CNS STIMULANT AND DEPRESSANT FOR EXPERIMENTAL ANIMALS

the above presentaion gives about the in vitro and in vivo methods for cns stimulant and depressant for pharmacological and toxicollogical screening methods 1

1. CNS STIMULANT AND DEPRESSANT N. VIJAYAKUMAR M.PHARM, DEPARTMENT OF PHARMACOLOGY PHARMACOLOGY AND TOXICOLOGY SCREENING METHOD CHETTINAD SCHOOL OF PHARMACEUTICAL SCIENCE

2. TABLE OF CONTENTs INTRODUCTION CNS STIMULANT CNS DEPRESSION in-vitro AND IN VIVO SCREENING METHODS

3. INTRODUCTION i INTRODUCTIO N              TO CNS

4. WHAT IS CNS ? CNS is made up of BRAIN and SPINAL CORD. The brain is divided into three parts : 1) Forebrain, 2) Midbrain,   3) Hindbrain.  Spinal cord begins from the brain stem and extends till the lowest end of the backbone.  Both the brain and spinal cord contain fluid filled spaces or cavitiy  Fluid in the space is called as cerebrospinal fluid(CSF), and contains nutrients, hormones, white blood cells to maintain the CNS.  Brain and Spinal cord mainly responsible for information processing, imagination, memory and communication.

5. CNS STIMULANT  Central Nervous System Stimulants may be used to reduce tiredness and increase alertness, competitiveness, and aggression.  CNS stimulants may be defined as "Drug substance that most specifically afford an enhancement in excitability either very much within the different portions of the brain or the spinal cord which may lead to convulsion . at higher dose .

6. BEHAVIORAL MANIFESTATION OF CNS STIMULATION Analeptic effects.  "Increase Alertness, Attension, Nervousness And Anxiety.    Can Also Lead to Convulsions due to high dose  Decrease Drowsiness and fatigue

7. Classification of Cns stimulant 1. Analeptics (convulsant) Doxapram (respiratory stimulant) Nikethamide (respiratory stimulant) Strychnine Picrotoxin Bicuculline 2. Psychomotor stimulants Amphetamine Methylphenidate Cocaine Phentemine Fenfluramine

8. 3. General cellular stimulants:- Methylxanthines derivatives Caffeine (Caffee) Theophylline (Tea) Theobromine (Chocolate) 4. Clinical antidepressants:- MAO inhibitors Catecholamine reuptake inhibitors 5. Psychotomimetic (Hallucinogenic)

9. IN VITRO METHODS OF EVALUATION : Neurotransmitter Release Assays Neurotransmitter Uptake Inhibition Assays Receptor Binding Assays Enzyme Inhibition Assays Electrophysiological Studies (Patch Clamp / Microelectrode) Synaptosomal Preparations (Neurotransmitter Turnover) Cell-based Reporter Assays

10. 1) Neurotransmitter Uptake Inhibition Assay (DAT/NET/SERT) AIM : To determine whether the test compound inhibits uptake of monoamine neurotransmitters (e.g., dopamine) by blocking presynaptic transporters (DAT/NET/SERT). PRINCIPLE : Radiolabeled neurotransmitter (e.g., [³H]-dopamine) is taken up by synaptosomes or transporter-expressing cells. A compound that inhibits uptake (typical of stimulants) reduces intracellular radiolabel accumulation. Percent inhibition relative to vehicle control quantifies activity. MATERIALS: Synaptosomal preparation (rat striatal synaptosomes) or HEK293 cells transfected with human DAT. [³H]-dopamine (radiolabeled substrate) Standard inhibitor: nomifensine or cocaine (positive control) Test compound dilutions, assay buffer, filtration plates or centrifuge tubes, scintillation cocktail

11. PROCEDURE : Prepare synaptosomes or transfected cells and keep on ice. Incubate tissue/cells with test compound (range of concentrations) or vehicle for 5–10 min at 25–37 °C. Add [³H]-dopamine (final tracer concentration) and incubate 2–5 min (time in linear uptake range). Rapidly terminate uptake by vacuum filtration or cold buffer washes. Lyse filters/tubes, add and count CPM in liquid scintillation counter. Calculate % uptake relative to vehicle; % inhibition = 100 × (1 − (CPM_test/CPM_vehicle)). Generate concentration–response curve and determine IC₅₀. RESULTS : Stimulant-like inhibitors (amphetamine, cocaine) show concentration-dependent decrease in uptake (higher % inhibition). IC₅₀ values for standards provide reference (e.g., cocaine IC₅₀ in 0.8µM range so it is a cns stimulant and it act as dopamine transport inhibitor)

