Assay Of Acetylcholinesterase Activity In The Brain
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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 to evaluate cholinergic function indirectly. Estimating the expression of choline acetyl transferase (ChAT) and acetylcholinesterase (AChE) by immunochemical and histochemical techniques provide information on the cholinergic function, but are tedious and time taking. Estimation of AChE activity provides a relatively easy and valuable assessment of cholinergic function. The method of AChE activity estimation is popularly known as Ellman’s method named after George Ellman who developed this method in 1961 (Ellman et al., 1961). The esterase activity is measured by providing an artificial substrate, acetylthiocholine (ATC). Thiocholine released because of the cleavage of ATC by AChE is allowed to react with the -SH reagent 5,5’dithiobis-(2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 412nm (Figure 1). The extinction coefficient of the thionitro benzoic acid is 1.36 × 104/molar/ centimeter. The concentration of thionitro benzoic acid detected using a UV spectrophotometer is then taken as a direct estimate of the AChE activity.
Figure 1: The steps involved in estimation of AChE activity using Ellman’s reaction. Acetylthiocholine is broken down in the presence of AChE to release thiocholine, which reacts with the -SH reagent 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) to form thionitro benzoic acid.
Methodology Reagents 1. 0.1M Phosphate buffer Solution A: 5.22g of K 2 HPO 4 and 4.68g of NaH2PO4 are dissolved in 150 ml of distilled water. Solution B: 6.2g NaOH is dissolved in 150ml of distilled water. Solution B is added to solution A to get the desired pH (pH 8.0 or 7.0) and then finally the volume is made up to 300ml with distilled water. 2. DTNB Reagent 39.6 mg of DTNB with 15 mg NaHCO 3 is dissolved in 10 ml of 0.1M phosphate buffer (pH 7.0). 3. Acetylthiocholine (ATC) 21.67 mg of acetylthiocholine is dissolved in 1 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
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 nonclassical 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 143
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. References 1.
Anuradha H, Srikumar BN, Deepti N, Shankaranarayana Rao BS, Lakshmana M (2004) Euphorbia hirta reverses chronic stress-induced anxiety through GABAA receptor benzodiazepine receptor-Cl - channel complex. Annals of Neurosciences 79.
2.
Appleyard ME (1992) Secreted acetylcholinesterase: non-classical aspects of a classical enzyme. Trends Neurosci 15: 485-490.
3.
Appleyard ME (1995) Acetylcholinesterase induces long-term potentiation in CA1 pyramidal cells by a mechanism dependent on metabotropic glutamate receptors. Neurosci Lett 190: 25-28.
4.
Ellman GL, Courtney KD, Andres V, Jr., Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88-95.: 88-95.
5.
Fishman EB, Siek GC, MacCallum RD, Bird ED, Volicer L, Marquis JK (1986) Distribution of the molecular forms of acetylcholinesterase in human brain: alterations in dementia of the Alzheimer type. Ann Neurol 19: 246-252.
6.
Hammond P, Brimijoin S (1988) Acetylcholinesterase in Huntington’s and Alzheimer’s diseases: simultaneous enzyme assay and immunoassay of multiple brain regions. J Neurochem 50: 1111-1116.
7.
Lakshmana MK, Raju TR (1996) 2, 4-dichloro phenoxy acetic acid alters monoamine levels, acetylcholinesterase activity & operant learning in rats. Indian J Med Res 104:234-9.: 234-239.
8.
Lakshmana MK, Rao BS, Dhingra NK, Ravikumar R, Govindaiah, Ramachandra, Meti BL, Raju TR (1998) Chronic (-) deprenyl administration increases dendritic arborization in CA3 neurons of hippocampus and AChE activity in specific regions of the primate brain. Brain Res 796: 38-44.
9.
Nagaraja TN, Desiraju T (1993) Effects of chronic consumption of metanil yellow by developing and adult rats on brain regional levels of noradrenaline, dopamine and serotonin, on acetylcholine esterase activity and on operant conditioning. Food Chem Toxicol 31: 41-44.
10. Nagaraja TN, Desiraju T (1994) Effects on operant learning and brain acetylcholine esterase activity in rats following chronic inorganic arsenic intake. Hum Exp Toxicol 13: 353-356. 11. Ramkumar, K., Shankaranarayana Rao, B. S., and Raju, T. R. (2003) Self-stimulation rewarding experience reverses stress induced behavioral deficits and cholinergic dysfunction. Proc.International IBRO/FENS Summer School “Development and Plasticity of the Human Cerebral Cortex”, Dubrovnik/ Zagreb, Croatia. 37. 12. Ravi SM, Prabhu BM, Raju TR, Bindu PN (2000) Longterm effects of postnatal aluminium exposure on acetylcholinesterase activity and biogenic amine neurotransmitters in rat brain. Indian J Physiol Pharmacol 44: 473-478. 13. Shankaranarayana Rao, B. S., Deepti, N., Prabhu, B. M., and Raju, T. R. (2003) Regional vulnerability in the levels of aminoacids and acetycholinesterase activity in different models of stress. Soc Neurosci Abs 33: 713.9. 14. Shankaranarayana Rao BS, Raju TR, Meti BL (1998) Self-stimulation of lateral hypothalamus and ventral tegmentum increases the levels of noradrenaline, dopamine, glutamate, and AChE activity, but not 5hydroxytryptamine and GABA levels in hippocampus and motor cortex. Neurochem Res 23: 1053-1059. 15. Sunanda, Rao BS, Raju TR (2000) Restraint stressinduced alterations in the levels of biogenic amines, amino acids, and AChE activity in the hippocampus. Neurochem Res 25: 1547-1552. 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 to evaluate cholinergic function indirectly. Estimating the expression of choline acetyl transferase (ChAT) and acetylcholinesterase (AChE) by immunochemical and histochemical techniques provide information on the cholinergic function, but are tedious and time taking. Estimation of AChE activity provides a relatively easy and valuable assessment of cholinergic function. The method of AChE activity estimation is popularly known as Ellman’s method named after George Ellman who developed this method in 1961 (Ellman et al., 1961). The esterase activity is measured by providing an artificial substrate, acetylthiocholine (ATC). Thiocholine released because of the cleavage of ATC by AChE is allowed to react with the -SH reagent 5,5’dithiobis-(2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 412nm (Figure 1). The extinction coefficient of the thionitro benzoic acid is 1.36 × 104/molar/ centimeter. The concentration of thionitro benzoic acid detected using a UV spectrophotometer is then taken as a direct estimate of the AChE activity.
