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Journal of Alzheimer’s Disease 45 (2015) 217–233 DOI 10.3233/JAD-142469 IOS Press

Neuroprotective Effects of Hydrated Fullerene C60: Cortical and Hippocampal EEG Interplay in an Amyloid-Infused Rat Model of Alzheimer’s Disease Vasily Vorobyova,∗ , Vladimir Kaptsova , Rita Gordona , Ekaterina Makarovab , Igor Podolskib and Frank Sengpielc a Institute b Institute

of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region,

Russia c School of Biosciences and Neuroscience & Mental Health Research Institute, Cardiff University, Museum Avenue,

Cardiff, UK Handling Associate Editor: Vladimir Buchman

Accepted 17 November 2014

Abstract. We studied the effects of fullerene C60 nanoparticles, namely hydrated fullerene C60 (C60 HyFn), on interrelations between EEG frequency spectra from the frontal cortex and the dorsal hippocampus (CA1) on an amyloid-␤ (A␤) rat model of Alzheimer’s disease (AD). Infusion of A␤1-42 protein (1.5 ␮l) into the CA1 region two weeks before EEG testing diminished hippocampal theta (3.8–8.4 Hz) predominance and eliminated cortical beta (12.9–26.2 Hz) predominance observed in baseline EEG of rats infused with saline (control) or with C60 HyFn alone. In contrast, these A␤1-42 effects were abolished in rats pretreated with C60 HyFn, 30 min apart. Dopaminergic mediation in AD has been shown to be involved in neuronal plasticity and A␤ transformation in different ways. To clarify its role in the cortex-hippocampus interplay in the A␤ model of AD, we used peripheral injection of a dopamine agonist, apomorphine (APO), at a low dose (0.1 mg/kg). In rats infused with C60 HyFn or A␤1-42 alone, APO attenuated the cortical beta predominance, with immediate and delayed phases evident in the A␤1-42 rats. Pretreatment with C60 HyFn diminished the APO effect in the A␤1-42 -treated rats. Thus, we show that intrahippocampal injection of A␤1-42 dramatically disrupts cortical versus hippocampal EEG interrelations and that pretreatment with the fullerene eliminates this abnormality. We suggest that some effects of C60 HyFn may be mediated through presynaptic dopamine receptors and that water-soluble C60 fullerenes have a neuroprotective potential. Keywords: Amyloid-␤, brain oscillation, dopamine agonist, neurodegenerative disorder, neuroprotection

INTRODUCTION Amyloid-␤ peptides (A␤) are hypothesized to play a central role in the pathogenesis of Alzheimer’s disease (AD) [1–3]. A␤ are well known to initiate a series of ∗ Correspondence

to: Dr. Vasily Vorobyov, PhD, Institute of Cell Biophysics, Pushchino, 142290, Russia. Tel.: +7 4967 739 468; Fax: +7 4967 330 509; E-mail: [email protected].

pathological processes in the hippocampus and in the cortex (for review, see [4, 5]). Although recent studies demonstrate that neurodegeneration and memory impairment are observed before development of amyloidosis [6], A␤ involvement in the pathogenesis of AD is still widely accepted (for review, see [7]). Transgenic animal models of AD may facilitate a breakthrough in the understanding of disease mechanisms [8–11]. On the other hand, microinjections of

ISSN 1387-2877/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved

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A␤ into the hippocampus allow investigation of direct effects of A␤ on the brain. In fact, intrahippocampal infusions of A␤1-42 revealed symptomatic and pathophysiological similarities of this model to AD and underline its usefulness for the development and evaluation of potential new drugs [12, 13]. A␤42/43 has been shown to initiate disruptions of hippocampal and cortical circuitries and their electrical oscillatory activities that are associated with cognitive dysfunctions and dementia typical of AD [6, 14]. In the electroencephalogram (EEG) frequency spectra of AD patients, an increase of power in the deltaand theta-bands (0.5–4.0 Hz and 4.0–8.0 Hz, respectively) and a reduction in the alpha- and beta-bands (8.0–12.0 Hz and 25–60 Hz, respectively) have been shown [15, 16]. In a few studies of the oscillatory processes in animal models of AD, reduced cortical theta versus enhanced beta-gamma activities in A␤PP/PS1mice [17] and increased delta versus reduced theta activities in the hippocampus and the cortex have been revealed [18]. In rats, a decrease in low-frequency theta-band oscillations and the weakening of binding between the dorsal hippocampus and the frontal cortex after microinjection of A␤25-35 into the lateral ventricle have been shown [19] that may underlie the spatial memory breakdown observed in AD. Fullerene C60 , a condensed ring aromatic compound with extended pi systems, is a novel carbon allotrope, whose water-soluble derivatives have potent and selective pharmacological effects on organs, cells, enzymes, and nucleic acids (for review, [20]). The main perspectives of water-soluble C60 are associated with its free radical-scavenging and direct nitric oxidequenching activities (for review, [21]), and with its ability to pass through lipid membranes into cells [22]. In the current study, we are using hydrated fullerene C60 (C60 HyFn) nanoparticles with combined colloidalhydrophilicpropertiesandhighantioxidantactivity[23] to inactivate the free radicals induced by A␤1-42 and linked to neurodegeneration in AD (for review, [24]). C60 HyFn has recently been shown to inhibit A␤ protein fibrillation in vitro and to improve spatial learning, disrupted by injection of A␤25-35 into the lateral ventricles in rats [25, 26]. These results are in line with those of subsequent studies (for review, [27]). The protective effect of C60 HyFn has been shown in ribosomal protein synthesis and in neurodegeneration [28]. However, how fullerenes affect oscillatory processes in the cortex and in the hippocampus remains unclear while an imbalance in network activities between brain areas has been shown to be important in cognitive processes [29] and in both AD [30] and Parkinson’s disease [31].

