A radioligand-binding assay for adenosine in tissue extracts

A radioligand-binding assay employing adenine analog-binding protein from rabbit crythrocytes and [³H]adenosine of high specific activity measures less than 1 pmol of adenosine in neutral HClO₄ extracts of tissue. Adenine and its nucleotides interfere with adenosine binding, so are removed by preliminary chromatography on PEI and phenyldihydroboryl cellulose columns. Free and bound adenosine are separated by filtration through cellulose acetate membranes or by absorbing free adenosine on charcoal. The recovery of 1.0–4.0 pmol of adenosine added to blood extracts and carried through purification and radioligand assay averaged 100% with an absolute error of ±0.23 pmol.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

Large-Scale Purification of Beta-Adrenergic Receptors from Mammalian Cells in Culture

S49 Mouse lymphoma wild-type cells were grown in spinner cultures of 40 liters to a density of approximately 3 million cells/ml. Growth of cells to high density (2–3 million cells/nil) required that the cell suspensions be bubbled with oxygen. Cells from 40 liter cultures were collected by centrifugation and disrupted by nitrogen cavitation. Highly purified membranes (0.35 g membrane protein) that were rich in beta-adrenergic receptor (0.4 – 0.7 pmol receptor/mg membrane protein) were prepared by differential centrifugation and then solubilized with the plant glycoside, digitonin (1.5% digitonin at 3 mg of membrane protein/ml). Beta-adrenergic receptors were isolated and purified by sequential affinity chromatography, ion-exchange chromatography, and steric exclusion high-pressure liquid chromatography. The extract was subjected to affinity chromatography on a dcrivatized Scpharose-4B CL column to which the high-affinity, beta-adrenergic antagonist (-)alprcnolol had been immobilized. Following extensive washing, the receptor bound to this matrix was eluted using a 0 – 100 micromolar linear gradient of (-)alprenolol. The receptor eluted as a sharp peak at 30 micromolar ligand and displayed a specific activity of 280 pmol receptor/mg of protein. Ion-exchange chromatography on DEAE-Sephacel increased the specific activity to 950 pmol/mg of protein. The final step in the purification, steric-exclusion high-pressure liquid chromatography on two TSK-3000 and one TSK-2000 columns, tandem linked, resulted in a beta-adrenergic receptor preparation with a specific activity of 6700 pmol/mg of protein (15, 900-fold purification). Autoradiography of the radioiodinated pure receptor, the receptor photolabcled with [¹²⁵l]iodoazidobenzylpindolol or silver-staining of chemical amounts of protein revealed that the M_r of the pure receptor is 66, 000 upon polyacrylamide gel electrophoresis in sodium dodccyl sulfate under reducing conditions. The receptor is a bcta₂-subtype adrenergic receptor.     

Differential assay for choline acetyltransferase

A rapid and sensitive radiochemical assay for choline acetyltransferase (EC 2.3.1.6) is reported. The assay allows for the fact that during incubation of [¹⁴C]acetyl-CoA and choline with a cell homogenate, at least one product is formed besides [¹⁴C]acetylcholine, which passes an anion exchange column. In contrast to [¹⁴C]acetylcholine, this major contaminant ([¹⁴C]acetylcarnitine) is not hydrolyzed apparently by Electrophorus acetylcholinesterase. Therefore, two types of assays are performed, the one in the presence of an acetylcholinesterase inhibitor, the other in the presence of acetylcholinesterase from Electrophorus. After passing the reaction mixtures over anion exchange columns, the radioactivities of the effluents are determined. Their difference is proportional to the choline acetyltransferase activity.     

Uptake and release of choline in cultures of human glioma cells

Human glioma cells (138MG) have a low-affinity uptake system for choline (Km = 20 µM; V_max = 56 pmol/min/10⁶ cells). The uptake is reduced by acetylcholine, hemicholinium-3, HgCl₂, and phosphodiesterase inhibitors. Release of [³H]choline from preloaded cultures showed two pools with half-lives of 1.3 and 160 min. Choline release was stimulated by 8-bromo-cAMP or isobutylmethylxanthine. The results suggest that release of choline occurs by a facilitated diffusion transport system and is increased by elevations of intracellular cAMP.     

