Neurochemistry Lab

8/8/2019 Neurochemistry Lab

1/13

An Introduction to Neurochemistry: Acetylcholinesterase Assayadapted from material presented at a PKAL conference on Neurobiology 1997

The ability of a neuron to communicate with other neurons through a chemicalsynapse is a source of endless fascination for neuroscientists. The chemical vehicles

that enable this communication are neurotransmitters. Neurotransmitters can becategorized into four groups monoamines, amino acids, peptides and acetylcholine.They are synthesized, packaged and transported (if necessary) to the terminal of thepresynaptic cell. The arrival of the action potential at the terminal of this cell triggersthe release of neurotransmitter into the synaptic cleft. After diffusion across thesynapse, the neurotransmitter can activate receptors on the postsynaptic cell resultingin excitation or inhibition of that cell. Finally, theneurotransmitter is inactivated throughenzymatic breakdown, re-uptake or by it's diffusion. Analysis of the variousbiochemical events associated with synaptic transmission is the domain of theneurochemist. This lab exercise concentrates on the neurotransmitter acetylcholine(ACh) and the enzyme which breaks it downsacetylcholinesterase (AChE), as an

introduction to neurochemical methods.

What you need to know..........

Acetylcholine (ACh) is one of the major neurotransmitters of the brain. It was firstdescribed in the PNS in a classic experiment by Otto Loewi (1921). While stimulatingthe vagus nerve of a frog to slow down its heart, Loewi collected the perfusate andapplied it to the heart of a second frog. This heart also slowed down! Loewi and hiscolleagues, "the first neurochemists", demonstrated that this inhibitory substance inthe perfusate was mimicked in every way by ACh. Then in 1936, H. H. Daledemonstrated that ACh is the neurotransmitter that has an excitatory effect in the

neuromuscular synapse. But how can ACh be inhibitory in the heart yet excitatory inthe muscle? It is now known that the effect of a neurotransmitter is not determined bythe neurotransmitter itself, but by its postsynaptic receptor. For example, the nicotinicACh receptor is an ionotropic receptor and a postsynaptic nerve will responddifferently than one containing the muscarinic ACh receptor (a metabotropic receptor).

The function of ACh in the PNS is well documented in the autonomic nervous systemand the neuromuscular junction. The autoimmune disease myasthenia gravis isthought to be due to a lack of cholinergic receptors in the PNS. You can read moreabout this disease in your book (Chapter 17). The function of ACh in the brain is lesswell defined, but ACh is thought to be related to motor activity, learning and memory,

and controlling sleep stages. Deficits of ACh are associated with Alzheimer's andHuntington's diseases. Figure 1 shows some of the regions in the brain where cellbodies and axonal projections that use cholinergic neurons are found.

1


2/13

Figure 1. Above is a map of the acetylcholine pathways in the rat brain. These pathways helpregulate global functions that rely upon the cerebral cortex; such functions include attention, arousal,motivation, memory and consciousness. You should observe where these reside in the brain. Thedetails of the different types of connections are beyond the scope of this lab. The basal forebraincontains two groups of cholinergic neurons: (1) the medial septal group (medial septal nucleus andvertical diagonal band) that project cholinergic axons to the hippocampus and parahippocampalgyrus and (2) the nucleus basalis group (yellow and orange; nucleus basalis, substantia innominata(not shown) and horizontal diagonal band) that project cholinergic axons to all parts of the neocortex,parts of limbic cortex and to the amygdala. The cholinergic pontomesencephalon neurons (blue;laterodorsal tegmental and pedunculopontine tegmental nuclei ) project onto hindbrain, thalamus,hypothalamus and basal forebrain. The red symbols indicate ACh cell bodies and projections (Textadapted from the web site of Dr. Nancy Woolfe .(http://www.bol.ucla.edu/~nwoolf/).

Acetylcholinesterase (AChE) is an important enzyme which regulates the effects ofacetylcholine at cholinergic synapses. AChE's main function is to terminate the effectsof ACh after it is released, acting like an off switch. AChE is one of the most efficientenzymes in existence, having a turnover time of 150 usec, which is equivalent to thehydrolysis of 5,000 ACh molecules/molecule of enzyme/sec. It is predominantly foundbound to the postsynaptic membrane. AChE has two sites which bind to the cationicand esteric domains of ACh, cleaving the molecule between these sites (see Fig. 2).

