A. Norepinephrine potentiation:

In –vivo: - Dogs anaesthetized with pentobarbital - Artery is cannulated for measuring blood pressure. - Vein is cannulated for i.v. administration of the drug. - Submaximal dose of NE is determined. - Test drug is injected and control dose of NE is repeated. - Procedure is repeated foll. Increasing doses of the test drug.

in dose ( 0f antidep. . .adrenergic receptors predominantly.In – vitro: . .Therefore dose which inhibits uptake does not cause increase in contraction of rat vas deferens.Isolated rat vas deferens.)which inhibits uptake and those which block α – receptors.No diff.contains α.

B.v. .ED 50 value less than that for NE potentiation. . Phenythylamine antagonism: -TCAs also antagonize the pharmacological effects of phenythylamine or tyramine.Measuring dog blood pressure. .I. administration of phenythylamine produces a pressor effect. .antagonised by low doses of TCAs.

Eg: isolated guinea pig atria. . Which contains mainly β receptors.In – vitro: -α blocking effect may complicate the result so use of a prepn.

Inhibition of NE uptake into cerebral cortex slices of - Mice: Test drugs are administered 1 hr.A. 100mg of tissue is incubated for 5min.1 ml of incubation medium. later and radioactivity determined in 0. containing 0. . weighed and homogenised with 2ml ethanol containing 0.1nmole NE-7-3 H per ml. Slices blotted. before mice are killed and brain removed.5ml of alcohol extract using a liquid scintillation counter. All extracts are centrifuged 30min. in 2ml Krebshenseleit soln.

administration of NE-3H . .Grs of 6 mice given the test drug 5min. . Inhibition of NE uptake in mouse heart: . .. before i. of NE injection – hearts are pooled and extracted into perchloric acid. Content of NE determined.% blocking= (Rc-Ri)100 Rc-1 B.v. Data is expressed as percentage of NE in controls.Ratio is NE in slice to that in medium.Killed after 30 min.

p.p.hydroxydopamine 7mg/kg i.Hydroxydopamine induced cardiac NE depletion: -Test drug i. . ferricyanide as oxidant.C. hearts are pooled and homogenised in butanol (6ml per gm of tissue) . . ----> after 16 hrs. –---->6. Antagonism of 6.Results are expressed as the ratio of NE in the heart of drug treated mice to that in the untreated mice. -Clear centrifugates back extracted with HCl -- NE determined by trihydroxyindole method with pot.

.Decrease in dopaminergic activity in basal ganglialeads to a relative excess of cholinergic activity. .Enhancement of dopaminergic transmission – can restore the motor function partially. .degeneration of neurons in substantia nigra – decrease in dopaminergic innervation. .GENERAL CONSIDERATIONS: .Parkinson’s disease.Dopaminergic agonists or cholinergic antagonists.

ataxia. lacrimation and hypothermia. prior to 0.c. Tremorine and Oxotremorine antagonism: Purpose and Rationale: .Muscarinic agonists tremorine and oxotremorine induce Parkinsonism like signs such as tremor. or std. . before administration of the compd. 1 hr. salivation. Measure rectal temp. . Procedure: .5 mg/ kg oxotremorine s. spasticity.Grs of 6-10 male mice weighing 18-22g. These signs are antagonized by anticholinergic drugs.IN – VIVO METHODS: 1. ( basal value) and 1.Dosed orally with the test comp. 2 & 3hr after oxotremorine.

are expressed as percentage of the number of the control gr.Scores for both symptoms. . periods are summarized. of body temp.Score tremor after oxotremorine in 10 sec obs. The numbers in the treated gr. at the 3 obs. after 1.Scores for all animals in each gr.Salivation and lacrimation are scored 15 and 30 min after oxotremorine. basal values.. Evaluation: Hypothermia. Tremor. 2 & 3h vs. period every 15min. for 1 hr.Diff. Salivation and lacrimation. .

MPTP 10.18 mg/kg i.Parkinsonism like disorder.reversed by L.v. .dopa. Procedure: .N-MPTP has been shown to cause symptoms of Parkinson’s disease .8 adult Rhesus monkey weighing 5-8 kg ----N.Given to primates – syndrome that resembles parkinson’s disease. .2. MPTP model in monkeys: Purpose and rationale: .8 days . for 5.

3.abnormal .absent .reduced 2.17( maximum severity) that assesses: .abnormal trunk .flexed posture .Posture: .Evaluation: Severity Rated on a scale of 0 (normal).0 – normal 1.Movement: 0.sleepy -Checking movements: 0 – present 1.abnormal trunk .normal 1.2.abnormal trunk and tail .reduced 2.Attention and blinking: 0 – normal 1. tail and limbs 4.

catatonia and immobility. rigidity.-Balance and Coordination: 0.Depletion of central catecholamines store.normal 1.Sedative effect foll.impaired 2 – unstable 4 –falls -Reactions: 0 –normal 1. . Reserpine antagonism: . . hypokinesia. by signs like ptosis. 3. This can be antagonized by dopamine agonists.reduced -Vocalization: 0 – normal 1 – reduced 2 – absent.

