Altered development in GABA co-release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

KEY POINTS: Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS). In vitro models allow the investigation of the genetically modified counter-regulator of motoneuron toxicity and may help in addressing ALS therapy. Spinal organotypic slice cultures from a mutant form of human superoxide dismutase 1 (SOD1G93A) mouse model of ALS allow the detection of altered glycinergic inhibition in spinal microcircuits. This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1(G93A) pathogenesis. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1(G93A) ). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild-type and SOD1(G93A) mice to investigate the development of glycine-receptor-mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co-release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1(G93A) developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1(G93A) spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1(G93A) glycinergic terminals in cultured and ex vivo spinal slices.

Carbon nanotube scaffolds tune synaptic strength in cultured neural circuits: novel frontiers in nanomaterial-tissue interactions.

A long-term goal of tissue engineering is to exploit the ability of supporting materials to govern cell-specific behaviors. Instructive scaffolds code such information by modulating (via their physical and chemical features) the interface between cells and materials at the nanoscale. In modern neuroscience, therapeutic regenerative strategies (i.e., brain repair after damage) aim to guide and enhance the intrinsic capacity of the brain to reorganize by promoting plasticity mechanisms in a controlled fashion. Direct and specific interactions between synthetic materials and biological cell membranes may play a central role in this process. Here, we investigate the role of the material's properties alone, in carbon nanotube scaffolds, in constructing the functional building blocks of neural circuits: the synapses. Using electrophysiological recordings and rat cultured neural networks, we describe the ability of a nanoscaled material to promote the formation of synaptic contacts and to modulate their plasticity.

A morphological analysis of growth cones of DRG neurons combining atomic force and confocal microscopy.

We have analyzed the morphology of growth cones of differentiating neurons from rat dorsal root ganglia (DRG) with conventional Laser Scanning Confocal Microscopy (LSCM) and Atomic Force Microscopy (AFM). Images of immunofluorescent DRG growth cones colabeled for actin and tubulin were superimposed to images obtained with AFM at different scanning forces. In order to reduce changes of the image surface caused by the pressure of the AFM tip, we have developed a procedure to obtain 0pN AFM images. Further analysis of these images revealed topographical structures with nanoscale dimensions, referred to as "invaginations" or "holes". These holes had an area varying from 0.01 to 3.5 microm(2) with a depth varying from 2 to 178 nm. Comparative analysis with LSCM images showed that these holes correspond to regions where staining of both actin and tubulin was negligible. Filopodia height varied from 40 to 270 nm and their diameter from 113 to 887 nm. These results show that the combination of LSCM and AFM reveal structural details with a nanoscale dimension of DRG growth cones, difficult to resolve with conventional microscopy.

Integration of confocal and atomic force microscopy images.

Atomic force microscopy (AFM) provides the possibility to map the 3D structure of viewed objects with a nanometric resolution, which cannot be achieved with other imaging methods such as conventional video imaging and confocal fluorescent microscopy. Video imaging with CCD cameras can provide an analysis of biological events with a temporal and spatial resolution not possible with AFM, while confocal imaging allows the simultaneous acquisition of immunofluorescence images. In this communication we present a simple method to combine AFM and confocal images to study differentiating embryonic stem (ES) cells-derived and dorsal root ganglia (DRG) neurons in culture. Neurons were grown on coverslips with micrometric markers that allow finding and imaging the same neuron with different microscopes. AFM and confocal images were registered using conventional methods used in Computer Science. The combination of these two techniques allows relating functional properties to morphological features of imaged neurons.

3D meshes of carbon nanotubes guide functional reconnection of segregated spinal explants.

In modern neuroscience, significant progress in developing structural scaffolds integrated with the brain is provided by the increasing use of nanomaterials. We show that a multiwalled carbon nanotube self-standing framework, consisting of a three-dimensional (3D) mesh of interconnected, conductive, pure carbon nanotubes, can guide the formation of neural webs in vitro where the spontaneous regrowth of neurite bundles is molded into a dense random net. This morphology of the fiber regrowth shaped by the 3D structure supports the successful reconnection of segregated spinal cord segments. We further observed in vivo the adaptability of these 3D devices in a healthy physiological environment. Our study shows that 3D artificial scaffolds may drive local rewiring in vitro and hold great potential for the development of future in vivo interfaces.