Biochemistry: ACh is synthesized in neuron terminals in a reaction catalyzed by theenzyme choline acetyltransferase (ChAT) (see Figure 2 below). The choline is derived

from membrane phospholipids whilethe acetyl CoA is a breakdown product ofglucose. ACh is degraded intocholine and acetate by acetyIcholinesterase (AChE).The choline is taken back into the presynaptic cell and reusedto produce additionalACh (see Figure 3 for how ACh is metabolized).

2


3/13

ChATacetyl CoA + choline =========> acetylcholine + CoA

AChEAcetylcholine choline + acetate

Figure 2. The structure of acetylcholine. The reaction that produces ACh is catalyzed by cholineacetyltransferase (ChAT) whereas the breakdown of ACh is catalyzed by acetyIcholinesterase (AChE)

The arrow indicates where ACh is cleaved AChE at the ester linkage.

Measuring AChE by the methods described in this lab exercise is a simple and quickway to look at ACh distribution, however, it is still unclear whether this enzymeconsistently co-localizes with ACh. So why not measure ACh itself as a marker for thecholinergic system? ACh degrades rapidly after death and it is very difficult to sacrificean animal quickly enough to preserve the acetylcholine.

Figure 3. Acetylcholine metabolism. Note that acetyl CoA is primarily found in the mitochondria butthat the synthesis of ACh is in the cytoplasm. It is unknown how the transport of acetylcholine out ofthe mitochondria occurs. Choline can be synthesized by the brain but it is thought to actually arrive inthe brain in either a phospholipid form (phosphatidylcholine) or as free choline (adapted from

Biochemical Basis of Neuropharmocology).

3


4/13

Enzyme Kinetics: The kinetics of enzyme/substrate interactions can be displayed in avariety of ways: rate of reaction against time; rate of reaction against enzymeconcentration; rate of reaction against substrate condensation The shape of all thesecurves is similar since they all start off linearly and then level off because either thesubstrate or the enzyme becomes limiting. The effect of substrate concentration on

the reaction rate is plotted in Fig. 4. This relationship is described mathematically inthe Michaelis-Menten equation (see Fig. 4). Note that there are two constants derived

from this curve: Vmax which is the maximum velocity of the reaction, and Km which Isthe substrate concentration at which half the maximum rate (1/2 Vmax) is achieved.These two constants are referred to frequently when dealing with enzyme kinetics.

Enzymes can be inhibited by a substancewhich can bind to the active site. Thisinhibitor will prevent the binding of the neurotransmitter (substrate); however if anexcess of neurotransmitter is present, it can out compete the inhibitor and the enzyme

will become active again. This is known as competitive inhibition. An inhibitor can alsobind elsewhere on the enzyme and change its shape so that the active site is no

longer available This inhibition is not effected by the concentration of substrate and iscalled noncompetitive inhibition. These two different kinds of inhibition can becompared mathematically by using the Lineweaver-Burke equation (which is derived

from the Michaelis-Menten equation). Based on what you have had in Bio220, youshould be able to diagram what a graph of the reaction with a competitive and anoncompetitive inhibitor would look like.

Figure 4. Plot of reaction rate versus increasing substrate concentration. From such a graph

you can calculate Vmax and Km as indicated.

A variety of compounds have been found that inhibit AChE. Nerve gases, a class oforganophosphorus compounds, such as sarin, are poisons which bind irreversibly toAChE and can have fatal consequences. The drug Cognex (tacrine hydrochloride) is a

4


5/13

drug used to treat patients with Alzheimer's disease. Tacrine and other AChEinhibitors prevent the degradation of ACh and thus prolong its action (see Figure 6 forsites of action of drugs that act on cholinergic synapes).

