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1.
Nat Rev Neurosci ; 20(5): 272-281, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30837689

RESUMEN

Cellular mechanisms that regulate the interplay of synaptic excitation and inhibition are thought to be central to the functional stability of healthy neuronal circuits. A growing body of literature demonstrates the capacity for inhibitory GABAergic synapses to exhibit long-term plasticity in response to changes in neuronal activity. Here, we review this expanding field of research, focusing on the diversity of mechanisms that link glutamatergic signalling, postsynaptic action potentials and inhibitory synaptic strength. Several lines of evidence indicate that multiple, parallel forms of plasticity serve to regulate activity at both the input and output domains of individual neurons. Overall, these varied phenomena serve to promote both stability and flexibility over the life of the organism.


Asunto(s)
Potenciales de Acción/fisiología , Encéfalo/fisiología , Neuronas GABAérgicas/fisiología , Inhibición Neural/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Animales
2.
Acta Chir Belg ; : 1-11, 2024 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-38547111

RESUMEN

INTRODUCTION: Post-operative hypocalcemia and postoperative persistent hypoparathyroidism remain the most common complications after thyroidectomy. Many approaches have been developed to prevent them, but actually, a common protocol is not yet individuated. MATERIALS AND METHODS: We retrospectively analyzed the results of a prospectively collected database. We dosed PTH preoperatively and 4 h after surgery (PTH_4); calcium was evaluated preoperatively, on the first (I_POD) and on the second postoperative day (II_POD). Hypocalcemia was defined when calcium <8 mg/dl. PTH_4 and I_POD calcium serum levels are identified to predict postoperative hypocalcemia. RESULTS: Three hundred and forty-eight patients were enrolled, 37 patients resulted as hypocalcemic on I_POD and 41 on the II_POD. PTH_4 is related to I_POD (p < 0.001, r = 0.45) and II_POD (p < 0.001, r = 0.44) calcemia. PTH_4-cut-off predicting I_POD hypocalcemia was 10.50 pg/ml (sensitivity: 78.7%, specificity: 72.7%). A PTH_4 value of 11.5 pg/ml is able to predict hypocalcemia during II_POD (sensitivity: 76.5%, specificity: 69.2%). We set up a combined test to predict II_POD hypocalcemia, using PTH_4 and I_POD calcium (sensitivity: 77.8%, specificity: 89.9%). CONCLUSION: This research shows the association between PTH_4 and postoperative hypocalcemia. The PTH_4 cut-off to predict I_POD-hypocalcemia was 10.5 pg/ml. We analyzed the calcemia trend during the postoperative period and we realized a combined test using PTH_4 and I_POD-calcemia. This test improves the accuracy of the previous test. Further studies, in particular multicentric, with a larger sample are necessary to validate the combined model.

3.
Infection ; 50(4): 989-993, 2022 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-35237950

RESUMEN

PURPOSE: The presence of the SARS-CoV-2 in the peritoneal fluid is a matter of debate in the COVID-19 literature. The study aimed to report the prevalence of SARS-CoV-2 in the peritoneal fluid of patients with nasopharyngeal swab tested positive for SARS-CoV-2 undergoing emergency surgery and review the literature. METHODS: The present study was conducted between March 2020 and June 2021. Diagnosis of SARS-CoV-2 positivity was confirmed by preoperative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Eighteen patients with positive nasopharyngeal swabs were operated in emergency in two third-level Italian hospitals. In 13 of these patients (72%), a peritoneal swab was analyzed: SARS-CoV-2 RNA was found in the abdominal fluid of two patients (15%). Neither of them had visceral perforation and one patient died. In ten patients with negative peritoneal swabs, visceral perforation and mortality rates were 30% and 20%, respectively. CONCLUSION: SARS-CoV-2 peritoneal positivity is rare. Abdominal surgery can, therefore, be safely performed in patients with COVID-19 using standard precautions. The correlation with a visceral perforation is not evaluable. The clinical outcomes seem uninfluenced by the viral colonization of the peritoneum. Assessment in large series to provide definitive answers about the involvement of the SARS-CoV-2 in the peritoneum will be challenging to coordinate.


Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genética
4.
Cell Mol Life Sci ; 78(5): 2279-2298, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32959071

RESUMEN

Learning and memory are known to depend on synaptic plasticity. Whereas the involvement of plastic changes at excitatory synapses is well established, plasticity mechanisms at inhibitory synapses only start to be discovered. Extracellular proteolysis is known to be a key factor in glutamatergic plasticity but nothing is known about its role at GABAergic synapses. We reveal that pharmacological inhibition of MMP3 activity or genetic knockout of the Mmp3 gene abolishes induction of postsynaptic iLTP. Moreover, the application of exogenous active MMP3 mimics major iLTP manifestations: increased mIPSCs amplitude, enlargement of synaptic gephyrin clusters, and a decrease in the diffusion coefficient of synaptic GABAA receptors that favors their entrapment within the synapse. Finally, we found that MMP3 deficient mice show faster spatial learning in Morris water maze and enhanced contextual fear conditioning. We conclude that MMP3 plays a key role in iLTP mechanisms and in the behaviors that presumably in part depend on GABAergic plasticity.


Asunto(s)
Hipocampo/fisiología , Metaloproteinasa 3 de la Matriz/metabolismo , Inhibición Neural/fisiología , Plasticidad Neuronal/fisiología , Aprendizaje Espacial/fisiología , Sinapsis/fisiología , Animales , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Masculino , Metaloproteinasa 3 de la Matriz/genética , Aprendizaje por Laberinto/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , N-Metilaspartato/farmacología , Inhibición Neural/genética , Plasticidad Neuronal/genética , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Sinapsis/genética
5.
J Neurosci ; 37(45): 10792-10799, 2017 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-29118207

RESUMEN

Inhibitory circuits are diverse, yet with a poorly understood cell biology. Functional characterization of distinct inhibitory neuron subtypes has not been sufficient to explain how GABAergic neurotransmission sculpts principal cell activity in a relevant fashion. Our Mini-Symposium brings together several emerging mechanisms that modulate GABAergic neurotransmission dynamically from either the presynaptic or the postsynaptic site. The first two talks discuss novel developmental and neuronal subtype-specific contributions to the excitatory/inhibitory balance and circuit maturation. The next three talks examine how interactions between cellular pathways, lateral diffusion of proteins between synapses, and chloride transporter function at excitatory and inhibitory synapses and facilitate inhibitory synapse adaptations. Finally, we address functional differences within GABAergic interneurons to highlight the importance of diverse, flexible, and versatile inputs that shape network function. Together, the selection of topics demonstrates how developmental and activity-dependent mechanisms coordinate inhibition in relation to the excitatory inputs and vice versa.


Asunto(s)
Sinapsis/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Humanos , Red Nerviosa/citología , Red Nerviosa/fisiología , Plasticidad Neuronal
6.
J Neurosci ; 37(7): 1747-1756, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-28073939

RESUMEN

Gephyrin is a key scaffold protein mediating the anchoring of GABAA receptors at inhibitory synapses. Here, we exploited superresolution techniques combined with proximity-based clustering analysis and model simulations to investigate the single-molecule gephyrin reorganization during plasticity of inhibitory synapses in mouse hippocampal cultured neurons. This approach revealed that, during the expression of inhibitory LTP, the increase of gephyrin density at postsynaptic sites is associated with the promoted formation of gephyrin nanodomains. We demonstrate that the gephyrin rearrangement in nanodomains stabilizes the amplitude of postsynaptic currents, indicating that, in addition to the number of synaptic GABAA receptors, the nanoscale distribution of GABAA receptors in the postsynaptic area is a crucial determinant for the expression of inhibitory synaptic plasticity. In addition, the methodology implemented here clears the way to the application of the graph-based theory to single-molecule data for the description and quantification of the spatial organization of the synapse at the single-molecule level.SIGNIFICANCE STATEMENT The mechanisms of inhibitory synaptic plasticity are poorly understood, mainly because the size of the synapse is below the diffraction limit, thus reducing the effectiveness of conventional optical and imaging techniques. Here, we exploited superresolution approaches combined with clustering analysis to study at unprecedented resolution the distribution of the inhibitory scaffold protein gephyrin in response to protocols inducing LTP of inhibitory synaptic responses (iLTP). We found that, during the expression of iLTP, the increase of synaptic gephyrin is associated with the fragmentation of gephyrin in subsynaptic nanodomains. We demonstrate that such synaptic gephyrin nanodomains stabilize the amplitude of inhibitory postsynaptic responses, thus identifying the nanoscale gephyrin rearrangement as a key determinant for inhibitory synaptic plasticity.


