9 research outputs found
Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABAA receptor clustering induced by inhibitory synaptic plasticity
Both excitatory and inhibitory synaptic contacts display activity dependent
dynamic changes in their efficacy that are globally termed synaptic
plasticity. Although the molecular mechanisms underlying glutamatergic
synaptic plasticity have been extensively investigated and described, those
responsible for inhibitory synaptic plasticity are only beginning to be
unveiled. In this framework, the ultrastructural changes of the inhibitory
synapses during plasticity have been poorly investigated. Here we combined
confocal fluorescence microscopy (CFM) with high resolution scanning electron
microscopy (HRSEM) to characterize the fine structural rearrangements of post-
synaptic GABAA Receptors (GABAARs) at the nanometric scale during the
induction of inhibitory long-term potentiation (iLTP). Additional electron
tomography (ET) experiments on immunolabelled hippocampal neurons allowed the
visualization of synaptic contacts and confirmed the reorganization of post-
synaptic GABAAR clusters in response to chemical iLTP inducing protocol.
Altogether, these approaches revealed that, following the induction of
inhibitory synaptic potentiation, GABAAR clusters increase in size and number
at the post-synaptic membrane with no other major structural changes of the
pre- and post-synaptic elements
Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABA A receptor clustering induced by inhibitory synaptic plasticity
Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABA(A) Receptors (GABA(A)Rs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABA(A)R clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABA(A)R clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements
Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces
Poly(3-hexylthiophene) (P3HT) is a hole-conducting polymer that has been intensively used to develop organic optoelectronic devices (e.g., organic solar cells). Recently, P3HT films and nanoparticles have also been used to restore the photosensitivity of retinal neurons. The template-assisted electrochemical synthesis of polymer nanowires advantageously combines polymerization and polymer nanostructuring into one, relatively simple, procedure. However, obtaining P3HT nanowires through this procedure was rarely investigated. Therefore, this study aimed to investigate the template-assisted electrochemical synthesis of P3HT nanowires doped with tetrabutylammonium hexafluorophosphate (TBAHFP) and their biocompatibility with primary neurons. We show that template-assisted electrochemical synthesis can relatively easily turn 3-hexylthiophene (3HT) into longer (e.g., 17 ± 3 µm) or shorter (e.g., 1.5 ± 0.4 µm) P3HT nanowires with an average diameter of 196 ± 55 nm (determined by the used template). The nanowires produce measurable photocurrents following illumination. Finally, we show that primary cortical neurons can be grown onto P3HT nanowires drop-casted on a glass substrate without relevant changes in their viability and electrophysiological properties, indicating that P3HT nanowires obtained by template-assisted electrochemical synthesis represent a promising neuronal interface for photostimulation
Three-dimensional multiple-particle tracking with nanometric precision over tunable axial ranges
The precise localization of nanometric objects in three dimensions is essential to identify functional diffusion mechanisms in complex systems at the cellular or molecular level. However, most optical methods can achieve high temporal resolution and high localization precision only in two dimensions or over a limited axial (z) range. Here we develop a novel wide-field detection system based on an electrically tunable lens that can track multiple individual nanoscale emitters in three dimensions over a tunable axial range with nanometric localization precision. The optical principle of the technique is based on the simultaneous acquisition of two images with an extended depth of field while encoding the z position of the emitters via a lateral shift between images. We provide a theoretical framework for this approach and demonstrate tracking of free diffusing beads and GABAA receptors in live neurons. This approach allows getting nanometric localization precision up to an axial range above 10 \ub5m with a high numerical aperture lens-quadruple that of a typical 3D tracking system. Synchronization or complex fitting procedures are not requested here, which leads to a suitable architecture for localizing single molecules in four dimensions, namely, three dimensions in real-time
The light-dependent pseudo-capacitive charging of conjugated polymer nanoparticles coupled with the depolarization of the neuronal membrane
