In the recent years a growing bulk of studies expanded knowledge about the positive impact of dietary phytochemicals (PhC), or nutraceuticals, on complex physiological processes and brain aging. Many of the potential health benefits from compounds extracted by edible vegetables are based on their biological activities and the possible modulation of cellular responses. Among the numerous effects exerted by PhC, cumulative evidence has highlighted their antioxidant and anti-inflammatory properties (Ghanim et al., 2010; Obrenovich et al., 2011). Given that inflammation and oxidative stress have been reported to sustain neurological alterations leading to neurodegeneration and cognitive decline (Lassmann, 2011), the use of nutraceuticals was suggested for therapeutic approaches. In this context, specific dietary regimens including plant-derived compounds have been suggested to enhance cognitive performances and reduce neurodegenerative impairments (Spencer, 2008, 2009; Rendeiro et al., 2012; Meng et al., 2015; Almeida et al., 2016), with a remarkable significance for age-related conditions (Joseph et al., 2005; Youdim and Joseph, 2001). In the brain, excitotoxicity represents a further risk for cell survival with possible involvement in neurodegeneration (Pallo et al., 2016; Wang and Reddy, 2017). Noticeably, sound evidence highlighted the influence of PhC on cell excitability and their positive implications on cognitive functions in aging (Williams et al., 2004). Notwithstanding, only a few researches have been provided on the modulation of neuronal excitability by nutraceuticals in order to unveil new cellular targets of PhC and related beneficial employments. The present study is focused on indicaxanthin, a yellow betalain pigment contained in cactus pear fruit (Opuntia ficus indica). This plant, distributed in Mexico, arid regions of America, Africa, Australia and Mediterranean area (de Wit et al., 2010; Albano et al., 2015) has recently emerged as a potential source of valuable components for human health and food industry (Feugang et al., 2006; Kim et al., 2012; Heinrich et al., 2014; Sánchez et al., 2014; Alencar et al., 2018). Indicaxanthin shares many of the favorable effects of PhC, as described in previous scientific reports (Butera et al., 2002). Indeed, indicaxanthin has proven its antioxidant potential both by preventing lipid peroxidation and reducing reactive oxygen/nitrogen species (Allegra et al., 2005, 2007; Tesoriere et al., 2006), but also by interfering with specific redox-dependent signaling pathways in experimental models of innate immunity in vitro (Allegra et al., 2014a; Tesoriere et al., 2015). Relevantly and in line with its redox-modulating effects, the pigment has also been shown to exert significant anti-inflammatory effects in vivo (Allegra et al., 2014b). All the effects exerted by indicaxanthin gain a more interesting value in the light of its remarkable bioavailability in vivo (Tesoriere et al., 2004). More recently a multidisciplinary approach has demonstrated that indicaxanthin orally administered at dietary-relevant doses is able to cross blood-brain barrier (BBB) and accumulate within the rat brain; beyond this, the intriguing ability of this phytochemical to modulate the bioelectric activity of hippocampal neurons for the first time has emerged (Allegra et al., 2015). In this light, the current research aimed to detect a brain distribution pattern of indicaxanthin, after oral administration of nutritionally-relevant amounts, by HPLC assay. Afterwards, microiontophoretic recordings were carried out to explore a possible influence of the indicaxanthin on the bioelectric activity of single neuronal units in specific brain areas.

Therefore, the present study is addressed to shed new light on the direct modulation of membrane excitability by edible PhC. In particular, this could unveil the nutritional importance of indicaxanthin and its influence on a pivotal aspect of neuron physiology, with a possible impact on the conditions of altered cell excitability that often underlie neuronal damages and, ultimately, cognitive dysfunctions.

