LECTURE 20-21: CELLULAR BASIS OF LEARNING & MEMORY

REQUIRED READING: Kandel text, Chapter 63, and Assigned Review Articles

Research on cellular basis of learning & memory mainly performed in three animal systems

Aplysia Drosophila Mouse

All neurons and synapsesin behavioral circuits are

identified and can be recorded easily

Ideal for detailing mechanisms underlying implicit learned motor

responses

Capable of learned behaviors

Amenable to randommutagenesis and

selection of mutantswith defective

behaviors

Similar anatomy to human

Amenable to study ofexplicit memory

Hippocampus amenableto electrophysiology

Behavior modification ofgenetically modified mice

APLYSIA SHORT-TERM LEARNED RESPONSES AFFECTING GILL WITHDRAWL REFLEX

HABITUATION SENSITIZATION CLASSICALCONDITIONING

Repeated tactile stimulation of siphon

depressesgill withdrawl response

Harmful stimulussensitizes

gill withdrawl responseto subsequent

harmful OR harmlessstimuli given to

same OR differentbody regions

Pairing harmful stimuluswith preceding harmless

conditioning stimulus sensitizesgill withdrawl response

to subsequentconditioning stimulus

but not to tactile stimuligiven to other body areas

HABITUATION IS DUE TO DEPRESSED NEUROTRANSMITTER RELEASE AT SEVERAL SITES

Rapidly repeated tactile stimulation of siphonattenuates gill withdrawl both during thetraining and for a short period afterwards.

Habituation is due to reducedneurotransmitter release by the

sensory neuron and by relevant interneuronsin response to the tactile stimulus.

I.e., the memory of habituationis distributed at various synapses

in the circuit

Whereas a rapid series of stimuli inducesshort-term habituation,

several sets of tactile stimuli distributedover several hours induces

long-term habituation that lasts for weeks.

Long-term habituation requiresnew protein synthesis and is due to

pruning of synaptic connections

SHORT-TERM SENSITIZATION IS MEDIATED THROUGH AXO-AXONICSEROTONERGIC SYNAPSES OF FACILITATING INTERNEURONS

Serotonergic facilitating interneuronssend axo-axonic connections to

broadly distributed sensory neurons

Unconditioned stimulus causes interneurons to release serotonin,which acts through metabotropic

HT receptors to increase thelikelihood of neurotransmitter release

following sensory neuron firing

Sensitization can be mimicked withoutsensitizing stimulus by local

experimental application of serotonin

Sensitization is mediated bypresynaptic elevation of

cAMP & PKA activity,which has three effects:

1) Greater proportion of vesicles in active zone

(synapsin phosphorylation?)

2) K+ channel inactivation increases duration of depolarization and

magnitude of Ca+2 influx

3) Activation of L--type calcium channels

CLASSICAL CONDITIONING EMPLOYS SEQUENCE-REINFORCED PRODUCTION OF cAMP

Conditioning is only effective when CS precedes US by a short interval (~ 0.5 sec)

CS elevates calcium in presynaptic terminal at moment of US.Calcium/CAM enhances the enzymatic activity of adenylate cyclase triggered by 5-HT.

Adenylate cyclase is a biochemical “coincidence detector”

TEMPORALLY SPACED SENSITIZATION OR CONDITIONING TRAININGSINDUCE LONG-TERM IMPLICIT MEMORY

Long-term sensitizationand conditioning are

also mediated through presynaptic cAMP production

and PKA activity

PKA induces specific CREB-dependent

gene transcription and protein synthesis:

Newly synthesized ubiquitinNewly synthesized ubiquitinhydrolase degrades hydrolase degrades

PKA regulatory subunits,PKA regulatory subunits, making the enzymemaking the enzymeconstitutively activeconstitutively active

Other newly synthesizedOther newly synthesizedproteins help build newproteins help build newpresynaptic terminalspresynaptic terminalsonto motor neuronsonto motor neurons

