Antonio Malgaroli

Loose-patch recordings of single quanta at individual hippocampal synapses

Synapses in the central nervous system are typically studied by recording electrical responses from the cell body of the postsynaptic cell. Because neurons are normally connected by multiple synaptic contacts, these postsynaptic responses reflect the combined activity of many thousands synapses, and it remains unclear to what extent the properties of individual synapses can be deduced from the population response. We have therefore developed a method for recording the activity of individual hippocampal synapses. By capturing an isolated presynaptic bouton inside a loose-patch pipette and recording from the associated patch of postsynaptic membrane, we were able to detect miniature excitatory postsynaptic currents (‘minis’) arising from spontaneous vesicle exocytosis at a single synaptic site, and to compare these with minis recorded simultaneously from the cell body. The average peak conductance at a single synapse was about 900 pS, corresponding roughly to the opening of 90 AMPA-type glutamate-receptor channels. The variability in this conductance was about 30%, matching the value reported for the neuromuscular junction. Given that our synapses displayed single postsynaptic densities (PSDs), this variability is larger than would be predicted from the random opening of receptor channels, suggesting that they are not saturated by the content of a single vesicle. Therefore the response to a quantum of neurotransmitter at these synapses is not limited by the number of available postsynaptic receptors.

Ero1α regulates Ca(2+) fluxes at the endoplasmic reticulum-mitochondria interface (MAM)

AIMS The endoplasmic reticulum (ER) is involved in many functions, including protein folding, redox homeostasis, and Ca(2+) storage and signaling. To perform these multiple tasks, the ER is composed of distinct, specialized subregions, amongst which mitochondrial-associated ER membranes (MAM) emerge as key signaling hubs. How these multiple functions are integrated with one another in living cells remains unclear. RESULTS Here we show that Ero1α, a key controller of oxidative folding and ER redox homeostasis, is enriched in MAM and regulates Ca(2+) fluxes. Downregulation of Ero1α by RNA interference inhibits mitochondrial Ca(2+) fluxes and modifies the activity of mitochondrial Ca(2+) uniporters. The overexpression of redox active Ero1α increases passive Ca(2+) efflux from the ER, lowering [Ca(2+)](ER) and mitochondrial Ca(2+) fluxes in response to IP3 agonists. INNOVATION The unexpected observation that Ca(2+) fluxes are affected by either increasing or decreasing the levels of Ero1α reveals a pivotal role for this oxidase in the early secretory compartment and implies a strict control of its amounts. CONCLUSIONS Taken together, our results indicate that the levels, subcellular localization, and activity of Ero1α coordinately regulate Ca(2+) and redox homeostasis and signaling in the early secretory compartment.

Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression.

Alphaviral vectors inhibit host cell protein synthesis and are cytotoxic. To overcome these limitations, we modified the nonstructural protein-2 (nsP2) gene in the Semliki Forest virus (SFV) vector, pSFV1. Packaging of SFV replicons with two point mutations in nsP2 resulted in high-titer recombinant SFV(PD) particles. SFV(PD) led to more efficient host cell protein synthesis, exhibited reduced cytotoxicity and improved cell survival, and allowed greater and prolonged transgene expression than the original vector, SFV. In dissociated hippocampal neurons and organotypic rat hippocampal slices, SFV(PD) infection preserved neuronal morphology and synaptic function more efficiently than SFV. Combination of the two point mutations with a replication-persistent mutation in nsP2 resulted in a highly temperature-sensitive vector, SFV(PD713P), which efficiently transduced neurons in hippocampal slice cultures. At 31 degrees C, SFV(PD713P) allowed continuous transgene expression in BHK cells, at amounts comparable to SFV(PD). These new SFV mutants are expected to substantially broaden the application of alphaviral vectors in neurons and other mammalian cells.

Proteomic analysis of protein S‐nitrosylation

Nitric oxide (NO) produces covalent PTMs of specific cysteine residues, a process known as S‐nitrosylation. This route is dynamically regulated and is one of the major NO signalling pathways known to have strong and dynamic interactions with redox signalling. In agreement with this scenario, binding of NO to key cysteine groups can be linked to a broad range of physiological and pathological cellular events, such as smooth muscle relaxation, neurotransmission and neurodegeneration. The characterization of S‐nitrosylated residues and the functional relevance of this protein modification are both essential information needed to understand the action of NO in living organisms. In this review, we focus on recent advances in this field and on state‐of‐the‐art proteomic approaches which are aimed at characterizing the S‐nitrosylome in different biological backgrounds.

Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins

BackgroundAll studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture.ResultsTo obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles.ConclusionBecause reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.

Protein homeostasis in neurons and its pathological alterations.

In neuronal cells, proteins are synthesized on ribosomes from the genetic information encoded in DNA. In some instances translation takes place at the neuronal cell soma but in other it occurs at distal location, such as in a dendritic spine. Folding is usually initiated before the completion of protein synthesis and its outcome strictly depends on the local environment in which the nascent protein is submerged. Incompletely folded proteins and, more importantly, misfolded proteins are under the surveillance of several quality control systems that re-establish the correct conformation or initiate protein degradation. Regulation and maintenance of these systems is a vital issue for neuronal and glial cells, and impairments at different levels leads to neurodegenerative diseases.

Silent synapses: I can't hear you! Could you please speak aloud?

Cyclic AMP application mimics the late phase of long-term potentiation in hippocampal CA3–CA1 cultures. Now imaging with FM1-43 shows that this activity-dependent change in synaptic strength involves the recruitment of pre-existing, presynaptically silent synapses.

Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways

The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signaling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterized by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological in vivo ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation.

Detection of fractal behavior in temporal series of synaptic quantal release events: a feasibility study

Since the pioneering work of Fatt and Katz at the neuromuscular junction (NMJ), spontaneous synaptic release (minis), that is, the quantal discharge of neurotransmitter molecules which occurs in the absence of action potentials, has been unanimously considered a memoryless random Poisson process where each quantum is discharged with a very low release probability independently from other quanta. When this model was thoroughly tested, for both population and single-synapse recordings, some clear evidence in favor of a more complex scenario emerged. This included short- and long-range correlation in mini occurrences and divergence from mono-exponential inter-mini-interval distributions, both unexpected for a homogeneous Poisson process, that is, with a rate parameter that does not change over time. Since we are interested in accurately quantifying the fractal exponent α of the spontaneous neurotransmitter release process at central synaptic sites, this work was aimed at evaluating the sensitivity of the most established methods available, such as the periodogram, the Allan, factor and the detrended fluctuation analysis. For this analysis we matched spontaneous release series recorded at individual hippocampal synapses (single-synapse recordings) to generate large collections of simulated quantal events by means of a custom algorithm combining Monte Carlo sampling methods with spectral methods for the generation of 1/f series. These tests were performed by varying separately: (i) the fractal exponent α of the rate driving the release process; (ii) the distribution of intervals between successive releases, mimicking those encountered in single-synapse experimental series; (iii) the number of samples. The aims were to provide a methodological framework for approaching the fractal analysis of single-unit spontaneous release series recorded at central synapses.

A pre-docking source for the power-law behavior of spontaneous quantal release: application to the analysis of LTP.

In neurons, power-law behavior with different scaling exponents has been reported at many different levels, including fluctuations in membrane potentials, synaptic transmission up to neuronal network dynamics. Unfortunately in most cases the source of this non-linear feature remains controversial. Here we have analyzed the dynamics of spontaneous quantal release at hippocampal synapses and characterized their power-law behavior. While in control conditions a fractal exponent greater than zero was rarely observed, its value was greatly increased by α-latrotoxin (α-LTX), a potent stimulator of spontaneous release, known to act at the very last step of vesicle fusion. Based on computer modeling, we confirmed that at an increase in fusion probability would unmask a pre-docking phenomenon with 1/f structure, where α estimated from the release series appears to sense the increase in release probability independently from the number of active sites. In the simplest scenario the pre-docking 1/f process could coincide with the Brownian diffusion of synaptic vesicles. Interestingly, when the effect of long-term potentiation (LTP) was tested, a ~200% long-lasting increase in quantal frequency was accompanied by a significant increase in the scaling exponent. The similarity between the action of LTP and of α-LTX suggests an increased contribution of high release probability sites following the induction of LTP. In conclusion, our results indicate that the source of the synaptic power-law behavior arises before synaptic vesicles dock to the active zone and that the fractal exponent α is capable of sensing a change in release probability independently from the number of active sites or synapses.