Lecture 4

Question 1

Models of research

Answer 1

In vitro:
-bacteria, yeast cells
-cell culture
-tissue slice

In vivo:
-invertebrates
-more complex animals
-human

In silico:
-computer

Question 2

What is a model in neuroscience?

Answer 2

A controlled environment that reflects and enables access to certain aspects of a system (represent a limited aspect of reality

Question 3

Purpose of low-level models

Answer 3

1. Limiting the complexity of the experimental environment to those aspects of the system that are directly relevant to the research question
2. Maximizing accessibility to the structure of interest

Question 4

Animals most commonly used in research

Answer 4

Rodents (90%)

Question 5

Cell culture

Answer 5

stem cells kept alive and grown on a petri-dish, they make connections with each other etc.

Question 6

Mice in research

Answer 6

Convenient genetic manipulation

Question 7

Experimental techniques

Answer 7

Manipulating
Monitoring

Question 8

Manipulation techniques

Answer 8

-Lesioning: mechanical lesions (not done anymore), electrolysis, chemical
-Electrical stimulation: to excite or inhibit neural functions
-Pharmacology (used especially when you want to know how specific receptors respond to specific drugs etc.)
-Genetic manipulation - altering genes to asses their functions
-Behavioural manipulation

Question 9

Monitoring techiques

Answer 9

-In vivo: electrophysiology, microdialysis, behavioural evaluation
-Ex vivo: localisation/quantification of tissue components in situ (histology, immunohistochemistry, in situ mRNA or DNA hybridisation, quantification of tissue components in tissue homogenates

Question 10

Stereotaxic brain surgery

Answer 10

A surgery where the head is fixated, so you can approach brain areas with very high precision. (human brain surgery, experimentation on animals). Good for: lesions, microinjections, cannula placement, microdialysis, electrodes, and headsets. Use it with stereotaxic brain atlas (3-axis coordinate system, brain is related to a 0 point first)- 0 point in rats: Bregma (at the connection point of three skull plates)

Question 11

Microdialysis

Answer 11

Insert thin catheter in the brain with an inner and outer tube. To the inner tube physiological salt solution flows in (perfusate) and it flows back out through the semi-permeable (permeable to some things but not others) outer part after compound exchange (dialysate).

Question 12

Different electrophysiology levels

Answer 12

Single ion channel, single unit (cell), multi-unit, field potentials, brain potentials

Question 13

What do electrophysiology techniques asses?

Answer 13

The electrical current (?) and potentials that are generated in the brain

Question 14

Patch-clamp recordings

Answer 14

Recording currents from a single channel or a few single channels.
How it is done: very thin glass pipette inside of which is an even thinner metal electrode. Lower it under the microscope to the surface of a cell, apply a bit of suction. Record electrical activity in just the little patch of membrane with a/more channels. Reference electrode in a bath –> measure the difference between 2 electrodes

Question 15

Intracellular recording

Answer 15

Electrode sticks into the cell. Extracellular reference electrode in bath. Records current or voltage over cell-membrane. In cell culture or slice

Question 16

Extracellular recordings

Answer 16

Recording electrode close to a cell recording its activity, often in vivo, reference electrode is grounded (at 0 mV) and is somewhere on the animal. Nowadays often a bundle of electrodes is stuck into a location not just one.

Question 17

Field potentials

Answer 17

Extracellular recording of synchronous activity of many cells. Reference electrode is grounded, commonly done in vivo.

Question 18

The signal you record from the inside and the outside of a cell are…

Answer 18

Different

Question 19

EEG

Answer 19

Recording potential differences between different electrodes on the scalp (and a reference electrode, usually behind the ear). Signal source: synchronous electrical activity of neurons that have a similar spatial orientation. Micropotentials have to summate to be able to be recorded from the outside. Summation can only occur if the currents all go in the same direction from parallel elements. –> Mostly recording signals from dendrites in cortical areas. (also because axons usually do not fire synchronously just sporadically, synchrony in axon firing occurs in epileptic seizures). Basically we are recording modulation from modulatory systems.

