Thesis
Reference
Spine dynamics, a postsynaptic point of view on synaptic plasticity in hippocampal slice cultures
KLAUSER, Paul
Abstract
La majorité des terminaisons axonales excitatrices du système nerveux central des mammifères contactent des excroissances de l'arborisation neuronale, appelées « épines dendritiques ». Les propriétés dynamiques de ces épines durant le développement ou suite aux modifications de l'activité sont encore incomprises, de même que leur implication dans les modifications de la connectivité du réseau. Grâce à la microscopie confocale à fluorescence et un modèle de cultures organotypiques d'hippocampe murin, nous avons d'abord montré que le rythme de renouvellement des épines diminue durant le développement, que la stabilisation des nouvelles épines est un événement rare et qu'il survient après une période critique de 24 heures coïncidant avec l'élargissement de la tête de l'épine et l'expression d'une densité post-synaptique. Finalement, nous avons observé que l'induction d'une activité rythmique comme lors des processus d'apprentissage, stabilise les synapses potentialisées et favorise la formation de nouveaux contacts à leur côté, tout en promouvant l'élimination des épines non-activées.
KLAUSER, Paul. Spine dynamics, a postsynaptic point of view on synaptic plasticity in hippocampal slice cultures. Thèse de doctorat : Univ. Genève et Lausanne, 2008, no. Neur.
32
URN : urn:nbn:ch:unige-130166
DOI : 10.13097/archive-ouverte/unige:13016
Available at:
http://archive-ouverte.unige.ch/unige:13016
Disclaimer: layout of this document may differ from the published version.
DOCTORAT EN NEUROSCIENCES des Universités de Genève
et de Lausanne
UNIVERSITÉ DE GENÈVE FACULTÉ DE MÉDECINE
Professeur Dominique Muller, directeur de thèse
TITRE DE LA THESE
SPINE DYNAMICS, A POSTSYNAPTIC POINT OF VIEW ON SYNAPTIC PLASTICITY IN HIPPOCAMPAL SLICE CULTURES
THESE Présentée à la Faculté de médecine de l’Université de Genève
pour obtenir le grade de Docteur en Neurosciences
par
Paul KLAUSER
de Sulgen (TG) et Genève
Thèse N° 32
Genève
Editeur ou imprimeur : Université de Genève
2008
Remerciements - Acknowledgments
Je voudrais remercier personnellement tous les membres du laboratoire de Dominique pour leur collaboration à ce travail. Ma reconnaissance est beaucoup plus grande que ces quelques mots de fin et j’espère que j’ai su vous l’exprimer tout au long de ces 4 années de thèse.
Merci Dominique de m’avoir offert une excellente place de travail dans ton équipe ! Merci Mathias de m’avoir encadré pendant ma formation scientifique ! Merci Lorenzo pour ta compagnie de tous les jours et la microscopie électronique !
Merci Laszlo pour ton enthousiasme et tes propositions de collaboration ! Merci Pablo pour ta participation à notre première publication !
Merci Bernadett pour les merveilleux outils de transfection ! Merci Marlys pour les belles tranches d’hippocampe !
Merci Lorena pour les soins prodigués aux cultures ! Merci Irina de m’avoir fait partager ta vision ultrastructurale !
Merci Sylvain pour tes explications biochimiques ! Merci Aline pour tes explications moléculaires ! Merci Stefano pour ton initiation à la recherche ! Merci Jozseph pour ton initiation aux neurosciences !
Dominique Muller, Mathias De Roo, Lorenzo Poglia, Pablo Mendez, Laszlo Vutskits, Bernadett Boda, Marlys Moosmayer, Lorena Jourdain, Irina Nikonenko, Sylvain Steen, Aline Dubos and Stefano Alberi took part into this work.
Thanks to Anthony Holtmaat, Jozsef Kiss and Thomas Oertner who accepted to be members of the jury.
Thanks to the Swiss National Science Foundation and the Velux Foundation for their generous 3-year MD-PhD grant (323600-115432).
Abstract
Within the brain, information that is encoded by neuronal activity can be transmitted but also processed and stored. At the microscopic level of the network, transmission occurs trough the saltatory conduction of action potentials along axons, processing through the spatial and temporal summation of synaptic signals along the dendritic tree and storage through modifications of excitatory synapses and more precisely of their postsynaptic elements: dendritic spines.
These microscopic protrusions that are visible at the surface of dendrites can undergo both
functional and structural changes. Functional plasticity is best illustrated by long-term potentiation (LTP) and long-term depression (LTD) which are fast but long-lasting alterations of synaptic transmission due to modifications of spine receptors. Structural plasticity involves fast
morphological changes like spine head enlargement and shrinkage or slower and more dramatic events like complete spine formation or elimination that rely on modifications of actin cytoskeleton.
Structural changes of dendritic spines are relatively poorly described and their relation with functional plasticity is not well understood. Therefore the objectives of this work are to define the properties of spine dynamics (formation and elimination) and of their related morphological changes (size variations) as well as to assess their contribution to functional plasticity and to modifications of network connectivity.
To investigate the properties of spine dynamics and of their related morphological changes, we applied repetitive confocal imaging to fluorescent pyramidal neurons in hippocampal slice cultures of 11 or 25 days in vitro. We found that, although the turnover rate of protrusions progressively decreases during development, the process of stabilization of new spines remains comparable both in terms of time course and low level of efficacy. Irrespective of the developmental stage, most new protrusions are quickly eliminated, in particular filopodia, which only occasionally lead to the formation of stable dendritic spines. We also found that the stabilization of new protrusions is determined within a critical period of 24 h and that this coincides with an enlargement of the spine head and the expression of PSD-95. Blockade of postsynaptic AMPA and NMDA receptors
significantly reduces the capacity of new spines to express PSD-95 and decreases their probability to be stabilized. These results suggest a model in which synaptic development is associated with an extensive, nonspecific growth of protrusions followed by stabilization of a few of them through a mechanism that involves activity-driven formation of a postsynaptic density.
To assess the contribution of spine dynamic to functional plasticity and to network connectivity, we combined electrophysiological methods and calcium imaging technics with repetitive confocal imaging of dendritic spines and found that learning-related patterns of activity that induce LTP act as a selection mechanism for the stabilization and localization of spines. Through a lasting NMDA receptor and protein synthesis–dependent increase in protrusion growth and turnover, induction of plasticity promotes a pruning and replacement of non-activated spines by new ones, together with a selective stabilization of activated synapses. Furthermore, most newly formed spines preferentially grow close to activated synapses and become functional within 24 h, leading to a clustering of functional synapses. Our results indicate that synaptic remodeling associated with induction of LTP favors the selection of inputs showing spatiotemporal interactions on a given neuron.
