The first antidepressant drugs were detected by scerendipity in clinical trials. Iproniazid was developed for the treatment of tuberculosis.
During clinical investigation of phenothiazine analogs as neuroleptics, imipramine was found to be relatively ineffective in agitated psychotic patients but showed remarkable benefit in depressed patient. Pre-clinical and clinical evidence indicate that an enhancement of 5-HT-mediated neuro-transmission underlie the therapeutic effect of most antidepressant treatments .
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| Male wistar albino rats |
IN VITRO METHODS
1) Inhibition of [3H]-Norepinephrine Uptake in Rat Brain Synaptosomes
PURPOSE AND RATIONAL: Inhibition of [3H]-Norepinephrine Uptake in Rat Brain Synaptosomes
Most important physiological process for removing and in activating norepinephrine in the synaptic cleft. Inhibited by cocaine certain phenylethylamines and antidepressants. In brain ,The hypothalamus shows the highest level and greatest uptake of noradrenaline. Therefore, this region is used for testing potential antidepressant drugs.
PROCEDURE: TISSUE PREPARATION
Male Wistar rats decapitated and brains rapidly removed.
Hypothalamic region is weighed, homogenized in 9 volumes of ice cold 0.32 sucrose sol using a Potter-Elvejhem homogenize.
This homogenate centrifuged at 1000g at 0–4 °C for 10 mins. Supernatant is decanted and used for experiment.
ASSAY: Inhibition of [3H]-Norepinephrine Uptake in Rat Brain Synaptosomes
200 μl of tissue suspension are incubated with 800 μl 62.5 nM 3H-norepinephrine in Krebs-Henseleit bicarbonate buffer.
20 μl of the appropriate drug concentration (or the vehicle) at 37 °C under a 95% O2/5% CO2 atmosphere for 5 min.
For each assay, 3 tubes are incubated with 20 μl of vehicle at 0 °C in an ice bath. After incubation all tubes are immediately centrifuged at 4000 g for 10 min.
The supernatant fluid is aspirated and the pellets dissolved adding 1 ml of solubilizer (Triton X-100 + 50% ethanol, 1 : 4).
The tubes are vigorously shaken, decanted into scintillation vials, and counted in 10 ml of liquid scintillation cocktail.
Active uptake is the difference between cpm at 37 °C and 0 °C.
EVALUATION: Inhibition of [3H]-Norepinephrine Uptake in Rat Brain Synaptosomes
- The percent inhibition at each drug concentration is the mean of 3 determinations.
- IC50 values are derived from log-probit analysis.
- IC50 values for the standard drugs desipramine and nortriptyline are around 20 nM.
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| Isolated brain of wistar albino rat |
2) Inhibition of [3H]-Dopamine Uptake in Rat Striatal Synaptosomes
PURPOSE AND RATIONALE:
High affinity, saturable, temperature and sodium-dependent transport of 3H-dopamine has been observed from different brain regions. Striata has a high content of dopamine and is suitable for uptake experiments.The 3H-dopamine uptake is inhibited by cocaine, certain phenylethylamines and antidepressants like nomifensine and bupropion, but not by tricyclic antidepressants.
The test can be used to characterize the mode of action of antidepressant drugs.
The test can be used to characterize the mode of action of antidepressant drugs.
PROCEDURE: TISSUE PREPARATION
Male Wistar rats are decapitated and the brains rapidly removed. Corpora striata are prepared, weighed and homogenized in 9 volumes of ice cold 0.32 M sucrose solution using a Potter-Elvejhem homogenizer. The homogenate is centrifuged at 1000 g at 0–4 °C for 10 min. The supernatant is decanted and used for the experiments.
ASSAY: Inhibition of [3H]-Dopamine Uptake in Rat Striatal Synaptosomes
100 μl of tissue suspension are mixed with 900 μl 55.5 nM 3H- dopamine solution in Krebs-Henseleit bicarbonate buffer and 20 μl of drug solution in appropriate concentration (or the vehicle as control). The tubes are incubated at 37 °C under a 95% O2/5% CO2 atmosphere for 5 min.
For each assay, 3 tubes are incubated with 20 μl of vehicle at 0 °C in an ice bath. After incubation all tubes are immediately centrifuged at 4000 g for 10 min. The supernatant fluid is aspirated and the pellets dissolved by adding 1 ml of solubilizer (Triton X-100 + 50% ethanol, 1 : 4).
The tubes are vigorously shaken, decanted into scintillation vials, and counted in 10 ml liquid scintillation counting cocktail. Active uptake is the difference between cpm at 37 °C and 0 °C.
EVALUATION: Inhibition of [3H]-Dopamine Uptake in Rat Striatal Synaptosomes
The percent inhibition at each drug concentration is the mean of 3 determinations. IC50 values are derived from log-probit analyses.
IC50 values for nomifensine are 460 nM, but >20 000 nM for tricycle antidepressants.
IN VIVO METHODS
1) Despair Swim Test
PURPOSE AND RATIONALE: Despair Swim Test
Behavioral despair was proposed as a model to test for antidepressant activity by Porsolt et al. (1977, 1978). It was suggested that mice or rats forced to swim in a restricted space from which they cannot escape are induced to a characteristic behavior of immobility. This behavior reflects a state of despair which can reduced by several agents which are therapeutically effective in human depression.
