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Soft Matter Physics Division - Biophysics at the University of Leipzig University of Leipzig
IntroductionNeurons and Neuronal Growth
 

Neurons and Neuronal Signalling

The human brain is a complex network of more than 100 billion interconnected neurons (from Greek "neuron" for "nerve") [1]. These extraordinary cells are specialized in information processing and transmission by electrochemical signalling. A mammalian neuron consists of a cell body, called soma or perikaryon, and several cellular processes, called neurites. Neurites are distinguished into multiple, widely ramified dendrites and one axon. The latter originates in the axon hillock and extends over 1 µm to 1 m or even longer, permitting signal transmission over long distances. The end of an axon is usually connected to dendrites of other neurons (sometimes to muscle or gland cells) via junctions called synapses. However, there are also axon-to-soma, axon-to-axon and dendrite-to-dendrite connections between neurons.
Synapses chemically transmit electrical or electrochemical signals between the two participating cells by release of neurotransmitters. A neuron receives the signal with special receptors on the membrane of its dendrites or the soma. Then, the signal is forwarded to the axon hillock, where it is decided based on an all-or-none principle if an action potential is initiated. The action potential travels along the axon as a pulse-like wave of voltage. This is accomplished by selective ion exchange across the membrane through voltage-gated ion channels (alteration of the transmembrane voltage or membrane potential). After reaching the presynaptic side of another synapse, the signal is passed to the next neuron.
 

Schematic representation of a neuron and its growth cone
Schematic representation of a neuron and detailed view of the growth cone with its main cytoskeletal components (actin filaments and microtubules). The fluorescence image on the right-hand side shows an NG108 growth cone. (Figure taken from diploma thesis of Steve Pawlizak, 2009.)
 

Growth Cones

Developing axons that are not yet synaptically connected have highly dynamic, motile structures at their leading edge. These structures are called growth cones. They guide axons to their synaptic target by transducing positive and negative cues into signals that regulate the cytoskeleton and thereby determine the course and rate of axonal outgrowth [1, 2]. Hence, growth cones are very important during embryonal and adult neurogenesis. They are also essential for regeneration of neuronal connections as well as for the increase of neuronal connectivity.
Growth cones have round or conical shape with two kinds of protrusions: thin fingerlike filopodia and flat lamellipodia between them. The growth cone palpates its immediate vicinity with this protrusions and reacts to attractive or repulsive guidance cues by means of outgrowth, growth cone turning or retraction. The membrane of growth cones contains special receptors and cell adhesion molecules that are sensitive to chemical gradients (chemotaxis) and mechanical substrate properties (durotaxis).
The morphology of growth cones is defined by the underlying structure of the cytoskeleton. While the axon is dominated by parallel aligned microtubules forming some kind of backbone, the growth cone mainly consists of actin filaments. In particular, filopodia contain F-actin bundles and the lamellipodium a dense F-actin meshwork. The filopodia may act as contractile elements while exploring their mechanical environment [3, 4].
 

Prenatal Neurogenesis

Neurons are created through a process called neurogenesis which mostly occurs during the prenatal development of the nervous system when the growing brain is populated [5]. The ventricular zone in the embryonic neural tube contains progenitor cells [6], which divide in mitotic cycles, diversify and give rise to neuroblasts and glioblasts. Eventually, neuroblasts and glioblasts will differentiate into neurons and glial cells respectively [7, 8, 1].
At first, radially oriented glial cells are produced, later the neurons and subsequently all other glial cells. [7, 8].
The radial glia span the thickness of the cortex from the ventricular zone to the outer, pial surface and provide guiding pathways for the migration of neurons outwards to their final locations in the gray and white matter of the nervous system [9, 10]. Once neurons have left the ventricular zone, they become permanently postmitotic, i.e. they do not divide anymore. On the other hand, glial cells do not lose their ability to multiply [5, 1].
The distances that neurons travel within the brain are in the range of several millimeters [9, 11], whereas cell size is just 10 to 30 µm. The migrating neurons form well-defined layers whose position is correlated with the date of birth of the neuron. Inner layers of the cerebral cortex are established first, outer layer last [1]. After arriving at their destination sites, neurons differentiate and extend axons, which precisely follow certain pathways to their connective targets. Within each layer, neurons acquire distinct morphologies and connections. Radially arrayed neurons in different layers become richly interconnected and functionally related [12].
When neuronal migration is complete and radial glia are not longer required as guides, they disappear or transform into astrocytes [13, 14].
 

