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ANXIETY It is an emotional state, unpleasant in nature, associated with un easiness, discomfort and concern of fear about some defined

or undefined future threat. Causes Biological Low levels of GABA, a neurotransmitter that reduces activity in the central nervous system, contribute to anxiety. A number of anxiolytics achieve their effect by modulating the GABA receptors. Selective serotonin reuptake inhibitors, the drugs most commonly used to treat depression, are frequently considered as a first line treatment for anxiety disorders. The effects of SSRIs in alleviating anxiety may result from a direct action on GABA neurons rather than as a secondary consequence of mood improvement. Severe anxiety and depression can be induced by sustained alcohol abuse,which in most cases abates with prolonged abstinence. Even moderate, sustained alcohol use may increase anxiety and depression levels in some individuals Amygdala The amygdala is central to the processing of fear and anxiety, and its function may be disrupted in anxiety disorders. Stress Anxiety disorders can arise in response to life stresses such as financial worries or chronic physical illness.

Screening of Anti anxiety drugs Invitro Assay for GABAergic compounds: - GABAa receptor binding - GABAb receptr binding Assay of histamine: H3 receptor binding in brain Serotonin receptor binding Benzodiazepine receptor: [3H]-flunitrazepam binding assay

Invivo 1. Exteroceptive stimuli models 2. Interoceptive stimuli models

1. Exteroceptive models

I. Response independent: a) Novel environment: Open field test Staircase exploration Y maze test Elevated plus maze test b) In escapable aversive events/conditioned aversive producers Conditioned emotional response Positioned startle Conditioned acceleration

II. Response contingent presentation of stimuli a) Behavioral suppression by aversive stimuli Geller-siefters conflict test Vogels conflict test 2. Interoceptive models: Electric stimulation of brain. Pharmacological manipulations (drug discrimination test). Caffeine induced anxiety Cocaine induced anxiety

Aggression / anxiogenesis Foot-shock induced anxiety Drug induced anxiety

1. Benzodiazepine receptor: [3H]-flunitrazepam binding assay PURPOSE AND RATIONALE Experiments using 3H-diazepam or 3H-flunitrazepam have demonstrated specific binding sites in CNS membrane preparations that satisfy the criteria for pharmacological receptors, e.g. saturability, reversibility, stereoselectivity and significant correlation with in vivo activities of the drugs in this class. Heterogeneity of BZD receptors has been reported. There are four classes of BZD and non-BZD high affinity ligands for BZD recognition sites associated with GABAA receptors: The first class (e.g. diazepam, flunitrazepam, alprazolam) facilitates the action of GABA, increasing the opening frequency of Cl channels. These ligands are called full positive allosteric modulators, or full agonists. A second class of ligands, which includes the -carbolines, can decrease the opening frequency of Cl channels. These ligands are known as full negative allosteric modulators, or full inverse agonists. A third class (e.g. flumazenil) binds with high affinity to BZD recognition sites, but it can also prevent the GABA modulations elicited by positive or negative allosteric modulators; this class is called a modulator antagonist. A fourth class of ligands for BZD recognition sites is known to elicit either partial amplification or partial attenuation of GABA action at various GABAA receptors, and comprises the class called partial positive and partial negative allosteric modulators or partial agonists and partial inverse agonists, respectively. The names 1, 2, and 3-receptor subtypes have been proposed to replace the nomenclature of BZD BZ1, BZ2, and BZp receptors.

PROCEDURE Male Wistar rats are decapitated and the brains rapidly removed. The cerebral cortices are removed, weighed and homogenized with a Potter-Elvejhem homogenizer in 20 volumes of ice-cold 0.32 M sucrose. This homogenate is centrifuged at 1 000 g for 10 min. the pellet is discarded and the supernatant is centrifuged at 30 000 g for 20 min. The resulting membrane pellet is resuspended in 40 volumes of 0.05 M Tris buffer, pH 6.9. The tubes containing 3H-flunitrazepam, buffer, drugs and H2O are incubated at 04 C in an ice bath. A 300 l aliquot of the tissue suspension is added to the tubes at 10-s intervals. The timer is started with the addition of the mixture to the first tube. The tubes are then incubated at 04 C for 20 min and the assay stopped by vacuum filtration through Whatman GF/B filters. This step is performed at

10-s intervals. Each filter is immediately rinsed with three 5-ml washes of ice-cold buffer, pH 6.9. The filters are counted in 10 ml of liquid scintillation counting cocktail. EVALUATION Specific binding is defined as the difference between total binding and binding in the presence of clonazepam. Specific binding is approximately 97% of total ligand binding. The percent inhibition at each drug concentration is the mean of triplicate determinations. IC50 calculations are performed using log-probit analyses.

2. Strychnine-induced-convulsions PURPOSE AND RATIONALE The convulsing action of strychnine is due to interference with postsynaptic inhibition mediated by glycine. Glycine is an important inhibitory transmitter to motoneurons and interneurons in the spinal cord, and strychnine acts as a selective, competitive antagonist to block the inhibitory effects of glycine at all glycine receptors. Strychnine-sensitive postsynaptic inhibition in higher centers of the CNS is also mediated by glycine. Compounds, which reverse the action of strychnine, have been shown to have anxiolytic properties. PROCEDURE Groups of 10 mice of either sex with a weight between 18 and 22 g are used. They are treated orally with the test compound or the standard (e.g. diazepam 5 mg/kg). One hour later the mice are injected with 2mg/kg strychnine nitrate i.p. The time until occurrence of tonic extensor convulsions and death is noted during a 1 h period. With this dose of strychnine convulsions are observed in 80% of the controls.

EVALUATION ED50-values are calculated using various doses taking the percentage of the controls as 100%. For time-response curves the interval between treatment and strychnine injection varies from 30 to 120 min.

3. Elevated plus maze test PURPOSE AND RATIONALE- Out of many possibilities to modify maze tests (e.g. water maze, the Y-mace, the radial maze, and the elevated plus maze have found acceptance in many laboratories. The test has been proposed for selective identification of anxiolytic and anxiogenic drugs. Anxiolytic compounds, by decreasing anxiety, increase the open arm exploration time; anxiogenic compounds have the

opposite effect. PROCEDURE- The plus-maze consists of two open arms, 50 10 40 cm, and two enclosed arms, 50 10 40 cm, with an open roof, arranged so that the two open arms are opposite to each other. The maze is elevated to a height of 50 cm. The rats (200250 g body weight) are housed in pairs for 10 days prior to testing in the apparatus. During this time the rats are handled by the investigator on alternate days to reduce stress. Groups consist of 6 rats for each dose. Thirty min after i.p. Administration of the test drug or the standard, the rat is placed in the center of the maze, facing one of the enclosed arms During a 5 min test period the following measures are taken: the number of entries into and time spent in the open and enclosed arms; the total number of arm entries. The procedure is conducted preferably in a sound attenuated room, with observations made from an adjacent room via a remote control TV camera. EVALUATION Motor activity and open arm exploratory time are registered. The values of treated groups are expressed as percentage of controls. Benzodiazepines and valproate decrease motor activity and increase open arm exploratory time.