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20th century was a century of __________ in Biology.
21st century is a century of ________ in Biology.
Describe Neural Circuits.
- They underlie all aspects of behavior.
- A wide variety of structures and regulatory proteins are required for a single circuit.
- Simple organisms have simple neural circuits and vice versa.
- Even subtle changes in expression of a single gene can affect behavior.
Subtle changes in the expression of a single gene can affect behavior, such as:
- Absence of expression
- changes in degree of expression
- expression of splice isoforms
- changes in expression pattern
- can overlap and take care of several functions
What is Phenylketonuria? What's it an example of?
- It is the inability to break down the AA phenylalaine.
- It is a recessive autosomal inherited trait.
- It's an example of a diseas caused by the change of a single gene.
What are some model organisms in Neural Science?
Mice, Elegans, Fruit flies, monkeys, frogs.
Journals that publish neuroscience:
- Journal of Neuroscience
- Nature Neuroscience
- Behavioral Neuroscience
Where is genetic information stored?
How many chromosomes do humans have? What kind?
46. 22 pairs of autosomes and a 2 sex chromosomes.
What do genes do?
- Regulate behavior
- Emotional regulation of behavior
Human brain and _____ brain react ______ to ________ stimuli in the ______ (region of the brain).
Mice ; Similar ; fear ; amygdala
Two major genetic approaches for studying specific DNA sequences
- 1. Specific amplification (DNA cloning)
- 2. Specific detection
Cloning of PCR products. (Types)
- Blunt-end cloning
- TA cloning (A- over hang)
- incorporation of restriction sites in PCR oligoprimers
Classical methods to detect and analyze gene transcription products
- Northern blotting
- cDNA library
- RNA in situ hybridization
- + Quantitative. Size. Integrity. Amount
- -Not sensitive. Need large amount.
- Beneficial because you can isolate small amounts of RNA.
- +Quantitative. Very Sensitive.
- -Need to know sequence by designing primers.
pros and cons of RNA in situ hybridization
- +Allows to analyze where a gene of interest is expressed in the brain. Can be semi-quantitive if radioactive.
- -Not always possible.
- Makes proteins.
- Use antisense probe.
- Doesn't make protein.
- Use sense probe.
Steps of RNA in situ hybridization
- 1. Create slides: Cut brain sections and place on glass slides using cryostat at -25oC
- 2.Creating a probe: cDNA or cRNA or DNA-oligo.
- 3.Labeling the probe4. Put on the brain slice and let it hybridize over night at 60oC. Then wash away probe.
- 5. Observe with radioactive x-ray film or under a microscope.
What 3 probes can be created for in situ hybridization? How is each created? What are the benefits of each?
- 1. cDNA: 200-500 bp long clone DNA using restriction enzyme for cleavage or PCR. Denature at high temperature for single strand.
- 2. cRNA: riboprobes. Sense or antisense. Can use ribonuclease to destroy cRNA that is undesired. -Degrades.
- 3. Oligonucleotides: you must know the target sequence. Rapid production and inexpensive= very common.
Why can't 32P be used for in situ?
- It is a very high energy isotope, too much for in situ.
- Can use for Northern and Southern blotting.
Radioactive oligo in situ hybridization
- DNA is labeled using 33P or 35S radioactive dNTP transferase enzyme.
- Oligo is applied to the brain sections in a hybridized buffer over night.
NeuN is a ___________ in ____ neurons.
- protein ; all.
- Serves as a marker.
Visualization of gene expression in vitro and in vivo uses
- GFP (green fluorescent protein)
________ is expressed in pyrimidial neurons in the amygdala.
Gene chip analysis is used for _________________.
analyzing gene transcription products (RNA).
What are the 2 methods used to create genetically altered animals?
- 1.Gene Targeting
- 2. Transgenics
What is "Gene targeting"?
- A method used to genetically alter animals.
- A homozygous animal is created.
- Has arms, selection cassette, some deleted sequences.
- Used for knockout and knock in.Transfer gene targeting vector into Embryonic stem cells. Implant this cell into a blastocyst and then into a female animal's uterus.
What is "Transgenics"?
- A method used to genetically alter animals.
- Has a promoter, cDNA, exon/intron, poly A tail.
- Using a pronuclei in a fertilized egg you select the desired gene with the vector and implant it into the uterus of your animal.
- A hemizygous animal is created.
- DNA is integrated randomly into the genome.
Most genes are ________. What does this mean?
Pleiotropic. A single gene can influence multiple phenotypic traits. They are expressed in different tissues in different ways at different times.
Transgenic DNA has _________ injection. What does this mean?
Random. It can go anywhere, except in the case of knockout. It has a positional affect. Depending on where it goes it may or may not be effective.
Cre- recombinase system is used for ________.
Cre mouse expresses the gene after __________.
How do you regulate transgene expression?
Using tTA system in the mouse brain. Insert tTA after the gene promoter before the poly A tail. If you want to turn the gene OFF, feed the mouse DOXYCYCLINE.
this is SPATIO-TEMPORAL control.
What does Doxycycline do? What is it used for?
It is used to turn of the desired gene in transgene expressive mice by blocking the bining site/changing its conformation.
What is CaMKII?
it is a protein kinase (Regulator of cell function). The CaMKII promotor from tTA controls expression. Involved in memory formation of mice (LTP).
The hippocampus is involved in __________.
Complex memory of directions. Spatial & learning memory.
Of various higher cognitive abilities, ______ is the only one that can be studied effectively in ______ __________ __________ that are accessible to ______ ______.
memory ; simple experimental organisms ; genetic manipulation.
In Drosophila, Which pathway is recruited during learning via Ca2+ stimulation? What stimulation does this result in? What does this lead to? What genes become active?
