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Depression symptoms indicate the Patho -physiology of CNS

Depression symptoms indicate the Patho -physiology of CNS. Heterogeneous disorder ( 5-6% population depressed at any given moment) 10% people – during their life time Function of the individual deteriorate the quality of life. Characterised:

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Depression symptoms indicate the Patho -physiology of CNS

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  1. Depression symptoms indicate the Patho-physiology of CNS • Heterogeneous disorder ( 5-6% population depressed at any given moment) • 10% people – during their life time • Function of the individual • deteriorate the quality of life Characterised: Apathy, low self esteem, insomnia, personal neglect, loss of appetite, loss of libido, pessimism, lack of motivation

  2. Cerebrum develops from Telencephalon hippocampus, amygdala, anterior thalamic nuclei, septum, limbic cortex and fornix, Abnormalities in in forebrain is responsible for negative thought emotion, behavior, motivation, long term memory, and olfaction

  3. Hypothalamus and Pituitary also may play important role in depression as they release hormone

  4. Classification of depression Major Depression Exogenous Depression: reactive or secondary depression. associated with a life crisis ‘event’ Endogenous Depression: genetical- biochemical disorder. Inability to experience ordinary pleasure. Not associated with any trigger Depressive Syndromes : Unipolar and Bipolar Affective Disorder (manic depression) • People with this type of illness change back and forth between periods of depression and periods of mania (an extreme high). • Symptoms of mania may include: • Less need for sleep • Overconfidence • Racing thoughts • Reckless behavior • Increased energy • Mood changes are usually gradual, but can be sudden

  5. Not been fully understood yet How ever the development of antidepressant drugs associated with Nor epinephrine Serotonin & Dopamine systems

  6. G-protein couple receptor α ( 1 ABD), (2ABC) Β (1,2,3) Dopaminergirc receptor D (1, 2,3,4,5) 2,34 decreased the level of cAMP, increased K+ channels 1, 5 increased cAMP Serotonin receptor 5HT 1(1 ABDEFP) 5HT 2(ABC) 5HT 3 5HT 4 5HT 5 (AB) 5HT 6, 7 Following are the receptor important in depression 5HT 1A 5HT 2A 5HT 6, 7 D2

  7. Metabolic pathway of 5HT and norepinephrin

  8. Amine theory 1950 after introduction of reserpine it is evident that the drug those used for treatment of hypertension or schizophrenia may induce depression. Pharmacological study reveal that reserpine inhibit storage of amine neurotransmitter such as serotonin and nor-epinephrine in the vesicles of presynaptic nerve ending Reserpine deplete- store of amine neurotransmitters such as serotonin and norepinephrine

  9. Treatment approach Block the reuptake pump, such action help in temporary stay of neurotransmitter (serotonin and norepinephrine) at the postsynaptic ending and facilitate neurotransmission through corresponding receptors. a. specific serotonin inhibition b. Non specific serotonin inhibition 2. MAO Inhibitor blocks major degradation pathway of neurotransmitter which permit more amount of neurotransmitter to accumulate and store at the presynaptic side. Tricyclic antidepressant Amitryptaline Nortryptaline Protryptaline Imepramine Desipramine Clopramine Trimipramine Doxepine Second and third generation Amoxipine Bupripion Meprotiline Mirtazapine Nefazodone Trazodon Venlafexine Selective 5HT reuptake Inhibitor Citalopram Esitalopram Fluoxetine Fluvoxamine Sertraline MAO inhibitor Phenelzine Tranylcypramine

  10. Preclinical Evaluation of antidepressant drugs Invivo Test Locomotors activity Open field Close field 2. Forced swim test 3. Rotarod performance test 4. Elevated Plus maze Test 5. Lower Lip retraction 6. Head twitching Response Invitro test 1. Radioligand binding Assay 2. Enzymatic fluromatric assessment of cAMP/Adenylecyclase activity

