Lecture 7 Flashcards
Chapter 5
what do allosteric modulators do
increase or decrease the endogenous neurotransmitter (the naturally occurring hormone?)
other than binding to the postsynaptic receptor proteins, how else can drugs increase or decrease postsynaptic activity
conventional neurotransmitters (dopamine and such) are made in the axon terminals, and from precursor molecules (generally amino acids flowing around, these are the building blocks to make the receptors)
turned into neurotransmitters by enzymes
then packaged into synaptic vesicles
these are all chances for a drug to alter the postsynaptic activity
all motor neurons release what as their main neurotransmitter
acutylcholine
all sensory neurons release what as their main neurotransmitter
glutamate
what is the CT scan
cheapest and fastest/… basically an X ray
what is the CT scan not good for
soft tissue like brain… doesn’t show up that clearly
what does MRI stand for
Magnetic resonance imaging
how does MRI work
When molecules (of the body) are in a strong magnetic field, the hydrogen atoms (i.e., single protons) spin with a particular orientation (defined by the magnetic field).
A radiofrequency wave (i.e., low energy electromagnetic radiation) is then passed through the body, which knocks the hydrogen atoms out of their orientation. As they return to the orientation set up my the magnet, they emit their own radio waves that are detected by the scanner.
The scanner can estimate the density of hydrogen atoms in each area of the brain (they are most prevalent in fat and water) by calculating the amount of radio waves that come out of each region after each pulse of radio wave energy is delivered. The result is a high spatial resolution, three-dimensional image of the brain.
what is the variant of an MRI
Diffusion Tensor Imaging (DTI)
what is the Diffusion Tensor Imaging (DTI)
an MRI technique that measures the direction and speed of the diffusion of water molecules
used to identify axon tracts
Colors indicate the direction
of water molecule diffusion
whats Macroelectrodes (EEG)
A macroelectrode is used to record activity of a large number of neurons in a particular region of the brain.
Macroelectrodes measure the net effect of action potentials and post synaptic potentials (synaptic transmission) from millions of cells located around the electrode.
Macroelectrodes are attached to an amplifier which records an electroencephalogram (EEG) which provides a measure of gross activity in the brain.
EEG’s provide a diagnostic tool with which particular states of consciousness or types of cerebral atrophy are associated with specific patterns of EEG waveforms.
what did people want that the EEG didnt give
analysis of not just the surface level activity but the deep down activity
what gives analysis of not just the surface level activity but the deep down activity
Positron Emission Tomography (PET)
what does the Positron Emission Tomography (PET)
do
The person is injected with radioactive compound. Early studies often used a radioactive sugar molecule (2-DG) to detect changes in energy use over time.
2-DG is similar to glucose, in that it is taken up by energy consuming cells in the body. However 2-DG is not broken down (metabolized) as easily as sugar is, so it stays around for hours.
The radioactivity of the 2-DG molecules can be detected by a scanner. The computer determines which regions of the brain have taken up the radioactive 2-DG and produces a picture of the brain showing different activity levels.
The disadvantages of PET scanners is their operating costs. For safety reasons, the radioactive molecules used in PET studies are designed to decay rapidly (over hours), thus they have to be made on site just before the experiment.
what is autoradiography
A comparable (but now outdated) approach in rodents was autoradiography.
Autoradiography
Procedure that to determine the location and quantity of radioactive substances in a piece of (brain) tissue
Radiation exposes photography film that is placed on top of the tissue
Measurements of 2-DG have been used to assess changes in energy use throughout the brains of rodents after they engage in different behaviours.
what is the best technique to scan the brain
Functional magnetic resonance imaging (fMRI)
what is the Functional magnetic resonance imaging (fMRI)
fMRI measures brain activity by taking a rapid series of MRI scans. In brief…
The amount of oxygen in blood influences the magnetic properties of blood cells.
With a series of MRI scans, it is possible to detect changes in blood oxygenation, which reflects blood flow, which correlates with neural activity.
