Simple spectroscopic Methods for estimating Brain Neurotransmitters, Antioxidant Enzymes of Laboratory animals like Mice: A review

About Authors:
Habibur Rahman1, M.C Eswaraiah2
1Department of Pharmacology,
2Dept. of Pharmacognosy,
Anurag Pharmacy College,
Ananthagiri (V), Kodad (M),
Nalgonda (Dt.), A.P-508206, India

ABSTRACT
Brain neurotransmitters like Dopamine, serotonin, Adrenaline, Glutamate and enzymes like Acetyl cholinesterase (AchE), Mono amino-oxidase (MAO-A & MAO-B) levels and antioxidant enzymes like Superoxide dismutase, catalase, peroxidase etc play important roles in different complex neurological disorders like Alzheimer’s disease(AD), Parkinsonism, depression etc. But biochemical estimation of these parameters is very difficult to conduct in simple laboratory. This review is a collection of simple spectroscopic methods to determine the common brain neurotransmitters and antioxidant enzymes.

Reference Id: PHARMATUTOR-ART-1244

Introduction
Neurotransmittersare endogenous chemical that transmit signals from a neuron to a target cell across a synapse. Glutamate is the major excitatory neurotransmitter in the brain and spinal cord and it involved in different disease like anxiety, epilepsy, psychosis, schizophrenia, Alzheimer’s disease and others common neurological disorders [1].  Excessive glutamate release can lead to excitotoxicity causing cell death.  GABA is used at the great majority of fast inhibitory synapses in virtually every part of the brain. Many sedative/tranquilizing drugs act by enhancing the effects of GABA [2].   Acetylcholine is distinguished as the transmitter at the neuromuscular junction connecting motor nerves to muscles.  Acetylcholine involved in memory, paralysis and others disorders.  Dopamine has a number of important functions in the brain. It plays a critical role in the reward system, but dysfunction of the dopamine system is also implicated in Parkinson's disease and schizophrenia.[3] Serotonin is a monoamine neurotransmitter to regulate appetite, sleep, memory and learning, temperature, mood, behavior, muscle contraction, and function of the cardiovascular system and endocrine system. It is speculated to have a role in depression, as some. The brain is uniquely vulnerable to oxidative injury, due to its high metabolic rate and elevated levels of polyunsaturated lipids, the target of lipid per oxidation.  Antioxidants appear to prevent oxidative stress in neurons and prevent apoptosis and neurological damage. Antioxidants are also being investigated as possible treatments for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [4, 5].

Estimation of Brain Neurotransmitters
Acetylcholineserase (AChE) enzyme determination

Acetylcholineserase (AChE) enzyme activity was estimated by Elman method [6].

Reagents
1. 0.1M Phosphate buffer
Solution A:  5.22g of K2HPO4 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 NaHCO3 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.

Procedure
The mice were decapitated; brains are removed quickly and placed in ice-cold saline. Frontal cortex, hippocampus and septum are quickly dissected out on a Petri dish chilled on crushed ice. The tissues are weighed and homogenized in 0.1M Phosphate buffer (pH 8). 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. The contents of the cuvette are mixed thoroughly by bubbling air and absorbance is measured at 412 nm in a spectrophotometer. When absorbance reaches a stable value, it is recorded as the basal reading. 20µl of substrate i.e., acetylthiocholine is added and change in absorbance is recorded. Change in the absorbance per minute is thus determined.

Reagents

Sample

Blank

Phosphate buffer solution

2.6 ml

2.7 ml

Supernatant

0.4 ml

0.4ml

DTNB

0.1 ml

…….

Calculations
The enzyme activity is calculated using the following formula [7].

                                                                        A/min X Vt
Achetylcholinestease activity (M/ml) =…………………………….
                                                                        ε X b X Vs

            Where,                                    

A/min= Change in absorbance per min

                                                ε= 1.361 X104 M-1cm-1

                                                b= pathlenth (1 cm)

                                                Vt= Total volume (3.1 ml)

                                                Vs= sample volume (0.4 ml)

The final reading of enzyme activity is expressed as μ moles/minute/mg tissue.

                                           μ moles /ml sample
μ moles/minute/mg protein = ……………………………
                                 mg protein/ml sample dilution       

Estimation of Monoamine Oxidase A and B
The MAO- A and B activity was estimated by the method of Charles and McEwen [8].

