Universidad Autónoma de Madrid
Faculty of Science Molecular Biology Department
Isoform-specific roles of GSK3 in synaptic transmission and plasticity
PhD thesis
Jonathan Evan Draffin
Madrid 2018
Universidad Autónoma de Madrid
Faculty of Science Molecular Biology Department
Programa de Doctorado en Biociencias Moleculares
Isoform-specific roles of GSK3 in synaptic transmission and plasticity
Doctoral thesis submitted by Jonathan Evan Draffin, Bachelor of Science in Biology and Master of Science in Genetics and Cell Biology, to obtain the degree of Doctor of Philosophy in Molecular Biosciences from the Universidad Autónoma de Madrid.
Thesis director: Dr. Jose Antonio Esteban García Thesis tutor: Dr. Francisco Zafra Gómez
Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM)
La realización de esta tesis doctoral ha sido posible gracias a la concesión de una ayuda predoctoral de Formación de Personal Investigador (FPI) del Ministerio de Economía y Competitividad.
Su desarrollo ha tenido lugar en el laboratorio del Dr. José Antonio Esteban García, bajo su supervisión y dentro de las instalaciones del Centro de Biología Molecular ‘Severo Ochoa’
(CBMSO), centro mixto de la Universidad Autónoma de Madrid (UAM) y del Consejo Superior de Investigaciones Científicas (CSIC).
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Acknowledgements
The people involved with this work in one way or another are too numerous to mention. Thanks to you all.
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Resumen
La plasticidad sináptica es un mecanismo fundamental por el que las neuronas alteran la fuerza de sus conexiones. Es probable que estos cambios codifiquen aspectos de las interacciones que tiene un organismo con su entorno. En efecto, formas paradigmáticas de la plasticidad, como la potenciación y depresión a largo plazo (LTP y LTD, respectivamente, del inglés long term potentiation/depression) se consideran como eventos moleculares importantes durante la formación y refinamiento de ciertos tipos de memoria. Tanto LTP como LTD se encuentra desregulado en patologías del sistema nervioso como la enfermedad de Alzheimer, lo que puede ser responsable por el fallo devastador de la memoria en esa condición. Glucógeno sintasa kinasa 3 (GSK3) juega un papel central en la enfermedad de Alzheimer, y se ha demostrado que se requiere para LTD y que controla el equilibrio entre distintas formas de plasticidad en la sinapsis. Por estas razones, se ha considerado como diana terapéutica en varios contextos.
GSK3 existe en dos isoformas, GSK3α y GSK3β, pero la importancia de la isoforma ‘α’ se ha pasado por alto frecuentemente. Se desconoce cuál de las dos isoformas de GSK3 está involucrado en mediar LTD.
En este trabajo, eliminamos específicamente la actividad de cada isoforma de GSK3 con ARN de interferencia y compuestos novedosos selectivos, para evaluar sus papeles en LTD. Empleando técnicas electrofisiológicas y de imagen in vivo, demostramos que GSK3α, pero no GSK3β, se requiere para LTD, y que se ancla de forma temporal en espinas dendríticas durante la inducción de LTD. Además, hemos encontrado que la proteína tau, que juega un papel fundamental en la enfermedad de Alzheimer, se requiere tanto para este anclaje de GSK3α, cómo para la LTD inducido por GSK3α. En presencia de actividad sináptica, entonces, GSK3α induce LTD a través de tau.
También investigamos el papel de GSK3 en controlar la transmisión sináptica en condiciones basales.
Sorprendentemente, la actividad de GSK3 fue necesaria para mantener la transmisión sináptica. GSK3 postsináptica estaba involucrado en este proceso, y la pérdida de transmisión debida a la inhibición de GSK3 se expresó postsinápticamente. Fue necesario bloquear a las dos isoformas de GSK3 en la postsinapsis para inducir la depresión sináptica, lo que implica que la actividad de cualquiera de las isoformas es suficiente para mantener a la transmisión basal.
Los resultados de esta tesis doctoral subrayan a GSK3α como diana terapéutica potencial en la mejora del déficit de memoria en la enfermedad de Alzheimer. También enfatizan la importancia de tratamientos selectivos por isoforma, considerando los papeles de GSK3 en funciones básicas de las sinapsis.
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Abstract
Processes of synaptic plasticity are fundamental mechanisms by which neurons alter the strength of their connections. Such changes are likely to encode aspects of an organism’s interaction with its environment.
Indeed, paradigmatic forms of plasticity such as long-term potentiation (LTP) and long-term depression (LTD) are thought to be important molecular events during the formation and refinement of certain types of memory. Both LTP and LTD are also frequently dysregulated in pathologies of the nervous system such as Alzheimer’s disease, which may be responsible for the devastating failure of memory in this condition. Glycogen synthase kinase 3 (GSK3) is a central player in Alzheimer’s disease, and has also been shown to be required for LTD and mediate the balance between different forms of plasticity at the synapse. For these reasons, it has been considered as a therapeutic target.
GSK3 exists as two isoforms, GSK3α and GSK3β, of which the importance of the α isoform has been frequently overlooked. A major unknown is which of the two GSK3 isoforms is involved in mediating LTD.
Here, we specifically target the GSK3 isoforms with shRNA knockdown and novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3β, is required for LTD, and is transiently anchored in dendritic spines during LTD induction.
Interestingly, we also find that the microtubule-binding protein tau, which plays key roles in Alzheimer’s disease, is required for this spine anchoring of GSK3α, and for GSK3α-induced LTD. Under conditions of synaptic activity, therefore, GSK3α induces LTD through tau.
We also examined the role of GSK3 in controlling synaptic transmission under basal conditions.
Surprisingly, we found that GSK3 activity is required to maintain synaptic transmission. Postsynaptic GSK3 was involved in this process, and loss of transmission following blockade of GSK3 activity was expressed postsynaptically. Both isoforms of GSK3 needed to be knocked down in the postsynapse for transmission to be depressed, implying that the activity of either isoform is able to maintain synaptic transmission.
The results in this PhD thesis highlight GSK3α as a potential therapeutic target for the alleviation of memory deficits during Alzheimer’s disease, and emphasise the importance of isoform-specific targetting of GSK3 more generally, given its vital roles in fundamental synaptic functions.