Résumé
Dans le cerveau, les informations qui sont encodées par l’activité neuronale peuvent être transmises mais aussi traitées et stockées. Au niveau microscopique du réseau de neurones, la transmission se fait grâce à la conduction saltatoire des potentiels d’action le long des axones, le traitement grâce à la sommation spatiale et temporelle des signaux synaptiques le long de l’arborisation dendritique et le stockage à travers les modifications des synapses excitatrices et plus précisément de leurs
éléments postsynaptiques: les épines dendritiques. Ces excroissances microscopiques visibles à la surface des dendrites sont sujettes à des changements fonctionnels et structuraux. La plasticité fonctionnelle est bien illustrée par la potentialisation à long terme (LTP) et la dépression à long terme (LTD) qui sont des modifications rapides mais durables de la transmission synaptique,
secondaires à des modifications de récepteurs glutamatergiques. La plasticité structurale qui dépend des modifications du cytosquelette d’actine implique des changements morphologiques rapides comme un élargissement ou un rétrécissement de la tête de l’épine ou bien des changements dynamiques plus lents mais plus importants comme l’élimination ou la formation de structures complètes. Cet aspect structural de la plasticité des épines dendritiques est mal décrit et sa relation avec la plasticité fonctionnelle incomprise. Ainsi, l’objectif de ce travail est de définir les propriétés de la dynamique des épines (formation et élimination) et des changements morphologiques
(variations de taille) qui leur sont associés, puis d’examiner leur contribution à la plasticité fonctionnelle (LTP) et aux modifications de la connectivité du réseau.
Pour étudier les caractéristiques des modifications dynamiques des épines dendritiques et de leurs changements morphologiques associés, nous avons observé de manière répétitive avec un
microscope confocal des neurones pyramidaux fluorescents dans des cultures organiques
d’hippocampes de 11 et 25 jours in vitro. Nous avons trouvé, bien que le rythme de remplacement des protrusions diminue progressivement durant le développement, que le processus de stabilisation des nouvelles épines reste comparable en terme de cinétique et d’efficacité. En effet,
indépendamment de l’âge développemental, la majorité des nouvelle protrusions sont éliminées rapidement et en particulier les filopodes qui ne conduisent qu’occasionnellement à la formation d’épines dendritiques stables. Nous avons ensuite démontré que la stabilisation des nouvelles protrusions est déterminée durant une phase critique de 24 heures et que cette période coïncide avec l’élargissement des la tête de l’épine et l’expression de PSD-95. Le blocage des récepteurs
glutamatergiques postsynaptiques AMPA et NMDA réduit de manière importante la capacité des nouvelles épines à exprimer PSD-95 et diminue leur probabilité d’être stabilisées. Ces résultats suggèrent une modèle dans lequel le développement synaptique est associé à une croissance importante et non spécifique de nouvelles protrusions suivie par la stabilisation d’un petit nombre d’entre elles grâce à un mécanisme qui implique la formation d’une densité postsynaptique en réponse à l’activité.
Afin d’analyser la contribution de la dynamique des épines à la plasticité fonctionnelle et aux modifications de la connectivité du réseau, nous avons combiné l’étude électrophysiologique et l’imagerie calcique avec l’observation microscopique répétée des épines dendritiques dans les cultures organotypiques d’hippocampes. Nous avons pu démontrer que des modes d’activité électrique présents durant les processus d’apprentissage et qui induisent de la LTP agissent comme un mécanisme de sélection pour la stabilisation et la localisation des épines. Suite à une
augmentation durable du rythme de remplacement des épines, dépendant de l’activation des
récepteurs NMDA et de la synthèse de nouvelles protéines, l’induction de plasticité fonctionnelle promeut l’élimination et le remplacement des épines non-activées, parallèlement à la stabilisation sélective des épines activées. De plus, la plupart des nouvelles épines croissent préférentiellement à proximité des synapses activées et deviennent fonctionnelles en 24 heures, favorisant un
regroupement de synapses fonctionnelles. Finalement, ces résultats suggèrent que le remodelage synaptique associé avec l’induction de la LTP favorise la sélection d’inputs montrant une cohérence spatiale et temporelle sur un neurone donné.
Table of contents
Introduction 7
From synapse to spine 8
Synapses through the brain 8
Microscopic organization of the nervous system 8
The synapse 11
Basic description of the glutamatergic synapse 14
Anatomical 14
Physiological 15
Spine description 17
Static point of view 17
Protrusion types 18
Spine organelles 19
Spine PSD 20
Actin cytoskeleton 23
Spine adhesion molecules 26
Dynamic point of view 27
Membrane oscillations 28
Size modifications (micro-changes) 28
Protrusions turnover (macro-changes) 28
Plasticity mechanisms 30
Functional plasticity 30
Structural plasticity 32
Micro-changes 32
Macro-changes 34
Plasticity conditions 36
Development 36
Synaptogenesis 36
Spinogenesis 37
Activity 39
LTP-LTD as paradigm for learning and memory 39
LTP-LTD and functional plasticity 40
LTP-LTD and micro-structural plasticity 43
LTP-LTD and macro-structural plasticity 44
Homeostasis 45
Definitions 45
Physiological conditions 47
Pathological conditions 48
Organotypic hippocampal slice cultures 51
Interest of the hippocampus 51
Central in learning and memory 51
CA3-CA1 synapses 51
Interest of the slice culture 52
Practical aspect of the slice 52
Developmental aspect of the culture 52
Line of research 54
Open questions 54
Development 54
Activity 54
Objectives 55
Model of development - first article 55
Model of activity - second article 55
Results 57
Research context 58
First article 59
Contributions 59
Main results 59
Appreciation 59
Second article 71
Contributions 71
Main results 71
Appreciation 71
Discussion 83
Legitimacy 85
Objectives 85
Methods 86
Data synergy 88
Micro-structural changes 88
Macro-structural changes 91
Conclusion 94
Bibliography 96
Appendix 113
Curriculum vitae 114
Introduction
From synapse to spine
Synapses through the brain
Microscopic organization of the nervous system
The following view of the cellular organization of the central nervous system is based on the Neuron Doctrine, but even if this modern theory is now accepted, it was not the first model proposed. Indeed, since the beginning of the nineteenth century, when the first microscopic observations of neuronal tissue were done, two radically opposed theories emerged and a long debate started.
Figure 1. Both illustrations are hippocampal representations made from observations at light microscopy using Golgi’s staining. The draw at the left (published in 1883) is from the inventor of the technic himself, Camillo Golgi and illustrates the Reticular Theory. Hippocampus is represented like a mesh network in which elements are arranged following the gross structure but without any polarity. Axons are missing and only the dendritic tree is represented. In this reticulum, putative ways of information are neither clearly delimited, nor oriented. The draw at the right (published in 1911) is from Ramon y Cajal who sustained the Neuron Doctrine. Hippocampus is represented as a polarized network in which neuronal elements are represented as distinct entities which are not only oriented, but also
dynamically polarized. Axons and dendrites are represented as distinct elements and ways of communication are even proposed by arrows.
Brief history
In 1838 Theodore Schwann and Mathias Schleiden proposed that the cell was the basic functional unit of all living organisms, but the Cell Theory was not believed to apply to the nervous system by some scientists. Then began fifty years of disputation that was dividing protagonists in two groups:
• The Reticularists who rejected the pertinence of the Cell Theory for the nervous system and suggested a reticular arrangement: a continuous network formed by the fuse processes of nerve elements.
