Autism subtypes identified using cross-species functional connectivity analyses Marco Pagani, Valerio Zerbi, Silvia Gini, Filomena Grazia Alvino, Abhishek Banerjee, Andrea Barberis, M. Albert Basson, Yuri Bozzi, Alberto Galbusera, Jacob Ellegood, Michela Fagiolini, Jason P. Lerch, Michela Matteoli, Caterina Montani, Davide Pozzi, Giovanni Provenzano, Maria Luisa Scattoni, Nicole Wenderoth, Ting Xu, Michael V. Lombardo, Michael P. Milham, Adriana Di Martino, Alessandro Gozzi Nature Neuroscience, 2026 It is often assumed that phenotypic heterogeneity in autism reflects underlying pathobiological variation. However, direct evidence supporting this link is lacking. Leveraging cross-species functional neuroimaging, we show that brain dysconnectivity patterns in autism can be parsed into biologically dissociable subtypes. Specifically, we found that functional magnetic resonance imaging (fMRI) connectivity alterations in 20 distinct genetic mouse models of autism cluster into hypoconnectivity-dominant and hyperconnectivity-dominant subtypes. These subtypes are linked to distinct biological pathways, with hypoconnectivity being associated with synaptic dysfunction and hyperconnectivity reflecting transcriptional and immune-related alterations. Here we identified analogous hypoconnectivity and hyperconnectivity subtypes in a multicenter human fMRI dataset of n = 940 individuals with idiopathic autism and n = 1,036 neurotypical individuals. The human autism subtypes are highly replicable, are associated with distinct functional network architectures and behavioral profiles and recapitulate the synaptic and immune-related pathways identified in the rodent dataset. Our work provides a new empirical framework for targeted subtyping of the autism spectrum.
Action Potentials as a Mean to Trigger the Specific Interaction of Negatively Charged Nanoparticles with Electrically Excitable Cells Amira El Merhie, Barbara Salis, Tiziana Ravasenga, Chiara Tramontano, Sahitya Kumar Avugadda, Giammarino Pugliese, Andrea Barberis, Teresa Pellegrino, Silvia Dante ACS Applied Nano Materials, 2024 In this study, we dig to understand the nanoparticle (NP)–cell interaction and the roles of the NP surface charge and the cell membrane potential. While the interaction between negatively charged cadmium selenide/cadmium sulfide (CdSe/CdS) NPs and primary neurons was determined in our previous study, here, by using differentiated mouse neuroblastoma (N2a) cells, nondifferentiated N2a cells, and Chinese hamster ovary (CHO) cells, we are able to understand the relation between electrically excitable cells and non-excitable cells with the same type of NPs in a more systematic way. Indeed, we demonstrate that highly negatively charged CdSe/CdS NPs (−50 mV) interact with the cell membrane of electrically active cells, such as those of primary neurons and differentiated N2a cells, while no interaction occurs with non-electrically active cells, such as CHO cells and nondifferentiated N2a cells. Notably, by ad hoc designed patch-clamp experiments, we confirm that the interaction of the negatively charged NPs with neuronal cells is triggered by the membrane electrical activity. In contrast, no interaction with the cellular membrane is noted in the control unstimulated patch-clamp neuronal cells performed with NPs bearing an almost neutral charge. Furthermore, we show that the interaction between electrically excitable cells occurs with NPs independently of their inorganic nature. Indeed, by replacing the inorganic rod-shaped NPs with negatively charged polymeric micelles made of the same polymer used for the CdSe/CdS NPs surface coating but without the inorganic core (CdSe/CdS NPs), the cell surface interaction still occurs with neuronal excitable cells. These data suggest that, independent of the organic or inorganic nature of the NPs tested here, the specificity of the NP–cell membrane interaction is still affected by the electric activity of the neuronal cells when negatively charged nanoplatforms are used.
Modified Carbon Nanotubes Favor Fibroblast Growth by Tuning the Cell Membrane Potential Giulia Suarato, Samuel Pressi, Enzo Menna, Massimo Ruben, Enrica Maria Petrini, Andrea Barberis, Dalila Miele, Giuseppina Sandri, Marco Salerno, Andrea Schirato, Alessandro Alabastri, Athanassia Athanassiou, Remo Proietti Zaccaria, Evie L. Papadopoulou ACS Applied Materials and Interfaces, 2024 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.
