Thesis
Reference
The role of cation-chloride cotransporter KCC2 in the development of pyramidal neurons in the cerebral cortex
BODOGAN, Timea
Abstract
Le développement du cerveux est un processus complexes, qui est très réglementé dans tous ses aspects. Pour la fonction, un équilibre précis en excitation et inhibition entre les circuits neuronaux est nécessaire. Le potassium-chlorure cotransporteur KCC2 spécifique au neurone a un rôle important au cours de la maturation de la neurotransmission inhibitrice GABAergique en contrôlant l'homéostasie du Cl-. Surtout, KCC2 est détecté à partir des premiers stades du développement du cerveau, et il y a plus en plus des preuves des études récentes indiquent la possibilité que KCC2 a d'autres fonctions au cours du développement, outre son caractéristique de transporteur. L'interaction récemment révélé avec le cytosquelette a ouvert la ligne des rapports décrivant son implication dans les processus non liés au transport de Cl- ou l'inhibition GABAergique. En accord avec ces observations, l'objectif principal de ma thèse était d'étudier le rôle de KCC2 dans le développement de neurones pyramidaux corticaux.
BODOGAN, Timea. The role of cation-chloride cotransporter KCC2 in the development of pyramidal neurons in the cerebral cortex. Thèse de doctorat : Univ. Genève et
Lausanne, 2015, no. Neur. 136
URN : urn:nbn:ch:unige-806631
DOI : 10.13097/archive-ouverte/unige:80663
Available at:
http://archive-ouverte.unige.ch/unige:80663
Disclaimer: layout of this document may differ from the published version.
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DOCTORAT EN NEUROSCIENCES des Universités de Genève
et de Lausanne
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Laszlo Vutskits, Directeur de thèse
THE ROLE OF CATION CHLORIDE COTRANSPORTER KCC2 IN THE DEVELOPMENT OF PYRAMIDAL NEURONS IN THE CEREBRAL CORTEX
THESE Présentée à la Faculté des Sciences de l’Université de Genève
pour obtenir le grade de Docteure en Neurosciences
par
Timea BODOGAN
de Hongrie
Thèse N° 136 Genève
Editeur ou imprimeur : Université de Genève 2015
1
2
A CKNOWLEDGEMENTS
I would like to thank all the people who supported and helped me during my thesis work.
First of all, I wish to thank Prof. Laszlo Vutskits, my thesis supervisor, for giving me the possibility to come to Switzerland and start my doctoral project in his laboratory and for his supervision and help during my PhD work.
Special thanks to Adrian Briner for the initial help and guidance in the beginning, for everything he taught me and for all the fun we had during working and building up the lab together.
Furthermore I want to thank:
• all the present and former members of our lab (the Vutskits lab) for all their help, especially Mari Virtanen for the great and inspiring discussions
• Beatrice King, Cathrine Fouda and Michèle Brunet for their technical assistance
• the members of the Kiss lab for sharing their knowledge, lab space and materials
• Hubert Fiumelli for his collaboration in preparing the plasmids and teaching me the electroporation
• Moritz Jacobshagen and Prof. Alexandre Dayer for their contribution in the time-lapse imaging
• the members of the Dayer and Jabaudon lab for the discussions and friendships
• Michiko Kanemitsu and Sahana Murthy for being there and encouraging me
• the members of the jury: Prof. Anthony Holtmaat, Prof. Jozsef Zoltan Kiss and Prof. Nicolas Toni
• my friends and family, especially Attila Heiczinger for all his kind support and endless patience during the difficult periods.
Without all these people, this four and half year in Geneva would not have been the same.
THANK YOU!
3
T ABLE OF CONTENTS
Acknowledgements ... 2
Table of contents ... 3
Summary ... 5
Résumé ... 6
List of Abbreviations ... 8
List of Figures ... 13
List of Tables ... 14
Introduction ... 15
1. Cortical development ... 16
1.1 Cell proliferation and Neurogenesis
... 16
1.2 Differentiation and Cell fate specification
... 18
1.3 Migration
... 22
1.4 Maturation
... 26
1. Axonal growth and Integration
... 26
2. Dendritic development and Spinogenesis ...
27
3. Synaptogenesis and Network formation ...
29
2. Development of the GABAergic system ... 32
2.1 The transition between excitatory vs. inhibitory neurotransmission
... 32
2.1 The role of cation-chloride cotransporter family
... 34
3. The K
+-Cl
-cotransporter 2 ... 36
4
3.1 The KCC2 molecule
... 36
3.2 KCC2 functions during development
... 40
Aim of the thesis ... 46
Materials and Methods ... 47
Results ... 58
The downregulation of KCC2 does not disturb normal cell proliferation and survival
... 59
KCC2 is involved in the early differentiation of pyramidal cortical precursor cells
. 61
The perturbed KCC2 expression leads to impaired cell distributions in the embryonic brain... 63
Radially migrating precursors containing shKCC2 have normal morphology
... 65
The low KCC2 level results disturbed migration of pyramidal precursors
... 67
shKCC2 electroporated pyramidal cells are able to reach layer II/III despite their delayed migration
... 69
The lack of KCC2 perturbs dendritic morphology of pyramidal neurons
... 72
The electroporation of shKCC2 affects spine distribution and spine head morphology of layer II/III pyramidal neurons
... 77
Discussion ... 89
References ... 89
5
S UMMARY
The development of vertebrate nervous system is a complex, multistep process, which is highly regulated in its every aspect. For normal brain functioning, a precise balance in excitation and inhibition between neural circuits is required. The neuron-specific potassium- chloride cotransporter KCC2 has an important role during the maturation of inhibitory GABAergic neurotransmission by controlling Cl- homeostasis. Importantly, KCC2 is detected from early stages of brain development, and growing body of evidences from recent studies point to the possibility, that KCC2 has other functions during development besides its transporter characteristic. The recently revealed interaction with the cytoskeleton opened the line of reports describing its involvement in processes unrelated to Cl- transport or GABAergic inhibition. In line with these recent observations, the primary goal of my thesis was to investigate the role of KCC2 in the development of cortical pyramidal neurons.
To this aim, I took advantage of in utero electroporation to express an shKCC2 vector in precursors of layer II/III pyramidal neurons in the rat somatosensory cortex. Using this technique, I examined the possible role of KCC2 in the proliferation, migration and differentiation of these principal cells. I found no difference in cell proliferation and early apoptosis between control and KCC2-downregulated progenitors. However, examination of early differentiation markers showed a decreased Sox2 (neural progenitor marker) and Tbr2 (intermediate precursor marker) expression in the ventricular-subventricular zone of shKCC2 electroporated animals compared to controls, hypothesizing an early, premature differentiation of neuronal precursors. Furthermore the downregulation of KCC2 led to perturbed intracortical distribution pattern of precursor cells in the embryonic cortex.
Morphological analysis of the migrating cells did not explain the disturbance in their distribution, but time-lapse imaging of cortical slices revealed an abnormal migration speed for the shKCC2 electroporated cells. Nevertheless these neuronal precursors were able to reach layer II/III, although at postnatal day 15 they demonstrated impaired dendritic arborisation, spine morphology and density compared to controls.