• The Neuronists who accepted the Cell theory and considered a nervous system that consists of independent cellular units called neurons.
It’s hard to think that it took so long for rejecting the reticular theory of the nervous system after the general Cell theory has emerged, but imagine that the microscopic technics used at this time were not good enough to consider soma, axons and dendrites as parts from the same entity. Further the
Introduction
specific morphology of neurons with their relatively small soma but long protrusions, rendered the association with cells described in other organs difficult.
The main proponent of the reticular theory was Joseph von Gerlach (1820-1896). In 1858 he developed two labeling methods, one using carmine mixed with gelatin and the other using gold chloride. The former was largely used among his contemporaries.
In 1865, was published, the posthumous work of Otto Friedrich Carl Dieters (1834-1863) who provided the first description of a complete nerve cell constituted of a soma, axon (axis
cylinder) and dendrites (protoplasmic processes). Although it allowed the first histological labeling, carmine staining revealed its limits quite early. The evolution of the microscopes that incorporated new achromatic lenses that delivered clearer images as well as the introduction of the microtome by Jan Evangelista Purkinje (1787-1869) that allowed the preparation of thin sections, greatly
improved the potential of light microscopy.
In 1873, an Italian physician named Camillo Golgi (1843-1926) published a new method to stain the nervous tissue. It involved hardening of the specimen in potassium dichromate and ammonia followed by its immersion in a silver nitrate solution to produce a random staining of some cells in their entirety by the micro-crystallization of silver chromate. This technique will be used for years under the name of Golgi’s staining and is probably the equivalent of modern fluorescent staining by the fact that it can resolve the eternal dilemma of labeling complex structures: rendering elements visible enough to appreciate correctly their complexity but not to much to avoid the loss of discrimination between them. Despite the fact that his new technique stained neurons in their entirety, Golgi confirmed the reticular theory by arguing that a so complex web of connections could not be the fact of distinct units. He also described connections through the fusion of protoplasmic processes (dendrites), revealing that despite the great improvement in
labeling offered by his new technic it still suffered from an evident lack of resolution.
In 1887, Ramon y Cajal (1852-1934), a Spanish physician, modified Golgi’s method by immersing tissue in fixative and silver nitrate a second time to stain the specimen more deeply and then improve the resolution. From his observations of the cerebellum Cajal formulated his law of dynamic polarization according to which information travels from dendrites to axons after passing through the soma. He described each nervous element as an autonomous unit that communicates through specialized junctions and finally concluded that the relationship between nerve cells was not continuity but contiguity.
It’s largely because of his work that neuron doctrine has finally been accepted and he can be considered as the father of modern neuroscience. In 1906 Ramon y Cajal and Camillo Golgi finally got the Nobel Prize of medicine together for their work on the structure of the nervous system.
Modern terminology was introduced only during the last decade of the ninetieth century. The term
“Neuron” was proposed in 1891. Then “Synapse” that is derived from Synaptein1 was proposed by Charles Scott Sherrington (1857-1952) in 1897 to name the theoretical connecting point between a neuron and another cell. In 1898 “Axon” was introduced by the Swiss physiologist Rudolph Albert Von Kölliker in replacement of “axis cylinder” and finally “Dendrites” by the Swiss physician Wilhelm His in 1889 in replacement of protoplasmic processes. (Glickstein, 2006) (Lopez-Munoz et al., 2006) (Agnati et al., 2007) (Costandi, 2007)
Introduction! From synapse to spine
1 Contraction of “syn” and “haptein” from Greek “together” and “to clasp”
Neuron Doctrine
The Neuron Doctrine, then, has four tenets:
1.The fundamental structural and functional unit of the nervous system is the neuron.
2.Neurons are discrete cells which are not continuous with other cells.
3.The neuron is composed of 3 parts - the dendrites, axon and cell body.
4.Information flows along the neuron in one direction - from the dendrites to the axon, via the cell body.
During the twentieth century, modern technics of imagery and electrophysiological studies
confirmed but also refined the Neuron Doctrine. The neuron is still the basic functional unit, but the discovery of electric synapses through gap junctions in some places validated at some point the concomitant existence of the reticular theory for a continuous network of fused cells. Moreover the polarized dynamic proposed by Cajal has been relativized by the discover of synapses at other places than only between axons and dendrites and the demonstration of back-propagating action potential (AP) within dendrites (Bullock et al., 2005).
Finally, Cajal was able to identify neuronal elements and to predict their general functioning rules through a simple static morphologic study. This exploit makes him famous and respected among scientists but allows also to draw two conclusions. First, there is an undeniable link between physiology and morphology: the function models the structure and reciprocally. Second, advances in scientific research are not simply made by the addition of facts that reinforce the evidence of conclusions, but are also and mainly the results of human intuition, that is characterized by emotion and incertitude. What is the most adequate tool to understand brain physiology than the brain itself?
Figure 2. Santiago Ramon y Cajal in his laboratory at the University of Valencia in 1887
Introduction! From synapse to spine
The synapse
As previously seen, the acceptation of the Neuron Doctrine revolutionized the end of the nineteenth century and marked the entry into the period of modern neurosciences. The notion of
communication between distinct entities, through contiguous structures that form synapses first asked the basic question of how transmission could occur through separated elements. Early,
exploration of the complex synaptic machinery raised the idea that synapses are not only devoted to the transferral of information from one cell to another but also and more curiously necessary to the regulation of the intercellular communication.
With the synapse, scientists have identified a key structure able to store and tune information within the cerebral cortex. This first step revolutionized the way of thinking the brain and brought new tools to investigate higher functions as learning or memory. The discovery of the transistor allowed the development of modern technics for information processing and storage. The discovery of the synapse allowed the emergence of the first valid hypothesis about thinking and memory.
Synapses can be classified according to their mode of transmission, their localization on the postsynaptic neuron or their polarity.
Transmission mode
The old debate about the modality of connections between cells has in fact never been completely closed and there are recent evidences supporting communication through cell continuity at electrical synapses. However, the role played by this kind of connections seems to be negligible in
comparison to the extend and the importance of the transmission through close contacts at chemical synapses.
Existence of electrical synapses in the CNS of mammals is finally well accepted but their importance and functional relevance are still debated. Cell-cell continuity is assumed by gap junctions that allow the current2 or small molecules to pass freely between them. Gap junctions are clusters of channels made of connexins. The functioning of electrical synapses is fast and
stereotyped because ions flow freely between cells. However there is no place for inhibitory action and the synaptic transmission could only be modified by a hypothetical phosphorylation of
connexins. In mammals, electrical synapses are best described at dendro-dendritic contacts between interneurons where they could participate in the generation of network oscillations in the theta and gamma range. The poor behavioral phenotype described in mice knockout for Cx36, the main neuronal connexins in adult, does not suggest a principal role for electrical synapses in the mammalian adult brain (Bennett and Zukin, 2004).