When a brain area is in use, blood flow to that region quickly increases (~5s lag).
This technique is popular because it doesn’t involve needles, surgery, or radioactivity. It provides both structural and functional information with decent spatial resolution (1 to 5 mm) and temporal resolution (several seconds).
Researchers are now trying to use fMRI to measure signaling molecules. The approach uses “enzyme-activated magnetic resonance contrast agents”. The goal is to develop molecules that differentially affect the magnetic properties of water when they undergo specific chemical (enzymatic) reactions.
The most direct measurements of neural activity are made how
with metal wires placed in the brain.
what are Microelectrodes
thin metal wires with a fine tip that can record the electrical activity of a single neuron (known as single-unit recording).
Electrodes are implanted in the brains of animals using stereotaxic surgery.
The wires are connected to a socket and the animals can be ‘plugged in’ to a recording system.
what are the 2 kinds of recording when it comes to neural activity
Chronic electrical recordings are made over an extended period of time.
Acute recordings are made over a relatively short period of time (often during surgery when the animal anesthetized).
To treat brain diseases, we often try to do what
change the activity of specific neurotransmitter receptors or specific cells or regions in the brain. Researchers test and study the effects of these manipulations on behaviour, often starting in rodents.
what are the 2 kinds of stimulation
Electrical stimulation
Chemical stimulation
what is Electrical stimulation
Involves passing an electrical current through a wire inserted into the brain. This will affect everything in the area (cell bodies and fibers of passage). Some electrical stimulation patterns, counterintuitively (often very high frequencies), tend to produce the same behavioral effects as lesioning the brain area.
what is Chemical stimulation
Is achieve with drugs. In rodents drugs are often administered through a guide cannula (hollow tube) implanted in a particular brain region. Anesthetics can be injected to shut down all neural activity. Alternatively, the drug may interact with receptor proteins, which likely won’t be located on fibers of passage (i.e., the axons just passing through the area).
In non-human animals we have developed ways to use light to depolarize and hyperpolarize neurons with millisecond precision– what does this allow us to do
We can now turn up and turn down the activity of specific cells and receptor signaling cascades in any given brain region or brain circuit
what is Optogenetics
refers to the use of light to control neurons which have been made sensitive to light through the introduction of foreign DNA. This foreign DNA encodes light-sensitive proteins known as opsins.Opsins are proteins that are sensitive to light.
what are opsins
We have opsins in our eye which we use for vision. Those particular opsins are metabotropic receptors and have a 30 millisecond delay. In contrast, the opsins we use for optogenetic experiments are typically ion channels with millisecond precision. The original ones were discovered in bacteria all over the world. Recently people have started to intelligently modify/design opsins for research purposes.
how do optogenetics work
Lots of different photosensitive ion channels evolved in bacteria and algae. A popular excitatory one named channelrhodopsin-II (ChR2) is permeable to sodium ions, so it depolarizes neurons and causes spiking when activated by blue light.
Other opsins, such as IC++ (designed by humans), are inhibitory light sensitive ion channels that pass chloride and hyperpolarize neurons when activated by blue light
what is virus
a smallinfectious agentthat replicates inside the cellsof other organisms. The DNA of a virus encodes instructions on how to make more virus.
what is Viral-Mediated Gene Delivery
Avirusis a smallinfectious agentthat replicates inside the cellsof other organisms. The DNA of a virus encodes instructions on how to make more virus.
We know how to remove the DNA from a virus, which renders the virus “replication-deficient”. We can also add foreign DNA to the virus, DNA that encodes things like fluorescent proteins or optogenetic proteins.
When a modified virus is injected into an animal’s brain, it infects the cells it comes into contact with. Some viruses infect cell bodies (e.g., AAV), others infect axon terminals (e.g., rabies). Once the virus gets its DNA into the infected cell’s nucleus, that cell will start to transcribe it and make the foreign protein.
