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LTP and LTD
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Historical influences – what is a memory?
Santiago Ramón y Cajal - proposed memories are formed by strengthening connections between existing neurons to increase the effectiveness of their communication. (1894) Hebb’s postulate “When an axon of cell A is near enough to excite a 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 efficiency, as one of the cells firing B, is increased.” -Donald O. Hebb (1949) To paraphrase: “Inputs that fire together, wire together.”
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LTP (long term potentiation) and LTD (long term depression)
Leading candidate molecular mechanisms underlying learning and memory. Fundamental forms of synaptic plasticity. Widespread in the brain, but may have different variations -therefore important to specify which neurons are being studied. LTP and LTD are models of what is occurring during learning and memory (proposed explanations)
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Laminar Structure of Hippocampus
Hippocampus is critical for formation of spatial and explicit memories Well characterized and highly organized, laminar cytoarchitecture and stereotyped wiring pattern Amaral and Witter, Neuroscience 31: (1989)
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Tri-synaptic Circuit of Excitatory Synapses in the Hippocampus
stim Intracellular recording fEPSP (“fields”) CA1 s. oriens s. pyramidale s. radiatum Schaffer Collaterals to cortex 3 CA3 from cortex Perforant Path 1 Mossy Fibers 2 Granule cell s. moleculare Dentate Gyrus Modified from Blitzer et al., Biol Psychiat. 57:113 (2005)
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Finding Hebb in the Hippocampus
“…repeatedly or persistently takes part in firing it…” Induction Stim. Schaffer Collateral at 100Hz, for 1 sec (tetanus) CA1 100Hz tetanic stimulation (High frequency stim (HFS) tetanus Baseline Test pulse - Stim. Schaffer Collateral at 0.2Hz CA1 (Bliss and Lømo, 1973)
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Producing LTP Baseline Induction Post-Induction
Stim. Schaffer Collateral at 0.2Hz CA1 Induction Stim. Schaffer Collateral at 100Hz, 1sec (tetanus) CA1 tetanus Post-Induction Stim. Schaffer Collateral at 0.2Hz CA1
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Weak HFS Induces Transient Potentiation; Strong HFS Induces Stable LTP
cooperativity Nature Rev. Neurosci. 4; (2003) (2x 100 Hz, 1 sec, D1.5mV) (1x, low intensity, D0.6mV) Tsokas et al., J. Neurosci. 25:5833 (2005)
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Repetitive stimulation induces stronger LTP
-One tetanus may cause LTP of several minutes to a couple of hours -Repeated trains (4) leads to longer-lasting LTP (many hours, or until the slice dies) -reminiscent of strengthening of memory with repetition
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Associativity of LTP Weak and strong inputs are both potentiated
-but only if they are associated (occurring within short time window) Three key properties of long-term potentiation (LTP) were elucidated in the 1970s and early 1980s. All of these can be explained mechanistically by the biophysical properties of the NMDA (N-methyl-D-aspartate) receptors that are required to trigger LTP. Cooperativity This term refers to the fact that when using high frequency stimulation to induce LTP, a crucial number of presynaptic fibres must be simultaneously activated — they must 'cooperate' to elicit LTP (that is, a threshold stimulation strength must be used). The frequency of stimulation interacts with the stimulus strength such that increasing one decreases the requirement for the other to trigger LTP. This property is explained by the fact that, to trigger LTP, the postsynaptic cell must be sufficiently depolarized to allow current (particularly Ca2+) to flow through the NMDA receptor channel. Input (synapse) specificity This property refers to the fact that, when LTP is elicited at one set of synapses on a postsynaptic cell, adjacent synapses that were not activated during the induction protocol do not show LTP. This property is explained by the requirement that, to elicit LTP, synaptic NMDA receptors must be activated, leading to a spatially restricted increase in intracellular Ca2+ in the relevant dendritic spine. Associativity This term refers to the fact that LTP can be elicited at synapses that are activated by low-frequency, sub-threshold stimuli if their activation is temporally concurrent with an LTP-inducing stimulus at another set of synapses on the same cell. This property is explained by the fact that the LTP-inducing stimulus provides the requisite depolarization, which is rapidly transmitted through the dendritic tree to those synapses in which the NMDA receptors were simultaneously activated by the sub-threshold stimulus. It is easy to envision how this property makes LTP an attractive mechanism for associating two pieces of information being conveyed by different sets of afferents that synapse on the same postsynaptic cell. Please close this window to return to the main article. Nature Rev. Neurosci. 4; (2003)