Chemical synapses represent the vast majority of contacts and their properties are suspected to be essential for information storage and processing within the brain. At chemical synapses, the two cells are physically separated by a cleft. An action potential in the presynaptic cell induces the liberation of neurotransmitters that flow through the cleft and link to receptors that polarize the postsynaptic cell. This complex mechanism allows the transmission to be modulated
presynaptically by the release of depolarizing or hyperpolarizing neurotransmitters that are
excitatory or inhibitory respectively and postsynaptically by modifications in receptors expression
Introduction! From synapse to spine
2 K+ ions
that can produce lasting changes in the transmission strength (Lisman et al., 2007). We will refer to chemical synapse when we will cite the general term “synapse” further in the text.
Localization
Synapses are principally established between an axon and a dendrite (axo-dendritic) as initially postulated by Cajal. However axons can also directly contact a soma (axo-somatic synapse) or an axon (axo-axonic synapse), but these last cases represent only a few contacts (Toni et al., 2007).
Dendrites can contact each other (dendro-dendritic synapse) in the case of electrical transmission (Haag and Borst, 2002).
Synapses are often grouped according to their function: axo-somatic synapses are mainly inhibitory, axo-dendritic excitatory and axo-axonic modulatory. The proximity of a synapse to the axon
hillock3 is important as the power of the signal decrease during its spreading. So axo-somatic synapses have a stronger influence on action potential generation than remote axo-dendritic (Spruston, 2008). We will refer to axo-dendritic contacts when we will cite the general term
“synapse” later in the text.
Polarity
Whether a synapse is excitatory or inhibitory depends on the receptors present on the postsynaptic side. Indeed, a neurotransmitter can be excitatory or inhibitory according to the reaction of the receptor to its binding. Then, a synapse is said excitatory if the binding of neurotransmitters to the receptors induces an influx of cations that depolarize the postsynaptic compartment and produces an excitatory post synaptic potential (EPSP). Conversely, a synapse is called inhibitory if the binding of neurotransmitters to its receptors induces an influx of anions that hyperpolarize the postsynaptic zone and produces an inhibitory post synaptic potential (IPSP).
The most abundant neurotransmitters in the CNS are the two amino acids glutamate and gamma- aminobutyric acid GABA, acting mainly on excitatory and inhibitory receptors respectively.
Transmitter gated channels on the postsynaptic cell can be of two types:
• ionotropic when the receptor is directly coupled with an ion channel;
• metabotropic when the receptor is indirectly coupled through a G-protein pathway to an ion channel.
In the brain, EPSPs are mainly mediated by ionotropic glutamate receptors which major subtypes are N-methyl-D-aspartate (NMDA), !-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate, named according to their synthetic agonist.
Non-NMDA receptors generate the early component of the EPSP and NMDA receptors contribute to the late component. NMDA receptors have some important characteristics:
• conductance of the cation channel is high for Ca++ 4, as well as for Na+ and K+;
Introduction! From synapse to spine
3 Trigger zone with low threshold for spike generation
4 Calcium influx is an important mediator of synaptic plasticity as it can trigger intracellular signaling cascades.
• permeability is voltage dependent as it requires the removing of the Mg++ block by previous depolarization5;
• opening and closing are relatively slow, retarding its contribution to the EPSP.
IPSPs are mainly mediated by GABA receptors in the brain. There are two types of GABARs:
• ionotropic receptors permeable to Cl-,called GABAa receptors;
• metabotropic receptors that generally activate K+ channels or inhibit voltage-gated Ca++ channels, called GABAb receptors.
Neurons from the central nervous system receive thousands of afferents which net effect depends on the postsynaptic integration of their strength6, polarity7, and location8. Postsynaptic potentials (PSPs) are processed temporally and spatially to reach the threshold necessary to make the postsynaptic cell to fire. Therefore, modulation of synaptic transmission is a key element for the regulation of individual cell firing rate and further network activity. Regulation of transmission strength, that depends on the capacity of the synapse to undergo changes is by far the most studied at excitatory synapses and will exclusively refer to plasticity at this kind of contacts later in the text.
(Kandel et al., 2000)
Figure 3. Synapses (adapted from Kim and Linden 2007)
Introduction! From synapse to spine
5 Significant level of activity is required before the opening of NMDARs. Immature synapses lacking non-NMDARs are called “silent synapses” due to their inability to reach this prerequisite.
6 Size of the postsynaptic potential
7 EPSP or IPSP
8 Dendrite, soma or axon terminal
Basic description of the glutamatergic synapse
Anatomical
From bouton to spine
Excitatory synapses are generally constituted by a presynaptic axonal bouton and a postsynaptic dendritic spine. This close apposition that is due to adhesion molecules leaves a gap in between: the synaptic cleft through which neurotransmitters diffuse. The synaptic cleft measures 24 nm in cryo- electron microscopy and contains electron-dense material probably consisting of the tight
arrangement of adhesion molecules and glutamate receptors (Zuber et al., 2005). Axons are neuronal efferent pathways that have to conduct AP quickly over long distances but also to
distribute information to thousand of targets along their path. To accomplish their task, axons make synapses along their branches through swelling of their structure or at the end of their branches (Anderson and Martin, 2001). These presynaptic elements are called varicosities or boutons “en passant” and terminal varicosities or boutons respectively.
Boutons contain round vesicles grouped in the active zone that faces the PSD. Boutons also contain dense core, coated or even double-walled vesicles. Mitochondria and endoplasmic
reticulum can also be found. Individual boutons form either single or multiple synapses, meaning that a single bouton can contact one or many spines arising from different dendrites. Generally boutons of excitatory synapses contact one of the small dendritic protrusions called dendritic spines and direct contacts with the dendritic shaft are less frequent (Shepherd and Harris, 1998).
Spines are highly specialized structures constituted most generally by a thin neck and a bulbous head that contain the postsynaptic receptors which are embedded in an electro-dense region called the postsynaptic density (PSD) (Harris and Stevens, 1989).
Gray’s classification
Excitatory synapses have ultrastructural characteristics that allow to differentiate them
morphologically from inhibitory synapses. These differences have been first categorized by Gray as type I and II (Gray, 1959). Then Colonnier resumed them slightly as asymmetric and symmetric respectively (Colonnier, 1968):
• Type I, asymmetric, excitatory synapses contain round synaptic vesicles in prominent dense presynaptic projections, have a wide synaptic cleft, a large active zone and concomitant PSD, and dense basement membrane;
• Type II, asymmetric, inhibitory synapses contain flattened synaptic vesicles in less obvious dense presynaptic projections, a narrow synaptic cleft, a small active zone and PSD, and a modest basement membrane.
Introduction! From synapse to spine
Physiological
From AP to EPSP
When a presynaptic AP that flows through the axon reaches a bouton, it induces an EPSP in the postsynaptic cell by the release of neurotransmitter in the synaptic cleft.
