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.

Graphene Oxide Nanosheets Reshape Synaptic Function in Cultured Brain Networks.

Graphene offers promising advantages for biomedical applications. However, adoption of graphene technology in biomedicine also poses important challenges in terms of understanding cell responses, cellular uptake, or the intracellular fate of soluble graphene derivatives. In the biological microenvironment, graphene nanosheets might interact with exposed cellular and subcellular structures, resulting in unexpected regulation of sophisticated biological signaling. More broadly, biomedical devices based on the design of these 2D planar nanostructures for interventions in the central nervous system require an accurate understanding of their interactions with the neuronal milieu. Here, we describe the ability of graphene oxide nanosheets to down-regulate neuronal signaling without affecting cell viability.

Graphene-Based Interfaces Do Not Alter Target Nerve Cells.

Neural-interfaces rely on the ability of electrodes to transduce stimuli into electrical patterns delivered to the brain. In addition to sensitivity to the stimuli, stability in the operating conditions and efficient charge transfer to neurons, the electrodes should not alter the physiological properties of the target tissue. Graphene is emerging as a promising material for neuro-interfacing applications, given its outstanding physico-chemical properties. Here, we use graphene-based substrates (GBSs) to interface neuronal growth. We test our GBSs on brain cell cultures by measuring functional and synaptic integrity of the emerging neuronal networks. We show that GBSs are permissive interfaces, even when uncoated by cell adhesion layers, retaining unaltered neuronal signaling properties, thus being suitable for carbon-based neural prosthetic devices.

Improving cardiac myocytes performance by carbon nanotubes platforms.

The application of nanotechnology to the cardiovascular system has increasingly caught scientists' attention as a potentially powerful tool for the development of new generation devices able to interface, repair, or boost the performance of cardiac tissue. Carbon nanotubes (CNTs) are considered as promising materials for nanomedicine applications in general and have been recently tested toward excitable cell growth. CNTs are cylindrically shaped structures made up of rolled-up graphene sheets, with unique electrical, thermal, and mechanical properties, able to effectively conducting electrical current in electrochemical interfaces. CNTs-based scaffolds have been recently found to support the in vitro growth of cardiac cells: in particular, their ability to improve cardiomyocytes proliferation, maturation, and electrical behavior are making CNTs extremely attractive for the development and exploitation of interfaces able to impact on cardiac cells physiology and function.

Adhesion to carbon nanotube conductive scaffolds forces action-potential appearance in immature rat spinal neurons.

In the last decade, carbon nanotube growth substrates have been used to investigate neurons and neuronal networks formation in vitro when guided by artificial nano-scaled cues. Besides, nanotube-based interfaces are being developed, such as prosthesis for monitoring brain activity. We recently described how carbon nanotube substrates alter the electrophysiological and synaptic responses of hippocampal neurons in culture. This observation highlighted the exceptional ability of this material in interfering with nerve tissue growth. Here we test the hypothesis that carbon nanotube scaffolds promote the development of immature neurons isolated from the neonatal rat spinal cord, and maintained in vitro. To address this issue we performed electrophysiological studies associated to gene expression analysis. Our results indicate that spinal neurons plated on electro-conductive carbon nanotubes show a facilitated development. Spinal neurons anticipate the expression of functional markers of maturation, such as the generation of voltage dependent currents or action potentials. These changes are accompanied by a selective modulation of gene expression, involving neuronal and non-neuronal components. Our microarray experiments suggest that carbon nanotube platforms trigger reparative activities involving microglia, in the absence of reactive gliosis. Hence, future tissue scaffolds blended with conductive nanotubes may be exploited to promote cell differentiation and reparative pathways in neural regeneration strategies.

Carbon nanotubes in neuroregeneration and repair.

In the last decade, we have experienced an increasing interest and an improved understanding of the application of nanotechnology to the nervous system. The aim of such studies is that of developing future strategies for tissue repair to promote functional recovery after brain damage. In this framework, carbon nanotube based technologies are emerging as particularly innovative tools due to the outstanding physical properties of these nanomaterials together with their recently documented ability to interface neuronal circuits, synapses and membranes. This review will discuss the state of the art in carbon nanotube technology applied to the development of devices able to drive nerve tissue repair; we will highlight the most exciting findings addressing the impact of carbon nanotubes in nerve tissue engineering, focusing in particular on neuronal differentiation, growth and network reconstruction.