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Input-specificity of LTP
A B specificity associativity Path A Path B A Only B Only A+B Baseline After HFS to Path A Blitzer, Long-term potentiation: Mechanisms of induction and maintenance. Sci. STKE 2005
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LTP Can Persist for Months / years In Vivo
(20-50T, dentate gyrus) Abraham et al., J. Neurosci. 22:9626 (2002)
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LTP has properties expected of a physiological basis for memory
1. RAPID – induced within seconds 2. GRADED – can be strengthened with repetition or intensity 3. ASSOCIATIVITY – can integrate inputs occurring at same time 4. SYNAPSE SPECIFIC - Induced only at appropriately stimulated synapses, and not neighboring synapses 5. PERSISTENT – can last up to months or longer in vivo 6. INDUCED BY PHYSIOLOGICAL STIMULI – “theta-burst stimulation” (TBS) mimics endogenous hippocampal rhythms
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Temporal stages in LTP (1) Induction (early phase, E-LTP) Analogous to “learning?” (2) Maintenance (late phase, L-LTP) Analogous to “memory?” Probably more stages exist Different stages involve distinct mechanisms
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Key Criteria for ‘Core’ events:
(1999) Key Criteria for ‘Core’ events: (1) Is the pathway activated upon LTP induction? (2) Is pathway activation required for LTP? (3) Does activation of the pathway mimic LTP? (4) Does activation of the pathway occlude LTP?
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Stimulation of NMDA-type Glutamate Receptors
LTP Induction : key steps Stimulation of NMDA-type Glutamate Receptors
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NMDARs are “coincidence detectors”
“Play The LTP Game” Glu Glu Glu Glu -glutamate binds AMPA and NMDA receptors but NMDARs are blocked at resting membrane potential by extracellular Mg2+ -AMPA receptors mediate most fast excitatory synaptic transmission -if glutamate is applied and membrane is depolarized at the same time, NMDA receptors are unblocked and allow calcium influx (Hebbian) -biophysical properties of NMDARs well suited for association
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NMDA receptors are critical for LTP and learning
I. Pharmacological inhibition Inhibition of NMDAr with APV abolishes LTP in vivo Nature 319, (27 February 1986)
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What about behavior? Morris water maze: test of spatial memory Requires hippocampal function (lesioned animals perform poorly) Nature 319, (27 February 1986) AP5 infusion into brain impairs performance on water maze II. Genetic inhibition Knockout of obligate GluN1 subunit of NMDARs in CA1 also blocks LTP and impairs spatial learning Tsien et al., Cell 87:1327 (1996)
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LTP Induction Requires Postsynaptic Ca2+
Malenka et al. Neuron 9:121 (1992) Requirement for Ca2+ is Brief photoactivated Ca2+ chelator diazo-4 Stimulation of NMDA-type Glutamate Receptors Increased Postsynaptic Ca2+
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During LTP, CaMKII is Activated in Dendrites
Phospho-CaMKII CONTROL LTP Stimulation of NMDA-type Glutamate Receptors Increased Postsynaptic Ca2+ Activation of CaMKII +LTP Giovannini et al. J. Neurosci. 21: 7053 (2001)
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CaMKII: How It Gets Activated
Persistent activation can be a form of “molecular memory” Binds directly to NMDA receptors Lisman et al. Nature Rev. Neurosci. 3:175 (2002)
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LTP Induction Regulates AMPAR channel properties and synaptic content
In 10 µM KN-62; 60 min after HFS: Glu Mostly GluA1/2 and GluA2/3 heteromers in adult hippocampus Stimulation of NMDA-type Glutamate Receptors Increased Postsynaptic Ca2+ Activation of CaMKII Enhanced AMPA-type Glutamate Receptor Function / Content Barria et al. Science 276:2042 (1997)
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Phosphorylation of AMPARs During LTP
YES CaMKII Site NO PKA site Lee et al. Nature 405:955 (2000)
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AMPAR delivery in LTP: Silent Synapses
functional LTP silent functional NMDAR AMPAR added LTP B1 B2 Isaac et al, 1997, Neuron, 18:269
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Building a Smarter Mouse Overexpress NMDA Receptors
Two relevant key points: 1. GluN2B expression is high early in development and decreases with age (opposite to GluN2A) 2. GluN2B allows greater calcium influx than GluN2A Nature 401: (2 September 1999)
