Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of mono- and diacylglycerol lipase activities in ibotenate-induced lesions of nucleus basalis magnocellularis.

Ibotenic acid was injected into the nucleus basalis magnocellularis region of rat brain in order to study whether an elevation of lipase activities was associated with the degeneration of cholinergic neurons in this potential animal model of Alzheimer's disease. Two plasma membrane fractions were prepared from different regions of ibotenate injected (right hemisphere) and non-injected (left hemisphere) rat brain. One plasma membrane fraction was from synaptosomes (SPM) and the other from glial and neuronal cell bodies (PM). Activities of mono- and diacylglycerol lipases in these plasma membrane fractions were markedly increased (3- to 5-fold) in hippocampus, midbrain and frontal cortical regions of rat brain at 10 days after the injection of ibotenate. The activity of choline acetyltransferase was decreased in frontal cortex but unchanged in hippocampus and midbrain. Our results suggest that the increase in lipase activity is much more widespread and non-specific than is the decrease in cholinergic function.     

Axonal transport in the electromotor nerves of Torpedo marmorata

Summary Studies on the axonal transport of cholinergic cell components were made on the electromotor nerves of Torpedo marmorata. Choline acetyltransferase was rapidly accumulated at ligatures on Torpedo nerves, both in vivo and in segments incubated in vitro. In vivo accumulation was maximal approximately one month after nerve interruption. Orthograde transport (both in vitro and in vivo) is calculated to have a velocity of 50–140 mm/day, if, as double-ligature experiments suggest, only about 15% of the axoplasmic enzyme is mobile. A small retrograde accumulation of the transferase was demonstrated. Lactate dehydrogenase did not accumulate but a slight reduction of its activity at ligatures was observed. In contrast to mammalian cholinergic nerves, no accumulation of esterase was observed. ACh accumulation proximal to a cut was apparent and may result in part from local synthesis in the presence of elevated levels of its synthesizing enzyme.Measurements have been made on the activity of choline acetyltransferase in the brain and all parts of the electric system. In view of these results it is difficult to see how the measured rate of axonal translocation is sufficient to supply the levels of the enzyme found within the electric organ. Within the electromotor cells, choline acetyltransferase is highly concentrated in the axon terminals.     

The use of antibody and complement to gain access to the interior of presynaptic nerve terminals

Summary Treatment of synaptosomes with sera containing antibodies (Ab) directed against synaptosomal membranes and complement (C) alters the plasma membrane so that it becomes selectively permeable to small molecules and ions but not to proteins. When synaptosomes are incubated with Ab and C, a rapid release of intracellular K occurs. This release does not occur after treatment with antiserum alone, or with normal serum + C. Ab + C treatment releases approximately the same amount of K as does detergent treatment or hypotonic lysis, two procedures that extensively disrupt the plasma membrane.The selectivity of the complement-induced lesion is consistent with the equivalent pore radius determined in other systems (Michaels and Mayer 1978; Sears et al. 1964). The lesions are large enough to allow the rapid permeation of small ions, but too small to permit the escape of the soluble cytoplasmic enzyme, lactate dehydrogenase. In addition, electron microscopic studies indicate that Ab+C treatment does not lead to gross morphological disruption of the synaptosomes. Ab+C treated synaptosomes are also permeable to calcium and ATP, as demonstrated by the stimulation of Ca sequestration into endoplasmic reticulum when ⁴⁵Ca and ATP are added to the incubation medium.     

The preparation of highly purified GABAergic and cholinergic synaptosomes from mammalian brain

Highly purified and metabolically viable gamma-aminobutyric acid (GABA)ergic and cholinergic synaptosomes were prepared from mammalian brain using antisera raised against glutamate decarboxylase and choline acetyltransferase respectively. These antisera appear to recognise specific outwardly facing components of GABAergic and cholinergic synaptic plasma membranes, as well as the specific neurotransmitter-synthesizing enzymes contained within the particular nerve terminal. After cell surface labelling had occurred, synaptosomes were incubated with magnetic microspheres which had previously been coupled with protein A. Labelled synaptosomes were then retrievable in a magnetic field. This separation technique allowed mg quantities of purified-synaptosomes to be prepared which contained less than 2% non-specific contaminants.     

