Anesthesia and neuronal circuitry development: molecular and cellular mechanisms

Thesis

Reference

Anesthesia and neuronal circuitry development: molecular and cellular mechanisms

BRINER, Adrian

Abstract

Les anesthésiques sont utilisés dans des millions de chirurgies pédiatriques chaque année.

Le bénéfice de leur action transitoire p est largement accepté, mais le cerveau humain en développement revient il à son état initial après une exposition aux anesthésiques? Comme les anesthésiques généraux modulent des systèmes de neurotransmetteurs qui sont cruciaux durant la formation du système nerveux central (SNC), les rôles neuro-protecteur et neurotoxique de ces médicaments ont été évoqués. Dans ma thèse, je me suis concentré sur les effets des anesthésiques généraux sur la formation des connexions corticales durant la phase de croissance rapide des neurites et synapses aussi appelée la période de poussée de cerveau. Nous démontrons de façon convaincante la vulnérabilité des réseaux neuronaux à l'exposition aux anesthésiques durant les périodes critiques du développement du cerveau des rongeurs. Il reste à démontrer si des effets similaires se produisent chez des espèces supérieures.

BRINER, Adrian. Anesthesia and neuronal circuitry development: molecular and cellular mechanisms. Thèse de doctorat : Univ. Genève et Lausanne, 2012, no. Neur. 90

URN : urn:nbn:ch:unige-217908

DOI : 10.13097/archive-ouverte/unige:21790

Available at:

http://archive-ouverte.unige.ch/unige:21790

Disclaimer: layout of this document may differ from the published version.

Faculté de Médecine

DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DE MEDECINE PD Laszlo Vutskits, directeur de thèse

Professeur Denis Morel, co-directeur de thèse

TITRE DE LA THESE

ANESTHESIA AND NEURONAL CIRCUITRY DEVELOPMENT – MOLECULAR AND CELLULAR MECHANISMS

THESE Présentée à la Faculté de médecine de l’Université de Genève

pour obtenir le grade de Docteur en Neurosciences

par

Adrian BRINER

de Fehraltorf ZH

Thèse N° # Thèse Université de Genève

Editeur ou imprimeur : Université de Genève 2011

!"#$%&'()*(+')*#,-*(

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

!"#$%&'()*(+')*#,-*(

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

2012 Thèse N° 90

to Marietta

Acknowledgments

I would like to thank personally all the people who contributed to this thesis by some means or other. It‘s difficult to express in words my gratitude to all those who supported me in any respect during the completion of the project. I really enjoyed the years in Laszlo‘s laboratory and I made good friends.

Thank you Laszlo for your excellent supervision and for the cycling on mount Salève!

Thank you Hubert and Jean-Luc for the nice collaboration!

Thank you Marc and Orbi for having been my roommates + for climbing!

Thank you Timi, Teddy, Horace, Catherine, Claudia & Kornel for your help to build up the lab!

Thank you Bea and Cynthia for your technical assistance!

Thank you Michiko, Eloisa, Gael, Michael, Inge, Ophelie, Patrick for your instructions!

Thank you Dominik and Jozsef for sharing part of your labspace with us!

Thank you Mathias, Sylvain and Paul for your help with the eletrophysiology setup!

Thank you Irina for your introduction to electron microscopy!

Thanks to Dayer and Jabaudon lab for the interlab meetings and retreat!

Thank you Denis for being my thesis co-directeur!

and finally thanks to the Confiseur at the cafeteria for preparing very tasty desserts!

Laszlo Vutskits, Hubert Fiumelli, Jean-Luc Martin, Alexandre Dayer, Mathias de Roo, Dominik Muller, Marc Giry-Laterriere, Marietta Meier, Irina Nikonenko, Timea Bodogan, Beatrice King, Teddy Belem and Jozsef Kiss participated in this work.

Thanks to the University Hospital of Genève and to Laszlo Vutskits for the funding.

Abstract

Anesthetic drugs are successfully used in millions of pediatric surgeries every year. The benefit of their transitory action during surgery is widely accepted, but recent animal studies raise concerns:

Does the developing human brain indeed come back to its initial state after anesthesia exposure?

Clinical studies demonstrated an association between major surgery with anesthesia at early ages and poor longterm neurocognitive outcome. Several factors such as coexisting pathology and the inflammatory response to surgery may be implicated. As general anesthetics modulate neuronal activity and neurotransmitter systems which are crucial during central nervous system (CNS) formation, both neuro-protective and neurotoxic roles of these drugs have been evoked. The goal of the present thesis is to examine the action of anesthetics on the growing brain and their role in the above mentioned clinical studies.

Proper brain development involves a series of precisely controlled steps. In my thesis I focus on the effects of general anesthetics on the formation of cortical connections during the phase of rapid neurite growth and synaptogenesis, also called the brain spurt period. There is extensive in vitro and in vivo evidence that anesthetic drugs affect neuronal survival. Little however is known about interference with the finely tuned mechanisms of neuronal network formation. The first objective of my work was to characterize the critical period during which the developing neuronal networks are particularly vulnerable to classical modulation of GABAergic and glutamatergic neurotransmitter systems by anesthetic drugs. Then we defined exposure time and dose necessary to induce measurable and potentially persisting structural changes in neuronal connectivity. As GABAergic signaling systems undergo major changes with development, I verified whether the effects of anesthetic drugs on the brain indeed depend on the maturational state of GABAergic neurotransmission.

I addressed these questions in a postnatal rodent model of cortical dendrito- and synaptogenesis. To evaluate the exposure time-dependent effects of anesthetic on synaptogenesis I first exposed 16- day-old Wistar rat pups to different volatile anesthetics and measured changes on layer 5 pyramidal neuron morphology in the cortex. I assessed important neuronal parameters such as dendritic structure and dendritic spines, which form the postsynaptic part of excitatory synapses. In this first study, volatile anesthetics did not induce apoptosis or alterations in gross dendritic arbor pattern, but spine density was significantly increased in a exposure-time and drug dependent manner. To further

test the developmental-stage dependent effects of anesthetics, I performed propofol anesthesia at distinct time-points between postnatal (PND) day 5 and 30 corresponding to the whole morphological maturation phase of layer 5 pyramidal neurons in the rat cortex. These treatment paradigms induced a decrease in dendritic spines at PND 5 and 10 while later on propofol significantly increased spine number, suggesting that the action of these drugs depends on the maturation state of neurotransmitter systems. Moreover, I could demonstrate that these structural changes persist for months. To verify our hypothesis that the state of GABAergic neurotransmission determines the action of propofol anesthesia on neuronal networks, we performed in utero electroporation of full length and truncated KCC2 constructs, a chloride co-transporter responsible for the GABAA reversal potential shift during neuron development. Surprisingly overexpression of KCC2 itself considerably increased spinogenesis in a chloride co-transport independent manner through interaction with the actin-cytoskeleton. These findings may explain the temporal synchronization between dendritic spinogenesis and the chloride reversal potential shift.

