Proactive and reactive inhibition during overt and covert actions. An electrical neuroimaging study.

Response inhibition is the ability to suppress inadequate but automatically activated, prepotent or ongoing response tendencies. In the framework of motor inhibition, two distinct operating strategies have been described: “proactive” and “reactive” control modes. In the proactive modality, inhibition is recruited in advance by predictive signals, and actively maintained before its enactment. Conversely, in the reactive control mode, inhibition is phasically enacted after the detection of the inhibitory signal. To date, ample evidence points to a core cerebral network for reactive inhibition comprising the right inferior frontal gyrus (rIFG), the presupplementary motor area (pre-SMA) and the basal ganglia (BG). Moreover, fMRI studies showed that cerebral activations during proactive and reactive inhibition largely overlap. These findings suggest that at least part of the neural network for reactive inhibition is recruited in advance, priming cortical regions in preparation for the upcoming inhibition. So far, proactive and reactive inhibitory mechanisms have been investigated during tasks in which the requested response to be stopped or withheld was an “overt” action execution (AE) (i.e., a movement effectively performed). Nevertheless, inhibitory mechanisms are also relevant for motor control during “covert actions” (i.e., potential motor acts not overtly performed), such as motor imagery (MI). MI is the conscious, voluntary mental rehearsal of action representations without any overt movement. Previous studies revealed a substantial overlap of activated motor-related brain networks in premotor, parietal and subcortical regions during overtly executed and imagined movements. Notwithstanding this evidence for a shared set of cerebral regions involved in encoding actions, whether or not those actions are effectively executed, the neural bases of motor inhibition during MI, preventing covert action from being overtly performed, in spite of the activation of the motor system, remain to be fully clarified. Taking into account this background, we performed a high density EEG study evaluating cerebral mechanisms and their related sources elicited during two types of cued Go/NoGo task, requiring the execution or withholding of an overt (Go) or a covert (MI) action, respectively. The EEG analyses were performed in two steps, with different aims: 1) Analysis of the “response phase” of the cued overt and covert Go/NoGo tasks, for the evaluation of reactive inhibitory control of overt and covert actions. 2) Analysis of the “preparatory phase” of the cued overt and covert Go/NoGo EEG datasets, focusing on cerebral activities time-locked to the preparatory signals, for the evaluation of proactive inhibitory mechanisms and their related neural sources. For these purposes, a spatiotemporal analysis of the scalp electric fields was applied on the EEG data recorded during the overt and covert Go/NoGo tasks. The spatiotemporal approach provide an objective definition of time windows for source analysis, relying on the statistical proof that the electric fields are different and thus generated by different neural sources. The analysis of the “response phase” revealed that key nodes of the inhibitory circuit, underpinning inhibition of the overt movement during the NoGo response, were also activated during the MI enactment. In both cases, inhibition relied on the activation of pre-SMA and rIFG, but with different temporal patterns of activation in accord with the intended “covert” or “overt” modality of motor performance. During the NoGo condition, the pre-SMA and rIFG were sequentially activated, pointing to an early decisional role of pre-SMA and to a later role of rIFG in the enactment of inhibitory control of the overt action. Conversely, a concomitant activation of pre-SMA and rIFG emerged during the imagined motor response. This latter finding suggested that an inhibitory mechanism (likely underpinned by the rIFG), could be prewired into a prepared “covert modality” of motor response, as an intrinsic component of the MI enactment. This mechanism would allow the rehearsal of the imagined motor representations, without any overt movement. The analyses of the “preparatory phase”, confirmed in both overt and covert Go/NoGo tasks the priming of cerebral regions pertaining to putative inhibitory network, reactively triggered in the following response phase. Nonetheless, differences in the preparatory strategies between the two tasks emerged, depending on the intended “overt” or “covert” modality of the possible incoming motor response. During the preparation of the overt Go/NoGo task, the cue primed the possible overt response programs in motor and premotor cortex. At the same time, through preactivation of a pre-SMA-related decisional mechanism, it triggered a parallel preparation for the successful response selection and/or inhibition during the subsequent response phase. Conversely, the preparatory strategy for the covert Go/NoGo task was centred on the goal-oriented priming of an inhibitory mechanism related to the rIFG that, being tuned to the instructed covert modality of the motor performance and instantiated during the subsequent MI enactment, allowed the imagined response to remain a potential motor act. Taken together, the results of the present study demonstrate a substantial overlap of cerebral networks activated during proactive recruitment and subsequent reactive enactment of motor inhibition in both overt and covert actions. At the same time, our data show that preparatory cues predisposed ab initio a different organization of the cerebral areas (in particular of the pre-SMA and rIFG) involved with sensorimotor transformations and motor inhibitory control for executed and imagined actions. During the preparatory phases of our cued overt and covert Go/NoGo tasks, the different adopted strategies were tuned to the “how” of the motor performance, reflecting the intended overt and covert modality of the possible incoming action.

Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.