Lecture 1 & First Week Assignments Flashcards
1
Q
What are in vitro models concerned with? (3)
A
- Bacteria/ Yeast cells
- Cell structure
- Tissue slice
2
Q
What are in vivo models concerned with? (3)
A
- Invertebrates
- Animal
- Human
3
Q
What are in silico models concerned with?
A
Computers
4
Q
Why are rats popular as study animals? (6)
A
- Cheap housing/ maintenance/ breeding
- Friendly disposition- easy handling
- Relatively intelligent and agile
- Resistant to infection
- Many inbred strains available
- A lot of information known
5
Q
Why are mice so popular?
A
-Convenient genetic manipulation
6
Q
What two broad types of experimental techniques are there?
A
Manipulation and monitoring
7
Q
What five types of manipulating experiments are mentioned?
A
Lesions Electrical stimulation Pharmacological Genetic manipulation Behavioural manipulation
8
Q
What types of lesions are there? (4)
A
Mechanical
Electrolysis
Chemical e.g Ibotenic acid- only cells are destroyed. Fibers of passage are spared
9
Q
What two broadtypes of monitoring techniques are there?
A
In vivo and Ex vivo
10
Q
What three types of In vivo monitoring techniques are there?
A
- Electrophysiology (electrical)
- Micro-dialysis (chemical)
- Behavioural evaluation (tasks)
11
Q
What types of ex vivo monitoring techniques are there?
A
Localisation/ Quantification of tissue components in situ
Quantification of components in tissue homogenates
12
Q
What does the stereotaxic apparatus entail?
A
A stereotaxic frame is a research (or medical) tool allowing fixation of the head in a standardised way. Vernier bars in three dimensions allow precision manipulation of drills, electrodes, liquid probes and other instruments in the brain.
13
Q
How can this apparatus be used on humans?
A
In neurosurgical practice, this allows a.o. for precision targeting of tumours or epileptic cells, or the insertion of electrodes for deep brain stimulation.
14
Q
How is the stereotaxic apparatus used on a rat?
A
In case of the rat; an fully anaesthetised specimen is fixated in the stereotaxic frame by way of an incisor bar (a bar below its upper jaw incisors) and a metal pin in each ear canal.
15
Q
Name and describe the three reference points on the skull of the rat
A
The interaural line, is one point of reference that can be used to navigate the rat brain. It is the line you can draw between the ear canals.
Bregma, is the second point of reference. It is the point on the skull where the frontal bone meets the parietal bones, or the intersection between the coronal and sagittal sutures.
In some cases, lambda (the point where the occipital bone meets the parietal bones, 9 mm posterior to bregma) may also be used.
16
Q
What are these three reference points used for?
A
On the basis of these reference points a three dimensional coordinate system is used to describe any location in the brain, with a anterior-posteror-axis (AP), a medio-lateral axis (ML) and the dorso-ventral axis (DV). Point Bregma (AP, ML, DV) has coordinates (0, 0, 0).
17
Q
How much do locations of brain nuclei vary between rats? What implications does this have?
A
In rats of a similar age class, variation between the location of brain nuclei with respect to bregma is below 0.5 mm. This coordinate system allows for a precise communication about the location of brain structures: e.g. the corpus callosum extends from bregma 2.2 to bregma -4.52. (in other words, it starts 2.2 mm anterior to bregma, and ends 4.52 mm posterior to bregma).
18
Q
What can be done for smaller or bigger rats
A
For smaller or bigger rats a simple scaling can be applied on the basis of the distance between Bregma and Lambda.
19
Q
In Paxinos and Watson’s atlas, what type of cuts and how far apart are they?
A
high-resolution drawings of 65 subsequent coronal sections (about 0.25 mm apart) , starting at the level of olfactory bulbs and extending to the posterior cerebellum and medulla oblongata.
20
Q
What in the level at which each section has been cut indicated by?
A
The level at which each section has been cut is indicated with bregma (dorsal) and interaural line (ventral) coordinates. If a section has been cut exactly in the coronal plane, the interaural line is always 9 millimeters behind bregma (Interaural -9 =bregma). However, if a section has been cut at an angle, reporting both bregma and interaural coordinates allows for precise reproduction of that angle.
21
Q
What do the capitals in the index indicate? How else is this represented in the drawings?
A
Grey matter (nuclei and laminae) are indicated with CAPITALS, white matter in lower case letters. In the drawings, white matter is outlined with a solid line, grey matter with dotted lines.
22
Q
How do you find the anterior posterior co-ordinates on a given page?
A
Top left hand corner shows where the section is cut
23
Q
How are ventricals drawn in diagrams?
A
Ventricles are depicted in solid blue or black.