In summary, these results indicate that KCC2 is required for the normal development of pyramidal neurons elucidating yet unknown functions and suggesting further studies to extend our knowledge about this transporter molecule.
6
R ÉSUMÉ
Le développement du système nerveux des vertébrés est un processus en plusieurs étapes complexes, qui est très réglementé dans tous ses aspects. Pour la fonction normale du cerveau, un équilibre précis en excitation et inhibition entre les circuits neuronaux est nécessaire. Le potassium-chlorure cotransporteur KCC2 spécifique au neurone a un rôle important au cours de la maturation de la neurotransmission inhibitrice GABAergique en contrôlant l'homéostasie du Cl-. Surtout, KCC2 est détecté à partir des premiers stades du développement du cerveau, et il y a plus en plus des preuves des études récentes indiquent la possibilité que KCC2 a d'autres fonctions au cours du développement, outre son caractéristique de transporteur. L'interaction récemment révélé avec le cytosquelette a ouvert la ligne des rapports décrivant son implication dans les processus non liés au transport de Cl- ou l'inhibition GABAergique. En accord avec ces observations récentes, l'objectif principal de ma thèse était d'étudier le rôle de KCC2 dans le développement de neurones pyramidaux corticaux.
Dans ce but, j’ai profité d’électroporation in utero à exprimer une shKCC2 dans les précurseurs de neurones pyramidaux de la couche II/III du cortex somatosensoriel de rat.
Grâce à ce technique, j’ai examiné le rôle possible de KCC2 dans la prolifération, la migration et la différenciation de ces cellules principales. J’ai trouvé aucune différence dans la prolifération cellulaire et l'apoptose entre contrôle et shKCC2 contenant progéniteurs.
Cependant, l'examen de marqueurs de différenciation ont montré une diminution de Sox2 (marqueur neuronale progénitrice) et Tbr2 (marqueur de précurseur intermédiaire) expression dans la zone du ventriculaire-subventriculaire de shKCC2 électroporées animaux par rapport aux contrôles, formulant l'hypothèses d’une différenciation précoce de précurseurs neuronaux.
En outre, la downrégulation de KCC2 a conduit à une distribution intracorticale perturbé de cellules précurseurs dans le cortex embryonnaire. L'analyse morphologique de ces cellules migrantes n'a pas expliqué la perturbation dans leur distribution, mais imagerie time-lapse de tranches corticales a révélé une vitesse de migration altérée des cellules contenant shKCC2.
Néanmoins, ces précurseurs neuronaux ont pu arriver á la couche II/III, bien que au jour postnatal 15 ils ont démontré anormal arborisation dendritique, puis morphologie et densité des épines par rapport aux contrôles.
7
En résumé, ces résultats indiquent que KCC2 est nécessaire pour le développement normal de neurones pyramidaux élucidant fonctions encore inconnues et en suggérant d'autres études d'étendre nos connaissances sur cette molécule transporteur.
8
L IST OF A BBREVIATIONS
4.1N: neuronal cytoskeleton-associated scaffolding protein 4.1 5-HT6R: serotonin receptor 6
AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid Astn1: astroactin 1
BDNF: brain-derived neurotrophic factor bFGF / FGF2: basic fibroblast growth factor
bHLH: basic helix-loop-helix DNA-binding domain BMP: bone morphogenetic protein
BrdU: 5-bromo-2'-deoxyuridine BSA: bovine serum albumin
[Ca2+]i: intracellular calcium ion concentration casp3: caspase-3
CC: corpus callosum
CAMK: Ca2+/calmodulin dependent protein kinase CCC: cation-chloride cotransproter
Cdc42: cell division control protein 42 Cdk5: cyclin-dependent kinase 5
[Cl-]i: intracellular chloride ion concentration CNS: central nervous system
CP: cortical plate
Cux1, 2: cut-like homeobox transcription factor 1 and 2 DIV: day in vitro
Dlx: distal-less homeobox gene family
9
E: embryonic day
ECl: chloride reversal potential EGABA: GABA reversal potential EGF: epidermal growth factor
EGFP: enhanced green fluorescent protein EGFR: epidermal growth factor receptor Em: resting membrane potential
Emx: empty spiracle homeobox gene family EphB: ephrin receptor B
Erg4: early growth response 4 FAK: focal adhesion kinase
FERM domain: protein 4.1–ezrin–radixin–moesin homology domain
FLN1: filamin 1
GABA: γ-aminobutyric acid
GABAAR: GABA activated receptor type A GFAP: glial fibrillary acidic protein GFP: green fluorescent protein GluR1: glutamate receptor subunit 1
Hp: hippocampus
IGF: insulin-like growth factor i.m.: intramuscular injection i.p.: intraperitoneal injection IPC: intermediate progenitor cell IPSP: inhibitory postsynaptic potential IUE: in utero electroporation
IZ: intermediate zone
ko: knockout
10 KCC: potassium-chloride cotranspoter
KCC2-CTD: potassium-chloride cotranspoter 2 C-terminal domain KCC2-C568A: KCC2, cystein substituted to alanine at point 568 KCC2-ΔNTD: KCC2 N-terminal deleted form
KCC2-FL: full length KCC2
LGE: lateral ganglionic eminence Lis1: lissencephalin 1
LY: Lucifer Yellow
LV: lateral ventricle (ventriculus lateralis) Mash1: mammalian achaete-scute homologue 1 MAP: microtubule associated protein
MGE: medial ganglionic eminence MZ: marginal zone
NBM: neurobasal medium
NCAM: neural cell adhesion molecule NCC: sodium-chloride cotransporter
NKCC: sodium-potassium-chloride cotransporter NeuN: neuronal nuclear marker
NGF: nerve growth factor Ngn1, 2: neurogenin 1 and 2
NKCC: sodium-potassium-chloride cotransproter NL2: neuroligin 2
NMDA: N-methyl-D-aspartate
NMDAR: NMDA receptor
NPC: neural progenitor cell NSC: neural stem cell NT: neurotrophin
11
P: postnatal day
PAR: partition-defective protein Pax6: paired box 6 transcription factor PBS: phosphate buffered saline
PBST: phosphate buffered saline with tween PKC: protein kinase C
PNS: peripherial nervous system
PP: preplate
PSA-NCAM: polysialylated neural cell adhesion molecule PSD: postsynaptic density
Rac1: Ras-related C3 botulinum toxin substrate 1 RC2: radial glial cell marker 2
RG: radial glia
Rho: Ras homolog protein family Robo1: Roundabout receptor 1
Satb2: special AT-rich sequence-binding protein 2 sema3: semaphorin III
SLC12a5: solute carrier family 12, member 5
Sox2: SRY (sex determining region Y)-box 2 transcription factor
SP: subplate
SSC: sometosensory cortex
SynCAM: synaptic cell adhesion molecule SVZ: subventricular zone
Tbr1, 2: T-box brain 1 and 2 transcription factors TF: transcription factor
TM: transmembrane domain
TrkB: Tropomyosin related kinase B, a tyrosine kinase receptor
12 TUJ1: class III β-tubulin
VDCC: voltage-dependent calcium channel VZ: ventricular zone
WNK: with no lysine kinase
13
L IST OF F IGURES
Figure 1. Major steps of pyramidal neuron development: ... 15
Figure 2. The three phases of stem cell development and lineages in vivo and in vitro. ... 17
Figure 3. Schematic summary of corticogenesis. ... 21
Figure 4. Expression patterns of different cell type-specific markers during cortical development in rodents. ... 25
Figure 5. GABAergic modalities in developing and mature neurons. ... 35
Figure 6. The KCC2 molecule and its expression pattern. ... 37
Figure 7. The role of KCC2 in dendritic spine development. ... 43
Figure 8. Methods. ... 53
Figure 9. The effectiveness of shKCC2 plasmid. ... 58
