Migrational waves from the SVZ built up the cortex

The cerebral cortex plays a key role in memory, attention, perception and awareness and is mainly composed of two types of neuronal cells: first projection (pyramidal) neurons which are glutamatergic and excitatory and second interneurons which are GABAergic and inhibitory. Cortical neurons arise from progenitor cells in the ventricular zone (Kriegstein and Noctor 2004). The first postmitotic cortical neurons that migrate out of the ventricular zone at E11 form a transient layer, the preplate. This preplate is split through subsequent waves of migrating cortical neurons at E13 into a superficial layer, called marginal zone and a deeper layer, the subplate. Both layers form the cortical plate. As additional waves of migrating neurons arrive in the cortical plate, they bypass the existing layers and migrate to the surface, where they form the cortical layers in an inside-out-fashion (Nadarajah and Parnavelas 2002). This means early-generated neurons are localized in deeper cortical layers (V-VI), while late-born-neurons migrate through the existing layers and form the more superficial layers (II, III and IV) (Ayala et al., 2007). A brain specific knockout of Cofilin 1 leads to the loss of cortical layers II, III and IV and a translucent appearance of the cortex (Bellenchi et al., 2007).

Fig. 9: Formation of the cortical layers during embryonic development. The cerebral neocortex is organized into 6 distinct neuronal layers. At E11 the preplate (PP) is established through the postmitotic migration of neurons from the ventricular zone (VZ) to the pial surface (PS). A second postmitotic wave migrates through the intermediate zone (IZ) and splits the preplate into the marginal zone (MZ) and the more superficial subplate (SP), which generates the cortical plate (CP) at E13.

During E14- E18 a subsequent wave of neurons from the subventricular zone (SVZ) reaches the cortical plate and expands it in an insight-out-fashion, which means that later born neurons migrate through the existing layers to the pial surface. In the adulthood the subplate degenerates and leaves behind a six-layered cortex (Gupta et al., 2002).

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In the adult stage the cortical plate contains six cortical layers. Each layer contains a characteristic distribution of neuronal cell types and connections with other cortical and subcortical regions, like the thalamus and the basal ganglia. Vertically connections of neurons in various layers form small microcircuits, called cortical columns. Thereby the cortex can be classified into three areas: sensory, motor and association. The sensory area receives and processes information about senses and receives sensory input from the thalamus. Control of voluntary movements are regulated by the motor areas, while the integration of sensory information with stored memory, as well as planning actions and movement is performed by the association area. To reach their position in the cortical plate neurons must be capable of sensing local microenvironmental cues, which establish layer fate. These microenvironmental cues change over time leading to different positions in the cortical plate. Important receptors to sense these guidance cues are localized in the membrane of a specific structure called growth cone, which will be explained further in the next chapter.

Actin assembly in the growth cone 1.3.4.1.

The establishment of highly ordered neuronal networks depends on the precise control of axon guidance during development. Growth cones at the distal tips of growing neurites sense a variety of attractive (BDNF) or repulsive (semaphoring 3A) cues from the environment, which induce changes in cell shape and motility (Pak et al., 2008). These changes are accompanied by alterations in the actin filament dynamics and reorganization, guiding the axon to its target. The axonal growth cone is composed of a central region filled with organelles and microtubules and a peripheral highly dynamic actin-rich region (Ishikawa and Kohama 2007; Tahirovic and Bradke 2009). At the periphery highly dynamic actin-based structures like filopodia and lamellipodia are located (figure 10). Both actin-rich structures vary in their actin arrangement. Lamellipodia are broad veil-like cellular protrusions with a branched actin network and can be further characterized by the absence of tropomyosins and the presence of Cofilin (Ishikawa and Kohama 2007). In contrast filopodia are thin protrusions containing unbranched and parallel F-actin bundles (Dickson 2002). The barbed ends of filaments are located to the ruffling membranes, whereas the pointed ends are located to the center of the growth cone. This guarantees a continuous in-cooperation of ATP-actin at the barbed ends, which drives the membrane further on and leads to the elongation towards the source of a guidance cue, and the dissociation of ADP-actin at the pointed end for the recycling of actin monomers.

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Fig. 10: Assembly of the growth cone. The growth cone at the tips of neurites is composed of a central domain, which contains microtubules and a peripheral domain, which consists of the actin cytoskeleton and shows the typical migrational protrusions at the ruffling membrane. Lamellipodia display a meshwork of actin filaments, while filopodia can be characterized through a parallel alignment of actin filaments. The assembly of actin filaments at the barbed ends is necessary for the outgrowth of growth cones, which drives the membrane further on. Based on this the barbed ends of filaments are always localized to the membrane , while the pointed end is directed to the transition zone, where microtubule- and actin-filaments meet (Dickson 2002). Blue: microtubule; orange: actin

Additionally the rearrangements of the actin cytoskeleton and microtubules are crucial for the initial establishment of polarity. The future axon is characterized by an enhanced growth cone dynamic (Bradke and Dotti 1999), whereas dendrites show very static growth cones.

