Detection of basal acetylcholine in rat brain microdialysate

A liquid chromatography-electrochemistry (LC-EC) method is described for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. This method is based on the separation of ACh and choline (Ch) by microbore liquid chromatography followed by passage of the effluent through a post-column immobilized enzyme reactor (IMER), containing acetylcholinesterase (AChE) and choline oxidase (ChO), and then the electrochemical detection of the hydrogen peroxide produced. Instead of the conventional platinum electrode used for the anodic detection of hydrogen peroxide, a peroxidase-redox polymer modified glassy carbon electrode operated at + 100 mV vs. Ag/AgCl has been used to detect the reduction of hydrogen peroxide. With this method, a detection limit of 10 fmol (injected) for ACh (S/N = 3:1) was obtained and the basal ACh concentration in striatal microdialysate was determined without using esterase inhibitors.     

Multienzyme microbiosensor based on electropolymerized o-phenylenediamine for simultaneous in vitro determination of acetylcholine and choline

The electrochemical biosensors based on poly(o-phenylenediamine) (PoPD) and acetylcholinesterase (AChE) and choline oxidase (ChO) enzymes were fabricated on carbon fibre (CF) substrate. The electropolymerized PoPD was used to reduce the interfering substances. The electrode assembly was completed by depositing functionalized carbon nano tubes (FCNTs) and Nafion (Naf). Amperometric detection of acetylcholine (ACh) and choline (Ch) were realized at an applied potential of +750 mV vs Ag/AgCl (saturated KCl). At pH 7.4, the final assembly, Naf-FCNTs/AChE-ChO((10:1))/PoPD/CF(Elip), was observed to have high sensitivity towards Ch (6.3±0.3 μA mM(-1)) and ACh (5.8±0.3 μA mM(-1)), linear range for Ch (K(M)=0.52±0.03 mM) and ACh (K(M)=0.59±0.07 mM), and for Ch the highest ascorbic acid blocking capacity (97.2±2 1mM AA). It had a response time of <5s and with 0.045 μM limit of detection. Studies on different ratio (ACh/Ch) revealed that 10:1, gave best overall response.     

A new microdialysis-electrochemical device for in vivo simultaneous determination of acetylcholine and choline in rat brain treated with N-methyl-(R)-salsolinol

Acetylcholine (ACh) and choline (Ch) play a critical role in cholinergic neurotransmission and the abnormalities in their concentrations are related to several neural diseases. Therefore, the in vivo determination of ACh and Ch is important to the research on neurodegenerative disorders. In this work, electrochemical biosensors based on poly(m-(1,3)-phenylenediamine) (pmPD) and polytyramine (PTy) modified enzyme electrodes were fabricated. The electropolymerized pmPD polymer was used to exclude interfering substances and the PTy layer facilitated the immobilization of acetylcholinesterase (AChE) and choline oxidase (ChOx). Then, ACh/Ch sensor and Ch sensor were coupled with microdialysis to produce a novel device, which provides a sensitive and selective method for simultaneous determination of ACh and Ch. This method has detection limits of 63.0 ± 3.4 nM for ACh and 25.0 ± 1.2 nM for Ch. The integrated device was successfully applied to assessing the impact of endogenous neurotoxin N-methyl-(R)-salsolinol [1(R),2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, (R)-NMSal] on ACh and Ch concentration, which is of great benefit to understand the pathogenesis of Parkinson's disease.     

High-performance liquid chromatography followed by peroxyoxalate chemiluminescence detection of acetylcholine and choline utilizing immobilized enzymes

A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.     

APPARENT IN VIVO ACETYLCHOLINE TURNOVER RATE IN WHOLE MOUSE BRAIN: EVIDENCE FOR A TWO COMPARTMENT MODEL BY TWO INDEPENDENT KINETIC ANALYSES

