Significantly reduced inflammatory foreign-body-response to neuroimplants and improved recording performance in young compared to adult rats.

The multicellular inflammatory encapsulation of implanted intracortical multielectrode arrays (MEA) is associated with severe deterioration of their field potentials' (FP) recording performance, which thus limits the use of brain implants in basic research and clinical applications. Therefore, extensive efforts have been made to identify the conditions in which the inflammatory foreign body response (FBR) is alleviated, or to develop methods to mitigate the formation of the inflammatory barrier. Here, for the first time, we show that (1) in young rats (74±8 gr, 4 weeks old at the onset of the experiments), cortical tissue recovery following MEA implantation proceeds with ameliorated inflammatory scar as compared to adult rats (242 ± 18 gr, 9 weeks old at the experimental onset); (2) in contrast to adult rats in which the Colony Stimulating factor 1 Receptor (CSF1R) antagonist chow eliminated ∼95% of the cortical microglia but not microglia adhering to the implant surfaces, in young rats the microglia adhering to the implant were eliminated along with the parenchymal microglia population. The removal of microglia adhering to the implant surfaces was correlated with improved recording performance by in-house fabricated Perforated Polyimide MEA Platforms (PPMP). These results support the hypothesis that microglia adhering to the surface of the electrodes, rather than the multicellular inflammatory scar, is the major underlying mechanism that deteriorates implant recording performance, and that young rats provide an advantageous model to study months-long, multisite electrophysiology in freely behaving rats. STATEMENT OF SIGNIFICANCE: Multisite electrophysiological recordings and stimulation devices play central roles in basic brain research and medical applications. The insertion of multielectrode-array platforms into the brain's parenchyma unavoidably injures the tissue, and initiates a multicellular inflammatory cascade culminating in the formation of an encapsulating scar tissue (the foreign body response-FBR). The dominant view, which directs most current research efforts to mitigate the FBR, holds that the FBR is the major hurdle to effective electrophysiological use of neuroprobes. By contrast, this report demonstrates that microglia adhering to the surface of a neuroimplants, rather than the multicellular FBR, underlie the performance deterioration of neuroimplants. These findings pave the way to the development of novel and focused strategies to overcome the functional deterioration of neuroimplants.

Significant Sex Differences in the Efficacy of the CSF1R Inhibitor-PLX5622 on Rat Brain Microglia Elimination.

Microglia play pivotal roles in central nervous system development, homeostasis, responses to trauma, and neurodegenerative and neuropsychiatric disorders with significant sex-bias in their symptoms and prevalence. Survival of the microglia in adult brains depends on the expression of the colony-stimulating factor 1 receptor (CSF1R). The inhibition of CSF1R by brain-permeant PLX5622 in the chow eliminates, within 5-10 days, ~90% of the microglia in female and male mice, thereby enabling the investigation of the roles of the microglia in health and pathological mice models. Because of a prevailing "impression" that PLX5622 is ineffective in rats, it has hardly been used in studies of adult rats. Here, we report that effective microglia elimination by PLX5622-chow in rats is highly sex-dependent. Our observations provide missing information for the limited use and interpretation of PLX5622 in biomedical studies of the microglia in rat models. The sex differences that are too often overlooked must be carefully considered and clearly emphasized.

Ultrastructural Analysis of Neuroimplant-Parenchyma Interfaces Uncover Remarkable Neuroregeneration Along-With Barriers That Limit the Implant Electrophysiological Functions.

Despite increasing use of in vivo multielectrode array (MEA) implants for basic research and medical applications, the critical structural interfaces formed between the implants and the brain parenchyma, remain elusive. Prevailing view assumes that formation of multicellular inflammatory encapsulating-scar around the implants [the foreign body response (FBR)] degrades the implant electrophysiological functions. Using gold mushroom shaped microelectrodes (gMµEs) based perforated polyimide MEA platforms (PPMPs) that in contrast to standard probes can be thin sectioned along with the interfacing parenchyma; we examined here for the first time the interfaces formed between brains parenchyma and implanted 3D vertical microelectrode platforms at the ultrastructural level. Our study demonstrates remarkable regenerative processes including neuritogenesis, axon myelination, synapse formation and capillaries regrowth in contact and around the implant. In parallel, we document that individual microglia adhere tightly and engulf the gMµEs. Modeling of the formed microglia-electrode junctions suggest that this configuration suffice to account for the low and deteriorating recording qualities of in vivo MEA implants. These observations help define the anticipated hurdles to adapting the advantageous 3D in vitro vertical-electrode technologies to in vivo settings, and suggest that improving the recording qualities and durability of planar or 3D in vivo electrode implants will require developing approaches to eliminate the insulating microglia junctions.

