Cell assemblies manipulation by optogenetics is pivotal to advance neuroscience and neuroengineering. In in vivo applications, photostimulation often broadly addresses a population of cells simultaneously, leading to feed-forward and to reverberating responses in recurrent microcircuits. The former arise from direct activation of targets downstream, and are straightforward to interpret. The latter are consequence of feedback connectivity and may reflect a variety of time-scales and complex dynamical properties. We investigated wide-field photostimulation in cortical networks in vitro, employing substrate-integrated microelectrode arrays and long-term cultured neuronal networks. We characterized the effect of brief light pulses, while restricting the expression of channelrhodopsin to principal neurons. We evoked robust reverberating responses, oscillating in the physiological gamma frequency range, and found that such a frequency could be reliably manipulated varying the light pulse duration, not its intensity. By pharmacology, mathematical modelling, and intracellular recordings, we conclude that gamma oscillations likely emerge as in vivo from the excitatory-inhibitory interplay and that, unexpectedly, the light stimuli transiently facilitate excitatory synaptic transmission. Of relevance for in vitro models of (dys)functional cortical microcircuitry and in vivo manipulations of cell assemblies, we give for the first time evidence of network-level consequences of the alteration of synaptic physiology by optogenetics

Neuronal function is highly sensitive to changes in oxygen levels, but how hypoxia affects dendritic spine formation and synaptogenesis is unknown. Here we report that hypoxia, chemical inhibition of the oxygen-sensing prolyl hydroxylase domain proteins (PHDs), and silencing of Phd2 induce immature filopodium-like dendritic protrusions, promote spine regression, reduce synaptic density, and decrease the frequency of spontaneous action potentials independently of HIF signaling. We identified the actin cross-linker filamin A (FLNA) as a target of PHD2 mediating these effects. In normoxia, PHD2 hydroxylates the proline residues P2309 and P2316 in FLNA, leading to von Hippel-Lindau (VHL)-mediated ubiquitination and proteasomal degradation. In hypoxia, PHD2 inactivation rapidly upregulates FLNA protein levels because of blockage of its proteasomal degradation. FLNA upregulation induces more immature spines, whereas Flna silencing rescues the immature spine phenotype induced by PHD2 inhibition.

While the design of closed-loop experimental protocols in cellular electrophysiology dates back more than 60 years, recent developments promise to significantly advance the field. We review a selection of recent applications of closed-loop methods in neurobiology, focussing on the intracellular and extracellular access to cellular excitability, employed to dissect the biophysical bases of information processing. We cover relevant methodologies targeting different levels of description, ranging from single ion channels to large ensembles of neurons, and extending across different time scales, ranging from milliseconds to longer intervals characteristic of the variability in the firing rate. We conclude by mentioning future perspectives and developments.

Brain α-synuclein deposits are the hallmark of various distinct neurodegenerative diseases, and it is proposed that α-synuclein assemblies with different structural characteristics or 'strains' (ribbons or fibrils) could account for pathological differences between these diseases; here different human α-synuclein strains are injected into rat brain, and are shown to propagate in a strain-dependent manner and cause different pathological and neurotoxic phenotypes. Synucleinopathies are neurodegenerative disorders characterized by α-synuclein-rich protein deposits which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. The discovery of α-synuclein assemblies with different structural characteristics has led to the hypothesis that different 'strains' could account for pathological differences between these different neurodegenerative diseases. This study reports that when different human α-synuclein strains — oligomers, ribbons or fibrils — are injected into rat brain in vivo, they propagate in a strain-dependent manner and cause different pathological and neurotoxic phenotypes. This work has implications for disease diagnosis and prognosis and for the prospects of developing therapeutic strategies tailored for specific synucleinopathies. Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions1. The molecular hallmark of synucleinopathies such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson’s disease and dementia with Lewy bodies2. The discovery of α-SYN assemblies with different structural characteristics or ‘strains’ has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies3,4. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson’s disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood–brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.

Micro-Electrode Arrays (MEAs) have emerged as a mature technique to investigate brain (dys)functions in vivo and in in vitro animal models. Often referred to as “smart” Petri dishes, MEAs have demonstrated a great potential particularly for medium-throughput studies in vitro, both in academic and pharmaceutical industrial contexts. Enabling rapid comparison of ionic/pharmacological/genetic manipulations with control conditions, MEAs are employed to screen compounds by monitoring non-invasively the spontaneous and evoked neuronal electrical activity in longitudinal studies, with relatively inexpensive equipment. However, in order to acquire sufficient statistical significance, recordings last up to tens of minutes and generate large amount of raw data (e.g., 60 channels/MEA, 16 bits A/D conversion, 20 kHz sampling rate: approximately 8 GB/MEA,h uncompressed). Thus, when the experimental conditions to be tested are numerous, the availability of fast, standardized, and automated signal preprocessing becomes pivotal for any subsequent analysis and data archiving. To this aim, we developed an in-house cloud-computing system, named QSpike Tools, where CPU-intensive operations, required for preprocessing of each recorded channel (e.g., filtering, multi-unit activity detection, spike-sorting, etc.), are decomposed and batch-queued to a multi-core architecture or to a computers cluster. With the commercial availability of new and inexpensive high-density MEAs, we believe that disseminating QSpike Tools might facilitate its wide adoption and customization, and inspire the creation of community-supported cloud-computing facilities for MEAs users.