Figure 5. Sites of action of various drugs at cholinergic synapses.Site 1: ACh synthesis is blocked by styryl pyridine derivatives such as NVP.Site 2: Transport of ACh into vesicles is blocked by vesamicol (AH5183).Site 3: Vesicle release is promoted by -bungarotoxin, black widow spider venom, an La3+. It

is blocked by botulinum toxin, cytochalisin B, collagenase pretreatment and Mg2+

.Site 4: The ACh receptors are activated by cholinometic drugs and anticholinesterases.Nicotinic receptors are blocked by rabies virus, curare, hexamethonium, ordihydroerythroidine. Agonists include n-methylcarbamylcholine, and dimethylphenylpiperazinium. Muscarinic receptors are blocked by atropine, pirenzepine, and quinuclidinylbenzilate.Site 5: Muscarinic receptors found in the presynaptic membrane may be blocked by AFDX-116, atropine, or quinuclidinyl benzilate.Site 6: AChE is inhibited by physostigmine (eserine), sarine, tacrine, DFP, or soman.

Site 7: Choline uptake blockers include hemicholinium-3, troxypyrrolium tosylate, or AF64A.(adapted from Biochemical Basis of Neuropharmocology)

In this lab..........

This experiment describes a method for measuring the activity of AChE in differentregions of the rat brain. The data can then be compared with AChE stained brainsections. The effect of the inhibitor tacrine will be examined to determine whether it isa competitive or a noncompetitive inhibitor.

5


6/13

THE PROTOCOL

The activity of AChE will be measured according to a method developed by Ellman etal. 1961. This method employs acetylthiocholine iodide (ATChI) as a syntheticsubstrate for ACh E. ATChI is broken down to thiocholine and acetate by AChE and

thiocholine is reacted with dithiobisnitrobenzoate (DTNB) to produce a yellow color.The quantity of yellow color which develops over time is a measure of the activity ofAChE and can be measured using a spectrophotometer (spec. 20).

These coupled reasons are represented by the following equations:

AChEacetylthiocholine iodide ==============> thiocholine + acetate

thiocholine + dithiobisnitrobenzoate ======> yellow colored products*

* produce of the reaction are 2-nitrobenzoate-5 mercaptothiocholine and 5-thio-2-nitrobenzoate (the latter is the

yellow product)

The activity of an enzyme is generally expressed as a rate: the quantity of substrate (inmoles) which is broken down by a known amount of enzyme per unit time. In thiscase, it will be the amount of ATChI which is broken down by AChE perminute.

1. Equipment and Solutions

A. Equipmentspectrophotometer scales

single edged razor blades kimwipesvortex pipettes - 5 mlice buckets and ice Pipetmen - various sizesrubber gloves capped 25 ml (centrifuge) tubesclean or disposable 13 x 100 mm glass test tubes and test tube rackaluminum foil stop watchessonicator or homogenizer Rat Atlas

B. Solutions0.1M phosphate buffer (PB), pH 8.00 075M acetylthiocholine iodide (ATCld)

0.01M dithiobisnitrobenzoate (DTNB)

You will prepare a brain homogenate and dilute it to approximately @5 mg/ml. The solutions, 0.1Mphosphate buffer (PB), pH 8.0 0.01M dithiobisnitrobenzoate (DTNB) and a stock of acetylthiocholineiodide (ATChI) at 0.1M will be provided. You will need to use the ATChI at the followingconcentrations: 0.1M; 0.075M; 0.05M: 0.025M; 0.01M and 0.005M. You may find it useful to

calculate how you will make the diluted ATChI solution prior to coming to class.

6


7/13

II. AChE assay in Different Brain Regions:

A. Prepare brain homogenates

TIP; Keep all tubes and solutions on ice during the experiment.

1. Weigh a sample of cortex (each student needs ~ 30 mg) and place it in alabeled tube. Add 1 ml of PB/30 mg tissue (30 mg/ml). Briefly sonicate orhomogenize this solution until the brain is uniformly dispersed in the buffer.Place the tube on ice.

2. Prepare the brain samples from cerebellum, striatum and hippocampus (asin #1 above). To help you identify these regions, refer to Figures 6 & 7. Clearlylabel the homogenates and store them on ice.

B. Assay (construct a flow sheet to help you stay organized flow sheet):

1. Turn on the spectrophotometer and set at 412 nm. Let it warm up for at least15 minutes prior to reading.

2. Label the assay tubes - four tubes (3 for the assay and one for a control) foreach brain region: cortex, cerebellum, hippocampus and stratum (4 x 4 = 16).

