Methods in Psychopharmacology - Behavioral Phenotyping PDF

-OMICS, IMAGING, BEHAVIOR OTHER FANCY Tools in SHIT Neuroscien ce: Behavior Phenotypin g Dr. Lisa Robison  Behavioral Neuroscience...

-OMICS, IMAGING, BEHAVIOR OTHER FANCY Tools in SHIT Neuroscien ce: Behavior Phenotypin g Dr. Lisa Robison  Behavioral Neuroscience Behavioral neuroscience = study of the underlying neural or brain mechanisms that guide behavior “the raison d'etre of the CNS is to optimize the organism's ability to interact with its environment” (Schneiderman, 1984, p. 199) How does the brain support sensorimotor function, emotion, cognition, etc.? How is brain function (and behavior) influenced by genetics, drugs, disease states A hitchhiker's guide to behavioral analysis in laboratory rodents - Sousa - 2006 - Genes, Brain and Behav Why study animals? Scientific Method Experimental control Invasive techniques Similar brain regions/neurochemical systems Determine behavioral output of brain function Developing animal models of psychiatric disorders Preclinical testing of drugs/other interventions – enables use of a large number of subjects Genetic models Animal models in behavioral neuroscience Rodents – rats and mice Things to consider: Size Depends on goals Genetic modifications Mice > Rats Handling Rats > Mice Social behavior Rats > Mice Cognitive function Rats > Mice Animal models in behavioral neuroscience General notes for behavior testing Calibration of behavioral assays increases reliability and replicability of results. Maximize differences between groups Manipulation of task difficulty Baseline performance vs. direction/strength of treatment Rodent sensory modality and function Performance on one task can aid in interpretation of others Battery! Think about motivating factors  Behavioral Tests & Respective Domains Motor Anxiety/Fear Learning and Memory function/activity Open field Novel object recognition Gait test Light/dark box test Beam walking test Elevated mazes Y-maze/T-maze tasks Radial arm maze Rotarod Depression/anhedonia Wire suspension Barnes maze Forced swim test test Water maze Sucrose preference Grip strength Fear conditioning test Open field Drug abuse Social Behavior Circadian activity liability/addiction Three-chamber social Nociception/Pain Locomotor sensitization interaction Von Frey fiber Conditioned place Aggression preference Hot plate & Tail flick Resident intruder test Self-administration  Motor function & Assessment General Coordination of precise neurological movements in order to tests perform an intended Gait action Balance Necessitates cooperation Coordination of the central nervous system (motor cortex, Dexterity basal ganglia, Strength cerebellum), the Locomotion & musculoskeletal system, ambulation as well as the sensory system Evaluating rodent motor functions: Which tests to choose? – S Tests to assess motor phenotype in mice: a user's guide (prin  Gait analysis: The Gait Test (Footprint test) A linear track with paper that leads to enclosed box. Animal walks down track after paws dipped in ink/paint.  Rotarod Rotating cylindrical rod Balance, motor coordination, physical condition assessed by: Latency to fall or max speed Measuring Motor Coordination in Mice | Protocol (jo  Beam walking test Beam walking test. (Feng et al. 2014) Measuring Motor Coordination in Mice | Protocol (jo  Wire Suspension Test (Wire hang) Measure: Latency to drop from a suspended wire Test for neuromuscular abnormalities of motor strength Grip Strength Test for neuromuscular abnormalities of motor strength Two versions Forelimb grip strength Four limb hangtime (Bonetto et al., 2015, IB The open field test One of the simplest behavior tests that can tell us a bit a rodent’s general locomotor activity levels and temperament (i.e. anxiety, willingness to explore). --Measure speed, distance **Helps to habituate to arena for other tests (e.g. novel object recognition, object place test) Square or circular arena can be used  Circadian activity Measurement of activity levels, usually in homecage over 24h Nocturnal Outputs are based on activity levels, but also provides info on circadian (Robison et al., 2017, Front Beh Neuro) rhythms (sleep/wake cycles) Amplitude of activity over the day Shifting of normal rhythms Homeostasis & Circadian (bvu.edu) Behavioral Tests for Pain/Nociceptio n Von Frey Test Hot Plate Tail Flick Methods Used to Evaluate Pain Behaviors in Roden Pain/Nociception Mechanical Sensitivity: Von Frey Fibers Calibrated Von Frey monofilaments are applied to hindpaw. The fibers vary in amount of force applied. Positive response = withdraw, lick, or shake of paw Blockade of Endocannabinoid-Degrading Enzym es Attenuates Neuropathic Pain | Journal of Phar macology and Experimental Therapeutics (aspe tjournals.org) Methods Used to Evaluate Pain Behaviors in Roden Thermal Sensitivity: Hot Hot plate Plate & Tail Flick Hot plate = Animals stand on a hot plate Tail Flick = Tail is subjected to heat source (e.g. hot water) Measure