PMCOL 343 - Animal Models

Animal Behavioural Models - Purpose

• Animal models are used to assess drug effects on behaviour and model aspects of human disease.

• Applications include antidepressants, anti-anxiety drugs, antipsychotics, cognitive enhancers, and addiction treatments.

• An animal model attempts to imitate selected aspects of a clinical condition.

• Models help identify potential therapeutic drugs and investigate the biological substrates of clinical conditions.

Animal Behaviour - Descartes

• Descartes viewed animals as automata whose behaviour could be reduced to simple reflex actions.

• He argued that humans have a rational soul, abstract thought, and minds, whereas animals act through instinct.

• Reluctance to compare humans with animals partly reflected beliefs that humans were God's creation and superior to animals.

Animal Behaviour - Darwin

• Charles Darwin and Alfred Wallace developed the theory of evolution through natural selection.

• Relevant works include On the Origin of Species and The Descent of Man.

• Darwin argued that humans descended from animals and that animals can inherit instincts.

• Darwin distinguished inherited instincts from learned habits, arguing that humans inherit instincts and learn habits.

Animal Behaviour - Historical Views

• Sherrington (1900) argued that animals act without minds and that there was no evidence of thought, feelings, or perception.

• Wundt (1911) suggested that basic sensations such as fear in animals may parallel human sensations.

• Pavlov (1897) observed that a trained dog's perception of feeding could cause salivation, indicating perception, memory, and organized thought.

4 McKinney Criteria

1. Etiological Validity: The cause or trigger used to induce the condition in the animal model mirrors the actual etiology in humans.

2. Face Validity: The outward behavioral symptoms and physical manifestations in the animal accurately resemble the clinical profile of human patients.

3. Construct Validity: The biological pathways, affected neural circuits, and underlying neurochemical mechanisms are identical between the model and the disease state.

4. Predictive Validity: Interventions that successfully treat or prevent the condition in humans produce the same therapeutic effect in the animal model.

Opioid Dependence - Rat Model

• Morphine is administered to rats through a slow-release subcutaneous capsule.

• Rats develop tolerance to morphine, find it rewarding, and develop opioid dependence.

• Withdrawal can be precipitated by removing morphine or administering naloxone, an opioid antagonist.

• Researchers observe withdrawal behaviours in saline-treated controls and animals given potential treatments.

Opioid Withdrawal - Testing Treatments

• Clonidine is used clinically to help treat opioid withdrawal and serves as a treatment control in the model.

• Wet dog shakes are a measured withdrawal behaviour: saline-treated rats show substantial behaviour, while clonidine inhibits it.

• BU224 is an experimental imidazoline receptor antagonist; it is ineffective against wet dog shakes but shows some effectiveness against mouth movements and diarrhoea at a higher dose.

• Opioid use is associated with constipation, while withdrawal can cause diarrhoea.

Opioid Model - Validity

• Inducing-condition similarity is limited because rats do not initially self-administer morphine in the same way that humans may use opioids.

• Behavioural similarity is strong because rats develop dependence and withdrawal.

• Neurobiological similarity is strong because the neurochemistry is similar and withdrawal can be severe.

• Clonidine works in both rats and humans, supporting treatment similarity.

Laboratory Animals - Species Differences

• Rat behaviour is strongly influenced by hunger, thirst, and the need to breed.

• Rats are small, clean, inexpensive, easy to handle, have short gestation periods, and live approximately three years.

• Mice are smaller, more affordable, and amenable to genetic manipulation, but their behavioural profile is less complex than that of rats.

• Guinea pigs have been used in experiments for centuries; they contributed to the discovery of vitamin C in 1907.

Laboratory Rats - Origins

• Donaldson established a breeding colony at the Wistar Institute in Philadelphia in 1906, supplying scientists until 1960.

• Wistar rats were bred specifically for research.

• Albino Wistar rats have poor vision.

• Wistar rats may be unsuitable for experiments requiring good visual acuity.

Rat Strains - Long-Evans

• Around 1915, researchers Long and Evans mated female Wistar rats with a wild male rat.

• The resulting Long-Evans strain has good vision.

• Long-Evans rats are useful for behavioural experiments requiring visual discrimination, including the Morris water maze.