In conclusion we convincingly demonstrate the vulnerability of neuronal networks to anesthesia exposure during critical periods of rodent brain development. Whether and to what extend similar effects occur in higher species and how these morphological changes modify long-term neurocognitive performance remains to be shown.

Résumé

Les anesthésiques sont utilisés dans des millions de chirurgies pédiatriques chaque année. Le bénéfice de leur action transitoire pendant la chirurgie est largement accepté, mais des études animales récentes soulèvent des préoccupations: le cerveau humain en développement revient il à son état initial après une exposition aux anesthésiques? Des études cliniques ont démontré une association entre chirurgie majeure avec anesthésie à un âge précoce et mauvais résultat neurocognitif à long terme. Plusieurs facteurs tels que la coexistence d’une pathologie et de la réponse inflammatoire liée à la chirurgie peuvent être impliqués. Comme les anesthésiques généraux modulent l'activité neuronale et les systèmes de neurotransmetteurs qui sont cruciaux durant la formation du système nerveux central (SNC), les rôles neuro-protecteur et neurotoxique de ces médicaments ont été évoqués. L'objectif de cette thèse est d'examiner l'action des anesthésiques sur le cerveau en croissance et leur rôle dans les études cliniques mentionnés ci- dessus.

Le bon développement du cerveau implique une série d'étapes contrôlées avec précision. Dans ma thèse, je me concentre sur les effets des anesthésiques généraux sur la formation des connexions corticales durant la phase de croissance rapide des neurites et de la synaptogenèse, aussi appelée la période de poussée de cerveau. Il est largement prouvé, à la fois in vitro et in vivo, que anesthésiques affectent la survie des neurones. Cependant peu d’informations existent sur les interférences avec les mécanismes minutieusement réglés de la formation des réseaux neuronaux.

Le premier objectif de mon travail était de caractériser la période critique durant laquelle le développement de réseaux neuronaux sont particulièrement vulnérables à la modulation classique de systèmes de neurotransmetteurs GABAergique et glutamatergique par les anesthésiques. Puis nous avons défini le temps d'exposition et la dose nécessaire pour induire des changements structurels mesurables et potentiellement persistants dans la connectivité neuronale. Comme les systèmes de signalisation GABAergique subissent des changements importants avec le développement, j'ai vérifié si les effets des anesthésiques sur le cerveau dépendent de l'état de maturation de la neurotransmission GABAergique.

J'ai abordé ces questions chez un modèle rongeur postnatal de dendritogénèse et synaptogenèse corticales. Pour évaluer les effets de l'exposition à l'anesthésie en fonction du temps sur la synaptogenèse, j’ai d'abord exposé des ratons Wistar âgés 16 jours à différents anesthésiques

volatiles et mesuré les changements sur la morphologie des neurones pyramidaux de la couche 5 du cortex. J'ai évalué certains paramètres importants tels que la structure des neurones dendritique et des épines dendritiques, qui forment la partie postsynaptique des synapses excitatrices. Dans cette première étude, les anesthésiques volatils n'ont pas induit d'apoptose ou d’altérations de la structure dendritique générale, mais la densité des épines était significativement augmentée en fonction de l’exposition et de l’anesthésique utilisé. Afin de mieux tester les effets des anesthésiques en fonction des stades du développement, j'ai réalisé des anesthésies au propofol à des temps distincts entre les jours postnataux (JPN) 5 et 30, correspondant à la phase de maturation morphologique des neurones pyramidaux de la couche 5 du cortex du rat. Ces paradigmes de traitement induit une diminution du nombre d’épines dendritiques au JPN 5 et 10, tandis que plus tard le propofol augmente considérablement le nombre d’épines, ce qui suggère que l'action des anesthésiques dépend de l'état de maturation des systèmes de neurotransmetteurs. Par ailleurs, j'ai pu démontrer que ces changements structurels persistent durant des mois. Pour vérifier notre hypothèse selon laquelle l'état de la neurotransmission GABAergique détermine l'action de l'anesthésie au propofol sur les réseaux neuronaux, nous avons effectué des électroporations in utero afin de surexprimer KCC2, un co-transporteur du chlorure responsable du changement du potentiel d’inversion de GABAA durant le développement neuronal, dans sa version entière ou tronquée. Étonnamment, la surexpression de KCC2 en soi augmente considérablement la spinogenèse indépendamment du co- transport de chlorure, par l'interaction avec l'actine du cytosquelette. Ces résultats peuvent expliquer la synchronisation temporale entre spinogenèse dendritiques et le changement du potentiel d’inversion du chlorure.

En conclusion, nous démontrons de façon convaincante la vulnérabilité des réseaux neuronaux à l'exposition aux anesthésiques durant les périodes critiques du développement du cerveau des rongeurs. Il reste à démontrer si, et dans quelle mesure, des effets similaires se produisent chez des espèces supérieures, et comment ces changements morphologiques modifient ils les performances neurocognitives à long terme.

Table of contents

Introduction 10

Changes in synaptic properties of neurons during development 13

Neuronal signaling in the brain 13

Inhibitory neurotransmitters in the mature brain 13 Neuronal network development and immature neuron synaptic properties 14 The GABA - glutamate sequence and generation of early network activity 15

Developmental changes in chloride homeostasis 16

Factors modulating KCC2 activity and the developmental switch 18

Premature expression and knock out of KCC2 19

KCC2 and excitatory synapse formation 19

Structural plasticity 21

Dendritic Spines 21

Synaptogenesis and synapse maturation 22

Spinogenesis 23

Spine homeostasis 24

The prefrontal cortex 26

Prefrontal cortex function 26

Perturbation of prefrontal connectivity 27

The vulnerability of immature brain to anesthesia exposure 28

Mechanisms of action of general anesthetics 28

Critical periods for anesthesia exposure 30

Control of systemic physiology 31

Effects on neuronal network formation 32

Debate about clinical relevance of animal data 33

Clinical studies 34

Line of research 36

Open questions 36

Objectives 37

Results 39

Research context 40

First article 41

Contributions, Main results & Appreciation

Second article 54

Contributions, Main results & Appreciation

Third article 67

Discussion 80

Legitimacy 81

General considerations 82

First article 83

Second article 85

Third article 87

General discussion 91

Future research 95

Single Cell Lucifer Yellow Injection Protocol 96

List of publications 99

Bibliography 100

Introduction

The main objective of my thesis was to study the impact of general anesthetics on neuronal network formation during the brain growth spurt with a special focus on dendritic arbor and spine development of layer 5 pyramidal neurons of the medial prefrontal cortex. In this introduction section, I will detail the scientific ratios behind this objective.

