Papers by Fabio Benfenati
Acta Physiologica Scandinavica, 1986
Nature Photonics, 2013
Interfacing organic electronics with biological substrates offers new possibilities for biotechno... more Interfacing organic electronics with biological substrates offers new possibilities for biotechnology by taking advantage of the beneficial properties exhibited by organic conducting polymers. These polymers have been used for cellular interfaces in several applications, including cellular scaffolds, neural probes, biosensors and actuators for drug release. Recently, an organic photovoltaic blend has been used for neuronal stimulation via a photo-excitation process. Here, we document the use of a single-component organic film of poly(3-hexylthiophene) (P3HT) to trigger neuronal firing upon illumination. Moreover, we demonstrate that this bio-organic interface restores light sensitivity in explants of rat retinas with lightinduced photoreceptor degeneration. These findings suggest that all-organic devices may play an important future role in subretinal prosthetic implants. E xtracellular electrical stimulation of neurons provides the basis for many implantable prosthetic devices, that help to restore motor activity (such as electrodes for functional electrical stimulation 1,2 ), to treat drug-resistant diseases (for example, deep brain stimulation in Parkinson's disease 3 ) and to restore sensory perception (such as in cochlear implants 4 or retinal prostheses 5-7 ). In the last decade, much effort has been dedicated to improve the interfaces between electrodes and neuronal tissues; indeed many issues related to biological affinity, biocompatibility, mechanical flexibility, ease of functionalization and cost-effectiveness have been investigated extensively. However, the use of light-enabled processes for cell stimulation has received much less attention, with the exception of the outstanding progress made in optogenetic techniques in recent years . Given this scenario, the interfacing of organic electronics and biological substrates offers substantial new possibilities 10-12 . Organic conducting polymers have been used widely as culturing substrates 13 , three-dimensional scaffolds 14 , electrode coatings 15,16 , organic biosensors 17,18 , actuators for drug release 19,20 and organic electrodes for controlling cell seeding 21 , growth 22,23 and activity detection 24 . Recently, an organic photovoltaic device has also been used for neuronal photostimulation 25,26 . This system provides interesting improvements when compared to inorganic semiconductors 5,27,28 and has great potential for in vivo biological applications, such as retinal prostheses.
Bioinspired Approaches for Human-Centric Technologies, 2014
Optics Letters, 2011
The acquisition of high-resolution images in three dimensions is of utmost importance for the mor... more The acquisition of high-resolution images in three dimensions is of utmost importance for the morphological and functional investigation of biological tissues. Here, we present a laser scanning two-photon microscope with remote and motionless control of the focus position. The movement of the excitation spot along the propagation direction is achieved by shaping the laser wavefront with a spatial light modulator. Depending on the optical properties of the objective in use, this approach allows z movements in a range of tens to hundreds of micrometers with small changes of the point spread function. We applied this technique for the three-dimensional (3D) imaging of fluorescent cells in the mouse neocortex in vivo. The presented system bypasses the limitations of microscopes based on moving objectives, enabling high-resolution inertia-free 3D imaging.
Nature Communications, 2012
GABA A receptors (GABA A Rs) exist as different subtype variants showing unique functional proper... more GABA A receptors (GABA A Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA A R are largely unknown. The intracellular concentration of chloride ([Cl − ] i ), the main ion permeating through GABA A Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl − ] i could modulate the sequential expression of specific GABA A Rs subtypes in primary cerebellar neurons. We show that [Cl − ] i regulates the expression of α3-1 and δ-containing GABA A receptors, responsible for phasic and tonic inhibition, respectively. our findings highlight the role of [Cl − ] i in tuning the strength of GABAergic responses by acting as an intracellular messenger.
Nature Communications, 2011
A key issue in the realization of retinal prosthetic devices is reliable transduction of informat... more A key issue in the realization of retinal prosthetic devices is reliable transduction of information carried by light into specific patterns of electrical activity in visual information processing networks. soft organic materials can be used to couple artificial sensors with neuronal tissues. Here, we interface a network of primary neurons with an organic blend. We show that primary neurons can be successfully grown onto the polymer layer without affecting the optoelectronic properties of the active material or the biological functionality of neuronal network. moreover, action potentials can be triggered in a temporally reliable and spatially selective manner with short pulses of visible light. our results may lead to new neuronal communication and photo manipulation techniques, thus paving way to the development of artificial retinas and other neuroprosthetic interfaces based on organic photodetectors. I nterfacing artificial functional materials and living neuronal tissues is at the forefront of bionanotechnology 1 . Attempts so far have been based on microscale processing of metals, inorganic semiconductors as electrodes 2 or photoactive layers in biased devices 3,4 , and more recently, nanomaterials have been investigated 5,6 . However, in spite of extensive research, the communication between biological tissues and artificial sensors is still a challenge. Constraints exist in the complexity of the fabrication processes (that is, metal and semiconductor lithography) and the mechanical properties of the implanted sensing/recording elements (poor flexibility and biocompatibility) that could elicit deleterious tissue reactions such as inflammation and gliosis. In addition, electrodes have fixed geometries that limit the location in space of the stimulus, and electrical currents are often detrimental to the overall system.
Journal of neurochemistry, 2007
The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, ... more The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH3 cells that express voltage-dependent calcium channels and are subjected to Ca2+ oscillations, we found that treatment with high K+-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH3 cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH3 cells and rat brain synaptosomes showed that K+-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH3 cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.