Asunto(s)
Proteínas Portadoras/metabolismo , Neuronas GABAérgicas/citología , Depresión Sináptica a Largo Plazo/fisiología , Proteínas de la Membrana/metabolismo , Densidad Postsináptica/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Algoritmos , Animales , Células Cultivadas , Simulación por Computador , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Hipocampo/citología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , N-Metilaspartato/farmacología , Péptidos/metabolismo , Polímeros , Densidad Postsináptica/efectos de los fármacos , Receptores de GABA-A/metabolismo , Valina/análogos & derivados , Valina/farmacología
7.
J Neurosci ; 36(20): 5437-47, 2016 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-27194325

RESUMEN

UNLABELLED: Phosphorylation of serine/threonine residues preceding a proline regulates the fate of its targets through postphosphorylation conformational changes catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. By flipping the substrate between two different functional conformations, this enzyme exerts a fine-tuning of phosphorylation signals. Pin1 has been detected in dendritic spines and shafts where it regulates protein synthesis required to sustain the late phase of long-term potentiation (LTP). Here, we demonstrate that Pin1 residing in postsynaptic structures can interact with postsynaptic density protein-95 (PSD-95), a key scaffold protein that anchors NMDA receptors (NMDARs) in PSD via GluN2-type receptor subunits. Pin1 recruitment by PSD-95 occurs at specific serine-threonine/proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. Upon binding, Pin1 triggers structural changes in PSD-95, thus negatively affecting its ability to interact with NMDARs. In electrophysiological experiments, larger NMDA-mediated synaptic currents, evoked in CA1 principal cells by Schaffer collateral stimulation, were detected in hippocampal slices obtained from Pin1(-/-) mice compared with controls. Similar results were obtained in cultured hippocampal cells expressing a PSD-95 mutant unable to undergo prolyl-isomerization, thus indicating that the action of Pin1 on PSD-95 is critical for this effect. In addition, an enhancement in spine density and size was detected in CA1 principal cells of Pin1(-/-) or in Thy-1GFP mice treated with the pharmacological inhibitor of Pin1 catalytic activity PiB.Our data indicate that Pin1 controls synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for LTP maintenance. SIGNIFICANCE STATEMENT: PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein at excitatory postsynaptic densities and a potent regulator of synaptic strength and plasticity. The activity of PSD-95 is tightly controlled by several post-translational mechanisms including proline-directed phosphorylation. This signaling cascade regulates the fate of its targets through postphosphorylation conformational modifications catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. Here, we uncover a new role of Pin1 in glutamatergic signaling. By interacting with PSD-95, Pin1 dampens PSD-95 ability to complex with NMDARs, thus negatively affecting NMDAR signaling and spine morphology. Our findings further emphasize the emerging role of Pin1 as a key modulator of synaptic transmission.


Asunto(s)
Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Región CA1 Hipocampal/citología , Células Cultivadas , Homólogo 4 de la Proteína Discs Large , Femenino , Células HEK293 , Humanos , Isomerismo , Potenciación a Largo Plazo , Masculino , Ratones , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Unión Proteica , Sinapsis/fisiología , Transmisión Sináptica
10.
J Clin Med ; 13(7)2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38610756

RESUMEN

Post-operative acute kidney injury (PO-AKI) is a frequent complication described in 15% of non-cardiac surgeries, 30% of cardiac surgeries, and 52% of patients requiring intensive post-operative care [...].

11.
ACS Appl Mater Interfaces ; 16(3): 3093-3105, 2024 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-38206310

RESUMEN

As is known, carbon nanotubes favor cell growth in vitro, although the underlying mechanisms are not yet fully elucidated. In this study, we explore the hypothesis that electrostatic fields generated at the interface between nonexcitable cells and appropriate scaffold might favor cell growth by tuning their membrane potential. We focused on primary human fibroblasts grown on electrospun polymer fibers (poly(lactic acid)─PLA) with embedded multiwall carbon nanotubes (MWCNTs). The MWCNTs were functionalized with either the p-methoxyphenyl (PhOME) or the p-acetylphenyl (PhCOMe) moiety, both of which allowed uniform dispersion in a solvent, good mixing with PLA and the consequent smooth and homogeneous electrospinning process. The inclusion of the electrically conductive MWCNTs in the insulating PLA matrix resulted in differences in the surface potential of the fibers. Both PLA and PLA/MWCNT fiber samples were found to be biocompatible. The main features of fibroblasts cultured on different substrates were characterized by scanning electron microscopy, immunocytochemistry, Rt-qPCR, and electrophysiology revealing that fibroblasts grown on PLA/MWCNT reached a healthier state as compared to pure PLA. In particular, we observed physiological spreading, attachment, and Vmem of fibroblasts on PLA/MWCNT. Interestingly, the electrical functionalization of the scaffold resulted in a more suitable extracellular environment for the correct biofunctionality of these nonexcitable cells. Finally, numerical simulations were also performed in order to understand the mechanism behind the different cell behavior when grown either on PLA or PLA/MWCNT samples. The results show a clear effect on the cell membrane potential, depending on the underlying substrate.


