Liliana Minichiello Mouse biology unit EMBL-MR Italy 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 terminal dendrite Myelin sheath Schwann cell Node of Ranvier Soma 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 Neurotransmitter receptors Postsynaptic density Synaptic cleft Dendritic spine synaptic vesicle Voltage-gated Ca++ channels Neurotransmitters re-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? CA1 CA3

Hippocampus-dependent learning should lead to observable LTP at hippocampal synapses in 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 functions of neurotrophin receptor tyrosine kinases and their signalling mechanisms 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 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 (trkB SHC and trkB PLC mutants) ….. (Minichiello et al, Neuron 2002) Background

SH2-B rAPSPPPP FRS2 SH2-B rAPS Shc SosRas/MAPKs Grab2 Gab1 AKT PI-3K Rsk PLC  1 Ca 2+ calmodulin kinase ? FRS2 PPPP creb Plasticity?

To interfere with either the Shc-site activated pathway/s or the PLC  -site activated pathway/s signalling point mutants

Summary Ras/MAPKs(Erk1/Erk2) P normal CaMKs (II/IV) P normal CREB P normal LTP (E-LTP; L-LTP) normal Spatial learning (behaviour) normal PI3K/AKT PI3K/AKT P normal Signaling molecules

This study implicates the PLC  /CaM kinase/CREB pathway/s in certain forms of hippocampal synaptic plasticity (E-and L-LTP), which require TrkB signalling In contrast, suppression of the SHC/Ras/MAPK pathway in trkB SHC/SHC had no effect on hippocampal LTP These results allow dissociation of the SHC/Ras/MAPK signalling from 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-B rAPS PP PP FRS2 SH2-B rAPS Shc SosRas/MAPKs Grab2 Gab1 AKT PI-3K Rsk PLC  1 Ca 2+ calmodulin kinase ? FRS2 PP PP creb Plasticity?

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 (trkB SHC and trkB PLC 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

trkB PLC/+ mutants showed fEPSP slope during conditioning not significantly different from baseline record, whereas fEPSP in controls and trkB SHC/+ 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