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Table of contents
Acknowledgements ... ix
Presentación ... Error! Bookmark not defined. Abstract ... xi
Table of contents ... xii
Abbreviations ... 1
Introduction ... 5
A history of remembering ... 6
Memory embodied ... 6
Holism reigns ... 6
HM freshens the stagnation ... 7
Multiple memory systems ... 7
‘Place sails’ - is the hippocampus also a navigator? ... 8
The mnemonic helmsman - squaring the two circles of research ... 8
Interlude: The hippocampus catches a current ... 8
Unearthing the sea-creature... 9
Hippocampal circuitry: direct and indirect ... 9
Functional significance of different hippocampal pathways ... 10
The hippocampus as an ideal preparation for neurobiological study... 10
Finding the engram ... 11
From engrams to plasticity ... 12
Excitatory synapses ... 12
Plasticity and memory ... 13
The plastic devil in the synaptic details: molecular mechanisms of plasticity ... 14
AMPA receptor structure and function ... 15
AMPA receptor function is determined by subunit composition ... 16
Mechanisms of LTP ... 17
Mechanisms of LTD ... 18
Structural plasticity ... 19
Signalling pathways controlling LTD ... 20
GSK3: lone wolf, and jack of all trades ... 20
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The challenge and solution of GSK3 regulation specificity ... 21
Post-translational regulation ... 21
Subcellular localisation ... 23
Alternative splicing ... 23
Isoforms ... 23
Doppelgangers and stereotypes ... 23
Postsynaptic roles of GSK3 ... 24
Control of receptor trafficking and function by GSK3 ... 24
Tau ... 24
GSK3 in synaptic plasticity ... 25
Differential isoform roles in synaptic plasticity ... 26
Objectives ... 27
Materials and Methods ... 28
Materials ... 29
Reagents ... 29
Antibodies ... 30
Drugs ... 30
Animals ... 30
Methods ... 31
Organotypic hippocampal slice cultures ... 31
Acute slice preparation ... 31
Primary hippocampal neuron culture ... 31
Lentivirus production ... 32
Sindbis virus production ... 32
Construction of DNA constructs ... 33
Expression of recombinant proteins ... 34
Electrophysiology ... 34
Whole cell recording ... 34
Field potential recording ... 35
Post-embedding immunogold electron microscopy ... 35
Slice treatment, dehydration and embedding ... 35
Post-embedding immunogold labelling of AMPA receptors ... 36
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EM image acquisition and quantification ... 36
Fluorescence Recovery After Photobleaching (FRAP) ... 37
Fixed-tissue confocal fluorescence imaging ... 37
Surface immunostaining of transferrin receptors... 38
Western blotting... 38
Statistical analysis ... 39
Results ... 40
Chapter 1: Basal synaptic transmission ... 41
GSK3 activity is required for the maintenance of basal synaptic transmission ... 41
The acute requirement for GSK3 in basal transmission has a partially presynaptic locus ... 43
Postsynaptic knockdown of GSK3 activity depresses basal synaptic transmission ... 45
The postsynaptic role of GSK3 in maintaining synaptic transmission is not mediated by a single isoform ... 47
Acute inhibition of GSK3ɑ depresses basal transmission ... 49
GSK3 inhibition induces a rearrangement of AMPA receptors in the spine ... 51
GSK3 inhibition results in a synaptic decrease in functional GluA2-containing AMPA receptors ... 55
Inhibition of GSK3 via Akt activation similarly depresses basal synaptic transmission 57 Dynamin is not required for the synaptic depression induced by GSK3 inhibition ... 59
Rab5 is not required for the depression induced by GSK3 inhibition... 61
PTEN is not required for the depression induced by GSK3 inhibition ... 62
Chapter 2: LTD ... 64
shRNA knockdown of GSK3ɑ, but not GSK3β, blocks NMDAR-dependent LTD ... 64
Pharmacological inhibition of GSK3ɑ, but not GSK3β, blocks NMDAR-dependent LTD 65 Increased GSK3 activity induces synaptic depression ... 66
GSK3ɑ, but not GSK3β, is transiently anchored in dendritic spines during LTD ... 69
GSK3ɑ spine anchoring requires tau ... 71
Synaptic depression induced by GSK3ɑ activity requires tau ... 73
Discussion ... 75
Chapter 1: GSK3 activity is required to maintain basal synaptic transmission ... 76
Partitioning of synaptic consequences of GSK3 activity changes ... 76
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Subcellular segregation of GSK3 functions... 78
Potential molecular targets of GSK3 in its role in controlling basal synaptic transmission ... 79
Mechanisms of isoform specificity during LTD ... 80
Upstream regulation of GSK3 isoforms during LTD ... 81
Inhibition of GSK3α during LTP ... 81
Subcellular localisation of isoforms ... 82
Structural locus of specificity: binding ... 82
Structural locus of specificity: activity towards substrate ... 83
GSK3α in Alzheimer’s disease ... 83
Tau in Alzheimer’s disease ... 84
LTD and Alzheimer’s disease ... 84
GSK3α in Alzheimer’s pathogenesis... 85
Tau in LTD: ideas from Alzheimer’s ... 85
Clinical perspectives: GSK3 as a therapeutic target ... 87
Isoform-specific therapy ... 87
GSK3α as a therapeutic target in Alzheimer’s disease ... 88
Conclusiones ... 89
Conclusions ... 90
Bibliography ... 91
Annex ... Error! Bookmark not defined.
1
Abbreviations
5HT - 5-hydroxytryptamine (serotonin)
ACSF - artificial cerebrospinal fluid AD - Alzheimer’s disease
ADBE - activity-dependent bulk exocytosis ADP - adenosine diphosphate
AMP - adenosine monophosphate
AMPAR - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AMPA - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
AMPK - AMP-activated kinase
APV - (2R)-amino-5-phosphonovaleric acid
AR-18 - AR-A014418 (N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea) Arf6 - ADP-ribosylation factor 6
ATP - adenosine triphosphate Aβ - amyloid-β
BCA - bicinchoninic acid BHK - baby hamster kidney BSA - bovine serum albumin
CA1 - Cornu Ammonis 1 CA3 - Cornu Ammonis 3 CaM - calmodulin
CaMKII - Ca2+/calmodulin-dependent protein kinase II cAMP - cyclic AMP
Cdk5 - cyclin-dependent kinase 5
CHIR - CHIR 99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2- pyrimidinyl]amino]
ethyl]amino]-3-pyridinecarbonitrile) cLTD - chemical long-term depression CME - clathrin-mediated endocytosis
CREB - cAMP response element-binding protein CRMP-2 - Collapsin response mediator protein 2
DG - dentate gyrus
DMEM - Dulbecco’s modified Eagle medium DMSO - dimethyl sulfoxide
DNA - deoxyribonucleic acid DN - dominant negative
2 Dyrk1A - dual-specificity tyrosine phosphorylation-regulated kinase 1A
EC - entorhinal cortex
EGFP - Enhanced Green Fluorescent Protein
EGTA - ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid eIF2α - eukaryotic initiation factor 2α
EM - electron microscopy
EPSC - excitatory postsynaptic current EPSP - excitatory postsynaptic potential EZ - endocytic zone
F-actin - filamentous actin
fEPSP - field excitatory postsynaptic potential
FRAP - Fluorescence Recovery After Photobleaching FTD - frontotemporal dementia
GABA - gamma-Aminobutyric acid G-actin - globular actin
GDP - guanosine diphosphate GDI - GDP dissociation inhibitor GFP - Green Fluorescent Protein GSK3 - glycogen synthase kinase 3 GTP - guanosine triphosphate
HEK - human embryonic kidney
HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HM - Henry Molaison
HRP - horseradish peroxidase
I1 - inhibitor-1 I2 - inhibitor-2
IRS - insulin receptor substrate
KLC2 - kinesin light chain 2
LFS - low-frequency stimulation L-LTP - late long-term potentiation LTD - long-term depression LTP - long-term potentiation
MCI - mild cognitive impairment
3 MCPG - (RS)-α-Methyl-4-carboxyphenylglycine
MEM - modified Eagle medium
mEPSC - miniature excitatory postsynaptic current mGluR - metabotropic glutamate receptor
MTR - microtubule-binding repeat
NFTs - neurofibrillary tangles NMDA - N-methyl-D-aspartate
NMDAR - N-methyl-D-aspartate receptor NSF - N-ethylmaleimide-sensitive factor
PBS - phosphate-buffered saline PCR - polymerase chain reaction
PDK1 - phosphoinositide-dependent kinase-1 PHF - paired helical filament
PH - pleckstrin-homology
PI3K - phosphoinositide 3-kinase
PIP3 - phosphatidylinositol (3,4,5)-trisphosphate PKA - cAMP-dependent protein kinase
PKC - protein kinase C PP1 - protein phosphatase 1 PP2A - protein phosphatase 2A PP2B - protein phosphatase 2B PPF - paired pulse facilitation PP - perforant path
PPR - paired-pulse ratio
PICK1 - Protein Interacting with C Kinase-1 PSD-95 - postsynaptic density protein 95 PSD - postsynaptic density
PTEN - phosphatase and tensin homolog
PVDF - polyvinylidene fluoride or polyvinylidene difluoride
RIPA - radioimmunoprecipitation assay RNAi - RNA interference
RNA - ribonucleic acid
S6K1 - p70 S6 kinase
SC79 - 2-Amino-6-chloro-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester SC - Schaffer collateral
SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis shRNA - short-hairpin RNA
4 SLM - stratum lacunosum moleculare
SNARE - SNAP (Soluble NSF Attachment Protein) REceptor SR - stratum radiatum
TBST - Tris-buffered saline/Tween-20 TfR - transferrin receptor
VO-OHpic - (OC-6-45)-Aqua(3-hydroxy-2-pyridinecarboxylato-κ-N1,κO2)[3-(hydroxy-κO)- 2- pyridinecarboxylato(2-)-κO2]oxovanadate(1-),hydrogen
VSV-G - vesicular stomatitis Indiana virus
5
Introduction
6 A history of remembering
A preoccupation with the workings of memory is one that considerably predates the history of psychology and neuroscience. Ancient Greek thought centred on the idea of ‘mimesis’; the imitation of that which really exists (the experienced world), just as a painting is a representation of the colour and form of the object that it depicts 1. Although this elegant notion has persisted, and flourished in modern scientific theories, the provenance of such representational knowledge was commonly taken to be divine, and its instantiation immaterial until much more recently.