Rat Strains - Sprague-Dawley

• Sprague-Dawley rats were developed by mating a hooded rat with a white female Wistar rat and then remating the offspring.

• The strain was selected for rapid growth, vigour, and good temperament.

• A company was established to market Sprague-Dawley rats.

• Sprague-Dawley rats have poor vision.

Rat Strains - Experimental Implications

• Different strains, including different Wistar colonies, can show substantial behavioural differences.

• Some Wistar strains drink little alcohol, while one inbred colony was found to drink ten times the normal amount.

• Long-Evans and other hooded rats have better eyesight for visual-discrimination tasks.

• Wistar and Sprague-Dawley rats have poorer vision, which can limit their suitability for such tasks.

Experimental Design - Variables

• The independent variable is the factor manipulated by the researcher, usually the type or amount of drug in behavioural pharmacology.

• The dependent variable is the observed outcome, such as a measurable behaviour.

• Experiments may compare conditions such as injury alone versus injury plus PPF to investigate relationships between events.

Experimental Design - Experimenter Bias

• Researchers can influence results if they know which condition a subject is in or which drug it receives.

• Blinding helps reduce bias by preventing the experimenter from knowing the experimental condition.

• Double-blind human studies conceal treatment assignments from both subjects and researchers.

• Blinding reduces, but does not eliminate, subject and experimenter bias; keeping the behavioural experimenter blind is an important control in animal studies.

Experimental Design - Three-Group Design

• Group 1 receives the experimental drug being tested.

• Group 2 receives a placebo or vehicle control.

• Group 3 receives an established drug with a known therapeutic effect.

• Experimental drug versus placebo tests whether the new drug improves outcomes; established drug versus placebo checks whether the model can detect improvement; experimental drug versus established drug tests whether the new drug offers an advantage.

Behavioural Models

• Antidepressant model: Porsolt forced swim test.

• Anxiety models: elevated plus maze, Vogel conflict test, and defensive burying.

• Learning and cognition models: Y-maze, Morris water maze, radial arm maze, and social interaction test.

Antidepressants - Porsolt Forced Swim Test

• Roger Porsolt developed the forced swim test following research on learned helplessness, associated with Seligman's work in 1975.

• Learned helplessness occurs when animals exposed to uncontrollable aversive events show decreased motivation to respond.

• In the forced swim test, behaviours such as immobility, swimming, and climbing are measured over a set interval.

• The test is used to assess the effects of potential antidepressant drugs on these behaviours.

Forced Swim Test - Drug Effects

• Fluoxetine (Prozac), an SSRI, decreases immobility and increases swimming.

• Reboxetine (Edronax), an NRI, decreases immobility and increases climbing.

• Immobility is used as a behavioural measure associated with learned helplessness or behavioural despair.

• Reminder: Review the example-data graph comparing immobility, swimming, and climbing.

Antidepressants - Tail Suspension Test

• Mice are suspended by the tail using a transducer.

• The amount of time spent immobile is measured as an indicator of behavioural despair.

• Antidepressants, including SSRIs, prolong attempts to right themselves.

• Why can antidepressants can act acutely in rodent tests when SSRIs generally take more than two weeks to work in humans.

Anxiety - Elevated Plus Maze

• Rodents generally prefer enclosed arms over open arms, making the elevated plus maze a test of anxiety-like behaviour.

• Researchers measure entries into open versus closed arms, time spent in open arms, and latency to enter an open arm.

• Lorazepam, a benzodiazepine, increases time spent in open arms and reduces the latency to enter them.

• Yohimbine, an alpha-2 adrenergic antagonist that can act as a stimulant and increase anxiety, decreases open-arm exploration and increases entry latency.

Elevated Plus Maze - Data Interpretation

• Greater time spent in open arms is interpreted as reduced anxiety-like behaviour.

• Longer latency to enter an open arm indicates greater avoidance of the exposed area.

• Heat maps can illustrate the animal's movement around the maze.

Anxiety - Vogel Conflict Test

• The Vogel test is a conflict test used to screen drugs for potential anxiolytic effects.

• Drinking is punished with mild electrical shocks, causing water consumption to decrease.

• Anxiolytic drugs can restore drinking responses despite the punishment.

• The animal must choose between satisfying thirst and avoiding the shock.