Adult Neurogenesis

By the end of the developmental period, the ventricular zone is depleted of all mitotic cells. However, neurogenesis and neuronal migration are not limited to embryonic development. It has been shown that even in the adult brain new neurons are generated [5, 11, 15, 16], even though the number of new neurons decreases while the organism ages [17]. This finding disproved the long-held theory that the nervous system is fixed and incapable of regeneration, but it does not refute the basic concept that a mature, differentiated neuron does not divide.
The new neurons originate from neural progenitor cells found in restricted brain regions, in particular the subventricular zone (SVZ) lining the lateral ventricles and in the subgranular zone (SGZ) which is part of the dentate gyrus of hippocampus. The precursor neurons from the SVZ migrate to the olfactory bulb, where they differentiate into interneurons. Cells form the SGZ migrate short distances into the granule cell layer of the dentate gyrus and subsequently differentiate into granule cells.
It has been reported that limited, localized neuronal injury and hypoxia induce neurogenesis and replacement of neurons in the adult cerebral cortex [17].
 

Neuronal Pathfinding

As already mentioned, both neuronal migration and neurite outgrowth follow specific pathways. Neurons and growth cones have to detect a variety of external signals and respond to them accordingly [18, 19, 2, 20]. Neuronal migration and neurite outgrowth are guided by the same molecular cues [21] with chemotaxis playing a major roll [22, 6, 18, 23]. Several classes of molecules, like diffusible secreted factors, adhesion molecules and cues from the surrounding extracellular matrix and adjacent cells are involved in the process of neuronal pathfinding and growth [18, 20, 21].
However, the complex migration and growth patterns of neurons and neurites cannot be completely explained with simple biochemical gradients, especially when considering the length of some pathways or the spread of some axons. In addition to biochemical cues, neurons are susceptible to their mechanical environment (durotaxis). For instance, enlarged filopodia tips of Aplysia growth cones are very sensitive to force [24], and it has been implicated that snail and leech neurons also feel and respond to external mechanical stimuli [25, 26]. Furthermore, it has been shown that neurite outgrowth and branching in vitro is influenced by the mechanical stiffness of the substrate [27, 28]. In particular, a preference for soft substrates has been observed. This characteristic behavior of neurons may be called inverse durotaxis, since most other cell types (e.g. fibroblasts) favor stiffer substrates [29, 30]. In vivo, neurons of the CNS grow along glial cells, which are significantly softer than their neighboring neurons [31]. These examples suggest an involvement of mechanics in neuronal and axonal guidance. However, the underlying mechanisms of neuronal mechanosensitivity are not yet understood.

Directed movement of neurons requires active recognition of their migration pathway. This motivates us to investigate the possibly active response of neurons to externally applied mechanical stress, in order to understand the growth cones’ sensing of mechanical properties of their environment (e.g. substrate stiffness). Our in vitro studies show that neurons actively palpate their mechanical environment with the help of their growth cones and retract their neurites from contacts they cannot mechanically deform [32]. After mechanical stimulation of the neuronal growth cones using a modified scanning force microscope (SFM) probe, the neurons retract their processes and re-extend them into a new direction when the exerted pressure exceeds approximately 300 Pa. This threshold corresponds to the maximum substrate stiffness that neurons can visibly deform. Furthermore, an immediate calcium influx through stretch-activated ion channels seems to be correlated with neurite retraction.

(This article is taken from the diploma thesis of Steve Pawlizak, University of Leipzig, 2009.)




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