• acetylcholine;
  • choline acetyltransferase;
  • hippocampus;
  • labeled choline;
  • NMR spectroscopy


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post-mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used 15N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before 1H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques.

Abbreviations used



acetylcholine esterase




Alzheimer's disease


choline acetyltransferase




[11C]N-methyl-4-piperidyl acetate


nucleus basalis of Meynert


nuclear magnetic resonance


Parkinson's disease




vesicular acetylcholine transporter


initial reaction rate

Acetylcholine (ACh) is a key modulatory neurotransmitter found in both the peripheral and central nervous systems. In the brain, the ascending cholinergic projections from the nucleus basalis of Meynert and the medial septum, in particular, play an important role in mediating aspects of learning and memory as well as attention (Deutsch 1971; Blokland 1995). The hippocampus and the neocortex receive rich cholinergic input from these nuclei. It has been suggested that the cognitive symptoms associated with Alzheimer's disease (AD) were attributed to a profound loss of cholinergic function in the CNS, giving rise to the ‘cholinergic hypothesis of a geriatric memory dysfunction’ (Bartus et al. 1982). In fact, there is evidence for alterations of the cholinergic neurotransmission in AD and Parkinson's disease (PD), in which cognition is impaired (even in the absence of frank dementia) (Ruberg et al. 1982; Whitehouse et al. 1982; Dubois et al. 1983; Perry et al. 1985; Mufson et al. 1989). These observations gave rise to experimental studies in rodents investigating the involvement of the cholinergic system on cognition by lesioning cholinergic pathways. Several studies reported impairment in learning and memory tasks following fimbria-fornix transection (Dunnett 1985), ibotenic acid lesions (Mayo et al. 1988), or intraventricular 192-IgG saporin injections (Leanza et al. 1995; Steckler et al. 1995). These findings led to the development of symptomatic treatment strategies, which aimed at restoring cholinergic neurotransmission through administration of cholinesterase inhibitors [for review see (Bosboom et al. 2004)].

The synthesis, storage, and turnover of ACh in the brain are dependent on the expression of specific enzymes. The ACh biosynthetic pathway involves an enzymatic reaction catalyzed by choline O-acetyltransferase (EC, ChAT). In this reaction, choline (Cho) is used as a substrate, whereas acetyl-CoA (ACoA) acts as a cofactor, donating an acetyl group (Cooper et al. 2003). The reaction catalyzed by ChAT has been extensively studied and follows a Theorell–Chance mechanism, with sequential binding of substrate and cofactor. ChAT has a much higher affinity for ACoA than for Cho, so that Cho binds predominantly to a ChAT–ACoA complex (Hersh 1982; Karczmar 2007).

In vitro, biochemical assays measuring the activity of ChAT have been used in several studies to assess the integrity of the cholinergic system. Data collected from AD and PD patients have reported dramatic decreases in ChAT activity in the cortex (Dubois et al. 1983; Bierer et al. 1995). These studies rely on a radiochemical method originally developed by Fonnum, which aims to reproduce the conditions necessary for ACh synthesis in vitro to assess the activity of the ChAT enzyme (Fonnum 1975). The tissue samples to be analyzed are incubated with 14C-ACoA, and the newly synthesized 14C-ACh is then separated from the precursor using a liquid cation exchange between organic and aqueous phases. The radioactivity subsequently measured in the organic phase is proportional to the ACh concentration and therefore reflects the activity of the enzyme. Although this well-established biochemical method can help assess the state of the cholinergic system in post-mortem tissue samples, it has no use in longitudinal monitoring of disease progression.

Established imaging methods monitoring the state of the cholinergic system are based on positron emission tomography (PET) or single-photon emission computed tomography (SPECT) and use radiotracers for in vivo imaging of acetylcholine esterase (EC, AChE) or vesicular acetylcholine transporter (VAChT), respectively. The SPECT tracer [123I]-iodobenzovesamicol binds to VAChT (the transporter responsible for storing ACh in vesicles) and is used to map pre-synaptic cholinergic terminal densities. PET studies typically use labeled ACh analogues with high selectivity and specificity for AChE, such as [11C]-N-methyl-4-piperidyl acetate (MP4A) and [11C]-N-methyl-4-piperidyl-propionate. Once hydrolyzed by AChE, these tracers release a hydrophilic metabolite that cannot cross the blood–brain barrier and therefore remains trapped at the site of the enzyme (Kuhl et al. 1994; Iyo et al. 1997). These modalities, however, do not provide a measurement of ACh synthesis (i.e., ChAT activity) in the brain, but rather reflect the distribution and density of cholinergic terminals. It is important to note that whereas AChE is found in both pre-synaptic cholinergic and post-synaptic cholinoceptive neurons, ChAT is present only in pre-synaptic cholinergic terminals, making it a more suitable and specific marker to identify cholinergic neurons and assess their functionality (Mesulam et al. 1983; Bloom and Kupfer 1995). However, there are currently no imaging ligands that can be used to directly investigate changes in the activity of the ChAT enzyme in the brain in vivo. A quantitative measurement of ACh synthesis in defined regions in the brain would enable early detection of cholinergic dysfunction and better assessment of disease state and progression in cognitively impaired patients.