Figure 10. The downregulation of KCC2 does not disturb cell proliferation and survival. .... 60
Figure 11. Changes in VZ/SVZ progenitor numbers after downregulation of KCC2. ... 62
Figure 12. The distribution of electroporated cells in E19 and E21 coronal sections. ... 64
Figure 13. Normal radial glia scaffold and morphology of migrating precursor at E21. ... 66
Figure 14. The downregulation of KCC2 perturbs the radial migration of pyramidal precursors. ... 68
Figure 15. The distribution of electroporated cells in postnatal coronal section. ... 71
Figure 16. The lack of KCC2 results impaired dendritic morphology of layer II/III pyramidal neurons. ... 73
Figure 17. Scholl analysis of apical and basal dendrites. ... 76
Figure 18. The downregulation of KCC2 leads to changes in the morphology and distribution of dendritic spines of layer II/III pyramidal neurons at P15. ... 78
Figure 19. Schematic illustration for the hypothetic role of KCC2 in the regulation and organization of actin. ... 87
14
L IST OF T ABLES
Table 1. List of primary antibodies used for immunohistochemistry of cryostat sections. ... 52
Table 2. List of secondary antibodies used for immunohistochemistry of cryostat sections. . 52
Table 3. MATLAB scripts to analyze cell distribution data. ... 56
Table 4. E19 distribution results per bin. *: statistically significant difference ... 63
Table 5. E21cell distribution results per bin. *: statistically significant difference ... 65
Table 6. P1 cell distribution results per bin. ... 69
Table 7. P3 cell distribution results per bin ... 70
Table 8. P15 cell distribution results per bin. *: statistically significant difference ... 70
Table 9. Results of the Scholl analysis of apical dendrites at P15. N: number of cells, *: statistically significant difference ... 74
Table 10. Results of the Scholl analysis of basal dendrites at P15. N: number of cells, *: statistically significant difference ... 75
Table 11. Number of spines with different spine head diameters. *: statistically significant difference ... 77
15
I NTRODUCTION
During vertebrate evolution, the central nervous system (CNS) became encephalised and formed a highly organized, complex structure: the brain. The most dynamic structural and functional development occurred in the telencephalon. Anatomically it can be divided to grey matter, containing cell bodies, and the white matter, which is made from axonal fibers (Cajal, 1899). The superficial part of grey matter reached its most developed level, the neocortex (or isocortex), in mammals (Northcutt and Kaas, 1995).
The mammalian cortex covers the whole surface of the telencephalon and is separated from deeper anatomical structures by white matter. It is a complex, integrative system that receives most of its afferents from the thalamus and other cortical areas. The cerebral cortex is formed by six specific layers, consisting of different neuronal populations and their connections, as well as supporting glial cell types. The generation of the layers in different brain regions occurs in regulated spatio-temporal sequences.
In the next chapters I would like to give a general overview on the major steps of corticogenesis (Figure 1), focusing on pyramidal neuron development; as well as introducing the potassium-chloride cotransporter 2 (KCC2) molecule and its contribution in development, system maturation and GABAergic transmission. The literature discussed here, is primarily from studies on rodents: mouse (Mus musculus) and rat (Rattus norvegicus).
Figure 1. Major steps of pyramidal neuron development:
cell proliferation, differentiation, migration and maturation.
CP: cortical plate, IPC: intermediate progenitor cell, IZ: intermediate zone, SVZ: subventricular
zone, VZ: ventricular zone.
16
1. Cortical development
1.1 Cell proliferation and Neurogenesis
In mammals, the neural plate (or neural ectoderm) is formed by neural induction of the ectoderm (Spemann and Mangold, 1924), that gives rise to all CNS components (Chang and Hemmati-Brivanlou, 1998). During the process of neurulation, the neural plate folds and closes to form the neural tube (Saxen, 1982). In the beginning, the wall of the neural tube consists of a single columnar layer of proliferative cells, the pseudostratified neuroepithelium.
The cells of the neuroepithelium are self-renewing, multipotent cells: neural stem cells (NSCs) (Davis and Temple, 1994; McKay, 1997), with the capacity to generate different cell types of the nervous system (Gage, 2000).
The embryonic development of the CNS has three cell proliferative phases (Figure 2):
expansion, neurogenesis and gliogenesis (Götz and Huttner, 2005; Temple, 2001).
Cortical neurogenesis takes place in the walls of the lateral ventricles (LV): in the ventricular zone (VZ), where columnar NSCs touch both the pial and ventricular surfaces in the beginning of development. At the time of mitosis NSCs undergo nuclear translocation (the nucleus moves to the ventricular surface) and morphological changes (from bipolar to round shape) (Sidman and Rakic, 1973; Takahashi et al., 1993). During the expansion, NSCs are dividing symmetrically to increase the number of proliferative cells and the size of the germinative layer. In addition, in the basal region of VZ, some of the progenitor cells divide asymmetrically to generate early born neurons: Cajal-Retzius cells (Haubensak et al., 2004).
These early neurons form a plexiform layer (Figure 3), the preplate (PP) by somal translocation to the pial surface. As VZ and the cortex wall thicken, neuroepithelial cells transform to elongated radial glial (RG) cells (Alvarez-Buylla et al., 2001; Misson et al., 1988a). RGs extend long (even 200-300µm) and thin fibers keeping the bipolar orientation and contact with the basal and apical surfaces (ventricle and pia, respectively) (Figure 1, 3, 4) (Kriegstein and Alvarez-Buylla, 2009; Noctor et al., 2002).
17 In the next phase RGs start to divide asymmetrically. During the cell-cycle they also show nuclear translocation, dividing at the ventricular surface like neuroepithelial NSCs (Misson et al., 1988b). One of the daughter cells remains RG with the inherited bipolar shape (Levitt et al., 1981), the other one becomes neural precursor (Noctor et al., 2001), which will differentiate into neuron (Kilpatrick et al., 1995; Temple and Qian, 1996). As development proceeds, intermediate progenitor cells (IPCs or transit amplifying progenitors) derived from RGs populate the region dorsally to VZ, to enlarge the neuronal progenitor pool by creating a second proliferative zone, the subventricular zone (SVZ), which persists until adulthood (Alvarez-Buylla and Temple, 1998). IPCs are loosely arranged multipolar cells without any connections to apical or basal membranes and usually divide symmetrically to produce restricted neural progenitor cells (NPC) or neuroblasts (Haubensak et al., 2004; Temple, 2001). NPCs are the source of different neuronal subtype expansion. They divide either asymmetrically to generate a single neuron and self-renew as IPC, or symmetrically to produce two new progenitors or in terminal division two neurons as daughter cells (Figure 1- 4) (Noctor et al., 2004).