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Overexpression of GluN2B Improves LTP …
Mutant Lines Controls Day 18 GluN2B Tg Tang et al., Nature 401:63 (1999) WT
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… and Improves Recognition/Spatial Memory
Preference for Novel Object Hidden Platform Tang et al., Nature 401:63 (1999) Been there, done that Caveat: mice may feel more inflammatory pain, which is NMDAR-dependent in pain-related forebrain areas Wei et al., Nat. Neurosci. 4, (2001)
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Summary: Core Signaling Events in Induction of LTP at CA3-CA1 Synapse
Baseline Synaptic Transmission (AMPAR-Mediated) During LTP Induction Blitzer, Long-term potentiation: Mechanisms of induction and maintenance. Sci. STKE 2005
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The Consolidation of LTP: Induction vs. Maintenance
Theta pulse stim (5 Hz) Stable reversal of LTP by theta pulse stimulation delivered 15 min after LTP induction in area CA1 of the freely moving rat. A, Group data from five animals showing that a 1 min train of TPS administered 15 min after TBS caused a lasting reversal of LTP. A second TBS episode administered 3 d after TPS produced a potentiation effect that was stable for the 4 d of recording thereafter. Each data point represents the group mean ± SEM (averages of 4 successive responses per animal and data point) of the initial slope of the dendritic field potential expressed as percentage of the baseline. Failure to obtain LTP reversal when theta pulse stimulation is administered 30 min after induction. A, Group data from six animals illustrating that LTP was not affected by a 1 min train of TPS delivered 30 min after induction and continued to persist for the 4 d of recording thereafter. Each data point represents the group mean ± SEM (averages of 4 successive responses per pathway and data point) of the initial slope of the dendritic field potential expressed as percentage of the baseline Similar to memory consolidation Staubli and Scafidi J. Neurosci. 19:8712 (1999)
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CaMKII Activity is Not Needed for LTP Maintenance
-Simiarly, NMDARs also are not required for LTP maintenance (after LTP is induced, APV has no effect) but induction still blocked Chen et al. J. Neurophysiol. 85:1368 (2001)
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LTP Maintenance Requires Protein Translation
(protein synthesis inhibitor) Osten et al. J. Neurosci. 16:2444 (1996)
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Requirement for transcription in L-LTP
(E-LTP) min ACT = actinomycin D, a transcriptional inhibitor Nguyen et al Science 265:
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LONG TERM DEPRESSION (LTD)
-Dudek & Bear, 1992 -Widespread--possibly found in all excitatory synapses -best studied in hippocampus, striatum, and cerebellum
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LTD in the Hippocampus CA1
Induction Low frequency stimulation (LFS) (e.g. 1 Hz stimulation for 15 minutes) J. Neurosci., July 1993, 13(7):
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NMDARs are required for LTD at CA1 synapses
J. Neurosci., 13: 2910 (1993) LTD is graded and saturable but does not occlude LTP PNAS 89: 4363 (1992) ‘De-depression’ HFS 1Hz
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How can NMDARs be required for both LTP and LTD?
Simple Model: Ca2+ Presynaptic Postsynaptic NMDA receptor HFS LFS Modifed from Lisman, 1989; Bear and Malenka, 1994. The level and pattern of calcium influx is the critical difference
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Bidirectional control of synaptic strength by kinases and phosphatases: Tri-state model
De-depression Potentiation PP2B Depotentiation Depression Lee et al., Nature (2000) 405: 955
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Clathrin-mediated endocytosis
LTD mechanisms II: AMPAR internalization Clathrin-mediated endocytosis Man et al., Neuron 25: 649–62, 2000 Insulin can also trigger endocytosis of ampa receptors for some bizarre reason – also clathrin coated pits. GST-amphiSH3 (glutathione –S- fusion protein amphiphysin SH3 domain) specifically interacts with endogenous dynamin and prevents it from taking in receptors– the 3m form fails to bind to dynamin and it cannot prevent dynamin from endocytosingS
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Bidirectional signaling in LTP and LTD
KINASES PHOSPHATASES Calcineurin/ PP1
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What are spines? Cajal, 1888, drawing of Purkinje cell, Golgi staining – “thorns or short spines” 1906 – Nobel awarded to Cajal + Golgi Golgi-stained modern day micrograph of cortical pyramidal neuron
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Morphology of spines 7 μm
Hippocampal CA1 neuron, calcein imaged using 2-photon laser scanning microscopy (2PLSM) Nimchinsky et al., 2002 Annu. Rev. Physiol.
