While nanosized particles (i.e., organic/inorganic particles having at least one dimension under 100 nm) have been present in our environment for thousands of years, their incidence has enormously increased since the first industrial revolution (e.g., combustion of fossil fuels) and has seen a further expansion with the diffusion of byproducts of manufacturing processes. However it is only after the appearance of engineered nanoscale materials, both in consumer products and for technological applications, that they have attracted widespread attention and increasing concern about their potential adverse effects on the environment and on human health. At the nanoscale, inorganic matter acquires unique properties that are not present when the same material is analyzed at a micro-macroscopic scale [1], and this poses new challenges for the understanding of the laws governing the interaction of nanoobjects with living cells and tissues.

The first systematic reports date from the early 80s (see e.g., [2]) and dealt with the health effects of ultrafine particles (UFPs) from ambient particulate matter (PM) on animals and humans. The term UFP refers to particles present in the environment, mainly from urban and industrial sources, and less than 100 nm in diameter, thus representing the nanosized component of PM; accordingly, the term “nanosized PM” (nPM) is currently used by some authors [3].

Around the same time, the first engineered nanoparticles (NPs) were developed for medical and industrial uses, and the issue of their potential toxicity was addressed [4]. However, only the last 10–15 years have witnessed a consistent surge in the number of papers related to this subject. These reports were mostly related to the cell types and tissues more likely to be exposed to the ultrafine pollutants, such as airway epithelial cells [5,6,7] and blood cells [8]; only in more recent years the nervous system has been taken into account as a potential target for NPs introduced into the organism from the external environment or following deliberate administration for diagnostic or therapeutic purposes [9,10].

The pioneering work of the Oberdöster group, started about 10 years ago [9,11,12] has set the basis for the understanding of the mechanisms that allow UFPs and NPs to access the nervous system (NS). Presently the literature is quite abundant, and solid evidence is available indicating that UFPs present in the environment can access the central nervous system (CNS) mainly through the olfactory and respiratory pathways; see also [13]. A comparable amount of data points to a complex set of adverse effects on the CNS. Most of these data have been obtained from both in vivo and in vitro experiments on animal models (rodents). Alterations in synaptic function, neuronal excitability and glial inflammatory responses have been reported [3,14]; these appear to be specific for some brain areas, such as hippocampus, one of the regions responsible for memory formation and learning. Prenatal exposure caused reduced neuronal differentiation and maturation [15]. Postnatal exposure caused locomotor dysfunction in brain regions involved in the development of neurodegenerative diseases such as Parkinson’s disease [16]; altered responses to behavioral tests were also observed [17]. Taken together these data, while based on animal research, suggest a link to potential involvement of nMP in human CNS disorders. A recent, comprehensive review, containing epidemiological data on air pollution and brain damage in humans, can be found in [18].

On the other hand, interactions between nanoparticles and neurons can happen by way of intentional introduction of engineered NPs into the animal (or potentially human) organism as probes or drug carriers, see e.g., [19]; for these NPs, evidence that they can pass the brain-blood barrier (BBB) has been provided [20,21]. These developments have created great expectations about the potential impact of nanoparticles in the approach to several neuropathologies, and have opened the way to the new discipline of “nanoneuromedicine” [22]; on the other hand, our understanding of the cellular parameters affected by the interactions between nanoparticles and neuronal cells and tissues is still preliminary and incomplete.

Changes in the cytosolic free calcium concentration, [Ca²⁺]_i, are key regulators of almost any cellular function: in eukaryotes, hundreds of proteins can bind calcium and consequently change their activity, thus modulating a myriad of intracellular signalling cascades. The gradient between extracellular (and organelle) and cytosolic calcium concentration is quite steep (more than 10⁵): thus even small changes in the permeability of the membranes to this ion can activate massive fluxes and induce fast and strong increases in [Ca²⁺]_i. Basically, two main mechanisms are involved: influx from the extracellular medium through different classes of ionic channels, and release form intracellular compartments and organelles, such as endoplasmic reticulum and mitochondria. Since these signals have to be controlled in space and time, a set of extrusion mechanisms (pumps and transporters), at the expense of metabolic energy, is in charge of extruding calcium back to the extracellular medium and to the intracellular compartments, acting as a homeostatic mechanism for this crucial functional parameter.