A single-vial biphasic liquid extraction assay for choline acetyltransferase using [3H]choline

A single-vial liquid extraction assay for choline acetyltransferase that uses [³H]choline as the labeled substrate has been devised. [³H]Choline is incubated with an excess of acetyl-CoA in a small reaction vial which also serves as a scintillation vial. After a suitable reaction period, unreacted [³H]choline is quickly and quantitatively converted to phosphoryl-[³H]choline by the addition of an excess of choline kinase. This treatment is followed by the addition of scintillation fluid containing sodium tetraphenylboron after which the vial is capped, shaken, and counted. A two-phase system is produced in which product [³H]acetylcholine is selectively extracted into the scintillation fluid, where it is counted. Phosphoryl-[³H]choline remains in the aqueous phase and is not counted. This assay is rapid, simple, and quite sensitive. In comparison to assays using acetyl-CoA as the labeled substrate, it is less sensitive to interference by other enzymes and thus more suitable for measuring choline acetyltransferase in crude extracts and in the initial stages of purification. Similar single-vial radiometric assays are described for choline kinase and acetyl-CoA hydrolases.     

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The peptides include bradykinin, Hyp³-bradykinin, des-Arg⁹-bradykinin and fragments of fibrinogen α-chain (Fib-α_[605–629]), inter-α-trypsin inhibitor heavy chain 4 (ITIH_4[666–687]) and complement component 4a (C4a_{[1337–1350]}). Ile¹³-ITIH_4[666–687], d20-C4a_{[1337–1350]} and Sar-D-Phe⁸-des-Arg⁹-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM d-l-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10–500 ng/ml), Hyp³-bradykinin (4–200 ng/ml), des-Arg⁹-bradykinin (2–100 ng/ml), Fib-α_[605–629] (120–3000 ng/ml), ITIH_4[666–687] (0.4–10 ng/ml) and C4a_{[1337–1350]} (1–25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.     

THE RELEASE IN VIVO OF [3H]ACETYLCHOLINE FROM CAT CAUDATE NUCLEUS AND CEREBRAL CORTEX BY ATROPINE, PENTYLENETETRAZOL, K+-DEPOLARIZATION AND ELECTRICAL STIMULATION

Abstract— In the present experiments, the resting and stimulus evoked release of newly synthesized [³H]acetylcholine from the caudate nucleus, the cerebral cortex and the cerebellar cortex into the perfusate of the push-pull cannula was studied in the unanesthetized, midpontine, pretrigeminally transected cat following infusion at the push-pull site of [³H]choline. Separation of the metabolites in the perfusate revealed that after 20 min, approximately 20% of the recovered radioactivity in the sample was in a lipid fraction, about 10% was found to be phosphorylcholine and around 3% was observed to be incorporated into acetylcholine. The rest of the recovered radioactivity remained as choline. Electrical stimulation applied directly to the caudate nucleus, local potassium depolarization, atropine and pentylenetetrazol were all observed to result in a significant and stimulus dependent increase in the levels of [³H]acetyIchoIine, but not [³H]choline or [¹⁴C]urea in the effluent of the push-pull cannula located in the caudate nucleus. A similar release of newly synthesized [³H]acetylcholine was observed following atropine and potassium stimulation in the cerebral but not the cerebellar cortex. The specificity of this evoked increase in the levels of [³H]acetylchoiine is substantiated by obtaining the release with stimuli having different modes of action, by the absence of stimulus evoked changes in the levels of other water-soluble elements found in the perfusate and by the absence of an observable release of [³H]acetylcholine in perfusion experiments involving the cerebellum, a tissue not thought to have strong cholinergic innervation. The percentage increases in release of [³H] acetylcholine over baseline levels evoked by the various methods closely corresponded to those reported in the literature for authentic acetylcholine. This was taken to suggest that the neuronal pools containing endogenous acetylcholine and those containing newly synthesized acetylcholine, if not identical, were disposed to behave in the same manner following the activation of the synapse.     