Biodistribution and neurophysiological data from this study fall within the remit of intense research on PhC and human health, focusing on the interaction of indicaxanthin with central nervous system. Within the immense pharmacological cornucopia of nutraceuticals, only few of them (curcumin, resveratrol, tea polyphenols) have been reported to cross the BBB and accumulate in different brain areas exerting neuroprotective effects (Milbury and Kalt, 2010; Vingtdeux et al., 2010; Pandareesh et al., 2015). In the present research, we demonstrate for the first time that indicaxanthin, a betalain pigment able to cross the BBB (Allegra et al., 2015), when administered at nutritionally-relevant amounts accumulates within different brain areas and remains unmodified at levels comparable or even greater than those showed by other PhC (Vingtdeux et al., 2010; Vanmierlo et al., 2012). Moreover, while some PhC distribute within the brain homogeneously (Milbury and Kalt, 2010), we here show that indicaxanthin gains a specific access to selected brain areas. This phytochemical was indeed found in different amount in cortex, hippocampus, diencephalon, brainstem, cerebellum, but not in the striato-pallidal complex. While indicaxanthin peculiar ability to cross BBB may be due to its amphiphilicity, high bioavailability and affinity to lipid membranes (Turco Liveri et al., 2009), a more complex scenario maybe envisaged to explain the selective accumulation of the pigment in some brain areas and the exclusion from others such as the striato-pallidal complex. Anatomically speaking, the discrepancy could be ascribed to the peculiar structural features of this subcortical region belonging to the basal ganglia; in particular, the presence of different fiber bundles, encapsulating the striatum and pallidum, could not allow indicaxanthin accumulation as easily as in the rest of the brain (Parent and Hazrati, 1995). The chemical structure of indicaxanthin could also account for its absence in some brain areas unlike, for example, the anthocyanins contained in the blueberry, capable to reach the striatum after oral consumption (Andres-Lacueva et al., 2005). Surely enough, concentrations of indicaxanthin necessary to enter in distinct brain structures can vary on the basis of the mechanism employed for the passage, for instance membrane transporters or endocytosis, and this could also be responsible for the lack of indicaxanthin accumulation both in the striatum and pallidum. This aspect surely deserves further studies focusing on the interaction of indicaxanthin with the different biological barriers lying in the brain.

Considering the permeability of indicaxanthin to BBB and its distribution, we extensively investigated the neuromodulatory potential of indicaxanthin. To this purpose, electrophysiological recordings were conducted in the brain areas mainly involved in forebrain cognitive functions, including striato-pallidal complex; single neuronal unit recordings associated with microiontophoresis represent an adequate and direct method to examine the electrophysiological outcomes of exogenous substances with unknown properties. Furthermore, administrations of indicaxanthin at three different amounts were carried out to trace a dose-response profile (Table 1). In detail, during cortical recordings, the evaluation of cell firing revealed that discharge frequency was inhibited by indicaxanthin at 0.34 and 0.17, but not at 0.085 ng/neuron. The inhibition induced by indicaxanthin at 0.34 appeared more intense than that at 0.17/ng neuron and characterized by an increased magnitude of effect accompanied by a lower latency and a greater duration of the effect, though statistical significance between effects at different amount of the molecule was observed only for the effect duration. This ability of indicaxanthin to modulate neuronal excitability emerges more clearly in the hippocampus, where the phytochemical always resulted effective. Indeed, the different amounts of indicaxanthin administered induced a dose-dependent significant inhibition of cell firing, especially on the magnitude of the effect. This was confirmed by the related changes in latency and duration of the effect. These data suggest that hippocampal neurons are more responsive to indicaxanthin with respect to cortical ones. In the striatum, we observed an opposite outcome since indicaxanthin determined the excitation of firing rate with an intensity related to the amount applied. In particular, the greater magnitude of the effect was associated to shorter latency and longer duration of the response, although statistical significance came out only for the latency between 0.34 vs. 0.085 ng/neuron. In the globus pallidus, belonging to basal ganglia circuit as well as striatum, indicaxanthin has instead induced the inhibition of neuronal firing, but only after the injection at 0.34 vs. 0.085 ng/neuron, that did not statistically differ between them. As for recordings performed in the thalamus, it was evidenced a marked reduction of neuronal excitability. Amount-related changes in magnitude were found and were confirmed by significant differences between doses in latency and duration. Finally, the subthalamic nucleus was pointed out as the areas that was less responsive to treatments since indicaxanthin was effective only at 0.34 ng/neuron in reducing cell firing.

This evidence sustains a prevalent inhibitory effect of indicaxanthin on discharge activity of neurons in rat brain (Table 1). As for the exclusive excitatory effect observed in the striatum, in order to explain this different result, it should be noted that all the other structures examined are mainly based on glutamatergic or GABAergic transmission, while in the striatum a relevant dopaminergic influence integrate glutamatergic input and contribute to the neuronal excitability (Parent and Hazrati, 1995; Michaelis, 1998). Therefore, the modulatory action of indicaxanthin occurred in a peculiar synaptic environment, more finely regulated, possibly underlying the increase in discharge frequency. Interestingly, in our previous researches the striatum already showed its particularity, given that the microiontophoretic administration of nitric oxide donors, likely intervening in glutamatergic and GABAergic transmission, induced excitation in all basal ganglia structures, except for the striatum where inhibition was observed (Sardo et al., 2003, 2006, 2009, 2011; Carletti et al., 2009, 2012).