Rearing and grooming episodes are registered by an experienced observer. with 5mg/kg reserpine and tested after 24hr. .30min. . . .Locomotor activity and grooming scores of drug treated animal compared with controls using ANOVA.Injected i.Procedure: . are injected.animals placed singly on floor of a perspex container placed upon Panlab proximity sensor unit. .p.Mice of either sex weighing 20 – 25g are used. prior to the obs. Evaluation: .Horizontal movements are recorded for 10min. test compds. .

250g .The rats rotate towards the opposite side when dopamine agonists and l. .lesion of the dopaminergic nigrostriatal pathway in the rat by neurotoxin 6.Male Wistar rats weighing 200. antagonists Procedure: .can be used for screening dopamine agonists. Circling behaviour in nigrostriatal lesioned rats: Purpose and rationale: . .dopa is given.hydroxydopamine induces hypersensitivity of the postsynaptic receptors in the striatum of the lesioned side. .4.

.rate of 1γ/L/min. Care is taken not to lesion the meninges. Allowed time for recovery and development of lesion..Anaesthetised with pentobarbital.Head is placed in a streotaxic device. Specially constructed plastic spheres serve as test chambers. a sagittal cut is - - - made in the skin and a 2mm wide hole is drilled . 8 microgm of 6 OHDA injected in 4 γ/L of saline. Wound is closed after the intracranial injection. A 30 gauge stainless steel cannula connected to a Hamilton syringe is aimed at the anterior zona compacta of the substantia nigra. .

Test compounds are given i. Evaluation: .p or s.Control value for ipsilateral turning-2. and animals placed in a circling chamber. for 1 0r 2 hrs. period.c. . . .Number of full turns ( ipsilateral or contralateral) are recorded every 15 min.Change of drug turns from control turns is recorded.Control value for contralateral turning apomorphine 1mg/kg ..5mg/kg damphetamine. . Circling is recorded over a 1 hr. Using various doses ED50 value can be calculated.

8 week old SD rats anesthetized.Lesioned by injection of 6.7 days after behavioural testing is performed. .% swing to each side.Male . . .5cm up.Held 2.5. .Bias – 70% or higher. . . .swing recorded.Mounted in a stereotaxic frame.At 30 and 45s right biased swing of 70 % or higher cf to normal rats. Elevated body swing test: Purpose: To study the asymmetrical motor behaviour.hydroxydopamine. Procedure: . .

Skilled paw reaching in rats: Purpose: -Injection of 6-OHdopamine results in impairment of paw reaching on both sides. Procedure: Apparatus: .Double staircase.Clear perspex chamber.each step contains a small well & 2 45mg saccharin flavored pellets are placed in each well. . A narrower compartment is connected to the chamber with a central platform running along its length. .6.

.w.4 weeks training – once each day for 10-15min.Familiarized with saccharin flavoured pellets.No.Food deprived.85% b. . conc.9% saline and 0. .Learning Procedure: . -1 i Lesions: . 4µg/µl 0.Lesioned by 6-OHDA into the medial forebrain bundle ( 1.deprived. .5 µl.Paw used: +1 c . .Missed pellets. .01% ascorbic acid). of pellets eaten – rat’s success in grasping and retrieving. . cf to non.lack of sensorimotor coordination.No of pellets on platform-index of attempt to reach and how far rat can reach.

Animals injected with test drug or saline i. Drug treatment: . as independent and weeks as dependent measure.p.5.Sham treatment.7 & 8 weeks after 6-OHDA lesion. 30 min. . Evaluation: -Test sessions 4. ..only saline and ascorbic acid. before 6-OHDA and 24 hr thereafter.Success. attempts and sensorimotor coordination – 2way ANOVA – grs.

7.Tests monitoring initiation time. Experimental setup: . stepping time and step length are performed on a wooden ramp.Unilateral model of akinesia. . . Stepping test in rats: Purpose and rationale: .marked and long lasting impairment in initiation of stepping movements with contralateral paw – can be ameliorated by the application of drugs. Procedure: Female SD rats -2 stereotaxic injections of 6-OHDA into dopamine pathway.6-OHDA lesion.

time to initiate a movement. step length. time for initiating movement with the other noted – 180sec cut off. time to cover a set dist.Hind part slightly raised. . with each forelimb.First 3 days familiarize with grip. .Smooth surfaced table is used for measuring adjusted steps. stepping time and step length: .2 parts: 1st. one forelimb fixed.2nd.2 tests per day – for 3 consecutive days. . .mean of 6 taken. Initiation time.2 days trained to run on the ramp.Next 1.initiation of adjusting steps when moved sideways.. . .

Test is repeated twice each day. repeated twice. of adjusting steps counted.total length of ramp by no. . . by left paw forehand.Step length.Sequence is right paw. .Sequence is right paw forehand and back hand foll. left paw..Stepping time – from movement initiation until the rat reaches the home cage. of steps.The no . . . and back hand. . Adjusting steps: -Rat is moved slowly sideways by the experimenter.Contralateral paw drags passively when rat is moved forehandwhile ipsilateral paw performs stepping .

Stepping tests are repeated as baseline weekly after the 6-OHDA lesion.Various drugs can be evaluated in weekly intervals. . Evaluation: .Drug application: .Drug tests are administered during 1 day only.Results are expressed as SEM. For statistical evaluation the data are subjected to one way ANOVA. . .

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