Figure 2. The three phases of stem cell development and lineages in vivo and in vitro.
First, stem cells expand by symmetric division. Second, during the asymmetric phase they generate neurons (N), than glial cells (third phase). Each phase occurs in strict temporal order. Modified from (Temple, 2001).
18 Before the third phase, there is a genetical switch point, when important genes start to be expressed and others are downregulated by the effect of intrinsic factors and extracellular signals (Sauvageot, 2002; Shen et al., 1998). At the end of these complex changes, around birth, neurogenesis switches to gliogenesis, meaning that RGs procreate IPCs restricted to produce glial progenitor cells (glioblasts) instead of NPCs (Figure 2, 3) (Levitt et al., 1981;
Parnavelas, 1999). The most intense proliferative phase is from embryonic day 11 (E11) in rat (Hockfield and McKay, 1985) and E8 in mouse (Figure 2). Neurogenesis peaks at E14 in rat, and by the time of birth, the vast majority of neurons are already born. The first astrocytes appear around E18 followed by oligodendrocytes generated postnatally (Bayer et al., 1991).
The proliferation of different cell types is temporally regulated: neurons born first, then glia (Qian et al., 2000). This temporal segregation makes sense, since the arriving neuronal subtypes establish local circuits first, than glial cells are incorporated into the network to support them (Miller and Gauthier, 2007). The birth of different neuron subtypes occurs in sequential order (Barbe and Levitt, 1991; Okano and Temple, 2009). Lower-layer neurons are born first, and then later born neuronal precursors are colonizing the upper cortical layers (Berry and Rogers, 1965). This fashion is called the inside-out generation of the laminar cortex.
1.2 Differentiation and Cell fate specification
The distribution of NSCs is region specific, since stem cells from certain regions generate cells, specific to the given region. The fate of the cells depends on their dorso-ventral and antero-posterial localization in the VZ, as well as on their sensitivity to extracellular matrix components (Hitoshi et al., 2002b). The gene expression patterns of different regions are restricted to determine progenitor identity (Schuurmans and Guillemot, 2002), their biological properties and capacity to generate different cell types. The main specialization of the cerebral cortex is that the dorsal telencephalon gives rise to glutamatergic pyramidal cells and different glial cells (Ayala et al., 2007); whereas most of the GABAergic (γ-aminobutyric acid) interneurons and oligodendrocytes originate from the basal forebrain (He et al., 2001), mainly from the medial and lateral ganglionic eminence (MGE and LGE, respectively). These neuronal precursors express, amongst others, the homeobox distal-less (Dlx) and Nkx gene
19 families, which contribute in their dorsal migration into the cortex and maturation into GABA releasing interneurons (Letinic et al., 2002); while dorsal progenitors express members of the empty spiracle (Emx) gene family (Anderson et al., 1997). Nevertheless, regional identity is also established by inductive signals.
NSCs and different progenitor cell types have different molecular characteristics (Hockfield and McKay, 1985; Temple, 1990). Cells in the VZ show high expression of adhesion molecules, such as NCAM (neural cell adhesion molecule) (Rutishauser and Jessell, 1988) and N-cadherin (Kadowaki et al., 2007; Tanriover et al., 2004). The polysialylated form of NCAM (PSA-NCAM), generally expressed during embryonic development and in restricted regions participating in adult neurogenesis, is important for the survival of proliferating cells (Vutskits et al., 2006). NSCs also express the intermediate filament nestin (Lendahl et al., 1990). When neuroepithelial cells turn into RGs, they begin to express glial markers (Doetsch, 2003), such as the glial fibrillary acidic protein (GFAP) (Woodhams et al., 1981) and the intermediate filament vimentin. RGs also start to express a specific marker: RC2 (radial glial cell marker 2) (Misson et al., 1988a). Notch signaling seems to promote transformation of neuroepithelial cells into RGs (Gaiano et al., 2000). Its important function also is to repress proneural genes, inhibiting neurogenesis, thus to increase and maintain the NSC pool (Hitoshi et al., 2002a; Lewis, 1998; Mizutani et al., 2007). Nestin is still present in RGs (Kawaguchi et al., 2001), but its expression drops down after a cell exit from the cell- cycle (Zimmerman et al., 1994).
Environmental factors are necessary to maintain the proliferative state and stimulate division, but they can also promote differentiation. The basic fibroblast growth factor (bFGF or FGF2) has mitogen function during early proliferation. With time, it has a concentration- dependent influence on different progenitor genesis. Low level FGF2 facilitates neurogenesis, while increasing FGF2 concentration together with high epidermal growth factor receptor (EGFR) and bone morphogenetic protein (BMP) level pushes into glial fate (Mehler et al., 2000; Qian et al., 1997). Others, for example brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3), also exhibit neuronal fate choice (Temple and Qian, 1995). BMP alone triggers differentiation and migration of neuroblasts (Li et al., 1998), but its effect can be downregulated by noggin (Li and LoTurco, 2000). Molecules released by early neurons have a feed-back on the progenitor pool. Some of these, e. g. gliogenic cytokines can facilitate the neuron-glia progenitor transition by directly starting glial protein transcription (Miller and Gauthier, 2007).
20 Essential factors are required to make the cells dividing asymmetrically instead of symmetrically, but lineage commitment is also genetically regulated. Intrinsic signals, like epigenetic regulators and the expression of specific transcription factors (TF, DNA-binding proteins) are also required to preserve the self-renewing potential. There are many genes and TFs described to be necessary to maintain consecutive mitosis. Presenilins (Kim and Shen, 2008) and members of the Sox family are important to keep cells in the cell-cycle through different signaling pathways. The paired box 6 (Pax6) homeodomain TF, expressed exclusively in VZ and SVZ, is required for proper cortical RG development (Götz et al., 1998). It regulates proliferation rates and adhesiveness of RGs from early stages (Warren et al., 1999). Proneural TFs are involved in neuronal subtype specification and their transient expression promotes cell-cycle exit and starts neuronal differentiation (Guillemot, 2007a).
The neurogenic basic helix-loop-helix (bHLH) TFs, like neurogenin 1, 2 (Ngn1, Ngn2) and mammalian achaete-scute homologue 1 (Mash1) are initiating neurogenesis and inhibit gliogenesis in the same time (Guillemot, 2007b; Parras et al., 2002). Cux1/Cux2 and Tbr1/Tbr2 are TFs specific to upper-layer neuronal precursors, already expressed in a subset of subventricular NPCs (Franco et al., 2012). Tbr2 is a specific marker of IPCs and required for their symmetric divisions (Englund et al., 2005). The decreasing level of neurogenic bHLHs (e.g. Ngn) permits the transcription of gliogenic genes (Bertrand et al., 2002). Notch inhibition promotes neurogenesis; contrary, the activation of Notch signaling leads to astrogliogenesis (Gaiano and Fishell, 2002; Mizutani et al., 2007).