Abstract— Acetylcholine turnover has been determined in whole mouse brain using a newly available high specific activity [³H]choline (70 Ci/mmol). Animals were killed at various time points (0.25–10 min) after pulse adminstration of [³H]choline (Ch) by microwave irradiation of the head. Steady-state levels of ACh were determined by radioenzymatic analysis as described by G oldberg & M c C aman (1973) as modified by M c C aman & S tetzer , 1977. Ch levels were determined by a modification of the method of M c C aman & S tetzer (1977). Radiolabelled metabolites of [³H]Ch were separated by selective extraction of [³H]Ch and [³H]ACh inio tetraphenylboron in 3-heptanone (C arroll et al. , 1977) coupled with an enzymatic separation of [³H]Ch from [³H]ACh. A precursor-product relationship was verified for Ch and ACh specific activities. Acetylcholine turnover rate was determined by the biosynthesis ratio method (S chuberth et al. , 1969, Method 1) and by the finite-differences method (N eff et al. , 1971, Method 2). Both methods of kinetic analysis revealed two distinct turnover rates for acetylcholine. In the first phase (0.25–1.5 min post-[³H]Ch), the ACh turnover rate averaged 22nmol/g/min (both methods). During the second phase, (2–10 min) acetylcholine turnover rates were significantly ( P < 0.05 and P < 0.01) lower; i.e. 7nmol/g/min (Method 1) and 5.9 nmol/g/min (Method 2). The data are consistent with a 2-compartment model for ACh turnover in whole mouse brain. Additionally, the method described for the separation of radiolabelled metabolites of [³H]Ch allows an accurate determination of ACh turnover in as little as 2 mg of tissue.     

An enzymatic method for measuring picomole quantities of acetylcholine and choline in CNS tissue

A rapid and sensitive enzymatic assay for measuring picomole quantities of both acetylcholine (ACh) and choline (Ch) in tissue extracts has been developed. After ACh and Ch were extracted into 15% 1 n formic acid/85% acetone by the procedure of Toru and Aprison, lipids were removed by a heptane-chloroform extraction. All quaternary ammonium compounds were isolated by precipitation with periodide. After the precipitate (including ACh and Ch) was dissolved in a known volume of water, aliquots were taken for both assays. In the ACh assay, endogenous Ch was removed after conversion to choline phosphate by choline kinase, whereas ACh was subsequently hydrolyzed by base. In the presence of [¹⁴C]acetyl-CoA and choline acetyltransferase, the choline moiety was converted into [¹⁴C]ACh. The labeled ACh was extracted into sodium tetraphenylboron/butenenitrile and then counted in a scintillation counter. In the Ch assay, the first enzyme reaction step is omitted and only the second is used. The lower limit of sensitivity in both assays is 20 pmoles. Once the tissue has been carried through the extraction step, over eighty determinations can be made in one day. In vivo levels of ACh and Ch in the cerebrum of rats are reported for totally frozen rats and for rats sacrificed by the near-freezing procedure of Takahashi and Aprison. Mean ACh values in the two groups statistically were the same (26.5 ± 2.2 and 25.3 ± 1.7 nmoles/g, respectively) whereas the mean Ch values were significantly different (25.7 ± 0.9 and 64.0 ± 3.6 nmoles/g, respectively). The difference in the Ch levels as well as the importance of specifying the conditions that effect the measurement of ACh and Ch are discussed.     

Choline Transport and Metabolism in Soman-or Sarin-Intoxicated Brain

The metabolism and blood-brain transport of choline (Ch) were investigated in perfused canine brain under control conditions and for 60 min after inhibition of brain cholinesterases by the organophosphorus (OP) compounds soman (pinacolylmethylphosphonofluoridate). Ch and acetylcholine (ACh) in blood and brain samples were analyzed using gas chromatography-mass spectrometry methods. Net transport of Ch was determined by Ch analysis in arterial and venous samples. Unidirectional transport of [3H]Ch was determined using the indicator dilution method. During control perfusion periods of 90 min, net efflux of brain Ch occurred at a rate of 1.6 +/- 0.4 nmol/g/min, and the Ch content of the recirculated perfusate increased 10-fold to approximately 8 microM. Brain Ch content increased in proportion to the increase in perfusate Ch level, but brain ACh was unaltered. Rapid administration of soman (100 micrograms) or sarin (400 micrograms) into the arterial perfusate after a 40-min control period resulted in a greater than 10-fold increase in ACh content in cerebral cortex, brainstem, and hippocampus. The ACh content of cerebellum increased only slightly. The Ch level in all four brain regions studied also increased two- to fourfold above control levels. Ch efflux from brain, however, decreased to 0.2 +/- 0.1 nmol/g/min during the 60 min after OP exposure. Unidirectional influx of [3H]Ch was 0.49 +/- 0.07 nmol/g/min before and did not change significantly 10 or 40 min after OP exposure, thus indicating that the Ch transporter of the brain endothelial cell is not directly inhibited.2+ Based on these results, it is proposed that (a) efflux of brain Ch occurs from the extracellular compartment, which becomes depleted when ACh breakdown is inhibited;(ABSTRACT TRUNCATED AT 250 WORDS)     

