Showing papers by "Fabio Benfenati published in 2009"

Opposite Changes in Glutamatergic and GABAergic Transmission Underlie the Diffuse Hyperexcitability of Synapsin I–Deficient Cortical Networks

Abstract: Synapsins (Syns) are synaptic vesicle (SV) phosphoproteins that play a role in synaptic transmission and plasticity. Mutation of the SYN1 gene results in an epileptic phenotype in mouse and man, implicating SynI in the control of network excitability. We used microelectrode array and patch-clamp recordings to study network activity in primary cortical neurons from wild-type (WT) or SynI knockout (KO) mice. SYN1 deletion was associated with increased spontaneous and evoked activities, with more frequent and sustained bursts of action potentials and a high degree of synchronization. Blockade of GABAA (g-aminobutyric acidA) receptors with bicuculline attenuated, but did not completely abolish, the differences between WT and SynI KO networks in both spontaneous and evoked activities. Patch-clamp recordings on cortical autaptic neurons revealed a reduced amplitude of evoked inhibitory postsynaptic currents (PSCs) and a concomitantly increased amplitude of evoked excitatory PSCs in SynI KO neurons, in the absence of changes in miniature PSCs. Cumulative amplitude analysis revealed that these effects were attributable to opposite changes in the size of the readily releasable pool of SVs. The results indicate distinct roles of SynI in GABAergic and glutamatergic neurons and provide an explanation for the high susceptibility of SynI KO mice to epileptic seizures.

ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I.

Abstract: Activity-dependent changes in the strength of synaptic connections in the hippocampus are central for cognitive processes such as learning and memory storage. In this study, we reveal an activity-dependent presynaptic mechanism that is related to the modulation of synaptic plasticity. In acute mouse hippocampal slices, high-frequency stimulation (HFS) of the mossy fiber (MF)-CA3 pathway induced a strong and transient activation of extracellular-regulated kinase (ERK) in MF giant presynaptic terminals. Remarkably, pharmacological blockade of ERK disclosed a negative role of this kinase in the regulation of a presynaptic form of plasticity at MF-CA3 contacts. This ERK-mediated inhibition of post-tetanic enhancement (PTE) of MF-CA3 synapses was both frequency- and pathway-specific and was observed only with HFS at 50 Hz. Importantly, blockade of ERK was virtually ineffective on PTE of MF-CA3 synapses in mice lacking synapsin I, 1 of the major presynaptic ERK substrates, and triple knockout mice lacking all synapsin isoforms displayed PTE kinetics resembling that of wild-type mice under ERK inhibition. These findings reveal a form of short-term synaptic plasticity that depends on ERK and is finely tuned by the firing frequency of presynaptic neurons. Our results also demonstrate that presynaptic activation of the ERK signaling pathway plays part in the activity-dependent modulation of synaptic vesicle mobilization and transmitter release.

Cognitive impairment in Gdi1-deficient mice is associated with altered synaptic vesicle pools and short-term synaptic plasticity, and can be corrected by appropriate learning training

Abstract: The GDI1 gene, responsible in human for X-linked non-specific mental retardation, encodes αGDI, a regulatory protein common to all GTPases of the Rab family. Its alteration, leading to membrane accumulation of different Rab GTPases, may affect multiple steps in neuronal intracellular traffic. Using electron microscopy and electrophysiology, we now report that lack of αGDI impairs several steps in synaptic vesicle (SV) biogenesis and recycling in the hippocampus. Alteration of the SV reserve pool (RP) and a 50% reduction in the total number of SV in adult synapses may be dependent on a defective endosomal-dependent recycling and may lead to the observed alterations in short-term plasticity. As predicted by the synaptic characteristics of the mutant mice, the short-term memory deficit, observed when using fear-conditioning protocols with short intervals between trials, disappeared when the Gdi1 mutants were allowed to have longer intervals between sessions. Likewise, previously observed deficits in radial maze learning could be corrected by providing less challenging pre-training. This implies that an intact RP of SVs is necessary for memory processing under challenging conditions in mice. The possibility to correct the learning deficit in mice may have clinical implication for future studies in human.

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals.

Abstract: Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.

Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

Abstract: An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/day. Depending on the lid membrane thickness, pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm2 through PDMS lids as compared with 18.69 g/day/dm2 with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

Growth Cone 3-D Morphology is Modified by Distinct Micropatterned Adhesion Substrates

Abstract: The development, connectivity, and structural plasticity of neuronal networks largely depend on the directional growth of axonal growth cones (GCs). The morphology and 3-D profile of axons and GCs of primary hippocampal neurons, grown onto glass surfaces coated with poly-D-lysine (PDL) and micropatterned with stripes of the adhesion molecule L1 by using the indirect microcontact printing, were investigated. Neurons were fixed at early stages (one to seven days) of in vitro development prior to synapse formation, and analyzed by fluorescence and atomic force microscopy. The latter technique allowed us to investigate the 3-D morphology of the GCs, and detect their morphological rearrangements during axon outgrowth and during contact with the underlying substrate. We found that axons decreased their height-to-width ratio over development in culture, and that this value became particularly low when the axon and the GC proceeded onto a surface containing attracting cues such as L1 with respect to GCs growing onto a nonspecific adhesion substrate such as PDL. Along with this shape change of the axons, GCs lying onto L1 tracks displayed a flattened shape, ideal for sensing and progression, whereas GCs onto areas of nonspecific adhesion displayed more prominent shapes and steeper edges.

Micro-electrode array based on optically transparent polymeric conductive materials, and method for the manufacturing thereof

Abstract: A micro-electrode array (A) is described comprising an insulating substrate (SUB) and a plurality of conductive paths (E, T, P) defined therein, each of which includes an electrode area (E) with microscopic dimensions, a connection area (P) with macroscopic dimensions, and a buried interconnection region (T), wherein the insulating substrate (SUB) is made of a first polymeric material, such as PDMS, and the conductive paths (E, T, P) are made of a second polymeric material, such as PEDOT. Furthermore, a method of manufacturing such micro-electrode array is described, based on the configuration of an insulating substrate (SUB) volume by replica molding on the first polymeric material through a prearranged master mould (M), bearing a configuration of microstructures (S) adapted to define a plurality of cavities (C); successively filling the cavities (C) with a second conductive polymeric material so as to constitute the electrode areas (E), the connection areas (P), and the interconnection regions (T); and arranging an insulating material sealing layer (B) for the filled cavities (C).

Imaging extracellular neuronal signaling on high resolution microelectrode arrays (MEAs) Hippocampal cultures coupled with a high resolution neuroelectronic interface

Abstract: This paper describes the experimental performances of a high-resolution CMOS integrated microelectrode array platform for acquiring extracellular neuronal signaling in-vitro. Electrophysiological signals of dissociated hippocampal cultures (14–30 DIVs, E18 from rats) are sampled at 7.7 kHz/channel from 4096 microelectrodes (21 μm inter-electrode separation). We show that the achieved spatial-temporal resolution together with data visualization as image sequences, enable the detailed observation of activation sites and burst activity propagations. This image-based approach is well adapted to high-resolution datasets in respect to raster plot visualization and opens the perspective of detailed image-based analysis of extracellular neuronal signals.

Prototyping all-polymer bioelectrical signal transducers

Abstract: For historical reasons, the signal transduction interface of bioelectronic devices is commonly based on metals or inorganic (semi-)conductors. This also applies to application areas where artificial components such as biomedical screening devices, in vitro microelectrode arrays and in vivo neuroprosthetics come into direct contact with biological tissue. In a proof-of-concept microelectrode array design study, we present an alternative all-polymer approach for the low-cost fabrication of bioelectrical signal transduction devices with adjustable flexibility, electrical impedance and transparency. The fabrication process entailed three steps. Firstly, by means of a replica-moulding strategy, different types of transparent polymers were microstructured by two-level SU-8 masters to create vias for contact pads and electrodes, and indentations for interconnecting microchannels. Secondly, recesses in the insulating polymer sheets were filled with conductive polymer composites based on quasi-transparent polystyrenesulfonate doped poly(3,4-ethylenedioxy-thiophene) (PEDOT:PSS). In a last step, the passive microelectrode arrays were backside-insulated by a second layer of a transparent polymer. The electrical properties of the resulting polymer microelectrode arrays were characterized by impedance spectroscopy, baseline noise measurements and recordings of bioelectrical signals from acute preparations of chicken cardiomyocytes. Biocompatibility was tested with in vitro cultures of cortical neurons derived from embryonic chicken.

A novel algorithm for burst and network burst detection Application to wild-type and SynI knockout mice cultures for the study of epileptogenesis

Abstract: In vitro networks of neurons typically exhibit bursting behavior. To help the experimenter in characterizing different preparations or identifying the changes possibly induced by specific protocols, we developed a new automated method for detecting both bursts and network bursts from electrophysiological activity recorded by means of micro-electrode arrays. The burst detection algorithm is based on the computation of the logarithmic inter-spike interval histogram to automatically detect the best threshold to distinguish between inter- and intra-burst spikes. We preliminary tested our new tools on spontaneous and chemically stimulated activity recordings from primary cultures of rat cortex neurons. Moreover, as a case study, we also applied our algorithms to recordings coming from embryonic primary cortical neurons of wild-type and SynI knockout mice. Our results show i) the flexibility and self-adapting properties of the proposed methods, by applying them to different kind of preparations; ii) their capability of revealing changes in the bursting activity due to different experimental conditions.