GENETIC SCREENS FOR GENES AFFECTING CONDITIONING IMPLICIT MEMORYALL AFFECT THE cAMP-PKA-CREB PATHWAY

FLY MUTANTS SELECTED FOR DEFECTS IN IMPLICIT MEMORY

DUNCE encodes cAMP phosphodiesterase

RUTABAGA mutant defective for Ca+2/CAM enhancement of cyclase

AMNESIAC encodes a peptide neurotransmitter acting on GS-coupled receptor

PKA-R1 encodes PKA

HIPPOCAMPAL NEURONS IN DIFFERENT RELAYS ARE ALLCAPABLE OF UNDERGOING SYNAPTIC LONG-TERM POTENTIATION

AXON STIMULATION PROTOCOL AMPLITUDE OF EPSCS

20 min 1 m 60 min

Once p

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6020 40 80““THETA” BURSTTHETA” BURST

One Theta burst gives what is sometimes calledEarly LTPEarly LTP,

which is less than doublingof EPSC which lasts for hours

Four Theta bursts spaced minutes apart generateLate LTPLate LTP,

with up to 4-fold EPSC stimulationthat lasts for days

INDUCTION AND EXPRESSION OF SYNAPTIC PLASTICITY

Prior synaptic activity can INDUCE long-term plasticity. Such plasticity can be INDUCED by molecular events occuring either presynaptically or postsynaptically.

The changes in transmission following synaptic plasticity can be EXPRESSED either presynaptically and/or postsynaptically, and need not correspond to the site of INDUCTION.

E.g., at a certain synapse, postsynaptic calcium influx can INDUCE plasticity which is then EXPRESSED as changes in presynaptic neurotransmitter release probability.

LTP AT MOSSY FIBER--CA3 SYNAPSES IS DUE TO PRESYNAPTIC CALCIUM INFLUXAND cAMP/PKA PATHWAY

LTP AT SCHAFFER COLLATERAL--CA1 SYNAPSES IS DUE TO POSTSYNAPTIC CALCIUM INFLUX AND CAM KINASE ACTIVITY

LTP at CA3-CA1 synapse is blocked byNMDAR antagonist APV and by inhibitors

of CAM kinase

PRESYNAPTIC COMPONENT OF EARLY AND LATE LTP AT CA3--CA1 SYNAPSES RESEMBLES SHORT- AND LONG-TERM SENSITIZATION

Late LTPLate LTPabsolutely requiresabsolutely requires

new protein synthesisnew protein synthesis

PRESYNAPTIC COMPONENT OF EARLY AND LATE LTP REQUIRES POSTSYNAPTIC CAMK ACTIVITY AND RETROGRADE SIGNALS

OTHER MECHANISMS OF PLASTICITY ENHANCING EPSPS

LTP can be expressed postsynaptically as a reduction of leak conductance in dendritic spine. This enables the EPSC to generate EPSP with greater length and time constants.

Excitatory transmission can be enhanced by HETEROSYNAPTIC INHIBITION OF INHIBITORY TRANSMISSION. This is mediated by endogenous cannabinoids acting on presynpatic terminals of nearby GABAergic synapses.

IS LTP REQUIRED FOR HIPPOCAMPAL CONSOLIDATION OF EXPLICIT MEMORY?CAMK AND NMDAR1 NEEDED FOR LONG-TERM SPATIAL REPRESENTATION IN HIPPOCAMPUS

Single pyramidal neuronin hippocampus

fires when mouse is incertain location(independent of

animal’s orientation)

Normal mouse remembers where it has been.spatial map in HC

does not change insubsequent chamber trials

Mice with hippocampus-restricted mutationsin CAMK or NMDAR1establish place fields,but do not remember

from day to day

IS LTP REQUIRED FOR HIPPOCAMPAL CONSOLIDATION OF EXPLICIT MEMORY?HIPPOCAMPAL CAMK AND NMDAR1 NEEDED FOR BOTH LTP AND SPATIAL MEMORY

SYNAPSES SENSITIVE TO NMDAR-MEDIATED LTP ARE ALSO SENSITIVETO NMDAR-MEDIATED LONG-TERM DEPRESSION (LTD)

