Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.

),
National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:142-144.

ASSAY OF ACETYLCHOLINESTERASE ACTIVITY IN THE BRAIN

Srikumar BN, Ramkumar K, Raju TR and Shankaranarayana Rao BS

Ever since the discovery of acetylcholine (ACh)

as a neurotransmitter by Sir Henry Dale and Otto
Loewi (for which they were awarded the Nobel
Prize in 1936), its function in health and
dysfunction in disease has been increasingly
recognized. In the recent past, the role of ACh in
learning and memory has been demonstrated
indubitably. Further, pharmacological
manipulation of cholinergic function has been
found useful in the treatment of CNS disorders like
Alzheimer’s and Parkinson’s disease. Thus,
assessing cholinergic function is considered as an
important tool in neuroscience research.
Acetylcholine per se has a very short half-life
and direct estimation of ACh is a little difficult in
brain homogenates. There are several approaches Figure 1: The steps involved in estimation of AChE activity
to evaluate cholinergic function indirectly. using Ellman’s reaction. Acetylthiocholine is broken down
Estimating the expression of choline acetyl in the presence of AChE to release thiocholine, which
transferase (ChAT) and acetylcholinesterase reacts with the -SH reagent 5,5’-dithiobis-(2-nitrobenzoic
(AChE) by immunochemical and histochemical acid) (DTNB) to form thionitro benzoic acid.
techniques provide information on the cholinergic
function, but are tedious and time taking. Methodology
Estimation of AChE activity provides a relatively Reagents
easy and valuable assessment of cholinergic 1. 0.1M Phosphate buffer
function.
Solution A: 5.22g of K 2 HPO 4 and 4.68g of
The method of AChE activity estimation is NaH2PO4 are dissolved in 150 ml of distilled
popularly known as Ellman’s method named water.
after George Ellman who developed this method
Solution B: 6.2g NaOH is dissolved in 150ml of
in 1961 (Ellman et al., 1961). The esterase activity
distilled water.
is measured by providing an artificial substrate,
acetylthiocholine (ATC). Thiocholine released Solution B is added to solution A to get the
because of the cleavage of ATC by AChE is desired pH (pH 8.0 or 7.0) and then finally the
allowed to react with the -SH reagent 5,5’- volume is made up to 300ml with distilled
dithiobis-(2-nitrobenzoic acid) (DTNB), which is water.
reduced to thionitrobenzoic acid, a yellow 2. DTNB Reagent
coloured anion with an absorption maxima at
39.6 mg of DTNB with 15 mg NaHCO 3 is
412nm (Figure 1). The extinction coefficient of
dissolved in 10 ml of 0.1M phosphate buffer
the thionitro benzoic acid is 1.36 × 104/molar/
(pH 7.0).
centimeter. The concentration of thionitro
benzoic acid detected using a UV 3. Acetylthiocholine (ATC)
spectrophotometer is then taken as a direct 21.67 mg of acetylthiocholine is dissolved in 1
estimate of the AChE activity. ml of distilled water.
142
ASSAY PROCEDURE
1. Dissection: Adult Male Wistar rats (250-300g
body weight) are used for the experiment. The
rats are decapitated; brains are removed quickly
and placed in ice-cold saline. Frontal cortex,
hippocampus and septum (and any other
regions of interest) are quickly dissected out on
a petri dish chilled on crushed ice.
2. The tissues are weighed and homogenized in
0.1M Phosphate buffer (pH 8).
3. 0.4ml aliquot of the homogenate is added to a
cuvette containing 2.6 ml phosphate buffer
(0.1M, pH 8) and 100µl of DTNB.
4. The contents of the cuvette are mixed
thoroughly by bubbling air and absorbance is
measured at 412 nm in a LKB
spectrophotometer. When absorbance reaches
a stable value, it is recorded as the basal reading.
5. 20µl of substrate i.e., acetylthiocholine is added
and change in absorbance is recorded for a
period of 10 mins at intervals of 2 mins. Change
in the absorbance per minute is thus
determined.
Calculations: The enzyme activity is calculated
using the following formula;
R = 5.74x 10-4 x A/CO
Where,
R = Rate in moles of substrate hydrolyzed /
minute / gm tissue
A = Change in absorbance / min
CO = Original concentration of the tissue
(mg / ml).
AChE ACTIVITY, CHOLINERGIC
FUNCTION AND COGNITION
Estimating the AChE activity provides valuable
information on cholinergic function. Studies on
brains from patients suffering from Alzheimer’s
disease (AD) have shown reduced AChE activity
in the hippocampus and cortex (Fishman et al.,
1986; Hammond and Brimijoin, 1988). Evidence
on role of AChE in cognitive function also comes
from studies in biopsy tissues of AD patients, which
143
show decreased AChE activity with a concurrent
loss of cognitive function (Hammond and
Brimijoin, 1988). Furthermore, AChE induces LTP
in hippocampal CA1 pyramidal neurons
(Appleyard, 1995). These studies demonstrate the
role of AChE in cognitive function unequivocally.
Further, AChE is known to have many non-
classical functions. There is a growing body of
evidence for morphogenic role of AChE. During
early development, AChE expression is tightly
correlated with neurite outgrowth, In addition
AChE role in cell survival and growth
(Appleyard, 1992).
Studies from our laboratory in the last decade
demonstrate that AChE activity is modulated by
several conditions that result in progressive and
regressive neuronal and behavioural plasticity.
Chronic (-) deprenyl administration-induced
increase in dendritic arborisation in the primate
brain is associated with an increased AChE activity
in the hippocampus and cortex (Lakshmana et al.,
1998). Intracranial self-stimulation treatment has
been shown to result in increased dendritic
arborisation, enhance learning in operant
conditioning tasks, and reverse chronic restraint
stress-induced behavioural deficits. This
enhancement of cognitive function is associated
with an increase in AChE activity (Ramkumar et
al., 2003; Shankaranarayana Rao et al., 1998).
Administration of substances like inorganic arsenic,
metanil yellow and 2, 4-dichloro phenoxy acetic
acid causes behavioural dysfunction in an operant
conditioning task. This has been found to be
coupled with decreased AChE activity
(Lakshmana and Raju, 1996; Nagaraja and
Desiraju, 1993; Nagaraja and Desiraju, 1994).
Aluminium toxicity is thought to be one of the
causative agents of Alzheimer’s disease. AChE
activity is decreased following long-term postnatal
exposure to aluminium (Ravi et al., 2000). Several
studies demonstrate that chronic stress leads to
cognitive dysfunction and results in disorders like
depression, anxiety and impairment of learning
and memory. Our studies show that restraint stress
for 21 days is concurrent with decreased AChE
activity (Shankaranarayana Rao et al., 2003;
Sunanda et al., 2000). Chronic immobilization
stress for 10 days followed by evaluation of anxiety
in an elevated plus maze and open field test results
in an increased AChE activity that is restored by
treatment with antianxiety drugs (Anuradha et al.,
2004). Thus, there is a tight correlation between
cholinergic function, AChE activity and cognition.
Accordingly, estimation of AChE activity provides
an important correlate of cholinergic activity and
cognitive function.
AChE inhibitors play an important role in
nervous system disorders owing to their potential
as pharmacological and toxicological agents. AChE
inhibitors are useful in the treatment of myasthenia
gravis. Recently, AChE inhibitors like tacrine and
rivastigmine are used in the treatment of
Alzheimer’s disease. Estimation of AChE activity
by Ellman’s method is also useful in the screening
of new molecules for possible AChE inhibitory
activity.
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