Liliana Minichiello Mouse biology unit EMBLMR Italy The

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 tongue nose ear

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……………

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

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

• 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

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

Is LTP triggered during learning? CA 3 • 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 CA 1 • Whether LTP would be triggered during learning and would be causally related to memory formation was debated topic still early 2000

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 hippocampusdependent task

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

Molecules of interest and appropriate mouse model Trk. B neurotrophin receptor Strategies used to understand the biological functions of neurotrophin receptor tyrosine kinases and their signalling mechanisms include: Ø Generation of a null allele Ø Generation of a conditional mutant allele Ø Generation of a point signalling mutant allele

Background We have previously shown that the neurotrophin receptor Trk. B, among other functions, plays an important role in complex learning particularly in hippocampal-related tasks Selective deletion of Trk. B from the postnatal adult forebrain Lox. P X trk. B-floxed mouse (Minichiello et al, Neuron 1999) To dissect the signal transduction pathway/s responsible for Trk. B-mediated hippocampal synaptic plasticity we have generated highly defined mouse models carrying point mutations on specific docking site of the Trk. B receptor (trk. BSHC and trk. BPLC mutants) …. . (Minichiello et al, Neuron 2002)

515 P SH 2 -B r. APS 816 P P FRS 2 Shc P PLC 1 ? Grab 2 Sos Ras/MAPKs Gab 1 Rsk PI-3 K Ca 2+calmodulin kinase AKT creb Plasticity?

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

Summary Signaling molecules Ras/MAPKs(Erk 1/Erk 2) PI 3 K/AKT Ca. MKs (II/IV) CREB P P LTP (E-LTP; L-LTP) Spatial learning (behaviour) P normal normal

Analysis of the different genetic models…. . • This study implicates the PLC /Ca. M kinase/CREB pathway/s in certain forms of hippocampal synaptic plasticity (E-and L-LTP), which require Trk. B signalling • In contrast, suppression of the SHC/Ras/MAPK pathway in trk. BSHC/SHC had no effect on hippocampal LTP • These results allow dissociation of the SHC/Ras/MAPK signalling from LTP induction downstream of the Trk. B receptor • Taken together, these results demonstrate that the PLC -site is necessary to mediate Trk. B-dependent synaptic plasticity

515 P SH 2 -B r. APS 816 P P FRS 2 Shc P PLC 1 ? Grab 2 Sos Ras/MAPKs Gab 1 Rsk PI-3 K Ca 2+calmodulin kinase AKT creb Plasticity?

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 Trk. B receptor (trk. BSHC and trk. BPLC mutants)

Procedure: associative learning task (a classical trace-conditioning paradigm of the eyelid response) and in parallel CA 1 hippocampal recordings (f. EPSP) recording electrode placed in the Hippocampal CA 1 region Bipolar recording electrodes placed in the ipsilateral orbicularis oculi muscle (electromyographic activity) Electrical shock (Orbicularis oculi muscle) Bipolar stimulating electrodes placed on the left supraorbitary branch of trigeminal nerve tone Gruart et al. LEAR&MEM, 2007

Point mutation at the PLC -docking site of Trk. B but not the Shcdocking site impairs acquisition of associative learning

In vivo recorded f. EPSP at the hippocampal CA 1 region during classical conditioning of eyelid responses Evolution of f. EPSP slope across hab. Cond. Ext. sessions trk. BPLC/+ mutants showed f. EPSP slope during conditioning not significantly different from baseline record, whereas f. EPSP in controls and trk. BSHC/+ increased progressively in slope during conditioning

Conclusion With this method we show that signalling through the PLC site of the Trk. B 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.