PROCEDURE: Despair Swim Test
Male Sprague- Dawley rats weighing 160–180 gr are used. They are brought to the laboratory at least one day before the experiment and are housed separately in Makrolon cages with free access to food and water. Naive rats are individually forced to swim inside a vertical Plexiglas cylinder (height: 40 cm; diameter: 18 cm, containing 15 cm of water maintained at 25 °C).
Rats placed in the cylinders for the first time are initially highly active, vigorously swimming in circles, trying to climb the wall or diving to the bottom. After 2–3 min activity begins to subside and to be interspersed with phases of immobility or floating of increasing length. After 5–6 min immobility reaches a plateau where the rats remain immobile for approximately 80% of the time. After 15 min in the water the rats are removed and allowed to dry in a heated enclosure (32 °C) before being returned to their home cages.
They are again placed in the cylinder 24 h later and the total duration of immobility is measured during a 5 min test.
Floating behavior during this 5 min period has been found to be reproducible in different groups of rats. An animal is judged to be immobile whenever it remains floating passively in the water in a slightly hunched but upright position, its nose just above the surface. Test drugs or standard are administered one hour prior to testing.
Since experiments with the standard drug (imipramine) showed that injections 1, 5 and 24 h prior the test gave the most stable results in reducing floating these times are chosen for the experiment.
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| Despair swim test |
EVALUATION: Despair Swim Test
Duration of immobility is measured in controls and animals treated with various doses of a test drug or standard. Antidepressant drugs, but also stimulants like amphetamine and caffeine, reduce duration of immobility. Dose-responses can be evaluated.
2)Tail Suspension Test in Mice
PURPOSE AND RATIONALE: Tail Suspension Test in Mice
The “tail suspension test” has been described by Steru et al. (1985) as a facile means of evaluating potential antidepressant. Clinically effective antidepressants reduce the immobility that mice display after active and unsuccessful attempts to escape when suspended by the tail.
PROCEDURE: Tail Suspension Test in Mice
Male Balb/cJ mice weighing 20–25 g are used preferentially. They are housed in plastic cages for at least 10 days prior to testing in a 12 h light cycle with food and water freely available. Animals are transported from the housing room to the testing area in their own cages and allowed to adapt to the new environment for 1 h before testing.
Groups of 10 animals are treated with the test compounds or the vehicle by intra-peritoneal injection 30 min prior to testing. For the test the mice are suspended on the edge of a shelf 58 cm above a table top by adhesive tape placed approximately 1 cm from the tip of the tail. The duration of immobility is recorded for a period of 5 min. Mice are considered immobile when they hang passively and completely motionless for at least 1 min.
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| Tail suspension test |
EVALUATION: Tail Suspension Test in Mice
The percentage of animals showing the passive behavior is counted and compared with vehicle treated controls. Using various doses, ED50 values can be calculated.
3) Muricide Behavior in Rats
PURPOSE AND RATIONALE: Muricide Behavior in Rats
Horovitz et al. (1965) described a selective inhibition of mouse-killing behavior in rat by antidepressants.The test can be used to evaluate antidepressants such as tricycles and MAO inhibitors.
PROCEDURE: Muricide Behavior in Rats
Male Sprague-Dawley rats (300–350 g) are isolated for 6 weeks in individual cages. They have access to food and water ad libitum. One mouse is placed into the rat’s cage. About 10 to 30% of rats kill the mouse by biting the animal through the cervical cord. Only rats consistently killing mice within 5 min after presentation are used for the test. The mice are removed 15 to 45 s after they have been killed in order to prevent the rats from eating them.
Drugs are injected i.p. to the rats before the test. Mice are presented 30, 60 and 120 min after drug administration.
EVALUATION: Muricide Behavior in Rats
Failure to kill a mouse within 5 min is considered inhibition of muricidal behavior. Performing dose-response experiments, the ED50 is defined as the dose which inhibits mouse killing in 50% of the rats.
4) Animal Model of Bipolar Disorder
PURPOSE AND RATIONALE: Animal Model of Bipolar Disorder
Bipolic disorder is a condition characterized by episodes of mania,depression and mood instability. Preclinical studies with animal models of mania and depression have been developed to evaluate the potencial efficacy of new psychotropics drugs.
The main problem is to find a model that mimics mood cyclicity,which is a hallmark of bipolar disorder. Cao and Pang and Arban et al described a rodent model of mania, in which hyperactivity by the combination of D-amphetamine and chlordiazepoxide.
PROCEDURE: Animal Model of Bipolar Disorder
Male CDI mice weighing 22-26g were housed at 21 +2 °C with 45% -75% humidity on a 12/12h light/dark cycle.
Test drugs were administered orally 60 min before test. D-amphetamine asulfate (1.25mg/kg i.p.) and chlordiazepoxide(6.25mg/kg i.p.) were injected 30 min before sesssion.
Locomotar activity was recorded by Digiscan Analyzer. Animals were placed in Plexigals cages equipped with 48 photocells and the distance traveled(cm) by each mouse over a 30 min period was determined.
EVALUATION: Animal Model of Bipolar Disorder
Statistical analysis was carried out using an ANOVA followed by Dunnett’s test. Results are expressed as mean+ SEM of the total distance traveled (in cm),with group size ranging from 9 to 12 mice.
Atrial Peptides: structure, function, pharmacology, modulation, antagonist and agonist .
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