3. Pipette 3 ml PB into each assay tube

4. Using a pipette add 200 uL of cortex homogenate to each of the four labeledassay tubes. Vortex each tube and return it to the ice. Repeat this step for theother three brain regions.

TIP: To ensure accurate results, remember to use good pipetting technique. Ifyou are not familiar with automatic pipetters, check with your instructor.

5. Zero the spec. 20 without a tube by setting the needle to O transmittance(use knob on the left front).

6. Add 100 uL DTNB to the first cortex tube, vortex, and place it in a test tuberack for five minutes. This allows the solution to reach room temperature.

TIP: To save time, 100 uL of DTNB can be added to the next tube which can be

stabilizing at room temperature while the protocol is followed for the first tube.

7. Vortex and quickly wipe the outside of the tube with a kimwipe (to removemoisture and fingerprints which could interfere with the passage of light). Placethe tube In the spec. 20 and zero the spectrophotometer to 0 absorbance usingknob on right) This will be your baseline reading before measuring productformation.

7


8/13

Figure 6. The rat brain. a - lateral aspect; b - ventral aspect; c - midsagittal view; and d - dorsalaspect of the brain stem. Refer to Figure 8 for a dissection plan. (Figure adapted from Experimental

Psychobiology: A laboratory manual, edited by Benjamin L. Hart, W. H. Freeman and Company 1976)

8


9/13

Figure 7. Dissection of the cerebral hemispheres to the rat brain. a. You will need to collect acerebellum sample by cutting the cerebellar peduncles to remove the cerebellum; b . You will alsoneed to collect a cerebral cortex sample which is a thin layer that should be gently pulled away. Theappearance of the brain with the cerebellum and the cerebral cortex removed is shown; c. Gentlyremove the corpus callosum to get at the underlying structures. The appearance of the brain with thecorpus callosum removed; d. You will need to collect the striatum sample which we can do by takingthe caudate-putamen nucleus (these comprise the striatum). Remove this portion. Next you canremove the hippocampus. You will need to cut it away from the fornix. Shown is removal of thehippocampus after the connections to the fornix have been cut. (Figure adapted from Experimental

Psychobiology: A laboratory manual, edited by Benjamin L. Hart W. H. Freeman and Company 1976)

9


10/13

8. Take the tube out of the spectrophotometer, quickly add 20 uL ATChl andvortex.

9. Immediately return the tube to the spec. 20. Note the time and take a zeroreading of absorbance. Take readings at 30 sec, 60 sec, 2 min., and 3 min.

and record the data in a table.

10. Repeat this procedure (steps 6-10) for the other 2 cortex l homogenates.Run thecontrol through the same procedure except do not add substrate(ATChI) but add 20 uL PB instead.

11. Assay the 3 cerebellum samples, the 3 striatum samples and the 3hippocampus samples and their controls.

TIP: Striatum rates may be very fast and go offthe scale of the spec. 20. If thishappens, dilute these samples by 2 but remember to double the rates when

doing the calculations.

C Calculate of the rate of the reaction:

1. Graph the data for the different brain regions - change in absorbance/min.against time. Are the graphs linear?

2. Calculate the rate of color change per minute for each reading and averagethe rates within each three minute run. Then average the rates between eachrun for each brain region, calculate the rate of the reaction according to thefollowing equation:

R = A/(1.36*104) x 1/(200/3320)Co = 1.22(10-3) A/Co

R = rate, in moles substrate hydrolyzed/min. g tissue

A = change in absorbance/min.

Co = original concentration of tissue (mg/ml)

200/3320 are volume corrections

1.36 (10

4

) is the extinction coefficient of the yellow product

3. Make a bar graph to show the enzyme activity of each brain region.

10


11/13

III. Comparison of the measurements of AChE distribution with AChE stainedslides (pictures)It is possible to dye thin sections of brain with a stain which is specificforAChE. A rat atlas has photographs of sections of rat brain stained for AChE inaddition to sections labeled with the Nissl stain (a stain for cell bodies). It is

interesting to compare these stained sections with the results from thepreceding protocol concerning concentration of AChE in different brain regions.The data should be complementary.