Anxiety - Defensive Burying

• Defensive burying measures a behaviour considered more ethologically relevant.

• Animals are habituated to the environment without a probe and then tested with the probe present.

• Initial contact with the probe delivers a mild shock, triggering defensive burying.

• Researchers measure latency to begin burying and the duration of burying.

Learning and Cognition - Y-Maze

• The Y-maze is a rapid test of spatial memory.

• A mouse starts in one arm and explores the other two arms, which should be visited in an alternating sequence such as A-B-C-A-B-C.

• Researchers measure the percentage of successful alternations.

• The test is sensitive to hippocampal lesions.

Learning and Cognition - Morris Water Maze

• The Morris water maze is a more complex test of spatial memory using a pool of water at 22°C with a hidden platform.

• The water is made milk-coloured so the platform is hidden, while the rat can use visual cues to learn its location.

• Rats typically receive four trials per day and have 90 seconds to find the platform.

• Performance depends on the hippocampus.

Morris Water Maze - Fragile X Model

• The fragile X mouse model involves deletion of the FMR1 gene and has learning problems.

• Fragile X mice show impaired Morris water maze performance, particularly when the task becomes more difficult during a reversal trial.

• Non-fragile X mice learn the new platform location more rapidly during reversal, while fragile X mice show a slower learning curve.

Morris Water Maze - Evidence and Applications

• Cortical lesions of similar magnitude do not impair performance in the same way as hippocampal lesions.

• The Morris water maze is useful for studying learning and memory.

• Scopolamine and benzodiazepines impair performance in this model, consistent with their effects on learning and memory in humans.

• Performance declines with age; older rats do not perform as well.

Cognitive Treatments - Memory and Thinking

• Cholinesterase inhibitors, including donepezil (Aricept) and rivastigmine (Exelon), prevent the breakdown of acetylcholine, which is important for memory and thinking.

• Memantine (Namenda) regulates glutamate activity, which contributes to processing, storing, and retrieving information.

Learning and Cognition - Radial Arm Maze

• The radial arm maze measures spatial reference memory and working memory.

• The task depends on the hippocampus and also involves the prefrontal cortex (PFC).

• The maze typically has eight arms, with four arms blocked and food placed in the other four.

• Animals learn to enter arms to retrieve food; the task is sometimes called a delayed match-to-sample test.

Radial Arm Maze - Memory Errors

• The test measures the incidence and type of memory errors during training and testing.

• A working memory error occurs when an animal re-enters an arm it has already visited and emptied; this can occur during both training and testing.

• A reference memory error occurs when an animal enters an arm that was baited during training but is no longer baited during testing.

Learning and Cognition - Social Interaction Test

• The social interaction test uses a three-chamber apparatus.

• After habituation to the environment alone, sociability is assessed by introducing an unfamiliar, non-cagemate mouse.

• Researchers measure how much time the test mouse spends exploring or interacting with the stranger.

• A social novelty test introduces a second stranger to assess the animal's response to a new individual.

Social Interaction Test - Drug Effects

• Anxiolytic compounds can dose-dependently increase social interaction.

• Examples include GABAergic agonists such as benzodiazepines, ethanol, and barbiturates.

• Corticotropin-releasing factor (CRF) produces an anxiogenic-like effect and reduces social interaction.

• The test is used in models of autism, fragile X syndrome, schizophrenia, and related conditions.

Behavioural Models - Interpreting Results

• In the forced swim test, reduced immobility is a behavioural response associated with antidepressant treatment in rodents.

• In the elevated plus maze, increased open-arm exploration is interpreted as reduced anxiety-like behaviour.

• In the Vogel test, restored drinking despite mild shock is interpreted as an anxiolytic-like effect.

• In defensive burying, researchers assess the timing and duration of the animal's defensive response to a threatening stimulus.

Antipsychotics - Startle Reflex

• The startle reflex occurs in both humans and animals.

• Repeated exposure to a startle stimulus produces a reduced response, called habituation.

• Prepulse inhibition (PPI) occurs when a mild stimulus presented before the startle stimulus further reduces the startle response.

• Patients with schizophrenia show deficient PPI, making it useful for modelling aspects of schizophrenia.

Antipsychotics - Apomorphine Model of Deficient PPI

• Apomorphine is a non-selective dopamine agonist that disrupts PPI in rodents.