Synaptic microarchitecture: the role of spatial interplay between excitatory and inhibitory inputs in shaping dendritic plasticity and neuronal output Dario Cupolillo, Vincenzo Regio, Andrea Barberis Frontiers in Cellular Neuroscience, 2024 Pyramidal neurons (PNs) receive and integrate thousands of synaptic inputs impinging onto their dendritic arbor to shape the neuronal output. The richness and the complexity of such input-output transformation primarily relies on the ability of neurons to generate different forms of dendritic local spikes, regenerative events originating in the dendrites profoundly influencing the probability and the temporal structure of somatic spiking. Extensive work during the last two decades has identified the impact of clustering and cooperative plasticity among glutamatergic synapse in promoting dendritic spikes. However, the role of inhibitory synapses in such processes remains elusive. In this opinion paper, following a general introduction on the impact of the synaptic input spatial distribution in neuronal activity, we highlight the coordinated plasticity of excitatory and inhibitory dendritic synapses as an emerging key factor in the organization of the dendritic input architecture. In particular we will emphasize that the relative positioning of diverse excitatory and inhibitory dendritic synapses at the microscale level is a major challenge for understanding how dendritic dynamics shape neuronal circuit function in the brain.In different brain areas, distinct synaptic inputs converging onto PNs show a macro-scale distribution across large dendritic compartments. For instance, in the hippocampal formation, excitatory fibers from entorhinal cortex (EC) project to the distal portions of apical dendrites of CA1 PNs through the perforant path (PP), while Schaffer collaterals (SCs) from the CA3 area mainly contact the proximal dendrites (Megias et al., 2001) (Fig. 1). Similarly, in the neocortex, intra-cortical layer 2/3 (L2/3) PNs axons (feedforward information) contact the proximal dendrites of layer 5 (L5) PNs, with cortico-cortical inputs from high-order cortical areas (feedback information) targeting their distal dendrites (Larkum, 2012). This illustrates a large-scale connectivity scheme wherein fibers from either distant or local brain regions preferentially contact distal or proximal dendrites, respectively (Felleman and Van Essen, 1991). Such broad-scale input organization reflects important functional properties where the activation of proximal dendrites typically produces single action potentials while co-activation of distal and proximal synaptic inputs can generate calcium plateau potentials -specific forms of dendritic spikes initiated in the distal dendritic region -leading the neuron to burst firing (Jarsky et al., 2005;Takahashi and Magee, 2009;Larkum et al., 1999). This supra-linear integration provides the biophysical basis for a fundamental associative process to combine and compare different types of information at the single cell level (Bittner et al., 2015;Larkum, 2012). Along the same line, the differential effect of distal feedforward inputs triggering single spikes and the combined activation of feedforward and distal feedback inputs inducing burst firing, provides the opportunity for the independent transmission of these two distinct signals through the same neuronal pathway (multiplexing) (Naud and Sprekeler, 2017).Intriguingly, GABAergic inputs are also non-randomly distributed along the axo-dendritic axis of PNs. Diverse subclasses of GABAergic interneurons (INs) target specific sub-regions of PNs including axon initial segment, soma, proximal dendrites and distal dendrites, with a specific temporal activation critically contributing to e.g. brain oscillations (Klausberger and Somogyi, 2007;Tzilivaki et al., 2023). In both hippocampus and neocortex, the proximo-distal dendritic compartmentalization of diverse GABAergic inputs creates a spatial pattern where distinct GABAergic fibers broadly align with specific subsets of excitatory inputs. For example, in the hippocampus, oriens-lacunosum-moleculare (O-LM), neurogliaform, and perforant path (PP)-associated INs target the distal dendrites of CA1 pyramidal neurons aligning with PP inputs from the EC. Comparably, bistratified, SC-associated and Ivy interneurons match glutamatergic inputs from CA3 onto proximal dendrites (Klausberger, 2009, Lovett-Barron et al., 2012). (Fig 1)The existence of structured patterns of synaptic inputs localization persists at smaller scales. At glutamatergic side, computational and experimental works showed that dendritic synaptic inputs clustering favors dendritic spikes initiation (Mel, 1993;Poirazi and Mel, 2001, Poirazi et al., 2003a, 2003b, Larkum et al., 2009). In L5 PNs, for instance the activation of glutamatergic inputs within a ~ 40 m range undergo supra-linear summation due to N-methyl-D-aspartate (NMDA) receptor-dependent regenerative mechanism, whereas inputs more than 80 m apart integrate linearly, indicating the key role of the spatial determinants in dendritic input summation (Polsky et al., 2004). The functional