In search of memory:from molecules to mind

By Vasyl Mykytyuk

Acknowledgement:

Professor Jerry Rudy of the psychology department at the University of Colorado at Boulder for his lectures and textbook “Neurobiology of Learning and Memory”

On average the human brain has 86 billion neurons

Glial cells are x10 to neurons

One neuron can have up to 10 000 synapses

During childhood and particularly during adolescence a process “synaptic pruning” occurs – up to 50% of neurons and synapses are reduced

Our brain always uses 20% of all energy

Brain functions

ArousalAttention

ConsciousnessDecision making

Executive functionsNatural language

LearningMemory

Motor coordinationPerceptionPlanning

Problem solvingThought

Mnemosyne (1881), mother of all Muses, goddess of memory by Dante Gabriel

Rossetti

Olin Levi Warner,Memory (1896). Library of Congress Thomas Jefferson Building, Washington, D.C.

The case of Henry Molaison

Henry Molaison 1926-2008

Forgetting curve by Herman Ebbinghaus (1913)

Two possible explanations of forgetting curve

Resting potential formation

ЕЛЕКТРИЧНЕ ПРОВЕДЕННЯ ІНФОРМАЦІЇ

• Membrane potential – Difference of charges across

the plasma membrane

• Resting potential– Resting cells are (-) inside

and (+) outside– Large amounts of Na+

outside the cell and K+ inside

• Action

potential/impulse– Rapid reversals in charges

across the plasma membrane– Caused by the exchange of

ions across the membrane of the neuron

– Threshold level (-55mV) needed to stimulate neurons ALL-OR-NONE principle

Sequence of event during synapse:

1. Arrival of action potential to the presynaptic axon terminal

2. Neurotransmitter release from synaptic vesicles into the synaptic cleft.

3. Molecules of neurotransmitter binding to specific receptors.

4 Induction of a chemical orelectrical signal in the postsynaptic cell.

The synaptic plasticity hypothesis, “the strength of synaptic connections—the ease with which an action potential in one cell excites (or inhibits) its target cell—is not fixed but is plastic and modifiable” (Squire and Kandel, 1999, p. 35).

Long-Term Potentiation (LTP) is produced by high-frequency stimulation (100 Hz = 100 times in a second, three times with 20 seconds between them)

Long-Term Depression (LTD) is produced by low-frequency stimulation (900 pulses of frequency 1–3 Hz ~ 15 minutes)

Dendrites are extensively populated with structures called dendritic spines. A spine is a small (submicrometer)membranous extrusion that protrudes from a dendrite. This figure illustrates a three dimensionalreconstruction of a section of a dendrite with spines of different shapes and sizes. Dendritic spines are ofspecial interest because (1) key receptors involved in the regulation of synaptic plasticity are located in spines and (2) changes in the composition and architecture of the spine are altered by neural activity.

(A) A schematic representation of an excitatory synapse. The PSD is opposed to the presynaptic active zone, attached to F-actin. (B) A schematic of the PSD. Core scaffold proteins of the PSD— PSD-95, GKAP, Shank, and Homer—interact with each other and other proteins and are thought to form a lattice for the assembly of postsynaptic membrane glutamate receptors and signaling molecules.

Mechanisms of ionotropic receptor action

Two types of ionotropic glutamate receptors: The a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor, The N-methyl-D-aspartate (NMDA) receptor

They are located in dendritic spines, play a major role in the induction and expression of LTP. When gluatamate binds to these receptors their channels open

and positively charged ions in the extracellular fluid (Na+ and Ca2+) enter the neuron.

APV, an NMDA receptor antagonist, prevents the induction of LTP but has no effect on its expression. CNQX, an AMPA receptor antagonist, prevents both the induction and expression of LTP.

NMDA receptor has two critical binding sites. One site binds to glutamate and the other site binds to magnesium - Mg2+.

Magnesium binds to a site in the pore, forming a magnesium plug.

Calcium enter requires two events:

1) glutamate has to bind to the NMDA receptor;

2) the synapse has to depolarize (by AMPA receptor).

The induction of LTP occurs when extracellular calcium enters the spine via the NMDA calcium channel. But entrance of calcium is not needed for LTP expression.

AMPA trafficking is regulated constitutively (double arrows) and by synaptic activity (single arrow). The second one is a mechanism for LTP-formation.

Changes in synaptic strength that support LTP evolve in stages that can be identified by the unique molecular processes that support each stage.

Generation and stabilization of trace

Constitutive trafficking of AMPA receptors

AMPA receptors become trapped in the PSD when (1) the influx of calcium into the spine via NMDA receptors (2) activates CaMKII. This kinase phosphorylates serine (P-serine) residues on the terminal of the TARP Stargazin. This activity releases the terminal from the membrane and (3) facilitates its binding to PSD-95 complexes, thereby trapping the receptor in the PSD. The stability of the trap increases as CaMKII phosphorylates more of the Stargazin serine residues.