Question 20

What are the frequency bands recorded by EEG associated with?

Answer 20

Specific arousal or cognitive states

Question 21

Genetic manipulation

Answer 21

The direct manipulation of an organism’s genes

Question 22

Related terms to genetic manipulation

Answer 22

Genetic engineering, Recombinant DNA technology, Genetic modification, Transgene technology

Question 23

Transgene

Answer 23

Foreign DNA inserted into the host organism

Question 24

Transgenic organism

Answer 24

The organism that has been transformed with the transgene

Question 25

In genetic manipulation the 2 main possibilities of inserting foreign DNA into host are:

Answer 25

1. Foreign DNA enters the host cells in form of a PLASMID - works in prokaryote hosts (bacteria and viruses)
2. Foreign DNA is integrated into the genome to survive and to be transcribed - in eukaryotes

Question 26

Plasmid

Answer 26

A circular strand of DNA that can sit in the other cell and can be processed in some organisms just like the host DNA (can be transcribed and proteins can be made)

Question 27

Common uses of genetic engineering

Answer 27

-Modification of crops (for resilience of pests and infection, stay fresh for longer)
-Genetically modified bacteria is used a lot in biomed to produce biomedical molecules: e.g. insulin, vaccines, enzymes for research
-Production of transgenic animals (for research)

Question 28

Principles of recombinant technology (in bacteria)

Answer 28

1. isolation of the gene of interest
2. modification of the gene
3. insert the gene into a vector (we get a plasmid that contains the gene for antibiotic resistance and the gene for insulin for example).Plasmid is put into a bath
4. Do procedures to help plasmid to slither into at least some of the bacteria - won’t work for all the bacteria. Antibiotic on the agar-coated petri dish kills the bacteria other than the one that contains the the gene of interest (bc it was coupled with a gene that has antibiotic resistance)
5. Isolate the genetically modified organism (and grow it further then)

Question 29

Application of recombinant technology in neuroscience

Answer 29

-Loss of function experiments (in mice alter the gene to cripple its function –> knock-out mice)
-Gain of function experiment (add copies of a gene) –> enhance function; or manipulate the gene to make it more transcribed
-Tracking experiments: when and where during development is a protein produced?-> Fusion gene

Question 30

Transgenic organism strict meaning

Answer 30

An organism that has had a DNA introduced into one or more its cells artificially

Question 31

Transgenic organism modern meaning

Answer 31

A transgenic organism that was transformed using a specific technique (the older one)
-The foreign DNA is integrated in the host genome in a random fashion by injecting it into the pronucleus of a fertilized ovum (problem: often poor expression of the gene, 10% disruption of endogenous gene important for development etc.)

Question 32

Newer gene manipulation technique

Answer 32

Work only in one strand of mice (knockout mice). take embryonic cells from a mouse fetus –> embryonic cells are transformed with foreign DNA. If that is successful, you select the embryos where this was successful, those embryos are placed in host mothers. Targeted DNA insertion: the experimenter is designing where in the host-DNA will the segment be inserted. Higher success rate. Done through HOMOLOGOUS RECOMBINATION: When double stranded DNA melts (i.e. the two strands come apart) it is possible for exogenous strands of DNA to pair with on of the ‘host’ strands.
This can happen when the ‘guest’ DNA is homologous with one of the original strands. (complementary code)

Question 33

Optogenetics

Answer 33

Used to manipulate the activity of specific cells in the brain through a genetic procedure. Use light-sensitive receptors coming from algae. If light hits them they activate the cell that they are attach to. Neurons can be activated or inhibited.

Question 34

Applications of microdyalisis

Answer 34

Continuous monitoring of chemical events in tissue
Used to continuously apply a drug
Contemporaneous drug delivery and monitoring its effects