Localization of enolase in synaptic plasma membrane as an alphagamma heterodimer in rat brain

Enolase, a glycolytic enzyme, is a multifunctional protein with location diversity. We revealed the intracellular distribution of enolase isozymes, such as alphaalpha-, alphagamma- and gammagamma-enolases, in rat brain synaptic terminals by biochemical and immunoelectron microscopic analyses. Specific activity of enolase of synaptic plasma membrane fraction (SPM2) obtained from synaptosomes was 23.2 +/- 4.4 x 10(-2) micromol/mg protein/min in the presence of 0.25% Triton X-100 and that of synaptosomal cytoplasm (LS) was 67.4 +/- 12.1 x 10(-2) micromol/mg protein/min. About half of enolase activity in synaptosomes was distributed to soluble fraction while the remaining stayed in particulate membrane fractions by ultracentrifugation. Immunoblot analysis of the fractions demonstrated both alpha and gamma subunits were distributed in SPM. In addition, immunoelectron microscopic analysis also revealed that both subunits were immunoreactive on the SPM. Using coimmunoprecipitation assay, we confirmed that the enolase was present not only as a homodimer form but also as an alphagamma hybrid form associated with membrane, where both subunits were coimmunoprecipitated from lysate of SPM2 in the presence of Mg(2+). These findings indicate that all forms (alphaalpha, alphagamma, and gammagamma) of enolase translocate to the plasma membrane and associate with some components in the SPM.     

Effect of aging and acetyl-L-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl-L-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl-L-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.     

Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.     

An assay of membrane-bound Trypanosoma brucei phospholipase using an integral membrane protein substrate and detergent phase separation

The technique of phase separation in a solution of the non-ionic detergent Triton X-114 was used to measure the enzymatic conversion of a membrane protein to a soluble product via removal of a hydrophobic moiety. The substrate was the major surface protein (p63), of Leishmania promastigotes and the enzyme was a phospholipase C purified from Trypanosoma brucei. This membrane-bound enzyme is responsible for the cleavage of the hydrophobic lipid membrane anchor of the variant surface glycoprotein (VSG), of T. brucei. The assay is fast, simple and uses small amounts of reagents. It has been used to determine the pH optimum, thermal resistance, and the sensitivity to inhibitors of the trypanosomal phospholipase.     

Regional and subcellular distribution of cholinergic enzyme and receptor activity in goldfish brain

The subcellular and regional distribution of markers for cholinergic activity was studied in goldfish brain. Nicotinic-cholinergic receptor ‘activity’ was analysed by the binding of [¹²⁵I]alphabungarotoxin (K_d = 0.5 nm) and muscarinic cholinergic ‘activity’ by binding of [³H]quinuclidinyl benzilidate (K_d= 0.4 nm). Choline acetyltransferase and acetylcholinesterase activities were also estimated. The subcellular distribution demonstrated an enrichment of the recovered activity in the synaptosomal fractions. In general, the pharmacological studies of receptor binding demonstrated an appropriate specificity for cholinergic receptors.The regional distribution of the markers was compared to data obtained from other species: while choline acetyltransferase in mammals is present in highest specific activity in the telecephalon, in the goldfish the activity is much greater in the hindbrain. Likewise, the binding of quinuclidinyl benzilidate by the diencephalon of the goldfish brain was greater than that by the tetencephalon; but the marked enrichment of choline acetyltransferase in the hindbrain was not accompanied by an enrichment in the material that bound quinuclidinyl benzilidate.     

3H]vesamicol binding in human brain cholinergic deficiency disorders

We measured the binding of the vesicular acetylcholine transport blocker [3H]vesamicol (2-[4-phenylpiperidino] cyclohexanol; AH-5183) to autopsied frontal cortex and amygdala of patients from 4 disorders having a marked brain cholinergic reduction, namely Alzheimer's disease, Parkinson's disease with dementia, dominantly inherited olivopontocerebellar atrophy and Down's syndrome. Although mean activity of the specific cholinergic marker enzyme choline acetyltransferase (ChAT) was markedly reduced by about 60% in frontal cortex in the 4 patient groups and by 80% or greater in amygdala of the Alzheimer's and Down's syndrome patients, [3H]vesamicol binding density was, on average, either normal or only slightly reduced as compared with the controls. This discrepancy suggests that in human brain [3H]vesamicol binding is either not preferentially localized to cholinergic nerve endings or, in these cholinergic deficiency syndromes, a substantial proportion of the vesamicol binding sites persist on cholinergic nerve terminals despite loss of ChAT activity.     

Presynaptic cholinergic mechanisms in brain of aged rats with memory impairments

Presynaptic cholinergic mechanisms were investigated in various brain regions of aged Fisher 344 rats with documented 24 hr retention deficits measured in a single-trial passive avoidance task. Sodium-dependent high affinity choline uptake was found to be decreased by 22% in hippocampus of 23–26 month old animals as compared to 6 month old controls. Prior depolarization of hippocampal or cortical synaptosomes with K⁺ resulted in stimulation of choline uptake which was similar in aged rats and young controls. No age-related differences were observed either in hippocampal, cortical, striatal acetylcholine or choline concentrations, or in the activity of choline acetyltransferase in hippocampus. Synthesis of acetylcholine in hippocampal and cortical slices under basal conditions, as well as under K⁺-stimulated conditions, did not differ in the two age groups examined. These neurochemical findings are consistent with an age-related decrease in hippocampal cholinergic neuronal activity without an actual loss in cholinergic neuron number. It is further suggested that this reduction in cholinergic neuronal activity may be related to the deficit in cognitive performance observed in aged Fisher rats.     

