1. 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 synapses in behavioral circuits are identified and can be recorded easily Ideal for detailing mechanisms underlying implicit learned motor responses Capable of learned behaviors Amenable to random mutagenesis and selection of mutants with defective behaviors Similar anatomy to human Amenable to study of explicit memory Hippocampus amenable to electrophysiology Behavior modification of genetically modified mice

2. APLYSIA SHORT-TERM LEARNED RESPONSES AFFECTING GILL WITHDRAWL REFLEX HABITUATIONSENSITIZATION CLASSICAL CONDITIONING Repeated tactile stimulation of siphon depresses gill withdrawl response Harmful stimulus sensitizes gill withdrawl response to subsequent harmful OR harmless stimuli given to same OR different body regions Pairing harmful stimulus with preceding harmless conditioning stimulus sensitizes gill withdrawl response to subsequent conditioning stimulus but not to tactile stimuli given to other body areas

3. HABITUATION IS DUE TO DEPRESSED NEUROTRANSMITTER RELEASE AT SEVERAL SITES Rapidly repeated tactile stimulation of siphon attenuates gill withdrawl both during the training and for a short period afterwards. Habituation is due to reduced neurotransmitter release by the sensory neuron and by relevant interneurons in response to the tactile stimulus. I.e., the memory of habituation is distributed at various synapses in the circuit Whereas a rapid series of stimuli induces short-term habituation, several sets of tactile stimuli distributed over several hours induces long-term habituation that lasts for weeks. Long-term habituation requires new protein synthesis and is due to pruning of synaptic connections

4. SHORT-TERM SENSITIZATION IS MEDIATED THROUGH AXO-AXONIC SEROTONERGIC SYNAPSES OF FACILITATING INTERNEURONS Serotonergic facilitating interneurons send axo-axonic connections to broadly distributed sensory neurons Unconditioned stimulus causes interneurons to release serotonin, which acts through metabotropic HT receptors to increase the likelihood of neurotransmitter release following sensory neuron firing Sensitization can be mimicked without sensitizing stimulus by local experimental application of serotonin Sensitization is mediated by presynaptic 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

5. 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”

6. TEMPORALLY SPACED SENSITIZATION OR CONDITIONING TRAININGS INDUCE LONG-TERM IMPLICIT MEMORY Long-term sensitization and conditioning are also mediated through presynaptic cAMP production and PKA activity PKA induces specific CREB-dependent gene transcription and protein synthesis: Newly synthesized ubiquitin hydrolase degrades PKA regulatory subunits, making the enzyme making the enzyme constitutively active Other newly synthesized proteins help build new presynaptic terminals onto motor neurons

7. GENETIC SCREENS FOR GENES AFFECTING CONDITIONING IMPLICIT MEMORY ALL 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 G S -coupled receptor PKA-R1 encodes PKA

8. HIPPOCAMPAL NEURONS IN DIFFERENT RELAYS ARE ALL CAPABLE OF UNDERGOING SYNAPTIC LONG-TERM POTENTIATION AXON STIMULATION PROTOCOL AMPLITUDE OF EPSCS 20 min1 m60 min Once per minute 10 Hz EPSP Slope (% original) 300 100 200 TIME (min) 60204080 “THETA” BURST One Theta burst gives what is sometimes called Early LTP Early LTP, which is less than doubling of EPSC which lasts for hours Four Theta bursts spaced minutes apart generate Late LTP Late LTP, with up to 4-fold EPSC stimulation that lasts for days

9. 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.

10. LTP AT MOSSY FIBER--CA3 SYNAPSES IS DUE TO PRESYNAPTIC CALCIUM INFLUX AND cAMP/PKA PATHWAY

11. LTP AT SCHAFFER COLLATERAL--CA1 SYNAPSES IS DUE TO POSTSYNAPTIC CALCIUM INFLUX AND CAM KINASE ACTIVITY LTP at CA3-CA1 synapse is blocked by NMDAR antagonist APV and by inhibitors of CAM kinase

12. PRESYNAPTIC COMPONENT OF EARLY AND LATE LTP AT CA3--CA1 SYNAPSES RESEMBLES SHORT- AND LONG-TERM SENSITIZATION Late LTP absolutely requires new protein synthesis

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

14. 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.

