Methods of Research in Neurobehavioral Pharmacology

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Methods of Research in Neurobehavioral
Pharmacology
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Neurobiological Techniques

• Sterotaxic surgery allows a researcher to implant
  a device into the brain in a very precise area
  according to a brain atlas
• Accuracy determined post-op by histological
  section
• Humans use halo device, with MRI or CT for
  placement
• Lesioning uses stereotaxic device to position
  electrode or cannula, and current or
  microinjection of neurotoxin is used to cause
  localized brain damage
• Toxins are more specific- can target specific
  cell types or soma (as with kainic acid for soma,
  or 6-hydroxydopamine for axon terminals)
• Gives way to assess what certain brain areas do
  in vivo

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Neurobiological Techniques

• Microdialysis uses stereotaxic surgery, lets
  researchers measure NT released in region of
  brain while animal is awake and active
• Uses specialized sealed cannula except at tip-
  can collect CSF and pump new CSF into area with
  pump
• CSF collected can be analyzed for amounts and
  types of NT via HPLC (high-performance liquid
  chromatography)
• Can monitor neurochemical levels during different
  types of behaviors (sleep, feeding, operant
  tasks, etc)
• In vivo voltammetry microelectrode passes small
  current into tissue, and changes in current flow
  give researcher idea of what NTs are being
  utilized. Very fast (15 ms delay) so can be used
  in real-time.

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Neurobiological Techniques

• Electrophysiological stimulation implant a
  macroelectrode and pass current into it, record
  animal behavior and electrophys response to
  stimulation
• Stimulates groups of cells, not individuals
• Should see results similar to administration of
  NT normally active in area
• Ex stimulate PAG to determine effect of pain
  reduction
• Can also implant microelectrodes into a cell or
  in surrounding ECF to monitor activity of single
  cell or small group of cells
• Intracellular recording more precise, but animal
  must be anesthetized, while in ECF animal can be
  awake and active- but it is less precise
• Patch clamping can measure the activity through a
  small section of axonal membrane, and can
  determine exactly how and when given ion channels
  respond to a given stimulus

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Neurobiological Techniques

• Radioligand binding assay homogenate area of
  brain tissue, then incubate with radiolabeled
  ligand. Then measure radiation with gamma
  counter, which reflects how much bound ligand is
  still in sample
• Must make sure binding is specific to receptor
  sites, so often add high conc of nonlabeled
  ligand to some samples to compete with labeled
  ligand- what remains can be subtracted out to
  determine actual binding
• Tissue sample can be saturated- there are finite
  number of receptors, so should see binding curve
  eventually level off
• Binding should be reversible since NT in vivo
  will bind and release many times. When unlabeled
  ligand added, the amount of bound ligand should
  decrease in predictable fashion
• Finally, binding of similar drugs should
  correlate with measurable biochemical or
  behavioral effect.
• Ex anti-schizophrenic drugs bind to D2 dopamine
  receptor, and their activity correlates to
  reduction of symptoms

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Neurobiological Techniques

• Receptor autoradiography gives info about
  receptor location and density for specific NT
• Use brain slices, and expose to radiolabeled
  ligand
• Slices places in cassettes, and exposed to
  autoradiographic film
• Radiation exposes film, and areas most exposed
  are highest in receptor for the ligand
• Ex cocaine binding in monkey brain

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Neurobiological Techniques

• In vivo receptor binding similar to receptor
  autoradiography, except radiolabeled drug is
  given to animal, then animal is sacrificed
• Shows where drug binds in intact animal
• Bioavailabilty and metabolism of drug can play
  role
• Use PET scans in humans for similar results
• Immunocytochemistry (ICC) brain is fixed in
  inert media, and tissue slices are cut and
  incubated with primary antibody
• Antibody attaches to antigen in tissue, but not
  to nonspecific proteins/molecules
• Secondary antibody applied specific to primary
  antibody, and secondary antibody contains
  radiolabel, enzyme, or fluorescent molecule that
  allows it to be located in tissue under
  microscope
• Many assays c-fos is very common