Journal of Neurochemistry, 2004
The Ca 2+ -sensor protein S100A1 was recently shown to bind in vitro to synapsins, a family of sy... more The Ca 2+ -sensor protein S100A1 was recently shown to bind in vitro to synapsins, a family of synaptic vesicle phosphoproteins involved in the regulation of neurotransmitter release. In this paper, we analyzed the distribution of S100A1 and synapsin I in the CNS and investigated the effects of the S100A1/synapsin binding on the synapsin functional properties. Subcellular fractionation of rat brain homogenate revealed that S100A1 is present in the soluble fraction of isolated nerve endings. Confocal laser scanning microscopy and immunogold immunocytochemistry demonstrated that S100A1 and synapsin codistribute in a subpopulation (5-20%) of nerve terminals in the mouse cerebral and cerebellar cortices. By forming heterocomplexes with either dephosphorylated or phosphorylated synapsin I, S100A1
European Journal of Neuroscience, 2000
Cytokines are extracellular mediators that have been reported to affect neurotransmitter release ... more Cytokines are extracellular mediators that have been reported to affect neurotransmitter release and synaptic plasticity phenomena when applied in vitro. Most of these effects occur rapidly after the application of the cytokines and are presumably mediated through the activation of protein phosphorylation processes. While many cytokines have an in¯ammatory action, interleukin-6 (IL-6) has been found to have a neuroprotective effect against ischaemia lesions and glutamate excitotoxicity, and to increase neuronal survival in a variety of experimental conditions. In this paper, the functional effects of IL-6 on the spread of excitation visualized by dark-®eld/ infrared videomicroscopy in rat cortical slices and on glutamate release from cortical synaptosomes were analysed and correlated with the activation of the STAT3, mitogen-activated protein kinase ERK (MAPK/ERK) and stress-activated protein kinase/cJun NH 2terminal kinase (SAPK/JNK) pathways. We have found that IL-6 depresses the spread of excitation and evoked glutamate release in the cerebral cortex, and that these effects are accompanied by a stimulation of STAT3 tyrosine phosphorylation, an inhibition of MAPK/ERK activity, a decreased phosphorylation of the presynaptic MAPK/ERK substrate synapsin I and no detectable effects on SAPK/JNK. The effects of IL-6 were effectively counteracted by treatment of the cortical slices with the tyrosine kinase inhibitor lavendustin A. The inhibitory effects of IL-6 on glutamate release and on the spread of excitation in the rat cerebral cortex indicate that the protective effect of IL-6 on neuronal survival could be mediated by a downregulation of neuronal activity, release of excitatory neurotransmitters and MAPK/ERK activity.
Annals of The New York Academy of Sciences, 1994
Proceedings of The National Academy of Sciences, 1996
The ability of neurotrophins to modulate the survival and differentiation of neuronal populations... more The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified
Proceedings of The National Academy of Sciences, 1998
Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have b... more Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuronspecific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12-13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.
The EMBO Journal, 2013
Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate... more Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/ Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na þ channels, thereby decreasing the neuronal Na þ current density. This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, reestablishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/ NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.
Biochemistry, 1992
Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are a... more Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.
Proceedings of The National Academy of Sciences, 1994
Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two fnking Src hom... more Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two fnking Src homology 3 (SH3) do . One hfnction of Grb2 is to couple tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors (tough its SH3 domains). Using an overlay assay, we have identified four major Grb2-binding proteins in synaptic fractions. These proteins interact with wild-type Grb2 but not with Grb2 containing point mutations in each of its two SH3 do corresponding to the loss of function mutants in the Caenorhabids ekgans Grb2 homologue sem-5. Two of the proteins, mSos and dynamin, were previously shown to bind Grb2. The third protein of 145 kDa is brain spedflc and to our knowledge has not been previously described. The fourth protein is synapsin I. Dynamin is required for synaptic vesicle endocytosis and synapsin I is thought to mediate the interaction of synaptic vesicles with the presynaptic cytomatrix. These data suggest that Grb2, or other protein con ing 513 doins, may play a role in the regulation of the exo/endocytotic cycle of synaptic vesicles and therefore of neurotransmitter release. Src homology 2 (SH2) and Src homology 3 (SH3) domains are protein modules involved in protein-protein interactions including those mediating regulatory cascades from cell surface receptors to intracellular effector proteins. SH2 and SH3 domains, which were originally recognized in c-Src (1), interact with phosphotyrosine-containing sequences and proline-rich sequences, respectively. SH2 and SH3 domains
Febs Letters, 1993
SNAP-25 a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinur... more SNAP-25 a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinurn neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln'97-Arg'98 peptide bond, while serotype E cleaves the Arg'80-Ile'8' peptide linkage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.
Science, 1993
Complex brain functions, such as learning and memory, are believed to involve changes in the effi... more Complex brain functions, such as learning and memory, are believed to involve changes in the efficiency of communication between nerve cells. Therefore, the elucidation of the molecular mechanisms that regulate synaptic transmission, the process of intercellular communication, is an essential step toward understanding nervous system function. Several proteins associated with synaptic vesicles, the organelles that store neurotransmitters, are targets for protein phosphorylation and dephosphorylation. One of these phosphoproteins, synapsin I, by means of changes in its state of phosphorylation, appears to control the fraction of synaptic vesicles available for release and thereby to regulate the efficiency of neurotransmitter release. This article describes current understanding of the mechanism by which synapsin I modulates communication between nerve cells and reviews the properties and putative functions of other phosphoproteins associated with synaptic vesicles.
Quantitative Neuroanatomy in Transmitter Research, 1985
Trophic Regulation of the Basal Ganglia, 1994
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Papers by Fabio Benfenati