neuropharm lecture 2

Question 1

How do we study the effects of drugs on the brain?

Answer 1

In vivo manipulations:
In vitro & In situ measurements:
Behavioral techniques:

Question 2

What do In vivo manipulations include

Answer 2

-Stereotaxic surgery;
-Micro-dialysis;
-Intracranial injections.

Question 3

In vitro & In situ measurements:

Answer 3

-Receptor bindings;
-Immunohistochemistry.

Question 4

Behavioral techniques:

Answer 4

-Operant conditioning;
-Place conditioning;
-Self-administration.

Question 5

Stereotaxic surgery

Answer 5

-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)

Question 6

In vivo manipulations- lesions and microinjections

Answer 6

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

Question 7

Option 1: Electrolytic lesions

Answer 7

-Using electrical current, you can destroy everything in proximity to the electrode (neurons, glia, tracts, etc.);
-Not very selective, but very effective.

Question 8

Chemical/Neurotoxic lesions

Answer 8

-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.

Question 9

Intracranial Self Stimulation

Answer 9

-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.);

Question 10

In vivo manipulations- microdialysis

Answer 10

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.

Question 11

In vivo manipulation- Electrophysiology

Answer 11

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).

Question 12

Single-cell recording

Answer 12

-Intracellular vs. extracellular;
-Anesthetized vs. freely moving/behaving;
-Measures the bioelectrical activity of the brain.

Question 13

In vitro manipulation- Radioligand binding and the process

Answer 13

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;

Question 14

In vitro manipulations- autoradiography and what does the process involve

Answer 14

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.

Question 15

In vitro manipulations- Immunohistochemistry and what does this process involve

Answer 15

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.

Question 16

In Situ Hybridization (ISH) and what does this process involve

Answer 16

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.

Question 17

2-Deoxyglucose Autoradiography:

Answer 17

-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.

Question 18

Imaging - computerized tomography

Answer 18

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.

Question 19

Imaging- Magnetic Resonance Imaging

Answer 19

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)

Question 20

Imaging- functional MRI and the principles

Answer 20

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.

Question 21

Imaging- Positron Emission Tomography and how does it work

Answer 21

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.

Question 22

How can we measure the locomotor response to drugs

Answer 22

-Beam breaks;
-Automated video tracking;
-Perimeter vs. center occupancy;

Question 23

How can measure the analgesic properties of drugs

Answer 23

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.

Question 24

How to measure and compare the rewarding properties of drugs.

Answer 24

Conditioned Place Preference (CPP)

Question 25

How can we measure fear and anxiety in an animal model?

Answer 25

The elevated plus maze is one really easy way to measure anxiety