Activation of tyrosine hydroxylase during electrical stimulation of isolated nerve endings of the rat hypothalamus

The effect of electrical stimulation on synaptosomal membrane-bound tyrosine hydroxylase (TH) activity in the rat hypothalamus was investigated. Electrical stimulation intensified respiration and glycolysis, evidence of excitation of the synaptosomes. Against this background, activity of membrane-bound TH was increased. The value of K_m for tyrosine fell from 0.091 to 0.026 mM. Inhibition of the enzyme activity by the end product of cathecholamine biosynthesis (noradrenalin) was reduced. It is postulated that the effect of depolarization on the rate of catecholamine synthesis in nerve endings is effected through modification of TH.Laboratory of Neurochemical Pharmacology, Institute of Pharmacology, Academy of Medical Sciences of the USSR. Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 543–545, May, 1977.     

Membrane-bound choline acetyltransferase is a cell surface marker of the differentiated NS-20Y cholinergic cell line

When differentiated cells of the cholinergic NS-20Y type were incubated with an antiserum to choline acetyltransferase (ChAT), in the presence of complement, immunolysis occurred as demonstrated by release of 20% of total lactate dehydrogenase and 70% of total ChAT. No significant immunolytic effects were observed when either undifferentiated (dividing or non-dividing) or partially differentiated cells were incubated under identical conditions. When the Triton X-114 phase separation technique was employed using membranes from differentiated cells, a small but significant proportion of choline acetyltransferase was recovered in the detergent-rich phase. These results suggest that a membrane-bound form of ChAT is a surface marker of the NS-20Y cell line.     

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine.

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.     

Decrease in choline acetyltransferase activity in the red nucleus of the cat after cerebellar lesion

The use of a microdissection technique and of a very sensitive radioisotopic assay for choline acetyltransferase, the enzyme that synthesizes acetylcholine, showed that the distribution of this enzyme in the feline red nucleus is uneven. Thus, the enzyme content progressively increased in the caudo-rostral direction, the level at the rostral aspect being about twice that at the caudal. Choline acetyltransferase activity in the cat red nucleus was examined after chronic lesions of the cerebellum. Lesions in the area of the dentate and interpositus nuclei were followed by a decrease in enzyme activity in the contralateral red nucleus, whereas no change was seen on the ipsilateral side. Since this enzyme is a very specific marker for cholinergic neurons, the decrease in enzyme activity caused by the lesions suggests the existence of a cholinergic cerebellorubral pathway. This is supported by the finding that the topographical effects on choline acetyltransferase activity in the red nucleus produced by selective lesions of either the dentate or the interpositus nucleus reflected the arrangement of the cerebellorubral pathways.Choline acetyltransferase activity also decreased in the nucleus ventralis lateralis and the nucleus ventralis anterior of the thalamus contralateral to the lesions. Since cerebello-rubral fibres are thought to be collaterals of cerebello-thalamic axons, at least in the case of these originating in the interpositus nucleus, this would indicate the presence of cholinergic fibres in the brachium conjunctivum (or superior cerebellar peduncle).The results are not in complete agreement with some of the previously reported pharmacological studies; this is discussed.     

Immunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method

Monoclonal antibodies to rat striatal choline acetyltransferase were produced by fusion of sensitized mouse lymphocytes with murine plasmacytoma (NS1) cells. Two stable anti-choline acetyltransferase lines were established by limiting dilution cloning. Specificity of antibody was established by the following criteria: (1) on an enzyme linked immunosorbant assay, antibodies reacted against choline acetyltransferase which was highly purified; (2) by immunoprecipitation, monoclonal antibody bound to its antigen and precipitated choline acetyltransferase activity from solution, when used in conjunction with rabbit antimouse IgG; and (3) monoclonal antibody was shown to specifically localize cholinergic neurons. The monoclonal antibody to choline acetyltransferase was radiolabeled in culture by incubating hybridomas in medium containing 3H-labeled amino acids. This 3H-labeled antibody was used for radioautography on cryostat sections of rat peripheral and central nervous systems. In a sampling of areas, highly specific labeling of cholinergic structures was afforded at both light and electron microscopic levels. Double labeling of tyrosine hydroxylase, a catecholaminergic marker, and choline acetyltransferase was carried out by reacting sections first with the 3H-labeled antibody to choline acetyltransferase and then with rabbit antibody to tyrosine hydroxylase. The choline acetyltransferase label was radioautographically processed and tyrosine hydroxylase was visualized by the peroxidase-antiperoxidase method. The combined techniques of peroxidase and radioautographic histochemistry provide permanent electron dense labels which can be examined simultaneously within a single histologic section.     

