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

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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

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