Taking into account the areas inhibited, indicaxanthin action could be framed within the context of neurotransmission systems exploiting glutamate and GABA, as already mentioned. In this view, the inhibition of neuronal activity could be due to the reduction of glutamate neurotransmission or the enhancement of GABA transmission. Although our data do not allow to assert the presence of a direct interaction with these systems, our previous article demonstrated the potential affinity of indicaxanthin to subunit N2A of NMDA glutamate receptor (NMDAR), that is pivotal for NMDAR function but is also involved in alteration linked to aging neurodegeneration (Gardoni et al., 2006; Allegra et al., 2015). Moreover, the ability of indicaxanthin to reduce glutamate-mediated neuronal excitability in the hippocampus was previously outlined (Allegra et al., 2015). In support of this interpretation, other researches propose the interaction between PhC and glutamate receptors and suggest, for example, that a diet enriched with blueberry ameliorated hippocampal learning process by preventing dysfunctions in the glutamatergic transmission (Williams et al., 2004; Coultrap et al., 2008). High expression of NMDAR subunit N2A in both the cortex and hippocampus suggests that this could be a potential site of action for indicaxanthin in these regions. Furthermore, the ubiquitous expression of this subunit in the entire adult brain could explain the widespread and reproducible effects of indicaxanthin. On the other hand, the implication of an interplay between indicaxanthin with GABA system should be also considered. In this regard, an enhancement of GABA-currents induced by the flavone hispidulin, exerting anticonvulsive outcomes, was evidenced in an animal model of seizure (Kavvadias et al., 2004). Furthermore, the finding that flavonoids produced anxiolytic effect by virtue of an affinity for benzodiazepine site on GABA receptor (Medina et al., 1998) suggests a fine addressed action of PhC in modulating neuronal excitability. However, the possible interaction of indicaxanthin with other channels determining the excitability state of neurons cannot be ruled out and deserves further studies.

In the light of the present results, a noticeable direct neuromodulatory action for the indicaxanthin emerges. This influence on the cell excitability could have implications in some cellular dysfunctions induced by alterations of membrane mechanisms. Indeed, increased excitation could bring to excitotoxicity, especially in cortical areas, that triggers damaging and neurodegenerative disorders, such as Alzheimer disease, above all, but also epilepsy (Lipton and Rosenberg, 1994; Danysz and Parsons, 2003; Dong et al., 2009; Mehta et al., 2013). Since many beneficial neuroprotective effects of nutraceuticals are almost exclusively ascribed to their anti-inflammatory and antioxidant properties, the findings of a direct influence of indicaxanthin on the bioelectric activity of neurons shed new light on the possible mechanisms lending to dietary vegetables their favorable and healthy features. In particular, considering the prevalent inhibitory effect of indicaxanthin, the potential brake to excitatory transmission could constitute a further factor underpinning the benefits for maintenance of cell function. In this framework, inhibitory action of indicaxanthin in cortical and hippocampal areas, involved in cognitive functions and often affected by neurodegenerative deficits, represents an intriguing perspective for the use of this and, possibly, other phyto-nutrients in the implementation of anti-neurodegenerative strategies. Indeed, several researches already reported the positive effects of diets enriched with PhC on the brain function by improving telencephalic cognitive processes (Spencer, 2008). For example, in aged rats, the chronic supplementation with flavonoids from gingko biloba increased hippocampal LTP via the reduction of neuronal excitability (Williams et al., 2004). The altered excitability underlying age-related amyloid pathologies could also lead to epileptogenesis and some dietary plant-derived nutrients already revealed anticonvulsant effects (Kavvadias et al., 2004; Nassiri-Asl et al., 2008; Gurevicius et al., 2013; Diniz et al., 2015; Aggleton et al., 2016), also in aberrant cortico-thalamic hyperexcited connections (Gurevicius et al., 2013). Since this detrimental link between neurodegeneration and altered neuronal excitability could constitute one of the factors concurring to brain diseases, this study opens novel therapeutic perspectives and prompts future investigations about mechanism of action of indicaxanthin in the neuronal environment. Together with the neuroprotective action of nutraceuticals due to their antioxidant and anti-inflammatory properties, a further cellular target at synaptic level was individuated with a view to deepen knowledge on beneficial effects of phytonutrients on neuronal function.

GG, FC, MA and AA conducted the experiments. GG, FC, MA and PS performed the data analyses. FC, MA, PS, GF, LT and MAL designed the experiments. FC designed and directed the project. GG, FC and MA wrote the manuscript with input from all the authors. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.