clustering of glutamatergic inputs has been observed directly in dendrites of both CA3 and L2/3 PNs, where spontaneous activity is more likely to co-activate neighboring glutamatergic spines rather than distant spines, thus forming glutamatergic synaptic "assemblets" within ~ 10 m (Takahashi et al., 2012). The clustered organization of glutamatergic inputs underpins an important role at the functional level. In the visual cortex, the clustering of similarly tuned inputs aids edge detection and contour integration (Iacaruso et al., 2017), while in the motor cortex, task-related inputs cluster within 10 μm subdomains to support decisionmaking (Kerlin et al., 2019). Besides the relevance of the tight spatial proximity between active glutamatergic synapses (synaptic clustering), the initiation of dendritic spikes strongly depends on the dendritic morphology. In thin and short dendritic branches, the high input resistance determines low attenuation of the depolarization produced by individual synapses thus promoting the signal summation within the branch (Kastellakis and Poirazi, 2019). For instance, the timely activation of ~ 20 glutamatergic inputs on a radial oblique dendritic branch of 100 m in CA1 PNs initiate a local sodium spike regardless of their spatial relationship along the branch, thus determining in-branch clustering (Losonczy and Magee, 2006). Anatomical studies of SCs synapses localization onto CA1 PNs dendrites have revealed a high nonuniform connectivity structure. In particular, the number of short inter-spine distances as well as the number of glutamatergic inputs per branch was greater than chance level, thus supporting both synaptic clustering and in-branch clustering modes, respectively (Druckman et al., 2014). Similar findings were observed for thalamocortical inputs onto L5 PN (Rah et al., 2013). Collectively, this evidence indicates that, at different scales, the spatial arrangement of glutamatergic synapses in dendrites of PNs crucially shapes the transfer function between synaptic activation and dendritic depolarization/spiking (Ujfalussy and Makara, 2020;Kastellakis and Poirazi, 2019).As with excitatory inputs, several lines of evidence show that inhibition depends on local spatial determinants at the microscale level, such as their fine relative positioning with respect to excitatory synapses (Boivin and Nedivi, 2018). Modeling studies suggest that GABAergic synapses positioned distally (off-path) from a cluster of glutamatergic synapses more efficiently raise the threshold for initiating a dendritic spike compared to proximally-placed ones (on-path), whereas the on-path location is more effective in shunting already-triggered dendritic spikes (Gidon and Segev, 2012). Both predictions have been corroborated experimentally ex vivo in L5 PNs, confirming that the specific spatial arrangement of GABAergic synapses in dendritic branches is an important determinant shaping dendritic excitability (Jadi et al., 2012). In this concern, studies report that diverse GABAergic inputs from specific interneurons are highly structured at branch and sub-branch levels. In CA1 PNs, O-LM interneurons (somatostatin+, SOM+) or neurogliaform interneurons (neural nitric oxide synthase+, nNOS+) preferentially target the ending or the intermediated region of the terminal domain of distal dendrites, respectively whereas bistratified interneurons (neuropeptide Y+, NPY+) target the origin of the terminal domain of proximal apical oblique and basal dendrites (Bloss et al., 2016). In addition, the study of the excitatory and inhibitory synapses distribution in the whole dendritic arbor in L2/3 PNs revealed that, while density of both synapses significantly vary in different neuronal sub-regions, its ratio was remarkably balanced at branch level (Iascone et al., 2020). Finally, inhibitory GABAergic synapses can be located directly on glutamatergic spines thus effectively controlling spine depolarization (Boivin and Nedivi, 2018;Chiu et al., 2013).Extensive work on glutamatergic spines reports that the expression of long-term potentiation (LTP) at an individual spine can lower the threshold for the induction of synaptic plasticity at neighboring spines by spreading signaling molecules such as small GTPases from the potentiated spine in dendritic stretches of ̴ 10 m: this establishes the coordinated potentiation of a subset of contiguous spines ultimately leading to the formation of a glutamatergic synaptic cluster (Harvey and Svoboda 2007;Harvey et al., 2008;Hedrick and Yasuda, 2017). On the other hand, the stimulation of a glutamatergic spine cluster can depress nearby spines through the diffusion of the phosphatase calcineurin, a mechanism that is expected to increase the structural and functional identity of specific clusters (Oh et al., 2017). Likewise, long-term depression (LTD) at an individual spines can either depress or potentiate neighboring spines (Chater and Goda, 2021). Overall, these observations suggest that short-range interplay between spines can define the spatial pattern of dendritic glutamatergic synapses. However, how GABAergic synapses contribute to these