Nuclear magnetic resonance (NMR) can be used to provide complementary information that is not accessible through PET. In fact, the high spectral resolution of NMR spectroscopy makes it a method of choice to monitor single metabolites non-invasively. It can provide measurement of concentration and synthesis rates of specific biochemical compounds in defined areas in the brain [for review see (Novotny et al. 2003; Hall et al. 2012)]. Monitoring ChAT activity in the living brain with NMR spectroscopy would provide an assessment of regional ACh synthesis rates and thereby reflect the functionality of cholinergic cells. In an effort toward the development of non-invasive techniques to assess brain ACh synthesis, the aim of this in vitro study was to use NMR spectroscopy to determine ChAT enzyme activity in rat brain homogenates. We used the radiometric assay of Fonnum as a gold standard and developed an NMR-based assay for real-time monitoring and quantification of enzymatic conversion of Cho into ACh.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tissue preparation

Adult female Sprague–Dawley rats were obtained from Charles River (Kisslegg, Germany). All the experimental procedures were carried out in compliance with the rules of the Ethical Committee for Use of Laboratory Animals in the Lund-Malmö region. The rats were killed by decapitation and the brains were rapidly removed, hippocampi were quickly dissected on ice, frozen in Eppendorf tubes, and stored at −80°C until processed for the determination of ChAT activity. At the time of the experiment, the samples were thawed and rapidly homogenized on ice with an ultrasonic disintegrator using a buffer containing 50 mM sodium phosphate pH 7.4, 0.5% Triton X-100, 10 mM EDTA at various concentrations as described below. The homogenized samples were kept on ice for 15 min before extraction of the supernatant by centrifugation at 7825 g for 10 min at 4°C.

Radiometric assay for determination of ChAT activity in vitro

The activity of the ChAT enzyme was determined using a modified version of the radiometric assay of Fonnum (Fonnum 1975), which uses [1-14C]-ACoA as a substrate (GE Healthcare Amersham, Buckinghamshire, UK; specific activity 56mCi/mmol). Briefly, standard assay conditions included mixing 10 μL of the tissue homogenate (5 mg/mL) with 20 μL of reaction buffer containing 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 8 mM Cho chloride, 0.1 mM physostigmine (AChE inhibitor), and 0.2 mM ACoA (with a 1 : 100 ratio of [1-14C]-ACoA). The samples containing Cho and ACoA at final concentrations of 5.3 mM and 0.13 mM, respectively, were immediately incubated for 15 min at 37°C. All samples were run in triplicates. Blanks were run substituting homogenate with water. The reaction was stopped by putting the tubes on ice and adding 4 mL of ice-cold 10 mM sodium phosphate buffer pH 7.4, followed by addition of 2 mL sodium tetraphenylborate (5 mg/mL in 15 : 85 acetonitrile:toluene; Sigma, St Louis, MO, USA). After shaking, the samples were centrifuged 2 min at 3400 g to separate the organic and inorganic phases. One milliliter of the organic phase (carrying the ACh) was mixed with 5 mL of LSC cocktail (Ultima Gold Plus; Perkin Elmer Inc. Waltham, MA, USA) and the radioactivity collected by means of liquid cation exchange was measured using a liquid scintillation counter (Beckman LS 6500; Beckman Inc. Fullerton, CA, USA) and quantified using an external standard calibration curve. ChAT activity was determined by measuring the conversion of [1-14C]-ACoA to [1-14C]-ACh and the results expressed as total ACh synthesized (in nmol) or ACh concentration in buffer (in μM) where appropriate.

Scale up of the radiometric microassay

The assay was optimized for NMR measurements by implementing a number of modifications in several steps. These included variation of the tissue concentration in the homogenate, variation of the concentration of ACoA, Cho, and of the [Cho]/[ACoA] ratio (Table 1). In addition, the kinetics of the reaction was determined by processing separate tubes (containing samples originating from the same pool) at different time points. The reaction was stopped at 0, 5, 10, 15, 20, 30, 45, 60, and 90 min. All conditions were applied to samples run in triplicates.

Table 1. Conditions tested for optimization of the radiometric assay
Tissue concentration in the homogenate (mg/mL)510202550100100200500
  1. At tissue concentration of 25 and 100 mg/mL, multiple [ACoA] were tested.