15. IS LTP REQUIRED FOR HIPPOCAMPAL CONSOLIDATION OF EXPLICIT MEMORY? CAMK AND NMDAR1 NEEDED FOR LONG-TERM SPATIAL REPRESENTATION IN HIPPOCAMPUS Single pyramidal neuron in hippocampus fires when mouse is in certain location (independent of animal’s orientation) Normal mouse remembers where it has been. spatial map in HC does not change in subsequent chamber trials Mice with hippocampus- restricted mutations in CAMK or NMDAR1 establish place fields, but do not remember from day to day

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

17. SYNAPSES SENSITIVE TO NMDAR-MEDIATED LTP ARE ALSO SENSITIVE TO NMDAR-MEDIATED LONG-TERM DEPRESSION (LTD) AXON STIMULATION PROTOCOL AMPLITUDE OF EPSCS 20 min1 m60 min Once per minute 10 Hz EPSP Slope (% original) 300 100 200 TIME (min) 60204080 LTP 20 min5 m60 min Once per minute 2 Hz EPSP Slope (% original) 300 100 200 TIME (min) 60204080 LTD

18. LTD HAS A LOWER CALCIUM CONCENTRATION THRESHOLD THAN LTP, BUT LTP IS DOMINANT LOW-FREQUENCY STIMULUS TRAIN LOW-LEVEL CALCIUM ENTRY CALCINEURIN ACTIVATION OF CALCINEURIN AMPA RECEPTOR INTERNALIZATIONLTD THETA- OR HIGH-FREQUENCY STIMULUS TRAIN GREATER CALCIUM ENTRY CALCINEURINCAMK ACTIVATION OF CALCINEURIN AND CAMK AMPA RECEPTOR INSERTION AND PHOSPHORYLATIONLTP

19. STRUCTURAL AND FUNCTIONAL FEATURES OF AMPA-TYPE GLUTAMATE RECEPTORS AMPA receptors are homo- or hetero-tetramers Restriction 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 from vesicles 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 synapse During LTP, GluR1-containing tetramers are added to the synapse

20. NMDAR-INDUCED CAMK ACTIVITY ACTS ON AMPA RECEPTORS IN TWO WAYS TO PROMOTE LTP CAMK PDZ-protein STG GRIP PSD-95 GRIP PSD-95 Calcineurin CAMK phosphorylates an unknown protein, enabling a PDZ-protein that interacts with long tail on GluR1 to deliver receptor TO EXTRASYNAPTIC SITE Delivered receptors migrate (randomly?) into post-synaptic density, where interactions of receptor- associated GRIP and STG and the major postsynaptic matrix protein PSD-95 anchor receptor to synapse Newly delivered GluR1-containing AMPA receptors can be phosphorylated directly by CAMK, which increases unitary conductance of the receptor Calcineurin activation promotes internalization of AMPA receptors containing only short-tail subunits, thereby promoting LTD WHEN HIGH CALCIUM ENTRY ACTIVATES BOTH CALCINEURIN AND CAMK, CAMK-MEDIATED GluR1-CONTAINING AMPAR EXOCYTOSIS EXCEEDS CALCINEURIN-MEDIATED SHORT TAIL-ONLY AMPAR ENDOCYTOSIS

21. HIGH CAMK ACTIVITY INDUCED DURING LATE LTP IS ALSO MEDIATED BY NEW 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 kinase AURORA called AURORA Aurora kinase activates translation of nearby dormant mRNAs ONE 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 by (CPEB) Cytoplasmic Polyadenylation Element Binding (CPEB) protein

22. 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’ CPE helps 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