EXPERIMENTAL SCREENING MODELS FOR VARIOUS DISEASES.pptx

SCREENING MODELS IN PHARMACOLOGY
DETAILED VIDEO ON EXPERIMENTATION MODELS.OBESITY
DIABETES
ALZHEIMER
PARKINSON
SCHIZPHRENIA
ULCER
PCOS
CARDIOVASCULAR DISEASE
ENDOCRINE DISEASE
NEUROLOGICAL DISEASE
PULMONARY DISEASE
RENAL DISEASE
HEPATOLOGICAL DISEAS
SAFETY PHARMAOCLOGICAL STUDIES
TOXICOLOGICAL STUDIES

1. EXPERIMENTAL SCREENING MODELS FOR VARIOUS DISEASES By Payaam VOHRA

2. Physical Chemical Mechanical Acoustic Vibrational Electrical BIOLOGICAL

3. Arrhythmia type Experimental model Wolff Parkinson white syndrome Transgenic PRKAG2 model Atrial Flutter Atrial flutter induced by Ach and rapid pacing. Atrial flutter by aconite. Canine right Atrial occlusion injury model. Atrial Fibrillation Atrial fibrillation in the isolated langendorff perfused heart. Canine model of chronic Atrial fibrillation. PACAP-27 induced Atrial fibrillation. Ventricular Fibrillation Ventricular fibrillation electrical threshold. Canine model of two stage ligation. Ventricular arrhythmia during exercise by ischemia. 6

4. DRUG ABUSE

5. In-Vivo METHODS Chemically induced arrhythmia Electrically induced arrhythmia Mechanically induced arrhythmia Exercise Induced arrhythmia 8

6. CHEMICALLY INDUCED ARRHTHMIA A large number of chemical agents alone or in combination are capable of inducing arrhythmia. These include 1 Aconitine antagonism in rats 2 Digoxin induced arrthymia in guinea pigs 3 Strophanthin or ouabain induced arrhythmia in dog 4 Adrenaline induced arrhythmia Coronary artery occlusion/reperfusion arrhythmia Arrhythmias induced directly by ischemia and reperfusion Coronary artery ligation in anesthetized dog results in ↑ in HR ↑in heart contractility ↑ in BP Ventricular arrhythmias MECHANICALLY INDUCED ARRHYTHMIA ELECTRICALLY INDUCED ARHYTHMIAS NERVE BLOOD FLOW CONDUCTION VELOCITY D-DIMER CRP 9

7. ALZHEIMER

8. DEPRESSION

9. STROKE

10. COLLAGENASE BCAO MCAO GLOBAL AND FOCAL EMBOLIC ENDOTHELIN MICROBALLON INFLATION MODEL Foot-Fault Test Motor function, Limb coordination Corner Test Sensorimotor asymmetry Adhesive Removal Test Sensorimotor dysfunction, Motor asymmetry

11. In vitro Models ADD A FOOTER 17 In vitro models in depression are generally used as preliminary high throughput screening for anti depressant and are usually receptor binding studies Inhibition of Dopamine/Serotonin uptake in rat striatal synaptosomes Binding to monoamine transporters Measurement of β-adrenoreceptor stimulated Adenylate cyclase Monoamine oxidase inhibition: Inhibition of type A and type B monoamine oxidase activities in rat brain synaptosomes Serotonin Binding Affinity NMDA receptor Antagonism

12. Learned Helplessness Model ADD A FOOTER 18 Following one or more sessions of inescapable shock, rats have been shown to develop persistent changes including weight loss, alterations in sleep patterns and HPA axis activity and loss of spine synapses in hippocampal regions In mice, the learned helplessness (LH) syndrome appears to be short-lived (2–3 days), and several mutant lines of mice have been phenotyped on the LH assay, with results largely compatible with their corresponding FST data. Like the FST or TST, both mice and rats display a considerable degree of interstrain variation, and escape deficits are reversed by a variety of antidepressants Following an uncontrollable and inescapable stress such as exposure to inescapable electric shocks, animals develop a state of “helplessness”

