NBL Module 13

Video Lecture 1

• LTP expression requires calmodulin and CaM Kinase II

• LTP early phase requires activation of specific kinases downstream of the calcium that is entering into the postsynaptic region via the NMDA receptors

  • Kinases- include several directly activated by Calcium or indirectly activated by Calcium

• One of the kinases activated by Calcium is CaM K2- calmodulin dependent Kinase II

  • Binds to Calmodulin

• Calmodulin- type of adaptor protein

  • Has 4 calcium binding domains

  • Interacts with specific targets and leads to their regulation

  • One of the targets is Cam Kinase II

  • Fairly small protein, binds 4 atoms of calcium

  • When calcium level rises postsynaptically, it undergoes a conformational change.

    • Allows it to interact with targets like CaM KII

• Cam KII has a regulatory domain and a catalytic domain

  • This is where the kinase domain is present

  • In the absence of activated calmodulin, CaM KII is autoinhibited

  • Cannot interact with and phosphorylate its substrate

• When Calcium levels rise and bind to Calmodulin, Calcium calmodulin and interact with and activate CaM K2

  • Binds to regulatory domain and releases catalytic domain

  • Now it’s able to phosphorylate its specific targets

  • These targets include proteins like AMPA receptors and proteins involved in the trafficking and tethering of AMPA receptors in the postsynaptic region

• Protein Kinase C, Protein Kinase A and ERK kinase

  • Have several complicated levels of regulation

  • Protein Kinase C binds Calcium and other cofactors

  • PKA is regulated indirectly through activation of cAMP levels downstream of Calcium and Calmodulin

  • ERK proteins- have very complicated mechanism of activation

• These 3 protein kinases are also activated in the postsynaptic region downstream of Calcium entry into the postsynaptic neuron

  • These protein kinases all lead to the regulation of AMPA receptors

• Protein kinases regulate AMPA receptors directly or indirectly

  • Directly- kinases lead to direct phosphoryulation of AMPA receptor alpha subunits

  • This modulates signal channel conductance- increases Calcium and Sodium ions that can flow through these channels in response to glutamate binding

  • Can also increase the density of AMPA receptors in postsynaptic region

    • Increase level of AMPA receptors through trafficking of receptors into plasma membrane and can also lead to tethering of receptors present in membrane and prevent their removal into endosomal pool

• These functions lead to increased AMPA receptor numbers and AMPA receptor activity

  • In response to glutamate, it’s the AMPA receptors that mediate the majority of the postsynaptic response

  • By increasing AMPA receptors→ leads to direct increase in postsynaptic EPSP produced in postsynaptic neuron

• Kinases can directly phosphorylate AMPA receptors and this leads to a change in the AMPA receptor conductance

• In addition, CaM KII can also phosphorylate a set of proteins called TARPs

  • Transmembrane AMPA receptor regulatory proteins

  • One of the TARPS well characterized is a protein called stargazin- identified originally in mutant strain of mice

• TARPs bind to AMPA receptors as they are trafficking

  • Following AMPA receptor biosynthesis and trafficking to and from the plasma membrane, what happens is by the phosphorylation of these TARP proteins, this leads to a tethering of the AMPA receptors that are localized at the postsynaptic region

    • Prevents endocytosis- there’s going to be a greater steady state level of AMPA receptors in the membrane

• Protein kinase ERK leads to an increase in the exocytosis or insertion of AMPA receptors through trafficking mechanism

  • Enhances the number of receptors that are going to be inserted into postsynaptic plasma membrane

  • Both increase AMPA receptor activity and can lead to increase in steady state levels of these receptors at postsynaptic plasma membrane

• Regulation of PKC and ERK kinases is very complicated

  • What is depicted is a very immediate effect of the activation of these kinases by the calcium-dependant mechanism

  • There are longer term effects in late phase of LTP

    • Primarily involve downstream targets of ERK kinase

  • Initial activation of protein kinases that leads to initial increase in postsynaptic response via increased AMPA receptors

    • ERK kinase can set in motion specific cascades involved in maintenance of specific increase of AMPA receptors

• E-LTP: activity of presynaptic axons

  • Transmit APs- arrive at presynaptic terminus

    • Leads to release of glutamate from presynaptic glutamatergic neuron

• Short term plasticity and longer-term effects- there can be an increase in the amount of glutamate release following the type of stimulation that induces LTP

• Once the glutamate is released into synaptic cleft, it can bind to both AMPA and NMDA receptor

  • Initially the AMPA receptors that mediate the response, EPSPs allowing Sodium to flow into postsynaptic membrane and depolarizing it