The gene expression profile of a progenitor regulates the intrinsic developmental program, which can change over time. The responsiveness of progenitor cells to certain cues can differ depending on their age. An example is the epidermal growth factor (EGF), which has no effect on cell proliferation during the expansion (Tropepe et al., 1999), but induces differentiation early during neurogenesis. Then, during the formation of SVZ, its effect switches to proliferation due to high EGF concentration and expression level of its receptor (EGFR), which increases with age (Kilpatrick et al., 1995; Qian et al., 2000). The change in EGFR level is important in the maturation of proliferating zones. By modulating responses to the environment, it has an impact not only on cell characteristics and phenotype, but also on cell differentiation and migration (Burrows et al., 1997; Lillien and Raphael, 2000). It has been also shown, that the unequal distribution of EGFR during mitosis leads to asymmetric division. The high EGFR containing daughter cell remains RG, while the other one becomes neural precursor (Sun et al., 2005) because of different downstream signaling pathways and functionality. With age, the length of the cell-cycle increases and it is also related to the
21 switch between symmetric and asymmetric divisions (Martens et al., 2000). All these facts together indicate that fate determination of a given progenitor can be modified by extrinsic signals, but the precise timing and coordination depends on its genetical program.
Gliogenesis, in general, repressed during neurogenesis by intrinsic mechanisms. In contrast, the switch to gliogenesis involves transcriptional repression of proneural genes and direct inhibition of proneural proteins. But the ability to respond to signals changes over time, creating windows for plasticity. Such epigenetic regulations, causing irreversible alterations in cell signaling and gene expression, are fundamental for lineage commitment. By which, after certain time point, regain of previous cell fate capacity is not possible in vivo. Late cortical progenitor cells are unable to react to early cues, since they are restricted to late progenitor fates (Desai and McConnell, 2000; Shen et al., 2006).
Figure 3. Schematic summary of corticogenesis.
First, neuroepithelial cells in the ventricular zone (VZ) divide symmetrically (red arrow) to increase the progenitor pool. They also generate early born neurons by asymmetric divisions (blue arrow), which colonize the mantle region or marginal zone (MZ). With time neuroepithelial cells turn into radial glias, which produce neurons and intermediate progenitor cells engaged to neuronal fate (nIPCs). nIPCs are located in the subventricular zone (SVZ), and they either self-renew or generate neurons by symmetric divisions. Immature neurons migrate radially through the intermediate zone (IZ), and differentiate into pyramidal cells after reaching the cortical plate (CP). Perinatally, radial glias switch to produce glia restricted gIPCs. The generation of different glial cell types occurs mostly after birth. NE: neuroepithelium. Modified from (Kriegstein and Alvarez- Buylla, 2009).
22
1.3 Migration
During early development, neuroblasts use somal translocation to change their location (Miyata et al., 2001; Nadarajah, 2003). Once the terminal division is finished, they lose their ventricular attachments and move the soma with the aid of the pial process (Figure 4). The pial process becomes continuously shorter as the soma advances to the surface with a relatively constant speed (Nadarajah et al., 2001). In contrast, later born postmitotic neuronal precursors are migrating radially up to the surface through RG guidance (Figure 1, 3, 4) (Hatten, 1990; Rakic, 1971). After the exit from the cell cycle, to leave the VZ/SVZ, cells need filamin 1 (FLN1), an actin binding protein (Gleeson and Walsh, 2000). FLN1 connects the actin cytoskeleton to membrane proteins which are important in neuron-glia interactions, e.g. integrins (Anton et al., 1999). Connexins and focal adhesion kinases (FAK) also help the attachment to the glia process (Matsuuchi and Naus, 2013; Valiente et al., 2011).
Neuronal precursors have to pass the intermediate zone (IZ) following their glia scaffolds, which provide a physical link between VZ and the pial surface. Some of the radially migrating cells have a pause at the border of the SVZ-IZ. They detach from the RG and become multipolar. Their processes show high motility and dynamic extensions and retractions, as if they explore the local microenvironment (Noctor et al., 2004). Some of these precursors translocate tangentially in the IZ (Tabata and Nakajima, 2003). Thereafter, they follow their migratory rout up to the surface. The reason of this transient stop is not completely clear yet. The most possible explanation is that these cells are searching for direction cues.
The IZ consists of pioneer axons from already settled neurons (Hatten, 1999) and innervating axons from other brain regions. These corticofugal axons are used for tangential migration from ventral areas across the RG system (O'Rourke et al., 1995). Tangentially migrating cells, coming from the basal forebrain, mostly differentiate into inhibitory interneurons, while radially migrating precursors are mainly excitatory projection neurons, pyramidal cells, from the dorsal VZ/SVZ (Tan et al., 1998). After arriving into the developing neocortex, interneurons seem to search for guidance cues or information about their laminar positions, often extending multipolar processes to the VZ/SVZ, similar to the abovementioned phenomena of pausing pyramidal precursors (Nadarajah et al., 2003). Then, to reach their appropriate place in the CP, interneurons also use radial migration (Kriegstein and Noctor,
23 2004). Interestingly, interneurons and projection neurons which are born at the same time, populate the same cortical layers (Ayala et al., 2007; Marin and Rubenstein, 2003).
Cyclin-dependent kinase 5 (Cdk5) and connexins are required for the multipolar-to-bipolar transition of postmitotic neurons in the SVZ (Liu et al., 2012; Ohshima et al., 2007). N- cadherin mediates their orientation along the RG guides in a reelin-dependent manner (Jossin and Cooper, 2011). After leaving the SVZ, cells have elongated bipolar shape. This polarity is established by the movement of the centrosome and its microtubular arrangement into the leading process, while the nucleus is anchored by cortical actin and a microtubular “cage”;
plus there is a thin trailing process at the posterior end (Solecki et al., 2006). The synergistic organization of the cytoskeleton (both actin and microtubule) is very important for proper locomotion. Independently of its proneural function, Ngn2 is also involved in the initiation of polarity and outgrowth of the leading process (Hand et al., 2005).
To commence displacement, BDNF and neurotrophin 4 (NF4) are important modulators, along with the expression of their receptor, the tyrosine kinase receptor TrkB (tropomyosin related kinase B) (Solecki, 2012). Neurons advance with discontinuous, salutatory movements (Tabata and Nakajima, 2001). At the level of the cell body, adherent junctions are formed to keep the migrating cell stable while it extends the leading process and projects filopodias along the glia fiber. The adherent junctions contain special neuron-glia adhesion molecules:
astroactin 1 (Astn1), neuregulin and integrins (Hatten, 2002; Marin and Rubenstein, 2003).
The leading process follows the trace of radial fibers (Campbell and Götz, 2002; Rakic, 1971) maintained by exploratory extensions of filopodias. Extensions and retractions are established by rapid microfilament, mainly actin reorganizations to search for attractive cues, while the length of the leading process remains relatively stable (Nadarajah et al., 2001). The extended leading process creates focal adhesions with the glial fiber. Nuclear translocation is initiated by the centrosome with the coordination of dynein. The soma and nucleus move forward with a sudden, burst-like “jump” with the help of the perinuclear tubulin cage (Solecki et al., 2004). The adherent junctions are released to form new ones at the new level of the cell body.