12. 2) Receptor Binding Assay (Dopamine D2): AIM: To measure affinity of a test compound for dopamine D₂ receptors by its ability to displace a radioligand. PRINCIPLE: Membrane preparations containing D₂ receptors are incubated with a radiolabeled antagonist (e.g., [³H]-spiperone). Test compounds that bind the receptor displace radioligand, reducing bound radiolabel. Ki values are calculated from displacement curves. MATERIALS: Brain membrane preparation (striatum) or cell membranes expressing D₂ receptor [³H]-spiperone (radioligand) Standard ligand: haloperidol (high-affinity D₂ antagonist) Non‑specific binding determined by excess cold ligand

13. PROCEDURE : Prepare membrane fraction and equilibrate in assay buffer. Incubate membranes with fixed concentration of [³H]-spiperone and various concentrations of test compound for 30–60 min at 25 °C. Separate bound from free ligand by rapid filtration and wash. Measure radioactivity retained on filters (CPM). Determine specific binding (total − nonspecific). Plot % displacement vs log[compound] and calculate IC₅₀; derive Ki using Cheng–Prusoff equation. Ki​= IC50​​/ 1+[L]​/ Kd​ where as Ki = inhibitation constant IC50 = Concentration of test compound that inhibit 50% of the ratioligand binding [L] = Concentration of Radioligand used in assay Kd = dissociation constant RESULTS : Compounds with affinity for D₂ will displace radioligand; antagonists/agonists both can bind (affinity differs). High affinity (low Ki) may indicate potential modulation of dopaminergic signaling, relevant to stimulant pharmacology.

14. EVALUATION METHODS IN CNS STIMULANT IN VIVO METHODS: Screening Of Analeptics By ACTO PHOT OMETER Sandauswurf' (displacement of sand) method Runway test Ptosis test Registration of motor activity Open field test Hole-board test Combined open field test

15. EVALUATION METHODS : IN VIVO MODELS: 1) Screening Of Analeptics By ACTO PHOT OMETER : PURPOSE : TO EVALUATE THE LOCOMOTOR ACTIVITY OF CERTAIN DRUGS PRINCIPLE : It consist of photocell , these photocell are activated when the rays of light fall on the photocell are observed and cut off due to movement of animal crossing the path of light beam. RODENT : Swiss Albino Mice (20 - 25g) DRUG : CHLORPROMAZINE HCL(3KG/MG) PROCEDURE : Take 6 albino rats and divided into 2 groups (3 std and 3 test) Place individiual each mice in the activity cage for 10min and note down the basal activity score After recorded inject the test drug to the mice with various interval Note the difference before and after administration of test dose

16. ACTOPHOTOMETER CONCLUSION: IT SHOWS INCREASE IN CNS STIMULANT IN LOCOMOTOR ACTIVITIES RESULT : Increased activity by number of counts compared to the control group. Therefore increase in cns activity.

17. 2. Runway test Aim: To study the effect of a drug on spontaneous activity and motor coordination. PRINCIPLE : The Y-shaped Runway is covered with paper that can indicate the foot prints of mice which is counted for evaluation. Material required : Wistar rats and Y Shaped runway apparatus Procedure: Wistar rats are used for the experiment. 8-19 rats are used for each dose the apparatus is symmetrical Y shaped runway made of wood and 13 inches high. Each arm is 15 inches long and 5 inches wide. A trial consists of placing a rat in the center of the Y and leaving it in the apparatus for 5 minutes. The number of times it enter the arms of the apparatus, is recorded as a measure of activity. In order to estimates the degree of ataxia , the rat is then placed on a runway covered with paper, so that footprint record of control rat shows that the regularity of spacing, is a measure of ataxia. The group of control rats had as the mean of spontaneous activity. Amphetamine at a dose of 0.19 mg/kg caused this number to increase to 20 times entries. Amylobarbitone sodium at a dose of 15 mg/kg caused it to increase to 22 times entries.

18. RESULT: There were increase in the mean value of various CNS stimulants at specific dose than the control animal group.