• At RMP, NMDA receptors have the Magnesium block

  • Even though they bind glutamate, they can’t participate in synaptic transmission

  • Once AMPA receptors have depolarized the membrane potential, that depolarization removes Mg block and NMDA receptors are activated by glutamate that has been bound to them

• NMDA receptors are permeable to both Sodium and Calcium but it’s the Calcium increase involved in idnuction of LTP

  • Calcium leads to activation of 4 different protein kinases in postsynaptic neuron

  • CaM KII, PKC, PKA, ERK

  • Lots of target for these protein kinases that are phosphorylated in response to activation downstream of calcium signal

• Early phase of LTP- lasts about 30 minutes between first 30 to 60 minutes

  • Involves transient Calcium signal

  • CaMKII and PKC are transiently activated

  • Once they do their job in phosphorylating their substrates, leads to downstream effects

    • Targets of these protein kinases in the early phase of LTP is the increase in AMPA receptor number and activity at the plasma membrane 

      • Indirectly occurs by phosphorylation of AMPA receptor- tethering proteins and indirectly by AMPA receptor trafficking proteins

• Initially, we have a number of AMPA receptors that mediate synaptic transmission at spine synapse

  • After repeated stimulation which is going to lead to induction and early phase of LTP- there is an increase in the number of AMPA receptors

  • AMPA receptors there bind glutamate better and produce greater responses when glutamate is bound

  • This produces a larger response after particular stimulation

• There is also more glutamate released presynaptically- contributes to increase in response as well

• Early phase of LTPs- this is an increase in synaptic strength and when we refer to synaptic strength, we refer to postsynaptic response

  • Though this was originally characterized electrophysiologically, we now know that there are many biochemical and morphological changes that occur

  • In LTP- lasts less than an hour, depends on activation of protein kinase activity- leads to increase in AMPA receptors

• Following initial 30 min- 60 min, we have late phase of LTP

  • Additional biochemical mechanisms come into play- lead to long term potentiation of the particular response

• LTD- similar features of LTP

  • Reversible, saturative, associative

  • Difference is what the stimulation was that leads to the long-term response

• LTP- high frequency stimulation

  • Depolarization of postsynaptic membrane at same time there is glutamate being released into synapse

• LTD- low frequency stimulation

  • Much lower number of inputs and input activity into postsynaptic

  • Also involves Calcium and it also involves NMDA receptor

  • Requires for induction activation of NMAD receptors and influx of Calcium

    • Major difference- much lower and sustained level of Calcium flowing into postsynaptic neuron

• During lTD, there is release of glutamate by low-frequency stimulation, enough depolarization too activate some of the NMDA receptors

  • During LTD- influx of Calcium is much lower

  • Low level of calcium influx activates calcium dependent protein phosphatases

    • Remove phosphate from substrates

  • Same type of substrates that were phosphorylated by Calcium dependant protein kinases and Calcium activated protein kinases are phosphorylated by Calcium dependant protein phosphatases

  • Once phosphate is removed from AMPA receptor, this leads to internalization of AMPA receptors via endocytosis of AMPA receptors into the pool

    • Leads to removal of AMPA receptors from postsynaptic plasma membrane

    • Fewer receptors = smaller response

• Protein phosphatases are activated by lower calcium levels than protein kinase

  • Small amount of calcium is sufficient to activate protein phosphatases

  • Not enough Calcium to activate protein kinases

  • Phosphatase activity dominates over kinase

  • Even though it uses similar kind of mechanism downstream, it involves completely different proteins dephosphorylating- opposite effect that leads to decrease in AMPA receptors

• Silent synapse is found between a presynaptic glutamatergic neuron and a postsynaptic neuron

  • CA3 and CA1 neurons in Schaffer collaterals

  • Only expresses NMDA receptors

  • Before LTP, these synapses have NMDA receptors but not AMPA receptors

  • Even though they receive presynaptic glutamate level at the synapse, they are unable to respond because NMDA receptors at RMP have the magnesium block

  • No postsynaptic response

• Silent synapses were originally discovered by depolarizing postsynaptic cell artificially

  • After you depolarize the response, you can measure the NMDA response- influx of Sodium and calcium in response to the glutamate

• Pairing paradigm- stimulate at low leve but depolarize membrane potential, you can measure postsynaptic response

  • If there is enough pressynaptic glutamate release and there is a pairing protocol to provide large depolarization of membrane potential and you provide a large presynaptic input of APs to release large amount of glutamate, after a few seconds to minutes, one can observe an AMPA receptor dependant current that is also present in the postsynaptic neurons

  • Silent synapse has now become unsilenced- AMPA receptors now are going to participate in postsynaptic response