13. Chronic Mild Stress Model 19 Chronic mild stress (CMS), better described as CUS, paradigms involve the application of varied intermittent physical stresses applied over a relatively prolonged time period (between 1 and 7 weeks Basically this model consist of various stressor applied throughout the weeks randomly to impart stress on mice which gradually develops anhedonic behaviour over the course of time CUS has also been shown to result in a number of other “emotional” changes that are difficult to objectively quantify, such as grooming deficits and changes in aggressive and sexual behaviour Aside from being a tool to study the physiological consequences of chronic stress, CUS has been applied recently to phenotype mouse mutants, study gender differences in stress responses, and validate novel antidepressants Despite all of its variability and disadvantages the CMS model simulates the human condition for Depression most closely than any other model singly While acute stress paradigms are used broadly for their ease, automation, and rapid phenotyping abilities, they offer singular readouts that often cannot be unambiguously interpreted

14. Genetic Models Forward Genetics 20 There are two main approaches for genetic models of depression Reverse Genetics It is and unbiased approach in which large number of random mutations are generated in the animal using simple mutagenic techniques This followed by breeding and screening for individuals with the desired aberrant phenotype After the generation of a mutant mouse line with the desired phenotype—in this case, depressive-like behaviour—the responsible gene can be identified. The reverse genetic approach is used more commonly in scientific practice and involves genetic manipulations that result in either loss- or gain of-function mutants. “Knockout mice” are the most well-known examples, in which a specific target gene is disrupted, resulting in a loss-of function mutant. However, loss of function can be achieved using other tools, such as insertion of transgenes that produce an antisense mRNA of the target gene or of short hairpin RNAs directed against the gene of interest For example, mouse lines expressing Cre recombinase, which is a tyrosine recombinase enzyme derived from the P1 bacteriophage, selectively in neurons of a specific neurotransmitter

15. ADD A FOOTER 21 Ontogenetic Mouse Model

16. Maternal Separation 22 In particular, maternal separation was proposed to represent an important animal model for investigation of the pathophysiology and treatment of major depression mong the paradigms used to study early adverse life events, long maternal separation in rodents mimics early life neglect/loss of parents in humans, and has been presented as one of the most potent natural stressors during development Maternal separation was developed to examine the consequences of early adverse experiences on behaviour and neurobiology, and this model has been described as a model of vulnerability to drug dependence, anxiety, stress-induced illness, and depression The mice are separated from their mother at the time of birth for unpredictable amount of time These mice when reach adult stage show hallmark signs of anhedonia and depression Early adverse life experiences represent one of the major risk factors for the development of mental disorders such as major depression.

17. Sleep Deprivation Model 23 Increased levels of messenger RNA for interleukin-1b (a pro-inflammatory cytokine) and for cortisol have been shown in rodents after sleep deprivation The procedure of this study consisted of handling the animals gently to prevent them from sleeping Furthermore, 72 hours of sleep deprivation in mice was induced using the platform method, which is accomplished by placing the animal on a platform submerged in water so that, when the animal falls asleep, it falls into the water and must then climb back onto the platform, thus forcing it to stay awake This study showed that after 72 hours of sleep deprivation, there was an increase of oxidative stress in the hippocampus Other sleep deprivation technique include the Flower Pot Technique wherein the mice is fitted in a hole in pot snuggly, when mice falls asleep the muscle relax which allows water to flow and thus keeps the mice awake Sleep has important homeostatic functions, and sleep deprivation is a stressor that has consequences for the brain as well as for many body systems.

18. Olfactory Bulbectomy (OBX) Model 24 OBX cause permanent changes in various behavioural parameters like alterations in regular sleep pattern dysregulated cognitive function bizarre sexual pattern, chronic stress hyperactivity The OBX model has been successfully detecting antidepressant drugs for decades now with the most reproducible effect, meaning the hyperactivity in the open field test This effect is also the confirmation test of successful surgery when the testing drug is meant to affect a different parameter of the OBX rats Many effects of the bulbectomy are reversible with the administration of antidepressant drugs and fail to return to baseline when given a drug lacking antidepressant activity Such effects include decrease in passive avoidance learning deviating leukocyte differential count and the microglial neuroinflammatory response These effects are reversible upon chronic administration of both selective serotonin reuptake inhibitors (SSRI) and tricyclic antidepressants (TCA) Bilateral olfactory bulbectomy (OB) results in endocrine, behavioral, immune system, and neurotransmitter changes that mimic many of the symptoms seen in human patients with major depression.