AP stimulate the release of the content of synaptic vesicles by triggering the opening of voltage-dependent calcium channels in the presynaptic terminal. As calcium enter, it can be effective immediately as calcium sensors are located within micro-domains near the calcium channels (Schneggenburger and Neher, 2000). Another consequence of this calcium gradient is that different vesicles have different release probabilities generating a spatial coupling between calcium channels and sites of vesicles release (Wadel et al., 2007). Synaptotagmin links four to five calcium ions and each vesicle contains fifteen synaptotagmin molecules but it is unclear how they work together to produce release. Vesicles are held at the synapse by the SNARE complex and the complex Ca-synaptotagmin induces their content release at the cleft (Tang et al., 2006). The initial opening of a vesicle may involve a small or a large fusion pore. Some openings are followed by closure, a process called “kiss and run” that allows the vesicle to be reused. The vesicle can also fuse completely with the plasma membrane and be subsequently reused by endocytosis at
extrasynaptic sites. Size of the fusion pore and mode of vesicle recovery are not linked (Lisman et al., 2007).
Glutamate initially at a vesicular concentration of some hundred of millimolar (Burger et al., 1989) diffuses through the pore and dilutes as its spreads through the cleft before meeting with AMPARs. Micromolar levels of glutamate can bind to single subunits of the channel and induce a desensitized state without opening. In contrast, millimolar concentrations induce a rapid opening of the channel followed by its desensitization. So the more glutamate that binds, the higher the
probability of opening before desensitization and then the higher the single channel conductance (Smith and Howe, 2000). Only a fraction of the synapse area is actually involved because there are large concentration gradients in the cleft and so the millimolar glutamate concentration required to open the AMPARs are only achieved within the 100 nm of the site of vesicle release. The tenth of channels opened during a quantal event are within a hot spot that cover only 25% of the area of the average CA1 synapse. Then average quantal current is far below saturation and the main
postsynaptic determinant of quantal size is AMPARs density and not the total channels number.
Number of vesicles released is certainly correlated with synapse size, multivesicular release being more frequent in large synapses (Raghavachari and Lisman, 2004).
Introduction! From synapse to spine
Figure 4. Synaptic transmission at glutamatergic synapses (for clarity NMDARs are omitted and only one vesicle is released) (Lisman et al., 2007)
The quanta theory
A single vesicle filled with neurotransmitter is the elementary unit of synaptic transmission that is defined as a quantum. The release of one quantum can be quantified when a spontaneous vesicular release occurs9 and then generates a miniature EPSP (mEPSP). Modifications of the synaptic transmission can be evaluated in quanta which are the elementary building blocks of EPSPs. The number of quanta is calculated from the fraction EPSP/mEPSP, mEPSP being uniquantal per
definition. The quantal size or mEPSC that is the response of the postsynaptic membrane to a single quantum is determined by AMPARs density, glutamate vesicle concentration, number of vesicles available and the mode of release (Edwards, 2007).
Introduction! From synapse to spine
9 Not evoked by an AP
Spine description
Spines are small membranous protrusions of 0.25 to 2.5 "m in length (Harris and Stevens, 1989), that arise from dendrites at a density until 15 per "m (Harris and Stevens, 1988) and at the tip of which are established most excitatory synapses in the CNS. These microscopic elements are more precisely examined at the electron microscope (EM) after chemical or cryogenic tissue fixation for the analysis of their ultrastructural components. Improvements in light microscopy specially with confocal or 2P microscopes10 and the development of various and effective fluorescent probes allows now to appreciate correctly the general shape of spines and even to make repeated
measurements in cultured tissues or living animals. These two approaches are complementary and the combination of the two allows to answer questions requiring both an ultrastructural and dynamic study.
The electrical function of dendritic spines has never been directly examined but experimental models refute their function as passive resistance and suggest active and more complex interactions with current (Tsay and Yuste, 2004). Dendritic spines are biochemical micro- compartment that house not only the synapse proper but also the essential postsynaptic components:
organelles involved in protein synthesis, membrane trafficking and ATP and calcium
metabolism(Alvarez and Sabatini, 2007). The principal function of dendritic spines is still debated even if it is proposed that they simply allow farther connections and a higher density of synaptic contacts (Sorra and Harris, 2000).
Static point of view
Since the first observation of dendritic protrusions by Cajal, their study often lead to contradictory conclusions and different classifications of spine type have been proposed. These divergencies are due first by the fact that spine population is quite heterogeneous and varies highly depending on physiological properties like the developmental stage of the specimen or the neuronal type that is observed (Nimchinsky et al., 2002). Second, the experimental approach is a supplementary and often underestimated source of variations. Origin of the tissue (Kirov et al., 1999) as well as its mode of preparation (Kirov et al., 2004) can modify profoundly spine appearance. Imaging sessions can also rapidly modify spine shape through light toxicity when fluorescence microscopy is
performed11. Differences concerning the type of information obtained with electron or light
microscopy also introduced variations in observations and conclusions. Finally, this is probably the intricacy between morphology and function that introduced the highest diversity because
observations have nearly always been done to serve hypothesis about physiology. So, criteria vary certainly as much among classifications as imagination among morphologists. For these reasons I will restrain my description to generally used criteria and to commonly seen spine types found at the CA1 region of the hippocampus.
Introduction
10 Light scanning microscopy (LSM) can be performed by a microscope that uses normal continuous laser light combined with a confocal aperture or a two-photon (2P) pulsed laser light alone(Helmchen and Denk, 2005).
11 Personal observations
The majority of dendritic protrusions consist of a “neck” that links a bulbous extremity called the
“head” to the parent dendrite. Because the neck hinders diffusion of molecules between the head and the dendrite, spines can be considered as biochemical compartments. Protrusions can be then classified depending on these two elements using qualitative or quantitative criteria but there is always a continuum between categories. Quantitative criteria are based on absolute measures or on their proportions while qualitative criteria are based on the simple presence or absence of the neck and the head.
Protrusion types
There is little consensus on a general category called “protrusions” that includes “spines” and
“filopodia”. No marker has been found to distinguish filopodia from spines and their identification is based on imprecise and subjective morphological features such as a long and thin neck combined with an absence of enlargement at the tip. Spines and filopodia can be considered as a
morphological continuum of the same entity or as completely distinct structures with different roles.
Two population of filopodia with different functions are described in the developing cortex.
Filopodia that are found in growth cones of developing neurons participate to dendritic growth and branching while filopodia that cover dendritic shafts are directly implicated in synaptogenesis and have a controversial role in spinogenesis. Filopodia at the tip of growth cones are excluded from the definition beneath because they differ from dendritic filopodia in terms of motility, structure and supposed function (Portera-Cailliau et al., 2003).
Filopodia
Filopodia are long and thin protrusions lacking a bulbous head at the tip. They are rare on mature neurons, so their function is probably related to development even if this role is controversial. At early postnatal ages, filopodia are abundant and precede the apparition of the first spines, but the proof of their direct transformation into spines is missing. In the adult, transformation of filopodia into spines is a very rare event (Majewska et al., 2006) that leads to spine loss in the vast majority of cases within the 2 days in vivo (Zuo et al., 2005a).
Spines
Spines are protrusion bearing a head at the tip. The most commonly used nomenclature was introduced by Peters and Kaiserman-Abramof in 1970 (Peters and Kaiserman-Abramof, 1970).