    • Reponse goes from 0 before LTP to a large increase in synaptic response

• Mechanisms involved in unsilencing of silent synapses are the same as the mechanisms involved in synaptic strengthening

  •  NMDA receptors are going to mediate Calcium increases, Calcium will phosphorylate AMPA receptors, and AMPA receptor tethering and trafficking proteins recruits AMPA receptors into the plasma membrane

  • Before there were no AMPA receptors and now there are AMPA receptors which are present

    • Contributes to increase in postsynaptic response

    • When measuring postsynaptic responses electrophysiologically using extracellular field potentials, we measure the response of thousands of synaptic spines

    • If we add more synaptic spines- synapses that are active- will lead to an increase in overall postsynaptic response

• Synapse- contains both AMPA receptors and NMDA receptors

  • Nearby- silent synapse

  • For silent to become unsilenced- needs incoming release from the presynaptic neuron of glutamate but it also requires concomitant depolarization of postsynaptic membrane- can’t provide on its own

  • Can depend on depolarization produced by nearby synapses

• When we have glutamate released by both axons at same time, glutamate will bind to AMPA receptors in mature synapse

  • Can passively spread to nearby synapses and depolarize that postsynaptic plasma membrane

  • Depolarization will remove the Magnesium block from NMDA receptors, then you have release of glutamate at the same time

  • Kicks off mechanism that lead to the increase in recruitment of AMPA receptors in postsynaptic plasma membrane

  • Silent synapse has been unsilenced and now there will be a response from both types of synapses

  • Hebbian mechanism- synapse specific

    • Have to have release of glutamate by presynaptic neuron

    • Once magnesium block has been removed, those receptors are going to allow for recruitment of AMPA receptors

• Saturable- physiological

• Input/synaptic specific- concomitant depolarization of postsynaptic membrane and release of glutamate at presynaptic terminus

• LTP is both cooperative and associative- similar in terms of concepts

  • Much more similar to physiological situation within hippocampal neuron

  • Non Physiological tetanus type of stimulation- incoming activity is sufficient to depolarize membrane

    • Have glutamate- produce potentiated response

    • Artificial situation

  • Within hippocampus- cooperative and associative properties work towards LTP

• Incoming presynaptic activity with postsynaptic response in normal synaptic transmission

  • Only one axon response

• Cooperativity- even though on their own they might not be able to produce LTP, if they are firing at the same time, they can cooperate to depolarize membrane potential so all 3 synapses can now be strengthened

• Associativity- incoming activity from synapse at same time associates responses

  • If there is a presynaptic input, 2 can associate to undergo LTP

• Specificity- requires input of glutamate

  • Always has to have presynaptic glutamate release

• Cooperativity- activation of multiple inputs which on their own are not sufficient to produce LTP

  • Can cooperate with each other to produce large enoug depolarization of membrane potential now that the NMDA receptors can become activated

• Selectivity- only synapses that receive incoming activity will be strengthened

• Associative- simultaneous pre and postsynaptic activity

• Late- develops during early LTP but late LTP actually lasts for very long time in hippocampal neuron

  • Hours to days to week depending on type of incoming info

• Requires new mRNA synthesis and transcription 

  • Translation of newly transcribed mRNAs as well

  • Requires activation of transcription and translation

  • Activation depends on protein kinases stimulated early phase of LTP and stimulated by that influx in Calcium

• Some of these kinases converge on Protein Kinase ERK

  • Phosphorylate and activate CREB and ERK

  • Leads to an increase of CREB and ERK dependent transcription

• Signal of activated kinase is somehow able to be transmitted to the transcription factors CREB and ELK which increase transcription of many genes

  • Some transcription factors lead to decrease of other genes

  • Transcrpition occurs, mRNA is transported to cyotplasm for translation

• Some can be transported to dendrite for local protein synthesis

  • Net effect- result in a persistent increase in AMPA receptor levels and AMPA receptor activity

  • Even though outcome in terms of synaptic response is similar to what occurs during early LTP, there are lots and lots of biochemical and structural changes that these dendritic spines undergo

    • Leads to an increase in dendritic spine volume

    • Can accommodate increase in AMPA receptors

  • Changes in the cytoskeleton, membrane excitability, continued activation of silent synapse

    • Increase in spine numbers- can be an addition of new spines to the particular dendritic region

Video Lecture 2

• Early LTP- occurs in first hour or so after LTP induction

  • There is an increase in postsytnaptic Calcium

    • Depends on NMDA receptors

  • Calcium goes on to activate specific protein kinases

    • These kinases phosphorylate AMPA receptors and AMPA receptor tethering and trafficking proteins that lead to increase in number of postsynaptic AMPA receptors that mediates early part of LTP