19. Chemical Models Of Depression Chronic Corticosterone 25 These models are generally based on pathophysiology of depression and unlike physical models employ one of the mechanism of the developing depression, generally used in mechanistic studies of novel antidepressants Reserpine Induced This model is based on the Hypothalamus – Pituitary – Adrenal Axis hyperactivity of depepression Chronic administration of corticosterone sends the HPA axis on a hyper drive and is unable to cope up even in mild stress condition Gradually these mice through even slightest of stressors or stimuli for short period can develop depression Reserpine was earlier widely used for treating hypertension and schizophrenia Observation show depressive like behaviour in rodent thus reserpine models are then develop Reserpine causes depletion of monoamine in brain, a classic pathology of depression Depressive symptoms are however accompanied by other symptoms like hypothermia and ptosis

20. ULCER

21. SCREENING METHODS IN VIVO METHODS Pylorus ligation in rat Isolated rat stomach Stress ulcer models Restraint- induced ulcer cold water immersion induced ulcer Histamine -induced ulcer Ethanol -induced ulcer NSAIDs- induced ulcer Acetic acid- induced gastric ulcer 31 IN VITRO METHODS [ 125 I] Gastrin Binding Assay Tiotidine Binding assay H + / K+ - ATPase inhibition Assay

22. OBESITY

23. Methods to induce experimental obesity Food induced obesity obesity can be induced in rats by offering a diet containing corn oil and condensed milk Male SD rats housed in controlled conditions At the age of 6 months rats, divided into 2 groups, ordinary Purina Rodent Chow is fed to G1, the G2 is fed with Purina Rodent Chow, corn oil and condensed milk Body weight and food intakes measured, sacrificed after 3 months Adipose tissue cell size, lipid content, hormones, plasma lipids are determined.

24. 2 . Hypothalamic obesity Hyperphagia in rats induced after hypothalamic lesions Female SD rats, fed HFD for 5-9 days, fasted over night, anesthetized with 35mg/kg pentobarbital sodium+1mg atropine methyl nitrate Bilateral knife cuts or electrolytic lesions sterotaxically positioned in the hypothalamus Histological verification of placement of knife ciuts is made in brain fixed in 10% buffered formalin and embeded in paraffin. serial sections of hypothalamus are examined histologically

25. 3. Goldthioglucose - induced obesity i.p or i.m injection of goldthioglucose induces obesity in mice Related to destruction of hypothalamic and extra thalamic areas of brain Swiss albino mice of either sex, fed with commercial mouse chow At the age of 6 months, single i.p injection of 30-40 mg/kg goldthioglucose Food intake and body weight determined for 3 months.

26. 4. Monosodium glutamate-induced obesity Adiposity induced in mice by repeated s.c injections of MSG Male charles river mice, treated immediately after birth with daily s.c MSG for 5 days Animals weaned at 3 weeks age, housed under controlled temp. Food consumption and body weight is measured at the regular intervals

27. Assays of anti-obesity activity Anorectic activity (food consumption in rats) Food intake and body weight measured in acute and semichronic experiments in normal or obese rats respectively Female zucker rats(250-350g), maintained under controlled conditions Rats fed with special dishes along with test compound or treated by i.p for 7 days Mazindol, 3mg/kg, a standard 2.APoE model

28. 2. Metabolic activity (GDP-binding in brown adipose tissue) Brown adipose tissue, a major site for non-shivering thermogenesis in rodents Obese male fatty Zucker rats(age; 13 weks , weight; 450g), receive multiple doses of test compound in the drinking or tap water for 21 days Food intake measured everyday, and bodyweight everyother day Rats sacrificed, brown adipose tissue minced, diluted with 250mM sucrose and homogenized supernatant collected, centrifuged, BSA 0.2% added After centrifugation, pellet suspended in albumin free-sucrose buffer Binding of GDP to mitochondria is determined by incubating mitochondria in basic medium containing 100mM sucrose, 20mM TES, 1mM EDTA, 1mM choline chloride, 2 μ M rotenone, and 10 μ M 3 H rotenone.