AXON STIMULATION PROTOCOL AMPLITUDE OF EPSCS

20 min 1 m 60 min

Once p

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10 Hz

10 Hz

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6020 40 80

LTP

20 min 5 m 60 min

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LTD

LTD HAS A LOWER CALCIUM CONCENTRATION THRESHOLD THAN LTP,BUT LTP IS DOMINANT

LOW-FREQUENCY STIMULUS TRAIN

LOW-LEVEL CALCIUM ENTRY

ACTIVATION OF CALCINEURINCALCINEURIN

AMPA RECEPTOR INTERNALIZATION

LTDLTD

THETA- OR HIGH-FREQUENCY STIMULUS TRAIN

GREATER CALCIUM ENTRY

ACTIVATION OF CALCINEURINCALCINEURIN AND CAMKCAMK

AMPA RECEPTOR INSERTION AND PHOSPHORYLATION

LTPLTP

STRUCTURAL AND FUNCTIONAL FEATURES OF AMPA-TYPE GLUTAMATE RECEPTORS

AMPA receptors are homo- or hetero-tetramersRestriction of calcium entry mediated by GluR2; tetramers containing >1 GluR2 subunit conduct only Na+/K+

AMPA receptors encoded by different genes or by alternative splicing have different C-terminal tails.Receptor tails contain phosphorylation sites for different protein kinases and binding sites

for PDZ-domain-containing proteins

Receptors containing only GluR2(short) and/or GluR3 subunits are delivered constitutively fromvesicles to synapse

Retention at synapse mediated by complex with Glutamate Receptor Interacting Protein (GRIP)

Receptors containing at least one GluR1(long) subunit are stored in intracellular vesicles near synapseDuring LTP, GluR1-containing tetramers are added to the synapse

NMDAR-INDUCED CAMK ACTIVITY ACTS ON AMPA RECEPTORS IN TWO WAYSTO PROMOTE LTP

CAMKPDZ-protein

STGGRIPPSD-95

GRIPPSD-95

Calcineurin

CAMK phosphorylates an unknownprotein, enabling a PDZ-protein

that interacts with long tailon GluR1 to deliver receptor

TO EXTRASYNAPTIC SITE

Delivered receptors migrate (randomly?)into post-synaptic density,

where interactions of receptor-associated GRIP and STG and themajor postsynaptic matrix protein

PSD-95 anchor receptor to synapse

Newly delivered GluR1-containingAMPA receptors can be phosphorylated

directly by CAMK, whichincreases unitary conductance

of the receptor

Calcineurin activation promotes internalizationof AMPA receptors containing only

short-tail subunits, thereby promoting LTD

WHEN HIGH CALCIUM ENTRY ACTIVATES BOTH CALCINEURIN AND CAMK,WHEN HIGH CALCIUM ENTRY ACTIVATES BOTH CALCINEURIN AND CAMK,CAMK-MEDIATED GluR1-CONTAINING AMPAR EXOCYTOSIS EXCEEDS CALCINEURIN-MEDIATEDCAMK-MEDIATED GluR1-CONTAINING AMPAR EXOCYTOSIS EXCEEDS CALCINEURIN-MEDIATED

SHORT TAIL-ONLY AMPAR ENDOCYTOSISSHORT TAIL-ONLY AMPAR ENDOCYTOSIS

HIGH CAMK ACTIVITY INDUCED DURING LATE LTP IS ALSO MEDIATED BYNEW CAM KINASE PROTEIN SYNTHESIS NEAR THE SYNAPSE

Most mRNAs have 3’ polyA tail, which is necessary for initiation of the mRNA’s translation

Neurons contain some mRNAs that are not polyadenylated, are not translated,and are transported along dendrites to areas near dendritic spines

NMDA receptor activation and calcium entry activates a protein kinasecalled AURORAAURORA

Aurora kinase activates translation of nearby dormant mRNAs

ONE OF THESE DORMANT RNAs ENCODES CAM KINASEONE OF THESE DORMANT RNAs ENCODES CAM KINASE

Because of its dendritic localizaation, new CAMK synthesis is restricted to the synapse undergoing LTP

The dendritic localization of dormant CAMK RNA and its activation during LTP are mediated byCytoplasmic Polyadenylation Element Binding (CPEB)(CPEB) protein

HOW DOES CPEB PROTEIN CONTROL RNA DORMANCY AND ACTIVATION IN NEURONS?

PolyA is needed for assembly of 5’translation initiation complex

CPEB protein binding to 3’ CPEhelps mask RNA 5’ end

CPEB phosphorylation by Aurora allows for recruitment of polyA polyermerase (PAP)

Polyadenylation of dormant RNA allows assembly of 5’ translation initiation complex