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What are protein kinases?
a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation.
(addition of a phosphate group)
What are protein phosphatases?
an enzyme that removes a phosphate group from its substrate by hydrolyzing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group.
(remove a phosphate group)
Name 5 Protein Kinases that are involved in memory.
Name 3 seperate events that activate CaMKII.
- A) Calcium influx:
- B) Self-Phosphorylation: CaMKII contains two parts, a catalytic region (preforms the phosphorylation reaction) and the regulatory region. Normally, the catalytic region is covered by the regulatory region, however, in LTP, it does not close. The exposed catalytic region continues to phosphorylate CaMKII substrates. Each subunit within CaMKII can be phosphorylated by a neighboring subunit. Presistent activity of CaMKII could contribute to the maintenance of snaptic potentiation.
- C)CaMKII calmodulin kinase II
Why is PKMζ different from many other molcules involved in memory?
It's involved in storage of the memory (and encoding) not acquisition.
Thought to be responsible for maintaining the late phase of long-term potentiation
The first molecule shown to be a component of the storage mechanism of long-term memory.
Which molecules does PKMζ help to keep at the synapse?
PKMζ perfused post synaptically intoneurons causes synaptic potentiation
- Inhibiting PKMζ in behaving animals erased spatial long-term memories in the hippocampus that were up to one month old, without affecting spatial short-term memories and erased long-term memories for fear conditioning and inhibitory avoidance in the basolateral amygdala.
- In the neocortex, thought to be the site of storage for most long-term memories, PKMζ inhibition erased associative memories for conditioned taste aversion in the insular cortex, up to 3 months after training.
- PKMζ is thus the first molecule shown to be a component of the storage mechanism of long-term memory.
What is PKMζ imortant?
Recent research has demonstrated alteration in PKMζ in Alzheimer's disease, providing a potential link between this kinase and neurodegeneration.
What are the main conclusions of the Alcino Silva and Susumu
Tonegawa two papers in Science in 1992?
What experiments were performed to show that α‐CaMKII is lacking in the α‐CaMKII mutant mice?
- Neuronal alphaCaMKII become known as a memory molecule and mice lacking this molecule became deficient in long term potentiation and spatial learning.
- To show that alpha camkII was lacking by giving a schematic representation of the construct used for the targeting experiement and compared wt mice to KO mice (Sections with alpha CAMKII) in Southern and Western blotting. They also showed the wt brain and the KO brain to show nothing else was affected.
What does it tell you if a gene knockout leads to deficits in
hippocampal LTP and context fear memory?
That the gene is important in LTP and contextual fear memory and that no other gene took over it's function.
Dependence of long-term memory on protein synthesis
- If you inject a protein synthesis inhibitor and
- train then test the mice you find out that long term memory is deficient but
- not short term memory.
Cued fear conditioning
In cued fear conditioning a mouse is placed in a box (that it has habituated to) and then a neutral tone is played, during the tone the mouse is shocked.After a few trials the mouse should learn to anticipate theshock from the tone (freezing). They are tested about 24hrs later and freeze at the sound of the tone. (uses only amygdala and auditory cortex)
Context fear conditioning
In context fear conditioning mouse is placed in a new box and given shock (training). After a few trails mouse will antecipate shock when entering new box. (uses amygdala and hippocampus)
Brain regions involved in learned fear
Auditory Danger Signal (CS)--> Auditory thalamus
- INDIRECT: auditory cortex, thalamo-cortico-amygdala pathway
- DIRECT: Thalamo-Amygdala pathway
--> lateral nucleus of amygdala--> central amgydala--> defense response (behavior, ANS, HPA)
Why are genes expressed selectively in brain areas important to find?
genes expressed selectively in the brain may be specified for particular behaviors and you can find a reason fr selective expression. Can make a KO to study what is effected (test function in the brain).
Name different types of neurons and their markers.
excitatory/pyramidal neurons- Glutamate --> camKII alpha is the markerinhibitory/interneurons- GABA --> GAD is a marker and GABA
How are amygdala and hippocampal probes for differential screenings made?
Make a radioactive probe for hybridization from rna --> pcr --> amygdala probe OR bacteriophage to RNA probe and view contrast of expression to differentiate (when placing cDNA from library on agarose plate bacteria will cover and holes will form).
stronger spots = stronger expression
Implicit Memory (non declaritive)
- memory for skills, associations and emotional responses; mainly dependent on the amygdala (learned fear and emotional responses), basal ganglia (habits), striatum (planning and modulation of movement pathways but is also involved in a variety of other cognitive processes involving executive function), and
- cerebellum (motor skills)
Explicit Memory (declaritive)
- Memory used for facts and events, mainly dependent on the medial temporal lobe
Provide a detailed diagram with explanations of
the cAMP‐PKA‐CREB pathway.
- 5-HT activates a G-coupled protein receptor
- The GPCR activates adenylin cyclase which produces cAMP
- cAMP activates Protein Kinase A
- PKA phosphoralates a K channel, closing the channel.
- This allows for more Ca to enter the cell during the AP.
- Protein Kinase A can also phosphoralate CREB which can than enhance synaptic activity.
Name protein kinases that directly activate CREB
- CaM Kinases: CaMP Kinase.
Describe the Dominant‐negative approach.
Dominant mutations in which the product of the mutant allele interferes with the function of the normal allele in the heterozygous state.A mutation whose gene product adversely affects the normal, wild-type gene product within the same cell. This usually occurs if the product can still interact with the same elements e the wild-type product but block some aspect of its function product.
DN is inhibitor that leads to lack of function (post translational interference), Binding is normal but you can't phosphorylate. Can have dominant ligand or receptor.functional knockout (not a real knockout)
Get 50% Aa, 25% aa, 25%
AAPKA R-subunit is usedto create a dominantnegative mutant
What is reconsolidation and fear extinction?
extinction- context dependent. weakens CR to CS. Inhibits original memory but it still exists/ Medial prefrontal cortex invovled heavily. reconsolidation- uses more of the amygdala. ability to retrieve/ move from/into long term memory
Name two genes involved in fear memory
GRPR involved in fear memory inhibition. (KO mice show enhanced memori in cued and fear conditioning. They have less GABA released and less inhibition than normal wt mice.)
Stathmin invovled in fear memory enhancement. (expressed in BLA stathmin deficient mice have deficits in innate fear and cued and contextual fear conditioning but normal spatial memory)