) were not pure and the antisera produced were not monospecific. The antisera inhibited and precipitated ChAc, but the precipitated enzyme-antibody complexes still retain ChAc activity. One millilitre of the most active serum precipitates 0–5 μmol/min of rat brain ChAc at the equivalence point. Its titre expressed in mg/ml of immunoglobulins precipitated with the antigen and the equivalence point was calculated at about 0.08 mg/ml of serum. This relatively low titre explains the lack of any visible ChAc immunoprecipitate in an immunodiffusion test. Cross-reactivity studies revealed that ChAc has undergone few changes during evolution, since antisera produced against rat brain ChAc still precipitate ChAc from fish (Torpedo).     

A histochemical study of the distribution of choline acetyltransferase and acetylcholinesterase activity in sensory ganglia and nerve roots of the bullfrog

Synopsis Histochemical techniques were employed for the localization of choline acetyltransferase (ChAc; EC 2.3.1.6.), acetylcholinesterase (AChE; EC 3.1.1.7) and cholinesterase (ChE; EC 3.1.1.8) activities in dorsal and ventral roots and dorsal root ganglia of the bullfrog. AChE activity was present in most of the neuronal elements of dorsal root ganglia, in some nerve fibres in the dorsal roots, and in all nerve fibres in ventral roots. ChE activity in dorsal root ganglia and in the dorsal roots was confined to non-neuronal elements. No ChE activity was demonstrable in the ventral roots. ChAc activity was localized in many neurons of the dorsal root ganglia and in some nerve fibres of the dorsal roots; however, none of the ventral root fibres were visibly reactive. Some supportive cells of the dorsal roots and ganglia contained small amounts of ChAc activity. Except for the ventral roots, the histochemical distribution of AChE and ChAc activity was similar. The results of solubility studies indicated that under the histochemical conditions, approximately 50% of the ChAc remained bound to the dorsal roots and ganglia, whereas more than 90% of the ChAc in the ventral roots was soluble. This would account for the lack of reactivity in ventral root fibres. Differences in ChAc solubility are discussed in relation to the interpretation of histochemical data and in relation to the concept of multiple forms of ChAc. The results of this study indicate that at least one-third of the neurons of the dorsal root ganglia contain significant levels of the enzymes involved in both the synthesis and hydrolysis of acetylcholine.     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

Ciliary ganglion neurons in cell culture: high affinity choline uptake and autoradiographic choline labeling

Chick ciliary ganglion neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline that has the properties expected for cholinergic neurons. The uptake has an apparent K_m of ca. 0.3 μM and is blocked by addition of 10 μM hemicholinium-3 or replacement of Na⁺ by Li⁺ in the uptake medium. When the choline uptake mechanism is used to label ciliary ganglion neuron-myotube cultures autoradiographically, over 99% of the neurons are labeled. A few cells with neuronal morphologies in such cultures (<1%) are labeled by γ-[³H]aminobutyric acid uptake. The number of [³H]choline-labeled neurons and the amount of Na⁺-dependent choline uptake is the same for ciliary ganglion neurons grown with and without skeletal myotubes. Rat superior cervical ganglion neurons, grown in cell culture under conditions that induce them to synthesize acetylcholine and form cholinergic synapses, are labeled by [³H]choline uptake, though not as heavily as ciliary ganglion neurons. In contrast, chick dorsal root ganglion neurons, a presumed population of noncholinergic neurons, are not labeled by [³H]choline uptake. Thus high affinity choline uptake can be used to label autoradiographically the cholinergic neurons tested, while at least one population of noncholinergic neurons remains unlabeled.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. II. Dependence on postnatal age