24
Q
What do bold lines indicate in diagrams
A
Borders of tissue.
25
In the anterior and posterior part of the brain, where does the corpus callosum split?
In the anterior part of the brain the corpus callosum splits into the forceps minor and in the posterior part of the brain into the forceps major.
26
In order to study tissue under a microscope, it has to be cut into thin slices. What is a pre-requisite for this and how can it be accomplished?
A prerequisite is that the tissue is, and stays, in prime condition. This can be accomplished by either freezing or fixating.
27
When is it preferred freezing and when fixated?
If retention of fatty tissue and certain heat-sensitive proteins is required, freezing techniques are generally applied.
when paraffin is used for embedding of the tissue, prior fixation is required. This is the case for the brain slices used in the 2nd practical with this course: the tissue has been fixated in paraformaldehyde, embedded in paraffin, sliced, and then deparafinnated.
28
After freezing, how can the tissue be cut?
After freezing, the tissue can be cut with a cryostate or a sled microtome
29
What is the purpose of fixation? (4)
- Giving the tissue a certain degree of rigidity
- Keeping intra- and extracellular structures in place
- Making the tissue fit for histological staining
- Making the tissue permeable for the substance used for embedding.
30
What may be used for fixating? What does this depend on?
Various compounds (e.g. ethanol, formaldehyde) or mixes (e.g. Bouin’s fixative) may be used for fixation; the selection depends on both the type of tissue and on what it is we need to be able to study
31
Considering bacteria and yeast cells do not have a nervous system, how is in vitro neuroscience research concerned with them?
They can be used to express molecules of the nervous system for example receptors such as yeast cells and dopamine receptors
32
How is studying cell cultures of interest to neuroscience research?
Neurons can be grown together on a petri dish connected to each other to form a network. This is handy to investigate interneuron communication. They can be reached very easily with micro electrodes and expose them to antibiotics, neurotransmitters etc to see how they react.
33
how are tissue slices of interest to neuroscience research?
They can be kept alive to stimulate some structures and record from other structures. This gives good access to cells without the complexity of recording from a brain that is in an animal
34
Why are computers used in neuroscience research?
To simulate neural structures and processes or other molecules to see how they interact with each other.
35
What percent of animals used in research are 1) rodents and 2) more complex animals (dogs, cats, monkeys)
1) 90%
| 2) 1%
36
How is electrical stimulation used in experimental manipulation?
To excite or inhibit neural function
37
Give three examples of pharmacological methods of experimental manipulation
=> Systemic, intracerebroventricular, or local intracerebral injection
=>Osmotic pumps
=>Microdialysis
38
How are organisms motivated in behavioural manipulation?
appetitive (rewarding) or aversive stimuli
39
Give three examples of techniques within Localisation/ Quantification of tissue components in situ
- Histology
- Immunohistochemistry
- In situ hybridisation for mRNA or DNA
40
What kind of techniques are used for the quantification of components in tissue homogenates
loads of techniques including genomics and proteomics
41
What is meant by the term "in situ"?
In their natural place
42
What are tissue homogenates
Brain yoghurt made from the tissue of interest through the vroom vroom vroom technique
brain tissue paste essentially
43
What kind've brain surgery is stereotaxic equipment used? (6)
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=> Lesions
=>Microinjections
=>Cannula placement
=>Micro-dialysis
=>Electrodes
=>Head sets
```
44
The atlas indicates that the amygdala target site is 2.8mm posterior to bregma, 4.5 mm lateral and 8.5 mm ventral.
Based on this information how would you place an electrode in the amygdala?
A hole is drilled 2.8 mm posterior to bregma and 4.5 lateral to it. Then, the electrode holder is positioned over the hole, and the electrode is lowered 8.5mm through the hole.
The electrode is anchored to the skull by letting dental acrylic harden around the electrode connector and several stainless steel skull screws.
45
Deacribe microdialysis in principle
The probe is lowered into the brain tissue. The probe has an inner tube and outer tube. There is a flow of liquid from the inner tube and a flow coming out of the outer tube. The membrane of the outer tube is semi-permeable, allowing water to freely flow in and out as well as certain molecules. The substance is chemically analysed.
46
What is the significance of the semi permeability of the probe?
It mimics a capillary in the brain (blood cells stay in, waste flows in and important substances for the brain flow out)
47
Give another name for the inflow and outflow of microdialysis
Outflow- Dialysate
| Inflow- Perfusate
48
What is contained in the perfusate and dialysate?
Perfusate- Physiological salt solution
| Dialysate- contains analyte
49
What value must the net inflow and outflow have ? why is this important?
Net value of 0, because otherwise liquid would be pumped into the brain (increasing pressure) or extracting from the brain (sucking it dry)
50
What is the purpose of the physiological salt solution?