  2. ACoA, acetylcoenzyme A; Cho, Choline.

    0.067 0.26   
[ACoA] (in mM)








[Cho] (in mM)

To assess ACh synthesis using NMR spectroscopy, some additional changes from the original radiometric protocol were needed. The NMR analysis requires the use of glass tubes, the diameter of which depends on the probe used in the magnet. The 13C analysis was performed using a 10-mm probe. In this case, to match the sensitive region of the radiofrequency coil, the optimal volume of the sample is 3 mL. Similarly, the 1H measurements were performed with a 5-mm probe, which requires a sample of 600 μL. We performed the assay under the radiometric conditions using the original microvolume and the largest volume that would be required by NMR, to compare assay performances and check whether scaling up the volume of the assay would affect the reaction. The reaction was carried out in a total volume of 30 μL or 3 mL on tissue homogenates (100 mg/mL) incubated with a reaction buffer at a final concentration of 0.53 mM ACoA and 5.3 mM Cho, whereas the other constituents were kept at concentrations similar to the standard conditions.

Optimization of ChAT assay for NMR experiments

The conditions for the 13C-Cho-based reaction assessed above in the scaled-up version of the assay were used and the reaction buffer prepared with 8 mM [1-13C]-Cho chloride (Isotec Inc., Miamisburg, OH, USA) and 0.8 mM ACoA ([1-14C] ACoA 1 : 200) (final concentration 5.3 mM and 0.53 mM, respectively). Samples were generated by combining 1 mL of 100 mg/mL tissue extract and 2 mL of the reaction buffer and incubated at 37°C for 120 min. At several time points (0, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min), 30 μL of sample were removed into a tube standing on ice and the reaction stopped by adding 4 mL ice-cold 10 mM phosphate buffer, pH 7.4. The reaction product was extracted as described above and the radioactivity was measured using a liquid scintillation counter.

The 15N-Cho-based assay was run by mixing 400 μL of homogenate (125 mg/mL) to 200 μL of reaction buffer (100 mM sodium phosphate buffer, pH 7.4, 600 mM NaCl, 0.2 mM physostigmine, 16 mM 15N-Cho chloride (Isotec Inc.,) and 1.6 mM ACoA with a 1 : 400 ratio of [1-14C] ACoA) to yield 5.3 mM Cho and 0.53 mM AcoA. The reaction was run as described in the 13C version of the assay.

Nuclear magnetic resonance spectroscopy

13C-NMR measurements were performed on a Varian Unity Inova 500 MHz spectrometer and 1H-NMR measurements were performed on an Agilent 500 MHz spectrometer (Agilent technologies, Santa Clara, CA, USA), both operating at 11.7 T. A 10-mm broadband probe and a 5-mm 1H/13C/15N triple resonance probe were used for 13C experiments and 15N-edited experiments, respectively. Probe performances were tested using standard procedures by the spectrometer manufacturer and were in accordance with manufacturer specifications.

Thin-wall 10-mm (Wilmad 513-7PP) and 5-mm (Wilmad 528-PP) NMR tubes (Wilmad-LabGlass, Vineland, NJ, USA) were used. The sample volume was adjusted to 3 mL for the 10-mm probe and 600 μL for the 5-mm probe, to match the optimal value for each probe. All samples analyzed contained 10% D2O for locking the signal. A liquid sample containing 5.3 mM of 13C- or 15N-labeled Cho in 10% D2O was used to optimize 90° pulse width, relaxation delay, decoupling power, and modulation frequency. Initial shimming was done on a sample including the brain homogenate, the buffer solution and containing 5.3 mM 13C- or 15N-Cho, prior to the dynamic reaction experiment.

Direct single-pulsed 13C-NMR measurements were performed at a center frequency of 125.7 MHz at 37°C. The reaction was initiated directly in 10 mm sample tubes immediately before placing the sample into the spectrometer, by mixing 1 mL of brain homogenate with 2 mL of phosphate buffer containing 13C-Cho (buffer composition same as in previous section). For each spectrum, 100 transients were accumulated using 60° pulses and 2.25-s repetition time. Waltz pulsed proton decoupling was applied during data acquisition.

15N-edited 1H-NMR measurements were performed at 37°C. The reaction was initiated directly in a 5-mm tube immediately before placing the sample into the spectrometer, by mixing 400 μL of brain homogenate with 200 μL of the reaction buffer containing 15N-labeled Cho (buffer composition same as in previous section). Because of low sensitivity of 15N, its detection at thermal equilibrium is not practical. Thus, magnetization transfer from nitrogen to protons is a preferred approach (known as inverse detection). The polarization was transferred from protons to 15N using Insensitive Nuclei Enhanced by Polarization Transfer, following which, a reverse Insensitive Nuclei Enhanced by Polarization Transfer sequence transferred the polarization back to protons. Two pulsed-field gradients were used to suppress the unwanted direct signals from protons not coupled to 15N. For each spectrum, 32 transients were accumulated using 7-s repetition time.

