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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. The method of AChE activity estimation is popularly known as Ellman's method named after George Ellman who developed this method in 1961.
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Assay of Acetylcholinesterase Activity in the Brain
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. The method of AChE activity estimation is popularly known as Ellman's method named after George Ellman who developed this method in 1961.
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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. The method of AChE activity estimation is popularly known as Ellman's method named after George Ellman who developed this method in 1961.
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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 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 show decreased AChE activity with a concurrent 1. Dissection: Adult Male Wistar rats (250-300g loss of cognitive function (Hammond and body weight) are used for the experiment. The Brimijoin, 1988). Furthermore, AChE induces LTP rats are decapitated; brains are removed quickly in hippocampal CA1 pyramidal neurons and placed in ice-cold saline. Frontal cortex, (Appleyard, 1995). These studies demonstrate the hippocampus and septum (and any other role of AChE in cognitive function unequivocally. regions of interest) are quickly dissected out on Further, AChE is known to have many non- a petri dish chilled on crushed ice. classical functions. There is a growing body of 2. The tissues are weighed and homogenized in evidence for morphogenic role of AChE. During 0.1M Phosphate buffer (pH 8). early development, AChE expression is tightly correlated with neurite outgrowth, In addition 3. 0.4ml aliquot of the homogenate is added to a AChE role in cell survival and growth cuvette containing 2.6 ml phosphate buffer (Appleyard, 1992). (0.1M, pH 8) and 100µl of DTNB. Studies from our laboratory in the last decade 4. The contents of the cuvette are mixed demonstrate that AChE activity is modulated by thoroughly by bubbling air and absorbance is several conditions that result in progressive and measured at 412 nm in a LKB regressive neuronal and behavioural plasticity. spectrophotometer. When absorbance reaches Chronic (-) deprenyl administration-induced a stable value, it is recorded as the basal reading. increase in dendritic arborisation in the primate 5. 20µl of substrate i.e., acetylthiocholine is added brain is associated with an increased AChE activity and change in absorbance is recorded for a in the hippocampus and cortex (Lakshmana et al., period of 10 mins at intervals of 2 mins. Change 1998). Intracranial self-stimulation treatment has in the absorbance per minute is thus been shown to result in increased dendritic determined. arborisation, enhance learning in operant conditioning tasks, and reverse chronic restraint Calculations: The enzyme activity is calculated stress-induced behavioural deficits. This using the following formula; enhancement of cognitive function is associated with an increase in AChE activity (Ramkumar et al., 2003; Shankaranarayana Rao et al., 1998). R = 5.74x 10-4 x A/CO Administration of substances like inorganic arsenic, Where, metanil yellow and 2, 4-dichloro phenoxy acetic R = Rate in moles of substrate hydrolyzed / acid causes behavioural dysfunction in an operant minute / gm tissue conditioning task. This has been found to be A = Change in absorbance / min coupled with decreased AChE activity CO = Original concentration of the tissue (Lakshmana and Raju, 1996; Nagaraja and (mg / ml). Desiraju, 1993; Nagaraja and Desiraju, 1994). Aluminium toxicity is thought to be one of the AChE ACTIVITY, CHOLINERGIC causative agents of Alzheimer’s disease. AChE FUNCTION AND COGNITION activity is decreased following long-term postnatal Estimating the AChE activity provides valuable exposure to aluminium (Ravi et al., 2000). Several information on cholinergic function. Studies on studies demonstrate that chronic stress leads to brains from patients suffering from Alzheimer’s cognitive dysfunction and results in disorders like disease (AD) have shown reduced AChE activity depression, anxiety and impairment of learning in the hippocampus and cortex (Fishman et al., and memory. Our studies show that restraint stress 1986; Hammond and Brimijoin, 1988). Evidence for 21 days is concurrent with decreased AChE on role of AChE in cognitive function also comes activity (Shankaranarayana Rao et al., 2003; from studies in biopsy tissues of AD patients, which Sunanda et al., 2000). Chronic immobilization 143 stress for 10 days followed by evaluation of anxiety 7. Lakshmana MK, Raju TR (1996) 2, 4-dichloro in an elevated plus maze and open field test results phenoxy acetic acid alters monoamine levels, in an increased AChE activity that is restored by acetylcholinesterase activity & operant learning in treatment with antianxiety drugs (Anuradha et al., rats. Indian J Med Res 104:234-9.: 234-239. 