It will flow from an area of high concentration (probe) to an area of low concentration (brain) and the molecules in the brain will do the same
51
Name some applications of microdialysis (3 kinda)
Continuous monitoring of chemical events in living tissue; gives temporal information (per minute etc) and allows sampling of multiple compounds contemporaneously
Continuous drug delivery
Contemporaneous drug delivery and monitoring of drug effects
52
What levels are there to electrophysiology? (5)
```
Single ion channel
Single uint (one cell)
Multi unit
Field potentials
'Brain potentials'
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53
Give three methods of measuring brain potentials
Electrocorticogram (ECG)
Electroencephalogram (EEG)
Magneto encephalography (MEG)
54
How can one record a single ion pump
Patch clamp recording: records current from a single channel
You examine the tissue using a high power microscope and bring the probe close to the surface of a single cell. This probe is a tiny glass pipette. You then apply a little suction (often via the mouth) to collect it onto the surface of the pipette and a tight seal is formed between the pipette and the cell (both fluid and electric).
The pipette is full of salt solution (conduction) and a hair (thinner than human hair) electrode is placed inside the pipette. Another electrode is placed in the bath outside the cell.
55
Where is does this ion pump recording take place ?
In cell cultures or slices
56
How would you move/ manipulate the pipette in this experiment?
By moving the wheels of a micro manipulator (moves it in micrometers or even more precise)
57
How is the electrophysiology of one cell recorded?
Intracellular recording:
A sharp electrode penetrates the cell membrane. The electrodes record voltage or current over cell membrane and these are contrasted against an extracellular electrode in a bath. A stimulating electrode can also be utilised to study the effects of such.
58
Where are these single-unit recordings usually made?
In tissue culture or slices (very hard to do in vivo)
59
In both these experiments name two pieces of equipment required to observe the recordings after they have been picked up by the electrodes
Amplifier and the oscilloscope
60
What happens when an electrode stimulates the cell so that (a) there are a set of gradually increasing hyperpolarising waves (b) gradually increasing depolarising waves
(a) the trend is reflected in the cell membrane where there are dips of gradually more negative voltage
(b) there are short hills of less negative voltage until it hits a threshold of around -55mV and a large spike is observed to around 40mV (action potential) then hyperpolarises back down to around -80mV before going back to the resting potential of around -70mV (depending on cell and reference electrode)
61
How are multi-unit recordings usually carried out?
```
Extracellular recordings (can be single or multiple)
A recording electrode (a little larger) is placed very close to a cell or a small bundle of electrodes (often 4) placed next to multiple cells. There are often multiple tips to the electrode at varying heights of the needle to make multiple recordings of very proximal cells. A grounded electrode is then used in reference to these recordings. Data analysis techniques are then used to disambiguate contribution of different cells to the recording.
```
62
Where do these multi-unit recordings usually take place
can take place in cell culture or slice or often in vivo.
63
How are field potentials recorded?
Also extracellular.
Records synchronous activity from many cells (around one or a few cubic mm of tissue) and compares this to a grounded electrode.
If an important stimulus hits a part of the cortex then a lot of the cells will be activated. If you measure just outside it, then you would be measuring the response of a whole lot of neurons that are opening their channels. If it is an excitatory channel, then when a positive charge arrives, the neurotransmitter will be released from the opposite synapse and trigger depolarisation of the cell which causes a release of negatively charged ions into the extracellular fluid. The electrode can measure this difference of voltage. The electrode therefore measures for negativity caused by a range of action potentials in proximal cells.
64
Where are these field potential recordings made?
Can be in slice but mostly in vivo
65
When these are measured in vivo, what differences does it make whether the animal is anaesthetised or not in regards to equipment utilised?
Anaesthetised- stereotaxic procedure
Awake animal- headset, electrode position adaptable with microdrives (i.e. screws to move the electrodes with micrometer precision.)
66
What is the lowest resolution to record electrical activity of the brain?
EEG- further away so electrodes get bigger, spatial resolution gets lower, recording from larger parts of the brain
67
What is used as a reference for EEG recordings?
The potential differences are recording between electrodes on the scalp. This could be for example, versus an a reference electrode or the average potential of all electrodes.
68
What does EEG source it's signal from? (2)
The synchronous activity of many (many!) neurons, especially from elements with similar spatial orientation.
Extracellular ion streams caused by dendritic electrical activity of cortical neurons (postsynaptic potentials)
69
Where does EEG get virtually no contribution from?
Action potentials
70
When this many synchronised epsp's or ipsp's that occur in a lot of cells in a particular area, what kind've activity are we generally talking about?
Modulatory activity (acetylcholine, dopamine etc), not so much one an one transmissions.