From 2D to 3D: novel nanostructured scaffolds to investigate signalling in reconstructed neuronal networks.

To recreate in vitro 3D neuronal circuits will ultimately increase the relevance of results from cultured to whole-brain networks and will promote enabling technologies for neuro-engineering applications. Here we fabricate novel elastomeric scaffolds able to instruct 3D growth of living primary neurons. Such systems allow investigating the emerging activity, in terms of calcium signals, of small clusters of neurons as a function of the interplay between the 2D or 3D architectures and network dynamics. We report the ability of 3D geometry to improve functional organization and synchronization in small neuronal assemblies. We propose a mathematical modelling of network dynamics that supports such a result. Entrapping carbon nanotubes in the scaffolds remarkably boosted synaptic activity, thus allowing for the first time to exploit nanomaterial/cell interfacing in 3D growth support. Our 3D system represents a simple and reliable construct, able to improve the complexity of current tissue culture models.

Spinal cord explants use carbon nanotube interfaces to enhance neurite outgrowth and to fortify synaptic inputs.

New developments in nanotechnology are increasingly designed to modulate relevant interactions between nanomaterials and neurons, with the aim of exploiting the physical properties of synthetic materials to tune desired and specific biological processes. Carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. Recent reports show that nanotubes can sustain and promote electrical activity in networks of cultured neurons. However, such results are usually limited to carbon nanotube/neuron hybrids formed on a monolayer of dissociated brain cells. In the present work, we used organotypic spinal slices to model multilayer tissue complexity, and we interfaced such spinal segments to carbon nanotube scaffolds for weeks. By immunofluorescence, scanning and transmission electronic microscopy, and atomic force microscopy, we investigated nerve fiber growth when neuronal processes exit the spinal explant and develop in direct contact to the substrate. By single-cell electrophysiology, we investigated the synaptic activity of visually identified ventral interneurons, within the ventral area of the explant, thus synaptically connected, but located remotely, to the substrate/network interface. Here we show that spinal cord explants interfaced for weeks to purified carbon nanotube scaffolds expand more neuronal fibers, characterized by different mechanical properties and displaying higher growth cones activity. On the other hand, exploring spontaneous and evoked synaptic activity unmasks an increase in synaptic efficacy in neurons located at as far as 5 cell layers from the cell-substrate interactions.

Mechanical computation in neurons.

Growth cones are the main motile structures located at the tip of neurites and are composed of a lamellipodium from which thin filopodia emerge. In this article, we analyzed the kinetics and dynamics of growth cones with the aim to understand two major issues: first, the strategy used by filopodia and lamellipodia during their exploration and navigation; second, what kind of mechanical problems neurons need to solve during their operation. In the developing nervous system and in the adult brain, neurons constantly need to solve mechanical problems. Growth cones must decide how to explore the environment and in which direction to grow; they also need to establish the appropriate contacts, to avoid obstacles and to determine how much force to exert. Here, we show that in sparse cultures, filopodia grow and retract following statistical patterns, nearly optimal for an efficient exploration of the environment. In a dense culture, filopodia exploration is still present although significantly reduced. Analysis on 1271, 6432, and 185 pairs of filopodia of DRG, PC12 and Hippocampal neurons respectively showed that the correlation coefficient |rho| of the growth of more than 50% of filopodia pairs was >0.15. From a computational point of view, filopodia and lamellipodia motion can be described by a random process in which errors are corrected by efficient feedback loops. This article argues that neurons not only process sensory signals, but also solve mechanical problems throughout their entire lifespan, from the early stages of embryogenesis to adulthood.

Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components.

During neuronal differentiation, lamellipodia and filopodia explore the environment in search for the correct path to the axon's final destination. Although the motion of lamellipodia and filopodia has been characterized to an extent, little is known about the force they exert. In this study, we used optical tweezers to measure the force exerted by filopodia and lamellipodia with a millisecond temporal resolution. We found that a single filopodium exerts a force not exceeding 3 pN, whereas lamellipodia can exert a force up to 20 pN. Using metabolic inhibitors, we showed that no force is produced in the absence of actin polymerization and that development of forces larger than 3 pN requires microtubule polymerization. These results show that actin polymerization is necessary for force production and demonstrate that not only do neurons process information, but they also act on their environment exerting forces varying from tenths pN to tens of pN.