Finally, the trailing process follows the movement in a slower motion, keeping the constant bipolar shape of the cell. As migration goes further, the trailing process progressively elongates and becomes the axon (Lewis et al., 2013).
With one cycle, cells are able to move ~1,5-2 µm. This short forward movement is followed by a longer stationary period (Nadarajah et al., 2003). Neuroblasts require microtubule- associated proteins (MAP), such as lissencephalin (Lis1) and doublecortin (DCX) for normal
24 migration (Gleeson and Walsh, 2000). DCX is important in microtubule stabilization, particularly in the phases of elongation and nuclear translocation when dynamic microtubule adaptation is essential (Francis et al., 1999; Gleeson et al., 1999). RGs do not retract their radial processes, maintaining the contact with the pial surface through the whole mitosis, thus supporting ongoing migration (Miyata et al., 2001). However, there are evidences for branching RGs at the superficial level (Misson et al., 1988a).
Multiple cells, usually descendants of the same RG, can migrate along the same fiber (Noctor et al., 2001). Hence, RGs form proliferative units which are linked to the correspondent cortical areas. Therefore, the structural distribution of VZ ancestors partially defines the cytoarchitecture and topographic map of the developing cortex through ontogenetic columns (Rakic, 1988). This protomap is than modified by tangentially moving neurons and dispersing interneurons. However cells are able to translocate from one fiber to another, most of them remain contacted to their original glia fascicle (formed by closely positioned RG fibers) after leaving the SVZ-IZ boundary. Gradient of chemoattractants secreted by CP neurons, like GABA and taurine, stimulate the migration and define the orientation of precursors (Behar et al., 2001).
Close to the surface, the arriving cells split the PP into marginal zone (MZ) and subplate (SP). The following cohorts are colonizing the space between SP and MZ, forming the cortical plate (CP) (Okano and Temple, 2009). As mentioned above, deep-layer neurons are born earlier, creating first layer VI and V. The subsequent waves of neurons are passing them to establish layer IV, than layer III and II (Desai and McConnell, 2000). Thereby, the cellular structure and laminar positioning tightly correlates with the birth date of different neuron populations.
The migration terminates in the presence of different stop signals. One important molecule in the extracellular matrix is reelin. It is first secreted by Cajal-Retzius cells and acts as guiding cue for migrating precursors. Reelin is necessary in numerous steps for the proper laminar organization of the cortex, starting from the cell transition from multipolar to bipolar and the orientation of the leading process (Förster, 2014). Reelin directly ends the migration of immature neurons at the MZ. By interacting with integrins, it enables neuron detachment from the glia scaffold (Dulabon et al., 2000). In the lack of reelin, cells are not able to penetrate the already settled neurons and accumulate under the PP in an inverted order (Gleeson and Walsh, 2000). It also leads to polarization deficit of migrating neurons, according to a most recent review (Förster, 2014). Neuron positioning is also controlled by
25 Cdk5 signaling, which partially overlaps the reelin pathway. Mice deficient in Cdk5 or in its activating subunits, p35 or p39, also develop inverted cortical lamination (Kanatani et al., 2005). Innervating axon terminals arrest neuron migration, too.
The speed of radial migration is estimated to 10-30 µm/h (Hicks and D'Amato, 1968;
O'Rourke et al., 1992), but it depends on the species, cell type and age. The arrival time of neurons relies on the distance they migrate and the migratory strategy they use. Neurons migrating dorsally reach their final destination within 2 days, whereas those, migrating laterally and/or ventrally need 3-4 days in rats (Bayer et al., 1991).
Figure 4. Expression patterns of different cell type-specific markers during cortical development in rodents.
Dividing progenitors in the ventricular/subventricular zones have different molecular characteristics than migrating or mature neurons. Radial glias (RG) express nestin, Sox2 and Pax6 among many other factors. In the subventricular zone Tbr2 regulates intermediate progenitor cell (IPC) divisions instead of Sox2. Migrating precursors loose progenitor properties; in contrast they show markers for neuronal maturation such as NeuN.
Fully mature neurons settled in the cortical plate show TUJ1 expression, additionally to NeuN. In the middle, one cell (red) is in the process of somal translocation. Modified from (Lui et al., 2011).
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1.4 Maturation
After arriving to their final positions, cells go through different steps of maturation:
development of cell-type specific cytoarchitecture, neurochemical maturation, dendritic arborization and synapse formation. At the end of these complex processes, neurons are integrated into the network. The beginning of this period is before birth and it persists up to adolescence (Levitt, 2003).
1. Axonal growth and Integration
Postmitotic cells start to differentiate into mature neurons after and during their migration.
Precursors migrating along the glia fiber already express class III β-tubulin (TUJ1) and NeuN (neuronal nuclear protein), markers for mature neurons (Figure 4) (Noctor et al., 2001).
Neurons have highly polarized morphology that is similar to the polarity established during migration (Barnes and Polleux, 2009). They extend axonal projection in response to guidance cues of different target areas already during their radial migration (Rutishauser and Jessell, 1988). The partition-defective proteins (PARs) are necessary for establishing polarity of migratory precursors. Thus for example, PAR3/6 forms a polarity complex with other proteins, which is an important regulator during migration (Solecki et al., 2004). These molecules are also involved in the initiation of axon from the trailing process (Lewis et al., 2013). Axon growth progress rapidly while the neuron migrates through the IZ. Different chemoattractors and repellents guide the growing axons toward the final targets. One of these molecules is semaphorin III (sema3), which is present in the extracellular matrix in a gradient starting from the MZ. Sema3 orients the growing axon towards the LV (Polleux et al., 1998).
The growth cone is especially sensitive to these cues, since it is the major navigator in axonal path finding. DCX is highly expressed at axonal growth cones to mediate the microtubular organization in emerging filopodias and lamellipodias (Francis et al., 1999). Actin is involved in their rapid turnover in order to explore for attractive signals. Additionally, stabilization by actin allows for microtubules to accomplish elongation and branching (Lewis et al., 2013).
Neurons reaching the MZ detach from their glia scaffolds and go through terminal translocation to settle in their final position. They often extend multiple branches to the MZ,
27 which help the movement of the soma (Nadarajah and Parnavelas, 2002). The surrounding extracellular matrix elements support this transition, too.
Soon after birth, RGs disappear from the mammalian brain. Some transform into astrocyte- like, GFAP-expressing type B cells in the SVZ (Merkle and Alvarez-Buylla, 2006), and they remain as a source for adult neurogenesis (Doetsch et al., 1999; Tramontin et al., 2003). SP neurons and their transient connections also disappear perinatally (Ferrer et al., 1992).
