Ada Dormann

Critical Calpain-Dependent Ultrastructural Alterations Underlie the Transformation of an Axonal Segment into a Growth Cone after Axotomy of Cultured Aplysia Neurons

The transformation of a stable axonal segment into a motile growth cone is a critical step in the regeneration of amputated axons. In earlier studies we found that axotomy of cultured Aplysia neurons leads to a transient and local elevation of the free intracellular Ca2+ concentration, resulting in calpain activation, localized proteolysis of submembranal spectrin, and, eventually, growth cone formation. Moreover, inhibition of calpain by calpeptin prior to axotomy inhibits growth cone formation. Here we investigated the mechanisms by which calpain activation participates in the transformation of an axonal segment into a growth cone. To that end we compared the ultrastructural alterations induced by axotomy performed under control conditions with those caused by axotomy performed in the presence of calpeptin, using cultured Aplysia neurons as a model. We identified the critical calpain‐dependent cytoarchitectural alterations that underlie the formation of a growth cone after axotomy. Calpain‐dependent processes lead to restructuring of the neurofilaments and microtubules to form an altered cytoskeletal region 50–150 μm proximal to the tip of the transected axon in which vesicles accumulate. The dense pool of vesicles forms in close proximity to a segment of the plasma membrane along which the spectrin membrane skeleton has been proteolyzed by calpain. We suggest that the rearrangement of the cytoskeleton forms a transient cellular compartment that traps transported vesicles and serves as a locus for microtubule polymerization. We propose that this cytoskeletal configuration facilitates the fusion of vesicles with the plasma membrane, promoting the extension of the growth cones lamellipodium. The growth process is further supported by the radial polymerization of microtubules from the growth cones center. J. Comp. Neurol. 457:293–312, 2003.

Calcium, Protease Activation, and Cytoskeleton Remodeling Underlie Growth Cone Formation and Neuronal Regeneration

The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca2+]i) to 200–300 μM induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca2+]i and of intraaxonal proteolyticactivity, we found that the elevated [Ca2+]i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca2+]i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.

Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices

Interfacing neurons with micro- and nano-electronic devices has been a subject of intense study over the last decade. One of the major problems in assembling efficient neuro-electronic hybrid systems is the weak electrical coupling between the components. This is mainly attributed to the fundamental property of living cells to form and maintain an extracellular cleft between the plasma membrane and any substrate to which they adhere. This cleft shunts the current generated by propagating action potentials and thus reduces the signal-to-noise ratio. Reducing the cleft thickness, and thereby increasing the seal resistance formed between the neurons and the sensing surface, is thus a challenge and could improve the electrical coupling coefficient. Using electron microscopic analysis and field potential recordings, we examined here the use of gold micro-structures that mimic dendritic spines in their shape and dimensions to improve the adhesion and electrical coupling between neurons and micro-electronic devices. We found that neurons cultured on a gold-spine matrix, functionalized by a cysteine-terminated peptide with a number of RGD repeats, readily engulf the spines, forming tight apposition. The recorded field potentials of cultured Aplysia neurons are significantly larger using gold-spine electrodes in comparison with flat electrodes.

Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro

The present report describes the experimental advantages offered by the combined use of Aplysia neurons and contemporary techniques to analyze the cellular events associated with nerve injury in the form of axotomy. The experiments were performed by transecting, under visual control, the main axon of identified Aplysia neurons in primary culture while monitoring several related parameters. We found that in cultured Aplysia neurons axotomy leads to the elevation of the [Ca2+]i in both the proximal and distal axonal segments from a resting level of 100 nM up to the millimolar range for a duration of 3-5 min. This increase in [Ca2+]i led to identical alterations in the cytoarchitecture of the proximal and distal segments. The formation of a membrane seal over the transected ends by their constriction and the subsequent fusion of the membrane is a [Ca2+]i-dependent process and is triggered by the elevation of [Ca2+]i to the microM level. Seal formation was followed by down-regulation of the [Ca2+]i to control levels. Following the formation of the membrane seal an increase in membrane retrieval was observed. We hypothesize that the retrieved membrane serves as an immediately available membrane reservoir for growth cone extension.