In neuronal cells, the calcium signalling toolbox is expressed to its full potential: the various mechanisms are finely regulated and specifically expressed in different cellular subcompartments, and can generate signals spanning a wide time range, form milliseconds to hours/days. In order to exert an accurate control on such neuronal functions as neuronal differentiation during development, synaptic transmission, electrical excitability, signal propagation, plasticity, calcium levels need to be finely tuned. Even minor perturbations in the mechanisms that underlie the complex pattern of signals induced either by the intrinsic neuronal activity or by extracellular cues (neurotransmitters, hormones, other signalling molecules) may have profound physio/pathological effects, either on a short or a long time scale. A relevant number of excellent general reviews is available; see e.g., [23,24,25,26].

Based on the above considerations, the calcium signalling machinery is a likely link between NPs entering the CNS and their potential neurotoxicity. A fair amount of data has accumulated on perturbation of calcium signalling and homeostasis by NPs in different types of cells and tissues, again mainly epithelial and blood cells, including some recent comprehensive reviews [27,28]. The information available on the perturbation of calcium homeostasis following the interaction of NPs with neurons and their internalization is more limited, but the pace is accelerating and some general features of the picture are now emerging. As for in vivo effects, there is a recent report [29] of the onset of a neuroinflammatory response and of anomalous calcium waves in microglia cells of the mouse brain following administration of lipid NPs. Another paper [30] has analyzed changes in basal calcium levels in the rat hippocampus following chronic administration of lead sulfide (PbS) NPs. All other data refer to in vitro measurements, even if in some cases they are accompanied by in vivo observations on neurotoxic effects; see e.g., [31].

Interestingly, while nanosized inorganic and organic particles can be internalized into cells, including neurons [32,33,34,35], most of the data discussed below refer to alterations in [Ca²⁺]_i due to calcium influx from the extracellular medium, with a minor contribution of release form intracellular compartments. This finding points to the plasmamembrane as the primary site of interaction of NPs with calcium mobilizing proteins and mechanisms.

One of the first reports dates from the end of the previous century, and is still one of the most relevant descriptions of the impact of fine and ultrafine environmental particulate on neuronal calcium signalling. In 1999, Veronesi group [36] reported that particulate matter (PM) and specifically residual oil fly ash (ROFA) induced inflammatory responses in human airway epithelial cells that was mediated by a rapid increase in [Ca²⁺]_i, totally dependent on influx from the extracellular medium. The response were completely abolished by capsazepine, a blocker of vanilloid receptors, and particularly of vanilloid receptor 1, VR1 [37]. These are polymodal membrane receptors, activated by irritants such as capsaicin, the active component of chili peppers, acidic pH, other intracellular and extracellular agonists and temperature. Notably, these receptors have subsequently been identified as calcium permeable transmembrane channels of the Transient Receptor Potential-Vanilloid (TRPV) family (specifically, VR1 is now called TRPV1, see [38]), part of the superfamily of TRP channels [39].

Of interest for this review, in the following year the same group [40] reported similar effects also on peripheral neurons, and, by comparing PM with micrometer sized polymeric particles, ascribed the activation of the VR1 receptor to the negative charge present on the surface of both classes of particles: the proton cloud collected around them was proposed to be the activator of acid sensing receptors, among them TRPV1 and other acid-sensitive channels (ASICS). In a subsequent paper [41] they described in more detail the calcium signals elicited by PM (size range from ≤0.2 µm to ≥10 µm), that ranged from single transients to oscillatory responses, in most cases fully reversible on a time span of minutes. Thus the scenario was set, and a molecular identity for the target of environmental particulate was proposed, even if the scale was not exactly defined. It must be emphasized that these data were mostly related to particulate and/or engineered particles in the micrometer range; however, in a recent paper [42] the same group has reported that statistically significant toxicity (neuronal loss) occurred only in the presence of ultrafine particles as compared to microparticles. These findings point to the need of a more precise distinction between acute neuroinflammatory responses and long-term toxic effects, and of the understanding of the underlying mechanisms.

Probably also because of the difficulties in dissecting and quantifying complex responses, apart for the above mentioned papers, most studies aimed at addressing the issue of nanoparticle-induced perturbation of intracellular calcium homeostasis have employed engineered NPs of controlled concentration, size and surface properties. The advantage of this approach is based on the possibility to assess, in a more quantitative and reproducible way, the influence of the physico-chemical properties of the NPs on the observed effects. On the other hand, it has to be stressed that environmental particulate is composed of both fine and ultrafine particles, and passing from the micro- to the nanoscale completely changes the properties and the potential interaction at the cell level.