Inflammatory Foreign Body Response Induced by Neuro-Implants in Rat Cortices Depleted of Resident Microglia by a CSF1R Inhibitor and Its Implications.

Inflammatory encapsulation of implanted cortical-neuro-probes [the foreign body response (FBR)] severely limits their use in basic brain research and in clinical applications. A better understanding of the inflammatory FBR is needed to effectively mitigate these critical limitations. Combining the use of the brain permeant colony stimulating factor 1 receptor inhibitor PLX5622 and a perforated polyimide-based multielectrode array platform (PPMP) that can be sectioned along with the surrounding tissue, we examined the contribution of microglia to the formation of inflammatory FBR. To that end, we imaged the inflammatory processes induced by PPMP implantations after eliminating 89-94% of the cortical microglia by PLX5622 treatment. The observations showed that: (I) inflammatory encapsulation of implanted PPMPs proceeds by astrocytes in microglia-free cortices. The activated astrocytes adhered to the PPMP's surfaces. This suggests that the roles of microglia in the FBR might be redundant. (II) PPMP implantation into control or continuously PLX5622-treated rats triggered a localized surge of microglia mitosis. The daughter cells that formed a "cloud" of short-lived (T 1 / 2 ≤ 14 days) microglia around and in contact with the implant surfaces were PLX5622 insensitive. (III) Neuron degeneration by PPMP implantation and the ensuing recovery in time, space, and density progressed in a similar manner in the cortices following 89-94% depletion of microglia. This implies that microglia do not serve a protective role with respect to the neurons. (IV) Although the overall cell composition and dimensions of the encapsulating scar in PLX5622-treated rats differed from the controls, the recorded field potential (FP) qualities and yield were undistinguishable. This is accounted for by assuming that the FP amplitudes in the control and PLX5622-treated rats were related to the seal resistance formed at the interface between the adhering microglia and/or astrocytes and the PPMP platform rather than across the scar tissue. These observations suggest that the prevention of both astrocytes and microglia adhesion to the electrodes is required to improve FP recording quality and yield.

Assessing the Feasibility of Developing in vivo Neuroprobes for Parallel Intracellular Recording and Stimulation: A Perspective.

Developing novel neuroprobes that enable parallel multisite, long-term intracellular recording and stimulation of neurons in freely behaving animals is a neuroscientist's dream. When fulfilled, it is expected to significantly enhance brain research at fundamental mechanistic levels including that of subthreshold signaling and computations. Here we assess the feasibility of merging the advantages of in vitro vertical nanopillar technologies that support intracellular recordings with contemporary concepts of in vivo extracellular field potential recordings to generate the dream neuroprobes that read the entire electrophysiological signaling repertoire.

Immunohistological and Ultrastructural Study of the Inflammatory Response to Perforated Polyimide Cortical Implants: Mechanisms Underlying Deterioration of Electrophysiological Recording Quality.

The deterioration of field potential (FP) recording quality and yield by in vivo multielectrode arrays (MEA) within days to weeks of implantation severely limits progress in basic and applied brain research. The prevailing hypothesis is that implantation of MEA platforms initiate and perpetuate inflammatory processes which culminate in the formation of scar tissue (the foreign body response, FBR) around the implant. The FBR leads to progressive degradation of the recording qualities by displacing neurons away from the electrode surfaces, increasing the resistance between neurons (current source) and the sensing pads and by reducing the neurons' excitable membrane properties and functional synaptic connectivity through the release of pro-inflammatory cytokines. Meticulous attempts to causally relate the cellular composition, cell density, and electrical properties of the FBR have failed to unequivocally correlate the deterioration of recording quality with the histological severity of the FBR. Based on confocal and electron microscope analysis of thin sections of polyimide based MEA implants along with the surrounding brain tissue at different points in time after implantation, we propose that abrupt FP amplitude attenuation occurs at the implant/brain-parenchyma junction as a result of high seal resistance insulation formed by adhering microglia to the implant surfaces. In contrast to the prevailing hypothesis, that FP decrease occurs across the encapsulating scar of the implanted MEA, this mechanism potentially explains why no correlations have been found between the dimensions and density of the FBR and the recording quality. Recognizing that the seal resistance formed by adhering-microglia to the implant constitutes a downstream element undermining extracellular FP recordings, suggests that approaches to mitigate the formation of the insulating glial could lead to improved recording quality and yield.