Improved Neuronal Adhesion to the Surface of Electronic Device by Engulfment of Protruding Micro-Nails Fabricated on the Chip Surface

One of the major problems in assembling efficient neuro-electronic hybrids systems is the low electrical coupling between the components. This is mainly due to the low resistance, extracellular cleft formed between the cells plasma membrane and the substrate to which it adhere. This cleft shunts the current generated by the neuron, or the device and thus reduces the signal to noise ratio. To increase the clefts electrical resistance we fabricated gold micronails that protrude from the transistor gate surface. The micronails were functionalized by phagocytosis facilitating peptides. Cultured neurons readily engulf the functionalized micronails forming tight physical contact between the cells and the surface of the device.

Electrically conductive 2D-PAN-containing surfaces as a culturing substrate for neurons

In the present contribution we report on a novel route to synthesize 2D-polyaniline (2DPAN) on sulfonated-poly(styrene) (SPS) templates by allowing first monomer assembly followed by chemical oxidation to achieve polymerization. We show that Aplysia neurons grown on 2D-PAN exhibit an unusual growth pattern and adhesion to this conducting substrate that is manifested by the formation of giant lamellipodia. The lamellipodial domains are characterized by small gap between the plasma membrane and the 2D-PAN substrate (ca. 30 nm) and actin rich skeleton resembling the skeleton of growth cones. This behavior is characteristic to uniform substrates containing only 2DPAN. However, in patterned substrates containing additionally poly(L-lysine) Aplysia neurons prefer to extend new neurites on the poly(L-lysine) domains.

Long Term Survival of Isolated Axonal Segments as Revealed by in Vitro Studies

When peripheral vertebrate axons are transected from their cell bodies, the isolated axons commonly degenerate within 3–4 days (Wallerian degeneration, 1, 2). In some lower vertebrates and invertebrates, however, some of the isolated axons maintain almost normal morphology, propagate action potentials and release transmitters for periods of several months (for reviews see 3,4).

High Calcium Concentrations, Calpain Activation and Cytoskeleton Remodeling in Neuronal Regeneration after Axotomy

A vast number of studies has demonstrated causal relations between excessive elevation of the free intra neuronal calcium concentration ([Ca2+] i ) and neurodegeneration. Calcium-induced neurodegeneration is believed to occur in acute conditions such as nerve-transection induced Wallerian degeneration (Waller, 1850), mechanical brain trauma, brain ischemia, hypoglycemic coma and status epilepticus. Calcium-induced neurodegeneration is also believed to participate in chronic conditions such as Alzheimer’s disease and aging. The degenerative effects of the elevated [Ca2+] i are thought to be mediated by the unbalanced activation of enzymes that take part in the normal neuronal function. These include proteinases, phospholipases, phosphatases and protein kinases. In turn, the unbalanced activation of these enzymes leads to cytoskeletal damage, membrane dysfunction, enhanced production of free radicals and, finally, neuronal degeneration (reviewed in Choi, 1994; Siesjo, 1994; Rothman and Olney, 1995; Kristian and Siesjo, 1998).

Neuronal architecture, receptor and Ca2+ channel distribution in regenerating giant interneurons.

During the course of normal development of the nervous system, individual neurons encounter various environmental signals. The nature of the signals, the spatial and temporal order in which they are presented to the developing neuron, together with the intrinsic properties of the neurons, determine the morphological, physiological and biochemical characteristics of the adult neuron. These properties in turn define the way in which individual adult neurons integrate incoming synaptic information and transmit it to other neurons. Among the extrinsic signals that participate in moulding the developing neuron into its adult form and function are growth factors which act as outgrowth promoting signals (Levi-Montalcini, 1976; Green & Shooter, 1980; Cowan et al„ 1984; Black, 1986; Varon et al, 1988). These growth factors are released from various sources such as glial cells, neurons and fibroblasts. Another class of signals are the neurotransmitters. Thus, growth cone motility of specific neurons can be inhibited by specific neu-