19. 3.Hole-board test : AIM: The evaluation of certain components of behavior of mice such as curiosity or exploration has been attempted. PRINCIPLE: An open field with holes on the bottom is used into which the animals could poke their noses which is a sign of Curiosity. PROCEDURE: Mice of either sex with a weight between 18 and 22 g are used. The hole-board has a size of 40 × 40 cm. Sixteen holes with a diameter of 3 cm each are distributed evenly on the floor. The board is elevated so that the mouse poking its nose into the hole does not see the bottom. Nose-poking is thought to indicate curiosity and is measured by visual observation in the earliest description and counted by electronic devices in more recent modifications. Usually, 6 animals are used for test and control group. Thirty minutes after administration of the test compound the first animal is placed on the hole-board and tested for 5 min. compare the control group and the test group.

20. CONCLUSION: INCREASE IN POCKING = HIGHER CURIOSITY DECREASE IN POCKING = LOWER CURIOSITY

21. CNS DEPRESSANT

22. WHAT ARE CNS DEPRESSANTS? Central Nervous System (CNS) depressants are type of drugs that slows down brain activity, which causes the muscles to relax ,calms and soothes a person making them useful for treating anxiety, panic, acute stress reactions, and sleep disorders .

23. CLASSIFICATION OF CNS DEPRESSANTS • These medicines include sedatives, tranquilizers, and hypnotics. 1) Sedatives- to treat sleep disorders like insomnia • Eg. Barbiturates such as Phenobarbital, pentobarbitalsodium . 2) Hypnotics- To Induce Sleep o Eg. Non-Benzodiazepines such as Zolpidem & zaleplon 3) Tranquilizers- to treat anxiety or to relieve muscle spasms. • Eg. Benzodiazepines such as Diazepam, clonazepam , Triazolam.

24. IN VITRO METHODS: 1)[3H]-GABA receptor binding GABAA receptor binding GABAB receptor binding 2)Benzodiazepine receptor: [3H]-flunitrazepam binding assay 3)Serotonin (5-HT1A) receptor: binding of [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin([3H]-DPAT)

25. 1)[3H]-GABA receptor binding: AIM: To study the binding of [³H]-GABA (radiolabeled GABA) to GABA receptors in brain tissue homogenates, and to evaluate the effect of test drugs on GABA receptor binding. PRINCIPLE: The GABA receptor is the major inhibitory receptor in the CNS. A radiolabeled ligand [³H]-GABA is used because it binds specifically to GABA receptors. When brain tissue homogenates (membrane preparations) are incubated with [³H]-GABA: Specific binding occurs at the receptor sites. Nonspecific binding (to other sites) is determined in the presence of excess unlabeled GABA. The amount of bound radioactivity is measured using a liquid scintillation counter. From this, binding affinity (Kd) and receptor density (Bmax) can be calculated. Test drugs that enhance binding usually act as CNS depressants (agonists or positive modulators). Drugs that inhibit binding act as CNS stimulants/convulsants (antagonists).

26. PROCEDURE: Reagents : 1)0.05 M Tris-maleate buffer (pH 7.1) Dissolve 6.05 g Tris-base in distilled water → make up to 1000 ml. Dissolve 5.93 g Tris-maleate in 500 ml water. Slowly add Tris-maleate solution into Tris-base solution until pH = 7.1. 2)0.32 M Sucrose solution Dissolve 109.5 g sucrose in distilled water. Make up volume to 1000 ml. Store at 4 °C. 3)[³H]-GABA (Radioligand) Stock: 40 Ci/mmol specific activity, made to 780 nmol in distilled water. Add 20 µl to each test tube → gives a final concentration of 15 nmol in assay. 4)Reference agonists (standard GABA mimetics) Isoguvacine: Dissolve 8.35 mg in 10 ml water. Muscimol: Dissolve 6.40 mg in 10 ml water. Add 20 µl of either solution to 1 ml incubation medium → final concentration = 0.1 mM. 5)Test drugs Prepare 1 mM stock solution. Make serial dilutions to get final concentrations in range: 2 × 10⁻⁸ M → 1 × 10⁻⁵ M (used for competition studies against [³H]-GABA).