Multisite Intracellular Recordings by MEA.

The enormous advances made over the last 50 years in materials science, microelectronics, and nanoelectronics, together with the acknowledgment that substrate-integrated planar multielectrode arrays (MEA) are limited to recording of extracellular field potentials (FPs) rather than the entire electrophysiological signaling repertoire of the brain, have prompted a number of laboratories to merge the advantages of planar MEA technologies (non-damaging and durable) with those of the classical sharp and patch electrodes for intracellular recordings. Unlike extracellular planar electrode-based MEAs, the new generation of three-dimensional (3D) vertical nanoelectrodes are designed to functionally penetrate the plasma membrane of cultured cells and operate in a similar manner to classical intracellular microelectrodes. Although only approximately 10 years has elapsed since the development of the first vertical 3D nanostructure-based MEAs, this technology has progressed to enable recordings of attenuated intracellular action potentials (APs) and synaptic potentials from individual neurons, cardiomyocytes, and striated myotubes. Furthermore, recently the scaling advantages of nanochip/microchip fabrication technologies enabled simultaneously intracellular recordings of APs from hundreds of cultured cardiomyocytes, thus heralding a new milestone in MEA technology.In this chapter we present the earliest and today's cutting-edge achievements of this "young vertical nano-sensors MEA technology" at the single-cell and network levels, explain the biophysical principles and the various configurations used to form functional nanoelectrode/cell hybrids, and describe the quality and characteristic features of the recorded intracellular APs and subthreshold synaptic potentials by the vertical nanoelectrode-based MEA. Basic cell-biological mechanisms that curtail the length of time intracellular access by the nanoelectrodes are discussed, and approaches to overcome this problem are offered.Recent development of biotechnologies that use induced human pluripotent stem cells taken from healthy subjects and patients, and in vitro drug screening for the development of personalized medicine as well as basic brain research will benefit tremendously from the use of MEAs that record the entire brain electrophysiological signaling repertoire from individual cells within an operational network rather than only extracellular FPs.

Multisite Attenuated Intracellular Recordings by Extracellular Multielectrode Arrays, a Perspective.

Multielectrode arrays (MEA) are used extensively for basic and applied electrophysiological research of neuronal- and cardiomyocyte-networks. Whereas immense progress has been made in realizing sophisticated MEA platforms of thousands of addressable, high-density, small diameter, low impedance sensors, the quality of the interfaces formed between excitable cells and classical planar sensor has not improved. As a consequence in vitro and in vivo MEA are "blind" to the rich and important "landscape" of sub-threshold synaptic potentials generated by individual neurons. Disregarding this essential fraction of network signaling repertoire has become the standard and almost the "scientific ideology" of MEA users. To overcome the inherent limitations of substrate integrated planar MEA platforms that only record extracellular field potentials, a number of laboratories have developed in vitro MEA for intracellular recordings. Most of these novel devices use vertical nano-rods or nano-wires that penetrate the plasma membrane of cultured cells and record the electrophysiological signaling in a manner similar to sharp intracellular microelectrodes. In parallel, our laboratory began to develop a bioinspired approach in-which cell biological energy resources are harnessed to self-force a cell into intimate contact with extracellular gold mushroom-shaped microelectrodes to record attenuated synaptic- and action-potentials with characteristic features of intracellular recordings. Here we describe some of the experiments that helped evolve the approach and elaborate on the biophysical principles that make it possible to record intracellular potentials by an array of extracellular electrode. We illustrate the qualities and limitations of the method and discuss prospects for further improvement of this technology.

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes.