2004). Thus, there is a tight correlation between 8. Lakshmana MK, Rao BS, Dhingra NK, Ravikumar R, cholinergic function, AChE activity and cognition. Govindaiah, Ramachandra, Meti BL, Raju TR (1998) Accordingly, estimation of AChE activity provides Chronic (-) deprenyl administration increases an important correlate of cholinergic activity and dendritic arborization in CA3 neurons of cognitive function. hippocampus and AChE activity in specific regions of the primate brain. Brain Res 796: 38-44. AChE inhibitors play an important role in 9. Nagaraja TN, Desiraju T (1993) Effects of chronic nervous system disorders owing to their potential consumption of metanil yellow by developing and as pharmacological and toxicological agents. AChE adult rats on brain regional levels of noradrenaline, inhibitors are useful in the treatment of myasthenia dopamine and serotonin, on acetylcholine esterase gravis. Recently, AChE inhibitors like tacrine and activity and on operant conditioning. Food Chem rivastigmine are used in the treatment of Toxicol 31: 41-44. Alzheimer’s disease. Estimation of AChE activity 10. Nagaraja TN, Desiraju T (1994) Effects on operant by Ellman’s method is also useful in the screening learning and brain acetylcholine esterase activity in of new molecules for possible AChE inhibitory rats following chronic inorganic arsenic intake. Hum activity. Exp Toxicol 13: 353-356. 11. Ramkumar, K., Shankaranarayana Rao, B. S., and References Raju, T. R. (2003) Self-stimulation rewarding 1. Anuradha H, Srikumar BN, Deepti N, experience reverses stress induced behavioral deficits Shankaranarayana Rao BS, Lakshmana M (2004) and cholinergic dysfunction. Proc.International Euphorbia hirta reverses chronic stress-induced IBRO/FENS Summer School “Development and anxiety through GABAA receptor benzodiazepine Plasticity of the Human Cerebral Cortex”, Dubrovnik/ receptor-Cl - channel complex. Annals of Zagreb, Croatia. 37. Neurosciences 79. 12. Ravi SM, Prabhu BM, Raju TR, Bindu PN (2000) Long- 2. Appleyard ME (1992) Secreted acetylcholinesterase: term effects of postnatal aluminium exposure on non-classical aspects of a classical enzyme. Trends acetylcholinesterase activity and biogenic amine Neurosci 15: 485-490. neurotransmitters in rat brain. Indian J Physiol 3. Appleyard ME (1995) Acetylcholinesterase induces Pharmacol 44: 473-478. long-term potentiation in CA1 pyramidal cells by a 13. Shankaranarayana Rao, B. S., Deepti, N., Prabhu, B. mechanism dependent on metabotropic glutamate M., and Raju, T. R. (2003) Regional vulnerability in receptors. Neurosci Lett 190: 25-28. the levels of aminoacids and acetycholinesterase 4. Ellman GL, Courtney KD, Andres V, Jr., Feather-Stone activity in different models of stress. Soc Neurosci Abs RM (1961) A new and rapid colorimetric 33: 713.9. determination of acetylcholinesterase activity. 14. Shankaranarayana Rao BS, Raju TR, Meti BL (1998) Biochem Pharmacol 7:88-95.: 88-95. Self-stimulation of lateral hypothalamus and ventral 5. Fishman EB, Siek GC, MacCallum RD, Bird ED, tegmentum increases the levels of noradrenaline, Volicer L, Marquis JK (1986) Distribution of the dopamine, glutamate, and AChE activity, but not 5- molecular forms of acetylcholinesterase in human hydroxytryptamine and GABA levels in brain: alterations in dementia of the Alzheimer type. hippocampus and motor cortex. Neurochem Res 23: Ann Neurol 19: 246-252. 1053-1059. 6. Hammond P, Brimijoin S (1988) 15. Sunanda, Rao BS, Raju TR (2000) Restraint stress- Acetylcholinesterase in Huntington’s and induced alterations in the levels of biogenic amines, Alzheimer’s diseases: simultaneous enzyme assay amino acids, and AChE activity in the hippocampus. and immunoassay of multiple brain regions. J Neurochem Res 25: 1547-1552. Neurochem 50: 1111-1116. 144
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