Memory embodied
Just after the turn of the 20th century, Richard Semon coined the term engram to denote “the enduring though primarily latent modification in the irritable substance produced by a stimulus (from an experience)” 2. According to this theory, interaction of an organism with the world would leave a physical trace in the excitable substance of its nervous system. Such modifications could accumulate to the point at which, once they had passed some threshold, future behaviour of the organism would demonstrate the existence of a lasting trace. This elaboration on the idea of memory in physical form had its roots in the thinking of English philosopher and physician David Hartley at the end of the 18th century 3. Hartley conceived of memory in a way a modern scientist could relate to:
‘The sensations remain in the mind for a short time after the sensible objects are removed. This is sufficiently evident in things visible and audible; one may infer it by analogy in other senses’.
Hartley also proposed a rudimentary physical basis for such a memory, whereby excitations in the medullary substance of the nerves produced by sensations leave behind a tendency for greater excitation
4.
Figure 1. Key figures in the scientific study of memory. From left to right: Plato, David Hartley (engraving by William Blake, 1791), Richard Semon, Brenda Milner and John O’Keefe.
Holism reigns
That memory was a physical entity, and that it was to be found in the brain, was a radical notion at the time. A century later, there was scientific consensus that memory and thought were inseparably intertwined with the motions of the brain. Studies on patients with brain lesions, with notable examples
7 by Paul Broca and Carl Wernicke, had established that not only did such faculties reside in the brain;
subsets of them could be localised to specific subregions, later with a remarkable degree of reproducibility (thanks to the legions of war casualties and their varieties of brain injury). As late as the mid-20th century, such a refutation of irreducibly distributed brain functions had not yet included memory research; following the systematic lesion of cortical areas in rats in the hunt for a region responsible for memory, Karl Lashley ceded that “It is not possible to demonstrate the isolated localization of a memory trace anywhere within the nervous system… The engram is represented throughout the region.” 5.
HM freshens the stagnation
This impasse to the reductive method wasn’t to last. In 1950, Wilder Penfield had the rare opportunity to perform the opposite manipulation: activation of a brain region by electrical stimulation, here in a human epilepsy patient being evaluated for surgery (and with the distinctly human capacity to verbally report on the subjective consequences of the stimulation). Penfield found that temporal cortex stimulation elicited the recall of episodic memories. Another epilepsy patient, Henry Molaison (HM), provided the first (and an immensely fruitful) case of surgical lesion in which memory was profoundly affected while other cognitive functions were left intact.
Neurosurgeon William Scoville identified the medial temporal lobe as the focus of Molaison’s seizures, and removed the anterior hippocampus, the amygdala and adjacent cortex from both hemispheres of the brain. Although HM’s seizures were palliated, the psychologist Brenda Milner showed that he suffered antero- and retrograde amnesia for declarative memories (which include both semantic - facts about the world; and episodic memories - re-experiencing an event in its original context), while neither his general intelligence not ability to learn motor skills were affected 6. This suggested that the hippocampus, or perhaps parts of the surrounding tissue, were responsible for forming new declarative memories.
Following this physical demarcation of a longstanding philosophical and psychological dichotomy of memory - between those memories accessible to conscious recollection, and those that are not 7–9 - the field blossomed into a biologically-grounded elaboration of multiple memory systems.
Multiple memory systems
The resulting picture of memory describes several complementary mechanisms in which different strategies can be applied to the same learning problem. It thus explains the otherwise surprising observation that some tasks that are learned declaratively by humans are nevertheless learned non- declaratively by experimental animals. For example, monkeys with medial temporal lobe lesions are as adept as healthy monkeys at learning to discriminate between similar visual stimuli, whereas amnesic human patients with similar lesions are unable to retain the learned memory. The explanation for this difference appears to be a treatment of the problem as a skill by the monkeys 10,11, but as a declarative memorisation by humans (dependent on the medial temporal lobe).
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‘Place sails’ - is the hippocampus also a navigator?
The human employment of declarative memory as a strategy in a broad range of tasks underlines the importance of understanding of medial temporal lobe function. For reasons explained later, the hippocampus, in particular, turned out to be an apt preparation in which to gain such an understanding - for in vitro and in vivo probing of cellular and circuit mechanisms underlying processes of memory. An early highlight of such research, and one of the catalysts of the field’s later explosion, was the discovery by John O’Keefe of ‘place cells’ 12. O’Keefe used miniaturised electrodes for extracellular single-cell recording, such that he could record activity from hippocampal neurons in awake and freely moving animals. Using this technique, he discovered that cells responded specifically to the current location of the animal; different place cells were found to have different firing locations (‘place fields’ 13). Although place was mapped non-topographically (place fields of neighbouring cells were no more similar than those of cells that were far apart), the combination of cells that were active at each location in the environment was unique, which led to the proposal that the hippocampus contained an internal representation of the external environment 14. This strong modulation of hippocampal cell activity by the location of the animal not only provided a direct link between the activity of single cells in the brain and a feature of the environment; it also suggested that space was a primary concern of the hippocampus. How could the dependence of declarative memory on the hippocampus be squared with this newly-described function?
The mnemonic helmsman - squaring the two circles of research
Episodic memory, the remembering component of declarative memory (cf. the knowing, or ‘semantic’
component 15) refers to the capacity to recall an event, usually as part of a larger sequence of events.