In contrast to the extensive use of microelectrode array (MEA) technology in electrophysiological studies of cultured neurons and cardiac muscles, the vast field of skeletal muscle research has yet to adopt the technology. Here we demonstrate an empowering MEA technology for high quality, multisite, long-term electrophysiological recordings from cultured skeletal myotubes. Individual rat skeletal myotubes cultured on micrometer sized gold mushroom-shaped microelectrode (gMµE) based MEA tightly engulf the gMµEs, forming a high seal resistance between the myotubes and the gMµEs. As a consequence, spontaneous action potentials generated by the contracting myotubes are recorded as extracellular field potentials with amplitudes of up to 10 mV for over 14 days. Application of a 10 ms, 0.5-0.9 V voltage pulse through the gMµEs electroporated the myotube membrane, and transiently converted the extracellular to intracellular recording mode for 10-30 min. In a fraction of the cultures stable attenuated intracellular recordings were spontaneously produced. In these cases or after electroporation, subthreshold spontaneous potentials were also recorded. The introduction of the gMµE-MEA as a simple-to-use, high-quality electrophysiological tool together with the progress made in the use of cultured human myotubes opens up new venues for basic and clinical skeletal muscle research, preclinical drug screening, and personalized medicine.

Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes.

Substrate integrated planar microelectrode arrays is the "gold standard" method for millisecond-resolution, long-term, large-scale, cell-noninvasive electrophysiological recordings from mammalian neuronal networks. Nevertheless, these devices suffer from drawbacks that are solved by spike-detecting, spike-sorting and signal-averaging techniques which rely on estimated parameters that require user supervision to correct errors, merge clusters and remove outliers. Here we show that primary rat hippocampal neurons grown on micrometer sized gold mushroom-shaped microelectrodes (gMµE) functionalized simply by poly-ethylene-imine/laminin undergo self-assembly processes to form loose patch-like hybrid structures. More than 90% of the hybrids formed in this way record monophasic positive action potentials (APs). Of these, 34.5% record APs with amplitudes above 300 µV and up to 5,085 µV. This self-assembled neuron-gMµE configuration improves the recording quality as compared to planar MEA. This study characterizes and analyzes the electrophysiological signaling repertoire generated by the neurons-gMµE configuration, and discusses prospects to further improve the technology.

A feasibility study of multi-site,intracellular recordings from mammalian neurons by extracellular gold mushroom-shaped microelectrodes.

The development of multi-electrode array platforms for large scale recording of neurons is at the forefront of neuro-engineering research efforts. Recently we demonstrated, at the proof-of-concept level, a breakthrough neuron-microelectrode interface in which cultured Aplysia neurons tightly engulf gold mushroom-shaped microelectrodes (gMµEs). While maintaining their extracellular position, the gMµEs record synaptic- and action-potentials with characteristic features of intracellular recordings. Here we examined the feasibility of using gMµEs for intracellular recordings from mammalian neurons. To that end we experimentally examined the innate size limits of cultured rat hippocampal neurons to engulf gMµEs and measured the width of the "extracellular" cleft formed between the neurons and the gold surface. Using the experimental results we next analyzed the expected range of gMµEs-neuron electrical coupling coefficients. We estimated that sufficient electrical coupling levels to record attenuated synaptic- and action-potentials can be reached using the gMµE-neuron configuration. The definition of the engulfment limits of the gMµEs caps diameter at ≤2-2.5 µm and the estimated electrical coupling coefficients from the simulations pave the way for rational development and application of the gMµE based concept for in-cell recordings from mammalian neurons.

Nanocrystalline diamond surfaces for adhesion and growth of primary neurons, conflicting results and rational explanation.

Using a variety of proliferating cell types, it was shown that the surface of nanocrystalline diamond (NCD) provides a permissive substrate for cell adhesion and development without the need of complex chemical functionalization prior to cell seeding. In an extensive series of experiments we found that, unlike proliferating cells, post-mitotic primary neurons do not adhere to bare NCD surfaces when cultured in defined medium. These observations raise questions on the potential use of bare NCD as an interfacing layer for neuronal devices. Nevertheless, we also found that classical chemical functionalization methods render the "hostile" bare NCD surfaces with adhesive properties that match those of classically functionalized substrates used extensively in biomedical research and applications. Based on the results, we propose a mechanism that accounts for the conflicting results; which on one hand claim that un-functionalized NCD provides a permissive substrate for cell adhesion and growth, while other reports demonstrate the opposite.

Rescue of tau-induced synaptic transmission pathology by paclitaxel.