  • Doesn’t require production of new poritne or new mRNA

    • After an hour this is where the late phase of LTP comes into play

• Late LTP develops during early LTP

  • Last for longer than an hour, can last up to weeks in hippocampus

  • Underlies storage of memories- involves systems that will be storing for a lifetime

  • Late LTP vs early LTP- mRNA and protein synthesis is required

  • Synapse and spine undergo changes in morphology and structure, as well as epigenetic changes to genome

• Late LTP involves activation of protein kinases like CaMKII, PKC, and PKA

  • All of these converge on one downstream kinase called ERK

• ERK- growth factor pathways

  • Extracellular regulated kinase

  • Originally identified in cells that undergo proliferation in cell culture in non-neuronal system

  • Critical for late LTP

• Kinases that phosphorylate and activate the ERK protein are going to have a transient activation

  • Early phase of LTP- kinases will be activated and after 30-60 min, go back to baseline activity

• ERK becomes activated

  • Once this is activated, it phosphorylates its downstream targets

  • 2 targets: CREB and ELK

    • Both of these are transcription factors

  • Activation of ERK leads to direct stimulation of transcription in neurons that undergo LTP

• During transcription- new mRNA synehsis

  • Get exported out of nucleus

  • Some will be translated directly in cytoplasm, some are transported to dendrite which are where the stnapses are that are undergoing increase in potentiation

    • Synapses that undergo plasticity

• Result- increase in specific genes

  • Memory related genes or Immediate-early

  • These are going to have effects on dendritic spines or morphology on synapse

  • Will lead to persistent increases in AMPA receptor activity or AMPA receptor levels

• Late phase of LTP- maintain increase in AMPA receptors that initially occurred during LTP

  • Also changes dendritic spine volume, changes in cytoskeleton, changes in membrane excitability through regulation of ion channels

  • Activation of silent synapses

• CREB- cyclic AMP response element binding protein

  • Phosphorylated by protein kinases downstream of ERK

  • FUnction- bind to specific region in promoter

  • CRE- cyclic AMP response element

• When CREB is phosphorylated- recruits protein called CREB binding protein- CBP

  • Leads to activation of complex

• CREB has to be phosphorylated to activate transcription because it has to recruit CBP/P300

• Histone acetyltransferase- HAT- CBP

  • Acetylation of histoen proteins

  • When this occurs- relaxes histones from binding to DNA, opens nucleosome to allow for recruitment of transcriptional activation machinery

• CREB is not directly phosphorylated by ERK protein

  • Uses different kinases in order to activate the CREB

• TGACGTCA- consensus sequence for CREB to bind to

  • Dna is double stranded- sense and antisense strain

  • DNA binds as a dimer- sequence is a palindrome

  • Requirement for the specific sequences- little bit of wiggle room 

  • Interesting genes in neuron that contain CRE sequence like BDNF, chromogranins, enkephalins, and others

• ERK is not a direct regulator for CREB

  • Uses RSK2 and MSK1 

• ERK is a MAP kinase- large gene family of kinase not only involved in LTP, but also cell growth and differentiation for cell survival

  • Phosphorylates downstream kinases- RSK and MSK

  • Once they become phosphorylated- enter into nucleus to phosphorylate CREB

  • Regujlation by ERK of CREB phosphorylation- requires intermediary kinases

• CREB can also be phosphorylated by PKA- protein kinase activated by cAMP

  • Initially identified in responses downstream of siganling that leads to increase in secondary messenger AMP

• CREB is also a substrate for CaM KII

  • CaM KII could phosphorylate CREB in LTP

  • CaM KII is activated by Calcium and calmodulin once calcium levels increase in postsynaptic neuron

  • Majority of CaM KII is localized in postsynaptic density- not a lot in the cytoplasm able to translocate into nucleus and cell body

  • Majority of phosphorylation occurs through RSK and MSK pathway

    • Could potentially lead to modulation

  • LTP could be modulated by signaling pathways that increase cAMP

    • This is one of the ways LTP can be modulated

• Once CREB is phosphorylated, it recruits CBP

  • HAT activity

  • Histones become acetylated

  • DNA opens up- can recruit basal transcriptional machinery for production of mRNAs

• Even though CREB binds to DNA- has to be phosphroyalted to recruit activity

  • Once DNA is relaxed, histones are not wrapped around- leads to increase in transcription of genes that bind CREB and have CRE in their promoters

• Increase in Calcium 

  • Through many different mechanisms- leads to activation of ERK

  • Once ERK is activated by phosphorylating RSK and MSK- translocate into nucleus- CREB becomes phosphorylated, recruits CBP- increase in transcription