Reagents
Sucrose (0.25M)
Tris buffer (0.1 M)
EDTA (0.02M)
Serotonin (4mM)
Sodium phosphate buffer (100 mM, pH 7.4)
Hydrochloric acid (1M)
Butyl acetate/cyclohexane

Preparation of sample
Mouse brain mitochondrial fraction are prepared by cutting the brain sample in to small pieces and rinsed in 0.25M sucrose, 0.1 M tris, 0.02M EDTA (pH 7.41) to remove blood. The pieces were homogenized for 45 sec in a potter-elvehjem homogenizer with 400 ml of the same medium. The homogenate was centrifuged at 800rpm for 10min and the pellets were discarded. The supernatant was then centrifuged at 12,000 rpm for 20 min in the same medium. The precipitate was washed twice more with 100ml of sucrose tris EDTA and resuspended in 50ml of the medium. The protein concentration was adjusted to 1 mg/ml.

Procedure for estimation of MAO- A
250 µl of the homogenate was added to 250 µl of serotonin and 250 µl of buffer. The reaction tube was placed at 37°C for 20 minutes and the reaction was arrested by the addition of 200 µl of 1M HCl. The reaction product was extracted with 5 ml of Butyl acetate. The organic phase was separated and measured at 280 nm using a spectrophotometer. Blank samples were prepared by adding 1M HCl (200 µl) prior to reaction and the reaction was carried out.
The MAO-A is expressed in nmoles/ mg protein.

Procedure for estimation of MAO- B
250 µl of the homogenate was added to 250 µl of serotonin and 250 µl of buffer. The reaction tube was placed at 37°C for 20 minutes and the reaction was arrested by the addition of 200 µl of 1M HCl. The reaction product was extracted with 5 ml of Cyclohexane. The organic phase was separated and measured at 242 nm using a spectrophotometer. Blank samples were prepared by adding 1M HCl (200 µl) prior to reaction and the reaction was carried out.

The MAO-B activity is expressed in nmoles/ mg protein.

Estimation of Dopamine, Adrenaline and Serotonin
Preparation of tissue extracts by method Schlumpf M [9].

Reagents:
1.      HCl – Butanol sol. : (0.85 ml of 37% hydrochloric acid in one-litre n-butanol)
2.      Heptanes
3.      0.1 M HCl:  (0.85 ml conc. HCl upto 100 ml H20)

Procedure:
On the day of experiment mice were sacrificed, whole brain was dissected out and the sub cortical region (including the striatum) was separated. Weight tissue was weight and was homogenized in5ml HCl–butanol for about 1 min. The sample was then centrifuged for 10 min at 2000 rpm. An aliquot supernatant phase (1 ml) was removed and added to centrifuge tube containing 2.5 ml heptane and 0.31ml HCl of 0.1 M. After 10 min of vigorous shaking, the  tube  was  centrifuged  under  the  same  conditions  as  above  in  order  to  separate the  two  phases,  and  the  overlaying  organic  phase  was  discarded.  The  aqueous  phase (0.2 ml) was  then  taken  either  for  5-HT  or  NA  and  DA  assay.  All steps were carried out at 00C.
(N.B: It taken in between 50-75 mg of tissue for homogenate with 5 ml of HCl-Butanol in correlation of same tissue concentration 1.5-5 mg/0.1 ml of HCl-butanol used in Schlumpf M et al, 1974. This is done to get adequate amount of supernatant liquid for analysis).

Estimation of Noradrenaline and dopamine [10].
Reagents

1.      0.4M HCl:  3.4 ml conc. HCl up to 100 ml H20
2.      Sodium acetate buffer (pH 6.9): 2.88 ml of 1M acetic acid (5.7 ml of Glacial acetic acid upto 100 ml with distilled water) +27.33 ml of 0.3M sodium acetate (4.08 g of sodium acetae 100 ml with distilled water) and volume is made up to 100 ml with distilled water). PH is adjusted with sodium hydroxide sol.
3.      5M NaOH : 20 g of sodium hydroxide pellets dissolved in distilled water and volume is made up to 100 ml with distilled water)
4.      0.1 M Iodine solution (in Ethanol): 4 g of pot. Iodide +2.6 g of iodine dissolved in ethanol volume is made up to 100 ml)
5.      Na2SO3  sol. ((0.5 g Na2SO3 in 2 ml H2O + 18 ml 5 M NaOH)
6.      10M Acetic acid: 57 ml of glacial acetic acid dissolved in distilled water up to 100 ml.