27. TISSUE PREPARATION: 1)Animal sacrifice & brain removal Use male Charles-River rats (100–150 g). Decapitate and quickly remove the whole brain. 2)Homogenization Homogenize brain in 15 volumes of ice-cold 0.32 M sucrose. 3)Centrifugation steps (fractionation) 1st spin: 1000 g for 10 min → discard pellet (nuclear fraction). 2nd spin: 20,000 g for 20 min → discard supernatant, keep pellet (crude mitochondrial fraction). 4)Pellet processing Resuspend pellet in 15 volumes distilled water (gentle homogenization). 3rd spin: 8000 g for 20 min → collect supernatant + buffy layer. 5)Synaptic membrane isolation 4th spin: 48,000 g for 20 min → collect pellet (crude synaptic membranes). Resuspend pellet in 15 volumes distilled water (without homogenization). 5th spin: 48,000 g for 20 min → final synaptic membrane pellet obtained. 6)Storage Discard supernatant. Seal tubes (parafilm). Store membrane pellets at –70 °C until use.

28. TISSUE PREPARATION:

29. ASSAY PREPARATION : Step 1: Preparation of Membranes Take a frozen synaptic membrane pellet (from rat brain). Resuspend in Tris-maleate buffer (pH 7.1) at 4 °C. Add Triton X-100 (0.05%) → this makes binding more specific. Incubate at 37 °C for 30 min. Centrifuge at 48,000 g for 10 min → discard supernatant. Resuspend pellet again in fresh Tris-maleate buffer at 4 °C. Step 2: Binding Assay Setup Each test tube contains: 1 ml of membrane suspension (Tris-maleate homogenate). 20 µl [³H]-GABA (radiolabeled GABA). 20 µl of test drug or 20 µl of standard drug (isoguvacine/muscimol, 0.1 mM). Step 3: Incubation Incubate mixture at 4 °C for 5 minutes.

30. Step 4: Termination of Reaction Centrifuge at 5000 rpm for 15 min. Remove supernatant. Wash pellet twice with Tris-maleate buffer to remove unbound [³H]-GABA. Step 5: Radioactivity Measurement Add 2 ml of scintillation fluid (Liquiscint) to each tube → vortex well. Transfer contents into scintillation vials. Rinse tubes with another 2 ml liquiscint and add to vials. Add extra 6 ml liquiscint to each vial. Measure radioactivity using a liquid scintillation counter. RESULT: High [³H]-GABA binding → suggests agonist/CNS depressant effect (drug enhances GABA activity). Low binding (displacement) → suggests antagonist/CNS stimulant effect.

31. 2)Benzodiazepine receptor: [3H]-flunitrazepam binding assay AIM: T he primary aim of the [3H]-flunitrazepam binding assay is to study and evaluate the interaction of benzodiazepine receptors with radiolabeled flunitrazepam. This assay helps identify and quantify the binding characteristics (such as affinity and density) of benzodiazepine receptors in brain tissues or membranes. PRINCIPLE: The principle behind the [3H]-flunitrazepam binding assay is based on the radiolabeled ligand binding technique, where [3H]-flunitrazepam (a radioactive form of the benzodiazepine flunitrazepam) is used to study the binding characteristics of benzodiazepine receptors in brain tissue.

32. Reagents : • [Methyl-3H]-Flunitrazepam (70–90 Ci/mmol) can be obtained from New England Nuclear. • Clonazepam HCl can be obtained from Hoffmann La Roche TISSUE PREPARATION: Male Wistar rats are decapitated and the brains rapidly removed. The cerebral cortices are removed,weighed and homogenized to 20 volumes of ice-cold 0.32 M sucrose. This homogenate is centrifuged at 1000 g for 10 min. The pellet is discarded and the supernatant is centrifuged at 30 000 g for 20 min. The resulting membrane pellet is resuspended in 40 volumes of 0.05 M Tris buffer, pH 6.9.

33. ASSAY: Materials: 0.05 M Tris Buffer (pH 6.9) – 1 mL 0.5 M Tris Buffer (pH 6.9) – 560 µL [3H]-Flunitrazepam – 50 µL Vehicle (for total binding) or 0.1 mM Clonazepam (for non-specific binding), or appropriate drug concentrations – 20 µL Tissue Suspension – 300 µL Ice-cold Buffer (pH 6.9) – for rinsing and washing the filters Whatman GF/B Filters – for vacuum filtration

34. ASSAY: Preparation of the Reaction Mixture: Mix the following in a test tube: 1 mL of 0.05 M Tris buffer (pH 6.9) 560 µL of 0.5 M Tris buffer (pH 6.9) 50 µL of [3H]-Flunitrazepam 20 µL of vehicle (for total binding) or 0.1 mM Clonazepam (for non-specific binding), or another drug you’re testing Add 300 µL of tissue suspension to the mixture (containing the target receptors) at 10-second intervals. Incubation: Incubate the tubes at 0–4°C in an ice bath for 20 minutes. Start the timer when the first tissue suspension is added. Stopping the Assay: After the incubation period, stop the reaction by performing vacuum filtration through Whatman GF/B filters. Perform filtration in 10-second intervals (i.e., filter each tube sequentially).