• RSK- ribosomal S6 kinase

  • MSK- mitogen and stress activated kinase

  • Involved in protein synthesis regulation in cultured cells responding to activation of cell proliferation

  • Kinases phosphorylate CREB protein at Serine 133- phosphorylation required for activation of transcription

• CaM KII and PKA can phosphorylate CREB on the same residue

  •  Can lead to alternative mechanisms for CREB regulation

• ERK can phosphorylate a different transcription factor called ELK1

  • Binds to a different element in DNA of specific genes in promoter- SRE

  • Serum response element- identifidd in culture cells that were stimulated to divide when serum was present

    • Has growth and trophic factors for cells- leads to activation of ELK

• ERK and ELK- surprise- involved in LTP

  • ERK and ELK were initially identified in non neuronal cell in growth and trophic stimulation in cell proliferation and survival

  • Type of plasticity where neurons undergo biochemical and morphological changes- also occurs in cell growth and survival

  • CREB and ELK bind to specific DNA elements

• Genes that contain either CRE or SRE are groups of genes upregulated in response to LTP

• Set of genes activated during late phase of LTP were actually identified in several different systems to be stimulated as a set of genes

  • IEGs- immediate early genes- activity regulated genes- plasticity related genes within neuronal plasticity research literature

• IEGs are a group of genes shown to be rapidly and transiently activated at transcriptional level following growth factor stimulation in cultured cells

  • Activity regulated genes- activated by seizure activity within rodent brain- very robust activity in specific regions of the brain

• Plasticity related genes are upregulated during synaptic/neuronal plasticity

• Common

  • Either gene itself has CRE in 5’ region of gene or have SRE within gene

  • Not all genes that contain these specific DNA elements are upregulated in response to LTP, but share

• Most genes contain not only one specific response element in their promoter, but several

  • Can bind transcription factors like NF Kappa B or AP-1

  • Lots of different types of transcription factors

  • Can contain additional transcription factor binding sites- have other response elements

  • Regulation of these plasticity related genes involves additional combinations of other transcription factor binding as well

• 2 major groups- transcription factors or Effector IEGs

• Transcription factors- activated by CREB

  • Go onto bind to response elements in other genes involved in plasticity- would have initial expression of transcription factors very rapidly during LTP

    • Would go on to regulate transcription of additional proteins like cFOS, cJUN, JUNB

    • Not initially identified in nervous systems- were identified in cell growth and survival pathways

• Effector IEGs- include many different types of genes

  • AMPA receptors- expression/basis of LTP involves increase in AMPA receptor numbers- present in postsynaptic regions

    • To replenish AMPA receptors to those regions- needs to be an increase in transcription of mRNAs that encode AMPA receptors and increase in AMPA receptor translation as well

  • BDNF- trophic factor in survival of neurons

    • Can bind to receptors and is involved in synaptic plasticity as well

    • Downstream is a signaling cascade that can lead to morphological changes that occur in the synapse and spine

  • Homer- GPCR scaffolding protein

    • Target- metabotropic glutamate receptor

    • Metabotropic receptors are also involved in LTP

  • ATC- activity regulated cytoskeletal proteins 

    • It has an important function in regulating postsynaptic and dendritic cytoskeleton- underlies morphological changes that occur in spines

• All have CRE or SRE in specific promoter regions- respond to activation of CREB or ELK transcription factors

• In addition to NMDA receptors, metabotropic glutamate receptors play an important role in modulation of LTP

  • Influx of Calcium- leads to stimulation of CaM Kinase

  • CaM Kinase leads to phosphorylation of AMPA receptors and AMPA receptor regulatory proteins

    • Also involved in pathways leading to regulation of CREB

  • In addition- ELK and AP1- other transcription factors involved in the pathway

• Increase in transcription of plasticity related genes

  • mRNAs will be produced by transcription, will be transported out of nucleus to be transcribed into cognate proteins

  • fMRP- peptide

• Proteins in cyotplasm of neuron- whose transcription is activated- biochemical and morphological changes that underlie late stages of LTP

• Synapse strengthening is synapse specific

  • How signal knows to strengthen only those synapses that have received incoming presynaptic activity

• How does the signal get from activated synapse to nucleus?