More recent work has shown that hippocampal neurons may also encode time during episodic events 16–
18. In this way, it appears that episodes in a specific memory are mapped to a continuous variable, or context. This variable may be space or time, but is not limited to these dimensions 17; sound 19, and even social space, provide striking examples of such an alternative dimensions 20. Although it appeared, then, that the hippocampus was specialised for spatial navigation, real-world ‘navigation’ requires a cognitive map 21. Such a ‘map’ must be learned. Indeed, place cells are also memory cells; the cells use environmental cues to set up their firing pattern, but place fields are maintained following the removal of these cues 22–24. A unified view is thus emerging in which the role of the hippocampus in navigation is memory 25.
Interlude: The hippocampus catches a current
Although the above-described function is undoubtedly a grand role for the hippocampus, it was by dint of being an apt preparation for in vitro probing of cellular and circuit mechanisms underlying processes of memory (apt for reasons we shall explore later) that research in the hippocampus took off. As a result, it is now the most well-studied area of the brain at the level of cellular and synaptic physiology.
9 Unearthing the sea-creature
Dissection of the medial temporal lobe unearths a curious structure quite distinct from the sulci of the surrounding cortex, one whose shape has been variously likened to a silkworm, and, perhaps most fittingly, a seahorse (Hippocampus). The peculiarity of the structure of the hippocampus at the gross anatomical level is equalled by the orderliness of its internal arrangement. Like other areas of the cerebral cortex, the hippocampus has a folded laminar structure, large pyramid-shaped projection neurons and smaller interneurons. In other respects, its anatomy is unique. It has three highly organised principal cell layers through which information flow is largely unidirectional (first described by Ramón y Cajal 26). This is in stark contrast to the cortex, in which reciprocal (both feedforward and feedback) connections between layers form the majority 27. Additionally, the majority of hippocampal fibres are aligned in a manner that is close to perpendicular to its long axis. As detailed below, each of these hippocampal peculiarities grants a specific advantage to the experimenter.
Figure 2. Cajal’s hippocampus. Left, a drawing (1909) by Santiago Ramon y Cajal (right) of the trisynaptic pathway in the hippocampus. Axons projecting from granule cells (green) in the dentate gyrus make synapse with dendrites of CA3 pyramidal cells (blue). The axons (Schaffer collaterals) projecting from CA3 neurons, in turn, make synapses with apical dendrites of CA1 pyramidal cells. Drawing, Lüscher and Malenka, 2012. Photo of Cajal, The Hippocampus Book.
Hippocampal circuitry: direct and indirect
For convenience, the entorhinal cortex can be considered the first step in the intrinsic hippocampal circuit.
Cells in the superficial layers of the entorhinal cortex project axons to the dentate gyrus, forming part of the major hippocampal input pathway, the ‘perforant path’. Likewise, the principal cells of the dentate gyrus, the granule cells, project axons (‘mossy fibres’) that synapse with pyramidal cells of the CA3 field of the hippocampus. The pyramidal cells of CA3, in turn, are the source of Schaffer collateral axons, the major input to the CA1 hippocampal field. The CA1 field of the hippocampus then projects unidirectionally to the subiculum, providing its major excitatory input. In none of these cases do receiving cells have major returns to projection cells, and thus each of these connections is unidirectional. Projections from CA1 and the subiculum make the intrinsic hippocampal circuit somewhat more intricate. For example, aside from
10 the subiculum, CA1 also projects to the entorhinal cortex, and the subiculum projects back to the entorhinal cortex. Through these latter connections both CA1 and the subiculum close the hippocampal signal-processing loop that begins in the superficial layers of the entorhinal cortex and ends in its deep layers.
In addition to this ‘trisynaptic’ (indirect) pathway (containing EC (layer II) → DG via the perforant path [synapse 1], DG → CA3 via mossy fibres [synapse 2], CA3 → CA1 via Schaffer collaterals [synapse 3]), information from the superficial layers of EC also reaches CA1 through a direct perforant path pathway, in which projections from EC synapse directly onto CA1 dendrites. These direct and indirect cortico- hippocampal inputs target distinct regions of the CA1 pyramidal neuron dendritic tree. Indirect pathway inputs, via CA3 Schaffer collaterals, form synapses onto more proximal regions (to the soma) of CA1 pyramidal neuron apical dendrites, which span a layer of CA1 known as stratum radiatum (SR). In contrast, direct PP inputs from (layer III) EC form synapses on the distal regions of CA1 pyramidal neuron apical dendrites in the stratum lacunosum molecular layer (SLM).
Functional significance of different hippocampal pathways
At a coarse level of granularity, the formation of a memory can involve the association of two stimuli, or an enhancement in the ability to discriminate between different stimuli. What, then, might be the computational (in David Marr’s sense of the term; 29) significance of the trisynaptic and direct entorhinal cortex connections to CA1? One proposal for CA1 function holds that CA1 acts as a novelty detector, comparing stored information (memory) in dentate gyrus and CA3 with ongoing direct sensory representations from the entorhinal cortex 30,31. The two extra synapses in the trisynaptic circuit introduce a delay in conduction and integration, meaning that information flowing through the trisynaptic path arrives at CA1 neurons some 15 to 20 milliseconds after the arrival of information through the direct EC inputs
32. Such a circuit design allows inputs coordinated in time to be integrated and compared in a manner that may contribute to the encoding of sequential episodic events 33–36. The functions of different pathways in the hippocampus impact on our interpretation of synaptic and molecular events. An understanding of such events has been aided by aspects of the structure of the hippocampus, as we shall explore below.
The hippocampus as an ideal preparation for neurobiological study
The principal cell layers in the hippocampus have a relatively simple organization, in which all cells reside in a single and compact layer. Coupled with the highly organised synaptic inputs to a single dendritic lamina, this spare architecture provides a particular advantage during the extracellular recording of local field potentials. Because current sources and sinks are well defined in certain regions of the hippocampus, the interpretation of the contributions to the field potential of current in different regions, forbidding in other regions of the brain, becomes a tractable task, and current flows in synaptic and somatic locales can be distinguished. Basic principles, such as excitatory synaptic transmission, and well- described phenomena like long-term potentiation were described through such studies. Since such recordings could be made almost as easily in vivo as in a hippocampal slice, the study of such processes in behaving animals has led to a rich understanding of them in the context of learning and memory.
11 An understanding of the coherent activity of populations of cells and synapses, gained in vivo, was complemented by cellular and molecular studies on pyramidal cells in slice preparations, which became feasible on an unprecedented scale. Another quirk of hippocampal anatomy - the highly parallel organisation of fibres within the trisynaptic circuit - means that effective synaptic activation is preserved in a slice, and also that slices survive for long periods of time ex vivo. In particular, the transverse hippocampal slice, in which CA3 → CA1 synaptic connections are exceptionally well preserved, has furnished an understanding of the functions, and the mechanistic basis thereof, of the various types of synapses, neurotransmitters and their receptors, neurotransmitter uptake mechanisms, neuronal transport mechanisms, and signalling pathways. In this well-controlled experimental system, recent experiments have taken advantage of technological innovations to answer the question of what material substrate actually learns in the hippocampus.
Finding the engram
By the mid-to-late 20th century, localism had supplanted holism in memory research, but holism was still alive and well in theories at the cellular and sub-cellular level. Are individual remembered concepts represented in the mechanisms of single neurons and/or their sub-cellular organs, or are they distributed in the neuronal activity among populations of neurons and synapses? ‘Grandmother cells’, observed in vivo in epilepsy patients 37, demonstrate the possibility of the localist conception, but the contingencies of technological progress have precluded the testing of holist theories. Only recently have the instruments been developed to address memory formation and recall at the level of cellular populations.