Asunto(s)
Nanotubos de Carbono , Humanos , Nanotubos de Carbono/química , Potenciales de la Membrana , Poliésteres/química , Polímeros/química , Fibroblastos
12.
Small ; 9(3): 402-12, 2013 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-23027505

RESUMEN

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.


Asunto(s)
Nanoestructuras/química , Neuronas/citología , Ingeniería de Tejidos/métodos , Animales , Adhesión Celular/fisiología , Células Cultivadas , Ratones , Neuronas/fisiología
13.
J Neurosci ; 31(5): 1752-61, 2011 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-21289185

RESUMEN

To reach the open state, the GABA(A) receptor (GABA(A)R) is assumed to bind two agonist molecules. Although it is currently believed that GABA(A)R could also operate in the monoliganded state, the gating properties of singly bound GABA(A)R are poorly understood and their physiological role is still obscure. In the present study, we characterize for the first time the gating properties of singly bound GABA(A)Rs by using a mutagenesis approach and we propose that monoliganded GABA(A)R contribute in shaping synaptic responses. At saturating GABA concentrations, currents mediated by recombinant GABA(A)Rs with a single functional binding site display slow onset, fast deactivation kinetics, and slow rate of desensitization-resensitization. GABA(A)Rs with two binding sites activated by brief pulses of subsaturating GABA concentrations (in the range of the GABA concentration profile in the synaptic cleft) could also mediate fast deactivating currents, displaying deactivation kinetics similar to those mediated by GABA(A)Rs with a single functional binding site. Model simulations of receptors activated by realistic synaptic GABA waves revealed that a considerable proportion of GABA(A) receptors open in the monoliganded state during synaptic transmission, therefore contributing in shaping IPSCs.


Asunto(s)
Inhibición Neural/fisiología , Receptores de GABA-A/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Electrofisiología , Células HEK293 , Humanos , Inmunohistoquímica , Microscopía Confocal , Plásmidos , Reacción en Cadena de la Polimerasa , Receptores de GABA-A/genética , Transfección/métodos
14.
Cell Rep ; 38(6): 110347, 2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35139381

RESUMEN

The induction of synaptic plasticity at an individual dendritic glutamatergic spine can affect neighboring spines. This local modulation generates dendritic plasticity microdomains believed to expand the neuronal computational capacity. Here, we investigate whether local modulation of plasticity can also occur between glutamatergic synapses and adjacent GABAergic synapses. We find that the induction of long-term potentiation at an individual glutamatergic spine causes the depression of nearby GABAergic inhibitory synapses (within 3 µm), whereas more distant ones are potentiated. Notably, L-type calcium channels and calpain are required for this plasticity spreading. Overall, our data support a model whereby input-specific glutamatergic postsynaptic potentiation induces a spatially regulated rearrangement of inhibitory synaptic strength in the surrounding area through short-range heterosynaptic interactions. Such local coordination of excitatory and inhibitory synaptic plasticity is expected to influence dendritic information processing and integration.


Asunto(s)
Región CA1 Hipocampal/fisiología , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Animales , Dendritas/fisiología , Ratones , Neuronas/fisiología
15.
Nat Commun ; 13(1): 7406, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36456575

RESUMEN

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.


Asunto(s)
Neoplasias de Células Escamosas , Neoplasias Cutáneas , Humanos , Microscopía Confocal , Fotones
16.
Front Immunol ; 13: 849140, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35222440

RESUMEN

We report a case of inflammatory colitis after SARS-CoV-2 infection in a patient with no additional co-morbidity who died within three weeks of hospitalization. As it is becoming increasingly clear that SARS-CoV-2 infection can cause immunological alterations, we investigated the expression of the inhibitory checkpoint PD-1 and its ligand PD-L1 to explore the potential role of this axis in the break of self-tolerance. The presence of the SARS-CoV-2 virus in colon tissue was demonstrated by qRT-PCR and immunohistochemical localization of the nucleocapsid protein. Expression of lymphocyte markers, PD-1, and PD-L1 in colon tissue was investigated by IHC. SARS-CoV-2-immunoreactive cells were detected both in the ulcerated and non-ulcerated mucosal areas. Compared to healthy tissue, where PD-1 is weakly expressed and PD-L1 is absent, PD-1 and PD-L1 expression appears in the inflamed mucosal tissue, as expected, but was mainly confined to non-ulcerative areas. At the same time, these markers were virtually undetectable in areas of mucosal ulceration. Our data show an alteration of the PD-1/PD-L1 axis and suggest a link between SARS-CoV-2 infection and an aberrant autoinflammatory response due to concomitant breakdown of the PD-1/PD-L1 interaction leading to early death of the patient.


