Cellular and molecular mechanism of learning and memory

1. Aplysia Mouse Cellular and molecular mechanism of learning and memory • All neurons in behavioral circuits are identified and can be recorded easily. • Ideal for studying mechanisms underlying learned motor responses • Similar anatomy to human • Can do electrophysiology and behavioral tests • Genetic modified technologies are available http://media.hhmi.org/hl/08Lect4.html

2. The synaptic plasticity for learning and memory The number of neurons in the adult brain did not increase significantly with age, so memories were not the result of new neuron production. • Changing the effectiveness of the synaptic communication (short term) • Protein synthesis and developing new synapses (long term)

3. Why use animals to study learning and memory? • Eric Kandel was originally interested in psychotherapy • Wanted to locate Freud’s id, ego, & superego • Plato’s appetitive, rational, & spirited souls • Harry Grundfest told him to start “one cell at a time” • Kandel decided that human brains are too complicated.

4. UsingAplysia to Study Learning • Only 20,000 neurons • Neurons are large and easy to identify. • The gill-withdrawal reflex can use to study both associative and nonassociative learning

5. Types of Learning • Associative learning involves a connection between two elements or events. • Classical conditioning • Operant conditioning • Nonassociative learning involves change in the magnitude of response to a environmental event. • Habituation • Sensitization

6. Nonassociative learning of aplysia Habituation When an animal repeatedly encounters a harmless stimulus, its reaction to it decreases. Sensitization When an animal repeatedly encounters a harmful stimulus it learns to respond more vigorously not only to that stimulus but also to other stimuli, even harmless ones.

7. Habituation Long term

8. Sensitization

9. Short Term Memory • Short Term Memory involves changing the effectiveness of a synapse. • More neurotransmitter is released from a synapses (presynaptic) • Increase the sensitivity of receptors (postsynaptic)

10. Short term effect for nonassociative learning Habituation • Decrease of neurotransmitters • Inactivation of receptors • decrease in Ca2+ influx Sensitization • Activation of interneurons with serotonin (5-HT) as transmitter. • 5-HT stimulate presynaptic neurotransmitter release.

11. Short term effect for nonassociative learning

12. Short-term sensitization is mediated through serotonergic synapses of facilitating interneurons Sensitization is mediated by presynaptic elevation of cAMP & PKA activity, which has 3 effects: More vesicles in active zone K+ channel inactivation increases duration of depolarization and magnitude of Ca+2 influx 3) Activation of L--type calcium channels

13. Long Term Memory • Protein synthesis • Increase number of neurotransmitter receptors • Structural changes • morphological reorganisation • change in the number of synapses Anisomycin (protein synthesis inhibitor)

14. Long term effect for nonassociative learning Habituation • decrease in the # and the area of active zones of the synapse • decrease in the total # of synapses per neuron and the extent of axonal branching • Sensitization • PKA activates transcription factor CREB to make new proteins • increase in the # and the area of active zones of the synapse • increase in the total # of synapses per neuron and the extent of axonal branching

15. Synapses changes during memory formation

16. Long term effect for nonassociative learning Aplysia experiencing sensitization had 2800 terminals. Aplysia experiencing habituation had 800 terminals.

17. Classical Conditioning in Aplysia

18. Classical Conditioning in Aplysia US – shock to tail UR – gill withdrawal CS – touch to mantle CR– learned gill withdrawal

19. Conditioning trainings induce long-term memory Long-term conditioning are also mediated through presynaptic cAMP production: Ubiquitin hydrolase degrades PKA regulatory subunits, making the PKA active PKA induces specific CREB-dependent gene transcription and protein synthesis Other newly synthesized proteins help build new presynaptic terminals onto motor neurons

20. Environmental enrichment increases the synapses connections standard enriched

21. Memory consolidation Storing knowledge into the long-term memory • “When an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A’s efficacy, as one of the cells firing B, is increased.” - Hebb (1949)

22. Long term potentiation (LTP) • the persistent improvement in communication between two neurons after stimulating them simultaneously • necessary for the encoding and storage of the memory • LTP has a transient early and a consolidated late phase