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Neurobiological Techniques

• Radioimmunoassay similar to ICC, but based on
  idea that there is competitive binding of
  antibody to its antigen
• A standard curve of known antigen concentration
  is prepared so unknown antigen concentrations can
  be compared
• Treat known antigen with radiolabeled antibody
  and record result, then add unknown antigen and
  see if it affects binding affinity, then plot as
  standard curve

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Neurobiological Techniques

• In situ hybridization lets researcher determine
  tissues manufacturing a certain protein- can
  detect specific mRNA
• If mRNA levels increase up-regulation, decrease
  down-regulation
• Very specific and sensitive technique
• Create probes by using labeled nucleotides
  complimentary to mRNA sequences
• Then expose tissue section to radiographic film
  and localize cells with those mRNA

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Neurobiological Techniques

• DNA microassay similar to ISH, but instead of
  measuring one mRNA level, researcher measures up
  to 20,000 short DNA sequences on a single chip
• Can screen entire genome of sequence researcher
  is interested in on one chip
• Scanner reads the amount of hybridization of each
  probe to the DNA on chip, and computer identifies
  pattern of gene activity

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Brain Imaging

• 2-deoxyglucose autoradiography (2-DG) 2-DG is
  radiolabeled and injected into the animal.
• 2-DG is partially metabolized by cells that use
  energy, but gives off radiation that can be
  measured from tissue sections via autoradiography
• Similar to PET scans in humans
• Computerized tomography (CT or CAT) pass X-rays
  into body from emitter and pick up radiation on
  photograpic plate
• Emitter and plate rotate around body part,
  allowing for a slices through tissue that can
  be reconstructed into 3D image
• Only really detects density of materials, so not
  very good for distinguishing individual brain
  structures

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Brain Imaging

• Magnetic resonance imaging (MRI) a magnetic
  pulse is directed at tissue, which makes
  hydrogens in tissue wobble, and emit radio waves,
  which is picked up by an emitter
• Allows for very good resolution of soft tissue,
  and can be reconstructed into 3D image
• Positron emission tomography (PET) can determine
  metabolic activity for tissue in vivo and during
  mental activity
• Use quickly decaying radioisotopes like 15O, 11C,
  or 18F (can also use radiolabeled drug)
• When isotope decays, a proton is emitted from the
  nucleus and strikes an electron, both particles
  are destroyed and emit gamma rays going in
  opposite directions
• Detectors pick up gamma rays, and make a map of
  where highest emissions come from (areas with
  most metabolic activity)

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Brain Imaging

• Functional MRI (fMRI) detects changes in blood
  oxygenation because oxygenated hemoglobin has a
  different MRI profile than deoxygenated
• Gives both anatomical AND functional info
• Doesnt require use of radioisotopes
• Electroencephalography (EEG) measures electrical
  events of large populations of neurons via
  electrodes on scalp
• Often used in studying consciousness, sleep,
  dreaming, and states of arousal
• Analysis of event related potentials (ERPs)
  allows the researcher to compare brainwave
  activity to known controls, and make comparisons
  between EEG of drug-induced state and other
  states of consciousness

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Genetic Engineering

• Targeted mutation (knockout) genetically alter
  an animal to delete a gene for a certain protein
  product, then insert gene into fertilized egg in
  mother
• By comparing behavior and drug response to
  unaltered mice, we can learn the fuction and
  importance of a protein (usually receptor,
  enzyme, or channel)
• Gene replacement replace one gene with another
  to make transgenic organism
• Can insert human gene that causes problem or
  disease into animal, and have viable animal model
  for experiments
• Can also insert gene into cells in cell culture,
  and make large number of clonal copies of cells,
  which can be used to screen new drugs in vitro
  (useful for identifying agonists and antagonists)
• Antisense nucleotides inject into ventricles of
  animal- the nucleotides bind to mRNA, delaying
  translation and possibly degrading mRNA
• Produces a reversible mutant animal whose
  behavior and drug responses can be observed
• Ex VIP antisense nucleotides in SCN of
  hypothalamus