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Large diversity exists in spine shape
Hippocampal CA1 neuron, GFP-filled: 3D reconstruction of a dendritic segment from confocal z-stacks McKinney, 2005, Biochem Soc Trans
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Spines and synapses >90% of excitatory synapses in CNS terminate on spines Neurotransmitter receptors are largely restricted to the surface of the spine synapse axon axon “en passant” dendrite dendrite Menahem Segal Nature Reviews Neuroscience 6, (April 2005)
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Dendritic spine development
“immature” “mature”
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Structural Synaptic Plasticity
Live confocal imaging of hippocampal neuron transfected with GFP-actin Dendritic spines are highly plastic and motile. This motility depends on actin cytoskeleton dynamics. Neuron. 1998 May;20(5): Rapid actin-based plasticity in dendritic spines. Fischer M, Kaech S, Knutti D, Matus A.
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Functions of dendritic spines – insights from biophysical properties
Spines allow biochemical compartmentalization: 1. high calcium concentrations possible without causing excitotoxicity 2. input-specific signaling 3. neck controls diffusional coupling to dendrite and provides electrical insulation 4. electrical insulation prevents saturation of signaling by a few inputs (thus allows integration of inputs in a more linear fashion) Spine head width Spine length Parent dendrite shaft Spine neck width
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Helical spine pattern maximizes connectivity
Purkinje cell dendrite Filtered (near face) schematic necks heads -Excitatory axons distribute to as many neurons as possible (minimize double hits) -Excitatory spines receive as many different inputs as possible onto a neuron -Creates a maximally distributed network with as many connections as possible to start with, which can later be modified by activity-dependent pruning and learning Neuron Sep 8;71(5):772-81
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Spine volume, postsynaptic area and vesicle number are correlated
What about synaptic strength? Postsynapse area Vesicle number KM Harris, 1989
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2-photon glutamate uncaging
720 nm Caged glutamate glu 2P-EPSC mimics native mEPSCs
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Spine volume correlates with synaptic efficacy
Matsuzaki et al., 2001, 2-photon glutamate uncaging, Hippocampal CA1 neuron
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LTP increases spine number
EPSP Engert & Bonhoeffer, 1999 Nature. LTP (using pairing protocol) increases spine number in hpc slice culture
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LTP increases spine size
1 μm (uLTP) (NMDAR unblocked) (HFS) Time-lapse images of dendritic spines on a hippocampal CA1 pyramidal neuron. Arrowhead indicates spot of two-photon uncaging of glutamate (stimulation for 1 min at 1 Hz). b, Time course of spine-head volume of stimulated (black circles) and neighbouring (green diamonds) spines shown in a. Matsuzaki et al., 2004.
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Larger spines are stronger
Spine-head volume (V, open symbols) Maximal AMPAR currents (I, filled symbols) All the small spines that showed enlargement immediately after pairing stimulation showed increase in synaptic strength as well. Matsuzaki et al., 2004
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Spine changes in long term plasticity
LTP LTD Smaller spines are weaker, but more easily potentiated Larger spines are stronger, but harder to potentiate further Small spines may be “plasticity” spines; large spines may be “memory” spines
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Are spines stable over time in vivo?