Behavioral and electrophysiological studies of Alzheimer's disease (AD) and other tauopathies have revealed that the onset of cognitive decline correlates better with synaptic dysfunctions than with hallmark pathologies such as extracellular amyloid-ß plaques, intracellular hyperphosphorylated tau or neuronal loss. Recent experiments have also demonstrated that anti-cancer microtubule (MT)-stabilizing drugs can rescue tau-induced behavioral decline and hallmark neuron pathologies. Nevertheless, the mechanisms underlying tau-induced synaptic dysfunction as well as those involved in the rescue of cognitive decline by MTs-stabilizing drugs remain unclear. Here we began to study these mechanisms using the glutaminergic sensory-motoneuron synapse derived from Aplysia ganglia, electrophysiological methods, the expression of mutant-human tau (mt-htau) either pre or postsynaptically and the antimitotic drug paclitaxel. Expression of mt-htau in the presynaptic neurons led to reduced excitatory postsynaptic potential (EPSP) amplitude generated by rested synapses within 3 days of mt-htau expression, and to deeper levels of homosynaptic depression. mt-htau-induced synaptic weakening correlated with reduced releasable presynaptic vesicle pools as revealed by the induction of asynchronous neurotransmitter release by hypertonic sucrose solution. Paclitaxel totally rescued tau-induced synaptic weakening by maintaining the availability of the presynaptic vesicle stores. Postsynaptic expression of mt-htau did not impair the above described synaptic-transmission parameters for up to 5 days. Along with earlier confocal microscope observations from our laboratory, these findings suggest that tau-induced synaptic dysfunction is the outcome of impaired axoplasmic transport and the ensuing reduction in the releasable presynaptic vesicle stores rather than the direct effects of mt-htau or paclitaxel on the synaptic release mechanisms.

Changing gears from chemical adhesion of cells to flat substrata toward engulfment of micro-protrusions by active mechanisms.

Microelectrode arrays increasingly serve to extracellularly record in parallel electrical activity from many excitable cells without inflicting damage to the cells by insertion of microelectrodes. Nevertheless, apart from rare cases they suffer from a low signal to noise ratio. The limiting factor for effective electrical coupling is the low seal resistance formed between the plasma membrane and the electronic device. Using transmission electron microscope analysis we recently reported that cultured Aplysia neurons engulf protruding micron size gold spines forming tight apposition which significantly improves the electrical coupling in comparison with flat electrodes (Hai et al 2009 Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices J. R. Soc. Interface 6 1153-65). However, the use of a transmission electron microscope to measure the extracellular cleft formed between the plasma membrane and the gold-spine surface may be inaccurate as chemical fixation may generate structural artifacts. Using live confocal microscope imaging we report here that cultured Aplysia neurons engulf protruding spine-shaped gold structures functionalized by an RGD-based peptide and to a significantly lesser extent by poly-l-lysine. The cytoskeletal elements actin and associated protein cortactin are shown to organize around the stalks of the engulfed gold spines in the form of rings. Neurons grown on the gold-spine matrix display varying growth patterns but maintain normal electrophysiological properties and form functioning synapses. It is concluded that the matrices of functionalized gold spines provide an improved substrate for the assembly of neuro-electronic hybrids.

Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone.

The transformation of a cut axonal end into a growth cone (GC), after axotomy, is a critical event in the cascade leading to regeneration. In an earlier series of studies we analyzed the cellular cascades that transform a cut axonal end into a competent GC. We found that axotomy of cultured Aplysia neurons leads to a transient elevation of the free intracellular Ca2+ concentration ([Ca2+]i), calpain activation and localized proteolysis of submembranal spectrin. These events are associated with the formation of distinct microtubule (MT) based vesicle traps that accumulate anterogradely transported vesicles that fuse with the spectrin free plasma membrane in support of the growth process (Erez, H., Malkinson, G., Prager-Khoutorsky, M., De Zeeuw, C.I., Hoogenraad, C.C., and Spira, M.E. 2007. Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J. Cell Biol. 176: 497-507.; Erez, H., and Spira, M.E. 2008. Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J. Comp. Neurol. 507: spc1.). Here we report that under conditions that limit calcium influx into the cut axonal end, axotomy leads to the formation of endbulbs (EBs) rather than to competent GCs. Under these conditions typical MT based vesicle traps are not formed, and Golgi derived vesicles concentrate at the very tip of the cut axon. Since under these conditions the spectrin barrier is not cleaved, vesicle fusion with the plasma membrane and actin polymerization are retarded and growth processes are impaired. We conclude that the immediate assembly of competent GC or an EB after axotomy is the outcome of autonomous local events that are shaped by the magnitudes of the [Ca2+]i gradients at the site of injury.