35. Washing the Filters: After filtration, immediately rinse the filters with three 5-mL washes of ice-cold buffer (pH 6.9) to remove any unbound ligand. Counting Radioactivity: Place the filters in a liquid scintillation counter. Add 10 mL of liquid scintillation cocktail to each filter to measure the radioactivity. RESULT: Specific binding was found to be approximately 97% of total [3H]-flunitrazepam binding. This means the assay is highly selective for benzodiazepine receptors, with only ~3% being non-specific binding. So it represent the cns depressant activity.

36. 3)Serotonin (5-HT1A) receptor: Binding of [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]-DPAT) AIM: To i dentify, quantify, and characterize the 5-HT1A serotonin receptors in brain tissues or cell membranes and to evaluate the binding affinity and density of these receptors using a radiolabeled selective agonist, [3H]-8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin). PRINCIPLE: The assay is based on the principle of radioligand binding, where a radioactively labeled compound selectively binds to a specific receptor, and the amount of binding is measured using a scintillation counter.

37. REAGENT: Tris Buffers (pH 7.7): a) 0.5 M: Tris HCl (57.2 g) + Tris base (16.2 g) → 1 L b) 0.05 M: 1:10 dilution of 0.5 M buffer c) 0.05 M + Additives: Pargyline HCl: 0.49 mg CaCl₂: 111 mg Ascorbic acid: 250 mg Make up to 250 mL with 0.05 M buffer [3H]-8-OH-DPAT : Radioligand (160–206 Ci/mmol) Stock: 10 nM → Add 50 µL → Final: 0.5 nM Serotonin (for non-specific binding): Stock: 0.5 mM in 0.01 N HCl Add 20 µL → Final: 10 µM .

38. Test Drugs: Stock: 1 mM → Serial dilutions Final assay concentrations: 2 × 10⁻⁵ to 2 × 10⁻⁸ M Use 7 concentrations for IC50 curve Tissue Preparation Steps: Sacrifice & Dissection: Rats are sacrificed by decapitation Hippocampi are quickly removed and weighed Homogenization: Homogenize in 20 volumes of 0.05 M Tris buffer (pH 7.7) First Centrifugation: Spin at 48,000 g for 10 minutes Discard supernatant, keep the pellet

39. Washing Step: Resuspend pellet in equal volume of 0.05 M Tris buffer Incubate at 37°C for 10 min (to remove endogenous ligands) Recentrifuge at 48,000 g for 10 min Final Resuspension: Resuspend the final pellet in 0.05 M Tris buffer containing: 4 mM CaCl₂ 0.1% ascorbic acid 10 µM pargyline (MAO inhibitor – prevents serotonin breakdown) Thus the clean membrane fraction from rat hippocampus containing functional 5-HT1A receptors for binding assays were prepared.

40. ASSAY: Incubate tubes at 25 °C for 15 minutes Stop the assay by vacuum filtration through Whatman GF/B filters Wash filters 2 times with 5 mL of ice-cold 0.05 M Tris buffer Place filters in scintillation vials Add 10 mL of Liquiscint scintillation cocktail Measure radioactivity using a liquid scintillation counter RESULT: The KD value for [3H] DPAT binding was found to be 1.3 nM by Scatchard analysis in a receptor saturation experiment. COMPONENT VOLUME TISSUE SUSPENSION 800 µL 0.05M tris buffer 130 µL vehicle/serotonin /test drug 20 µL [3H]-8-OH-DPAT 50 µL total volume 8mL

41. EVALUATION OF CNS DEPRESSANTS In-vivo methods:- 1. Forced Swim Test 2. Benzodiazapine Induced Sleeping Time 3. Motor-Incoordination 4. Tail Suspension Test 5. Rota-Rod Test 6. Tread-Wheel Method 7.CORNEA AND PINNA REFLEXES