Filament actin in the spine head is crosslinked with spectrins. (B) The protease calpain is activated when calcium enters the spine through the NMDA receptors. Calpain degrades spectrins and facilitates the dissembling of actin. C. This activity contributes to the rapid

insertion of additional AMPA receptors in the PSD.

Stabilizing the Trace

(A) (B)

(A) When drugs such as latrunculin and cytochalasin that prevent actin polymerization are applied before TBS, they do not prevent the induction of LTP, but it rapidly decays.

(B) LTP is maintained if these drugs are applied 15 minutes after LTP is induced.

N-cadherins are reorganized by an LTP-inducing stimulus. (A) Illustration of an unpotentiatedsynapse with the presynaptic terminal and postsynaptic spine weakly bonded by cadherin monomers. (B)A high-frequency stimulation is delivered to this synapse to induce LTP. (C) The high-frequency stimuluspromotes cadherin dimerization so the now-enlarged spine containing additional AMPA receptors istightly coupled to the presynaptic terminal and well positioned to receive glutamate released from thepresynaptic terminal.

An LTP-inducing stimulus enhances the function of integrins in two ways. (A) Prior to induction integrins are below the plasma membrane. Integrin interacting epitopes are encrypted and the activity of metaloproteinases (MMP9s) in the extracellular matrix (ECM) is low. (B) The induction stimulus increases the expression of integrins in the spine head and the activity of MMPs in the ECM. The proteolytic activity of MMPs directed at encrypted epitopes increases availability of integrin interacting epitopes.

Consolidation of Synaptic Changes: Translation and Transcription

Consolidation of Synaptic Changes: Translation and Transcription

New protein synthesis is essential for consolidation of synaptic changes

Synaptic activity simultaneously induces local mRNA translation and genomic signaling cascade, that trigger transcription of new mRNA

Messenger RNA and protein translation machinery, such as endoplasmic reticulum (ER) and ribosomes, is present locally in the dendritic spine region. Synaptic activity can activate this machinery and translate this mRNA.

Consolidation of Synaptic Changes: Specific Mechanisms

BDNF activates local protein synthesis through PI3K/mTOR pathway

(A) Release of caged glutamate, coupled with a spike-time protocol, produces sustained growth and maintenance of a single spine. (B) Inhibiting the TrkB response to BDNF prevents sustained growth and maintenance. This result indicates that this protocol releases BDNF into the extracellular space.

General models of how the signals reach the nucleus: synapse-to-nucleussignaling and soma-to-nucleus signaling

Synapse-to-Nucleus Signaling

Different signaling cascades lead to cAMP-responsive, element-binding (CREB) protein phosphorylation and transcription initiation

Soma-to-Nucleus Signaling. Calcium enters through Voltage-Gated Calcium Channels in soma of neuron and activates calmodulin and Calcium/calmodulin-dependent protein kinase type IV which in its turn activate CREB

“cAMP Response Element-Binding Protein Is a Primary Hub of Activity-Driven Neuronal Gene Expression”

Eva Benito, Luis M. Valor, Maria Jimenez-Minchan, Wolfgang Huber and Angel BarcoThe Journal of Neuroscience, 14 December 2011

CREB was found to occupy ≈4,000 promoter sites in vivo, but many of them are cell-specific

http://in.umh-csic.es/NADtranscriptomics/tablas/CREB_regulon.txt

http://in.umh-csic.es/NADtranscriptomics/tablas/CREB_timecourse.txt

Calcium entering the neuron through NMDA receptors is critical for the production ofLTP but this source of calcium alone may not be sufficient to produce any form of LTP

Ryanodine receptors (RyRs)Calcium-induced calcium release (CICR) - extracellular Ca2+ enters through the NMDA receptor and binds to ryanodine receptors (RyRs) to release additional Ca2+ from the ER

Glutamate binds to a subtype of metabotropic receptor called mGluR1, which is coupled which G-protein, which in its turn activate an effector-enzyme (Phospholipase C) . When IP3 binds to IP3R calcium is released from ER in the dendritic compartment.

A single TBS train produces a short-lasting form, LTP1; four trains produce amoderately lasting form, LTP2; and eight trains produce a very long-lasting form, LTP3.

• LTP1 depends only on RyRs releasing ER calcium in the dendritic spinecompartment.• LTP2 depends on IP3 receptors releasing calcium from ER in the dendriticcompartment.• LTP 3 depends on calcium entering the soma through vdCCs.

How do mRNAs transcribed in response to synaptic activity find their way to the correct synapses?