Prior to analysis, all NMR spectra were manually phased and baseline corrected. A line broadening of 3 Hz was applied and quantitative measurements of peak integrals were performed using the spectrometer software.

Data analysis

Data are presented as mean ± SEM, unless stated otherwise. The correlation in Fig. 4 was estimated using the Pearson's product-moment correlation coefficient with two-tailed significance. An exponential model for a first-order reaction was fitted to the time-course data displayed in Figs 2, 3, 5, and 6, using non-linear regression analysis. The fit was performed using the Statistical Package for the Social Sciences version 19 (spss Inc., Chicago, IL, USA) according to the following equation:

  • display math

where A is the amplitude of the ACh signal, Amax the maximum amplitude of the ACh signal at the end of the reaction, k the reaction rate constant, and t the time. The initial reaction rate (Vi) was calculated as the product of Amax and k. r2 is indicated as a measurement of the goodness of fit of the model.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, our aim was to establish a quantitative assay for monitoring ACh synthesis from a non-radioactive labeled Cho, based on ultra-high field NMR spectroscopy. For this purpose, we started with the well-known radiometric assay of Fonnum (1975), and varied the enzyme, cofactor, and substrate concentrations as well as the volume of the reaction to determine the conditions suitable for NMR measurement of ACh synthesis. We then carried out proof-of-concept kinetic studies using 13C- or 15N-labeled Cho as a precursor and measured the activity of the ChAT enzyme. Below, each parameter leading to the optimized assay conditions is described.

Effect of tissue concentration on measured ChAT activity

The original assay for determination of ChAT activity is designed to consume a small amount of tissue and takes advantage of the very high sensitivity of the radioactive detection of an incorporated 14C label to the final product. However, as NMR is known to be inherently insensitive, it was important to identify the maximum amount of ChAT enzyme (here varied by adjusting the concentration of hippocampal tissue in the homogenate) that can maintain the reaction rate obtained in the original assay formulation. For this purpose, we determined the correlation between ACh synthesis and increasing amounts of rat hippocampal tissue (homogenate concentration ranging from 5 to 500 mg/mL) while maintaining the concentration of Cho (5.3 mM) and ACoA (0.13 mM) as well as the volume of the reaction (30 μL) and the reaction time (15 min) constant. These results were then plotted as concentration curves (Fig. 1). Increasing the amount of homogenate from 0.05 to 0.25 mg had only a minimal effect on ACh synthesis rate as the amount (shown on the left y axis in Fig. 1) or the concentration (shown on the right y axis) was increased, that is, a five-fold increase in the homogenate amount (from 0.05 to 0.25 mg) resulted in approximately four-fold high ACh levels generated over 15 min. Increasing the amount of tissue from 0.25 to 0.5 and 1 mg did result in higher ACh synthesis. However, the increase in the product generated gradually declined, for example, an increase from 0.25 to 1 mg provided only a 2.5-fold increase. Further increasing the homogenate amount appeared to be inefficient as a five-fold increase in tissue amount (from 1 to 5 mg) resulted in less than a two-fold increase in ACh synthesis. We concluded that the original assay conditions could maintain the ACh synthesis rates for tissue amounts up to 0.25 mg, whereas further increases would require modification of other parameters in the assay.


Figure 1. Relationship between the amount of hippocampal tissue and acetylcholine (ACh) synthesis. The assay conditions are as indicated in the chart. The amount of tissue (in mg) present in the total reaction volume is indicated on the x axis. Data points are means of four independent experiments each run in triplicates and presented as total amount of ACh (in nmol, left y axis) or concentration of ACh (in μM, right y axis) synthesized. Error bars are SEM. Cho, choline; ACoA, acetylcoenzyme A; t, time; Vr, reaction volume.

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Effect of cofactor concentration on ChAT activity over time