  11. 1. a. Open Field Locomotors Activity • Perform with rodent, serve as good preliminary test to determine motor deficit and anxiety • Parameters are measured • a. amount of distance traveled • b. Observation of various horizontal, vertical and stereotype behavior. • c. grooming (removal of dirt) • d. Rearing

  12. Each Open Field monitor consists of sets of 16 light beams arrays in the horizontal X and Y axes. The hardware detects beams broken by the animal so that the software can determine the location of the rodent within the cage. Additionally, each cage contains Start/Stop buttons on each side. These buttons may be used to start and stop experiments for individual rodents when the researcher is not in proximity to the PC. Each monitor consists of a node which is physically attached to the cage frame and has a small unobtrusive profile. The node's simple connections to the PC and sensors make set up effortless. Each node captures 100 frames per second from its respective monitor and supports up to 12 sensor pairs. Sensor pairs have 16 infrared light beams that traverse the animal monitor and can be arranged in an unlimited number of configurations. Only three pairs are needed to track an animal in the horizontal plane and record rearing behavior. Additional sensor pairs can be used to reveal hole poke behavior, behavior in a multi level cage etc.

  13. b. Closed field Locomotors activity measurements Using Opto M3 Multichannel activity monitor- Swiss albino mice were individually placed in plastic cages and then the crossing of each individual channel (ambulation) were counted from 2 to 6 minutes.

  14. 2 Forced Swim Test of Mice (Porsolf et. al) A new behavioral method for inducing a depressed state in mice. One hour after I. P administration of anti-depressant drugs mice were dropped into the cylinder and left for 6 minutes. The immobility of last 4 minutes is counted. Cylinder : Height- 25, diameter-10cm, depth of water- 6 cm , temp-21-23 C. Antidepressant drug will reduce the immobility

  15. 3. Rotarod Performance Test Rotarod performance test is based on a rotating rod with forced motor activity being applied, usually by a rodent. In the test , a rodent is placed on a horizontally oriented rotating cylinder (rod) suspend above a cage floor which is low enough not to injure the animals but high enough to induce avoidance of fall. Rodent naturally try to stay on the rotating cylinder and avoid falling to the ground. The length of the time that a given animal stay on this rotating rod is a measure of their balance, coordination, physical condition and motor planning Animal use: Hamster Gerbil Mouse Scientific uses: Used to asses the motor function. Drugs such as Anti-depressant Anxiolytic( Benz) Serotonin agonist and antagonist

  16. Reliability of the Test depends on: 0. Size of the cylinder 0. Speed of the cylinder 0. Composition material of the surface 0.Amount of practice /training given to the animals. Specification: Animal diameter width number of animal Mice 30mm 60mm 4 Rats 60mm 85mm 4 Falling distance Mouse-top edge drum – top edge floor grid 14.7/15.8 cm Rat -- top edge drum- top edge floor grid- 29.5/27.2 cm 3. Rotarod Performance Test Speed Constant mode- 1-60rpm Accelerating mode- same as above Shock intensity- 0.1-3mA Shock length-- 0.1 10 Sec

  17. 4.Elevated Plus Maze Test (EPM): ( File and coworker) Widely used behavioral assay for rodent and validated to assess the anti-anxiety effects of pharmacological agents. Initial Y shape maze were modified to four arms ( two open and two enclosed) that are arrange to form a plus shaped. (Handly and Mitani) These author described the assessment test of anxiety behavior of rodent by using the ratio of time spent on the open arms to closed arms. Y

  18. Unlike other behavioral assays used to assess anxiety responses that rely upon the presentation of noxious stimuli ( electric shock, food /water deprivation, loud noise, exposure to predator etc) that typically produced conditioned response, the elevated maze relies upon rodent proclivity towards dark , enclosed space (approach) and open space (avoidance) and unconditioned fear of height/open space avoidance. Rodents are usually placed in the intersection of the four arms of elevated maze and their behavior is typically recorded for 5 minutes. Other ethiologicalmeasures that can be observed in the rodents in the maze are Number of rearing Head dips defection Freezing or stretched. Uses: Evaluation of anxiolytic drugs such as Benz 2. anti-depressant 3. Drug of abuse 4. Hormone Corticoid, adrenalin, estrogen, progestin and androgens