• This disruption is used to develop a rat model of deficient PPI.

• Most antipsychotics can reverse the apomorphine-induced PPI deficit.

Stroke - Middle Cerebral Artery Occlusion

• Middle cerebral artery occlusion (MCAO) can be permanent or temporary.

• MCAO restricts blood flow to brain tissue and can lead to cell death.

• The model is used to screen drugs that may reduce damage following stroke.

• NMDA antagonists and free-radical scavengers have been investigated using these models; edaravone (MCI-186) is shown as a free-radical scavenger with neuroprotective effects in mice with permanent focal brain ischemia.

Stroke - Tissue Analysis

• MCAO tissue images distinguish redox-active, viable tissue from necrotic or dead tissue.

• TUNEL staining marks apoptosis.

• Individual cell types can also be stained to examine the tissue response.

Multiple Sclerosis - Disease Mechanism

• Multiple sclerosis (MS) is a CNS-directed autoimmune disease.

• It involves T-cell-mediated inflammation, demyelination, and axonal injury.

• The animal model is experimental autoimmune encephalomyelitis (EAE).

• EAE is induced by immunizing a rat or mouse with a specific myelin peptide that triggers an immune response against myelin in the brain and spinal cord.

MS - EAE Model Components

• Myelin antigens used include MOG35-55, PLP139-151, and MBP84-104.

• The immune response involves inflammatory cells, including T cells and macrophages, in the spinal cord.

• Tissue images show immune-cell markers such as CD3 and CD45, along with myelin staining and demyelination.

MS - EAE Disease Activity

• Disease activity is assessed using a weakness or paralysis score.

• Clinical signs typically appear 10–14 days after immunization.

• The EAE model has been instrumental in the development of MS therapies.

Modern Approaches - Cre-Lox System

• Cre-Lox is a gene-manipulation strategy that is more precise than a global knockout.

• It can knock out or knock in a specific gene in a defined population of cells.

• The gene of interest is flanked by loxP sites, and Cre recombinase acts at those sites to enable the genetic modification.

Cre-Lox - KCNMA1 Example

• Cre recombinase is driven by the AdVil promoter, identified as being expressed only in sensory neurons of the dorsal root ganglion (DRG).

• These mice are crossed with mice carrying loxP sites around the KCNMA1 gene, producing a floxed KCNMA1 arrangement.

• KCNMA1 encodes the BK subtype of potassium channel; the resulting mice lose BK channels in sensory neurons while other cells retain normal expression.

• This reduces off-target effects and the system can be inducible and reversible, allowing manipulation at selected times using drugs such as tetracycline-related compounds or tamoxifen.

Gene Editing - CRISPR-Cas9

• CRISPR stands for Clustered Regularly Interspersed Short Palindromic Repeats.

• These DNA sequences occur in prokaryotes and form part of a prokaryotic immune system that identifies genetic sequences for cleavage by Cas9.

• Jennifer Doudna and Emmanuelle Charpentier pioneered the use of CRISPR as a gene-editing tool in eukaryotic cells.

Optogenetics - Mechanism

• Four main steps.

Optogenetics - Basic Principle

• Optogenetics controls neuronal activity using light.

• Channelrhodopsin (ChR2) is activated by blue light and is excitatory.

• Halorhodopsin (NpHR) is activated by yellow light and is inhibitory.

Optogenetics - TRPV1 Pain Model

• A TRPV1-Cre mouse is crossed with a mouse carrying a loxP-flanked STOP cassette that prevents expression of ChR2-EYFP.

• In the offspring, Cre recombinase removes the STOP cassette, allowing ChR2-EYFP expression in TRPV1-lineage neurons.

• EYFP-positive DRG neurons show functional ChR2 expression in nociceptors.

• Blue light at 470 nm evokes currents in the same neurons that respond to capsaicin, a TRPV1 channel agonist.

Optogenetics - Evoked Pain Behaviour

• Blue light activates ChR2 in TRPV1-positive pain-sensing neurons and evokes a strong nocifensive response.

• Delivering local anaesthetic to TRPV1-positive pain-sensing neurons prevents blue light from activating the cells.

• When the cells are no longer activated, the behavioural response is absent.