- cAMP signaling pathway
- stimulation of the cAMP-dependent protein Kinase (PKA)
- increase in CREB phosphorylation
- the genes that stabilize memory become active
Generally the activation/induction of __________ ultimately leads to _________ of ______.
CREB ; stabilization of memory
Which promoter from tTA controls expression?
Long term memory for learned fear in human and mice requires what 2 things?
PKA & CREB
CaMKII can be regulated with what?
Is the relationship between transgene size and number of copies equally or inversely proportionate?
Inversely. The smaller the transgene the more copies you can make.
True or False: in Gene Targeting you can control expression.
True. However, no such control is available in transgenics; the integration is random.
PKA ____ CREB.
How do you alter a mouse so that you can have spatio-temporal control of the desired gene?
Create a transgenic mouse using the tTA system (in which you can feed the mouse Dox to turn off the gene of interest).
What is the difference between tTA and rTA systems?
In tTA systems, your gene of interest is shut OFF through Doxycycline ingestion. However, in rTA systems Doxycycline will turn the gene ON.
What is the main benefit of using an rTA system of the transgenic approach?
Without the drug, the animal will act as the wild type.
Ca2+ induces ________.
How is a conditional/region specific knockout generated?
- Using CRE recombinase you can generate a conditional knockout by mating a mouse with a loxP->cDNA->loxP and a Promoter->CRE->PolyA tail mouse.
- Area between the loxP sites will be deleted.
What is the most popularly used strain of mice?
C57BL/6J or just C57.
True or False: all strains of mice have the same results to the same mutations.
FALSE! Different strains can react differently to the same mutations and comparing strains is important.
What is are the functions of microtubules?
Transport (walk along them).
What should you do to your mice before you conduct behavioral experiments?
strain 129 x C57, then that strain x C57, etc. for 5 generations.
How do you conduct a Morris Water Maze test? (5 steps)
- 1. get 2 groups (KO and wt)
- 2. for the first few days have a visible platform (KO and wt mice should have similar results)
- 3. make the platform hidden
- 4. remove the platform
- 5. replace platform in different location
Name mice's 2 reactions to fear.
- 1. Flee
- 2. Freeze then fight
What kind of task is a non-spatial test that requires hippocampal function?
Novel-Object recognition task.
Describe the novel object recognition task. What does it test?
- A mouse is placed in a box with 2 familiar objects, then the mouse is removed. When placed back in the box there is one familiar object and one novel onject. Usually mice stay more towards the novel object.
- This is a non-aversive, non-spatial task that requires hippocampal function.
What aspects and areas of the brain does the Morris Water maze test?
- Spatial and learning memory
- Hippocampal function
What does the Radial arm maze test?
Spatial learning, long term and working memory that requires hippocampal and prefrontal cortex function.
What does tTA control?
What are phosphotases?
Protein Enzymes that dephosphorylate various proteins and enzymes usually turning off pathways.
What are Kinases?
Protein enzymes that phosphorylate various proteins and enzymes and usually turn on pathways.
PKA has 2_______ and 2________. cAMP ________ to which 2? What do the other units do?
- 2 catalytic subunits
- 2 regulatory subunits
- Binds to the regulatory subunits which stay in the cytoplasm while to other leave to phosphorylate transcription factors like CREB.
Which receptors are activated by Calcium influx?
How is CaMKII activated?
Calcium influx activates NMDA receptors which activate CaMKII.
What part of the brain is used to learning and memory?
Hippocampus (prefrontal cortex for working memory)
What aspect of memory is the prefrontal cortex involved in?
What area of the brain is responsible for memory and learning of fear?
Describe fear conditioning.
A mouse is placed in a box. A tone is played for a few seconds and then while the tone continues to play the mouse is shocked. Repeat this until the mouse is conditioned. Later the mouse can be moved to a different box, with the same tone, and it will freeze. This is a fear response due to reactions in the amygdala. When a mouse is placed in the same box and it freezes this is due to context memory of the hippocampus.
Describe conditioned taste aversion.
A mouse is fed saccharine (sweet water) which it really likes. A few hours later it's injected with LiCL or some other chemical that will make it sick. Later, the mouse will avoid saccharine. If given water and saccharine it will chose water.
Describe ihibitory avoidance.
A mouse is on a box surrounded by metal grids, when the mouse steps on a grid it is shocked. The mouse learns not to step on the grid. Memory is assessed by time spent on the grid.
Describe passive avoidance.
Here mice learn to inhibit a natural tendency like sleeping in the dark because it has been associated with shock.
Desribe latent inhibition.
Extended preexposure to a safe stimulus makes it difficult for the mouse to associate danger with it. For exampled, a cage that it has been safe it many many many times and is shocked once, when placed back in the cage it won't exhibit fear.
CREB is phosphorylated by ____________
catalytic subunits of PKA in the nucleus.
CREB is involved in _________ formation.
The first transcription factors to be expressed are what? What does this suggest they are useful for?
- IEGs (immediate early genes).
- Short term memory formation
What happens in Trace fear conditioning?
The tone isn't played simultaneously with the shock it is played after so it is harder for the mouse to become conditioned to expect a shock with the tone.
What is Kinesin?
Kinesin is a transport molecule that moves along microtubules.
Short term memory is ________ dependent.
Long term memory is _______,___________, and ________ dependent.
Protein; transcription; translation
What is a Prion?
A protein that can change conforation and aggregate for changes of the brain.
If you add _________, what happens as new __________ grow? Is this indefinite?
- CPEB3 ; dendritic spines.
- Long term memory improves.
- But only to a certain extent, too much of a good thing isn't always good.
Ubiquitination facilitates _______________________ and ____________ by modulating the activity of CPEB3.
hippocampal plasticity ; hippocampal memory storage.
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