2. Dendritic development and Spinogenesis
There are different neuron subtypes in the mammalian neocortex, but the dominant cell type is pyramidal neuron (~80% of all cortical neurons). To develop their identical morphology, primary neurites emerge from the soma of the immature pyramidal cells. The apical dendrite occurs first, followed by basal dendrites. The position of the centrosome together with polarity proteins and the Golgi apparatus coordinates the outgrowth of the dendrites and the axon (Lewis and Polleux, 2012; Solecki et al., 2006). N-cadherin has an important role in neurite induction, direction and elongation, as well as in axon fasciculation (Tanriover et al., 2004). The phosphorylation of Ngn2 triggers the initiation of the apical dendrite, as it does for the leading process outgrowth in migrating neurons (Hand et al., 2005). Astn1 and Cdk5 are also required for normal dendritic development (Ohshima et al., 2007). Interestingly, sema3 has an opposite effect than on axonal development. It acts as chemoattractant for apical dendrite orientation (Polleux et al., 2000). The apical dendrite extends towards layer I, where it ends in tuft branches. Often it crosses several layers, but later the apical dendrite can be retracted, depending on layer specifications. Basal dendrites, in contrast, are shorter and usually form dense arborization in the same layer as the cell body.
Dendrites are essential in signal processing and transduction toward the soma. Their complex structure is established by multiple branches. The development of the arborization is regulated by neurotrophins, like nerve growth factor (NGF), insulin-like growth factors (IGFs), BDNF, NT3 and NT4 (Niblock et al., 2000). These factors can stimulate dendritic branching and elongation. They activate signaling pathways through Trk receptors, which can promote modification in actin cytoskeleton dynamics (McAllister et al., 1995). Therefore, NTs have crucial role not only in branching, but in dendritic remodeling, too. Notch signaling
28 is a key element of branch initialization. Its overexpression dramatically increases the number of branching points and the total dendritic length (Whitford et al., 2002). Microtubules and their regulators (e.g. MAPs) are responsible for elongation and the stabilization of newly formed branches. As development proceeds, the dendritic structure becomes more and more complex. Around adolescence branching terminates (Briner et al., 2011), but elongation continues until adulthood (Petit et al., 1988). Although the pyramidal morphology is relatively conserved, the proportion and arrangement of the dendrites might be different in each cortical areas and even within layers of the same area (Benavides-Piccione et al., 2006).
For completing mature morphology, pyramidal neurons develop spines. Spines are tiny protrusions from the dendrites and are the major sites of excitatory synapses. They form specialized compartments for synaptic transmission and to isolate inputs (Yuste and Denk, 1995). In rats, the first spines appear after birth, around postnatal day 4 (P4) (DeFelipe et al., 1997). There are different evidences and hypothesized models for spinogenesis (Ethell and Pasquale, 2005; Yuste and Bonhoeffer, 2004). It has been described that spines can autonomously emerge from the dendritic shaft without previous connections, but spines generated from shaft synapses are also observed. A transition from filopodia to spine is another possible way of spinogenesis. Filopodias are long, thin protrusions with high motility and short lifetime, often considered as immature or precursor spines (Dailey and Smith, 1996;
Fiala et al., 1998). They are able to search for and initiate synaptic contacts with surrounding axons (Bhatt et al., 2009). The base of their dynamic motility is intensive actin remodeling (Bonhoeffer and Yuste, 2002).
Mature spines vary in size and shape. The literature describes stubby, mushroom and thin spines based on their morphology (Harris et al., 1992). This classification is widely used, although intermediate and atypical forms also exist. The morphology has a huge impact on spine functionality. The length of the spine neck is proportional to the degree of electrical and biochemical filtering and isolation by propagating voltage pulses (Araya et al., 2006); while the volume of the head corresponds to the number of presynaptic vesicles, the size of the postsynaptic density (PSD) and the receptive field, thereby determining the strength of the synapses (Arellano et al., 2007; Harris et al., 1992). Small changes in spine dimensions can influence electric properties and synaptic transmission, which are very important for local signal integration. The family of small GTPases, including RhoA, RhoB, Rac1 and Cdc42, modulates spine dynamics (e.g. turnover, motility, changes in size and shape) and synaptic activity through actin cytoskeleton regulation (Bonhoeffer and Yuste, 2002; Ethell and Pasquale, 2005; Saneyoshi et al., 2010). All mature spines are believed to form a synapse with
29 an axonal bouton, but the existence of more than one synapse on the same spine is also possible (Holtmaat and Svoboda, 2009).
3. Synaptogenesis and Network formation
The normal neuronal network is a complex system of axonal and dendritic connections achieved by synapses.
Excitatory synapses are asymmetric, glutamatergic connections, mainly formed on spines with a dense aggregation of synaptic proteins under the postsynaptic membrane (so called postsynaptic density, PSD) and round vesicles at the presynaptic membrane of the axon. In contrast, symmetric GABAergic synapses are mostly formed on the dendritic shaft with oval presynaptic vesicles and without PSD. The distribution of the two types is changing with age (DeFelipe et al., 1997). During the early postnatal period the majority of synapses are formed on the dendritic shaft, while later the number of asymmetric synapses increases as spinogenesis proceeds (Ethell and Pasquale, 2005).
Filopodias are important elements of synaptogenesis. These dynamic, but transient structures often establish nascent synaptic contacts with surrounding axon terminals (Dailey and Smith, 1996). Some of these contacts develop into mature synapses by stabilization, but the rest is retracted (Fiala et al., 1998). Hence, spine dynamics can facilitate synapse initiation. The formation of a new excitatory synapse is a rapid and controlled multistep process starting with the initial target recognition and arrangement of pre- and postsynaptic membranes next to each other (Akins and Biederer, 2006). Adhesion molecules stabilize the contact, and their interaction activates actin cytoskeleton rearrangement (Takai et al., 2003).
Cadherins are key mediators of synaptic specificity and are expressed both pre- and postsynaptically (Obst-Pernberg and Redies, 1999). Synaptic cell adhesion molecules (SynCAMs), neurexins and neuroligins trigger synaptic development by mediating cell-cell interactions and intracellular processes (Missler et al., 2012). After the assembly of pre- and postsynaptic zones, scaffolding proteins (e.g. PSD95) are transported and linked to adhesion molecules or the membrane. The membrane stabilization process is followed by glutamatergic receptor recruitment. Young synapses contain less α-amino-3-hydroxy-5-methyl-4-isoxazole- propionate (AMPA) receptors negatively regulated by N-methyl-d-aspartate (NMDA)
30 signaling, but this composition changes during synaptic maturation (Hall and Ghosh, 2008).
Protein trafficking is important to maintain synapse polarity and to further stabilize the connection. The transport of synaptic vesicles, formation of active zones (with channels) and the membrane incorporation of additional elements are required for a functional synapse.
Note that inhibitory synapses are generated in a similar fashion, but with different molecular composition and before the appearance of excitatory synapses, since GABAergic interneurons become postmitotic and start network activities earlier than pyramidal cells (Represa and Ben- Ari, 2005).
