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
Axonal terminal
Soma
Node of
Ranvier
Schwann cell
Nucleus
Myelin sheath
Dendritic spines in 3D
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?
•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
CA1
•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
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
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
Selective deletion of TrkB from the postnatal adult forebrain
LoxP
LoxP
X
trkB-floxed mouse
(Minichiello et al, Neuron 1999)
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)
515 P
SH2-B
rAPS
816 P
P FRS2
Shc
P
PLC1
?
Grab2
Sos
Ras/MAPKs
Gab1
Rsk
PI-3K
Ca2+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(Erk1/Erk2)
PI3K/AKT
CaMKs (II/IV)
CREB
P
P
LTP (E-LTP; L-LTP)
Spatial learning (behaviour)
P
normal
P
normal
normal
normal
normal
normal
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
515 P
SH2-B
rAPS
816 P
P FRS2
Shc
P
PLC1
?
Grab2
Sos
Ras/MAPKs
Gab1
Rsk
PI-3K
Ca2+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 TrkB receptor (trkBSHC and trkBPLC mutants)
Procedure: associative learning task (a classical trace-conditioning
paradigm of the eyelid response) and in parallel CA1 hippocampal
recordings (fEPSP)
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
Gruart et al.LEAR&MEM, 2007
Point mutation at the PLC-docking site of TrkB but not the Shcdocking site impairs acquisition of associative learning
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
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.