processes remains largely obscure. Traditionally, inhibition has been considered poorly plastic and to take part to plasticity phenomena mainly by adjusting the threshold for the induction of glutamatergic plasticity (Steele et al., 1999). In this concern, modeling studies report that specific placement of GABAergic synapses with respect to either excitatory synapses or dendritic branches can spatially constraint glutamatergic plasticity hence influencing the degree of glutamatergic synapses clustering (Bar-Ilan et al., 2012). Similarly, the activation of GABAA receptors by GABA uncaging leads to the shrinkage of nearby glutamatergic spines within a range of ̴ 15 m, reinforcing the spatial role of inhibition in promoting the competitive selection of dendritic spines (Hayama et al., 2013).Nevertheless, several lines of evidence indicate that GABAergic synapses express several forms of plasticity (Chiu et al., 2019). This prompts the questions of how glutamatergic and GABAergic plasticity interact at dendritic level at the microscale level and how this can shape synaptic clustering -topics that have thus far been investigated mainly through indirect approaches (Chapman et al., 2022). After the induction of spike-timing-dependent plasticity at a specific synaptic population subset in an auditory cortex PN, the plasticity of excitatory and inhibitory plasticity at distinct unstimulated synaptic population subset was found to be co-tuned to achieve a precise excitation-to-inhibition set point (Field, 2020). Interestingly, the plasticity-induced remodeling of excitatory and inhibitory synapses on dendrites of L2/3 PNs in the visual cortex is spatially coordinated in dendritic portions of ̴ 10 m suggesting short-range interplay between inhibitory and excitatory synapses (Chen et al., 2012). In addition, the stimulation of thalamic afferents to distal dendrites of cortical L2/3 PNs induces inhibitory LTP at GABAergic synapses formed by SOM+ interneurons in the same dendritic portion, thus hinting to local interaction between excitatory and inhibitory synapses (Chiu et al, 2017). Extending this framework, a modeling study identifies the presence of plastic GABAergic synapses as important organizers of dendritic glutamatergic synaptic clustering (Kirchner and Gjorgjieva, 2021).A more recent work investigated the spatial determinants for the interaction between individual dendritic glutamatergic and GABAergic synapses in hippocampal neurons (Ravasenga et al., 2022). By inducing single-spine LTP through the pairing of glutamate uncaging with somatic action potential train, they observed that GABAergic synapses located within a spatial range of ̴ 3-4 m around the potentiated spine were depressed. Although several factors could limit the generalization of finding including the poorly physiological induction of LTP and the lack of in vivo data, the spatial dependence of the interaction between excitation and inhibition likely plays an important role in the organization of dendritic synaptic inputs. First, by considering the local effect of inhibition (Gidon and Segev, heterosynaptic interplay is expected to disinhibit specific potentiated glutamatergic inputs through a winner-takes-all process, with e.g. other concurrent plasticity phenomena maintaining the global dendritic homeostatic balance. Second, the activity-dependent depression of a neighboring GABAergic synapse can contribute to the formation of glutamatergic synaptic clusters thus complementing the cooperative plasticity phenomena between glutamatergic inputs mentioned above. Finally, in the light of this short-range interplay, the convergence of diverse excitatory and inhibitory inputs within the same dendritic stretch can crucially impact at the network level, allowing, for instance, specific glutamatergic inputs to differentially control inputs from different interneuron subtypes. For example, PP and thalamic inputs contact the distal apical dendrites of CA1 PNs together with inputs from O-LM and PP-associated interneurons, which primarily mediate feed-back and feed-forward inhibition, respectively. If ,differently from thalamic inputs, EC inputs are consistently located within the "interplay range" with inputs from O-LM interneurons, EC activity could weaken neighboring O-LM inputs (Fig. 1). This could bias the balance of inhibition from feedback to feed-forward, thereby altering how these dendrites process and integrate incoming signals. Thus, in analogy with the aforementioned large-scale matching between excitation and inhibition in proximal and distal dendritic compartments, it is important to define the co-alignment between excitatory and inhibitory inputs at the microscale level. The spatial pattern of diverse excitatory and inhibitory inputs along the dendrites may serve as a 'fingerprint' for PN subtypes, where the consistent pairing of particular excitatory inputs with inhibitory inputs from specific interneurons could act as structural 'synaptic motifs'. In a broader framework, the impact of excitatory-inhibitory short-range synaptic interplay can be assessed by including specific synaptic topology and plasticity