42. 1.ROTA ROD APPARTUS : PURPOSE : SKELETAL MUSCLE RELAXANT AND CNS DEPRESSANT by using rota rod apparatus Drug : Diazepam (1mg/kg) Principle: Reduction of motor co-ordination, CNS depression and skeletal muscle relaxation lead to decrease in the fall off time and decrease in number of free ridings of animal balancing on the rotarod. PROCEDURE: Animals are divided into two groups Administer one group with the drug to be tested and other with vehicle by intraperitoneal route. Pick rodents one by one. Put the rodents on rotarod apparatus, rotating at the speed of 25 rotations/60 sec. When the animal fall off respective time will be displayed on the timer. Compare the test and the standard group

43. ROTA ROD APPARATUS FREE RIDING OF RAT BEFORE FALL OFF CONCLUSION : The observed reduction in fall off time and free riding shows that Diazepam at 1 mg/kg i.p. dose produces decrease in motor co-ordination and decrease in muscle strength. there fore it shows an cns depressant.

44. 2. FORCED SWIMMING : PURPOSE: C haracteristic behavior of immobility PRINCIPLE: To measure an animal's behavioral despair in response to an inescapable, aversive stimulus, typically water, to model depressive-like behavior Apparatus : Vertical Plexiglass cylinder: 40 cm height × 18 cm diameter Filled with 15 cm water maintained at 25 °C PROCEDURE : Pre-Test (Day 1) Place rats individually into the cylinder. Initially, rats swim actively (circling, climbing, diving). After 2–3 minutes, activity decreases and periods of immobility appear. By 5–6 minutes, immobility reaches a plateau (~80% of the time). After 15 minutes, rats are removed. Dry them in a heated enclosure (32 °C), then return to home cages. Test Session (Day 2) 24 h later, place rats again in the same cylinder. Record total duration of immobility during a 5-minute test. Immobility is defined as Passive floating with minimal movements Upright position, nose above water.

45. Drug Administration : Test drug/standard (e.g., Imipramine) given 1 h before test. For stable results, injections at 1, 5, and 24 h before the test can also be used. CONCLUSION : Duration of immobility between the test and the standard group should be evaluated d. Antidepressant drugs, but also stimulants like amphetamine and caffeine, reduce duration of immobility. Dose-responses can be evaluated.

46. 3.TAIL SUSPENSION METHOD : PURPOSE : TO MEASURE STRESS IN THE RODENTS PRINCIPLE: B ehavioral test useful in the screening of potential antidepressant drugs. PROCEDURE : Divide animals into groups of 10. Administer test compound or vehicle via intraperitoneal injection. Wait 30 minutes before testing. Suspend each mouse by the tail using adhesive tape, placed ~1 cm from the tip. Hang the mouse on the edge of a shelf, 58 cm above the surface. Record immobility time for 5 minutes. A mouse is considered immobile when it hangs passively and remains completely motionless for at least 1 second (some protocols use ≥1 min, but standard scoring is based on total immobility duration).

47. Interpretation Longer immobility → depressive-like behavior. Reduced immobility after drug treatment → antidepressant activity. CONCLUSION: Compare the test and standard groups . The percentage of animals showing the passive behavior is counted and compared with vehicle treated controls. Using various doses, ED50 values can be calculated

48. REFERENCES: 1)https://www.ncbi.nlm.nih.gov/ 2)Drugabuse.com 3)researchgate.net 4) Screening Methods in Pharmacology by Robert Turner 5) Drug Discovery and EvaluationPharmacological Assays Second Completely Revised, Updated, and Enlarged Edition by H.GERALD VOGEL. 6) Rang HP, Dale MM, Ritter JM, and Flower RJ. CNS stimulants and psychotometric drug, 6'th edition 7) Vogels 8) Pharmacological Screening Methods and Toxicology by Avanapu Srinivasa Rao Namburi Bhagya Lakshmi 9) Drug Discovery and Evaluation by H. Gerhard Vogel (Ed.) 10) JoVE protocols and JoVE figures illustrating synaptosomal dopamine uptake/release workflows and practical steps. (useful procedural images). 11)Literature reviews on stimulant mechanisms (e.g., amphetamine/cocaine effects on DA release/uptake). (https://pubmed.ncbi.nlm.nih.gov/11071707/) 12) Screening models for CNS stimulant drugs: A Review ( https://asianjpr.com/HTML_Papers/Asian%20Journal%20of%20Pharmaceutical%20Research__PID__2013-3-3-10.html)

49. THANK YOU