4-(1-Naphthylvinyl)pyridine Decreases Brain Acetylcholine In Vivo, but Does Not Alter the Level of Acetyl-CoA

The effects of intraperitoneally administered 4-(1-naphthylvinyl)pyridine (NVP; 200 mg/kg) on the concentrations of acetylcholine (ACh), choline (Ch), and acetyl-CoA (AcCoA) in rat striatum, cortex, hippocampus, and cerebellum were investigated. Twenty minutes after treatment, the content of ACh was significantly diminished, whereas that of Ch was increased. In response to stress (swimming for 20 min), these changes were enhanced. However, the AcCoA content did not change in any of the brain regions. It is thus very likely that the decrease of brain ACh concentration induced by NVP is due to the drug's effect on choline acetyltransferase (ChAT) and/or the reduction of the high-affinity Ch uptake, and not on the availability of AcCoA. Presumably, the pharmacologically diminished activity of ChAT may become the rate-limiting factor in the maintenance of ACh levels in cholinergic neurons.     

Effects of oxotremorine on synthesis of acetylcholine in striatum and whole brain of mice killed by various techniques

Levels of acetylcholine (ACh) and choline (Ch) and turnover of ACh have been studied in whole brain and striatum of mice by mass fragmentography, employing either spinal dislocation or microwave irradiation to kill the animals. Oxotremorine (OT) was found to increase levels of ACh and Ch both in whole brain and striatum regardless of the way of killing. In whole brain turnover of ACh was decreased after OT independently of the way of killing, but in striatum a decrease was observed only if microwave irradiation was used, which is in contrast to previous findings. The discrepancy between whole brain and striatum may be explained by the preserving effect of microwave irradiation on a very fast turning-over pool of ACh in striatum.     

Improved method for the routine analysis of acetylcholine release in vivo: quantitation in the presence and absence of esterase inhibitor

An improved high-performance liquid chromatographic (HPLC) method using electrochemical detection (ED) is described capable of routinely measuring the low levels of acetylcholine (ACh) typically found in rat brain microdialysis samples. Microdialysis was performed in the striatum of the urethane anesthetized rat using a 4-mm membrane length, high recovery (40% at 1.0 μl/min; ambient conditions), loop-design probe perfused with an artificial cerebrospinal fluid (aCSF) solution containing physiologically normal calcium levels (1.2 mM). The HPLC method utilizes a polymeric stationary phase to resolve choline (Ch) from ACh. These analytes are then converted to hydrogen peroxide (H₂O₂) by a solid-phase reactor (containing immobilized choline oxidase and acetylcholinesterase enzymes). The H₂O₂ is detected amperometrically and quantitated on a platinum (Pt) working electrode (+300 mV; with a unique analytical cell featuring a solid-state palladium reference electrode). Two designs of the Pt working electrode were examined, differing only in the support material used (Kel-F or PEEK). The Kel-F/Pt electrode had a limit of detection (LOD) for both analytes of <30 fmol per 10 μl with a signal-to-noise ratio of 3:1. Striatal microdialysis perfusates were monitored for ACh and Ch over a 0–1000 nM range of neostigmine (NEO) in the CSF perfusion medium. Using the 4-mm probe, basal ACh and Ch levels were detected with a NEO level as low as 10 nM and were found to be 37 ± 3 fmol and 22 ± 1 pmol per 10 μl (mean ± S.E.M., n = 6 replicates) respectively. In similar experiments using 3-mm concentric probes comparable (lower) levels of ACh were found with the 50 and 1000 nM NEO doses (n = 4–21 animals). ACh could not be reliably quantitated when animals were perfused with the 10 nM dose of NEO (n = 4). The PEEK/Pt electrode had an improved LOD of < 20 fmol per 10 μl due to a two- to three-fold decrease in the background noise component. Basal striatal levels of ACh in the absence of NEO approached the LOD and were found to be 15 ± 2 fmol per 10 μl; Ch was 5 ± 1 pmol per 10 μl (n = 2, mean of five basal samples). The analytical system requires very little maintenance; a simple electrochemical electrode cleaning step eliminates the need for routine polishing of the Pt electrode and the mobile phase is stable for up to one week. Both ACh and Ch are resolved in under 7 min making this method highly suitable for analysis of microdialysis samples.     