Since anesthetics have major impact on GABAergic and glutamatergic neurotransmission, I am going to overview the biology of GABA and glutamate signaling during central nervous system development. I believe that this chapter will help the reader to realize the important developmental differences in the functional modalities of these neurotransmitter systems between early and later developmental stages and thus to understand why one has to probe different developmental time points when evaluating the effects of anesthetics on circuitry development. As we will see, there is a sequential appearance of these two neurotransmitter systems during corticogenesis, changing from a GABA dominated early network activity to a precisely controlled balance between glutamatergic excitation and GABAergic inhibition, crucial for the establishment of proper neuronal connectivity of the developing brain. Receptors of these neurotransmitters undergo mayor changes in subunit composition, which alters their electric properties from slow and long currents to fast and short ones. Moreover the characteristics of GABAergic neurotransmission are dramatically modified by the upregulation of the chloride co-transporter KCC2 during the brain growth spurt period. The resulting alterations in chloride reversal potential changes the action of the GABAAreceptor from excitatory to inhibitory and thereby probably also the effect of those anesthetic drugs acting by this receptor type on the developing neuronal networks.

One major readout of my work was the analysis of dendritic spines. Hence, the second part of this introduction is devoted to the physiology of these tiny structures, representing the primary sites of excitatory synaptic inputs to principal neurons. These small protrusions of dendrites are believed to be one of the information storage site in the cortex. Potential changes in dendritic spines induced by anesthetic drugs would thereby also alter the computational properties of a given neuron and network. Interestingly, at a very early age before spinogenesis starts, excitatory synapses are formed directly on dendritic shafts. Later on, spines with specialized structural organization emerge. They contain a more complex synapse machinery and allow spatial and temporal information segregation, indispensable for the maturation of cortical networks. While spine turnover was shown to be high early in development the dynamic subsequently decreases, leading to a more stable network, potentially less vulnerable to external manipulations. We used classical histological techniques to

analyze dendritic spines post-hoc in anesthetized animals. In this way we could study spine density and other important morpho-functional parameters such as spine head diameter, which correlates with synapse strength and number of AMPA receptors; giving us a rather precise idea about the excitatory synaptic connections in the cortical network before and after anesthesia.

By evaluating the effects of general anesthetics on dendritic arbor and spine development, I focused on the prefrontal cortex. This cortical area has been extensively studied in the context of psychiatric disorders and is particularly vulnerable to the detrimental effects of stress. The rat medial prefrontal cortex, considered a homologue of the human prefrontal cortex, has an important function in working memory and higher order cognitive tasks. Chronic stress exposure in rodents leads to alterations of network inputs onto dendritic spines of principal neurons, what results in a long term impairment of accurate information representation in the working memory. For this reason the prefrontal cortex was especially suitable to study anesthetic drug induced network changes.

Finally, in the last part of this introduction, I summarize recent research on experimental and clinical toxicity. Paradoxically, concerns about the safety of currently used anesthetic drugs haven‘t been risen by clinical studies first, but by fundamental work in animals demonstrating increased neuronal cell death in the developing rodent brain. This increase in neuronal apoptosis has been reproduced in several other species including non human primates showing also impairment of long term neuro-cognitive outcome. The question whether these findings are relevant for daily pediatric anesthetic practice remains to be answered. Indeed, clinical studies especially designed to address this issue are contradictory. Longer and repeated anesthesia exposure during the first postnatal years seems to affect a child‘s long term cognitive performance while short exposure does not.

Additionally, clinical studies have the inherent limitation how to separate the effects of general anesthesia from other important concomitant parameters which are perturbed in neonates and children undergoing surgery. Furthermore, the exact period of vulnerability in humans and the dose threshold are not known, leading to a high variability in different study designs, which makes conclusions even more complicated. Recently the link between neuro-apoptosis and impaired cognitive performance in animals has also been challenged, proposing reduced synaptogenesis and neurogenesis as causal factors in this findings.

Changes in synaptic properties of neurons during development

In this chapter I will discuss the activity-dependent formation of neuronal networks in the central nervous system (CNS), and the role of cation-chloride co-transporters during this critical period of CNS development with a focus especially on inhibitory neurotransmission. For this topic see also two excellent recent reviews: (Ben-Ari et al., 2007) and (Blaesse et al., 2009)

Neuronal signaling in the brain

A well-controlled balance between excitation and inhibition maintains neuronal network activity in the mature brain (Liu, 2004). Glutamate is by far the most important excitatory neurotransmitter while gamma-aminobutyric acid (GABA) and glycine are the main inhibitory transmitters. GABA and glutamate act either through ionotropic receptors, which directly open ion channels or trough metabotropic receptors, which indirectly activate ion channels by a coupled G-protein pathway.

Ionotropic receptors for glutamate are named after their specific agonists NMDA, AMPA and kainate, and are always excitatory. There are 3 mGluR metabotropic G-protein coupled receptors which differ in their intracellular signaling mechanisms. AMPA and Kainate receptors generate the early component for the Na+ and K+ -induced fast depolarizing responses, while NMDA receptors mediate the slower and late postsynaptic depolarization (Cull-Candy et al., 2001). NMDA receptors are special glutamate receptors having additional high conductance for Ca²⁺. Their opening is voltage-dependent since it requires the removal of the Mg²⁺ block by previous depolarization (Dingledine et al., 1999). NMDA receptor activation is crucial for the induction of AMPA-mediated synaptic plasticity such as LTP and LTD (Whitlock et al., 2006; Rao and Finkbeiner, 2007).