Demonstration of choline acetyltransferase activity in the caotid body of the cat

1. The distribution of choline acetyltransferase in the carotid body of the cat has been investigated with the electron microscope to determine sites of enzymic activity. This is of relevance to the possible role of acetylcholine as a transmitter in the carotid body.2. Tissues were fixed for short periods and incubated by the method of Kasa, Mann & Hebb, for the fine structural localization of choline acetyltransferase.3. The enzyme was found in the cytoplasm of the type I cells and seemed to be associated with vesicles. No enzyme was found in the large nerve endings synapsing with the type I cell.4. Whole carotid bodies were assayed for their choline acetyltransferase activity and significant amounts were found.5. It is concluded that acetylcholine may be a transmitter in the carotid body and that it is synthesized in type I cells. A possible mode of initiation of chemoreceptor afferent impulses is suggested.     

Neocortical cholinergic innervation: A description of extrinsic and intrinsic components in the rat

Electrothermic lesion of the peri-pallidal region of the rat caused a marked reduction in the activity of choline acetyltransferase in the ipsilateral fronto-parietal cortex without affecting the activity of glutamate decarboxylase. Only lesions that involved the ventral globus pallidus significantly reduced cortical choline acetyltransferase activity; and lesions limited to the thalamus, internal capsule, pyriform cortex or zone incerta were ineffective. Excito-toxin lesions of the ventral globus pallidus caused 45-5% reductions in all presynaptic markers for cholinergic neurons but did not significantly decrease presynaptic markers for noradrenergic, serotonergic or histaminergic neurons in the cortex. The maximal reductions in cortical choline acetyltransferase activity achieved with the pallidal lesion was 70%; and enzyme activity reached its nadir by four days after placement of the lesion. The pallidal lesion, which ablated the large isodendritic acetylcholinesterase positive neuronal perikarya, resulted in a profound loss in histochemically stained acetylcholinesterase-reactive fibers in the fronto-parietal cortex but not in the cingulate, pyriform and occipital cortex or hippocampal formation; analysis of the subregions in choline acetyltransferase activity. The kainate lesion of the parietal cortex to ablate intrinsic neurons did not reduce the activity of tyrosine hydroxylase, a marker for noradrenergic terminals, but depressed glutamate decarboxylase by 68%; in contrast choline acetyltransferase activity fell only 29%. The results indicate that approximately 70% of the cholinergic innervation in the fronto-parietal cortex is derived from acetylcholinesterase positive neurons in the peripallidal nucleus basalis, whereas the remainder appears to be localized in cortical intrinsic neurons.     

Ultrastructural, functional and biochemical characteristics of mouse and human neuroblastoma cell lines

Three commercially available neuroblastoma cell lines were analyzed to determine their ultrastructural, functional and neurochemical characteristics. The cell lines were Neuro-2a and NB41A3, derived from mouse neuroblastoma, and IMR-32, derived from a human neuroblastoma.Ultrastructurally, the neuroblastoma exhibited two major differences. Both mouse neuroblastoma cell lines contained virus-like particles, whereas the human neuroblastoma was free of virus particles. Scanning electron-microscopy revealed that the external morphology in the mouse cell lines was heterogeneous, suggesting the presence of more than one cell type, while the external morphology of the human cell line was homogeneous, suggesting the presence of a single cell type.Neurochemically, choline acetyltransferase, tyrosine hydroxylase and glutamate decarboxylase, synthetic enzymes for acetylcholine, catecholamines and γ-aminobutyric acid, respectively, were present in each cell line. Mean choline acetyltransferase activity was highest in NB41A3, but differences in this activity among the three lines were not statistically significant. Tyrosine hydroxylase activity in IMR-32 was significantly higher than in the mouse neuroblastoma cells. Glutamate decarboxylase activity was equivalent in all three cell lines.Functionally, each cell line was tested to determine its ability to form cholinergic synapses on cultured striated muscle cells. Only the IMR-32 cells formed appreciable numbers of synapses with muscle cells as detected by electrophysicological recording.The delineation of several characteristics of these three neuroblastoma cell lines will allow other investigators to choose the most appropriate of these cell lines for their studies. The heterogeneity of cell types of the mouse neuroblastoma may compromise biochemical measurements as each cell type may have different characteristics. Since only the IMR-32 form cholinergic synapses and have no more choline acetyltransferase activity than the other two cell lines, the presence of this enzyme is not a reliable marker for the ability to form synapses.