In the following paragraphs, also in the light of the increasing interest for their diagnostic and therapeutical use in neuromedicine, we will focus on engineered NPs and briefly review the available knowledge on the calcium-mobilizing effects of different classes of NPs.

The most relevant data presented in this and in the following two chapters are summarized in Table 1 and in the cartoon of Figure 1.

Dendrimers (nanosized protein-like polymers) have been used for drug delivery and have been shown to be able to cross the blood-brain-barrier [61]; therefore, the understanding of the mechanisms activated following their interactions with neurons is of particular relevance. In rat hippocampal slices, Nyitrai et al., [62] have investigated the calcium signals elicited by polyamidoammine (PAMAM) dendrimers. After 30 min incubation, dendrimers could be detected on the cell surface of both neurons and astroglial cells, with weak internalization in neurons. Acute application of the dendrimers (0.1 mg/mL) induced fast and mostly reversible oscillations of [Ca²⁺]_i in astrocytes, while in neurons the increase was long lasting, both at the somatic and at the dendritic level. These increases in cytosolic calcium were paralleled by a strong mitochondrial depolarization, that in astroglial cells appeared subsequently to the calcium signal, pointing to a causal relationship between the two events. Moreover, when neuronal activity was blocked by means of a pharmacological approach, the number of glial cells showing mitochondrial depolarization was unchanged, while duration and intensity of the response were reduced: thus, dendrimers can induce mitochondrial depolarization in astroglial cells both directly and as a consequence of neuronal activation. The more transient depolarization observed in astroglial cells may explain why the neurotoxic effects were less severe in these cells as compared to neurons. Overall, these observations, albeit quite preliminary, evidence the need of more detailed investigations of the potential toxicity of these nanoparticles.

Most of the data discussed in the preceding paragraphs have been obtained by means of calcium imaging techniques from neuronal (or glial) cells in culture. This approach gives information on the behavior of a large number of individual components of a population, but is somehow indirect and cannot provide the identification of the specific targets affected by the interaction of NPs with the plasmamembrane: for this purpose electrophysiological measurements are needed. The patch clamp technique [63] has been widely used to obtain direct biophysical information of the changes in the properties of membrane channels following interaction with agonists and antagonist and also with nanoobjects [53,54,55,59]. However, only a cell at a time, and for a limited time span (up to tens of minutes) can be recorded: this limits the usefulness of the approach when delicate matters have to be investigated on a large number of cells and the effects of chronic administrations have to be assessed. For this reason, while not directly related to calcium signalling, it is worth mentioning a report on the effects of NPs on the activity of a neuronal network of mouse cortical neurons cultured on multi-electrode arrays, MEAs [64]. The authors analyzed the changes in firing pattern of the network following perfusion with three different types of nanoparticles: carbon black (CB), Fe₂O₃, and TiO₂. All induced a reduction in the firing activity, those of greater negative superficial charge (CB) having more marked effects. Since the firing of action potentials is directly related to calcium influx through voltage dependent calcium channels, this approach may prove highly useful for assessing effects of NPs on neuronal channels on a medium to long time scale: MEAs allow to record neuronal activity and its evolution in a neuronal population on a time of days/weeks, thus providing an additional and relevant level of information.

From the schematic and far from exhaustive review of the available data on perturbation of neuronal calcium signalling induced by acute and chronic interaction with nanoparticles, in particular engineered ones, it becomes evident that the picture is quite complex and still incomplete. A few points can be set: the role of concentration, size and surface properties; the specificity of each type of NP, related to differences in the physico-chemical properties (this point is well evidenced by the dramatic differences between Ag and Au NPs); the specificity of molecular targets involved and of the cellular model under investigation.

A few general features can anyway be extracted from the available information: -

in general, most NPs interfere with neuronal calcium homeostasis by interactions at the plasmamembrane; only a few reports of a role of internalization in this context are available.

Looking at the references included in this review, it becomes evident that the field is quite young: most of the papers related to NPs and neuronal calcium signalling, if we exclude the early ones on environmental particulate, date from the last four years. The ensemble of all these data has to be taken as preliminary and in need of more careful investigations: however, considering the pace at which new information is added, we can expect a burst of relevant and exciting news in the very near future.