These tools include optogenetics, which involves the transgenetic expression of light-sensitive excitatory ion channels such as channelrhodopsin or inhibitory ion pumps, such as halorhodopsin. When expression of a light-sensitive ion channel is placed under the control of an endogenous promoter, its expression can be limited to certain cell types, or to cells with a certain history of genetic activity. Exposing neurons expressing these rhodopsins to light of the right wavelength results in the de- or hyper-polarisation of the cell, in the case of channelrhodopsin or halorhodopsin expression, respectively.
In such a study 38, expression of a channelrhodopsin transgene was tied to the promoter of the intermediate-early gene cFos, which is turned on immediately after neuronal activation. With this set-up, channelrhodopsin, labelled with GFP, is generated uniquely in neurons that fire, producing activity- dependent labelling of cells. The expression of channelrhodopsin was limited to a time window during which (fear) learning occurred, by suppressing expression outside of the window with an antibiotic administered through the animal’s diet. With this experimental configuration, it is possible (by observing GFP) to see which neurons participated in the learning. Then, by activating channelrhodopsin (and host neurons) with blue light, it can be determined whether those particular neural ensembles are sufficient
38,39, or necessary 40,41, for the reactivation of the memory (identified by freezing of the animal). These and other optogenetic studies have shown that only a subset of neurons is active during the learning of a single memory. If these cells are activated (by light), the behavioural consequence of the memory is elicited. If they are inhibited, recall is impaired.
12 From engrams to plasticity
Studies in which engrams were genetically labelled, then, have shown that certain neuronal populations encode specific memories in mice. How exactly do the cells represent the memory? Does a neuron
‘contain’ a whole memory? Or is the memory in the location of the neurons, the spiking order, or the relative firing rates? Although all of these possibilities have been taken seriously in one form or another, the answer might entail a more direct connection to biology. Ramon y Cajal speculated that structural changes in neurons were the mechanism of learning:
“The organ of thought is, within certain limits, malleable and capable of perfection.… The cerebral cortex is similar to a garden filled with trees, the pyramidal cells, which, thanks to an intelligent culture, can multiply their branches, sending their roots deeper and producing more and more varied and exquisite flowers and fruits.” 42
Synapses, the impressively dynamic connections between neurons, have long caught the attention of those studying memory 43,44. Each neuron has thousands of connections, and if one considers them at various scales, they are as dynamic as almost any mental process we might wish to find a physical basis for. Almost 70 years ago, Donald Hebb theorised that synaptic connections between active cells change in a manner depending on their activity 45, leading to the postulate that: ‘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’ 45, that is, that neurons that “fire together, wire together” 46 into cellular assembles.
Early studies in the sea slug ‘Aplysia’ demonstrated that synapses did in fact possess associative properties 47, lending credence to the Hebbian postulate and the synaptic plasticity research program.
Since then, a vast amount of evidence has been gathered that suggests that that neuronal connectivity through synapses is indeed altered in an activity-dependent manner. Such alterations can occur according to the rules of spike timing-dependent plasticity 48–50 such that synapses that were likely to have contributed to the generation of action potential (in practice, those that fired just beforehand) are potentiated, and those that weren’t (that is, those that fired shortly after) are depressed 51. Plasticity can also be induced not according to rules based on relative timing, but on input intensity (firing frequency).
Such long-lasting enhancements or diminishments of the effectiveness of synaptic transmission are known as long-term potentiation (LTP) and long-term depression (LTD), respectively 52 These forms of synaptic strengthening and weakening, experimentally robust in the hippocampal slice, establish patterns of connectivity that are commonly envisioned as encoding ‘information’ relevant to the organism 53–55.
Excitatory synapses
Studies in the hippocampus have focused mostly on excitatory DG → CA3 or CA3 → CA1 synapses. In contrast to inhibitory synapses, which form contacts mostly with the cells soma, excitatory synapses are found predominantly on dendritic spines and adjacent dendritic shafts. At such (chemical) synapses, neurotransmission is mediated by neurotransmitter release from the presynaptic bouton into the synaptic
13 cleft, and neurotransmitter detection by postsynaptic receptors, which have excitatory or inhibitory effects on the postsynaptic cell. Most excitatory neurotransmission is mediated by the amino-acid glutamate, which acts as a ligand for the ionotropic AMPA, NMDA and kainate receptors (as well as metabotropic glutamate receptors; mGluRs) anchored in the postsynaptic density (PSD), before being taken up by neuronal or glial transporters. The PSD is formed mostly of scaffolding (mainly PSD-95) and signalling molecules (among which CaMKIIα and CaMKIIβ are the most abundant 56), and serves to bring together receptors and their downstream effectors. Ion flux through ionotropic receptors therefore has not only fast effects on graded and action potentials in the neuron, but also slower effects mediated by signalling cascades.
All excitatory ionotropic receptors selectively pass sodium and potassium ions, and thereby contribute to the excitatory postsynaptic potential (EPSP) elicited by glutamate release. Most such basal ion flux is mediated by AMPA and NMDA receptors, with a much smaller contribution from kainate receptors. Some 20-40 copies of both AMPA and NMDA receptors may be present at a single CA1 synapse 57. These two receptor types differ in their kinetics (AMPA receptors have short decay times compared to NMDA receptors 58); in the degree to which they flux Ca2+ (NMDA receptors are highly permeable to calcium ions, whereas most AMPA receptors are not); and in the voltage-dependence of their activity (NMDA receptors are blocked by Mg2+ at negative membrane potentials, whereas AMPA receptors have a fairly linear current-voltage relationship). Both AMPA and NMDA receptors play fundamental but distinct roles in synaptic plasticity. These are explored below.
Plasticity and memory
Owing to the blossoming of cellular and molecular studies in the hippocampus, insights into the mechanisms of synaptic plasticity - in particular, that NMDA receptors (NMDARs) are required for the induction of LTP and LTD - preceded a more direct linking of plasticity with behaviour. The first correlation between LTP and spatial memory was provided when Richard Morris showed that pharmacological blockade of NMDARs prevents spatial memory formation by rats in the Morris water maze - without impairing the animal’s learning to swim to the platform when it is visible, a task that does not require the hippocampus 59. Consistent with a role for the hippocampus in mapping episodes in a specific memory to a continuous variable such as space or time, NMDAR blockade does not prevent memory formation in the water maze if animals are pretrained on the task in a different spatial context 60,61.
An alternative form of evidence involves showing that LTP is co-present with behaviour, which exists in the form of in vivo extracellular recordings during learned behaviours. For example, Whitlock et al (2006) found that one-trial inhibitory avoidance learning produces an enhancement in synaptic responses evoked by electrical stimulation of SC → CA1 pyramidal neuron inputs. Moreover, learning behaviours brought about the same changes in AMPAR phosphorylation and membrane trafficking as seen during induction of LTP by tetanic stimulation. Although less thoroughly examined than LTP, evidence for the importance of LTD in both learning and the extinction of learned behaviours has also accumulated. LTD is facilitated by learning; Kemp and Manahan-Vaughan (2004) showed an enhanced induction of CA1
14 LTD by low-frequency stimulation when rats explored novel objects in a new environment, which suggests that learning recruits overlapping mechanisms.