Inhibitory neurotransmitters in the mature brain

GABA acts on ionotropic GABAA and on metabotropic GABAB receptors. The GABAA receptor is composed of five different subunits which form a ligand gated ion channel (Fritschy and Brunig, 2003). The channel is permeable for Cl^- and to a lesser extend to HCO3-; the direction of the induced postsynaptic current depends directly on the electrochemical gradient and the concentrations of these two ions. There are important changes in neocortical and hippocampal GABAA receptor subunit composition during brain maturation (Yu et al., 2006). This heterogeneity is a major facet of their regulation, since distinct GABAA receptor subtypes are localized at different subcellular sites (e.g. synaptic/extrasynaptic) and are integrated in specific neuronal circuits subserving distinct brain functions and pharmacological roles (Tyagarajan and Fritschy,

2010). In line with this, Okada et al. demonstrated that the α2-to-α1 subunit switch underlies the developmental speeding in the decay time of GABAergic IPSCs in the thalamus (Okada et al., 2000).

The intracellular chloride concentration is low in mature neurons, thus GABAA receptor activation generates membrane hyperpolarization and therefore a reduction in action potential firing at this developmental stage. Since reversal potential for chloride currents is close to the resting membrane potential, small changes in intracellular chloride concentration can dramatically affect the strength and even polarity of GABA/glycine-mediated transmission (De Koninck, 2007).

Neuronal network development and immature neurons synaptic properties

Things are different in the developing brain: already 20 years ago Ben-Ari and coworkers demonstrated developmental changes of GABAergic signaling in neonatal rat hippocampal slices using intracellular recordings (Ben-Ari et al., 1989). Their major findings were that GABA acting via GABAA receptors depolarizes and excites the immature neurons due to an elevated intracellular chloride concentration, which is reduced progressively during the second postnatal week. The neuronal network activity in the hippocampus during the early postnatal period is driven by synchronized GABAA-mediated giant depolarizing potentials (GDPs). Although the situation in the immature cortex is slightly different, Ben Ari suggests in his review a common feature for both structures: the transition from silent neurons to networks that generate a multitude of behaviorally relevant patterns is not direct (Ben-Ari et al., 2007). It includes a transition dominated by the presence of a primitive pattern sharing many common mechanisms with hippocampal GDPs. The depolarizing GABAA receptor responses increase intracellular Ca²⁺ load in different types of immature neurons. In immature hippocampus cultures, the Ca²⁺ elevation induced by GABA was greater than that of equimolar concentrations of the excitatory transmitter glutamate (Obrietan and van den Pol, 1995). The same research group showed that the intracellular Ca²⁺ increase is mainly due to the activation of voltage-gated calcium channels as it persists when Na⁺ channels are blocked and is suppressed by blockers of calcium channels such as nimodipine. Based on these results the above cited authors speculated that GABA may serve as an excitatory intercellular messenger involved in developmental signaling prior to the time when its primary function is to inhibit neuronal activity. The voltage-dependent calcium conductance is developmentally regulated: in organotypic slices application of extracellular potassium evokes robust increase in intracellular Ca²⁺

in P7 neurons but no response at P13 (Ganguly et al., 2001). In the same time, excitatory GABA

action reduces the voltage-dependent Mg²⁺ block of NMDA channels leading thereby to a synergistic action of GABA and glutamate in immature neurons (Leinekugel et al., 1997).

The GABA - glutamate sequence and generation of early network activity

During brain development, transmitter-gated postsynaptic receptors are expressed and operative well before synapse formation and the presence of presynaptic structures, suggesting that their action is not restricted to synaptic transmission (Represa and Ben-Ari, 2005). These receptors on immature neurons typically have slow kinetics, which will facilitate the generation of long-lasting currents and increase intracellular calcium concentrations (Owens and Kriegstein, 2002). GABA release is tonic at an early developmental stage and acts as a trophic factor to modulate several essential developmental processes such as neuronal migration (Heck et al., 2007), proliferation (Nguyen et al., 2003) and differentiation (Borodinsky et al., 2003). In developing neurons in culture GABA facilitates glutamate-mediated depolarization to fire action potentials under conditions where glutamate by itself can not evoke an action potential. This facilitation has a broad time window during the decaying phase of the GABA-mediated depolarization (Gao et al., 1998).

Depolarizing extra-synaptic (paracrine) and synaptic GABAergic signaling promotes not only action potential activity, but also the opening of voltage-gated Ca²⁺ channels and activation of NMDA receptors (Ben-Ari et al., 2007). Elevated intracellular Ca²⁺ levels would then activate

Fig. 1

Figure 1 adapted from (Ben-Ari et al., 2007) shows some of the most important steps on the timeline for rodent hippocampal development.

GABA synapses are formed before glutamate synapses. Giant

depolarizing potentials (GDP) are characteristic for the immature pattern during the most intense phase of dendritic growth and spinogenesis in the first postnatal week.

intracellular signaling cascades, which exert the trophic effects of GABA during development.

Since synchronous neuronal activity enhances the release of trophic factors, it is not surprising that BDNF has been proposed to be implicated in these trophic actions of excitatory GABA. Actually, GABAergic stimulation switches from enhancing to repressing BDNF expression in rat hippocampal neurons during maturation in vitro (Berninger et al., 1995).

GABAergic activity precedes glutamatergic (AMPA receptor-mediated) synaptic transmission during development and early glutamatergic synapses are predominantly based on postsynaptic NMDA receptors (Ben-Ari et al., 2007). Interestingly, there is a delay in the expression of GABA transporters in the early postnatal period, leading to higher GABA concentration and wider diffusion compared to glutamate and to a dominance of local spontaneous GABAA receptor mediated transmission over glutamate-mediated synaptic activity during the first postnatal week in rodents (Represa and Ben-Ari, 2005). This seems to be a general intrinsic programmatic feature of neuron development as the GABA-glutamate sequence is respected both in embryonic and in adult born neurons (Overstreet-Wadiche et al., 2006). Even in the adult brain GABA first depolarizes newly generated neurons in the dentate gyrus and this action seems to be crucial for the synaptic integration into the existing neuronal network (Ge et al., 2006). On this newly generated neurons in the adult brain anesthetics may play a pathogenic role even after the closure of the so called critical periods during development (Jiang et al., 2005).

Neurons with small dendrites tend to be silent, more developed neurons often have GABA but not glutamate PSCs and neurons with more complex dendritic arborisation have both GABA and glutamate PSCs. The important role of early GABAergic innervation is also stressed by the fact that in the postnatal period GABA-releasing synapses comprise close to 50% of the total number of synapses in the cortex, a number which declines to 15% in the adult brain (De Felipe et al., 1997).

