Micha E. Spira

Assembly of a new growth cone after axotomy: the precursor to axon regeneration.

The assembly of a new growth cone is a prerequisite for axon regeneration after injury. Creation of a new growth cone involves multiple processes, including calcium signalling, restructuring of the cytoskeleton, transport of materials, local translation of messenger RNAs and the insertion of new membrane and cell surface molecules. In axons that have an intrinsic ability to regenerate, these processes are executed in a timely fashion. However, in axons that lack regenerative capacity, such as those of the mammalian CNS, several of the steps that are required for regeneration fail, and these axons do not begin the growth process. Identification of the points of failure can suggest targets for promoting regeneration.

Real Time Imaging of Calcium-Induced Localized Proteolytic Activity after Axotomy and Its Relation to Growth Cone Formation

The emergence of a neuronal growth cone from a transected axon is a necessary step in the sequence of events that leads to successful regeneration. Yet, the molecular mechanisms underlying its formation after axotomy are unknown. In this study, we show by real time imaging of the free intracellular Ca2+ concentration, of proteolytic activity, and of growth cone formation that the activation of localized and transient Ca2+-dependent proteolysis is a necessary step in the cascade of events that leads to growth cone formation. Inhibition of this proteolytic activity by calpeptin, a calpain inhibitor, abolishes growth cone formation. We suggest that calpain plays a central role in the reorganization of the axons cytoskeleton during its transition from a stable differentiated structure into a dynamically extending growth cone.

Low mobility of the Ca2+ buffers in axons of cultured Aplysia neurons

Cellular Ca2+ buffers determine amplitude and diffusional spread of neuronal Ca2+ signals. Fixed Ca2+ buffers tend to retard the signal and to lower the apparent diffusion coefficient (D(app)) of Ca2+, whereas mobile buffers contribute to Ca2+ redistribution. To estimate the impact of the expression of specific Ca2+-binding proteins or the errors in Ca2+ measurement introduced by indicator dyes, the diffusion coefficient De and the Ca2+-binding ratio kappa(e) of endogenous Ca2+ buffers must be known. In this study, we obtain upper bounds to these quantities (De < 16 microm2/s; kappa(e) < 60) for axoplasm of metacerebral cells of Aplysia california. Due to these very low values, even minute concentrations of indicator dyes will interfere with the spatiotemporal pattern of Ca2+ signals and will conceal changes in the expression of specific Ca2+-binding proteins, which in the native neuron are expected to have significant effects on Ca2+ signals.

Penicillin decreases chloride conductance in crustacean muscle: a model for the epileptic neuron.

The effects of penicillin were studied on the neuromuscular preparation of the ghost crab, Ocypoda cursor. Penicillin in doses lower than 2 mM reduced both the amplitude of inhibitory junction potentials and conductance increases induced by external application of GABA. The nature of the latter effect appears to be 2-fold, a weaker competitive inhibition and a more powerful non-competitive effech which may be ionophore blockade. Penicillin in concentrations above 2 mM diminished resting conductance, especially that of chloride. The action of penicillin is, in general, to decrease chloride conductance in this preparation. The crustacean neuromuscular preparation may provide a useful analogue for understanding penicillin evoked epilepsy. The reduced chloride conductance could explain decreased inhibition, increased excitation and depolarization shifts in cortical neurons.

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.

Spatiotemporal Distribution of Ca2+ Following Axotomy and Throughout the Recovery Process of Cultured Aplysia Neurons

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca2+ concentration ([Ca2+]i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans‐membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura‐2, and monitoring the neurons’resting potential and input resistance (Ri) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca2+]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating [Ca2+]i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post‐axotomy, [Ca2+]i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca2+]i recovers to the control level 7 – 10 min post‐axotomy. In Ca2+‐free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca2+]i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca2+]i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca2+, Mg2+ and Na+ into intact axons do not induce the release of Ca2+ from intracellular stores, indicates that Ca2+ influx through voltage gated Ca2+ channels and through the cut end are the primary sources of [Ca2+]i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca2+]i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.

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.

Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy

Transformation of a transected axonal tip into a growth cone (GC) is a critical step in the cascade leading to neuronal regeneration. Critical to the regrowth is the supply and concentration of vesicles at restricted sites along the cut axon. The mechanisms underlying these processes are largely unknown. Using online confocal imaging of transected, cultured Aplysia californica neurons, we report that axotomy leads to reorientation of the microtubule (MT) polarities and formation of two distinct MT-based vesicle traps at the cut axonal end. Approximately 100 μm proximal to the cut end, a selective trap for anterogradely transported vesicles is formed, which is the plus end trap. Distally, a minus end trap is formed that exclusively captures retrogradely transported vesicles. The concentration of anterogradely transported vesicles in the former trap optimizes the formation of a GC after axotomy.

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.

Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy

In differentiated axons almost all microtubules (MTs) uniformly point their plus ends towards the axonal tip. The uniform polar pattern provides the structural substrate for efficient organelle transport along axons. It is generally believed that the mass and pattern of MTs polar orientation remain unchanged in differentiated neurons. Here we examined long-term effects of the MTs stabilizing reagent paclitaxel (taxol) over MTs polar orientation and organelle transport in cultured Aplysia neurons. Unexpectedly, we found that rather than stabilizing the MTs, paclitaxel leads to their massive polar reconfiguration, accompanied by impaired organelle transport. Washout of paclitaxel does not lead to recovery of the polar orientation indicating that the new pattern is self-maintained. Taken together the data suggest that MTs in differentiated neurons maintain the potential to be reconfigured. Such reconfiguration may serve physiological functions or lead to degeneration. In addition, our observations offer a novel mechanism that could account for the development of peripheral neuropathy in patients receiving paclitaxel as an antitumor drug.