In the original radiometric assay, the Cho precursor is present in excess, and therefore the reaction proceeds with maximum rate as long as the cofactor remains available. When the reaction time is set to 15 min (as is done typically in this assay), the cofactor concentration (0.13 mM) is sufficient to maintain the reaction at or close to its initial rate for the whole assay time. At this stage, we extended the reaction to 90 min to collect data longitudinally at multiple time points. We investigated whether the concentration of ACoA was sufficient to maintain the reaction rate beyond the initial 15 min or if a higher amount of cofactor would be necessary. For this purpose, we selected two amounts of homogenates: 0.25 mg (determined to maintain the highest reaction efficiency in the first set of experiment), and 1 mg (for which the reaction efficiency started to decline notably). We then varied the concentration of ACoA (either double or half of the standard amount used, and similarly adjusted to match the four-fold increase from 0.25 to 1 mg tissue) and determined the total amount of ACh synthesized over 90 min of reaction (Fig. 2a and b) as well as the Vi, k, and Amax values (Fig. 2c). In both cases, the concentration of ACoA corresponding to the original assay is shown in black, whereas the other conditions are shown in gray symbols. The reaction proceeds well beyond the first 15 min and can be followed for 90 min, albeit at a slower rate (Fig. 2a and b). Reduction of ACoA concentration by half has a detrimental effect as can be seen by a separation of the ACh synthesis curves at around 30 min, beyond which the reaction slows in the corresponding group (gray squares). Increasing ACoA concentration two-fold did not appear to result in an increase in the amount of ACh synthesized during the time of the experiment (gray circles) (Fig. 2a and b). k, Amax, and Vi were obtained by fitting an exponential model for a first-order reaction to the data and are displayed in panel C. Most notably, varying ACoA concentration in both conditions (0.25 and 1 mg tissue) did not affect Vi, but resulted in a decrease of the reaction rate constant k. In addition, we found that Vi increased proportionally with the amount of tissue, which suggested a linear scalability of the assay as long as the relationship between homogenate and cofactor was maintained (Fig. 2c).


Figure 2. Synthesis of acetylcholine (ACh) as a function of time at various acetyl-CoA concentrations. Amount of ACh synthesized by 0.25 (a) or 1 mg (b) of hippocampal tissue. Three concentrations of ACoA were tested for each tissue amount, as indicated in the chart labels. Data are expressed as total amount of ACh (in nmol). Data points are means of five (a) or three (b) independent experiments each run in triplicates. Error bars are SEM. (c) The kinetic parameters (k, Amax, and Vi) obtained from an exponential equation for a first-order reaction fitted to the data are displayed for each reaction condition, along with the r2 value measuring the goodness of fit.

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Next, we investigated how the reaction would proceed when changing the [Cho]/[ACoA] ratio (Fig. 3). For this experiment, 1 mg of tissue was used and the initial concentration of cofactor was set to 0.53 mM, which is identical to the most favorable condition found in the previous set of data described above (shown in Fig. 2b in black diamonds). In this set of experiment, we varied Cho and ACoA concentrations and compared the kinetics of the reaction at three different [Cho]/[ACoA] ratios. The estimated k, Amax, and Vi parameters obtained as described above are displayed in Fig. 2b. A 10-fold decrease in Cho concentration (from 5.3 to 0.53 mM) resulted in a 17-fold reduction in Vi as well as in a 40% decrease in the reaction rate constant k, clearly indicating that excess of Cho is necessary for the reaction to proceed at maximum rate. At this low Cho concentration, we ran the reaction in 10-fold excess of ACoA and observed that this was even more detrimental as this reaction had the lowest Vi and k values of the three. The estimated Amax value of the reaction ran under this condition (with 5.3 mM of ACoA) is 18.3 nmol, which is double the value obtained at low ACoA concentration, regardless of Cho concentration. This approximates the highest theoretical Amax achievable if the reaction would consume all Cho present in the test tube (15.9 nmol). However, the large standard error associated with the Amax estimate suggests that the data obtained under these conditions (and within that time frame) were no longer sufficient for the non-linear model. Of note, a linear regression can be fitted to the data set measured between 0 and 90 min at low [Cho] and high [ACoA] (r = 0.983, r2=0.966, p < 0.001; two-tailed significance of the Pearson's product-moment correlation coefficient r) according to the following equation: A = 0.051t + 0.124.


Figure 3. Synthesis of acetylcholine (ACh) as a function of time and its dependence on initial substrate and cofactor concentrations. (a) Amount of ACh synthesized by 1 mg of hippocampal tissue over time. Three [Cho]/[ACoA] ratios were tested, as indicated in the chart labels. Data are expressed as total amount of ACh (in nmol). Data points are means of three independent experiments each run in triplicates. Error bars are SEM. (b) The kinetic parameters (k, Amax, and Vi) obtained from an exponential equation for a first-order reaction fitted to the data are displayed for each reaction condition, along with the r2 value measuring the goodness of fit.

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Establishment and validation of the liquid NMR-based ChAT activity assay

Translation of the in vitro ChAT assay from the commonly used microassay format (suitable for radiometric analysis) to an NMR compatible format requires a scale up to a reaction volume of approximately 3 mL for 10-mm probes and 600 μL for 5-mm probes. This represents up to a 100-fold increase in reaction volume. We therefore tested if the conditions optimal for the microassay above could be reproduced in a 3 mL reaction volume. Fig. 4 shows the correlation between the time course of the ChAT assay run in either 30 μL or 3 mL total volume. As indicated by the strong linear correlation between the rates measured under the two conditions (r = 0.997, p < 0.001; two-tailed significance of the Pearson's product-moment correlation factor), the reaction rate of the assay (measured as concentration of ACh in the buffer at a given time point) remains identical between the two assay volumes. This indicates that the NMR assay can be performed in the volume required by the radiofrequency coil without compromising the efficiency of the reaction.