Carbon nanotubes: artificial nanomaterials to engineer single neurons and neuronal networks.

In the past decade, nanotechnology applications to the nervous system have often involved the study and the use of novel nanomaterials to improve the diagnosis and therapy of neurological diseases. In the field of nanomedicine, carbon nanotubes are evaluated as promising materials for diverse therapeutic and diagnostic applications. Besides, carbon nanotubes are increasingly employed in basic neuroscience approaches, and they have been used in the design of neuronal interfaces or in that of scaffolds promoting neuronal growth in vitro. Ultimately, carbon nanotubes are thought to hold the potential for the development of innovative neurological implants. In this framework, it is particularly relevant to document the impact of interfacing such materials with nerve cells. Carbon nanotubes were shown, when modified with biologically active compounds or functionalized in order to alter their charge, to affect neurite outgrowth and branching. Notably, purified carbon nanotubes used as scaffolds can promote the formation of nanotube-neuron hybrid networks, able per se to affect neuron integrative abilities, network connectivity, and synaptic plasticity. We focus this review on our work over several years directed to investigate the ability of carbon nanotube platforms in providing a new tool for nongenetic manipulations of neuronal performance and network signaling.

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.

Interfacing neurons with carbon nanotubes: (re)engineering neuronal signaling.

Carbon nanotubes (CNTs) are cylindrically shaped nanostructures made by sheets of graphene rolled up to form hollow tubes. Owing to their unique range of thermal, electronic, and structural properties, CNTs have been rapidly developing as a technology platform for biological and medical applications, including those designed to develop novel neuro-implantable devices. Depending on their structure, CNTs combine an incredible strength with an extreme flexibility. Further, these materials exhibit physical and chemical properties which allow them to efficiently conduit electrical current in electrochemical interfaces. CNTs can be organized in scaffolds made up of small fibers or tubes with diameters similar to those of neural processes such as axons and dendrites. Recently, CNT scaffolds have been found to promote growth, differentiation, and survival of neurons and to modify their electrophysiological properties. These features make CNTs an attractive material for the design of nano-bio hybrid systems able to govern cell-specific behaviors in cultured neuronal networks. The leading scope of this short review is to highlight how nanotube scaffolds can impact on neuronal signaling ability. In particular, we will focus on the direct and specific interactions between this synthetic nanomaterial and biological cell membranes, and on the ability of CNTs to improve interfaces developed to record or to stimulate neuronal activity. CNTs hold the potential for the development of innovative nanomaterial-based neurological implants. Therefore, it is particularly relevant to improve our knowledge on the impact on neuronal performance of interfacing nerve cells with CNTs.

The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinement.

Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca(2+) signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca(2+) imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca(2+) transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca(2+) oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca(2+) confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca(2+).

Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in Ca(v)2.1 knockin migraine mice.

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 1: FHM1) is caused by mutations in Ca(V)2.1 (P/Q-type) Ca(2+) channels. Knockin mice carrying a FHM1 mutation show increased neuronal P/Q-type current and facilitation of induction and propagation of cortical spreading depression (CSD), the phenomenon that underlies migraine aura and may activate migraine headache mechanisms. We studied cortical neurotransmission in neuronal microcultures and brain slices of FHM1 mice. We show gain of function of excitatory neurotransmission due to increased action-potential-evoked Ca(2+) influx and increased probability of glutamate release at pyramidal cell synapses but unaltered inhibitory neurotransmission at fast-spiking interneuron synapses. Using an in vitro model of CSD, we show a causative link between enhanced glutamate release and CSD facilitation. The synapse-specific effect of FHM1 mutations points to disruption of excitation-inhibition balance and neuronal hyperactivity as the basis for episodic vulnerability to CSD ignition in migraine.

Neutralization of nerve growth factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons.

The molecular mechanisms of migraine pain are incompletely understood, although migraine mediators such as NGF and calcitonin gene-related peptide (CGRP) are believed to play an algogenic role. Although NGF block is proposed as a novel analgesic approach, its consequences on nociceptive purinergic P2X receptors of trigeminal ganglion neurons remain unknown. We investigated whether neutralizing NGF might change the function of P2X3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X3 receptor-mediated currents and Ca2+ transients, an effect opposite to exogenously applied NGF. Recovery from receptor desensitization was delayed by anti-NGF treatment without changing desensitization onset. NGF neutralization was associated with decreased threonine phosphorylation of P2X3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X2/3 receptors, consistent with enhanced membrane location of P2X2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X3 subunits and their membrane assembly with P2X2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X3 receptor activity, indicating separate NGF- or CGRP-mediated mechanisms to upregulate P2X3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X3 receptor activation. Our findings outline the important contribution by NGF signaling to nociception of trigeminal sensory neurons, which could be counteracted by anti-NGF pretreatment.