latency to remove/lick paw (hot plate) or flick tail (tail flick) TAIL FLICK 09.2018 STD PROCEDURE - Behavior - Hot Plate Test.pd (ucsf.edu) Frontiers | Methods Used to Evaluate Pain Behaviors in Rodents | Frontiers in Molecular Neuroscience Models of Nociception: Hot‐Plate, Tail‐Flick, and Form alin Tests in Rodents - Bannon - 2007 - Current Proto cols in Neuroscience - Wiley Online Library Tests for “Anxiety-like” Behavior Open field Light/dark box Elevated mazes What can you test in the Open Field? General locomotor activity/exploration Distance traveled Speed Anxiety-like behavior (Motivator = agoraphobia) Center activity  LIGH DAR T K Light/Dark Box (LDB) LDB has 2 compartments Light (2/3) – open and brightly lit Dark (1/3) – dark and covered Motivated by: Agoraphobia. Similar to OF, conflict between exploration and preference for safe dark areas Measures anxiety-like behavior Time in light compartment = less anxious Short latency to enter light = less anxious Elevated Mazes Elevated Plus Maze (EPM) Cross-shaped platform consisting of two open and two closed arms Elevated Mazes Elevated Plus Maze (EPM) Cross-shaped platform consisting of two open and two closed arms placed 50–70 cm above the ground Elevated Zero Maze Avoids confusing interpretation of center time  Elevated Mazes Conflict between curiosity to explore & fear of open spaces allows assessment of anxiety Open vs. closed activity More time in open = less anxious Shorter latency to open area = less anxious  Tests for “Depressive- like” Behavior Forced swim test Sucrose preference test https://www.sciencedirect.com/science/article/pii/S016643281 Categories of Tests for Depressive-like Behavior Behavioral despair = active attempt to respond to a threat; interpreted as susceptibility to negative mood (negative reinforcement-based) Forced swim test Reward-based/Anhedonia = reduced motivation or inability to experience pleasure (positive reinforcement- based) Sucrose Preference Test  Forced swim test (FST; behavioral despair) Immobile Note: Better to view from side Most widely used test for depressive-like behavior Animals placed in an inescapable cylinder filled with water. Measure time spent: Immobile = passive stress- Swimming/climbing coping strategy or depressive- like behavior Swimming & climbing = active escape strategy Cons = can induce “depression”  Sucrose Preference Test (SPT; Anhedonia) Relies on innate interest for sweet food/solutions. Measure intake of sweet solution vs. plain water in home cage Lower intake of sucrose (%) = Figure 2. Sample data from sucrose preference test performed on anhedonia control (Cntr), chronically despaired mice (CDM) and CDM treated for 4 weeks with classical antidepressant (CDM+AD) Sucrose Preference Test to Measure Anhedonic Behaviour in... (bio-protocol.org)  Behavioral Tests for Social Behavior Direct social interaction test  PINNIN Direct Social Interaction Test G Animals individually WRESTLIN G habituated to open field box Pairs of rats (same sex, treatment group) placed in open field box for set duration. Measure frequency and FOLLOWIN G MUTUAL duration of behaviors: SNFFING Play-related Pinning Boxing/wrestling Following/chasing Unrelated to play CLIMBING Climbing over/under Mutual sniffing Behavioral Tests for Aggression/ Social Dominance Resident-intruder test Resident-intruder RESIDEN Test T Similar to direct social interaction test, but “intruder” placed into homecage of “resident”, with the latter having territorial advantage Only works with males Measure attack latency + frequency and duration of behaviors like: Pouncing INTRUDE Following R Wrestling Pinning Sniffing The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress (n ih.gov) Behavioral Tests for Learning & Memory Fear conditioning Novel object recognition test Y-maze/T-maze tasks Radial arm maze Barnes maze Water maze  “referen ce Frontal memory” “working cortex – memory” generally – across generally trials within trials Hippocamp us/MTL Motor Neostriatum learning & cerebellum (Rotarod) Spatial learning &  Novel object recognition Rodent habituated to arena, then two trials: 1) Training (2 same objects) 2) Testing (old/familiar + novel object) Measure # interactions and/or proportion of time spent exploring novel object  intact memory 2-Object Novel Object Recognition (non-spatial; episodic/object recognition | Behavioral and Functional Neuros cience Laboratory | Stanford Medic memory) ine Dependent on temporal lobes (hippocampal + cortical lesions impair; perirhinal cortex) A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data - PubMed (nih.gov) The novel object recognition memory: neurobiology, test procedure, and its modifications | Spring Barnes Maze (Spatial Learning + Memory) Apparatus = circular table with circular holes around the circumference. Goal is for animal to reach box that is positioned beneath one of the holes with aid of visual cues. Motivator(s) = agoraphobia (can use bright light & other aversion) and/or