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Behavioral Pharmacology

• Most behavioral pharmacology done on animals
• Can have rigorous controls that reduce
  variability and confounds
• Can more ethically do dissections, lesions, and
  electrophysiological measures of activity
• In humans, can often only imply correlational
  relationships between drug and effect/behavior,
  in animals, we can move past this and establish
  causal relationships
• In many cases, animal and human brain,
  physiological, and pharmacological measures are
  very similar, so they make good models
• Face validity relationship between physiological
  effect in animal and humans. Mostly for directly
  observed phenomena BP, body temp, heart rate,
  etc.
• Construct validity describes relationship
  between testing procedure done on animals and its
  ability to predict clinical effects in humans,
  regardless of similarity of test responses

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Behavioral Pharmacology

• Animal behavior tests should be
• Specific to the class of drug screened (dont
  want to see antidepressant drug producing same
  behaviors as analgesics)
• Sensitive so that doses are in a normal
  therapeutic range and show dose-response
  relationship
• Demonstrate the same rank order of potency
  (ranking drugs according to effective dose) as
  the drug order of potency in therapeutic
  situations

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Behavioral Pharmacology Assessment

• Behavioral observation (tremors, reflexes, temp,
  heart rate, etc)
• Measures of motor activity (urine marking,
  crossing IR light beams, etc)
• Measures of analgesia (tail-flick test)

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Behavioral Pharmacology Assessment

• Learning memory usually a training stage
  followed by test stage
• T-maze animal navigates a maze for a food/drug
  reward, mistakes are counted
• Radial arm maze spatial memory task, animal
  placed in central chamber with radial arms. At
  end of each arm is reward- animal should only go
  down each arm once for reward
• Morris water maze spatial memory task, circular
  pool of water with submerged platform and
  landmarks around it. Very little pretraining
  required

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Behavioral Pharmacology Assessment

• Delayed response test animal is shown a cue,
  then a delay, then must remember appropriate cue
  for reward
• Measures of anxiety animal is placed in
  situation that provokes anxiety, and behavior is
  monitored
• Light/dark box
• Elevated plus maze
• Measures of fear
• conditioned emotional response- stimulus is
  paired with unavoidable electric shock, so when
  stimulus by self is presented, animal freezes
• Fear-potentiated response as above, but if
  stimulus is paired with new novel stimulus,
  animal shows enhanced startle response
• Measures of reward
• Conditioned place preference animal is injected
  with either drug or saline over period of days
  and placed in one of two chambers so it
  associates environment with drug response. Then
  place animal in large box with both chambers and
  see which one it spends more time in.
• Animal spends more time in area that is
  associated with most rewarding stimulus
• Can also pretreat animal with agonists or
  antagonists for drug to modify place preference

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Behavioral Pharmacology Assessment

• Operant conditioning techniques idea is
  consequences control behavior
• Ex animal learns to press lever for
  reinforcement (food/drug)
• Once behavior is established, a schedule for
  reinforcement is put in place (either fixed ratio
  or fixed interval)
• The degree to which the animal wants the reward
  is measured by how often it seeks to press the
  lever
• This is the basis for the self-administration
  method of drug use
• Animals (and humans) will self-administer drugs
  that are rewarding (cocaine, alcohol, etc), but
  not those that are not (aspirin, antidepressants,
  etc).
• Can also train animal to press several levers for
  different drugs, and see which one animals find
  most rewarding
• Can also set up microelectrode in brain to allow
  animal to self-administer small electric shock to
  stimulate rewarding neuronal pathways and monitor
  activity

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Behavioral Pharmacology Assessment

• Negative reinforcement variable of variable
  ratio schedule which increases probability of a
  response that ends a noxious stimulus
• Ex operant analgesia testing animal is trained
  to push lever in response to increasing voltage
  foot shock. Then animal given analgesia, and
  threshhold for lever pulling is monitored
• Ex learned helplessness close analogue to human
  depression
• Animal is placed in cage with floor that shocks
  periodically, but no way to stop it
• After period of time, animal is put in another
  cage where there is an easy escape route- animals
  trained for learned helplessness will not try to
  escape, but WILL show signs of anxiety
• Giving animal antidepressant drug eliminates
  learned helplessness response