Neurobehavioural methods and drug design for the CNS

Question 1

Why are pharmaceutical companies investing less in CNS research programmes?

Answer 1

 Poor understanding of mechanisms underlying the disease
 Limited human brain tissue for research
 Lack of biomarkers to measure biological states and therapeutic effects
 Heterogeneity within the population that is affected by CNS disorders
 High failure rates even after drugs reach clinical trials – costs money
 Lack of clinical phenotyping and endophenotyping data
• Endophenotype – hereditary characteristic associated with a condition but isn’t a direct symptom of the condition
 Sex differences, circadian rhythms, and strain differences – aren’t always controlled for in animal models
 Animal models that don’t fully recapitulate the disorder
 Low predictive validity or reproducibility of animal models

Question 2

List the advantages and disadvantages of using rodents as a model

Answer 2

Advantages:
	Mammalian
	Well-characterised
	Genetically tractable
•	Allows them to be ‘humanised’
	Genome has been mapped
Disadvantages:
	Difficult to model many elements of CNS disorders:
•	Delusions
•	Mood
•	Memory
	Models/assays generally only test a single element of a phenotype
	Findings don’t always translate to humans

Question 3

List 2 non-genetic uses for rodent models

Answer 3

Behavioural:
•	Maternal separation
•	Acute stress – restraint; predator exposure
•	Chronic stress
	Pharmacological/surgical:
•	Neurochemical lesions – MPTP and Parkinson’s 
•	Middle cerebral artery occlusions
•	Drug administration

Question 4

Explain the open field test and state which disorders it assesses

Answer 4

o Open-field
 Mouse in large box
 Normal mice will go to the middle; anxious mice stick to the edge

used for depression and anxiety, unconditioned test

Question 5

Explain the social interaction maze and state which disorder it assesses

Answer 5

o Social interaction
 1-3 chambers
• Stranger chamber + empty chamber, or stranger chamber + novel object chamber
 Anxiety = reduced social interaction, prefers object over new rodent

assesses anxiety
unconditioned test

Question 6

Explain the forced swim test and which disorder it assesses

Answer 6

 Place rodent in water; see how it responds
 Depressed rodents give up struggling more easily
 Rodents are not allowed to drown
 Important to assess the frequency of behaviour and the duration of behaviour

assesses depression
unconditioned test

Question 7

Explain the conditioned startle

Answer 7

o Conditioned startle
 Relies on Pavlovian (cued) or Skinner-like (contextual) conditioning
 In anxiety, fear takes longer to extinguish

Extinction = how long it takes to un-learn the behaviour

conditioned: electrical footshock (US) in specific context (CS) (US-CS association) –> exposure to same context (CS) without footshock (fear expression, freezing behaviour) –> fear extinction (repeat CS without shock)
cued: electrical footshock (US) in specific context + auditory cue (CS) (association) –> exposure to auditory cues (CS) under different context without footshock delivery (freezing behaviour) –> fear extinction (repeated exposure to auditory cues (CS) without footshock delivery)

Question 8

Explain the conditioned place preference as an assay for addiction

Answer 8

o ‘will press lever for food’ – up to 50 times for object of addiction
o Takes advantage of stimulus pairing:
 Drug paired with environment; will learn to go to/avoid environment depending on the effect of the drug administered
 Prefers areas where addictive substance was administered to areas where the substance wasn’t administered
• Patterned floors/walls help show which area the rodent prefers

Pre-test accounts for any inherent bias towards rooms

Question 9

Explain the novel object recognition assay for memory

Answer 9

 Give animal box with 2 identical objects in it
 Delay
 Move to box with 1 copy of the original objects and a novel (new) object
• Measure how long the animal spends exploring each object
o Relies on natural behaviour – novelty preference (preferential exploration of new things)
 During ‘test’ phase, animals spend more time with the new object than with the familiar one
• Can be expressed as a ‘discrimination ratio’
o Discrimination = (novel time – familiar time)/*(novel time + familiar time)
o If D=0, animals explore the 2 objects equally. If D>0, animals explore novel object more
 Lesions to the perirhinal cortex abolish novel object preference for both short and long durations
• Perirhinal cortex is adjacent to + highly connected with the hippocampus; damage disrupts transmission
 Object preference task can test spatial memory
• Lesions to the hippocampus but NOT the perirhinal cortex disrupt performance

Question 10

Explain the Morris water maze, and state what it is used to test

Answer 10

Tests memory.
 Classical test of memory in rodents
• Rodent is given spatial cues to aid navigation
 Relies on rodent’s desire to get out of the water
• Thus avoiding the ‘dangerous’ situation
 Train rodent over the course of a week; in this time, path length (‘escape latency’) decreases
• Due to animal learning the location of the hidden platform
 Platform then removed from quadrant – animal spends more time in the quadrant where the platform was
 Rodents with hippocampal lesions take longer to learn the location of the hidden platform and learn it less effectively – latency period is still longer than normal rodent
• Cortical lesions seem to have no significant effect on memory acquisition
 We can pharmacologically inactivate the hippocampus; this blocks the retrieval of the spatial memory
• E.g., can use muscimol. Muscimol agonises GABAA-Rs; thus can temporarily ‘inactivate’ a brain region
o Muscimol infusion to CA1 region blocks the retrieval of previously learned information – e.g., the hidden platform location
 We can block hippocampal NMDA with AP5
• D-AP5 isomer  no LTP in rodents
o L-AP5 (not recognised by body)  no effect on LTP
• D-AP5 isomer also blocks spatial memory
o Blockade of hippocampal NMDA receptors blocks the ‘preference’ for the target quadrant
 Rodents cannot remember the location of the hidden platform when given D-AP5

Question 11

Name 3 other assays for depression and/or anxiety

Answer 11

o Elevated plus maze/zero maze
o Barnes maze
o Tail suspension

Question 12

Name 3 other assays for conditioned depression/anxiety

Answer 12

o Learned helplessness
o Active/passive avoidance
o Defensive burying

Question 13

Which mechanisms of disease can neurobehavioural tests be used to identify?

Answer 13

• drug effects
• KO/KI mice
• Optogenetic stimulation
o Activating genes for conditions with specific wavelengths of light
• DREADDs
o Designer Receptors Activated by Designer Drugs

Question 14

What are the limitations of neurobehavioural tests?

Answer 14

• Typically depend on the animals not having locomotor changes (impaired movement/coordination, hyper-/hypo-activity
• Initial assessment of locomotor activity should be performed – open-field test/rota-rod
o If a drug induces sedative effects, this will impact the results
• No single test is sufficient and multiple related tests for a phenotype should be performed to confirm the effect of the drug

Question 15

Why control for sex differences?

Answer 15

• Half the population is female
o Women experience more adverse drug reactions than men
• Sex differences in experimental outcomes
• Improves data applicability

Question 16

Name 2 experimental methods for controlling cyclical variations in female mice, and evaluate the necessity of this

Answer 16

• Staging
o Noting the stage of the oestrus cycle of the female mice used
• Ovariectomy and oestrogen replacement
• Necessity – no more variation in female animals than males
o Differences between sexes, but no more variance in females than males

Question 17

Define endophenotype

Answer 17

Endophenotype – hereditary characteristic associated with a condition but isn’t a direct symptom of the condition