food in goal box Measures Time or pathlength to find hole # Errors (incorrect hole)  Morris water maze (Spatial Learning + Memory) “Can I remember and find where I parked my car?” Apparatus = water-filled pool with a hidden escape platform beneath the surface. When the animal is released into the water, it will swim around the pool to look for the platform to escape from the water. Visual cues are placed around the maze. Motivator: Rodent aversion to water  DAY 2 DAY 3 DAY 1 Hidden Trials (5) Probe Trial (1) Visible Visible Trials Trials (5) Hidden Trials Probe Trial SPATIAL LEARNING SPATIAL MEMORY 5 x 3 min trials 5 x 3 min trials 1 x 3 min trial 24 24 24h 24h hr hr TARGET QUADRAN T Morris Water Maze Spatial Learning & Memory Test Morris water maze (Spatial Learning + Memory) HIDDEN TRIALS Fig.1 NO2 inhalation deteriorates spatial learning and memory in C57BL/6J mice. (a) Learning curve for PROBE 5 days of invisible training to find the TRIAL hidden platform in the Morris water maze. (b) The number of times crossing the target zone. (c) Percentages of time stayed in the target quadrant. (Wei et al. 2016) Radial Arm Maze (Spatial Learning + Memory) Apparatus = 8 (or more) arms spaced equidistant apart. Rodent trained on task to learn that a food item (motivator) placed at end of certain arms. Reference memory = rodent chooses to visit only arms that contain rewards on a Radial Arm Maze trial Working memory = rodent chooses a single arm only once. Re-entry = working memory error. Y-maze for Spontaneous Alternation (Spatial Working Memory) Animal placed in center of Y maze and allowed to freely explore the 3 arms in a single trial Motivated by: natural curiosity/ preference to explore new arms (Momeni et al. 2015) Measures: # Arm entries = general levels of exploration/activity % alternation = intact spatial working memory Spontaneous alternation  Dependent on hippocampus + PFC T-maze Spontaneous Alternation (Spatial Correc Working Memory) t arm Can also use Y-maze shape Animal placed in start arm (base) of T maze and allowed Forced choice with food to explore motivator Free choice OR forced choice Measures: % correct arms Trial duration Motivated by: natural curiosity/ preference to explore new arms; can use a food motivator (requires food deprivation) Fear Conditioning Based on associative learning (classical conditioning)  CUE CONTE Fear Conditioning XT Conditioned to associate a cue and/or context with aversive outcome (shock) Measure freezing behavior in response to conditioned stimulus Cue = amygdala Contextual = amygdala + hippocampus Oxford Research Encyclopedias  Drug Abuse Liability/Addiction Locomotor sensitization Conditioned place preference Self-administration Improving translation of animal models of addictio n and relapse by reverse translation | Nature Revie Animal Models of Addiction - Science Types of Learning Locomotor sensitization Animals administered a drug over multiple sessions (psychostimulant) Sensitization = early index of abuse liability  sign of neural plasticity (not active drug- seeking) Measured as increased activity or stereotypy over time to same or different (cross- sensitization) drug Locomotor Sensitization Study | Melior Conditioned Place Preference (CPP) Based on Pavlovian/Classical conditioning (Associative learning)  Conditioned Place Preference (CPP) Animal is given drug pairings in one side of set of chambers Preference is then tested for the previously drug-paired chamber Preference for drug-paired side = abuse liability (reward) (Mu et al., 2014 Frontiers | Drug-Induced Conditioned Place Preference and Its Practical Use in Su bstance Use Disorder Research | Behavioral Neuroscience (frontiersin.org) Molecular Brain Conditioned Place Preference (CPP) This model allows measurement of the reinforcing properties of a drug in the absence of the drug itself. Dependent on association between drug and context Can also test aversion (avoidance), in addition to preference. CON: Looks at drug-seeking behavior, but drug administration is PASSIVE. Low face-validity. Extinction behavior = loss of preference/drug seeking after drug pairings end (analogous to “quitting”) Reinstatement behavior = reinstatement of drug-seeking behavior after exposure to the drug, a cue, or stress (analogous to “relapse”)  Drug self-administration Based on operant conditioning (positive reinforcement) Animals trained to make a behavioral response (lever press, nose pose) to get an i.v. infusion of drug Positive responses indicative of reinforcing value/abuse liability Most drugs abused in humans are self- administered in rodents Drug self-administration How does this compare to CPP? Can run fixed ratio (FR) or progressive ratio (PR) PR = increasing # responses needed; motivation (Smith et al., 2011, Psychopharmacology) Can assess compulsive drug seeking despite negative consequences (e.g. shock) Can look at escalation, extinction & reinstatement behavior Can use food as reinforcer (Sharma et al., 2012, JO