Superior cervical ganglion (SCG) neurons taken from perinatal rats and dissociated in culture develop cholinergic properties. This report examines this "plasticity" of neurotransmitter function with regard to its dependence on the stage of neuronal development. Explants of SCG from rats ranging in age from 2 d to adult were cultured, and the number of neurons surviving after 6 wk in culture was evaluated. The activities of choline acetyltransferase (ChAc) and DOPA decarboxylase (DDC) were assayed for each age group over time in culture, and the cytochemistry of the synaptic vesicle population was studied after norepinephrine loading and KMnO4 fixation. The specific activity of ChAc in all explants fell during the first 3--4 d in culture (secondary to degeneration of presynaptic terminals), with an increase during the next 30 d in explants from all age groups except in those from the 22-d- old and adult rats. The highest activity found after 1 mo in culture was in explants from 2-d-old rats (62.5 mmol per kg dry wt per h); the lowest was in explants from adults (1.3 nmol per kg dry wt per h). After 1 mo in vitro, there were no significant differences in DDC activity among explants from animals of any age (similar to approximately 220 mmol per kg dry wt per h). Co-culture of the SCG explants with heart muscle increased even further the ChAc activity in explants from 2-d-old rats but not in explants from 16-d-old and 6.5-wk- old animals. The cytochemistry of the synaptic vesicle population in 1- mo-old cultures correlated well with the ChAc activity; when the ChAc activity was high, the proportion of synaptic vesicles with clear centers was 71--88%. In explants from adult animals, only 12% of the vesicles contained clear centers. From these data we conclude that the maturity of the SCG neuron influences the degree to which it is able to adjust its neurotransmitter mechanisms. That the axons of this neuron are interacting with target tissues during the time that neurotransmitter plasticity is retained suggests that interaction with the target may play a role in the determination of transmitter type.     

Alterations in dopaminergic receptors in Huntington's disease.

To detect variations in dopaminergic receptors and cholinergic activity in regions of postmortem Huntington's diseased brains, ³H-spiroperidol binding assays and choline acetyltransferase (ChAc) activities were carried out. A significant reduction in ³H-spiroperidol binding in the caudate nucleus, putamen and frontal cortex of choreic brains was detected which appeared to be due to a decrease in the total number of binding sites rather than to a decrease in affinity of ³H-spiroperidol for the dopaminergic receptor. In choreic brains, there were also significant reductions in ChAc activity in the caudate nucleus and putamen. The decreases of both ³H-spiroperidol binding and ChAc activity in the neostriatum suggest that the dopaminergic receptors are localized postsynaptically on cholinergic interneurons. Dopaminergic receptor alterations in the basal ganglia may be one of the causes of the abnormal motor movements found in HD while alterations of these receptors in the frontal cortex may be associated with the neuronal degeneration found in that area of choreic brains.     

Head and trunk neural crest in vitro: Autonomic neuron differentiation

Isolated cultures of premigratory neural crest cells were used to study the initial stages of autonomic neuron development. Autonomic neurons are phenotypically characterized on the basis of their neurotransmitter synthetic enzymes, dopamine β-hydroxylase (DBH) and choline acetyltransferase (CAT). DBH converts dopamine to norepinephrine in noradrenergic neurons while CAT synthesizes acetylcholine from choline in cholinergic neurons. Activities of both enzymes were detected in isolated cultures of trunk neural crest and head neural crest. DBH was detected at all culture ages examined (from 1 to 20 days) whereas CAT activity was first detected only after 5 days in vitro. While specific enzyme activity of DBH peaks on Day 6 and specific enzyme activity of CAT peaks on Day 10, absolute activity for both enzymes increases throughout the 20-day culture period. DBH and CAT develop in vitro without any spinal presynaptic input, without typical target tissue interactions (such as blood vascular elements or heart tissue), and without addition of conditioned medium factors.     

Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina

Background  

Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT).     

Multiple Forms of Choline Acetyltransferase from Rat Brain

WE have found that several different forms of choline acetyl-transferase (ChAc) from rat brain can be separated by isoelectric focusing. Such heterogeneity of ChAc is of particular interest in the context of its ultrastructural localization. Subcellular fractionation^1–4 and histochemistry⁵ have shown that the enzyme in rat, in conditions of low ionic strength and pH, adhered to several different membranous structures.     

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 μM) or higher (150 μM) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 μM choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K_m's with different affinities.The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.