Synaptic tag and capture hypothesis(Frey, Morris, 1998)

What is the molecular basis of the synaptic tag?

Changes in functional and structural properties of spines that depend on actin filaments are a major component of the tagging operation.

Potentiated spines contain more AMPA receptors and an enlarged actin cytoskeleton. Enlarged spines are better traps or tags for capturing new mRNAs/proteins needed for consolidation. One possibility is that actin-associated proteins interact with microtubules to cause them to invade the spine and permit motor proteins to cargo the relevant mRNAs/proteins to the spines that have been potentiated. In addition, once consolidated, spines with an enlarged actin cytoskeleton might successfully compete for the molecules that are needed to self-maintain and thus provide part of the solution to the molecular turnover problem.

The degradation of existing proteins is important for LTP production.

Components of the UPS are localized in dendrites, synapses, and the nucleus. Glutamate transmission activates the ubiquitination processes and the activation of CaMKII plays acritical role in recruiting proteasome into dendritic spines. Moreover,stimulation that induces LTP activates this system and results in protein degradation

Protein Degradation Influences Three Phases of LTP: (a) an early stage that involves only post-translation modifications, (b) a later stage that depends on local protein synthesis in dendritic-spine regions, and (c) a second wave of proteins synthesized from new mRNA that is a product of genomic signaling cascades

SUMMARY OF CONSOLIDATION PROCESS

Maintaining the Consolidated Trace

Maintaining the Consolidated Trace

the molecular turnover problem. The synaptic molecules that support memory traces are shortlived in comparison to the duration of our memories.

Crick, however, suggested ageneral answer: “… the molecules in synapses interact in such a way that they can be replacedwith new material, one at a time, without altering the overall state of the structure.”

(A) An unpotentiated synapse is depolarized. (B) As the synapse is potentiated, AMPA receptors are added to the postsynaptic density. (C) Two processes—(1) degradation of AMPA receptors and (2) receptor endocytosis — then pressure the synapse to (D) return to its pre-potentiated state.

(A) Kinases like PKC have aninhibitory–regulatory domain and a catalytic domain. Such kinases require a second messenger (SM) to remove the inhibitory domain and expose the catalytic domain.

(B) PKMζ has no inhibitory domain so it does not require second messenger to be catalytic. It is perpetually active.

(C) PKMζ mRNA is present in dendritic spine regions and its translation requires synaptic activity to activate several kinases and actin filaments.

(D) Once translated, PKMζ self perpetuates; it interacts with other proteins to remove the translator repressor and thereby facilitates translation of PKMζ mRNA in dendritic spines where it was initially translated.

(A) As noted, PKMζ has no inhibitory domain; however, the peptide ZIP functions as an inhibitory domain and inactivates it. (B) ZIP applied to a slice prior to the inducing stimulus does not affect the early induction phase of LTP but does prevent the late phase. (C) ZIP applied 2 or 5 hours following induction reverses late-phase LTP. (After Serrano et al., 2005.)

A pool of GluA2 AMPA receptors is trapped outside of the synapse by protein interacting with C kinase 1 (PICK1). Release of this pool depends on atrafficking protein enzyme, NSF. When PKMζ interacts with the NSF–PICK1 complex, the receptors are released for entry into the PSD.

(A) The activation of Trk (tyrosine kinase) receptorsphosphorylates tyrosine sites on GluA2 AMPA receptors. This activity releases the receptors from the PSD into the endocytotic zone where they bond with the NSF–PICK1 complex for recycling.

(B) Thepresence of PKMζ disrupts the normal endocyticcycle—the receptors remain trapped in the PSD andthe synapse remains potentiated.

PKMζ maintains potentiated synapses through two parallel and complementary actions: (1) it facilitates the release of nonsynaptic pools of GluA2 receptors to replace GluA1 receptors and (2) it interferes with the endocytic cycle that normally removes GluA2sfrom the synapse.

(A) Once synaptic activity induces PKMζ synthesis in a spine compartment (spine 2 above) in response to synaptic activity, continued synthesis could lead to PKMζ spreading from the original compartment to neighboring spines (1 and 3), where it could potentiate these spines by releasing GluA2 receptors (not shown).

(B) The spread of PKMζ is minimized because once the GluA2 receptors are released into the spine (1) they are free to recapture the PKMζ–PICK1 complex (2). In principle this autotagging process traps PKMζ in the spines where it was synthesized. Because PKMζ disrupts the endocytic cycle, an additional outcome is that

GluA2s remain in the PSD region and the spine remains potentiated.