29. 3 . Uncoupling protein and GLUT4 in brown adipose tissue Uncoupling proteins, family of inner mitochondrial membrane membrane transporters dissipate the proton gradient UCP1 expressed exclusively in brown adipocytes Male fatty rats, 10 weeks age, given s.c injection of test compound Sacrificed after 14 weeks treatment , brown and white adipose tissue removed Northern and western blotting is performed , the total RNA and GLUT4 proteins determined

30. 4. Resting metabolic rate RMR, influenced by various drugs both in normal and obese animals Female yellow KK mice and female C57B1 mice, age 12 weeks, housed under controlled conditions, fed with commercial powdered chow and tap water Test compound given i.m for 2 weeks Daily food intake and eight is measured RMR estimated by means of closed-circuit metabolic system, consists of chamber, circulating pumps, desciccant and CO 2 absorbant canisters

31. SCREENING OF SYMPATHOMIMETICS AND SYMPATHOLYTICS

32. CARDIOVASCULAR ANALYSIS IN VIVO α- and β- adrenoreceptors in the mouse iris α2-adrenoreceptor blockade measured in vivo by clonidine -induced sleep in chicks Activity at β1- and β2-adrenoreceptors in the rat β1- and β2-Sympatholytic activity in dogs Intrinsic β- sympathomimetic activity in reserpine -pretreated dogs

33. SYMPATHOMIMETIC DRUGS MAY RESULT IN ANY ON FOLLOWING EVENTS LIKE Mydriasis in the eye Enhanced stroke volume Acceleration of the heart rate Dialation of the coronary arteries Constriction of the pulmonery vessels Relaxation of bronchial muscles Inhibition of gastric secretion Constriction of gastrointestinal sphincters Stimulation of uterus and constriction of spleen capsule

34. INFLAMMATORY

35. ANTIPROLIFERATIVE ASSAY (I) Seed the cells in 24-well tissue culture plates at a density of 10 4 cells/cm 2 . That means 1 ml/well at a concentration of 2x10 4 cell/ml (one well is considered 2 cm 2 ). Prepare 3 wells/experimental point. For each experiment, blank and solvent controls must be assayed along with at least 3 doses for the test compound. Incubate the cells at 37 o C in 9% CO 2 -air for a 24 h-recovery period. Treat the test cells with serial concentrations of drug/test compound in an appropriate vehicle, in triplicate. The vehicle should not exceed 1% of the tissue culture medium. 45

36. Carrageenan-Induced Paw Edema DEXTRAN INDUCED PLETHYSMOMETRY HISTAMINE BRADYKININ Pleurisy model Ctoron oil Cotton Pellet Granuloma Test Granuloma Pouch tes Formalin induced

37. CNS DRUGS

38. Screening models for CNS stimulant/depressant drugs In-vivo methods Run away Test or Y Maze Test Actophotometer Open Field Test Hole Board Test Elevated Plus Maze Forced Swim Test Light Dark Test Barbiturate induced sleeping time Motor coordination Tail Suspension test

39. Run away Test or Y Maze Test This test is used to study the effect of a drug on spontaneous activity and motor coordination. Swiss albino rats of either sex were selected. The mice were placed individually in a symmetrical Y–shaped runway (33 cm x 38 cm x 13 cm) for 3 min and the number of the maze wall 4 ft (an' entry’) were counted

40. Actophotometer The locomotor activity can be easily studied by using Actophotometer . Swiss Albino mice of either sex (20 – 25g ) were randomly divided into three groups of six animals. The rats were placed individually inside the chamber of actophotometer for 10 min and basal activity score was noted. The animals were treated with drug and after 30 min of mice are placed again in actophotometer for 10 min and the activity was monitored. Percentage increase(in case of CNS stimulant)/ decrease(in case of CNS depressant) in activities were calculated.

41. Open Field Test Mice were carried to the test room in their home cages and were handled by the base of their tails at all times. Mice were placed into the center or one of the four corners of the open field and allowed to explore the apparatus for 5 minutes. After the 5 minute test, mice were returned in their home cages and the open field was cleaned with 70 % ethyl alcohol and permitted to dry between tests. The Open Field Test provides simultaneous measures of locomotion, exploration and anxiety .

42. Hole Board Test Rats were transported from housing room to testing room inside their home-cages to minimize transfer effect. To avoid possible visual and/or olfactive influences, animals were allowed to acclimate for 30 minutes far from observational apparatus. Each subject, experimentally naïve at test beginning, was placed in the arena centre and allowed to freely explore for 10 min and this gives the idea about CNS stimulant/depressant activity of drugs. Experiments were recorded through a digital video camera and video files stored in a personal Computer.