The GABA - glutamate sequence first takes place in interneurons followed by pyramidal neurons in the cortex. These observations have important clinical implications considering the role of neuronal development in mental disorders and the effects of widely used transmitter-acting drugs during pregnancy (Ben-Ari et al., 2007).

Developmental changes in chloride homeostasis

Among different chloride co-transporters the potassium-chloride co-transporter KCC2 plays a crucial role during the maturation of GABAergic neurotransmission. KCC2 is part of a transporter family which has nine members in mammals (Blaesse et al., 2009). Cation chloride co-transporters

are essential for controlling intracellular chloride homeostasis; NCCs and NKCCs are involved in Cl^- uptake whereas KCCs are responsible for Cl^- extrusion out of neurons. They are electrically silent, do not directly act on the neuronal membrane potential and were first described for their role in volume regulation (Weisman et al., 1989). KCC2 is a glycoprotein with a molecular weight of 140 kDa and a plasmalemmal ion transporter. Structural analyses suggest that all these transporters have a similar transmembrane topology consisting of relatively large intracellular N and C termini and a central hydrophobic domain containing 12 membrane-spanning segments (Gerelsaikhan et al., 2006). Other potassium chloride co-transporters (KCC1, KCC3 and KCC4) are expressed in various cell types (Payne et al., 2003), the expression of KCC2 however is exclusively limited to CNS neurons (Williams et al., 1999). The mammalian KCC2 gene (alias Slc12a5) generates two neuron- specific isoforms by using alternative promoters and first exons (Uvarov et al., 2007). Both isoforms (termed KCC2a and KCC2b) are present at similar levels in the neonatal mouse. While the expression level of KCC2a remains relatively constant during postnatal development in rodents, the expression of KCC2b is strongly up-regulated during the first two postnatal weeks, especially in the cortex. This means that the KCC2b isoform is responsible for the „developmental shift“ from depolarizing to hyper-polarizing GABAergic responses in the same period (first description of the molecular mechanisms related to this shift by (Rivera et al., 1999)). See also Figure 2 for a more detailed description.

As stated above, GABA has a depolarizing action on immature neurons, mainly due to an outward chloride gradient generated by the early expression of NKCC1, a sodium chloride co-transporter which provides the driving force for Cl^- efflux through GABAA receptors (Marty et al., 2002).

Nevertheless, depolarizing action does not automatically mean excitation since there can also be a shunting inhibition by a decrease in the temporal and spatial summation of excitatory inputs. The opening of GABAA receptors could have a shunting inhibitory action even without a voltage inhibition by a value of EGABAA more negative than resting Vm (Riekki et al., 2008). To summarize, we can state that functional inhibition is largely determined by the efficacy of Cl^- extrusion, which has to be high enough to maintain a level of EGABAA sufficiently negative (but not necessarily hyper-polarizing) to prevent the cell from firing (Blaesse et al., 2009).

In more mature neurons however, the up-regulation of KCC2b produces a negative shift in GABA reversal potential and reduces GABA-elicited calcium responses. GABAergic stimulation induces then mainly IPSC due to a Cl^- equilibrium potential more negative than the membrane potential.

This shift falls into the early postnatal period in rodents, while in humans the KCC2 upregulation starts much earlier during gestation as in other species with precocious neonates (Vanhatalo et al., 2005).

Development

Epileptic activity trauma, axiotomy Fig. 2

Figure 2 illustrates the changes in chloride reversal potential induced by chloride co-transporters NKCC1 and KCC2. Early in development, EGABA is higher than threshold and resting membrane potential leading to an excitatory action of GABAA agonists on immature neurons and an increase in intracellular Ca through removal of Mg block from NMDA receptors, adapted from (Fiumelli and Woodin, 2007)

Factors modulating KCC2 activity and the developmental switch

The functioning of the chloride pump depends not only on phosphorylation (Wake et al., 2007), but also on the maintenance of a strong K+ gradient by the plasmalemmal Na-K ATPases. Precise chloride transport capacity of KCC2 can be measured by dirty ⁸⁶Rb+ assays (Hartmann et al., 2010) but most people prefer indirect electrophysiological techniques. Currently, the gramicidin- perforated patch clamp technique is often used to measure the presence of a functional chloride co- transport because it has the advantage of not altering intracellular chloride concentrations (Kyrozis and Reichling, 1995). Even very low amounts of KCC2 could be sufficient to maintain a hyper- polarizing EGABAA provided that the net influx into a neuron (i.e., the cellular Cl^- load) is small (Blaesse et al., 2009). Consequently agents which rapidly and constantly increase Cl^- influx (such as anesthetic drugs) could completely perturb EGABA in the aforementioned situation and thereby

affect the activity in immature neuronal networks. One of the aim of the present thesis is to investigate this hypothesis.

Ganguly and colleagues showed that chronic blockade of GABAA receptors with bicuculline methoiodide (BMI) prevented the upregulation of KCC2 expression and delayed the GABA shift in hippocampal cultures (Ganguly et al., 2001). In the same time, blocking Na+ spikes with tetrodotoxin affects neither the developmental shift nor the up-regulation of KCC2 (Ludwig et al., 2003). Together these observations suggest that the shift is triggered by miniature GABAA

postsynaptic potentials generated without action potentials and GABA could play a self-regulatory role. Additionally, high-frequency stimulations (Ouardouz and Sastry, 2005) and BDNF acting via tyrosine kinase receptors were also able to increase the level of KCC2 co-transporters (Aguado et al., 2003; Ludwig et al., 2011). Whether anesthetic drugs by their strong GABAergic action induce a premature developmental shift of GABAA responses is an intriguing possibility and remains to be determined.

Premature expression and knock out of KCC2

Complete developmental deletion for KCC2 leads to death immediately after birth due to disturbance in respiratory rhythmogenesis (Hubner et al., 2001). On the other hand, in mice which lack only the KCC2b isoform there is no negative shift of GABAA responses from depolarizing to hyper-polarizing during neuronal maturation and these mice survive about two to three weeks postnatally before dying of recurrent seizures (Zhu et al., 2005). Cancedda and coworkers showed that over-expression of KCC2 does not affect the radial migration of neuronal progenitors to layer 2-3 of the somatosensory cortex but their morphological maturation would be markedly impaired (Cancedda et al., 2007). As stated by Blaesse et al in their review in 2009, KCC2 has obviously other still unknown roles in CNS development and function (Blaesse et al., 2009). One of the aim of the present thesis is to investigate this issues and the role of anesthetic drugs in this context.