Biochemical studies on muscaranic receptors in the salamander olfactory epithelium

Muscarinic cholinergic receptors in the olfactory epithelium of the salamander, Ambystoma tigrinum, were studied via binding of 3-[³H]quinuclidinyl benzilate. The receptors are present on the olfactory receptor cells in the epithelium to an amount of 0.08 pmol/mg homogenate protein. Both choline acetyltransferase and acetylcholine esterase are present in the salamander olfactory epithelium.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Quantitative analysis of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol in human plasma using high-performance liquid chromatography

A highly sensitive and precise high-performance liquid chromatography (HPLC) assay was developed and validated for the quantitation of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl) phenoxy]ethanol (FC-1271a) in human plasma. Plasma samples (1.0 ml) containing FC-1271a and internal standard (toremifene citrate; Fareston^®) were extracted using a 2% 1-butanol, 98% hexane solution with an extraction efficiency of >97%. Samples were reconstituted in methanol, irradiated with high intensity ultraviolet light (254 nm) for 1 min, and injected onto a C₁₈ reverse phase column. Samples were eluted isocratically at a flow-rate of 0.5 ml/min with a mobile phase consisting of 6.5% water and 0.5% triethylamine in methanol. The fluorescence of photochemically activated compounds was detected using a fluorometer set at an excitation wavelength of 266 nm and emission wavelength of 370 nm. Under these assay conditions, standard calibration curves were linear through a concentration range of 10–400 ng/ml. In summary, we have developed and validated an HPLC assay to quantitate FC-1271a in human plasma.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

Acetyl-l-carnitine as a precursor of acetylcholine

Synthesis of [³H]acetylcholine from [³H]acetyl-l-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [³H] and [¹⁴C] labeled acetylcholine were produced when [³H]acetyl-l-carnitine andd-[U-¹⁴C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-l-carnitine to acetylcholine was dependent on concentration of acetyl-l-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-l-carnitine in the treatment of age-related cholinergic deficits.     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes

Sodium-dependent [3H]choline uptake and coupled [3H]acetylcholine synthesis were inhibited in rat cerebral cortical synaptosomes in a dose- (1-10 micrograms/ml) and time-dependent manner by affinity-purified antibodies directed against ubiquitin (anti-Ub). Neither sodium-independent [3H]choline uptake nor [3H]acetylcholine release was affected by up to 10 micrograms/ml anti-Ub, indicating that the cholinergic terminals were not depolarized by the anti-Ub. Binding of anti-Ub to synaptosomes, as measured with 125I-protein A, was saturable and occurred over the same concentration range (1-10 micrograms/ml) at which uptake inhibition was observed. Although preimmune IgG bound to the synaptosome preparation to a greater extent and was apparently not readily saturable, this fortuitous binding was without effect on high affinity choline uptake and conversion to acetylcholine. The results suggest the presence of a ubiquitin-protein conjugate on the synaptosomal surface and a functional relationship between this protein conjugate and the sodium-dependent choline transport system.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Acetylcholine formation from glucose following acute choline supplementation

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-¹⁴C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-¹⁴C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-¹⁴C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that shorttern elevation of brain choline does not enhance ACh formation from [U-¹⁴C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.Special issue dedicated to Dr. Louis Sokoloff.     

Metabolism of acetylcholine in the nervous system of Aplysia californica. IV. Studies of an identified cholinergic axon

[3H]Choline, injected directly into the major axon of the identified cholinergic neuron R2, was readily incorporated into [3H]acetylcholine. Its metabolic fate was similar to that of [3H]choline injected into the cell body of R2. Over the range injected, we found that the amounts of acetylcholine formed were proportional to the amounts injected; the synthetic capability was not exceeded even when 88 pmol of [3H]choline were injected into the axon. Newly synthesized acetylcholine moved within the axon with the kinetics expected of diffusion. We could not detect any selective orthograde or retrograde transport from the site of the injection. In contrast, as indicated by experiments with colchicine, 30% of the [3H]acetylcholine formed after intrasomatic injection was selectively exported from the cell body and transported along the axon. Most of the [3H]acetylcholine was recovered in the soluble fraction after both intra-axonal and intrasomatic injection of [3H]choline; only a small fraction was particulate. The significance of large amounts of soluble acetylcholine in R2 is uncertain, and some may occur physiologically. The concentrations of choline introduced by intraneuronal injection into both cell body and axon were, however, greater than those normally available to choline acetyltransferase in the cholinergic neuron; nevertheless, these large concentrations were efficiently converted into the transmitter. The synthetic capacity of the neuron supplied with injected choline may exceed the capacity of storage vesicles and of the axonal transport process.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.