Depending on the absolute or proportional size of the head and on the presence or absence of a constricted neck spines are classified as follow:
• Stubby spines: absence of constricted neck or ratio width over length> 0;
• Thin spines: absolute head width < 0.6 "m or proportionally small head;
• Mushroom spines: absolute head width > 0.6 "m or proportionally big head.
Introduction! Spine description
Figure 5. Protrusion types (Ethell and Pasquale, 2005)
Spine organelles
There is growing evidence that the different spine shape and size reflect different developmental stage and strength of synapses. The best example is the strong correlation between spine size and the supposed level of synaptic activity. Spine head is increased at strengthen synapses while
decreased at weaken synapses. Large presynaptic active zones bearing a high number of vesicles are generally apposed in front of large PSDs with a high density of glutamate receptor on large spine heads (Harris and Stevens, 1989) (Takumi et al., 1999b). Further, larger spines are more likely to content organelles that can improve their efficacy (Cooney et al., 2002) ( Bourne et al., 2007).
RER, Golgi and polyribosomes
Rough endoplasmic reticulum (RER), Golgi compartments and polyribosomes are ubiquitous in proximal dendrites and cell body but a strong synaptic activation can redistribute polyribosomes in distal dendrites and large spines suggesting a role of local translation in synapse strengthening (Bourne et al., 2007). In general, proteins are synthesized in the soma and then transported through the Golgi to dendrites and spines (Horton and Ehlers, 2004), but mRNAs for synaptic proteins like calcium/calmodulin-dependent protein kinase II (CaMKII) (Martone et al., 1996) or AMPARs (Ju et al., 2004) are found in distal dendrites and spines where they can be locally translated in response to activity.
SER
Smooth endoplasmic reticulum (SER) is a continuous reticulum that predominates in distal
dendrites but can protrude into about 20% of spines (Cooney et al., 2002) (Sorra and Harris, 2000).
SER plays a role in calcium handling and in the transport of lipids and proteins (Horton and Ehlers, 2004). In more than 80% of large mushrooms, SER is organized in laminae separated by dense material that is called spine apparatus (Spacek and Harris, 1997). Its function is unknown, but mice lacking synaptopodin, an actin-associated protein that localizes the spine apparatus show altered synaptic plasticity (Deller et al., 2003).
Non-ER compartment
This category comprise membranous compartments that do not belong to the ER system and are found in half of spines. Morphologically we can distinguish vesicular, multivesicular, tubular or amorphous vesicular compartments but functionally it seems that there are only two categories:
endosomes and small vesicles that take part to endocytosis and exocytosis processes respectively
Introduction! Spine description
(Cooney et al., 2002). Exo-, or endocytosis systems traffic receptors to and from the synaptic membrane during plasticity mechanisms (Kopec et al., 2006) (Kim et al., 2007). All these compartments constitute also the membrane pool used to enlarge or shrink the spine head in response to activity (Park et al., 2006).
Mitochondria
Mitochondria are rare in spines despite their abundance in dendrites, but synaptic activation can drive their translocation into larger spines. Besides providing ATP, mitochondria play a role in calcium buffering and apoptosis, however their postsynaptic function in spines is unclear. A curious mechanism to produce ATP locally at the PSD without mitochondria has even been described12 (Wu et al., 1997). Anyway, dendritic mitochondria appear to be essential for formation and maintenance of dendritic spines (Li et al., 2004).
Proteasomes
Activity-dependent ubiquitination can leads to the proteosomal degradation of some postsynaptic scaffolding proteins (Ehlers, 2003). This process could mediate the activity-dependent
internalization of AMPARs (Colledge et al., 2003). Taken together it seems that ubiquitination of postsynaptic proteins can alter strength and stability of synapses (Cline, 2003).
Spine PSD
Presence of a PSD is a characteristics of asymmetric glutamatergic synapses that are mainly formed onto dendritic spines of pyramidal neurons in the neocortex (Spacek and Hartmann, 1983),
hippocampus (Harris and Stevens, 1989) and cerebellum (Harris and Stevens, 1988), but also directly onto dendritic shafts of interneurons in the neocortex and hippocampus. However the basic structure of the PSD is indistinguishable between dendritic spines or dendritic shafts suggesting that gross structure of the PSD is probably the same in all excitatory synapses, independently of their localization.
PSD is an electron-dense thickening of the spine head that faces the presynaptic zone and contains glutamate receptors, scaffolding proteins and signaling proteins. The typical PSD is disk- like structure with a diameter of 200-800 nm and a thickness of 30-50 nm (Carlin et al., 1980), made of more than 450 different proteins (Yoshimura et al., 2004). PSD size and shape are well correlated with spine head diameter and synaptic efficacy. PSD is macular in small spines while annular or irregular and segmented in large spines (Sorra and Harris, 1998). Morphological features are distinct at the cleft and cytoplasmic surface of the PSD. The cleft surface is characterized by a dense layer of small particles while the cytoplasmic surface is convoluted in appearance with irregular protrusions (Petersen et al., 2003). This difference is due to differences in the molecular composition of the two surfaces.
Glutamate receptors in the PSD
At hippocampal CA3-CA1 synapses, the PSD generally contents between 20 and 300 glutamate receptors which AMPARs, NMDARs, mGluRs and associated proteins (Okabe, 2007) belong.
Introduction! Spine description
12 Presence of glycolytic enzymes in isolated PSDs
NMDARs are abundant, and constituted mainly of subunits NR1 and NR2A-B. NMDARs are motile and their subunits composition change during development with a switch from NR2B to NR2A as synaptic activity increase (Bellone and Nicoll, 2007). Their transport to synaptic site is thought to be due to intracellular trafficking for NR2A (Janssen et al., 2005) or lateral diffusion from extrasynaptic sites for NR2B (Choquet and Triller, 2003). Experiments with NR1 knockout mice suggest that NMDA receptors are not required for development and maintenance of the PSD.
Even in the absence of NMDARs, levels of GluR1 and PSD-95 are unaltered and gross anatomy of the synapse is normal (Forrest et al., 1994).
In hippocampus AMPAR containing GluR1 and GluR2 are higly expressed (Martin et al., 1993).
GluR2 subunit confers a low calcium permeability to the receptor (Hollmann et al., 1991). Double knockout GluR2/3 and simple knockout GluR1 experiments suggest that both continuous recycling of GluR2/3 and activity-dependent insertion of GluR1 are not required for the basic PSD
morphology but are essential to maintain basic AMPA currents and plastic changes respectively (Meng et al., 2003) (Zamanillo et al., 1999). Number of AMPARs per spine increases with distance from the soma in a process called distance-dependent scaling that is thought to compensate for distance-dependent decrease of synaptic signals (Smith et al., 2003).
G-protein-coupled metabotropic glutamate receptors (mGluRs) of the group I are constituted by subunits GluR1/5 and activate the phosphatidylinositol pathway. They are scarce but can be found at the borders of the PSD (Sheng and Hoogenraad, 2006).