43. Elevated Plus Maze This test has been widely validated to measure anxiety in rodents. This apparatus was made of Plexiglas and consisted of two open arms (30cm × 5cm) and two closed arms (30cm × 5cm) with 25cm walls. The arms extended from a central platform (5cm ×5cm). The maze was elevated 38.5cm from the room floor. Albino rats of either sex (150 - 200 g) were randomly divided into three groups of six animals. Each animal was placed at the center of the maze, facing one of the enclosed arms. Number of entries and the time spent in enclosed and open arms was recorded for 5 min test. Entry into an arm was defined as the animal placing all four paws onto the arm. All tests were taped by a video camera.

44. Forced Swim Test Albino rats of either sex (150 - 200 g) were selected. Rats were placed individually in a transparent glass cylinder (12 cm in diameter, height 25 cm), which was filled with water to a height of 15 cm. Two swim sessions were conducted. An initial 15-min pre-test followed 24 hr later by a 6 min test. In the pre test session, the mice which have not yet treated were forced to swim in a glass cylinder for 15 min. In the second session, each mouse received a respective dose of sample 1 hour prior to test, and placed in the cylinders again for 6 min. The following behaviors were recorded during the last 4 min. 1. Immobility: floating in water without swimming. 2. Swimming: active movements of extremities and circling in the container. 3. Climbing: active movements of forelimbs on the container wall

45. Light Dark Test The apparatus consists of a plexiglass box with two compartments (20cm × 20cm each), one of which illuminated compartment, facing one of the dark areas. The time spent in illuminated and dark places, as well as the number of entries in each space, was recorded for 5 min And this gives us an idea about the CNS stimulant/depressant property of the drug. White rats-black background Black rats-White background

46. Motor coordination Muscle coordination was determined via the use of Ugo Basile Rota rod bar. Swiss albino mice were placed on rota rod prior to treatment and at 0.5, 1, 2, 3, 4 and 5 hrs after treatment. Any mouse that fell off before the cut off time 2 min were excluded from the experiment

47. Tail Suspension test For the test, mice were suspended on the edge of a shelf, 58 cm above the ground with adhesive tape placed approximately 1 cm from the tip of the tail. The duration of immobility was recorded for a period of 5 min after the drug treatment.

48. Anxiety models In vitro assay for GABAergic compounds: [3H]-GABA receptor binding GABAA receptor binding GABAB receptor binding Benzodiazepine receptor: [3H]-flunitrazepam binding assay Serotonin receptor binding General considerations Serotonin (5HT1A) receptor: binding of [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]-DPAT) . Serotonin (5HT1B) receptors in brain: binding of [3H]5-hydroxytryptamine ([3H]5HT) 5HT3 receptor in rat entorhinal cortex membranes: binding of [3H]GR 65630 Histamine H3 receptor binding in brain In vitro models 58

49. Effect on behaviour Methods based on unconditioned (spontaneous) response Exploratory activity Elevated plus-maze light-dark (two compartment box) open field, closed field, etc. Social behavior social interaction maternal separation, ultrasonic distress calls Predator mouse defen s e test battery human threat (primates) predator’s call, odor associated avoidance response Methods based on unconditioned (spontaneous) response Conflict models Vogel punished drinking Geller-Seifter conflict marmoset, pigeon confl i ct models Other four plate test active/passive avoidance learning conditioned ultrasonic vocalization 59

50. Anticonvulsant activity Pentylenetetrazole (Metrazol) induced convulsions Strychnine-induced convulsions Picrotoxin-induced convulsions Isoniazid-induced convulsions Yohimbine-induced convulsions PURPOSE AND RATIONALE This assay has been used primarily to evaluate antiepileptic drugs. However, it has been shown that most anxiolytic agents are also able to prevent or antagonize chemical-induced convulsions 60

51. E levated plus-maze Purpose The test has been proposed for selective identification of anxiolytic and anxiogenic drugs Source of anxiety : open space, height, new environment Procedure : The rats (200–250 g body weight) are housed in pairs for 10 days prior to testing in the apparatus Groups consist of 6 rats for each dose. Thirty min after i.p. administration of the test drug or the standard, the rat is placed in the center of the maze, facing one of the enclosed arms. Parameters measured : Time spent in the open arms entries into the open arms time spent in the closed arms entries into the closed arms total entries central time Anxiolytic effect : Statistically significant increase e in open time or open arm entries 61

52. Dimensions: The plus-maze consists of two open arms, 50 × 10 × 40 cm, and two enclosed arms, 50 × 10 × 40 cm, 50cm 62