23. LTP A demonstration of long-term potentiation LTP happens when electrical impulses are fired successively at a high rate so that the postsynaptic neuron is depolarized High-frequency stimulation baseline 30 min 60 min (at least) http://www.learner.org/courses/biology/units/neuro/images.html

24. Main actors control LTP: Glutamate receptors • AMPA receptor –Na+ channels • NMDAreceptor– allows Na+ and Ca2+ ions to enter the neuron • responds to glutamate ONLY when the membrane is partly depolarised by Na+ • glutamate excitation of NMDA receptors opens NMDA-dependent Ca2+ channels

25. Basics of NMDA receptor-dependent CA1 LTP Schaffer collaterals glutamate + - AMPA NMDA 2+ Mg

26. Basics of NMDA receptor-dependent CA1 LTP single stimulus Schaffer collaterals glutamate + - AMPA NMDA 2+ Mg + Na

27. Basics of NMDA receptor-dependent CA1 LTP tetanic stimulation Schaffer collaterals glutamate + depolarized membrane AMPA NMDA - 2+ Mg + Na Protein phosphorylation, gene transcription etc. LTP 2+ Ca

28. Basics of NMDA receptor-dependent CA1 LTP Weak stimulus Schaffer collaterals Cooperativity glutamate + depolarized membrane - NMDA 2+ AMPA Mg + Na 2+ Ca AMPA Protein phosphorylation, gene transcription etc. LTP Schaffer collaterals + Na glutamate

29. Evidence for an LTP/Learning Link • Mice with abnormal NMDA receptors have difficulty learning • Mice with more than normal NMDA receptors have “super” memory • Drugs that block LTP block learning • Drugs that facilitate LTP facilitate learning AMPA agonists: AMPA, glutamate antagonists: CNQX, NBQX NMDA agonists: glutamate, aspartate, NMDA antagonists: APV, AP5, MK-801, Ketamine, Phencyclidine, Mg++

30. The NMDA receptor and spatial learning Infusion NMDA receptor antagonist APV.

31. Deletion of the NR1 subunit in CA1

32. Deletion of the NR1 subunit in CA1 Water maze

33. Ampakines enhance AMPA receptor function

34. AMPA receptors and memory formation • NMDA receptor-dependent AMPA receptor synaptic delivery in LTP. • Calcium influx through NMDA receptors initiates the delivery of AMPA receptors from the recycling endosome to the postsynaptic site. • NMDA receptors are central to producing LTP but AMPA receptors maintaining it.

35. Mechanisms of Synaptic Plasticity • CaM-KII (calcium-calmodulin kinase type II ) • Enzyme present in dendrite, activated by Ca++ • Ca++ binds with CaM-KII, linking proteins attach to NMDA receptor • AMPA receptors are linked to NMDA receptor by protein.

36. NMDAR and AMPAR can be positively regulated by phosphorylated αCaMKII

37. Using tTA system to control the location and timing of mutant CaMKII expression

38. Mutant TG off Mutant TG on

39. Early LTP is protein synthesis independent • Last 1-3 hours • Ca++ entry through NMDA channels triggers LTP • Results in activation of protein kinase C (PKC) • Insertion of new AMPA receptors into the postsynaptic • Retrograde signal (NO) enhances glutamate release

40. Late LTP is protein synthesis dependent • Last >24 hours • Facilitated by dopamine • Repeated Ca2+ influx recruits an adenylyl cyclase, which activates cAMP-PKA-MAPK-CREB signaling and synthesis new proteins

41. Steps in the neurochemical cascade during the memory formation Chemical or electrical activation • Increase intracellular Ca+2 concentration • Activate kinases (CaMKII PKA, PKC etc) • Active transcription factor such as CREB binding to specific DNA sequence (CRE) • Active transcription to make new protein Genes required for memory formation

42. NMDAR-dependent signalling

43. cAMP response element-binding protein (CREB) Ca cAMP • CREB transcription factor: • Transcription factor that binds to the cAMP response element (CRE) • Phosphorylation by CaMKII is necessary for its nuclear translocation and DNA binding • CRE (CREB response element): • DNA sequence TGACGTCA in the promoter region of a gene NMDA D1 AMPA A Cyclase CaMKII PDE CREB phosphorylation and translocation to nucleus

44. Regulation of CReB-dependent gene expression involved in memory formation