Asunto(s)
COVID-19/inmunología , Colitis/inmunología , Colon/metabolismo , Síndrome de Liberación de Citoquinas/inmunología , Inflamación/inmunología , SARS-CoV-2/fisiología , Anciano , Antígeno B7-H1/metabolismo , Colon/patología , Resultado Fatal , Femenino , Humanos , Receptor de Muerte Celular Programada 1/metabolismo , Autotolerancia , Transducción de Señal
17.
Nanoscale Horiz ; 8(1): 95-107, 2022 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-36426604

RESUMEN

Over the last few years it has been understood that the interface between living cells and the underlying materials can be a powerful tool to manipulate cell functions. In this study, we explore the hypothesis that the electrical cell/material interface can regulate the differentiation of cancer stem-like cells (CSCs). Electrospun polymer fibres, either polyamide 66 or poly(lactic acid), with embedded graphene nanoplatelets (GnPs), have been fabricated as CSC scaffolds, providing both the 3D microenvironment and a suitable electrical environment favorable for CSCs adhesion, growth and differentiation. We have investigated the impact of these scaffolds on the morphological, immunostaining and electrophysiological properties of CSCs extracted from human glioblastoma multiform (GBM) tumor cell line. Our data provide evidence in favor of the ability of GnP-incorporating scaffolds to promote CSC differentiation to the glial phenotype. Numerical simulations support the hypothesis that the electrical interface promotes the hyperpolarization of the cell membrane potential, thus triggering the CSC differentiation. We propose that the electrical cell/material interface can regulate endogenous bioelectrical cues, through the membrane potential manipulation, resulting in the differentiation of CSCs. Material-induced differentiation of stem cells and particularly of CSCs, can open new horizons in tissue engineering and new approaches to cancer treatment, especially GBM.


Asunto(s)
Glioblastoma , Humanos , Electricidad Estática , Ingeniería de Tejidos/métodos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Diferenciación Celular , Microambiente Tumoral
18.
Front Synaptic Neurosci ; 13: 727406, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34899260

RESUMEN

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

19.
Biophys Rep (N Y) ; 1(2): None, 2021 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-34939046

RESUMEN

The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy. However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark noise, photon-detection efficiency, after-pulsing probability, and optical cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present, to our knowledge, a novel SPAD array detector with an active cooling system that substantially reduces the dark noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to -15°C significantly improves the signal/noise ratio due to a 10-fold decrease in the dark count rate compared with room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing green-fluorescent-protein-tagged proteins. For fluorescence fluctuation spectroscopy, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements for which dark noise is substantially higher than the median value. Overall, this detector represents a further step toward the integration of SPAD array detectors in any FLSM system.

20.
Neuropharmacology ; 169: 107643, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-31108109

RESUMEN

The flexibility of neuronal networks is believed to rely mainly on the plasticity of excitatory synapses. However, like their excitatory counterparts, inhibitory synapses also undergo several forms of synaptic plasticity. This review examines recent advances in the understanding of the molecular mechanisms leading to postsynaptic GABAergic plasticity. Specifically, modulation of GABAA receptor (GABAAR) number at postsynaptic sites plays a key role, with the interaction of GABAARs with the scaffold protein gephyrin and other postsynaptic scaffold/regulatory proteins having particular importance. Our understanding of these molecular interactions are progressing, based on recent insights into the processes of GABAAR lateral diffusion, gephyrin dynamics, and gephyrin nanoscale organization. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.


Asunto(s)
Plasticidad Neuronal/fisiología , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Potenciales Sinápticos/fisiología , Animales , Agonistas de Receptores de GABA-A/metabolismo , Agonistas de Receptores de GABA-A/farmacología , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacología , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Receptores de GABA-A/análisis , Sinapsis/química , Sinapsis/efectos de los fármacos , Potenciales Sinápticos/efectos de los fármacos
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