Depression symptoms indicate the Patho- physiology of CNS Heterogeneous disorder ( 5-6% population...
-
Upload
clarissa-morris -
Category
Documents
-
view
218 -
download
1
Transcript of Depression symptoms indicate the Patho- physiology of CNS Heterogeneous disorder ( 5-6% population...
Depression symptoms indicate the Patho-physiology of CNS
Heterogeneous disorder ( 5-6% population depressed at any given moment)10% people – during their life timeFunction 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
hippocampus, amygdala,
anterior thalamic nuclei, septum, limbic cortex
and fornix, emotion, behavior,
motivation, long term memory, and olfaction
Abnormalities in in forebrain is
responsible for negative thought
Cerebrum develops from Telencephalon
Hypothalamus and Pituitary also may play important role in depression as they release hormone
Classification of depressionMajor DepressionExogenous 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 sleepOverconfidenceRacing thoughtsReckless behaviorIncreased energyMood changes are usually gradual, but can be sudden
Not been fully understood yet
How ever the development of antidepressant drugs associated with
Nor epinephrineSerotonin&Dopamine systems
G-protein couple receptor
α ( 1 ABD), (2ABC)Β (1,2,3)
Dopaminergirc receptorD (1, 2,3,4,5) 2,34 decreased the level of cAMP, increased K+ channels1, 5 increased cAMP
Serotonin receptor5HT 1(1 ABDEFP)5HT 2(ABC)5HT 3 5HT 45HT 5 (AB)5HT 6, 7
Following are the receptor important in depression5HT 1A5HT 2A5HT 6, 7D2
Metabolic pathway of 5HT and norepinephrin
Amine theory1950 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
Treatment approach1. 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 antidepressantAmitryptalineNortryptalineProtryptalineImepramineDesipramineClopramineTrimipramineDoxepine
Second and third generationAmoxipineBupripionMeprotilineMirtazapineNefazodoneTrazodonVenlafexine
Selective 5HT reuptake Inhibitor
CitalopramEsitalopramFluoxetine
FluvoxamineSertraline
MAO inhibitorPhenelzineTranylcypramine
Preclinical Evaluation of antidepressant drugs
Invivo Test1. Locomotors activity Open field Close field2. Forced swim test3. Rotarod performance test4. Elevated Plus maze Test5. Lower Lip retraction 6. Head twitching Response
Invitro test1. Radioligand binding Assay2. Enzymatic fluromatric assessment of cAMP/Adenyle cyclase activity
1. a. Open Field Locomotors ActivityPerform 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
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.
b. Closed field Locomotors activity measurementsUsing 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.
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
2
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: HamsterGerbilMouse
Scientific uses:Used to asses the motor function.
Drugs such asAnti-depressant
Anxiolytic ( Benz)Serotonin agonist and
antagonist
SpeedConstant mode- 1-60rpmAccelerating mode- same as aboveShock intensity- 0.1-3mAShock length-- 0.1 10 Sec
Reliability of the Test depends on:0. Size of the cylinder0. Speed of the cylinder0. Composition material of the surface0. Amount of practice /training given to the animals.
Specification:Animal diameter width number of animalMice 30mm 60mm 4Rats 60mm 85mm 4
Falling distanceMouse-top edge drum – top edge floor grid 14.7/15.8 cmRat -- top edge drum- top edge floor grid- 29.5/27.2 cm
3. Rotarod Performance Test
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
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 ethiological measures that can be observed in the rodents in the maze are a. Number of rearingb. Head dipsc. defectiond. Freezing or stretched.
Uses:1. Evaluation of
anxiolytic drugs such as Benz
2. anti-depressant
3. Drug of abuse
4. Hormone
Corticoid, adrenalin, estrogen, progestin and androgens
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 50X10cmTwo enclosed arms- 50X 10X (30h) cmHeight 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
5. Lower Lip Retraction (LLR)
Seretonergic compounds such as8-OH-DPATBuspironIpsapiron 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.
0 0.5 1
Lower incisorVisibility
not or hardly visible
Partially visible Completely visible
Score
Both seretonergic agonist and their interaction can be observed by this method.
Head Twitching Response (HTR) using DOI
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.
DOI- 1(2,5 dimethoxy 4- iodophenyl) 2 amino propane is 5HT2A/2C agonist)
In Vitro Pharmacological Test
Receptors name Radioligands Site of the brain
5HT1A (3H)-8 OH-DPAT Rat hippocampus
5HT2A (3H)-Ketanserin Rat cortex
5HT6 (3H)-LSD HEK293
5HT7 (3H)-LSD HEK 293
D2 (3H)-Spiperon Rat striatum
1. Radioligand Binding Assay
Total binding= specific binding + non specific binding
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.
2. Measurement of cAMP production/ Adenyl cyclase activity
Steps for reaction1. Conversion of cAMP
2. Enzymatic destruction of non cyclic adenosine nucleotide and phosphorylation3. Conversion of cAMP into ATP4. Enzymatic amplification of ATP5. Conversion of F6P to NADPH
References:1. Katzung , basic and clinical Pharmacology2. Pharmacology by Rang and Dale
3.
4.
5
6
7
8
9