Activity-independent intracellular Ca2+ oscillations are spontaneously generated by ventral spinal neurons during development in vitro.

Within the CNS, distinct neurons may rely on different processes to modulate cytosolic Ca2+ depending on the network developmental phase. In particular, in the immature spinal cord, synchronous electrical discharges are coupled with biochemical signals triggered by intracellular Ca2+ waves. Nevertheless, the presence of neuronal-specific Ca2+ elevations independent from synaptic activity within mammalian spinal networks has not yet been described. The present report is the first description of repetitive calcium events generated by discrete ventral spinal neurons maintained in organotypic culture during in vitro maturation stages crucial for network evolution. Ventral interneurons in one-third of slices displayed spontaneous intracellular calcium transients suppressed by calcium-free extracellular solution or by application of cobalt, and resistant to blockers of network activity like TTX, CNQX, APV, strychnine or bicuculline. Our data suggest a primary role for mitochondria in intracellular calcium oscillations, because CCCP, that selectively collapses the mitochondrial electrochemical gradient, eliminated the ability of these neurons to show activity-independent calcium oscillations. Likewise, CGP-37157, a blocker of mitochondrial Na+/Ca2+ exchanger, inhibited oscillations in the majority of neurons. We propose that spontaneous Ca2+ transients, dynamically regulated by mitochondria, occurred in a discrete cluster of interneurons possibly to guide the development of synaptic connections.

Delayed upregulation of ATP P2X3 receptors of trigeminal sensory neurons by calcitonin gene-related peptide.

Recent evidence indicates a key role for the neuropeptide calcitonin gene-related peptide (CGRP) in migraine pain, as demonstrated by the strong analgesic action of CGRP receptor antagonists, although the mechanisms of this effect remain unclear. Most trigeminal nociceptive neurons releasing CGRP also express ATP-activated purinergic P2X3 receptors to transduce pain. To understand whether the CGRP action involves P2X3 receptor modulation, the model of trigeminal nociceptive neurons in culture was used to examine the long-term action of this peptide. Although 79% of CGRP-binding neurons expressed P2X3 receptors, acute application of CGRP did not change P2X3 receptor function. Nevertheless, after 1 h of CGRP treatment, strong enhancement of the amplitude of P2X3 receptor currents was observed together with accelerated recovery from desensitization. Receptor upregulation persisted up to 10 h (despite CGRP washout), was accompanied by increased P2X3 gene transcription, and was fully prevented by the CGRP antagonist CGRP(8-37). Surface biotinylation showed CGRP augmented P2X3 receptor expression, consistent with confocal microscopy data indicating enhanced P2X3 immunoreactivity beneath the neuronal membrane. These results suggest that CGRP stimulated trafficking of P2X3 receptors to the cell-surface membrane. Using pharmacological tools, we demonstrated that this effect of CGRP was dependent on protein kinase A and PKC activation and was prevented by the trafficking inhibitor brefeldin A. Capsaicin-sensitive TRPV1 vanilloid receptors were not upregulated. The present data demonstrate a new form of selective, slow upregulation of nociceptive P2X3 receptors on trigeminal neurons by CGRP. This mechanism might contribute to pain sensitization and represents a model of neuronal plasticity in response to a migraine mediator.

Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin.

BACKGROUND: Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated. RESULTS: Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X(3) and TRPV1 receptors after 1-4 days in culture (together with their more frequent co-localization), while P2X(2) ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca(2+) imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist alpha, beta-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X(3) receptors (selectively antagonized by A-317491) and heteromeric P2X(2/3) receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X(3) receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X(3) receptors. CONCLUSION: Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors.

Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+-sensing modulatory sites.