Stargazin (!-2) is one of the transmembrane AMPA receptors regulating proteins (TARPs, also !-3,
!-4, !-8) which are major constituents of the PSD where they regulate fast excitatory transmission by interacting with AMPARs (Nakagawa et al., 2005).
Signaling proteins in the PSD
CaMKII is one of the most abundant protein in neurons and is highly enriched in PSDs (Kennedy et al., 1983) where it represents about 6% of the total mass (Chen et al., 2005). Two isoforms ! and #- CaMKII form holoenzymes of 12-14 subunits.
This serine/threonine-specific kinase translocates so massively to the PSD after calcium influx through NMDARs that it can alter the PSD volume (Shen and Meyer, 1999) by forming huge clusters of 100 nm diameter at the cytoplasmic side. (Dosemeci et al., 2001) (Petersen et al., 2003).
When activated, CaMKII interacts with NMDARs through NR2B subunit (Bayer et al., 2001).
CaMKII can also interact with !-Actinin13 and Densin-18014 that could orientate its activity-induced localization to the PSD (Walikonis et al., 2001). Phosphorylation of SynGAP by CaMKII increases its GTPase activity on Ras to promote spine head shrinkage (Oh et al., 2004). CaMKII holoenzymes can bind to F-actin through their #-subunit but the couple calcium/calmodulin promote their
dissociation (Shen and Meyer, 1999). CaMKII can also be activated independently of Calmodulin by interacting with Myosin Va15 that is enriched in the PSD (Costa et al., 1999). This activation by
Introduction! Spine description
13 F-actin cross-linking protein
14 Transmembrane protein
15 Brain Myosin V
Myosin Va seems to play an important role in the translocation of GluR1 to the synapse when triggered by activity (Correia et al., 2008). In summary CaMKII is central in the PSD trough its interaction with at least three different kind of proteins: glutamate receptors (NR2B),
transmembrane proteins (Densin-180) and actin interacting proteins (!-Actinin and Myosin Va).
Scaffolding proteins in the PSD
Although lacking catalytic domains, PSD scaffolding proteins can influence spine development and morphology by recruiting effectors proteins.
Membrane associated guanylate kinase proteins (MAGUKs) is a family of scaffolding proteins sharing a common domain organization: three N-terminal PSD-95/Discs large/Zona occludens-1 (PDZ) domains, a Src-homology 3 (SH3) domain, and a carboxy terminal catalytically inactive guanylate kinase (GK) domain. PDZ domain of MAGUK family proteins interacts with a variety of membrane proteins present in the PSD (Elias and Nicoll, 2007). PSD-95 (also known as SAP-90) was the first MAGUK to be identified followed by SAP-97, SAP-102 and PSD-93. In immature spines, the principal MAGUK is SAP-102 that probably targets AMPARs to the plasma membrane via TARPs. In more mature spines, this role is taken over by PSD-95 and PSD-93 while SAP-97 would deliver AMPARs to the plasma membrane in an activity-dependent manner (Fitzjohn et al., 2006). PSD-95 interacts not only with TARP but also with NR2 for the postsynaptic clustering of AMPARs and NMDARs respectively but experiments with KO mice suggest that some of these functions can be substituted by other MAGUKs (Elias et al., 2006). PSD-95 can also interact with postsynaptic signaling molecules like ErB416, SynGAP17, SPAR18, Kalirin-719 and cell adhesion molecules like #-Neurexin20 (Irie et al., 1997). Taken together, MAGUK family proteins are implicated in PSD organization (glutamate receptors clustering) and function (interaction with signaling molecules). Moreover, their similar organization domains probably allow overlapping or redundant functions.
Homer proteins (Homer1-3) bind principally to group I mGluRs and inositol triphosphate receptors (IP3Rs). They appear to regulate the coupling between membrane mGluRs and ER-associated intracellular IP3R (Tu et al., 1998).
Shank proteins (Shank1-3, also known as ProSAP, SSTRIP, CortBP, Synamon and Spank) contain multiple domains for protein-protein interactions, including Ankyrin repeats, SH3 domains and PDZ domains. They have multiple binding partners that include both membrane-associated proteins and cytoskeletal proteins as well as other scaffolding proteins (Homer and GKAP). As Shank3 can interact with itself to form large sheet-like structures of helicoidal fibers, it could form a platform for the construction of the higher order PSD structure (Baron et al., 2006). In agreement, Shank
Introduction! Spine description
16 Receptor tyrosine kinase
17 GAP for Ras
18 GAP for Rap
19 GEF for Rac1
20 Ligand for Neuroligin
overexpression modifies deeply the morphology of the synapse by promoting spine enlargement (Sala et al., 2001).
Guanylate kinase-associated protein (GKAP, also known as SAPAP) consists of four members (SAPAP1-4) with similar domain organization: 14 amino-acid repeats in the middle of their
sequence and a PDZ domain. In addition to binding multiple proteins, GKAP can cross-bridge two large scaffolding proteins by interacting with Shank and PSD-95 (Sala et al., 2001).
Organization of the PSD
Hundreds of different components of the PSD are distributed radially and tangentially within this highly organized molecular meshwork.
Tangential distribution of glutamate receptors is determined by interactions with submembranous scaffolds molecules and their mode of delivery. Consistent with that point NMDARs are abundant at the center of the PSD while AMPAR show an even distribution (Racca et al., 2000). It could be also that NMDARs are more tightly anchored to synaptic proteins than AMPARs which show a continual recycling. Anyway, the removing of both AMPARs and NMDARs seems to take place at peripheral zones where molecules involved in endocytosis are localized (Racz et al., 2004).
Laminar organization of the PSD molecules is revealed by peak concentration of each protein in different layer. NR2 and PSD-95 are close to the plasma membrane with an average depth of 12 nm.
GKAP and Shank are shifted toward the cytoplasmic side with an average depth of 25 nm
(Valtschanoff and Weinberg, 2001). At most cytoplasmic sides, CaMKII and Homer are abundant.
As the postsynaptic cytoplasm is enriched in F-actin and as association of F-actin with PSD components is reported, it is likely that F-actin is implicated in maintenance and turnover of
scaffolding proteins of the deepest layer of the PSD like GKAP, Shank and Homer. Moreover, there is a positive correlation between proximity with actin at the cytoplasmic side of the PSD and mobility of scaffolding proteins (Kuriu et al., 2006). Concerning glutamate receptors, GluR1 is not surprisingly shown to be more mobile than NR1 (Sharma et al., 2006). Cytoskelton-related
molecules like !-Actinin or Cortactin show kinetics comparable to actin.
Actin cytoskeleton
Mice lacking PSD-95 exhibit normal spine morphology, despite altered learning patterns (Migaud et al., 1998), but disruption of actin during spine morphogenesis results in the disassembly of synaptic elements suggesting that actin cytoskeleton is more responsible for spine morphogenesis than PSD (Zhang and Benson, 2001). Actin is one of the most abundant proteins in neurons and is mainly concentrated in spines where it turns over with a time constant of some minutes. (Star et al., 2002).