KINETICS OF PLASMA CHOLINE IN RELATION TO TURNOVER OF BRAIN CHOLINE AND FORMATION OF ACETYLCHOLINE1

—[²H₄]Ch (2 μmol kg^-1 min^-1) was infused into both anaesthetized and conscious rats to study the kinetics of plasma and brain choline (Ch) and brain acetylcholine (ACh). A larger amount of endogenous Ch was found to leave the brain than enter, even in conscious animals. [²H₄]Ch was taken up into the brain where a portion was converted to [²H₄]ACh. Upon stopping the infusion, however, more [²H₄]Ch was found to leave than enter, indicating a source capable of generating Ch in brain which is labelled by infusion for 32 min. There appears, however, to be more than one source of Ch in the brain since the post mortem increase is not labelled following prolonged infusion. Thus, the brain Ch pool appears to be continually diluted by the sources within the brain to the extent of 93 per cent. During the infusion of [²H₄]Ch, the total levels of brain Ch and ACh did not increase. The brain Ch and ACh specific activities rose exponentially and appear to approach an asymptote at about 4 h. The source or sources of Ch within the brain produce Ch at a rate of 26·3 nmol g^-1 min^-1. The turnover of free Ch in the rat brain is 28·4 nmol g^-1 min^-1.     

The source of choline for acetylcholine synthesis in brain

More is known about the synthesis and metabolism of acetylcholine (ACh) than other choline (Ch) containing compounds in the brain in spite of the fact that ACh represents only a small fraction of the total Ch esters. This review will attempt to summarize the evidence for the source of Ch in the brain and its relation to the turnover of ACh. Ch is a precursor not only for ACh but also for phosphoryl Ch and phospholipids. It appears that in the rat a bound form of Ch in the brain can produce free Ch which can leave the brain, be converted to ACh or be reutilized for phospholipid synthesis. There is evidence that one of the sources of free Ch that is utilized for ACh synthesis is outside the cholinergic nerve terminal.     

Improved method for the routine determination of acetylcholine and choline in brain microdialysate using a horseradish peroxidase column as the immobilized enzyme reactor

A modified microbore high-performance liquid chromatography-immobilized enzyme reactor-electrochemical detection system for acetylcholine (ACh) and choline (Ch) was developed. The system used the horseradish peroxidase and a solution mediator ferrocene to convert the analyte into an oxidized ferrocene species which was detected electrochemically by reduction at 0 mV. There was an excellent linear relationship between the concentration of ACh/Ch and the peak height over the range of 1-5000 nmol/l. The limit of detection for ACh was 2 fmol/5 microl (S/N=3:1). Compared with the common method recommended by Bioanalytical System Inc. (BAS), this method exhibits a 200-fold improvement in the detection limit. The ACh and Ch levels in rat brain microdialysate were examined.     

Synaptosomal transport and acetylation of choline

Acetylcholine (ACh) synthesis can be impaired by reduction of the availability of either of its precursors, choline (Ch) or acetylcoenzyme A (AcCoA). The high affinity transport of Ch is inhibited by hemicholinium-3 and this results in reduced synthesis of ACh (1–3). Under some circumstances ACh metabolism in the brain appears to be affected by parenteral (4) or dietary (5, 14) administration of Ch. The production of AcCoA can apparently be reduced by inhibition of the utilization of pyruvate or glucose, which also decreases the synthesis of ACh (6, 7). Recent experiments by Barker and Mittag (8, 9) led them to propose that the high affinity transport of Ch and the subsequent transfer of an acetyl group from AcCoA, catalyzed by Ch acetyltransferase (CAT), were directly coupled. We have tested this hypothesis by reducing the availability of AcCoA and measuring both the rate of transport of Ch by the high affinity system and the rate at which it is converted to ACh.     