  19. Utility of a single test session in the elevated plus maze test has decay effects. Therefore a period of three week required to provide animal in-between the test. Handling of animals before testing: Experience with handling , stress or injections can alter behavioral response of rodents in the elevated plus maze test. It is important to ensure that in the experiments using the elevated plus maze, handling of rodents and any experience with prior stress, particularly before testing is constant across animals and treatment groups. Specification: Automated device produced by Campden Instrument LTD. Two open arms 50X10cm Two enclosed arms- 50X 10X (30h) cm Height from the floor grid- 50cm Experimental requirements: The plus maze place in the dark room and center of the apparatus required to illuminate with 25 W electric bulb hanging above 100cm. This apparatus may connected to PC to facilitate the counting

  20. 5. Lower Lip Retraction (LLR) Seretonergic compounds such as 8-OH-DPAT Buspiron Ipsapiron or RH-24969 can induce LLR. LLR can prevent by 5HT antagonist At least 10-20 animal are required for this exp. This experiment is based on the fact that 5HT1A agonist can affect the musculature of lower Lips in rats, a symptom which is referred to the lower lip retraction. Animals (Wister Rats) weighing about 200-250gm prior to experiments should be housed as per guideline in clean cage in a group maintaining 12 h day-night cycle. All group of animal should be provided with free access of food and water. Drugs (5HT1A agonist) are injected to animals S.C . Within an hr after injection the lower lip of the rats are retracted so that the lower incisors become completely visible. The extent of lower lip retraction depend upon dose and time. Usual observation time are 15, 30 and 45 minutes after S.C injection. Best time for experiments 9- 14 h.

  21. Score Both seretonergic agonist and their interaction can be observed by this method.

  22. Head Twitching Response (HTR) using DOI DOI- 1(2,5 dimethoxy 4- iodophenyl) 2 amino propane is 5HT2A/2C agonist) Head Twitching behavior of animal is a distinctive behavior. It is an easily monitored function and usually mistaken with other head shake of jerks. DOI causes head twitching via activation of serotonin receptors and is measured in glass container containing sawdust (12cm in diameter). Five minutes after IP injection of DOI (5mg/kg) the number of the head twitches are counted for five minutes.

  23. In Vitro Pharmacological Test 1. Radioligand Binding Assay Total binding= specific binding + non specific binding

  24. Sample are required to collect from the respective site in the isotonic phosphate buffer ( 50mMNa2/K phosphate buffer containing 37.8mM Na2HPO4 and 12.2 mM KH2PO4), PH -7.4 and homogenized using polytron homogenizer. The homogenate centrifuges at suitable (18000rmp) for 30 minutes at 4 0C. The pellets are resuspended in Ice cold buffer (50 mM Na2/K- phosphate) and served as receptor sample for radioligand binding assay. In case of HEK 293 cells , are lysed using hypertonic buffer and samples are used for radioligand binding assay. Full length of 5HT6, 7 receptor gene can be obtained from PCR from human brain cDNA library, sub-cloned into a mammalian vector, PcDNA (Invitrogent) and stably expressed in HEK 293 cells.

  25. 2. Measurement of cAMP production/ Adenylcyclase activity Steps for reaction Conversion of cAMP 2. Enzymatic destruction of non cyclic adenosine nucleotide and phosphorylation 3. Conversion of cAMP into ATP 4. Enzymatic amplification of ATP 5. Conversion of F6P to NADPH

  26. References: 1. Katzung , basic and clinical Pharmacology 2. Pharmacology by Rang and Dale 3. 4.

  27. 5 6

  28. 7 8 9

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