Procedure
To the 0.2 ml of aqueous phase, 0.05 ml 0.4 M HCl and 0.1 ml of EDTA / Sodium acetate buffer (PH6. 9)were added, followed by 0.1 ml iodine solution(0.1 M in ethanol) for oxidation. The reaction was stopped after 2 min by addition of 0.1 ml Na2SO3 solution. 0.1 ml Acetic acid is added after 1.5 min. The solution was then heated to 100°C for 6 min when the sample again reached room temperature, excitation and emission spectra were read from the spectrofluorimeter. The readings were taken at 330-375 nm for dopamine and 395-485 nm for nor-adrenaline.

Estimation of Serotonin
The serotonin content was estimated by the method of Schlumpf[9].

Reagents
1.      O-phthaldialdehyde (OPT) reagent: (20 mg in 100 ml conc. HCl)

Procedure
To 0.2 ml aqueous extract 0.25 ml of OPT reagent was added. The fluorophore was developed by heating to 100°C for 10 min. After the samples reached equilibrium with the ambient temperature, readings were taken at 360-470 nm in the spectrofluorimeter. Tissue blanks for Dopamine and nor-adrenaline were prepared by adding the reagents of the oxidation step in reversed order (sodium sulphite before iodine). For serotonin tissue blank, 0.25 ml cont. HCI without OPT was added.Internal Standard: (500 µg/ml each of noradrenaline, dopamine and serotonin are prepared in distilled water: HCl-butanol in 1:2 ratio.

Estimation of Glutamate
The level of Glutamate was estimated by multiple development paper chromatography as described by Raju [11].

Reagents
1.butanol: acetic acid: water: 12: 3: 5
2. Ninhydrin reagent : 0.25%
3. Copper sulphate solution: 0.005%
4. Standard glutamate: 2.942 mg of glutamate in 10 ml distilled water.

Procedure
1.0 ml of the supernatant from brain homogenate was evaporated to dryness at 70°C in an oven and the residue is reconstituted in 100 ml of distilled water. Standard solutions of glutamate and GABA at a concentration of 2mM along with the sample are spotted on Whatman No. 1 chromatography paper using a micropipette. It was placed on a chamber containing butanol: acetic acid: water (12: 3: 5 v/v) as solvent. When the solvent front reached the top of the paper, it was removed and dried. A second run is performed similarly, after which the papers are dried sprayed with ninhydrin reagent and placed in an oven at 100°C for 4 minutes. The portions which carry glutamate corresponding with the standard are cut and eluted with 0.005% CuSo4 in 75% ethanol. Their absorbance is read against blank at 515 nm in spectrophotometer.

Calculation
The levels of glutamate and GABA are calculated by using the following formula;

            Unknown OD X  Standard in mg (3µg) X 1000
A=        ……………………………………………………
          Standard OD   X Volume spotted (10µl) X    W

Where,

A = Aminoacid content in umoles/gram wet weight tissue

1000 = Conversion factor for gram wet weight tissue

W = weight of the tissue in gram

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Comments

I am a student of M.Sc. Semester IV and i am doing my dissertation work on activity of neurotransmitters on chicken brain intoxicated by fugal food. in that my one protocol is study of MAO-A and B , i have fulfill my this protocol by the method of Charles, M. and J. McEwen, 1977. Mao Activity in Rabbit Serum. In: Methods in Enzymology, XVIIB, Tabor, H. and C.W. Tabor (Eds.). Academic Press, New York and London, pp: 692-698. in this method i have got my readings but i do not know the formula to convert it into nmoles/mg protein. so, if u will email me formula for that and related articals it will help me in my dissertation work & i will be thank full to you for that.

Calculations are the biggest headache for rookie researcher. Your instructions are easy to comprehend. Thanks a lot...

dpsingh's picture

Please send the calculation of MAO-A and MAO-B activity in nmol/mg protein.
I have absorbance for the same.
 
Thanks.

SirI AM DOING mpharm in trivandrum medical college. This protocol is very useful for my study and i got the results. but i dont have the formula for calculating the amount of neurotransmittors. I request u to kindly send me the calculations for determination of neurotransmittors. if u send this as soon as possible It will definetly help me to complete my work and am will be very thankful to you.
Thanks
Athira

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