  • Mm away from nucleus

  • There could be diffusion or trafficking of kinases from postsynaptic region to nuclear region for activation

    • Motoring of specific kinases or complexes 

  • ERK complex- could be specifically transported into neuronal cell body to phosphorylate MSK and RSK to be translocated into nucleus

• Types of eresponses that lead to LTP lead to depolarization of postsynaptic membrane

  • If that depolarization is great enough- Sidium ions diffuse along emmbrane and passively diffuse cell bodies

  • Enough incoming depolarizations- neuron fires AP

• Back propagating AP- travels in forward direction along axons

  • Due to voltage gated ion channels in cell body- AP can be propagated back into dendrites

  • Once this AP has been produced- could lead to opening of voltage gated channels found in the membrane within cell body

• Candidate channels activated by back propagated by AP- VGCa2+Cs

  • Initial production of large response within neuron leads to AP

  • AP flows back, leads to activation of VGCa2+Cs, influx of Calcium near cell body and nucleus

    • This Calcium signal leads to activation of ERK and RSK and MSK found near cell body- downstream activation of kinases in region, that would be the signal leading to activation of transcription

• BDNF- identified as a trophic factor, survival factor for neurons that express receptor for BDNF- TrkB

  • Also shown that BDNF is synthesized in response to LTP

  • Using exogenous system- addition of BDNF leads to synapse strengthening and contribute to activity

  • Knockout/reduction of BDNF levels or receptor- synapses do not undergo typical type of LTP

• BDNF- Survival factor and plasticity in development of synapse

  • Binds to TrkB- receptor tyrosine kinase 

  • BDNF can also activate some of the same types of activities and functions such as AMPA receptor trafficking- similar to CaM Kinase

  • Might be contributing later on to recruitment of AMPA receptors and tethering of AMPA receptors at postsynaptic plasma membrane

• BDNF works presynaptically and postsynaptically

  • Presynaptic- regulators in increase of glutamate release that occurs during LTP

• BDNF release location has not been clearly established

  • Could be either presynaptic or postsynaptic neuron or both

• Group of genes whose expression decreases in LTP- have been named memory suppressor genes

  • 2 have been well studied

• Protein phosphatase 1- activity is decreased in LTP

• Calcineuron- PP2B- dephosphorylate proteins

• At the same time that kinase activities are activated during LTP, there is reduction in proteins that dephosphrylate same groups of substrate- lead to net increase in protein phosphorylation during LTP

• CREB2- inhibitor of CRE protein and CREB activities

  • Decreasing inhibitor of CREB contributes to activation of CREB dependant RNA transcription

Video Lecture 3

• Changes work together for expression of LTP

  • Increase in AMPA receptor (Density)

  • Long term biochemical and morphological changes

• Newly transcribed mRNA can be transported from cell body to dendrites

  • Can be synthesized in both locations

  • If you block protein synthesis- leads to inhibition of LTP

• Protein synthesis in cell body has been characterized well

  • In dendritic spines- local protein synthesis

  • Syntehssi of proteins within dendrite provides a pool of newly synthesized and critical proteins that can travel a short distance and be incorporated into the changes occurring in dendritic spines

• ERK and other kinases activate translation by phosphorylating translating proteins

  • RSK- ribosomal S6 kinase- protein synthesis

  • Upregulation of protein synthesis in cell soma and dendritic spines

• Translation of proteins involves not only soluble cytoplasmic proteins synthesized on free polyribosome

  • Like CaM KII

  • Translation of transmembrane proteins involves rough ER and golgi complex

• There are rough ER and smooth ER

  • Small golgi like structures called dendritic golgi outputs are localized to golgi

  • Synthesis of transmembrane proteins like AMPA receptors and ion channels that regulate excitability of membrane within dendritic spine- can be translated close to site of where they will be inserted

• Part of the secretory pathway- small secretory vesicles are present in dendritic spines

  • Deliver transmembrane proteins into dendritic spine membrane

• Increase in number of proteins with mRNAs being shipped to dendirtes and newly translated proteins will be incorporated

  • Enlargement of spine volume

  • Increase is required to accommodate newly syntehsized transmembrane and cytoplasmic proteins present in dendritic spine

• AMPA receptor increase that mediates increase in response of postsynaptic region within spine

  • Increase in AMPA receptors- delivered to postsynaptic region

  • AMPA receptors will be tethered so they won’t be endocytosed easily

  • EPSPs

  • If you block AMPA receptor synthesis- blocks late phase of LTP

• Switch in AMPA receptor subunit

  • There are some changes to individual gene components- affects AMPA receptor specificity and activity

• NMDA receptors- involved in induction of LTP, lead to increase in Calcium levels which kicks off LTP program

• ARC- activity regulated cytoskeletal associated protein

  • Spines undergo changes in actin cytoskeleton

  • One of the changes thought to be regulating this is ARC- essential for LTP, if you inhibit arc activity, you will block LTP

  • Plays prominent role in transition phase from early to late LTP

• ARC is not just dedicated to dendritic spine plasticity- neuron in general

  • Levels are affected in neuropsychiatric disorders like depression

• Hebbian nature/synaptic specificity

  • Only synapses with incoming stimulation will be strengthened (nearby ones won’t)

• Activation of kinases, increase in transcription, mRNA translation, some will be translated in cell body, some in dendrites

• How does it know which specific synapse?