Although studies in the hippocampus constitute the bulk of work on synaptic plasticity, some of the best evidence linking LTP and LTD to learning and memory comes from studies in amygdala, in which the relatively simple circuitry and its role in well-established behavioural paradigms provide a decisive experimental advantage 64. Early studies showed that cued fear conditioning, in which animals learn to associate a tone with a shock, led to an LTP-like potentiation of synaptic responses, both to the auditory conditioned stimulus, in vivo 65, and to electrical stimulation of thalamic input to the amygdala in an ex vivo slice 66. That is, plasticity was co-present with such behaviour. Cued fear conditioning also occluded subsequent induction of LTP at cortico-amygdala synapses in acute slices 67, suggesting that behaviourally- and electrically-induced LTP share mechanistic commonality.
However, clear-cut evidence that causal intervention provides was again constrained by technological innovation, and previous experiments claiming to demonstrate the necessity of LTP or LTD were hampered by concerns of specificity (of NMDARs for LTP). Correlative claims, although specific in their measurement of the process in question (that is, LTP or LTD), were unable to demonstrate sufficiency.
Recent in vivo experiments, however, used optogenetics to directly stimulate the corticothalamic auditory inputs to amygdala. By inducing LTD at conditioned synapses, the experimenters were able to suppress fear conditioning memory, whereas the subsequent induction of LTP restored the fearful memory 68. Such experiments constitute some of the strongest evidence linking memory to plastic changes that enhance or diminish synaptic responses.
The plastic devil in the synaptic details: molecular mechanisms of plasticity
There are thousands of synapses per neuron, and depending on the particular brain region and cell type in question, the strength of each one can change through a wide variety of mechanisms. Presynaptically, these mechanisms include the release of more neurotransmitter 69,70 and changes in the properties of the release machinery 71–73. Facets of such mechanisms are manifest in phenomena of short-term plasticity, such as paired-pulse facilitation (PPF). Here, residual presynaptic calcium 74 during the arrival of the second of two pulses delivered in quick succession (that is, typically when separated by up to 400 ms at CA3 → CA1 synapses) results in facilitation of the second pulse in a manner that depends on initial presynaptic strength (to the extent that this is determined by the probability of release) 75. Specifically, residual calcium (from the arrival of the action potential elicited by the first pulse) in the presynaptic terminal when the second action potential arrives, causes more calcium sensors involved in vesicle release to reach threshold, leading to an enhanced release of neurotransmitter vesicles. Experimentally, therefore, to the extent that changes in presynaptic strength have occurred via changes in the mechanisms involved in determining calcium sensitivity (quantal content, for example, tends not to vary
76), a large increase in the second response as compared to the first (paired pulse ratio; PPR) indicates a low release probability at the recorded synapse(s), and vice versa. Other forms of short-term plasticity shape synaptic activity over the course of seconds to minutes.
15 During longer-term alterations at CA3 → CA1 synapses in the hippocampus, however, delivery and removal (or changes in the properties) of AMPA receptors in response to NMDA receptor activation is the canonical mechanism that constitutes plasticity 77–83. Such delivery or removal may be accompanied by the formation or elimination of dendritic spines 84,85. Alternatively, structurally mature but functionally silent synapses can be unsilenced via (AMPA) receptor delivery 71,86–90. A hippocampal cell is able to coordinate trafficking of a single receptor subtype in order to express, without mutual interference, such divergent forms of plasticity as LTP and LTD, as well as regulate basal synaptic strength. Molecular differences among the subunit components of AMPA receptors provide one source of specificity to accomplish this task.
AMPA receptor structure and function
AMPA receptors (AMPARs) are ionotropic glutamate receptors that mediate fast excitatory synaptic transmission in the mammalian brain. Four highly homologous genes code for the subunits that assemble to form the tetrameric receptor, denominated GluA1-4. There exists considerable heterogeneity between AMPAR subunits among both the (intracellular) carboxy-terminal (C-terminal)91 and N-terminal (extracellular) domains 92. GluA2, GluA3 and an alternatively spliced form of GluA4 (GluA4S) have short C-terminal domains, and GluA1, GluA4, and an alternatively spliced form of GluA2 (GluA2L) have a long version. The N-terminal domain comprises ~50% of the AMPA receptor and is also highly diverse between subunits 93. Such C- and N-terminal differences determine differential binding of various AMPAR interacting proteins, which regulate the subunits’ highly dynamic trafficking in and out of synapses alongside a variety of post-translational modifications. Such trafficking is modulated in response to synaptic activity and is an important mechanism in the activity-induced changes in synaptic transmission of which plasticity consists. All else being equal, enhanced AMPAR function at synapses underlies long- term potentiation of synaptic strength (LTP), whereas lessened function underlies long-term depression (LTD).
16 Figure 3. The structure of AMPA receptors. A. Schematic diagrams of AMPA receptor structure (top) mapped onto a domain diagram (below). N-terminal domain (NTD), ligand-binding domain (LBD), trans- membrane domain (TMD), membrane domains 1-4 (M1-4) and C-terminal domain (CTD) are indicated. Also shown are the Q/R site, which determines calcium permeability and rectification properties, and R/G and flip/flop sites, which affect desensitisation and kinetics properties of the receptor 94,95. B. Structure of a GluA2 homomer. Each individual polypeptide chain in the tetramer is coloured (labelled A –D). An agonist is shown bound to the LBD. Yellow arrows denote dimerization between subunits. The polypeptide chains involved in dimerization differ from layer to layer, which defines the distinction between NTD and LBD. A schematic showing top-view arrangement of individual receptor monomers is shown on the right. After 96.
AMPA receptor function is determined by subunit composition
AMPA receptor subunits are typically assembled into identical pairs of heterodimers to form the final tetrameric receptor, although homomeric pairs of GluA1, GluA2 and GluA3 are all possible 97. Not all possible subunit combinations are equally likely; rather, the vast majority of AMPA receptors in hippocampal pyramidal neurons exist as GluA1/2 (~80%) or GluA2/3 heterodimers (most of the remaining 20% 97). Receptors assembled in these configurations appear to obey simple rules that govern their trafficking; Due in part to C- and N-terminal differences between AMPA receptors, delivery of GluA1/2 heteromers occurs in response to activity 79,80, whereas GluA2/3 receptors maintain the surface pool of synaptic AMPA receptors by constitutively cycling in and out of the membrane 79,98 (Fig. 4). Internalisation of both forms of AMPA receptors may occur during activity-dependent synaptic depression, during LTD
99.
A small proportion (fewer than 10%) of endogenous AMPA receptors exist in subunit combinations that lack a GluA2 subunit 100. Such receptors thereby lack the positively charged arginine residue in the pore region that GluA2 invariably carries 101,102, and have markedly divergent electrophysiological properties as a result. Firstly, they are permeable to calcium (whereas GluA2-containing receptors are not).
Secondly, they have an increased conductance. Finally, they are vulnerable to blockade by intracellular positively-charged polyamines at positive membrane potentials, and therefore conduct current less effectively than at negative potentials. That is, they have an inwardly rectifying current-voltage relationship. Remarkably, the arginine in the GluA2 pore region (encoded at position 607 in GluA2 mRNA) is not coded in the DNA 103. Instead, ~99% of GluA2 transcripts are subject to adenosine-to-inosine editing at this site 104, producing a glutamine-to-arginine change in the translated protein. Although endogenously extremely rare, AMPA receptors containing unedited GluA2 subunits (either as a homomer, or combined with a non-GluA2 subunit) will therefore behave as ‘GluA2-lacking’ receptors, with their unique electrophysiological idiosyncrasies. The functional relevance of GluA2-lacking receptors is poorly understood. There is some evidence that certain forms of synaptic potentiation are expressed primarily through an alteration in AMPA receptor conductance, rather than insertion of new receptors 105. In such cases, GluA2-containing receptors are exchanged for GluA2-lacking receptors, whose higher conductance will potentiate transmission even without a change in total receptor content at the synapse.