71
Why does EEG not pick up on the action potentials? (2)
Mostly recording the dendritic part, not the action potentials because they are too scarce and axons go in many directions, even if there were enough action potentials they would have to be going the same directions but they don't, and therefore cancel each other out.
72
What trend is also seen in the observed potentials?
They are oscillating
73
How may these oscillating patterns differ
There are characteristic frequency bands with different spatial distributions.
They are associated with different arousal/ cognitive states.
74
Where do these oscillating patterns arise from?
Neuronal and network characteristics (some are understood, others not)
75
What is meant by genetic manipulation? give some alternate names
The direct manipulation of an organisms genes
76
Which of these is not synonymous with genetic manipulation? explain
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genetic engineering,
Spontaneous mutations,
recombinant DNA technology,
Inbred strains,
genetic modification,
transgene technology.
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Inbred strains are essentially selective breeding to obtain selective traits but this is not what is meant by genetic manipulation as it is not manipulating the genes directly the the DNA level
Spontaneous mutations occur naturally when genes mutate, which is the basis of our genes mutating to adapt to their environment. (although are often maladaptive)
77
What is meant by the transgene?
The foreign DNA inserted into the host organism
78
What is meant by the transgenic organism?
The organism transformed by the transgene
79
What are the two main possibilities in genetic manipulation? When are these cases most often true?
=> Foreign DNA exists in the cell in the form of a plasmid (more common in prokaryote hosts)
=> Foreign DNA is integrated into the host genome (Always the case in mammals)
80
What is significant about the DNA existing in a plasmid in prokaryote hosts?
DNA is more resilient to degradation in the circular structure of a plasmid than a linear one ( this is only the case in prokaryote organisms- not so much in eukaryote hosts)
81
What common uses are there for genetic manipulation? (3)
=>Modification of crops, for resilience to pests/ infections and longer freshness
=> Use of GM bacteria to produce biomedical molecules e.g insulin, vaccines, enzymes for research
=>Production of transgenic animals (mice) for research
82
Describe the principle of recombinant technology in basteria (in 5 steps)
1. Isolation of the gene of interest
2. modification of the gene
3. insertion of the gene into a vector
4. Insertion of the vector into cells of the organism to be modified => transformation
5. Tests to isolate genetically modified organisms
83
What is used to isolate the gene (and free a segment of the plasmid)
the enzyme EcoRI
84
How is the gene fused with the plasmid? (name the technique and substance used)
Hybridisation and DNA ligase
85
What happens directly after the vector is inserted in a bacteria cell
The cell is cloned and the bacteria is platted on medium to grow, then into a culture before it is 'purified' (isolated)
86
What is done to ensure that only bacteria containing the recombinant DNA grows?
Plasmids are used which contain the gene for antibiotic resistance and the genes are platted with antibiotics (as well as nutrients) when on a medium for growing.
87
How can you insert the plasmid into a bacteria cell?
Heat them up a little to make the membrane more permeable and slide it in before cooling it again
88
How is recombinant technology applied in neuroscience? (3)
=> Loss of function experiments (Knock out mice- gene altered to cripple its function)
=> Gain of function experiments (knock in mice- Increased function of a gene, via extra copies or increased synthesis.)
=> Tracking experiments/ Expression studies
89
Describe what tracking experiments/ expression studies examine and how this can be carried out
When (in development) and where is a protein produced? (such as a dopamine receptor.)
- 'fusion' gene: a juxtaposition of the wild-type gene with a reporting element (something that you can see or track) such as Green Fluorescent Protein (GFP)
- Promoter is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyses the production of a dye
90
How may the term 'transgenic organism' have different connotations?
It has the strict meaning of an organism that has had DNA introduced into one or more of its cells artificially
However it also has the modern connotation of a transgenic organism that was transformed using a specific technique (the oldest technique.) The foreign DNA is integrated in the host genome in a random fashion, by injecting it into the pronucleus of a fertilised ovum.
91
What negative effects could this old transgenic technique have? (3)
+/- 10% disrupt an endogenous gene important for normal development
Often poor expression of the gene
Multiple copies
92
What new transgenic technique is now used?
"Knockout"(technique): DNA is introduced first into embryonic stem cells. Transformed ES cells are identified and injected into a 4 day old mouse embryo - a blastocyst.
93
How is this better than the old transgenic technique (2) and what drawback does it have (1)
- Targeted DNA insertion => through homologous recombination
- Much higher success rate
However it can only be carried out on a specific strain our mice as this is a procedure that most species don't survive.
94
This transgenic animal will now be a chimeric animal, what does this mean?
It only has modified dna in some of its cells
95
How do you go from a chimeric animal to one which has the dna in all of its cells?
Inbreeding