• Synaptic tagging and capture

  • Early LTP- something happens in synapse- early info that leaves behind specific tag

  • Remains within activated synapse, captures newly synthesized protein or mRNAs

  • Strengthening only occurs in newly activated synapse

• CaM KII- activated by Calcium flowing in through NMDA receptors

  • Calcium binds to calmodulin, activates CaM KII

  • CaM KII binds to and synthezed the protein

  • CaM KII serves as the tag- leads to changes in actin cytoskeleton

    • Indirectly leads to capture of specific plasticity molecules

• CaM KII is soluble- how could it be involved in tagging? 

  • Not soluble- becomes tethered at synapse when activated

  • NMDA receptor is a binding partner

  • Tag of the specific synapse which has undergone activation

• Not the only mechanism- different manipulations of CaM KII

  • Certainly not the lone protein

• PKM zeta is a member of the PKC family

  • PKC family is one of the proteins involved in early LTP

  • PKM zeta- following LTP, influx of Calcium

  • Rapidly synthesized in dendritic spine and PKM synthesized in response to LTP- newly synthesized PKM zeta- happens in early phase of LTP

    • Depends on translation, not transcription 

• PKM zeta- synthesis will be regulated

  • Could regulate types of trafficking proteins

  • Remains at synapse

  • Essential for LTP, synthesized early on

  • Identifies it as a candidate

  • Can capture newly synthesized proteins 

• Concomitant increase in spine volume

• Both ERK and PKM zeta are involved in increase of spine volume

  • Synthesis of newly synthesized proteins could lead to enlargement of spine

  • Some proteins and mRNAs- captured by synapses to provide synapse specificity

• NMDA receptor functions as ionotropic receptor- Calcium and Sodium into postsynaptic neuron

  • Structural and scaffolding role

  • Lots of other signaling and scaffolding proteins binds to complex

• PSD-95- ampa receptor tethering at synapse

  • Kalirin- regulation of membrane trafficking

• NMDA receptor and calcium influx- could lead to changes in dendritic cytoskeleton

  • Additional trafficking receptors and proteins involved in actin cytoskeleton

  • Large protein complex- NMDA receptor serves both signaling and scaffolding role

• In addition to postsynaptic density proteins- have tethering of AMPA and NMDA receptors- synapse is a region where there are cell adhesion molecules expressed and bind to each other 

  • Neurolignads, postsynaptic cell adhesion proteins- binding partners of nurexins and cadherins

    • Cadherins bind to each other

  • As spines get larger, presynaptic area increases

    • Expression of cell adhesion molecules is also upregulated

• Synapse- ability of pre and post to bind to each other increases

  • Strengthening of synapses- potent strength of post synaptic response

  • Synapse can get stronger as well through cell adhesion molecules binding to each other as postsynaptic region enlarges, will have more proteins to keep it stably attached to presynaptic region

• Silent synapses- become strengthening

  • As AMPA receptors are recruited to post synaptic region

• During LTP- increase in total number of spines

  • Formation of new spines during LTP process

  • 1 week after LTP- density of spines

    • Following LTP- increase in density

    • Statistically significant increase

• How could this be Hebbian- how would neuron know which specific spine/area to produce?

  • Could be non-input specific

• Within dendrite, spines are developing

  • Filopodia- mature and developing into mature spine

  • Small regions of contact between pre and post synaptic target- there could be activity like glutamate release

    • Filopodia- immature spine

    • Very early spine- can’t measure synaptic contacts between presynaptic and postsynaptic

  • Could be non-hebbian plasticity

• If there is robust activity in areas close to spine, regions close to it- not specific, some of plasticity proteins- could spill out in regions close to activity dependent processes

  • Not 100% fidelity- secondary mechanism to increase spine density

• Downstream of changes- increase of AMPA receptors, spine volume increases in morphological changes

• Unsilencing of silent synapses- can increase spine volumes

• Hippocampus is’t the place where long term memory is stored

  • Transmits info into other regions of cerebral cortex

  • Called systems consolidation

  • We don’t know the mechanisms, but involves similar synaptic and molecular changes that occur within hippocampus itself

• Once hippocampus has transmitted info about which incoming info for declarative and spatial memory is important, the hippocampus goes back to prememory state