The upstream signals that lead to these and other changes that result in synaptic strength alterations are explored below.
17 Figure 4. AMPA receptor trafficking is determined by subunit composition. AMPA receptors formed of GluA2/3 dimers cycle constitutively to and from the membrane, in the absence of synaptic activity (lower left).
NMDA receptor activity, via the induction of chemical LTD, or electrical LTD or LTP, signals to recruit other pools of AMPA receptors to be removed from or delivered to the membrane during LTD or LTP, respectively.
These receptors are preferentially GluA1/2 in the case of LTP, and both GluA2/3 and GluA1/2 in the case of LTD, although other receptors can be delivered or removed with lower priority 106.
Mechanisms of LTP
The initial stimulus for both LTP and LTD is the activation of NMDA receptors. Classically, the relevant function of NMDA receptors in this context is fluxing calcium into the cell. Since the same qualitative stimulus is the trigger for synaptic strength changes of the opposite sign, the cell must embody a mechanism that discriminates based on the intensity of the stimulus. Experimentally, indeed, LTP is typically induced by one or more bursts of high-frequency (tetanic) stimulation (commonly at 100 Hz), whereas LTD is induced with low-frequency stimulation (typically at 1 Hz, for 5 - 15 minutes). As we shall see later, this selectivity is likely to be encoded in the differing sensitivities of calcium sensors involved in the two forms of plasticity.
During LTP, calcium activates calcium/calmodulin-dependent kinase II (CaMKII). CaMKII is found at high concentrations in dendritic spines and is required for the expression of LTP 107. A hallmark of the regulation of CaMKII is autophosphorylation at Thr286, which generates ‘autonomous’ calcium and calmodulin (CaM)-independent kinase activity that persists even when the initial Ca2+-stimulus subsides
108–110. Such autonomy has been thought of a form of molecular memory; indeed, the Thr286 auto- phosphorylation event is required for memory per se (Giese 1998), as well as for LTP 111. A comparable autonomy is also achieved through CaMKII binding to the GluN2B subunit of NMDA receptors 112, and such binding is required for the maintenance of synaptic strength 113 and for LTP 114. CaMKII autonomy is a parsimonious explanation for memory, although there is a large amount of evidence for synaptic changes capable of maintaining increased synaptic strength even when CaMKII activity.is no longer
18 active. At the very least, though, CaMKII is capable of triggering the LTP cascade in response to a signal that may have left little other cellular evidence of its presence, so CaMKII autonomy may rather serve as a temporary tag for synapses undergoing potentiation. The activation of CaMKII leads to the phosphorylation of a variety of proteins, including AMPA receptor-associated proteins such as TARPs
115,116, and AMPA receptors themselves 82. In turn, such direct AMPA receptor phosphorylation can cause in increase in the conductance of the phosphorylated receptor 83, and influence its trafficking and eventual insertion into the synaptic membrane 117.
Aside from CaMKII, a number of other protein kinases, such as cAMP-dependent protein kinase (PKA), protein kinase C (PKC), tyrosine kinases, and mitogen-activated protein kinases, have all been suggested to contribute to LTP 118–121. On the whole, these kinases organise the initiation of, or intermediate steps in, the trafficking of AMPA receptors to and from the synapse. In one recent model of this process 122, AMPA receptors are continuously endocytosed from the perisynaptic membrane under basal conditions through an Arf6-dependent (and clathrin/dynamin- independent) recycling endosome pathway, and recycled back to the cell membrane away from the synapse. This receptor removal is approximately balanced by a parallel (and clathrin/dynamin- dependent) pathway that recruits surface receptors from sites distal from the spine, and transports them in transferrin receptor-associated endosomes for exocytosis at or near the synapse. During LTP, the clathrin/dynamin-dependent recruitment is upregulated, resulting in a net insertion of receptors 122.
Mechanisms of LTD
Two major forms of LTD have been studied in the hippocampus: NMDA receptor-dependent LTD, and (to a lesser extent) mGluR-LTD. Although both forms of LTD can probably occur at the same synapse, the mechanisms involved in each may diverge considerably at the level of protein kinase and phosphatase signalling, and in the pools of AMPA receptors whose endocytosis they control. The focus here will be on NMDA receptor-dependent LTD.
NMDA receptor-dependent LTD is commonly induced experimentally by low frequency stimulation (LFS) of afferent fibres or bath application of NMDA (‘chemical LTD’; cLTD, 123). These two forms of induction are mutually occlusive 124, suggesting they share common mechanisms. Classically, an influx of calcium through NMDA receptors has been understood to be the stimulus that triggers the signalling cascade that leads to AMPA receptor endocytosis. In this vision, calcium entry is detected by calmodulin (CaM), which binds to the protein phosphatase calcineurin (CaN; also known as protein phosphatase 2B – PP2B). The affinity of CaN for calcium/CaM is higher than that of CaMKII, which is the neat molecular explanation of how an influx of calcium through NMDA receptors is able to trigger either LTP or LTD; modest calcium influxes resulting from low-frequency stimulation will preferentially activate CaN. The situation appears to be more complex, however: CaMKII is required for both LTP and LTD, through GluA1 phosphorylation at Ser831 and Ser567, respectively 111. In any case, recent work has cast doubt on the requirement for calcium entry downstream of NMDA receptor activation. Using drugs that block the NMDA receptor channel pore but spare ligand binding, Nabavi et al. (2014) failed to see a blockade of LTD induction, suggesting that a non-ionotropic function of NMDA receptors was responsible for LTD. Indeed, a
19
‘metabotropic’ function of NMDA receptors has since been described in the context of excitotoxic NMDAR receptor signalling 125. Perhaps owing to experimental differences (see ref 130), another lab failed to reproduce these results 127. In both calcium-dependent and independent scenarios, PP1 is activated, either through calcium-bound CaM activating calcineurin, or through direct interaction with the NMDA receptor 128. PP1, in turn, then dephosphorylates its substrates, which include GluA1 at the Ser845 site
129, leading to LTD. AMPA receptors may be stabilised at the postsynaptic membrane through an interaction between GluA2 and N-ethylmaleimide-sensitive factor (NSF; an ATPase whose activity is required for membrane fusion) 129. The clathrin adaptor protein AP2, which also binds to the NSF site on GluA2, may then compete with NSF for GluA2 binding to destabilise AMPA receptors and enable endocytosis during LTD 129.
Clathrin- and dynamin-mediated endocytosis of AMPA receptors is required for LTD 130 and mainly occurs in the endocytic zone (EZ) adjacent to the postsynaptic density (PSD) following cLTD induction 131,132. Particularly important for controlling the synaptic localisation of AMPA receptors during LTD is the protein PSD-95. PSD-95 anchors AMPA receptors at the synapse and during LTD is dephosphorylated at its Ser295 reside to enable its synaptic removal and accompanying AMPA receptor endocytosis 133. Such endocytosis requires the activity of the small GTPase Rab5, which stimulates the formation of endocytic pits and shuttles endocytic vesicles into early endosomes 134. For LTD to be stable beyond its initial phase, endocytosed AMPA receptors must be prevented from returning to the synaptic locale.