Acetylcholine and Choline in Neuronal Tissue Measured by HPLC with Electrochemical Detection

Abstract: A simple, rapid method is presented for the determination of acetylcholine (ACh) and choline (Ch) in neuronal tissue using HPLC with electrochemical detection. The method is based on the separation of ACh and Ch by reverse-phase HPLC and mixing the effluent as it emerges from the column with acetylcholinesterase and Ch oxidase, which converts endogenous Ch and Ch produced by the hydrolysis of ACh to betaine and hydrogen peroxide. Production of hydrogen peroxide is continuously monitored electrochemically. The sensitivity of the procedure is 1 pmol for Ch and 2 pmol for ACh. Specificity of the method is based on HPLC, two specific enzymatic reactions, and the detection of hydrogen peroxide.     

Acetylcholine release and choline uptake by cuttlefish (Sepia officinalis) optic lobe synaptosomes

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K+ depolarization was found to be very high (0.04 pmol ACh.s(-1).mg(-1) protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na+ channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Ch.min(-1).mg(-1) protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe.     

In Vivo Effects of β-Bungarotoxin on the Acetylcholine System in Different Brain Areas of the Rat

The in vivo effects of beta-bungarotoxin (beta-BT) on the acetylcholine (ACh) system were studied in the whole cerebrum and in different brain regions. The effect of beta-BT on cerebral ACh and choline (Ch) contents was time-dependent. The results show that a single intracerebroventricular injection of 1 microgram toxin increased both the ACh and Ch contents in the cortex, hippocampus, and cerebellum, while in the striatum the ACh level was decreased. Ten nanograms of toxin injected into the lateral ventricle twice, on the first and third days, led to a reduced ACh level 2 days after the last treatment. In animals treated with the same dose three times, on the first, third, and fifth days, and sacrificed 2 days after the last injection, the choline acetyltransferase and acetylcholinesterase activities were reduced and the number of muscarinic acetylcholine receptors was decreased. A biphasic effect of the toxin was therefore demonstrated. It is suggested that in the first phase of the toxin effect the increased levels of ACh and Ch may be due to the inhibition of neuronal transmission, while in the second phase, when the elements of the ACh system are reduced, the neuronal degenerating effect of beta-BT plays a significant role.     

Studies on a Possible Functional Coupling Between Presynaptic Acetylcholinesterase and High-Affinity Choline Uptake in the Rat Brain

The relationships between presynaptic acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 microM [N-methyl-3H]ACh was two-thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was 1/25 of the amount passing through the synaptosomal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.     

Acetylcholine biosensor involving entrapment of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane

A bienzymatic sensor for the determination of acetylcholine was prepared by physical coimmobilization of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane obtained by a cyclic freezing-thawing process. The enzyme-modified polymer was applied on a platinum electrode to form an amperometric sensor, based on the electrochemical detection of enzymatically developed hydrogen peroxide. The analytical characteristics of this sensor, including calibration curves for choline and acetylcholine, pH, and temperature effects, and stability are described.     

Measurement of Acetylcholine Turnover Rate in Brain: An Adjunct to a Simple HPLC Method for Choline and Acetylcholine

Abstract: An existing method for measuring acetylcholine (ACh) and choline (Ch) is shown to be useful formeasuring the turnover rate of ACh in mouse brain. Methl-[3H]Ch is injected into mice. They are killed atdifferent times by microwave irradiation and Ch and AChextracted and separated by reverse-phase HPLC. Ch andACh are converted to hydrogen peroxide by a post-column enzyme reaction. Hydrogen peroxide, which isdirectly related to the tissue content of Ch or ACh, isdetermined electrochemically. The fractions that corre-spond to the detector response for Ch and ACh are col-lected for the measurement of radioactivity. In this wayspecific radioactivities of endogenous Ch and ACh areestimated in the same sample. We used the specific ra-dioactivity values determined by this procedure to esti-mate the turnover of ACh for striatum, cerebral cortex, and hippocampus of the mouse.