Imaging results from Karel Svoboda’s lab say ‘no’ Imaging results from Wen-Biao Gan’s lab say ‘yes’ Conclusion: depends
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In vivo imaging of dendritic spines
Images from somatosensory cortex (barrel cortex)
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Are spines stable over time? (no, not really)
Pyramidal neuron in barrel cortex 17% of spines are transient 23% of spines are semi-transient 60% of spines are stable Even “stable” spines are unstable 15% of “stable” spines disappear within 30 days Trachtenberg et al., Nature 2002
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Sensory deprivation enhances spine turnover
Barrel cortex Trachtenberg et al., 2002 Deprived (black circles) more unstable and fewer stable spines vs. control (white circle)
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Are spines stable over time? (yeah, sorta)
Pyramidal neurons in visual cortex Grutzendler et al., 2002
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Spine turnover decreases with animal age
Pyramidal neurons in visual cortex More stable with age… …but fewer in number Grutzendler et al., 2002
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Persistence of spines depends on their volume
persistent spines transient spines Spine volume and spine brightness are tightly correlated Larger spines are more stable Holtmaat et al., 2005
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Spines form excitatory synapses
Gray’s Type I, asymmetric synapse Kennedy, 2000
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The Postsynaptic Density (PSD)
An electron dense area on tip of spine head, occupying ~10-15% of total spine surface area. Adjacent to the cytoplasmic face of postsynaptic membrane Close apposition to the presynaptic active zone and docked synaptic vesicles This organization focuses the synaptic signal, increasing its efficiency, accuracy and speed Highly insoluble in nonionic detergents due to tight association with actin cytoskeleton – allows biochemical purification PSD
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PSDs have a large variety of shapes and sizes
PSDs can be continuous or “perforated”
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PSD components Contains hundreds to thousands of different proteins:
1. Receptors and voltage gated ion channels (NMDAR, AMPAR, mGluR, calcium channels) 2. Scaffold proteins (PSD-95 family, GKAP, Shank, Homer) 3. Signaling and regulatory proteins (CamKIIa, PKC, PKA, MAP kinases, Ras/Rho/Rac family small GTPases) 4. Cytoskeletal proteins, crosslinkers, regulator (F-actin, cortactin, myosin, cofilin, ARP2/3, alpha-actinin, spectrin) 5. Cell adhesion molecules (cadherins, neurexin/neuroligins, Eph/ephrins)
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Postsynaptic density (PSD) protein networks
Calabrese B et al. Physiology 2006;21:38-47
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PSD-95 scaffold protein PSD-95 is one of the major components of the PSD Member of the MAGUK (membrane-associated guanylate kinase) family Other family members: PSD-93/Chapsyn-110, SAP97/hDlg, SAP102 Scaffold proteins contain several protein-protein interaction domains to tether multiple components Domains of PSD-95 PDZ1-3
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Signaling/cytoskeletal protein assembly by PSD-95
NMDAR NMDAR PSD-95 Fyn nNOS SynGAP SPAR PSD-95 CRIPT GKAP band 4.1 a-actinin MAP1A tubulin F-actin
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Postsynaptic glutamate receptors interact with distinct scaffolding / anchoring proteins
Heterogeneous Dynamic Less tightly associated with PSD Consistent Rel. Stable NMDAR AMPAR PSD-95 Homer GRIP PDZ domain mGluR peri- synaptic (D. Bredt, C. Garner, S. Grant, R. Huganir, M. Kennedy, P. Seeburg, P. Worley, E. Ziff, M. Sheng)
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Many neurological diseases have spine defects
Fragile X Mental retardation Autism spectrum disorders Alzheimer’s disease Schizophrenia
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Fragile X has supernumerary, thin spines
+Pak inhibitor FRAX486 WT FMR1 KO FMR1 gene is silenced in Fragile X long / thin spines, too many spines FMR1 KO mice have too many spines, and they are long and thin (immature) (also show repetitive movements, hyperactivity, reduced anxiety and LTP, excessive LTD) P21-activated kinase (PAK) regulates actin dynamics and spine morphology Dominant negative (dn) PAK kinase has too few spines, and they are short and stubby (opposite of FMR1 KO) FMR1 KO x dnPAK rescues spines and behavior (Proc Natl Acad Sci U S A Jul 3;104(27): FMR1 KO given PAK kinase inhibitor FRAX486 has rescued spines and behavior Proc Natl Acad Sci U S A Apr 2;110(14):5671-6
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Spine abnormalities in many diseases
Nature Neuroscience 14, 285–293 (2011)
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Learning objectives LTP and LTD
1. Understand what synaptic plasticity is, and why it is important for nervous system function. 2. Understand the role of the hippocampus in memory, and why it is a good model system for learning and memory studies. 3. Understand the concept of Hebbian plasticity and its relation to LTP associativity and NMDA receptor coincidence detection. 4. Understand why LTP and LTD are attractive models of synaptic plasticity. 5. Understand how different stages of LTP are induced and the signaling proteins involved. 6. Understand the mechanisms that maintain LTP for different durations. 7. Understand how LTD is induced and expressed. 8. Have an idea of how synaptic plasticity (in models) relates to learning and memory (in animals), and how this can be experimentally tested. Different dendritic spine morphologies Functions of dendritic spines Morphological plasticity in response to stimuli Rapid actin-based motility of spines Spine turnover rates in vivo What is the PSD and basic constituents Structure and functions of scaffold proteins at the Dendritic spine defects in neurological diseases
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