Internalised AMPA receptors pass through endosomal sorting checkpoints that redirect receptors back to the plasma membrane or organise their degradation via the lysosomal pathway, thereby preventing synaptic return 117,135,136.
Overall, then, a complex set of mechanisms regulates the up- and down-regulation of receptor content at the synapse. These, however, are not the only means by which synaptic strength is dynamically modulated over the long term.
Structural plasticity
Although a synapse of given size can vary in receptor content, functional changes mediated by AMPA receptor insertion or removal are often accompanied by structural changes in the spines on which the altered synapses are found. Spines with synapses that undergo LTP are enlarged 49,81, and spines with synapses at which LTD is induced, shrink 137–141. At least to some degree, functional and structural plasticity overlap in their signalling mechanisms: they both require Ca2+ influx through postsynaptic NMDA receptors, activation of CaMKII and small GTPases, and actin polymerisation 81,142–145. A crucial molecular axis that determines the sign of structural plasticity is the equilibrium between F- and G-actin
139. Tetanic stimulation causes a rapid and persistent shift toward F-actin polymerisation, resulting in an enlarged spine. Low-frequency stimulation, by contrast, shifts the equilibrium toward G-actin, resulting in a loss of postsynaptic actin and a decrease in spine size 139.
As we have seen, the mechanisms involved in potentiative and depressive forms of functional plasticity (LTP and LTD) are not mere inversions of the same molecular cascades. Although there is overlap in the
20 mechanisms of functional and structural plasticity, the two forms can be dissociated under certain conditions 141,146,147. This raises the question of the degree to which functional and structural plasticity differ in their mechanistic symmetry and in the particular higher level processes that are explained by each of them.
Signalling pathways controlling LTD
A notable target of PP1 dephosphorylation during NMDA receptor-dependent LTD is the protein glycogen-synthase kinase (GSK3). The activity of GSK3 is required for LTD induction (148; described further below), and components of the primary signalling pathway controlling the activation of GSK3 have been directly implicated in LTD in their own right. In this pathway, the PI3K-Akt axis, phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) control the synthesis and degradation, respectively, of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3
provides membrane anchoring for phosphoinositide-dependent kinase-1 (PDK1), which activates another PIP3-anchored protein, Akt, which in turn acts as a tonic inhibitor of GSK3. In fitting with this scheme, the activity of PTEN was found to be required for LTD 149. A PDZ domain-dependent interaction with PSD-95 recruits PTEN to the synaptic membrane and is required for the eventual expression of LTD 149.
The longstanding generalisation that LTP recruits protein kinases and LTD recruits phosphatases, although easily falsifiable in principle and non-trivial to explain, has mostly held true. Aside from CaMKII 111, in a systematic investigation of the requirement of 57 Ser/Thr protein kinases for LTD, only GSK3 was necessary 150. Several unusual characteristics of GSK3 may have allowed it to be seamlessly co-opted into the mechanisms of LTD. These properties are explored below.
GSK3: lone wolf, and jack of all trades
Glycogen-synthase kinase (GSK3) refers to two paralogs (derived from different genes) that are commonly (and herein) referred to as isoforms, GSK3α and GSK3β (Fig. 5A). GSK3 is a serine/threonine kinase so named for its phosphorylation activity towards glycogen synthase 151, the rate-limiting enzyme in glycogen metabolism, although the known cellular repertoire of GSK3 has now burgeoned to include over 100 reported (and 500 predicted) substrates, more than any other kinase 152. Such promiscuity gives GSK3 the ability to influence many cellular processes, including cell fate determination 153, cardiovascular processes 154, metabolism 155, and, in mammals, oncogenesis 156, inflammation 157 and neurological diseases 158–160. From an evolutionary perspective, therefore, an uncommon cellular burden has been laid at the feet of GSK3, despite the existence of a host of other kinases that could, in principle, have borne some of the load. It appears, then, that some property of GSK3 is sufficiently unique so as to make profitable the functional complexities required to ensure temperance of GSK3 towards its many substrates.
Given this broad sphere of influence, an enigma surrounding GSK3 has been how regulatory signals that converge on GSK3 can remain specific in their consequences downstream of GSK3. Plausible, if merely
21 partial, answers to this conundrum can be found in the understanding of the post-translational regulation of GSK3, and in the way this mechanism is intertwined with its substrate specificity.
The challenge and solution of GSK3 regulation specificity Post-translational regulation
In contrast to most other kinases, GSK3 has the unorthodox characteristics of being constitutively active, of having substrates that usually need to undergo priming phosphorylation by another kinase, and of being inhibited, rather than activated, in response to stimulation of the two main signalling pathways known to impinge on it, the insulin and Wnt pathways. The latter two characteristics are consequences of a single regulatory mechanism linked to the serine-21/9 site of GSK3α and GSK3β, respectively (Fig.
5B). Two functional domains of GSK3 are involved in this mechanism: a substrate binding domain that recruits primed substrates to GSK3, and a kinase domain that phosphorylates the substrate 161–163. Only
‘primed’ substrates, pre-phosphorylated at a serine/threonine site commonly four amino acids C-terminal to the eventual (serine/threonine) GSK3 phosphorylation site, are able to bind to the substrate binding domain. Such binding aligns the target GSK3 site on the substrate with the kinase domain of GSK3, enabling subsequent substrate phosphorylation. For some substrates, this mechanism enables the sequential phosphorylation of several sites, given a single phosphorylated priming site, such as in the case of residues Thr41, Ser37, and Ser33 in β-catenin, a specific substrate of GSK3. This priming mechanism underlies inhibitory GSK3 serine phosphorylation: phosphorylation of serine-21 in GSK3α or of serine-9 in GSK3β allows the N-terminal tail of GSK3 to act as a primed pseudo-substrate 161, competing with the binding of primed substrates and impeding their phosphorylation. Another aspect of the regulation of GSK3 activity is also explained by this mechanism: since generally only primed substrates can be phosphorylated by GSK3, the buck of specificity can be passed to the priming kinase for any given substrate.
In general, the serine-9/21 phosphorylation of GSK3 serves as a valuable reporter of conditions that regulate the activity of GSK3 via this mechanism. However, a few caveats should be issued regarding its interpretation. Firstly, when GSK3 is bound in certain protein complexes (another mechanism for achieving GSK3 specificity), the phosphorylation status of the serine site may be inconsequential for GSK3 activity. This appears to be the case for tau phosphorylation, where GSK3 is in complex with 14- 3-3 protein 164,165, and is also true for β-catenin phosphorylation, when GSK3 is in complex with Axin (166; reviewed in 167,168). In this latter case, it should be noted that only a small amount of β-catenin is associated with the Axin destruction complex, and therefore changes in serine-phosphorylated GSK3 may often affect total β-catenin phosphorylation 169. Secondly, since the serine phosphorylated N-terminal tail of GSK3 acts as a pseudo-substrate, binding to the substrate binding domain is competitive with primed substrates 161. As a result, primed substrate phosphorylation by GSK3 will increase with increasing substrate concentrations.
The requirement for substrate priming, then, mediated by kinases other than GSK3, combined with sufficient GSK3 serine dephosphorylation, serves as a spatiotemporal coincidence-detection mechanism that limits GSK3 activity to appropriate channels. Specificity is also achieved by having several different