Figure 4. Correlation between acetylcholine (ACh) synthesis under macroassay (reaction volume Vr = 3 mL) and microassay (reaction volume Vr = 30 μL) conditions over 90 min. The assay was run using pre-established conditions (5.3 mM choline and 0.53 mM acetyl-coA). For each incubation volume, data were obtained at time points varying from 0, 5, 10, 15, 20, 30, 45, 60, and 90 min, and plotted against each other. Data are expressed as means of three independent experiments each run in triplicates. Error bars are SEM. Pearson's correlation showed a linear correlation profile (r = 0.997, p < 0.001; two-tailed significance of the Pearson's product-moment correlation factor). Dash line represents y = x and is displayed for comparison purpose.

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On the basis of the assay conditions established above, we utilized 13C-Cho and attempted to monitor the emergence of a 13C-ACh signal using direct 13C NMR (Fig. 5). We found that despite a large reaction volume (3 mL) used in this assay (and therefore a larger quantity of product generated per unit time), the first quantifiable signal [defined as a signal for which the signal-to-noise ratio was greater than 3], was obtained only after 32 min of reaction. From 32 to 130 min, although the signal-to-noise ratio calculated for each data point was greater than 3, it remained low (between 4 and 10). The noise is well represented by the spread of the data points in Fig. 5c. An exponential equation for a first-order reaction was fitted to the time course of the 13C data collected over 2 h (k = 0.015 min−1, SE: 0.004, r2=0.765) (Fig. 5c). The noisy signal measured over the time course of the experiment may explain the low r2, which represents the proportion of the variability in the ACh signal that is accounted for by the exponential fit. A radiometric measurement performed on a 30-μL aliquot collected from the NMR tube after 100 min of reaction showed that [1-13C, 2-14C]-ACh concentration was at a level expected at this time point (0.34 mM), and confirmed that ACh synthesis had taken place.


Figure 5. Direct 13C-NMR experiment. (a) ACh synthesis catalyzed by ChAT using 13C-choline as a substrate (visible by NMR). (b) 13C spectrum acquired during the synthesis of ACh and obtained after 96 min of reaction. Inset in (b) shows the evolution of ACh signal over time. (c) Time course of [1-13C, 2-14C]-ACh synthesis using the 13C signal measured by NMR. Data points were calculated as integrals of single ACh NMR peaks obtained at every time point. Open circles represent signals for which signal-to-noise ratio ≤ 3, whereas filled circles represent signals for which signal-to-noise ratio > 3. An exponential model for a first-order reaction was fitted to the data points and the fitted line, the estimated parameters Amax and k, along with the r2 value (representing the proportion of variance in the data that is explained by the fit) are displayed in (c).

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In a separate experiment, the conversion of 15N-Cho into ACh was monitored with 15N-edited 1H-NMR. Twelve minutes after the reaction was initiated, ACh signal was detectable and quantifiable as a small peak at 3.21 ppm through the resonance of the trimethylamine head group [N(CH3)], and adequately resolved from the adjacent Cho peak at 3.19 ppm (Fig. 6b). The increase in ACh signal was monitored over 2 h and an exponential equation for a first-order reaction fitted to the data (k = 0.022 min−1, SE: 0.001, r2=0.976) (Fig. 6c). As the reaction buffer contained both 15N-Cho and 14C-ACoA, it was possible to provide accurate and absolute quantification of the [15N,14C]-ACh product using the radiometric method on an identical sample. An exponential equation for a first-order reaction was fitted to the time course of the 14C data (k = 0.024 min−1, standard error: 0.003, r2=0.985) (Fig. 6d). A normalized plot of the exponential fits shows that the two detection methods (radiometric and NMR based) provide nearly identical results in terms of product formation over time (Fig. 6e).