Tau-induced traffic jams reflect organelles accumulation at points of microtubule polar mismatching.

It is currently accepted that tau overexpression leads to impaired organelle transport and thus to neuronal degeneration. Nevertheless, the underlying mechanisms that lead to impaired organelle transport are not entirely clear. Using cultured Aplysia neurons and online confocal imaging of human tau, microtubules (MTs), the plus-end tracking protein - end-binding protein 3, retrogradely and anterogradely transported organelles, we found that overexpression of tau generates the hallmarks of human tau pathogenesis. Nevertheless, in contrast to earlier reports, we found that the tau-induced impairment of organelle transport is because of polar reorientation of the MTs along the axon or their displacement to submembrane domains. 'Traffic jams' reflect the accumulation of organelles at points of MT polar discontinuations or polar mismatching rather than because of MT depolymerization. Our findings offer a new mechanistic explanation for earlier observations, which established that tau overexpression leads to impaired retrograde and anterograde organelle transport, while the MT skeleton appeared intact.

Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones.

Following axotomy, both the proximal and distal cut axonal ends transform into growth cones (GCs). Whereas the GCs formed by the tip of the proximal segment branch to form neurites, the structure formed by the distal cut end fails to grow. The mechanisms underlying the formation of an aberrant GC by the distal cut end are not understood. Earlier we described the cascade that transforms the tip of the proximal cut axon into a GC. This involves microtubule (MT) polar reorientation, which culminates in the formation of two MT-based vesicle traps, one for Golgi-derived vesicles and the other that retains retrogradely transported vesicles. The formation of these traps is the outcome of local interactions between dynamically repolymerizing MTs and molecular motors. The concentration of Golgi-derived vesicles in the plus-end trap is essential for the successful generation of a functional GC. By using online confocal imaging of transected cultured Aplysia neurons, we analyzed here the restructuring of the distal cut end after axotomy. We found that initially the proximal and distal cut ends undergo identical alterations. Nevertheless, in contrast to the proximal end, the distal cut axon forms only a minus-end MT-based trap that concentrates endocytotic vesicles driven by minus-end oriented motors. Whereas the MTs forming the trap polymerize pointing their plus-ends centrifugally to form finger-like protrusions, the trapped vesicles cannot translocate out to fuse with the plasma membrane. Thus, the structure formed at the distal cut axon is incompetent to support growth processes.

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 microm 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.

On-line confocal imaging of the events leading to structural dedifferentiation of an axonal segment into a growth cone after axotomy.

The transformation of a transected axonal tip into a growth cone (GC) after axotomy is a critical step in the cascade of events leading to regeneration. However, the mechanisms underlying it are largely unknown. In earlier studies we reported that axotomy of cultured Aplysia neurons leads to a transient and local increase in the free intracellular Ca2+ concentration, calpain activation, and localized proteolysis of the submembranal spectrin. In a recent ultrastructural study, we reported that calpain activation is critical for the restructuring of the microtubules and neurofilaments at the cut axonal end to form a compartment in which vesicles accumulate. By using on-line confocal imaging of microtubules (MTs), actin, and vesicles in cultured Aplysia neurons, we studied the kinetics of the transformation and examined some of the mechanisms that orchestrate it. We report that perturbation of the MTs' polymerization by nocodazole inhibits the formation of an MT-based compartment in which the vesicles accumulate, yet actin repolymerization proceeds normally to form a nascent GC's lamellipodium. Nevertheless, under these conditions, the lamellipodium fails to expand and form neurites. When actin filament polymerization is inhibited by cytochalasin D or jasplakinolide, the MT-based compartment is formed and vesicles accumulate at the cut axonal end. However, a GC's lamellipodium is not formed, and the cut axonal end fails to regenerate. A growth-competent GC is formed only when MT restructuring, the accumulation of vesicles, and actin polymerization properly converge in time and space.