Liliana MinichielloMouse biology unit

EMBL-MRItaly

The molecular basis of learning and memory

How do we perceive the outside world?

• In the 1760's, the famous philosopher Immanuel Kant proposed that our knowledge of the outside world depends on our modes of perception

• There are five senses: sight, smell, taste, touch, and hearing

sight, smell, taste, touch, and hearing

eye

ear

tongue

nose

Each of these senses consists of specialized cells that have receptors for specific stimuli

The receptors for specific signals have links to the nervous system and thus to the brain

…….and brain cells communicate this information between each other using electrical signals……………

How do we perceive the outside world?

Axonal terminaldendrite

Myelin sheathSchwann cell

Node of RanvierSoma

Nucleus

Dendritic spines of neuron cells play a key role in neuronal network connections

Dendritic spines in 3D

Structure of a typical neuron

Synapses allow nerve cells to communicate with one another through axons and dendrites, converting electrical impulses

into chemical signals

Axonal terminal

Neurotransmitterreceptors

Postsynaptic density

Synaptic cleft

Dendritic spine

synaptic vesicle

Voltage-gatedCa++ channels

Neurotransmitters

Neurotransmittersre-uptake pump

How neurons make connections

•The ability of the connection, or synapse, between two neurons to change in strength is known as synaptic plasticity

•As memories are postulated to be represented by interconnected networks of synapses in the brain, synaptic plasticity is one of the important neurochemical foundations of learning and memory

•A well studied form of synaptic plasticity is long-term potentiation (LTP)

•LTP is considered to be the mechanism for the acquisition and storage of information by synapses in the hippocampus

•Basic properties of LTP•Triggering mechanisms•Signal transduction mechanisms•Expression mechanisms•Maintenance of LTP

Long Term Potentiation: 30 years of progress

•The fact that LTP could be reliably generated in brain regions involved in learning and memory (such us the hippocampus) was used as evidence for its functional relevance

•Whether LTP would be triggered during learning and would be causally related to memory formation was debated topic still early 2000

Is LTP triggered during learning?

CA1CA

3

•Hippocampus-dependent learning should lead to observable LTP at hippocampal synapsesin vivo

•Lack of an appropriate ‘tricky technique” made this question difficult to answer! •Earlier last year Gruart et al., showed an LTP-like increase in hippocampal synaptic responses in awake mice that where trained in a hippocampus-dependent task

Little evidence

Having established an innovative method to measure in vivo recordings during learning, we asked whether molecular pathways required for learning are also those generating LTP when measured directly on a relevant circuit of a learning animal

Molecular mechanisms of learning

TrkB neurotrophin receptor

Strategies used to understand the biological functionsof neurotrophin receptor tyrosine kinases and their signallingmechanisms include:

Generation of a null allele Generation of a conditional mutant allele

Generation of a point signalling mutant allele

Molecules of interest and appropriate mouse model

We have previously shown that the neurotrophin receptor TrkB, among other functions,plays an important role in complex learning particularly in hippocampal-related tasks

(Minichiello et al, Neuron 1999)

Selective deletion of TrkB from the postnatal adult forebrain

trkB-floxed mouse

LoxP LoxP X

To dissect the signal transduction pathway/s responsible for TrkB-mediated hippocampal synaptic plasticity we have generated highly defined mouse models carrying point mutations on specific docking site of the TrkB receptor (trkBSHC and trkBPLC mutants) …..

(Minichiello et al, Neuron 2002)

Background

SH2-BSH2-BrAPSrAPS

PPPP

PPPP

515515

816816

FRS2FRS2SH2-BSH2-BrAPSrAPS

ShcShc SosSos Ras/MAPKsRas/MAPKsGrab2Grab2

Gab1Gab1

AKTAKT

PI-3KPI-3K RskRsk

PLCPLC11

Ca2+calmodulin kinase

?

FRS2FRS2PPPP

PPPP

515515

816816

crebPlasticity?

To interfere with either the Shc-site activated pathway/s or the PLC-site activated pathway/s

signalling point mutants

SummarySummary

Ras/MAPKs(Erk1/Erk2)Ras/MAPKs(Erk1/Erk2) P normalnormal

CaMKs (II/IV)CaMKs (II/IV) P normalnormal

CREBCREB P normalnormal

LTP (E-LTP; L-LTP)LTP (E-LTP; L-LTP) normalnormal

Spatial learning (behaviour)Spatial learning (behaviour) normalnormal

PI3K/AKTPI3K/AKT P normalnormal

Signaling molecules

•This study implicates the PLC/CaM kinase/CREB pathway/s in certain forms of hippocampal synaptic plasticity (E-and L-LTP), whichrequire TrkB signalling

•In contrast, suppression of the SHC/Ras/MAPK pathway in trkBSHC/SHC had no effect on hippocampal LTP

•These results allow dissociation of the SHC/Ras/MAPK signallingfrom LTP induction downstream of the TrkB receptor

•Taken together, these results demonstrate that the PLC-site is necessary to mediate TrkB-dependent synaptic plasticity

Analysis of the different genetic models…..

SH2-BSH2-BrAPSrAPS

PPPP

PPPP

515515

816816

FRS2FRS2SH2-BSH2-BrAPSrAPS

ShcShc SosSos Ras/MAPKsRas/MAPKsGrab2Grab2

Gab1Gab1

AKTAKT

PI-3KPI-3K RskRsk

PLCPLC11

Ca2+calmodulin kinase

?

FRS2FRS2PPPP

PPPP

515515

816816

crebPlasticity?

•We asked whether molecular pathways required for learning are also those generating LTP when measured directly on a relevant circuit of a learning animal

•We have applied an innovative method to measure in vivo recording during learning in heterozygous mice carrying point mutations on specific docking sites of the TrkB receptor (trkBSHC and trkBPLC mutants)

Molecular mechanisms of learning

Gruart et al.LEAR&MEM, 2007

Bipolar recording electrodes placed in the ipsilateral orbicularis oculi muscle

Bipolar stimulating electrodes placed on the left supraorbitary branch of trigeminal nerve

recording electrode placed in the Hippocampal CA1 region

(Orbicularis oculi muscle)

(electromyographic activity)

tone

Electrical shock

Procedure: associative learning task (a classical trace-conditioning paradigm of the eyelid response) and in parallel CA1 hippocampal

recordings (fEPSP)

Point mutation at the PLC-docking site of TrkB but not the Shc-docking site impairs acquisition of associative learning

trkBPLC/+ mutants showed fEPSP slope during conditioning not significantly different from baseline record, whereas fEPSP in controls and trkBSHC/+

increased progressively in slope during conditioning

Evolution of fEPSP slope across hab. Cond. Ext. sessions

In vivo recorded fEPSP at the hippocampal CA1 region during classical conditioning of eyelid responses

With this method we show that signalling through the PLC site of the TrkB receptor is key to both processes (associative learning and parallel LTP) indicating that the same molecular mechanism forms the basis for learning a task and for changes in synaptic plasticity seen in awake animals.

Conclusion