neuropharm lecture 2 Flashcards
How do we study the effects of drugs on the brain?
In vivo manipulations:
In vitro & In situ measurements:
Behavioral techniques:
What do In vivo manipulations include
-Stereotaxic surgery;
-Micro-dialysis;
-Intracranial injections.
In vitro & In situ measurements:
-Receptor bindings;
-Immunohistochemistry.
Behavioral techniques:
-Operant conditioning;
-Place conditioning;
-Self-administration.
Stereotaxic surgery
-Implant electrodes or needles into a specific brain region;
-3D target is calculated using a brain atlas; a brain atlas provides a
standardized set of coordinates to determine specific sites within the brain
-All distances are measured from sutures on the skull: bregma (bone
Directly above the brain)
In vivo manipulations- lesions and microinjections
Lesions and ablations are a technique used to destroy a very specific part of the brain by inserting an electrode into the brain, aiming the tip at your 3D target;
Option 1: Electrolytic lesions
Option 2: Chemical/Neurotoxic lesions
Option 1: Electrolytic lesions
-Using electrical current, you can destroy everything in proximity to the electrode (neurons, glia, tracts, etc.);
-Not very selective, but very effective.
Chemical/Neurotoxic lesions
-Using chemicals, can start to be more selective & strategic;
-Destroy neurons, but leave axons/tracts intact;
-Can be used to target specific neural pathways;
-E.g. 6-OHDA destroys norepinephrine and dopamine cells selectively.
Intracranial Self Stimulation
-Animals are implanted with electrode, coupled to a lever (or other mechanism);
-They can press the lever to receive electrical stimulation (or food, water, mate, etc.);
In vivo manipulations- microdialysis
Technique used to measure neurotransmitter release in specific brain regions while the subject behaves.
Insert cannula(e) in 3D target stereotaxically.
-Cannula is a sophisticated instrument with a semi-permeable membrane;
-Filled with artificial CSF.
Based on concentration gradients, chemicals will move across the cannula membrane, and can be sampled by experimenter.
In vivo manipulation- Electrophysiology
Electrophysiology involves implanting electrodes to stimulate specific cell(s) with tip of electrode and evaluate the consequent change in behavior.
Electrophysiology can also be used to record (using a recording electrode) the activity of single cells à populations of neurons (in vitro).
Single-cell recording
-Intracellular vs. extracellular;
-Anesthetized vs. freely moving/behaving;
-Measures the bioelectrical activity of the brain.
In vitro manipulation- Radioligand binding and the process
Radioligand binding can be used to study the number of receptors in any given brain region/tissue;
-This techniques can provide information on # of receptors and receptor affinity in each region.
The process involves:
-Grinding tissue into a homogenate;
-Adding radioactively-labeled drug (i.e. radioligand) into homogenate;
-Let incubate (cook);
-Wash the sample to get rid of unbound radioligand;
-You can then quantify the # of bound radioligands using a scintillation counter;
In vitro manipulations- autoradiography and what does the process involve
Autoradiography allows you to visualize the distribution of receptors across the brain.
Process involves:
-Removing, fixing, slicing and mounting brain tissue on to microscope slide;
-Place an autoradiographic film on top of slides;
-Chemicals (e.g. NT’s, drugs, etc.) bound to receptors will react with film;
-Particles emitted from the radioactive tissue expose the film;
-This will tell us how many receptors there are, and where the receptors are.
In vitro manipulations- Immunohistochemistry and what does this process involve
Your immune system produces antibodies à proteins produced by white blood cells that target and destroy foreign substances.
Immunohistochemistry involves:
-Raising antibodies for a particular antigen;
-Isolating antibodies from blood plasma;
-Tagging antibodies with chemicals that produce color;
-Applying antibodies to brain slices mounted on a slide;
-Only neurons (or glia cells) which contain the antigen will have antibodies bound to them.
Through a series of chemical reactions, we can make the chemically-tagged antibodies emit color à quantify amount and location of color.
In Situ Hybridization (ISH) and what does this process involve
ISH is used to identify tissue that manufactures a specific protein. This is different then identifying tissues that contains said protein (or peptide)!
This process involves:
-Making probes/primers that contain complementary base-pair sequence to protein of interest;
-Label these radioactively (or with dyes);
-Apply labeled probe to slides with brain tissue;
-Labeled probes will attach (hybridize) with complementary base-pair sequence;
-Expose tissue to film and quantify.
2-Deoxyglucose Autoradiography:
-When cell firing increases, its metabolic rate increases à it must!
-Injecting 2-deoxyglucose into an animal will allow us to visualize (and quantify) the activity of cells;
-Therefore, inject animal with 2-DG, test the animal, sacrifice and prepare brain tissues.
-Cells that were active will have taken up 2-DG. Cells that were more active will have taken up more 2-DG.
-c-Fos is a transcription factor that increases during protein synthesis.
-We can raise antibodies for c-Fos and apply these antibodies to our prepared brain tissues;
-Brain regions that contain antibodies for c-Fos must have been active at time of sacrifice.
Imaging - computerized tomography
Computerized Tomography (CT) Scans
-X-Rays are passed through the brain (or body);
-Based on the density of the tissues, the X-Rays will be absorbed differently;
-Sensitive detectors will illustrate 2D structures based on their densities;
-Can visualize damaged areas, bone fractures, tumors, cannula/implants, etc.
Imaging- Magnetic Resonance Imaging
Magnetic Resonance Imaging (MRI) is a way to take static images of the brain.
MRI is based on the principle that atoms will emit waves (measurable) when placed in a strong magnetic field.
Hydrogen atoms (e.g. water) in your brain will stand in formation in the presence of a strong magnet.
Radio pulses can be delivered to the brain which bumps these atoms out of place à they wobble irregularly and produce a tiny electrical field.
Brain areas with high H2O content (neuron-rich areas) will stand out from areas with low H2O content (axons)
Imaging- functional MRI and the principles
The principles of functional magnetic resonance imaging (fMRI) are:
-When a neuron is active, it uses oxygen (more);
-Active neurons thus signal the blood vessels to dilate to increase blood flow to activated regions;
-Changes in oxygen content in blood alters magnetic properties of water;
-fMRI signals which areas are displaying changes in activity;
-Can be performed in real time.
Imaging- Positron Emission Tomography and how does it work
Positron Emission Tomography (PET) scans measure the metabolic activity of neurons.
How does it work?
-Patients are injected with radioactively labeled oxygen (15O);
-Radioactive 15O releases positrons (because they are neutron deficient);
-Emitted positrons are neutralized by electrons in the brain which produces energy in the form of photons;
-PET cameras detect photon emissions;
-Activated brain regions use more 15O, emit more photons, and this can be captured by the PET camera.
How can we measure the locomotor response to drugs
-Beam breaks;
-Automated video tracking;
-Perimeter vs. center occupancy;
How can measure the analgesic properties of drugs
Using the tail-flick test,
In the tail-flick test, we shine a high-intensity light beam on the tail of the animal;
The light gets hot, and eventually becomes uncomfortable. The animal will flick his/her tail to escape the sensation.
Tail-flick latency can be quantified and used as a measure of analgesia.
How to measure and compare the rewarding properties of drugs.
Conditioned Place Preference (CPP)