In recent years Multi-Electrode Arrays (MEAs) have emerged as a powerful tool to study brain (dys)functions in-vivo and in in-vitro animal models. Typically, each session of electrophysiological experiments with such MEAs generate large amount of raw data (e.g., 60 channels/MEA, 16 bits A/D conversion, 20 kHz sampling rate: approximately 8 GB/MEA, h uncompressed) and inferring meaningful conclusions from them require rigorous and automated processing. To this goal, the current work proposes a cloud-computing based software workflow, QSpikeTools for preliminary preprocessing and analysis of neuronal activities recorded from MEAs with 60 recording sites. It exploits the facilities provided by some open-source tools to delegate CPU-intensive and independent operations to be performed on individual recorded channels (e.g., signal filtering, multi-unit activity detection, spike sorting, etc.) to a multi-core computer or a computer cluster to be executed in parallel. We report that the required time in performing the desired processing and analysis decreases significantly with increasing number of employed cores. With the commercial availability of new, sophisticated, and inexpensive high-density MEAs, we believe that widely dissemination of QSpikeTools may facilitate its adoption and customization, and possibly inspire the creation of community-supported cloud-computing facilities for MEAs users.

The price of peat, one of the most important constituents of growing media in pot plant industry (at least in Europe), is increasing and there is an emergent interest on peat-lands for their natural and/or archaeological value. This has produced an 'anti-peat' campaign in many European countries and is expected to lead, in the near future, to a drastic reduction in the use of peat in favour of alternative materials. The paper reports the results of three simple experiments performed to test two peat substitutes in growing media for outdoor or greenhouse container plants:coconut coir fibre and organic green compost (two commercial products from green and urban organic wastes were evaluated). In the first experiment, two shrubs (Photinia x fraseri 'Red Robin' and Viburnum tinus L.) were grown in four different mixtures:peat-pumice (1:1, v:v; standard substrate), pumice-coconut coir (1:1, v:v), pumice-compost-peat (0.50:0.30:0.20, v:v) and pumice-compost-coconut (0.50:0.30:0.20, v:v). In Photinia leaf area and/or the dry weight of whole plant and its main organs were significantly reduced when the pots were filled with substrates other than the standard one. By contrast, in Viburnum plant growth was stimulated by the use of alternative substrates. In the second and third experiment, other four different substrates were tested for greenhouse production of pot geranium (Pelargonium peltatum L.) or tray plugs of two bedding species (Calendula officinalis L. and Ageratum houstonianum L.) and tomato (Solanum lycopersicum L.):peat-perlite (0.75:0.25, v:v), coconut:perlite (075:0.25, v:v), compost-peat-perlite (0.375:0.375:0.250, v:v) and compost-perlite (0.75:0.25, v:v). In these experiments, compost was provided by a different supplier with quality certification and no important effect of growing medium on plant growth characteristics or seedlings emergence (exp. 3) was observed. These findings confirmed that compost and coconut coir may contribute to reduce, at least partially, the use of peat in professional horticulture, provided the producers have a well established form of quality assurance to ensure the consistency and safety of the product, which starts with strict selection of raw materials.

A simple spreadsheet (SIMULHYDRO) was designed to predict the consumption of both water and fertilisers, and the environmental impact associated to nutrient leaching, in greenhouse soilless cultures on the basis of a limited number of variables (such as global radiation, air temperature, and the ion composition of irrigation water) and parameters, the most important of which is the ion uptake concentration (i.e. the expected ratio between ion and water uptake by the crop). SIMULHYDRO aggregates three major models that run on a daily basis to estimated: i) crop water uptake (V-U); ii) the ion composition and the electrical conductivity of recycling nutrient solution (ECNS); iii) the ion composition of drainage water in open (free-drain) and semi-closed (with periodical discharge of the recirculating water) growing systems. SIMULHYDRO was used to simulate the water and mineral relations of greenhouse tomato plants grown in semi-closed substrate (rockwool) culture using saline water (approx. 9.5 mmol L-1 Nacl) and three different fertigation strategies: A) V-U was systematically compensated with nutrient solution at full strength (ECNS = 2.5 dS m(-1)) in order to maintain a (relatively) constant nutrient content; mostly due to the accumulation of ballast ions (Na+ and CT), this strategy resulted in a progressive increase of ECNS till it reached a ceiling value (ECNSMAX = 4.5 dS m(-1)) tolerated by the crop, afterwards the recirculating water was flushed out; B) an EC set-point of approx. 3.0 dS m(-1) was maintained and the nutrient solution was flushed out whenever the N-NO3- concentration decreased below 1.0 mmol L-1; C) V-U was initially compensated with nutrient solution at full strength, as in Strategy A; when ECNS reached 4.5 dS m(-1), the mixing tank was refilled with fresh water only, in order to withdraw N from the nutrient solution before discharge. The experiment included also an open system to verify the possible influence of salinity build-up and/or nutrient depletion on crop yield. A good agreement was found between simulated and measured data. No important effects of the adopted strategies were observed on V-U and fruit yield, although the former was significantly higher in open system than in the Strategies A and B. Strategies A and C reduced total water use compared to the others. The results confirmed that a semi-closed system conducted following the strategy of full nutrient solution replenishment (Strategy A) may produce a massive environmental pollution due to nutrient (nitrogen) leaching, although to a much lesser extent than open growing system.