These dynamic properties principally rely on Actin “treadmilling” of existing filaments but severing, branching or nucleation of new filaments contributes also to actin rearrangement.
Accumulating evidence suggests that actin dynamics could drive formation and loss of protrusions as well as their morphological plasticity.
Introduction! Spine description
Actin polymerization
Actin is present as monomeric (G-actin; globular) or filamentous form (F-actin). There is a natural tendency of G-actin to polymerize into double helical filaments but binding proteins can sequester monomeric actin and suppress the polymerization of G-actin into F-actin (Safer and Nachmias, 1994). Interestingly, the ratio of G-actin over F-actin is regulated by activity (Okamoto et al., 2004).
Actin “treadmilling”
F-actin has a structural polarity with two ends that lengthen and shorten at different rate:
“treadmilling”. This produces a continuous exchange of actin molecules without change in total length. Assembly and disassembly of actin molecules take place at the barbed and pointed end respectively. Most of F-actin in dendritic spines undergo “treadmilling” as attests its rapid turnover (Honkura et al., 2008) and actin-binding proteins can accelerates this process21.
Actin-binding proteins
Dendritic spines are highly enriched in F-actin that can form bundles in spine neck and core of spine head or a fine meshwork at the periphery of the spine head (Landis and Reese, 1983). The organization of F-actin in bundles or networks is determined by binding proteins and each F-actin organization is accompanied by a certain set of actin-binding proteins which subcellular localization can be changed by extracellular signals (Sekino et al., 2006). Many actin-binding proteins are present in dendritic spines. Some proteins regulate actin length by severing F-actin and/or regulating actin “treadmilling”. Other cross-linking proteins organize F-actin into bundles or networks. At last, side-biding proteins modify the structural properties of F-actin and modulate its interactions with other binding proteins.
Cofilin and its related actin depolymerizing factor (ADF) bind cooperatively to actin filament. They decrease actin length by severing and increasing the rate of dissociation at the pointed end (Carlier and Pantaloni, 1997). ADF/Cofilin are thought to play a role in spine head shrinkage (Ethell and Pasquale, 2005).
Gelsolin can have opposite effects on actin stability depending on calcium levels. Gelsolin severes actin filaments when calcium concentration is very high like during excitotoxicity (McGough et al., 2003). Conversely, Gelsolin stabilizes actin by capping the barbed end when calcium increase is moderate like during synaptic plasticity (Star et al., 2002).
Actin-related proteins 2 and 3 (Arp2/3) complex is regulated by many signaling pathway that affect spine shape and motility like Rho family GTPases and other calcium regulated actin-binding proteins like Profilin (Higgs and Pollard, 2001). Arp2/3 complex enhances actin nucleation, causes branching and cross-linking of F-actin (Mullins et al., 1998) and is thought to induce enlargement of the spine head (Ethell and Pasquale, 2005).
Profilin increases actin length by promoting polymerization trough the exchange of ADP for ATP bound to actin (Kovar et al., 2006) and nucleation trough Arp2/3 (Yang et al., 2000). Profilin is
Introduction! Spine description
21 Like ADF/Cofilin
redistributed from dendrites to spines after NMDA activation (Ackermann and Matus, 2003) and could contribute to stabilize actin filaments (Finkel et al., 1994) and maintain a mature spine morphology (Ackermann and Matus, 2003).
Cortactin is an activator of the Arp2/3 complex that interacts with scaffolding protein Shank.
Cortactin redistributes from spines to dendrites during NMDARs activation. Suppression of Cortactin suppresses spines whereas overexpression causes spine elongation (Hering and Sheng, 2003).
Drebrin is a side-binding protein which presence in dendritic spines is regulated in an activitiy- dependent manner (Fujisawa et al., 2006). Drebrin is thought to promote actin assembly and synaptic clustering of PSD-95 at postsynaptic sites (Takahashi et al., 2003).
RhoA-specific kinase (ROCK) allows regulation of F-actin organization in a RhoA activity- dependent manner. Active RhoA activates ROCK and stabilizes F-actin and inversely (Schubert et al., 2006).
!-Actinin organizes F-actin in networks or bundles depending on the low or high concentration of the cross-linking protein respectively (Wachsstock et al., 1993) and could anchor NMDARs to cytoskeleton (Wyszynski et al., 1998).
Spinophilin (Neurabin II) and Neurabin I are a cross-linking proteins that bundle F-actin and bind to protein phosphatase 1 (PP1) for modulating actin organization in dendritic spines (Satoh et al., 1998).
Myosins are ATP-driven and actin-based motor proteins that move organelles along F-actin or F- actin themselves. Myosin VI is involved in spine formation (Osterweil et al., 2005), Myosin Va in the trafficking of spine proteins (Yoshimura et al., 2006) and Myosin IIB in spine morphology (Ryu et al., 2006). Myosin IIB is activated in two ways: first, by a myosin-linked regulation22 (Moussavi et al., 1993); second by an actin-linked regulation23 (Hayashi et al., 1996). Finally, spine
morphogenesis could be regulated by small GTPases via a myosin-linked regulation24 (Zhang et al., 2005), or by variations in calcium concentration via an actin-linked regulation25.
Other actin-binding proteins which function is very uncertain in spines include Synaptopodin that associates with spine apparatus or SER (Deller et al., 2000), Fodrin and actin-binding protein 1 (Abp1) that associate with Shank family scaffolding proteins (Bockers et al., 2001) (Haeckel et al., 2008).
Introduction! Spine description
22 Inhibition of Myosin ATPase activity by myosin light chain (MLC) is relieved by MLC phosphorylation
23 Prevented access of myosin to F-actin by Drebrin is relieved by a candidate protein
24 Phosphorylation of MLC
25 A candidate protein could play a role similar to Tropomyosin in the muscle
Signaling to actin
Number of intracellular signaling cascades originate from receptors at the cell surface and converge on actin-binding proteins to control actin cytoskeleton. The best characterized are small GTPase of the Rho family including Cdc42, Rac, RhoA (Newey et al., 2005) and small GTPases of the Ras family with Ras and Rap (Kennedy et al., 2005). Additionally, some actin-binding proteins like Gelsolin are sensitive to calcium levels and can be activated or inhibited through changes in calcium concentration (McGough et al., 2003). Thus, alternative calcium kinetics from different sources can also modulate actin dynamics (Brunig et al., 2004).
Figure 6. Signaling to actin through calcium and small GTPases (Ethell and Pasquale, 2005)
Spine adhesion molecules
Synaptic cell adhesion molecules, are not merely structural components that stabilize the two synaptic membranes in front of each other trough homo- or heterophilic interactions, but are also dynamic regulators of spines by controlling intracellular signaling cascades or secondary protein- protein interactions.
Neurexins (presynaptic) and their partners Neuroligins (postsynaptic) contain both a PDZ domain that allows interactions with synaptic scaffolding proteins. Neuroligins-Neurexins are probably implicated in synaptogenesis by recruiting PSD-95 that clusters NMDARs at the postsynaptic membrane (Irie et al., 1997).
Introduction! Spine description