Figure 6. 15N-edited proton NMR experiment. (a) ACh synthesis catalyzed by ChAT using 15N-choline as a substrate (visible by NMR) and 14C-ACoA as a cofactor (detectable by liquid scintillation). (b) Top: applied pulse sequence. τ1 = 200 ms, τ2 = 92 ms, black rectangles represent 90° pulses, white rectangles represent 180° pulses, PFG: pulse field gradient (10 : 1 ratio). Bottom: 1H spectra acquired during the synthesis of ACh. The spectra displayed show the chemical shift region of interest. Choline was identified through the resonance frequency of the protons linked to the 15N nucleus [N(CH3)] (3.19 ppm) and ACh signal was resolved from Cho at 3.21 ppm. See inset for evolution of ACh peak over time. (c) Time course of [15N,14C]- dual-labeled ACh synthesis using either the 15N-edited proton signal measured by NMR or (D) the 14C beta decay measured by liquid scintillation. NMR data in (c) represent the integrals of single ACh NMR peaks obtained at every time point. 14C ACh levels in (d) were quantified using external standards. An exponential model for a first-order reaction was fitted to data points obtained with both methods. The two fitted lines and the estimated parameters Amax and k, as well as the r2 value (representing the proportion of variance in the data that is explained by the fit) are displayed in each panel accordingly (c and d). (e) Comparison between ACh signal measured by the radiometric method (red line) and proton NMR (blue line). For comparison, fitted models were normalized to estimated Amax in each case. Open circles correspond to experimental data points plotted in (c and d).

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Early studies investigating the state of the cholinergic system have relied on the use of post-mortem anatomical or biochemical methods. Data collected in human and rat studies have established the importance of measuring ChAT activity to assess the presence and function of cholinergic neurons [for review see (Contestabile and Ciani 2008)]. However, one caveat of data acquired post-mortem is the time point of the recorded events. They provide information at the time of death, but fail to measure events occurring during the progression of the disease, where neurons in distinct areas of the brain might initially become dysfunctional and later degenerate. Exploring the dynamics of neurotransmitter handling (synthesis, storage, release, and breakdown) may help the detection of these early metabolic abnormalities preceding frank neurodegeneration. In fact, PET studies using MP4A have shown reduction in cortical AChE activity of 30% in patients with mild AD, that is, early in the course of the disease (Iyo et al. 1997). However, considering that AChE is also present on post-synaptic cholinoceptive neurons, decrease in AChE activity might be insensitive and underestimate the loss of cholinergic neurons. In addition, SPECT studies using radioligands targeting VAChT can provide measurement of cholinergic terminal densities and have indicated a negative correlation between cortical binding of the ligand and dementia severity in AD (Kuhl et al. 1996). Although ChAT is the most specific and selective marker for cholinergic neurons, there is currently no radioligand that can be used to measure its activity in vivo.

Here, we demonstrate the possibility to directly monitor ACh synthesis in hippocampal homogenates using 15N-edited 1H-NMR spectroscopy. For this purpose, we first optimized the parameters of the reaction to yield maximum ACh. This was of particular importance for adaptation of the radiometric assay to an NMR detection-based assay as the sensitivity of conventional NMR methods is low. Through the real-time monitoring of the ACh peak by NMR, our study showed that the ChAT enzymatic reaction could be followed in vitro by NMR spectroscopy in a dynamic fashion.

The activity of ChAT at physiological intracellular concentrations of substrates (which are non-saturating) is far less than its maximal rate as reported in vitro at saturating concentrations and corresponds to the maximum rate of ACh release (Karczmar 2007). Earlier studies have established that ChAT itself is not the rate-limiting factor in ACh synthesis, but rather Cho availability (Tucek 1984). In vivo, this would be reflected by Cho uptake rate into the target tissue compartment. Our results confirmed the importance for Cho to be provided in large excess so that the reaction could proceed at its maximum rate. If Cho needs to be delivered in excess to saturate ChAT and reliably assess the activity of the enzyme, it remains to be determined what amount of Cho could be safely delivered to the brain and what would be the best way to do so. A recent study from Cudalbu and colleagues showed that 2.5 mL of Cho at 90 mM could be administered intravenously in rats without serious physiological disturbances, and that Cho was subsequently detectable in the brain (Cudalbu et al. 2010).

The low intrinsic sensitivity of NMR hinders its use for studies of naturally low-abundant neurotransmitters. For example, it has not been possible to detect any of the key modulatory neurotransmitters (ACh, dopamine, noradrenaline, or serotonine) by 1H-NMR spectroscopy in vivo. Although 1H-NMR spectroscopy can detect signal from choline-containing compounds, 98% of the signal originates from glycerophosphocholine and phosphocholine (Boulanger et al. 2000). To bypass the problem of signal overlap in 1H-NMR, studies have resorted to the use of 13C-NMR with 13C-labeled compounds to follow their metabolic conversion into end products. In a proof-of-concept study, Katz-Brull and colleagues reported an NMR spectroscopic method to monitor ACh synthesis in rat brain slices perfused with enriched 13C-Cho. By taking advantage of the high spectral resolution of 13C-NMR, they were able to detect 13C-ACh in this in vitro preparation. Signals from both 13C-Cho and 13C-Ach were visible and therefore demonstrated that the ChAT enzyme present in the tissue slices converted 13C-Cho to 13C-Ach. The need for signal accumulation (the product formation was assessed on a spectrum acquired in 4 h after 2 h of slice perfusion with 13C-Cho) prevented any dynamic analysis of ACh synthesis over time (Katz-Brull et al. 2005).