1. Look carefully at the AChE stained sections and draw representativesections from each slide. Make sure to label the brain structures and indicatehow they are stained. Note in the drawing which areas of the brain containrelatively more and relatively less AChE.

2. Compare the distribution of AChE as determined lay staining patterns in thesections with your estimates of AChE distribution from the neurochemical

method.

WHERE DO YOU GO FROM HERE?

Collect and organize your data in a lab report. The following questions should helpyou focus on the meaning of your data:

1. Are your calculated reaction rates linear? If not, can you explain what is happening?

2. Does the distribution of AChE determined by the Ellman method correlate to thedistribution of AChE on the stained slides (or in the atlas)?

3. Were you pleased with how you designed your experiment? Can you think of waysto improve it?

POINTS TO PONDER

1. From your findings in this lab, is it possible to relate the patterns of enzymedistribution in different structures of the brain to functions associated with thesestructures?

2. It is important to consider whether the concentration of an enzyme such as AChE is

necessarily indicative of the concentration of the neurotransmitter ACh. Assuming thatyou have the knowledge and skills to measure ACh directly and there is a poorcorrelation between the concentration of ACh and AChE, can you think of a reason whythis should be so?

3. If you were designing a drug to enhance ACh levels, would you want to use acompetitive or a noncompetitive inhibitor? Is this an important feature to be

11


12/13

considered when designing a drug?

4. Consider a neurological disorder that is thought to involve the cholinergic system.Could tacrine be used to treat the condition? Suggest other chemicals that could beused to treat the disorder and where they would have their effect in the life cycle of The

neurotransmitter.

REFERENCES

Cooper, J.R., F.E. Bloom, and R.H. Roth. 1992. The Biochemical Basis ofNeuropharmacology, 6th ed. Oxford University Press, New York, NY.

Dale, H. H., W. Feldberg, and M. Vogt. 1936. Release of acetylcholine at voluntarymotor nerve endings. J. Physiol., 86- 353-380.

Ellman, C.L., D. Courtney, V. Andres, and R. Featherstone. 1961. A new and rapidcolorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7:8895.

Fonnum, F. 1975. A rapid radiochemical method for determination ofcholineacetyltransferase. J. Neurochem., 24: 407-409.

Greenfield, S. 1983. Acetylcholinesterase may have novel functions in the brain. TlNS,7: 364-368.

Hardy, J., L Heimer, R. Switzer, and D. Watkins. 1976. Simultaneous demonstration ofhorseradish peroxidase and acetylcholinesterase. Neuroscience, 3: 1-5.

Hoover, D. B., E. A. Mum, and P. M. Jacobowitz. 1978. A mapping of the distribution ofcholineacetyltransferase and acetylcholinesterase in discrete areas of the brain. BrainRes., 153 295-300.

Koelle, G. B. 1955. Histochemical identification of acetylcho]inesterase in cholinergic,adrenergic and sensory neurons. J. PharmRcol. and Exper. Ther., 114: 167-184.

Lexrey, A. I., B. H. Wainez, E.J. Mufson and M. M. Mesulam. 1983. Co-localization of

acetylcholinesterase and chose acetyltransferase in the rat cerebrum. Neuroscience,9: 922.

Loewi, O. 1921. Pfgers. Arch., 189: 239;193: 201.

Paxinos, G. and C. Watson. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed.Academic Press Inc. San Diego, CA.

12


13/13

Quinn, D. M. 1987. Acetylcholinesterase: Enzyme structure, reaction dynamics, andvirtual transition states. Chem. Rev., 87: 955-979.

Robertson, R.T., C. F. Holunann, J.L. Bruce and J.T. C:oyle. 1988. Neonatal

enucleation reduces specific activity of acetylcholinesterase in developing rat visualcortex. Devel. Brain Res., 39: 298302.

Sussman, I. L., M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, and I. Silman.1991. Atomic structure of acetylcholinesterase from Torpedo california; Aprototypic acetylcholine-binding protein. Science, 253: 872-B79.

Tallarida R. J., and L. S. Jacob. 1979. A Dose-response relation in pharmacology.Springer-Verlag, New York.

Thompson, R. F. 1993. The Brain: A Neuroscience Primer, 2nd ed. W. H. Freeman

and Co., New York,

13

Documents

Neurochemistry Lab