During postnatal development, there is a continuous increase in spine and synapse number, followed by rapid spine loss. This period of extensive refinement involves elimination of existing connections and lasts until adolescence. The intensive spine turnover in younger animals is due to activity-dependent plasticity during the first few postnatal weeks (Butz et al., 2009). During this critical time period, the rapid phase of dendritic growth occurs parallel with very high synapse density (Briner et al., 2010), followed by a period of synapse elimination during which synaptic density and synapse number significantly decrease (Hensch, 2004). Structural plasticity allows retraction of unstabilized dendrites in contribution to dendritic remodeling. Spine composition of a dendrite also changes over time (Harris et al., 1992). In younger brains long and thin spines are found in higher percentage (Fiala et al., 1998), while later the number of mature spine types (often classified as stubby and mushroom) increases, related to stability (Dailey and Smith, 1996). In adults, spine number and density are relatively stable due to slower synaptic turnover, namely the formation- elimination of synapses is less intense in adults. Despite that, new spines and synapses can be formed in relation to adult learning and memory.
Neurotransmitter-induced activity is vital in synaptic maturation, plasticity and dendritic growth (Wong and Ghosh, 2002). Ca2+ influx induced by NMDA and AMPA type glutamate receptors activates calcium/calmodulin-dependent protein kinases (CaMKs) (Ethell and Pasquale, 2005). CaMKs (e.g. CAMKII and CAMKIV) are crucial in dendrite and spine/synapse formation and maintenance, since they directly control actin polymerization (Redmond et al., 2002). CaMKs are involved in synaptic circuit formation and functioning by coordinating signaling cascades. AMPA and NMDA trafficking to the postsynaptic membrane is a dynamic process mediated by Rho GTPases. Receptor activation induces actin remodeling, which leads to stabilization and changes in spine and synapse properties (Saneyoshi et al., 2010). Excitatory GABA signaling also influences synaptic integration via NMDA receptor mediation (Figure 5) (Akerman and Cline, 2007; Wang and Kriegstein,
31 2008). In summary, synaptic inputs are necessary for dendrite and spine stabilization, thus having an impact on the final morphology of the cell. Additionally, dendrites and spines have their own mRNA pool and elements of translation for local protein synthesis, thereby to independently control synaptic strength (Martin et al., 2000).
During synaptogenesis, extensive axonal branching occurs, offering possible targets for synapse formation. Actually, synaptic activation is required for normal axonal branching patterns (Lewis et al., 2013).
At the end of development, layers reach their functional segregation. Deeper layer neurons of the cortex (layer V, VI) mainly project to subcortical regions, while upper layers (layer II, III, IV) are responsible for intracortical circuits. The initial axonal connections arise subcortically from the brainstem, basal forebrain and the thalamus. Subplate neurons are essential for the formation of thalamocortical circuits, since thalamocortical axons develop connections first with SP neurons early during development, and then they invade layer IV as SP disappears (Kanold, 2009). These early afferents also triggers dendritic development of postmigratory neurons (Wong and Ghosh, 2002).
Elimination of unactivated postmitotic cells, which have not been established stable connections, occurs during postnatal development, most prominently during the first week (Ferrer et al., 1992). It is part of the normal differentiation program. Apoptosis or programmed cell death plays pivotal role in the regulation of final cell number. Both interneurons and projection neurons go through cell-intrinsic cascades leading to cell death (Southwell et al., 2012).
The fine tuning of neuronal activation depends on the precisely controlled balance of excitation and inhibition, and the computation of glutamatergic and GABAergic synapses which highly depends on their structural arrangement along the dendrite (Liu, 2004).
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2. Development of the GABAergic system
There are two main activity systems in the CNS: the excitatory and inhibitory transmitter systems, which are precisely controlling brain functions together. The principal neurotransmitter molecule of the excitatory system is glutamate, while GABA and glycine are for inhibitory transmission. GABA is the first neurotransmitter becoming functional during development, however, receptors of the neurotransmitters are already expressed during different phases of development highlighting other important roles before the beginning of their contribution in synaptic transmission (Behar et al., 2001; LoTurco et al., 1995; Represa and Ben-Ari, 2005; Wang and Kriegstein, 2008).
2.1 The transition between excitatory vs. inhibitory neurotransmission
In the adult telencephalon, GABA which is released primarily by interneurons, exerts fast inhibition through ionotropic GABAA receptors (GABAARs) of the postsynaptic cell. When GABA binds to postsynaptic GABAARs, it results inward Cl- flow, since GABAAR functions as a specific ion channel. It allows ion flow to both directions, but the direction of Cl- depends on its electrochemical gradient (Kaila, 1994). In mature cells, the intracellular Cl- concentration ([Cl-]i) is relatively low compared to the extracellular space, which leads to passive Cl--efflux through the activated GABAARs. This results generation of inhibitory postsynaptic potentials (IPSPs) and membrane hyperpolarization (Kaila, 1994; Payne et al., 1996; Zhu et al., 2005). The inhibitory action of GABA is a very important controller of normal brain function and network activity.
In turn, during early development GABA evokes depolarizing response (Owens et al., 1996), which has a role in several Ca2+-dependent developmental processes (Figure 5). It has been described that GABA act as trophic factor, and modulate neuronal proliferation and differentiation by controlling mitotic activity of VZ/SVZ progenitors (Ben-Ari et al., 1994;
Kriegstein and Owens, 2001; LoTurco et al., 1995; Young et al., 2012). Additionally, GABA facilitates neuronal migration (Represa and Ben-Ari, 2005). Ionotropic GABA receptor type
33 A and C are involved in the initial movement of progenitors from the proliferative zones to the IZ (Heck et al., 2007). Then metabotropic GABAB receptors influence their migration to the CP through additional modulation of the intracellular Ca2+ concentration ([Ca2+]i) (Behar et al., 2001). GABA also initiates dendritic arborization (Young et al., 2012) and synaptogenesis (Kriegstein and Owens, 2001). These actions are due to elevated [Cl-]i, thereby GABA activation produces outward movement of the Cl- and membrane depolarization, i. e. GABA transmission has excitatory modalities in immature neurons (Cherubini et al., 1991). This early depolarization stimulates NMDA receptors, thus GABA serves as an important modulator in the maturation of glutamatergic transmission and development of early network activity (Akerman and Cline, 2007).
During neuronal maturation, a constant decrease in Cl- concentration can be measured, which mediates electrochemical potential changes (DeFazio et al., 2000). As a result, GABA signaling has reverse effect in mature neurons. When [Cl-]i reaches such low level, where the Cl- reversal potential (ECl, a membrane voltage at which Cl- currents change their direction) is less than the normal resting membrane potential (Em), it negatively shifts GABA reversal potential (EGABA) leading to membrane hyperpolarization after GABAA receptor opening (Owens et al., 1996).
The change in [Cl-]i starts during the second postnatal week in rodents (Figure 5), leading to the switch in GABAA-mediated response from depolarization to hyperpolarization (Delpire, 2000; Tyzio et al., 2007). But GABAergic system maturation occurs gradually throughout the brain until the end of the first month of postnatal life (Ben-Ari et al., 2012; Ikeda et al., 2003).
The intracellular Cl- level is maintained by different transporters which expression and composition in the cell changes over time. Members of the cation-chloride cotransporter superfamily are described to be primarily involved in this maturation process (Kaila, 1994;
Payne et al., 2003).