Mice were genetically modified to lack PKMζ. However, established LTP was maintained and, moreover, ZIP reversed the LTP maintenance.These unexpected results support three conclusions: (1) enduring memories and LTP can be established independent of the presence of PKMζ, that is, the maintenance of LTP does not require PKMζ, (2) ZIP is not selective for PKMζ as it also interacts with other molecules, and (3) there must be other kinases or other molecules that can participate in the maintenance of LTP and memories. There are at least two alternative explanations of these data.• PKMζ is the primary memory maintenance molecule in normal rats, and the genetic studies have uncovered redundant compensatory molecules whose contribution is masked by the normal presence of PKMζ.• PKMζ is not involved in the maintenance of LTP and maintenance depends on other kinase targets of ZIP.

The field will now need to answer several questions:• What are these new targets of ZIP that must be engaged to support maintenance?• Are the processes these new targets regulate redundant with PKMζ?• What if any role does PKMζ play in maintenance?Until these questions are answered it may be appropriate to conclude that PKMζ and other kinasetargets of ZIP maintain the synaptic changes that support LTP.

Prion-like protein (cytoplasmic polyadenylation element bindingprotein (CPEB)) is a candidate for maintenance molecule

(A and B) Schematic models of pathogenic (A) and functional (B) prions.(C) Antibody that is specific for the aggregated (functional prionic) form of ApCPEB selectively blocks the maintenance of long-term facilitation produced by 5HT.

Large spines become resistant to modifications by calcium efflux out of the neck

Some spines are large (A) – they remember, but there are always new filopodium-like spines (C) grow to find synapses-contact, thus there can not be problem of dendritic saturation with stable spines

Epigenetics:

our experience can affect our DNA

Memory formation involves epigenetic mechanisms,

* 5-AZA – 5-azacytidine

Epigenetics: The sins of the fatherThe roots of inheritance may extend beyond the genome, but the mechanisms remain a puzzle.

Virginia Hughes

Every day we change our brain, that`s why we will never be the same tomorrow…

Molecular mechanisms of forgetting

Figure 7. Model of Dopamine-Mediated Learning and Forgetting The left panel illustrates dopamine-mediated learning. Upon electric shock, a US signal is delivered when DANs release large amounts of dopamine (red spheres) onto the MBs. Binding of dopamine to the dDA1 receptor (green) leads to increased cAMP, while the CS odor stimuli results in increased Ca2+ signaling. Coincident Ca2+ and cAMP signaling leads to strong memory formation within the MBs (Tomchik and Davis, 2009). The right panel illustrates dopamine-mediated forgetting. After learning, a low level of ongoing dopamine release is sensed by the DAMB receptor (red) and weakens the memory over time. Differences in affinity of the two receptors for ligand, compartmentalization, coupling to downstream signaling pathways, or signaling kinetics may account for the dominant action of dDA1 in acquisition and DAMB in forgetting.

2012

Spike-timing-dependen-plasticity (STDP)

those who fire together, wire together

NMDA receptor activation through depolarization after presynaptic activation results in enhancement, NMDA receptor activation through depolarization before presynaptic activation results in weakening of synaptic strength, both thought to be mediated by different levels of calcium [34].

Memory modulation

NMDA receptor can include either GluN2A or GluN2AB

Receptors with GluN2AB take more time to close, so much more Calcium is influxed

During our maturation our GluN2AB are replaced by GluN2A, so it is more difficult to strenghten our synapses

Joe Tsien genetically engineered mice, overexpressing GluN2AB in hippocampus and cortex.

Gary Lynch and his colleagues have developed a class of drugscalled ampakines that may enhance cognitive function, AMPAr are also critical for retrieval

Sunifiram, Unifiram

Memory modulation framework

Epinephrine does not cross the blood brain barrier. However, when it is releasedfrom the adrenal medulla it binds to adrenergic receptors on the vagal nerve. In response to activation, the vagal nerve releases glutamate on neurons in the solitary tract nucleus (NTS). Activated NTS neurons release glutamate onto neurons in the locus coeruleus, which in turn release norepinephrine that binds to adrenergic receptors in the basolateral amygdala (BLA). Disrupting any component of this neuro-hormonal circuit will prevent arousal from enhancing memory.

The Fate of Retrieved Memory

retrieving or reactivating the memory can return it to an active, labile state

Active trace theory

Reconsolidation theory

the synapses underlying the trace become unbound or weakenedretrieval also initiates another round of protein synthesis so that the trace is “reconsolidated.”

Model representation of reconsolidation cascade(Tronson, Nature reviews, 2007)

Multiple memory systems

Hierarchy of Episodic memory system

The indexing theory of hippocampus role in Episodic memory

Hippocampus against entropy

Ribot`s Law explanation

Frederick Bartlett`s serial reproduction

Memory during sleep

2004

Ribeiro, Nicolelis, 2004

Memory manipulations

Nature, 01 June 2014

2014

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