Almost all lab-made viral constructs contain a section of DNA that encodes a fluorescent protein (e.g., GFP). Fluorescent proteins are used to later identify the infected cells.
If you want to know what an area of the brain is good for, what should you do
cut it out of a rodent and see how their behaviour changes.
what is Experimental ablation
Experimental ablation (lesion study) involves the removal or destruction of a portion of the brain. Presumably the functions that can no longer be performed following the surgery are the ones the brain region normally controls.
This approach is much cheaper and more straightforward than optogenetics and the other similarly fancy modern techniques.
How do scientist create small lesions in the brain (without using a knife or icepick)?
One approach is to burn the tissue…
what is Radiofrequency Lesions and how does it wotk
Small lesions can be made by passing radiofrequency current through a metal wire that is insulated everywhere but the tip.
This electric current produces heat that burns cells around the tip of the wire.
The size and shape of the lesion is determined by the duration and intensity of the current.
A downside to this approach is that axons just passing through will also be burned
what is Excitotoxic lesion
Brain lesion produced by intracerebral injection of a glutamate receptor agonist, such as kainic acid. These drugs cause so much excitation (calcium influx) that the affected neurons often undergo apoptosis, while axons passing through (fibers of passage) are usually spared.
what is Sham lesion
“placebo” procedure that duplicates all sets of the producing brain lesion except for one that actually causes extensive brain damage
what is Reversible lesion
A temporary brain lesion can be achieved by injecting drugs that block or reduce neural activity in a given region. Common drugs include…
GAGA receptor agonists (which hyperpolarize cell bodies)
Voltage-gated sodium channel blockers (stops all action potentials)
what is Retrograde labeling
tracing afferent axons
explain Retrograde labeling
What brain areas send their axons here? Retrograde labeling is used to label the cells that innervate (project to) a given region
Various chemicals, such as fluorogold can be used as a retrograde tracer
Fluorogold molecules are taken up by axon terminals and transported back to the cell body.
what is Anterograde labeling
(tracing efferent axons)
explain (tracing efferent axons)
Where do the axons from these cells go? Anterograde labeling is used to label where axons from a particular location go to.
Various chemicals, such as PHA-L, can be used as an anterograde tracer.
PHA-L molecules are taken up by cell bodies and transported down to axon terminal
what is Immunohistochemistry
Also known as, immunochemistry, immunocytochemistry, immunos, etc.
a histological method that is used to label proteins and peptides in biological tissue.
give an in-depth explanation of Immunohistochemistry
The immune system of mammals produces antibodies, which are proteins that circulate within the blood.
Antibodies are designed to identify and bind short amino acid sequences (i.e., proteins) that are foreign to the animal. The protein sequences that antibodies bind to are known as antigens.
When an antibody binds to an antigen, it signifies to the body that this protein should be broken down and destroyed. But before destroying the foreign protein, the immune system will first make lots of different kinds of antibodies that target/bind to different portions of the foreign protein to prepare for a potential invasion.
Biochemists know how to extract and purify specific kinds of antibodies from mammals, antibodies that bind particular proteins. They also know how to modify antibodies to make them fluorescent (i.e., by conjugating them to fluorescent protein such as GFP).
If somebody gives you a fluorescent antibody that selectively binds to a specific receptor, you could wash that antibody over a brain slice to identify where that receptor is located.
So, the trick is simply that we need some mammal to make antibodies that bind to particular neurotransmitter receptors (or any protein or peptide of interest).
How to make an antibody for a specific protein (like a specific serotonin receptor) to label it in brain slices:
How to make an antibody for a specific protein (like a specific serotonin receptor) to label it in brain slices:
Synthesize or purify the protein you want to label (e.g., the serotonin receptor 5HT2B).
Attach (conjugate) this protein to an antigen, which is simply a protein that is known to elicit a large immune response in a mammal, such as rabbit.
Inject the mammal (rabbit) with this combo protein (the antigen conjugated to the 5HT2B receptor protein).
