Screening of Anxiolytic Agents

February 28, 2022 Rima Pharmacological Screening 0

  • Anxiety disorder are most common psychiatric disorder and can be categorized into different types including post-traumatic stress disorder (PTSD), generalized anxiety disorder, social anxiety disorder, obsessive compulsory disorder (OCD), panic disorder, agoraphobia, and specific phobias. According to data published by WHO in 2020, around 264 million people worldwide are suffering from anxiety disorder. 
  • Different kinds of drugs which can be used as anxiolytic include:

Screening of anxiolytic agents

Pre-clinical screening model of anxiolytic agents include in-vivo and in-vitro methods.

In-vitro methods for screening of anxiolytic agents

  1. In-vitro assay for GABAergic compounds: 3H-GABA receptor binding
  2. GABAA receptor binding
  3. GABAB receptor binding
  4. Benzodiazepine receptor: [3H]-flunitrazepam binding assay 
  5. Serotonin receptor binding
  6. Serotonin (5-HT1A) receptor: binding of [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin([3H]-DPAT)
  7. Histamine H3 receptor binding in brain

In-vivo methods for screening of anxiolytic agents

Effects on behavior

  1. Anti-anxiety test (light-dark model)
  2. Anticipatory anxiety in mice
  3. Social interaction in rats
  4. Elevated plus maze test
  5. Water maze test
  6. Staircase test
  7. Cork gnawing test in rats
  8. Distress vocalization in rat pups
  9. Schedule induced polydipsia in rats
  10. Four plate test in mice
  11. Footshock induced freezing behavior in rats
  12. Ultrasound induced defensive behavior in rats
  13. Novelty suppressed feeding
  14. Acoustic startle response in rats
  15. mCPP induced anxiety in rats
  16. Marmoset human threat test

Anti-aggressive activity

  1. Foot shock induced aggression
  2. Maternal aggression in rats
  3. Isolation induced aggression
  4. Resident intruder aggression test
  5. Water competition test
  6. Rage reaction in cats

Anti-convulsant activity

  1. Strychnine induced convulsions
  2. Isoniazid induced convulsions
  3. Picrotoxin induced convulsions
  4. Yohimbine induced convulsions
  5. Pentylenetetrazole (Metrazole) induced convulsions

Elevated Plus Maze test

Purpose and rationale

  • It is one of the most popular behavioral tests used for screening of anxiolytic agents in rodents. This method was initially developed for rats.
  • It is based on natural aversion of rodent for open and elevated area and natural aversion on their exploratory behavior in novel environment. Use of anxiolytic agents may increase number of entry and time spent in open arm and anxiogenic agents may do viceversa. 

Apparatus used

Figure- Non-automated elevated plus maze is used in screening of anxiolytic agents (Source- Lafayetteneuroscience.com)

  • The apparatus consists of plus shaped maze with two open arms and two closed arms of the same dimension. Two open arms are opposite to each other and so are closed arms. The maze is elevated from the ground.

Procedure

  • Before 10 days of experiment, rats are housed in pair. They are divided into groups of 6 each for standard drug and different doses of test agent. After 30 minutes of i.p. administration of drug or test agent, rat is placed in center of maze with face towards close arms. 
  • Test is conducted for 5 minutes during which number of entries into open or close arm, time spent in them, and total number of arm entries are observed. Usually, trained observer observes their movement and score either live or from a video image. 
  • Entry in an arm- Animal places all four paws into the arm.
  • It is conducted in soundproof room. 

Evaluation

  • Motor activity and open arm exploratory time are calculated. Percentage of entry and time spent in each arm is primary index of anxiety. 

Modification

  • Several modifications of derivatives of EPM have been developed like zero maze, T maze and unstable elevated exposed plus maze. 

Anti-anxiety test (Light-dark model)

Purpose and rationale

  • It is also commonly used murine model of anxiety which is based on innate aversion of rodent to bright light and aversion on exploratory behavior in novel environment. 

Apparatus

Figure- Light-dark apparatus for screening of anxiolytic (source- fischersci.com)

  • It consists of two interconnected chambers which vary in illumination (bright and dim), color (white and dark) and size (2:1). Under normal condition, mice tend to spend more time in dark area which may be decreased by anxiolytic agents. 
  • There are photocells across the apparatus which counts movement and time spent in light and dark chamber. 

Procedure

  • Mice or rats are divided into group of 6-7 animals each for standard drug and different doses of test agent. They are treated with standard and test agent and after 30 minutes, placed in cage. 
  • The test is continued for 10 minutes in which movement and time spent in dark and light chamber is measured. 

Evaluation

  • Following parameters are accessed:
    • Latency time for first passage from light to dark chambers
    • Number of transitions between two chambers
    • Movement and time spent in each compartment. 

References

  1. Griebel g etal. 50 years of hurdles and hope in anxiolytic drug discovery. Nat Rev Drug Discov. 2013 Sep; 12(9): 667–687.
  2. Garakani A etal. Pharmacotherapy of Anxiety disorders: Current and Emerging Treatment options. Front. Psychiatry. 2020: 11(595584); 1-21.
  3. Bourin M. Animal model for screening anxiolytic like drugs: a perspective. Dialogues Clin Neurosci. 2015 Sep; 17(3): 295–303.
  4. https://lafayetteneuroscience.com/products/no-auto-elevated-plus-mice
  5. https://www.slideshare.net/AdvaithaMv/screening-of-anxiolytics-44529278
  6. https://www.fishersci.com/shop/products/light-dark-box/10000355
  7. Lippincott’s Illustrated Reviews Pharmacology, 6th edition. 
  8. Drug Discovery and Evaluation, Pharmacological Assay. 2nd edition.