KCC2 and excitatory synapses formation

KCC2 is expressed in the plasma membrane of somata and dendrites in different brain regions, e.g.

cerebellum (Williams et al., 1999) and the cortex (Szabadics et al., 2006); the density of KCC2 in hippocampal principal cells increases along the axo-somato-dendritic axis with cell type-specific distribution profiles within the dendritic tree. These distinct subcellular expression patterns may result in steady-state chloride gradients and compartmentalization of EGABAA within an individual neuron (Baldi et al., 2010). In cortical neurons these gradients can reach a value of up to 15 mV

(Khirug et al., 2008). Gulyas and coworkers found a high expression of this co-transporter in the vicinity of excitatory synapses in the rat hippocampus, which is somehow unexpected since GABAergic synapses are mainly located on dendritic shafts (Gulyas et al., 2001). This observation was confirmed by Rivera and coworkers, who demonstrated in vitro that KCC2, independently of its Cl^- transport function, is a key factor in the maturation of dendritic spines. The morphogenic role of KCC2 in the development of excitatory synapses would be mediated by structural interactions between KCC2 and the spine cytoskeleton (Li et al., 2007). In line, Wang et al. showed that GABA depolarization cooperates with NMDA receptor activation to regulate excitatory synapse formation (Wang and Kriegstein, 2008).

Structural plasticity

Neuronal networks are defined by the structure of axons and dendrites and the synapses that connect them (Holtmaat and Svoboda, 2009).While the formation of synapses starts already around birth in the rodent cortex (De Felipe et al., 1997), the most intense phase of synaptogenesis (at least for excitatory glutamatergic synapses) is during the time of rapid spinogenesis between the end of the first until the third postnatal week. These steps in neuronal network formation are under the control of synchronized spontaneous neuronal activity (Stellwagen and Shatz, 2002), while the refinement of the connections by spine pruning needs appropriate sensory input (Zuo et al., 2005b).

Dendritic Spines

Dendritic spines are tiny protrusions of dendritic shafts on pyramidal cells in the cortex which appeared to be the main postsynaptic target for asymmetric excitatory glutamatergic synapses (Harris et al., 1992). Excitatory synapses are characterized by a thickened PSD on the spine head adjacent to a widened cleft with dense staining material and a presynaptic axonal varicosity containing round clear vesicles. Synapses on spines represent only a small percentage of the total synapse number early in life; shaft synapses and filopodia dominate during this period, whereas the proportion of spine synapses dramatically increases later on (Fiala et al., 1998). As excitatory synapses are also formed on dendritic shafts and by aspiny neurons, the spines postsynaptic biochemical compartment may be responsible for a specific function that is particular for pyramidal cells (Arellano et al., 2007). Yuste and Denk demonstrated that spines separate synaptic inputs from each other by compartmentalizing calcium responses and therefore also restrict local biochemical reactions (Yuste and Denk, 1995). Later on, the same group showed that even the spine neck plays an electrical role in the transmission of membrane potentials, as voltage pulses propagating to the spine or from the spine are attenuated in proportion to the length of the spine neck, isolating synapses electrically (Araya et al., 2006).

For many year, spines have been classified in categories as thin, stubby and mushroom shaped, with the filopodia put apart (Ethell and Pasquale, 2005). A practice that has recently been abandoned by several authors because of the difficulty to find convincing selection criteria. Nevertheless, newly formed spines tend to be thin and long, sometimes without a clearly distinguishable head while older spines are often of mushroom shape. Arellano and coworkers stated that the most striking feature of the morphologies of spines is the continuum of their variability in shape and size

(Arellano et al., 2007). No clear subgrouping of spines can be detected in the distributions of morphological variables. However, this variables allow us to gain important functional information since the head volume has been shown to be directly proportional to the size of postsynaptic density (Harris et al., 1992) and to the number of a- amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (Matsuzaki et al., 2001), and therefore to the strength of the synapse. Figure 3 summarizes the relation between morphology and functional parameters.

Synaptogenesis and synapse maturation

The generation of new synapses is a tightly-controlled multistep process. It starts by the arrangement of pre- and postsynaptic membranes next to each other and their stabilization through adhesion molecules within minutes (Takai et al., 2003). Adhesion molecule interaction leads also to actin cytoskeleton rearrangements and the assembly of pre- and postsynaptic zones (Biederer et al., 2002). Scaffolding proteins are delivered and inserted in the pre- and postsynaptic membrane (PSD 95 as one of the first in the postsynaptic side), followed by the recruitment of glutamatergic receptors (Hall and Ghosh, 2008). Early synapses often have a very low amount of AMPA receptors and are essentially composed of NMDA receptors with a NR1 and NR2B subunits composition.

These synapses are called silent and are not detectable at resting membrane potential, as NMDA receptors activity needs the removal of voltage-dependent magnesium block by prior AMPA receptor activation. With the maturation of the synapses, more AMPA receptors are inserted in the postsynaptic compartment and the subunit composition in NMDA receptors changes, leading to a decrease in the duration of NMDA currents and in the number of silent synapses (Bellone and Nicoll, 2007). Additionally, the probability of transmitter release decreases with synapse maturation (Lauri et al., 2006) but there is an increase in pooled vesicles at the presynaptic side (Mozhayeva et al., 2002).

Nature Reviews | Neuroscience LTP

AMPAR NMDAR

Spine volume Spine brightness (integrated flurescence)

Spine volume

PSD size, AMPAR content LTP

LTD Spine

head

Spine head LTD

PSD Synaptic vesicles

PSD size Number of docked synaptic vesicles

Optical point spread function

The point spread function (PSF) describes the response of an imaging system to a point object. In microscopy the PSF is a measure of the resolution.

Optophysiological recording Optical microscopy-based imaging of cellular function, such as calcium imaging.

function^98,99. Spine motility decreases with develop- mental age in vitro^94,100 and in vivo, and is regulated by sensory experience^56,78,81. Changes in spine motility might be triggered by synaptic activity and precede more prominent morphological changes, such as spine retraction or stabilization^56,78.