  • Once synapse has become strengthened, to go back to prememory activity- has to be reversed

  • Reverse is related to LTD

  • Hippocampus can now be used for encoding of future long-term memory

Video Lecture 4

• Memories are present in cerebrocortical networks

• Synaptic.cellular consolidation

  • 2 places where it can occur- hippocampus during memory encoding process

    • Cerebrocortical circuits once memory has been transferred from memory

  • Late phase of LTP

• Systems consolidation

  • Independent of hippocampus in a scale of a few weeks

  • Synaptic/cellular consolidation- consequence of biochemical and morpholiglcal changes initiated by original experience

    • In hippocampus- hours 

  • Neocortical interaction between that and hippocampus

    • Days to years interaction

  • Can take many periods for memory to be consolidated

• Consolidation takes place during LTP in hippocampus

• Synaptic consolidation- produces changes in efficacy, whether in hippocampus or cortex

• Synapses within neocrtical circuits will be modified

• 2 stage- hippocampus is involved in initial stage, transmits info into hippocampus

  • Once hippocampus has transmitted info and relative activity- undergoes synaptic renormalization (LTD)

  • Reorganized and renormalized back to prememory state

• Declarative memories- stored in cortical networks

  • For recall- hippocampus is not involved

  • Episodic memory- hippocampus is involved

• Systems consolidation- LTP and synaptic consolidation in process- we don’t know much about mechanisms involved

  • One activity- sleep

• Info is going to be transmitted from cortical networks to hippocampus for encoding of relevant info that is stored in long term memory

  • Hippocampus transmits info back to cortical networks which eventually become independent

• Role of hippocampus- connect pieces of info initially transmitted to hippocampus from sensory and association cortices

  • Binds info together, connects different cortical regions for storage

• Sleep- defined as process in which body is paralyzed, brain different stages of activity

• SWS- slow in frequency

• Sleep is important for memory

  • One original hypothesis- Different phases of sleep are dedicated to different types of memory

• Both stages are involved

• Sequential hypothesis

  • Waking- acquisition of memory, info is transmitted from cerebral cortex to store in hippocampus

• As transition between SWS to REM sleep- rapid eye movement- strengthening of neocortical memory representations- consolidation of synapses within neocortex

• Several cycles of slow wave sleep and REM sleep in 8 hour cycle

• Sleep spindle- hippocampal dependent LTM

• Occurs before slow wave sleep

• Info gets transmitted from cortical networks to hippocampus

  • Function- help transmit info, replay back from cerebral cortex to strengthen activity

  • Initial parts of transmission of strengthened circuit activity is transmitted back to neocortical circuits

• SWS- systems consolidation, followed by rescaling activity

  • Hippocampal dependent activity would be rescaled

• Wake state- memories stored and encoded to cerebral cortex can be retrieved back to short term memory, will be recalled to use

• Cortical networks can do 2 things: encode new memories using hippocampus

  • Previously strengthened neocortical networks will be used for retrieval of newly encoded info

  • Synapses are modulated and strengthened- not much info about system consolidation and retrieval

• During systems consolidation- initial activity can be recorded in cortex

  • Cortex contacts hippocampus to gauge info available to transfer

• Theory one- Hippocampus is not required to encode memories after several weeks or months

• HM- multiple trace model- declarative memories separated to semantic and episodic memory

  • Semantic memories- have standard theory, memories become independent of hippocampus as a function of tiem

  • Episodic memories- autobiographical, episodic rich memory require hippocampus for longer periods of time

  • Recall/retrieval of info depend on hippocampus

  • There may be different types of systems involved in ongoing encoding of memories and retrieval as well

• After memories are stored- reactivation period

  • Become restored and unstable

  • Memory reconsolidation- restabilizes memory that was previously destabilized

  • If reconsolidation is modulated in this time by brain memory or drugs- can change negatively 

  • Individuals who have had traumatic events- has been developed as a therapy to help them forget those

• Once CaM Kinase becomes activated- autophosphorylation

  • Persistently activated, doesn’t need calmodulin for activation

  • Remains persistent even after original signals have diminished

  • Can phosphorylate other CaM Kinases

• Prion proteins- has normal cell shape, interactions with mutated prion protein- can bind to normal protein and convert it into disease causing prion

  • Transforamtion of proteins between conformations

  • Converts protein to prion protein mutation

  • Creutzfeld-Jacob disease- mutation

  • Other proteins could also change conformation

  • Memory causing prion- would be stable, anytime newly synthesized prememory is produced- memory causing prion could convert a protein into a memory prion

  • Alzheimer’s and Parkinson’s- persistent changes downstream