Wait for the rabbit’s immune system to create natural antibodies that target this foreign combo protein. These newly made antibodies will bind to different parts of the 5HT2B-antigen combo protein.
Draw blood from the rabbit and purify the recently created antibodies that bind to your protein of interest.
Attach (conjugate) this newly created serotonin receptor antibody to a dye or fluorescent marker.
Now you have a fluorescent antibody that can be used to label the protein you are interested in.
So, simply wash some brain slices with a solution containing this antibody and all the 5HT2B receptor protein in the brain slices will become visible.
Neural Activity Often Correlates with what
Changes in the Expression of Immediate Early Genes
what is meant by Neural Activity Often Correlates with Changes in the Expression of Immediate Early Genes
Immediate Early Genes are genes that tend to be expressed following periods of elevated spiking activity.
c-Fos is an immediate early gene. c-Fos protein levels rise in the nucleus of neurons in the minutes after increases in neural activity.
Levels of c-fos protein are often measured with immunohistochemistry.
how to Measuring signaling molecules in the Brain
It is also possible to measure changes in the amount of signaling molecules in the extracellular space of particular brain regions during certain behaviors.
Dialysis refers to the use of an artificial semipermeable membrane to either deliver molecules to or measure the amount of molecules in some solution or brain area.
Microdialysis probe
Small metal tube that holds dialysis tubing. These can be placed in the animal’s head.
what is Dialysis
refers to the use of an artificial semipermeable membrane to either deliver molecules to or measure the amount of molecules in some solution or brain area.
what is Microdialysis probe
Small metal tube that holds dialysis tubing. These can be placed in the animal’s head.
what is Stereotaxic Surgery
Stereotaxic surgery is a surgical intervention that uses a stereotaxic apparatus. This is a device that permits a surgeon to put something into a very specific part of the brain.
It is used to inject things into the brain, such as drugs, viruses, or tracer molecules (dyes).
It is also used to permanently implant things, like cannula, electrodes, or fiber optic cable.
what is Stereotaxic Atlas
A page from a stereotaxic atlas of a rat. A stereotaxic atlas is a collection of drawings and pictures of brain slices from a particular animal that provides coordinates for stereotaxic surgery.
what is Bregma
The junction point where pieces of skull fuse together. Bregma is often used as a reference point for stereotaxic brain surgery.
what are Common Reasons for Stereotaxic Surgery:
Stereotaxic surgery is commonly used for one time injections of drug or virus to:
Lesion a brain area (e.g. excitotoxic lesion)
Lesion a specific type of cell in a particular brain area (e.g. inject a compound that specifically kills dopamine neurons)
To change gene expression (e.g. to remove a gene needed for serotonin synthesis or to deliver DNA that encodes a foreign protein). This is typically accomplished with viral-mediated gene delivery.
Stereotaxic surgery is also used to:
Implant cannula (hollow tubes) to allow for later infusions of drugs. E.g., temporary (or reversible) lesions can be made by infusing a local anaesthetic (e.g. lidocaine) which blocks action potentials.
Implant microelectrodes for stimulation or recording experiments.
Implant fiber optic cables to allow for imaging or stimulation using optogenetic techniques.
what is Fixative
Chemical like formaldehyde; used to preserve body tissue
what is Microtome
Instrument that produces very thin slices of body tissues. It is also called a vibratome (vibrating microtome). Another version is the cryostat, which keeps the tissue frozen during slicing (for thinner slices).
what is Nissl Staining
Without some contrast agent (dye or stain), it is hard to see what you are looking at in a brain slice.
A Nissl stain refers to a particular type of blue dye, but the term is also used in general to refer to dyes that stick to DNA and thus stain the nucleus of cells.
One of the most commonly used stains in neuroscience is the. It stains cell bodies
what is Genome
Complete set of genes that compose DNA of particular species
what is Allele
A specific version of a gene. For example, in the general population, there are three different versions of the serotonin 5HT-2B receptor that are commonly observed. This means that there are three common alleles of that gene in the population.