Synapse stability. An emerging consensus is that a subpopulation of dendritic spines and axonal boutons is remarkably stable, with lifetimes on the order of the lifespan of the mouse. Even more remarkable is that the relative sizes of individual dendritic spines and bou- tons can be maintained for months ^{(FIG. 1)}, suggesting that synaptic weights could also be stable for months

(BOX 2). By contrast, in vitro studies indicate that synaptic protein complexes are highly labile, with protein life- times of a day or two, orders of magnitude shorter than synapse lifetimes¹⁰¹. In addition, synaptic molecules (such as Ras, PSD-95, Shank3, bassoon and synapto- physin) continuously redistribute between synapses on the same neurite over minutes to hours44,102,103104. How can stable synapses exist in the context of high pro- tein turnover either through redistribution or through unstable protein components? Answers to this ques- tion might come from experiments that track the fates of synaptic proteins in vivo. For example, PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organ- izes the associated signalling complexes¹⁰⁵. PSD-95 is thought to determine the size and strength of synapses.

Using two-photon photo-activation of PSD-95 tagged with photo-activatable GFP (paGFP), the trafficking of PSD-95 molecules in and out of single PSDs was measured in vivo⁴⁴. Synaptic PSD-95 in single PSDs in vivo turned over remarkably quickly (with a half- life of approximately one hour) and exchanged with PSD-95 in neighbouring spines by diffusion ^{(FIG. 1g)}. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. In other words, the kinetic interactions between PSD-95 molecules and individual PSDs are tuned to regulate and maintain synapse size.

Spine growth and synapse formation

High-resolution optical microscopy alone typically cannot detect synapse formation and elimination. Contact of dendrite and axon is a poor predictor of synapses, as sev- eral non-synaptic contacts occur per actual synapse^24,26. Furthermore, as a volume corresponding to the ^optical point spread function often contains multiple synapses, optical overlap of fluorescent presynaptic and postsynap- tic molecules does not provide proof of a synapse. Instead, detection of synapses requires retrospective analysis using EM^13,106, array tomography¹⁰⁷, direct imaging of synaptic proteins in vivo (FIG. 1e,f), or perhaps optophysiological recordings with single synapse sensitivity^28,108–110.

To investigate the relationship between spine growth and synapse formation, in vivo time-lapse imaging experiments have been followed up by serial section EM analysis of previously imaged structures13,14,23,54. Both, new dendritic spines and new axonal terminaux boutons were found to bear synapses ^{(FIG. 2)}. An analy- sis of the time-course of spine maturation in the adult somatosensory cortex revealed that synaptogenesis could be remarkably slow: spines that were older than four days always had a synapse, however only 30% of spines that were two days old formed a synapse (^{FIG. 2 b,c} and ^{FIG. 3}). The other ~70% of the newly formed spines probably corresponds to the small steady-state popula- tion of spines lacking synapses^11,12. Consistent with the in vivo studies, an EM study of cultured hippocampal brain slices showed ultrastructural hallmarks of synapses only 15–19 hours after the stimulus³⁶; another study in Box 2 | Correlates of synaptic strength

High-resolution in vivo imaging can be used to track the appearance and disappearance of synaptic structures. Can imaging also be used to measure the strengthening or weakening of existing synapses? There are indications that measurements of spine volume could provide an excellent indication of synaptic strength.

Spine volume is proportional to the area of the postsynaptic density (PSD)⁶, which in turn is proportional to the synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor (AMPAR) content^168–170 and to the postsynaptic sensitivity to glutamate¹¹⁰. These quantities also co-vary with presynaptic parameters that indicate the efficiency of glutamate release, including the size of the active zone and the number of docked vesicles¹⁷¹. Spine volumes increase after long-term potentiation (LTP)^172–175, as does the postsynaptic sensitivity to glutamate uncaging^172,174. Similarly, long-term depression (LTD) causes spine head shrinkage¹⁷⁶. Spine volumes can be estimated from in vivo images by measuring spine brightness, the fluorescent signal integrated over a dendritic spine^22,177,178.Changes in spine volume tracked in vivo^23,179 can thus be interpreted as changes in synaptic strength (see figure).

Synaptic strength might also be tracked more directly with recombinant fluorescent synaptic proteins. For example, the AMPAR subunit GluR1 tagged with pH-sensitive green fluorescent protein (GFP) has been used to report the synaptic GluR1 content in the membrane of single spines^173,180. Postsynaptic density protein 95 (PSD-95) tagged with GFP can be used to measure the size of the PSD^44,104. Furthermore, synaptic transmission is associated with Ca²⁺ influx through glutamate receptors and voltage-gated Ca²⁺ channels. Thus, genetically encoded Ca²⁺ indicators targeted to synaptic compartments^163,181 can directly report synaptic transmission in single synapses^108,109.

In summary, imaging experiments could be used to directly read out the weights of single synapses in vivo.

NMDAR, N-methyl- -aspartate receptor.

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NATURE REVIEWS | NEUROSCIENCE VOLUME 10 | SEPTEMBER 2009 |⁶⁵¹

Figure 3 taken from (Holtmaat and Svoboda, 2009). Spine head diameter and brightness can be measured by in vivo imaging.

This parameters correlate with PSD size, AMPAR content and with the number of docked synaptic vesicles, which allows to follow LTP and LTD in vivo.

Fig. 3

Spinogenesis

Different models were proposed for the generation of spines during development and in the mature brain and these models may not be mutually exclusive (Ethell and Pasquale, 2005):

The Miller-Peter model of spine growth suggests that spines would arise from shaft synapses. This model was supported by findings that both excitatory and inhibitory synapses are mostly located on dendrite shafts rather than on filopodia early during development and that there is a direct extension of spines or spine precursors carrying preformed PSDs from the shaft in developing hippocampal slice cultures (Marrs et al., 2001). The model, however, was challenged by Knott and coworkers who showed that spine growth precedes synapse formation in the adult neocortex in vivo and new spines often make synapses on boutons that also contain synapses with other spines (multi-synapse boutons) (Knott et al., 2006).

The Sotelo Model postulates the direct formation of spines based on the fact that in the Reeler mutant mice Purkinje cells develop morphologically normal spines despite of a massive reduction of presynaptic terminals (Sotelo, 1990). In organotypic hippocampal slices spines that are only a few hours old rarely form synapses, whereas older spines, ranging from 15 to 19h, consistently have ultrastructural hallmarks typical for synapses (Nagerl et al., 2007). This reinforces the idea that spinogenesis is not directly induced by the presynaptic partner but rather through a intrinsic, cell autonomous mechanism of the neuron.

A third possibility would be the dendritic filopodia to spine transition. In this case filopodia, these long, thin, headless and highly motile structures are considered as spine precursors. Dendritic filopodia have a relative short lifetime in the range of minutes to hours (Dailey and Smith, 1996).

