Liliana Minichiello Mouse biology unit EMBL-MR Italy

1. Liliana Minichiello Mouse biology unit EMBL-MR Italy The molecular basis of learning and memory

2. 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

3. nose eye tongue ear sight, smell, taste, touch, and hearing

4. How do we perceive the outside world? 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……………

5. dendrite Axonal terminal Node of Ranvier Soma Schwann cell Myelin sheath Nucleus Dendritic spines in 3D Structure of a typical neuron Dendritic spines of neuron cells play a key role in neuronal network connections

6. Neurotransmitters synaptic vesicle Neurotransmitters re-uptake pump Voltage-gated Ca++ channels Axonal terminal Synaptic cleft Dendritic spine Postsynaptic density Neurotransmitter receptors How neurons make connections Synapses allow nerve cells to communicate with one another through axons and dendrites, converting electrical impulses into chemical signals

7. 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

8. Long Term Potentiation: 30 years of progress Basic properties of LTP Triggering mechanisms Signal transduction mechanisms Expression mechanisms Maintenance of LTP

9. CA1 CA3 Is LTP triggered during learning? 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

10. Little evidence 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

11. Molecular mechanisms of learning 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

12. 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 TrkB neurotrophin receptor Strategies used to understand the biological functions of neurotrophin receptor tyrosine kinases and their signalling mechanisms include:

13. LoxP LoxP Selective deletion of TrkB from the postnatal adult forebrain trkB-floxed mouse X (Minichiello et al, Neuron 1999) Background We have previously shown that the neurotrophin receptor TrkB, among other functions, plays an important role in complex learning particularly in hippocampal-related tasks 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)

14. FRS2 FRS2 515 515 Grab2 Shc Sos Ras/MAPKs SH2-B rAPS Gab1 816 816 PLC1 Rsk PI-3K ? Ca2+calmodulin kinase AKT P P P P P P P P creb SH2-B rAPS Plasticity?

15. signallingpoint mutants To interfere with either the Shc-site activated pathway/s or the PLC-site activated pathway/s

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

17. Analysis of the different genetic models….. • 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 • trkBSHC/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

18. FRS2 FRS2 515 515 Grab2 Shc Sos Ras/MAPKs SH2-B SH2-B rAPS rAPS Gab1 816 816 PLC1 Rsk PI-3K ? Ca2+calmodulin kinase AKT P P P P P P P P creb Plasticity?

19. Molecular mechanisms of 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 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)

20. recording electrode placed in the Hippocampal CA1 region Bipolar recording electrodes placed in the ipsilateral orbicularis oculi muscle (electromyographic activity) Electrical shock (Orbicularis oculi muscle) tone Bipolar stimulating electrodes placed on the left supraorbitary branch of trigeminal nerve Procedure: associative learning task (a classical trace-conditioning paradigm of the eyelid response) and in parallel CA1 hippocampal recordings (fEPSP) Gruart et al.LEAR&MEM, 2007

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

22. In vivo recorded fEPSP at the hippocampal CA1 region during classical conditioning of eyelid responses Evolution of fEPSP slope across hab. Cond. Ext. sessions 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

23. Conclusion 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.