Figure 1: The steps involved in estimation of AChE activity using Ellman’s reaction. Acetylthiocholine is broken down in the presence of AChE to release thiocholine, which reacts with the -SH reagent 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) to form thionitro benzoic acid.
Methodology Reagents 1. 0.1M Phosphate buffer Solution A: 5.22g of K 2 HPO 4 and 4.68g of NaH2PO4 are dissolved in 150 ml of distilled water. Solution B: 6.2g NaOH is dissolved in 150ml of distilled water. Solution B is added to solution A to get the desired pH (pH 8.0 or 7.0) and then finally the volume is made up to 300ml with distilled water. 2. DTNB Reagent 39.6 mg of DTNB with 15 mg NaHCO 3 is dissolved in 10 ml of 0.1M phosphate buffer (pH 7.0). 3. Acetylthiocholine (ATC) 21.67 mg of acetylthiocholine is dissolved in 1 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
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 nonclassical 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 143
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. References 1.
Anuradha H, Srikumar BN, Deepti N, Shankaranarayana Rao BS, Lakshmana M (2004) Euphorbia hirta reverses chronic stress-induced anxiety through GABAA receptor benzodiazepine receptor-Cl - channel complex. Annals of Neurosciences 79.
2.
Appleyard ME (1992) Secreted acetylcholinesterase: non-classical aspects of a classical enzyme. Trends Neurosci 15: 485-490.
3.
Appleyard ME (1995) Acetylcholinesterase induces long-term potentiation in CA1 pyramidal cells by a mechanism dependent on metabotropic glutamate receptors. Neurosci Lett 190: 25-28.
4.
Ellman GL, Courtney KD, Andres V, Jr., Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88-95.: 88-95.
5.
Fishman EB, Siek GC, MacCallum RD, Bird ED, Volicer L, Marquis JK (1986) Distribution of the molecular forms of acetylcholinesterase in human brain: alterations in dementia of the Alzheimer type. Ann Neurol 19: 246-252.
6.
Hammond P, Brimijoin S (1988) Acetylcholinesterase in Huntington’s and Alzheimer’s diseases: simultaneous enzyme assay and immunoassay of multiple brain regions. J Neurochem 50: 1111-1116.
7.
Lakshmana MK, Raju TR (1996) 2, 4-dichloro phenoxy acetic acid alters monoamine levels, acetylcholinesterase activity & operant learning in rats. Indian J Med Res 104:234-9.: 234-239.
8.
Lakshmana MK, Rao BS, Dhingra NK, Ravikumar R, Govindaiah, Ramachandra, Meti BL, Raju TR (1998) Chronic (-) deprenyl administration increases dendritic arborization in CA3 neurons of hippocampus and AChE activity in specific regions of the primate brain. Brain Res 796: 38-44.
9.
Nagaraja TN, Desiraju T (1993) Effects of chronic consumption of metanil yellow by developing and adult rats on brain regional levels of noradrenaline, dopamine and serotonin, on acetylcholine esterase activity and on operant conditioning. Food Chem Toxicol 31: 41-44.
10. Nagaraja TN, Desiraju T (1994) Effects on operant learning and brain acetylcholine esterase activity in rats following chronic inorganic arsenic intake. Hum Exp Toxicol 13: 353-356. 11. Ramkumar, K., Shankaranarayana Rao, B. S., and Raju, T. R. (2003) Self-stimulation rewarding experience reverses stress induced behavioral deficits and cholinergic dysfunction. Proc.International IBRO/FENS Summer School “Development and Plasticity of the Human Cerebral Cortex”, Dubrovnik/ Zagreb, Croatia. 37. 12. Ravi SM, Prabhu BM, Raju TR, Bindu PN (2000) Longterm effects of postnatal aluminium exposure on acetylcholinesterase activity and biogenic amine neurotransmitters in rat brain. Indian J Physiol Pharmacol 44: 473-478. 13. Shankaranarayana Rao, B. S., Deepti, N., Prabhu, B. M., and Raju, T. R. (2003) Regional vulnerability in the levels of aminoacids and acetycholinesterase activity in different models of stress. Soc Neurosci Abs 33: 713.9. 14. Shankaranarayana Rao BS, Raju TR, Meti BL (1998) Self-stimulation of lateral hypothalamus and ventral tegmentum increases the levels of noradrenaline, dopamine, glutamate, and AChE activity, but not 5hydroxytryptamine and GABA levels in hippocampus and motor cortex. Neurochem Res 23: 1053-1059. 15. Sunanda, Rao BS, Raju TR (2000) Restraint stressinduced alterations in the levels of biogenic amines, amino acids, and AChE activity in the hippocampus. Neurochem Res 25: 1547-1552. 144
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