rules in available biophysical computational models predicting the spiking output of PNs receiving realistic excitatory and inhibitory temporal activity patterns at cellular level. This will allow to understand how short-range plasticity contribute to modulate specific network oscillations by tuning at dendritic level the contribution of diverse interneuron subtypes, or how it could enable associative learning by differentially gating information from distinct brain areas. Importantly, this could also inform or how aberrant short-range plasticity would lead to the disruption of coordination between different interneurons subtypes activity ultimately causing pathology. In the long run, the refined information about the dendritic synaptic spatial arrangement and short-range interaction could inform computational models that include dendritic computation in large-networks functions and will also contribute designing more neuromorphic and efficient deep neuronal networks (DNNs) (Pagkalos et al., 2024(Pagkalos et al., , 2023)). (A) Representative selection of excitatory and inhibitory inputs received by CA1 dendrites. Specific subsets of excitatory inputs are aligned with distinct GABAergic fibers. Proximal dendrites in the stratum radiatum are targeted by SC (orange), amygdala projections (green), as well as local SC-associated interneurons (pink) and bistratified interneurons (purple). In contrast, distal dendrites in the stratum lacunosum moleculare receive inputs from the thalamus (yellow), the EC through the PP (red), O-LM interneurons (dark blue), and PP-associated interneurons (light blue). Dashed box delineates a distal dendritic portion represented in B. (B) Two different possible spatial arrangements of excitatory and inhibitory inputs on a distal dendritic segment. Left panel: GABAergic inputs from either O-LM or PP-associated interneurons (striped light-dark blue) are positioned within an "interplay range" with thalamic or PP inputs (d1) or located beyond this range (d2). This points to the existence of excitatory-inhibitory spatial combinations, wherein certain inhibitory inputs consistently spatially paired with specific subsets of glutamatergic inputs. Right panel: excitatory and inhibitory inputs are randomly distributed along a dendritic segment. In this spatial arrangement, there are no consistent rules determining the pairing of specific GABAergic and glutamatergic inputs at the microscale level. (s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum; s.lm., stratum lacunosum moleculare; PP, perforant path; SC, Scaffer Collaterals; Thal, Thalamic; Amyg, Amygdala)
The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy Alessandro Rossetta, Eli Slenders, Mattia Donato, Sabrina Zappone, Francesco Fersini, Martina Bruno, Francesco Diotalevi, Luca Lanzanò, Sami Koho, Giorgio Tortarolo, Andrea Barberis, Marco Crepaldi, Eleonora Perego, Giuseppe Vicidomini Nature Communications, 2022 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.
Electrostatic polarization fields trigger glioblastoma stem cell differentiation Tamara Fernandez Cabada, Massimo Ruben, Amira El Merhie, Remo Proietti Zaccaria, Alessandro Alabastri, Enrica Maria Petrini, Andrea Barberis, Marco Salerno, Marco Crepaldi, Alexander Davis, Luca Ceseracciu, Tiziano Catelani, Athanassia Athanassiou, Teresa Pellegrino, Roberto Cingolani, Evie L. Papadopoulou Nanoscale Horizons, 2022 Glioblastoma cancer stem-like cells seeded on substrates exhibiting surface potential differences, undergo differentiation due to the forced hyperpolarization of the membrane potential at the cell/substrate interface.
Spatial regulation of coordinated excitatory and inhibitory synaptic plasticity at dendritic synapses Tiziana Ravasenga, Massimo Ruben, Vincenzo Regio, Alice Polenghi, Enrica Maria Petrini, Andrea Barberis Cell Reports, 2022 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.
Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy Eli Slenders, Eleonora Perego, Mauro Buttafava, Giorgio Tortarolo, Enrico Conca, Sabrina Zappone, Agnieszka Pierzynska-Mach, Federica Villa, Enrica Maria Petrini, Andrea Barberis, Alberto Tosi, Giuseppe Vicidomini Biophysical Reports, 2021 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.
Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy Alexander Kuhlemann, Gerti Beliu, Dieter Janzen, Enrica Maria Petrini, Danush Taban, Dominic A. Helmerich, Sören Doose, Martina Bruno, Andrea Barberis, Carmen Villmann, Markus Sauer, Christian Werner Frontiers in Synaptic Neuroscience, 2021 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.
Long-term plasticity of inhibitory synapses in the hippocampus and spatial learning depends on matrix metalloproteinase 3 Grzegorz Wiera, Katarzyna Lebida, Anna Maria Lech, Patrycja Brzdąk, Inge Van Hove, Lies De Groef, Lieve Moons, Enrica Maria Petrini, Andrea Barberis, Jerzy W. Mozrzymas Cellular and Molecular Life Sciences, 2021 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.
Changes of GABAA receptor activation kinetics in hippocampal neurons cultured for different periods of time Cellular and Molecular Biology Letters, 2004