Our 13C analysis on rat brain homogenate showed similar limitations to follow the synthesis of ACh. Although the 13C-ACh signal was detectable during the course of the reaction, it was quantifiable only after 32 min of reaction and the data acquired were noisy. Although the reaction volume analyzed by 1H-NMR was five-fold smaller than the one analyzed by 13C-NMR, using 15N-enriched Cho and 15N-edited proton spectroscopy, we could detect ACh 12 min after the reaction was initiated, placing this method in a time window compatible with imaging applications. Of note, the amount of hippocampus used in the experiment (100 mg) is very close to the weight of the hippocampus in an adult rat brain.

Novel techniques based on hyperpolarization methods are capable of further enhancing weak NMR signals and are currently under development. They can provide major increase in NMR sensitivity by enhancing the polarization of nuclear spins (Golman et al. 2003). Using dynamic nuclear polarization (DNP) to hyperpolarize deuterated 13C-Cho, Allouche-Arnon and coworkers provided evidence for the metabolic acetylation of 13C-Cho to 13C-ACh by using purified carnitine O-acetyltransferase (EC and showed improved sensitivity and temporal resolution compared with thermal equilibrium NMR spectra. The product was detectable within 15 s and the T1 relaxation time was found to be sufficiently long to suggest that it may be suitable for in vivo applications (Allouche-Arnon et al. 2011). It is important to note that the metabolic process was catalyzed by purified carnitine acetyltransferase extracted from pigeon breast muscle, and therefore differs from the synthesis of ACh in the brain by the ChAT enzyme. There are presently no reports demonstrating if ACh synthesis in the brain can be monitored in vivo.

Other studies have reported on the hyperpolarization of 15N-labeled Cho in vitro, taking advantage of the longer T1 relaxation time (100–200 s) of 15N. Using 15N-NMR, Gabellieri and colleagues described a method to monitor the in vitro conversion of hyperpolarized 15N-Cho to phosphocholine by choline kinase (Gabellieri et al. 2008). More recently, hyperpolarized 15N-Cho was detected by 1H-NMR after polarization transfer from 15N to protons, which is made possible by the small 3J(15N,1H) couplings between 15N and methylene and methyl protons in Cho (Sarkar et al. 2009). With this method, this study took further advantage of both the high sensitivity of 1H-NMR and the long T1 relaxation time of 15N. In addition, hyperpolarized 15N-Cho was successfully detected by 15N-NMR for the first time in vivo in a voxel placed over the rat head after administration of a rapid bolus through the femoral vein. Although the signal from the hyperpolarized substrate was clearly seen, none of the Cho metabolites could be detected (Cudalbu et al. 2010). The authors conceded that a high fraction of the signal might be attributed to the blood pool. The proportion of Cho bolus that did effectively cross the blood–brain barrier and reached the brain remains to be precisely determined. It should be noted here that in that experiment, AChE inhibitors were not administered along with Cho. After its synthesis and release in the synaptic cleft, ACh is rapidly hydrolyzed by AChE (Tucek 1984). It is therefore very likely that ACh hydrolysis and turnover took place at a rate too fast for the signal to be detected by NMR. Nevertheless, whether the signal from brain 15N-ACh could be detected in the presence of AChE inhibitors remains unclear. Future in vivo studies based on methods, such as described here, and resorting to the use of AChE inhibitors may be able to detect newly synthesized ACh in the brain. This would offer a unique estimation of neurotransmitter synthesis capacity and thereby reflect the functionality of cholinergic neurons in potentially defined areas of the brain.

In summary, this study constitutes a proof of principle for the real-time detection of ChAT-mediated ACh synthesis using an NMR method based on 15N-edited proton detection. Parallel assessment of [15N,14C]-ACh with the radiometric method confirmed the ability of the technique to measure ChAT activity over time. However, improving the sensitivity of the detection appears to be necessary to successfully translate this assay in vivo. One of the promising methods bearing potential to achieve sufficiently high sensitivity for in vivo application is hyperpolarization through DNP. There are, however, several biological (amount of Cho and AChE inhibitors that can be safely administered, uptake of Cho into the brain), and technical (design of pulse sequence, volume of polarized sample) aspects that need to be optimized for this to be successful. Nevertheless, this study constitutes a step toward the development of in vivo DNP–NMR coupled studies, which could develop into a new diagnostic tool for exploring cholinergic dysfunction in the brain.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by grants from the European Research Council (TreatPD 242932), the Swedish Research Council (2009-2318, 2008-3092), and the Knut and Alice Wallenberg Foundation. The authors wish to acknowledge Lund University Bioimaging Center (LBIC) at Lund University for providing experimental resources. The authors declare no financial conflict of interest.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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