Filopodia would thereby explore the surrounding extracellular space for potential presynaptic targets probably guided by the release of glutamate or other molecules. Ziv and Smith reported that dendritic filopodia in hippocampal cell cultures may actively initiate synaptogenic contacts with nearby axons and thereafter evolved into dendritic spines (Ziv and Smith, 1996). Similar result were reported in vivo (Zuo et al., 2005a),(Trachtenberg et al., 2002). However, these conclusions were made without a specific markers for a distinction between filopodia and spines. Further evidence for this model was brought by the discovery of molecules which accelerate or slow down the transformation from filopodia to dendritic spines. Telencephalin (TLCN) deficiency for example

causes the acceleration of spine maturation with decreased number of dendritic filopodia in developing hippocampal neurons while over-expression of TLCN induces a dramatic increase in the density of dendritic filopodia and a concomitant decrease in the density of spines (Matsuno et al., 2006). Kayser and colleagues further strengthened evidence that the filopodia to spine sequence is particularly important during development, since reduction of filopodia motility without changes in spine motility results in reduced synaptogenesis early in development but not later on.

Spine homeostasis

Dendritic spines contribute to the development and remodeling of synaptic networks through their constant formation and elimination (Yang et al., 2009). Their turnover rate is higher during development and is cell type- and brain region-dependent. Spine formation is a highly inefficient process as irrespective of the developmental stage, most new protrusions are quickly eliminated, in particular filopodia (De Roo et al., 2008a). While thin spines show less stability, newly generated thick spines tend to persist for months. The fraction of persistent spines increases from 35% at PND 16-25 to 73% at PND 175-225 (Holtmaat et al., 2005). Neuronal activity and sensory deprivation can dramatically influence dendritic spines turnover (Oray et al., 2004). Theta burst stimulation

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spontaneous activity in spinogenesis would be to block all activity during uterine development. This has recently been achieved in munc18-knockout mice⁵¹.Munc18 is necessary for transmitter release throughout the CNS and peripheral nervous system (PNS),but surprisingly, mice that lack the protein are reported to have relatively normal neocortical synapses and circuits at birth.

Unfortunately,these mice die shortly after birth,before spinogenesis has taken place.Interestingly,there is mas- sive apoptosis in many regions ofthe nervous system (although not in the cortex), implying that neurotrans- mitter release might be necessary for neuronal survival throughout the CNS. Further analysis ofthese mice — for instance,by culturing tissue from newborn mice — or other transmitter-release-deficient mice that survive into the period of spinogenesis is likely to be pertinent to the understanding ofthe role of activity in spine formation.

Neuronal activity and pyramidal spine maintenance.

The role of activity in the maintenance of connections has been investigated extensively¹⁴, and we will only touch on this issue as it relates to spinogenesis. As we have already stated, spine density (and synapse density) seems to follow a stereotypical developmental pathway, with initial overproduction followed by a reduction to a plateau level that persists through adulthood14,15,39,42. There is ample evidence that, after the overproduction stage, spine density can be affected by sensory depriva- tion or by experimental paradigms that modify synaptic activity40,67,75–78. Spine density can increase as well as decline, implying that spinogenesis can also occur at later stages in development.

Another interesting insight has come from recent molecular studies, which indicate that the Rho family of small GTPases is an important potential contributor to Neuronal activity and pyramidal spine development.

What is the influence ofneuronal activity on the develop- ment of spines from pyramidal neurons? Do pyramidal neuron spines behave like parallel fibre spines or like climbing fibre spines from Purkinje cells? We will first focus on the role of sensory activity, and we will then discuss a potential role for spontaneous activity.

Comparative developmental studies have provided some interesting insights into the role of sensory activity.

Some species, such as rats and mice, are born with rela- tively immature brains (altricial), whereas others are born with more developed brains (precocious).In the guinea-pig, spinogenesis has already occurred by birth, and these animals are born with an essentially mature complement of spines and synapses⁷¹. This simple fact has an important implication — that in some species at least, spinogenesis and even synaptogenesis occur in the absence of environmental influences¹¹. Even in rats and mice, a large proportion of spinogenesis and synapto- genesis in the primary visual cortex occurs before eye opening⁵², and the only morphological event that seems to correlate with eye opening is the elongation ofthe spine neck⁵⁵.Moreover,between different individuals of the same species,there is a less than one-day offset in the overall time course of spinogenesis and synaptogenesis¹⁴. These observations indicate that many aspects of this programme are genetically determined, and that sensory evoked activity is not essential for ontogenetic spinogenesis in pyramidal neurons.

However,there is increasing evidence that the devel- oping brain in uterohas robust patterns of spontaneous activity, which are potentially important for circuit rearrangements^72–74. So, it remains possible that sponta- neous activity is necessary for normal spinogenesis in pyramidal neurons.An ideal experiment to test the role of

a Sotelo model

b Miller/Peters model

c Filopodial model

Figure 3 |Three models for spinogenesis. This diagram illustrates the essential features of the three models of spinogenesis. In the Sotelo model (a), spines emerge independently of the axonal terminal. In the Miller/Peters model (b), the terminal actually induces the formation of the spine. Finally, in the filopodial model (c), a dendritic filopodium captures an axonal terminal and becomes a spine.

The 3 spinogenesis models illustrated by (Yuste and Bonhoeffer, 2004), The Sotelo Modell with direct spine growth, the Miller Model - shaft to spine synapse and the filopodia to spine transition model.

Fig. 4

induced LTP leads to new spine formation that peaks at around 2h (Nagerl et al., 2004) and these newly formed spines can have functional synapses. De Roo and colleagues used learning-related patterns of activity known to induce long-term potentiation to demonstrate selective stabilization of activated spines (De Roo et al., 2008b). They also showed that these mechanisms are NMDA receptor and protein synthesis dependent. Even specific learning paradigms selectively alter spine homeostasis by a transient increase in spine turn over, which is repeated if another new task is learned (Xu et al., 2009; Yang et al., 2009). In summary, neuronal activity leads generally to higher spine turnover, increase of PSD expression on newly formed spines and increase in stability for spines with a functional synapse.

Spine homeostasis is affected in several mental illnesses. In a mouse model for fragile X syndrome for example, knock out for the X-linked fragile X mental retardation gene resulted in a heightened spine turnover due to a larger pool of short-lived spines (Pan et al., 2010), (Penzes et al., 2011).