53. 2.IN VIVO MODELS: Haffner’s Tail Clip Method Hot - plate test Electrical stimulation of the tail Grid - shock test Formalin test in rats Chemotherapy-Induced Pain Spinal Cord Injury Radiant heat method Tail immersion test

54. Ultra sonic distress calls Source of anxiety: maternal separation Parameters measured: time spent with ultrasonic vocalization total number of ultrasonic vocalization Anxiolytic effect: statistically significant increase in either parameters measured ; 64

55. Light-dark model Source of anxiety : light , n ovelty , Procedure An electronic system using four sets of photocells across the partition automatically counts movements through the partition and clocks the time spent in the light and dark compartments. Naive male mice or rats are placed into the cage. The animals are treated 30 min before the experiment with the test drugs or the vehicle intraperitoneally and are then observed for 10 min Groups of 6–8 animals are used for each dose Parameters measured : Time spent in both area(horizontal, vertical activity) movement time in both area number of transitions Anxiolytic effect : Statistically significant increase e in light movement time or number of transition 65

56. Soci al intera ction PURPOSE AND RATIONAL In an unfamiliar and brightly lit environment, the normal social interaction of rats (e.g. sniffing, nipping, grooming) is suppressed. Anxiolytics counteract this Suppression SOURCE OF ANXIETY : presence of an unfamiliar social partner PROCEDURE Male Sprague-Dawley rats (225–275 g body weight) are housed in groups of 5 animals The apparatus used for the detection of changes in social behaviour and exploratory behaviour consists of a Perspex open-topped box (51 × 51 cm and 20 cm high) with 17 × 17 cm marked areas on the floor. One hour prior to the test, two naive rats from separate housing cages are treated with the test compound orally. They are placed into the box (with 60 W bright illumination 17 cm above) and their behaviour is observed over a 10-min period by remote video recording.. 66

57. Purpose In the staircase paradigm, step-climbing is purported to reflect exploratory or locomotor activity, while rearing behaviour is an index of anxiety state. The number of rearings and steps climbed are recorded in a 5 min period. Parameter measured The number of steps climbed and the number of rears are counted over a 3-min period Anxiolytic effect The number of rearings and steps climbed are recorded in a 5 min period. The dissociation of these parameters is considered to be characteristic for anxiolytic drugs. The test was modified for rapid screening of anxiolytic activity in mice Staircase test 67

58. Organ anatomy Hormone prenatal development embryogeniss organogenesis MICRONUCLEUS DROSOPHILIA CHRMOSOMAL ABBERATIONS AMES Sex linked Assays

59. Marble burying Source of anxiety : Presence of an unfamiliar objects (potential source of danger) Parameters measured : N umber of buried marbles Anxiolytic effect: Statistically significant decrease in the number of buried marbles 70

60. Stress-indu ced h y pert h ermia (Handling order) Source of anxiety: Anticipatory anxiety, handling, new environment Parameters measured: Core temperature in the first three and last three animals in each group of 15 mice Anxiolytic effect: Body temperature of the last three animals are not significantly different from the first three 71

61. Vogel lick-c onfli ct Source of anxiety : Stressful situation (48 h water depr .) conflict between thirst and punishment after drinking Parameters measured : Number of accepted punishment (electric shock) Anxiolytic effect : Statistically significant increase in the accepted shocks 72

62. mCPP-indu ced anxiety Source of anxiety: Chemically induced anxiety Parameters measured: Time spent in both side (horizontal, vertical activity) frequency of motion number of transition Anxiolytic effect: Statistically significant increase parameters measured in the lit compartment or in number of transition 73 Methylchloro phenoxy propionic acid

63. Geller conflict paradigm PURPOSE AND RATIONALE Experimentally induced conflict by punishing food rewarded behaviour has been used to differentiate between various psychoactive drugs by Geller and Seifter EVALUATION The total number of lever presses during the conflict periods (CRF) and the non-conflict periods (VI) are counted An increase of lever presses in the conflict period is regarded as indication of an anti-anxiety effect, a decrease of lever presses in the non-conflict period as an indication for a sedative effect 74

64. CENTRAL ANALGESIC ACTIVITY IN VITRO MODELS: * 3 H-Naloxone binding assay * 3 H-Dihydromorphine binding to 𝜇 opiate receptors in rat brain * Receptor binding of nociceptin * Bioassays for nociception * Receptor binding of cannabinoids * Vanilloid receptor binding