On nociceptive neurons, one important mechanism to generate pain signals is the activation of P2X(3) receptors, which are membrane proteins gated by extracellular ATP. In the presence of the agonist, P2X(3) receptors rapidly desensitize and then recover slowly. One unique property of P2X(3) receptors is the recovery acceleration by extracellular Ca(2+) that can play the role of the gain-setter of receptor function only when P2X(3) receptors are desensitized. To study negatively charged sites potentially responsible for this action of Ca(2+), we mutated 15 non-conserved aspartate or glutamate residues in the P2X(3) receptor ectodomain with alanine and expressed such mutated receptors in human embryonic kidney cells studied with patch clamping. Unlike most mutants, D266A (P2X(3) receptor numbering) desensitized very slowly, indicating that this residue is important for generating desensitization. Recovery appeared structurally distinct from desensitization because E111A and D266A had a much faster recovery and D220A and D289A had a much slower one despite their standard desensitization. Furthermore, E161A, E187A, or E270A mutants showed lessened sensitivity to the action of extracellular Ca(2+), suggesting that these determinants were important for the effect of this cation on desensitization recovery. This study is the first report identifying several negative residues in the P2X(3) receptor ectodomain differentially contributing to the general process of receptor desensitization. At least one residue was important to enable the development of rapid desensitization, whereas others controlled recovery from it or the facilitating action of Ca(2+). Thus, these findings outline diverse potential molecular targets to modulate P2X(3) receptor function in relation to its functional state.

Quantal release of ATP from clusters of PC12 cells.

Although ATP is important for intercellular communication, little is known about the mechanism of endogenous ATP release due to a dearth of suitable models. Using PC12 cells known to express the P2X2 subtype of ATP receptors and to store ATP with catecholamines inside dense-core vesicles, we found that clusters of PC12 cells cultured for 3-7 days generated small transient inward currents (STICs) after an inward current elicited by exogenous ATP. The amplitude of STICs in individual cells correlated with the peak amplitude of ATP-induced currents. STICs appeared as asynchronous responses (approximately 20 pA average amplitude) for 1-20 s and were investigated with a combination of patch clamping, Ca2+ imaging, biochemistry and electron microscopy. Comparable STICs were produced by focal KCl pulses and were dependent on extracellular Ca2+. STICs were abolished by the P2X antagonist PPADS and potentiated by Zn2+, suggesting they were mediated by P2X2 receptor activation. The highest probability of observing STICs was after the peak of intracellular Ca2+ increase caused by KCl. Biochemical measurements indicated that KCl application induced a significant release of ATP from PC12 cells. Electron microscopy studies showed narrow clefts without 'synaptic-like' densities between clustered cells. Our data suggest that STICs were caused by quantal release of endogenous ATP by depolarized PC12 cells in close juxtaposition to the recorded cell. Thus, STICs may be a new experimental model to characterize the physiology of vesicular release of ATP and to study the kinetics and pharmacology of P2X2 receptor-mediated quantal currents.

Chronic NGF treatment of rat nociceptive DRG neurons in culture facilitates desensitization and deactivation of GABAA receptor-mediated currents.

1 The present study tested the hypothesis that nerve growth factor (NGF) could affect presynaptic inhibition mediated by GABAA (GABA-sensitive ionotropic receptors) receptors on the afferents of nociceptive dorsal root ganglia (DRG) neurons, thus reducing the filtering of central nociceptive signals. 2 To investigate this issue, small-diameter, nociceptive DRG neurons were cultured for 48-72 h either in the normal medium or in the presence of NGF (50 ng ml(-1)). After 15 min washout, cells were patch clamped with Cs+ containing electrodes to block GABAB (GABA-sensitive metabotropic receptors) receptor-activated currents. 3 Chronically treated DRG neurons showed no difference in the peak amplitude of GABA-induced currents. However, NGF-treated cells exhibited increased fading of the response to continuous GABA application, with faster desensitization onset, decreased residual current at the end of agonist application and slower recovery from desensitization. Moreover, the deactivation phase after brief agonist pulses was also accelerated. 4 Unlike responses to GABA, chronic NGF treatment had no effect on the desensitization process to the excitatory transmitter ATP, as no difference in peak amplitude, fast and slow time constants of current decay was found. 5 Experimental tests indicated that the observed effects on GABA currents were not a reactive process triggered by washing out NGF after its long application. Acutely applied NGF did not change GABAA receptor-mediated responses. 6 NGF-treated neurons showed decreased sensitivity to the antagonist picrotoxin. The action of pentobarbitone, midazolam, bicuculline or gabazine was, however, unchanged. 7 These observations suggest that the modulation of GABAA receptor function of DRG nociceptors by NGF may contribute to the algogenic action of this neurotrophin.