The local exposure of extracellular signals to one neurite leads to the activation of signaling pathways, which alter the activity of actin-binding proteins and therefore promote actin remodeling. Extracellular signals are propagated by the Rho family of GTPases (Rac1, Cdc42 and RhoA), which induce the activation of ROCK that regulates the activation state of LIMK, controlling ADF/Cofilin activity (Endo et al., 2003). In this one neurite the actin cytoskeleton is more permissive for microtubule protrusion from the core, which drives the center of the growth cone further on (Bradke and Dotti 1999). This suggests that actin filaments in dendritic growth cones form a barrier for the protrusion of microtubules and therefore the growth cone remains static. Studies have shown that Cofilin 1 is highly active in the axonal growth cones compared to non-growing dendritic growth cones (Garvalov et al., 2007). The severing activity of the ADF/Cofilin family generates more available barbed ends, which is important for the branching of filaments in lamellipodia and leads to an increase in motility (Krause et al., 2004). The loss of filopodia and lamellipodia from growth cones, by application of cytochalasin to disrupt actin filaments, decreases axon growth rates and leads to a complete loss of guidance (Bentley and Toroian-Raymond 1986).

An impact on the polarization process could also affect the branching and dendritic arborization of cortical pyramidal neurons resulting in a reduced neurite length or dendritic arborization (Li et al., 2000). The role of actin during dendritic development will be further discussed in the next chapter.

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Actin is involved in dendritic arborization 1.3.4.2.

During development the nervous system must progress from `disconnected` neurons to a network of neuronal circuits that is able to dynamically process information and generate an appropriate output. Proper dendritic arborization is a critical process for accurate development of neuronal circuits and for activity-dependent plasticity of mature neurons (Scott and Luo 2001). Dendrites are the site of 90% of all excitatory synaptic contacts and dendritic development determines the number and pattern of synapses (Hume and Purves 1981). Therefore dendritic abnormalities are the most common pathological correlation for mental retardation. Extension, retraction and branching of dendritic processes are complex functions that are regulated by an interaction between intrinsic developmental programs and local environmental cues, and additionally the level of neuronal activity. Dendritic growth is dependent on the stabilization of a subset of highly motile and protrusive dendritic structures.

Real-time imaging has demonstrated that dendritic elaboration occurs through a net growth of highly dynamic filopodia that are actin-based structures (Dailey and Smith 1996). The Rho family of small GTPases (Rho, Rac and Cdc42) is known to be a regulator of signaling pathways to the actin cytoskeleton (Luo 2000). A blockade of Rac or Cdc42 reduces the number of primary dendrites, while an increase in RhoA activity causes a retraction of dendrites (Li et al., 2000). Actin binding proteins, like ADF/Cofilins are known regulators of actin depolymerization, which are controlled by RhoA. Additionally RhoA mediates the increase in dendritic growth caused by NMDA receptor activation (Nimchinsky et al., 2002).

Therefore neuronal activity has a major effect on dendritic arborization, as shown by the fact that a decrease in activity results in reduced dendritic outgrowth (McAllister 2000). Repetitive depolarization activates ERK and CaMKII, which promote dendritic outgrowth by enhancing transcription of Wnt. Wnt in turn stimulates the Rac pathway that modulates the actin cytoskeleton (Rosso and Inestrosa 2013). Additionally neuronal activity also regulates the number of dendritic filopodia, which may be a substrate for new branches. Kossel et al (1997) showed that cultured hippocampal neurons form dendritic branches only in the presence of afferent innervations, showing that new dendritic branches are stabilized by synapses (Kossel et al., 1997). It was proposed that the formation of synaptic contacts may guide dendritic growth (Vaughn 1989). The loss of Cofilin 1 alters spine density and influences receptor motility to dendritic spines and could therefore contribute to alterations in dendritic arborization (Rust et al., 2010). A direct link between the ADF/Cofilin family and dendritic branching was made by Bläsius (2012), who showed that the ablation of Cofilin 1 leads to a reduced dendritic branching of cortical neurons in culture (Bläsius 2012).

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1.4. Actin in synapses

Synapses are specialized morphological structures, which reside between two synaptic cells to enable the communication through the release of neurotransmitters from the presynaptic neuron, which bind to receptors on the postsynaptic cell. During development and throughout life, synapses are highly dynamic structures which are modulated by activity to alter the synaptic strength and morphological plasticity, which build the basis for learning and memory processes (Holtmaat and Svoboda 2009). In the human brain a number of 10¹⁶ neurons are estimated, which exhibit a pattern of synaptic connectivity (Munno and Syed 2003). Actin is the most abundant cytoskeletal protein in presynaptic terminals as well as in postsynaptic dendritic spines (Fifkova and Delay 1982). The contribution of actin to the establishment of a functional synapse, as well as its important role in the assembly and functionality of the pre- and postsynaptic site will be discussed in the next chapters.