: The mechanism underlying visual restoration in blind animal models of retinitis pigmentosa using a liquid retina prosthesis based on semiconductive polymeric nanoparticles is still being debated. Through the application of mathematical models and specific experiments, we developed a coherent understanding of abiotic/biotic coupling, capturing the essential mechanism of photostimulation responsible for nanoparticle-induced retina activation. Our modeling is based on the solution of drift-diffusion and Poisson-Nernst-Planck models in the multi-physics neuron-cleft-nanoparticle-extracellular space domain, accounting for the electro-chemical motion of all the relevant species following photoexcitation. Modeling was coupled with electron microscopy to estimate the size of the neuron-nanoparticle cleft and electrophysiology on retina explants acutely or chronically injected with nanoparticles. Overall, we present a consistent picture of electrostatic depolarization of the bipolar cell driven by the pseudo-capacitive charging of the nanoparticle. We demonstrate that the highly resistive cleft composition, due to filling by adhesion/extracellular matrix proteins, is a crucial ingredient for establishing functional electrostatic coupling. Additionally, we show that the photo-chemical generation of reactive oxygen species (ROS) becomes relevant only at very high light intensities, far exceeding the physiological ones, in agreement with the lack of phototoxicity shown in vivo
Visualization 2: Three-dimensional multiple-particle tracking with nanometric precision over tunable axial ranges
3D tracking of a GABAA receptor in the membrane of a living neuron Originally published in Optica on 20 March 2017 (optica-4-3-367
Ca2+ binding to synapsin I regulates resting Ca2+ and recovery from synaptic depression in nerve terminals
Synapsin I (SynI) is a synaptic vesicle (SV)-associated phosphoprotein that modulates neurotransmission by controlling SV trafficking. The SynI C-domain contains a highly conserved ATP binding site mediating SynI oligomerization and SV clustering and an adjacent main Ca(2+) binding site, whose physiological role is unexplored. Molecular dynamics simulations revealed that the E373K point mutation irreversibly deletes Ca(2+) binding to SynI, still allowing ATP binding, but inducing a destabilization of the SynI oligomerization interface. Here, we analyzed the effects of this mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses from primary hippocampal neurons. Patch-clamp recordings showed an increase in the frequency of miniature excitatory postsynaptic currents (EPSCs) that was totally occluded by exogenous Ca(2+) chelators and associated with a constitutive increase in resting terminal Ca(2+) concentrations. Evoked EPSC amplitude was also reduced, due to a decreased readily releasable pool (RRP) size. Moreover, in both excitatory and inhibitory synapses, we observed a marked impaired recovery from synaptic depression, associated with impaired RRP refilling and depletion of the recycling pool of SVs. Our study identifies SynI as a novel Ca(2+) buffer in excitatory terminals. Blocking Ca(2+) binding to SynI results in higher constitutive Ca(2+) levels that increase the probability of spontaneous release and disperse SVs. This causes a decreased size of the RRP and an impaired recovery from depression due to the failure of SV reclustering after sustained high-frequency stimulation. The results indicate a physiological role of Ca(2+) binding to SynI in the regulation of SV clustering and trafficking in nerve terminals
Selective Targeting of Neurons with Inorganic Nanoparticles: Revealing the Crucial Role of Nanoparticle Surface Charge
Nanoparticles
(NPs) are increasingly used in biomedical applications,
but the factors that influence their interactions with living cells
need to be elucidated. Here, we reveal the role of NP surface charge
in determining their neuronal interactions and electrical responses.
We discovered that negatively charged NPs administered at low concentration
(10 nM) interact with the neuronal membrane and at the synaptic cleft,
whereas positively and neutrally charged NPs never localize on neurons.
This effect is shape and material independent. The presence of negatively
charged NPs on neuronal cell membranes influences the excitability
of neurons by causing an increase in the amplitude and frequency of
spontaneous postsynaptic currents at the single cell level and an
increase of both the spiking activity and synchronous firing at neural
network level. The negatively charged NPs exclusively bind to excitable
neuronal cells, and never to nonexcitable glial cells. This specific
interaction was also confirmed by manipulating the electrophysiological
activity of neuronal cells. Indeed, the interaction of negatively
charged NPs with neurons is either promoted or hindered by pharmacological
suppression or enhancement of the neuronal activity with tetrodotoxin
or bicuculline, respectively. We further support our main experimental
conclusions by using numerical simulations. This study demonstrates
that negatively charged NPs modulate the excitability of neurons,
revealing the potential use of NPs for controlling neuron activity