We therefore focus our efforts on estimating relative differences in frequency spectra of cortical and hippocampal EEGs in rats, intrahippocampally infused either with A␤1-42 alone or pretreated with hydrated fullerene C60 . Given a powerful A␤ regulation of synaptic transmission (for review, see [5]) and close association between cognitive dysfunction and receptor modification in AD and Parkinson’s disease (for review, see [21]), we are also interested in the EEG balance between these brain structures before and after peripheral injection of apomorphine (APO), an agonist of dopamine D1 /D2 receptors [32, 33]. Dysfunction of dopaminergic transmission has been shown to alter long-term depression-like plasticity in AD patients [34],whereasdopaminereceptoractivationbyAPOwas accompanied by promotion of A␤ degradation [35, 36]. We show for the first time that intrahippocampal injection of A␤1-42 dramatically disrupts cortical versus hippocampal EEG interrelations and that pretreatment with the fullerene eliminates this abnormality. We propose that effects of C60 HyFn may, at least in part, be mediated through presynaptic dopamine receptors and that water-soluble fullerenes C60 have a neuroprotective potential. MATERIALS AND METHODS Animals Twenty two Wistar male rats (290–330 g) obtained from the vivarium of the Institute of Theoretical and Experimental Biophysics of Russian Academy of Sciences were used in the EEG study. An additional thirty rats were involved in morphological and histochemical evaluation of neuronal viability. During one-month adaptation to the experimental conditions and consequent one-month experimentation, the animals were kept in a standard regime, with food and water ad libitum and a 12-h/12-h day/night cycle. The procedures were carried out in accord with the principles enunciated in the Guide for Care and Use of Laboratory Animals, NIH publication No 85-23, and approved by the Institutional Ethical Review Committee. Intrahippocampal infusions Anesthesia was induced with subcutaneous (s.c.) injection of tiletamine/zolazepam (Zoletil® , Virbac, France) in combination with xylazine (Rometar® , Bioveta, Czech Republic) at doses of 25 mg/kg and 1 mg/kg, respectively. Saline, C60 HyFn (at 0.5 nmol/␮l), and A␤1-42 protein (Sigma, USA, at 2 nmol/␮l), in

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volumes of 1.5–2.0 ␮l, were slowly infused (for ∼2 min) symmetrically into the dorsal hippocampus (CA1, AP −3.8, L 2.5, H 3.5) [37]. C60 HyFn was obtained courtesy of Dr. G. Andrievsky who developed this highly stable water-soluble form of the fullerene C60 (MER Corp, USA) [23]. A temporarily inserted guide cannula (blunt stainless steel needle, 0.8 mm inner diameter) with flexible silastic tubing attached to a 5-␮l Hamilton syringe was used for the infusion. The needle was kept in place for 3 min before withdrawal. Before injection of A␤1-42 , its aliquots were defrosted and incubated at 37◦ C for two days to aggregate the protein into fibrils [38] that were assessed by electron microscopy. Four groups of rats with two intrahippocampal injections (30 min apart) were studied. They were arbitrarily allocated to “saline + saline”, “C60 HyFn + saline”, “saline + A␤1-42 ”, and “C60 HyFn + A␤1-42 ” (4 rats in each group). In control (“na¨ive”) rats (n = 6), no cannulation was performed. Electrode implantation Immediately after the intrahippocampal injections, monopolar recording electrodes (nichrom wires, 0.1 mm in diameter, with tips free from insulation for 0.1 mm) were implanted symmetrically over the frontal cortex and underlying the dorsal hippocampus, CA1, of both hemispheres (AP −2.8, L 2.0, H 0 and 3.0, respectively) [37]. A reference electrode (stainless steel wire 0.4 mm in diameter) was placed in the nasal bone close to the midline and at a distance of 8-9 mm in front of bregma to minimize an influence of oscillations from the olfactory bulbs. Its tip was positioned at the optimal depth to minimize respiration artefacts. The implanted electrodes were linked to a female connector (Sullins Connector Solutions, CA) and fixed to the skull with dental cement. The electrode positions were verified postmortem in brain slices prepared with a freezing microtome.

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was temporarily interrupted when they woke up. This ensured that, before commencing the main part of the experiment, the initial state of each animal was similar to enable correct comparison of the results obtained in different experimental groups. After the baseline interval, EEG recordings were continued either for 60 min on Day 11 or for 120 min after injection of APO (Sigma, Milan, Italy) at a dose of 0.1 mg/kg (s.c.) on Day 14. To minimize the effect of oxidation only freshly dissolved APO was used. Computation of EEG spectra Thefrequencyspectraofsuccessive12-sEEGepochs were analyzed on-line in the range of 0.5–25 Hz (bandpass filtered at 0.1–50 Hz) via a computerized system. Each epoch was digitized with a multichannel A/D DT2814 converter (Data Translation, Inc., Marlboro, MA, USA) using a sampling rate of 2 kHz. Taken into account the non-stationarity of EEG signals [39], we have used a modified version of period-amplitude analysis [40] which, contrary to the Fourier transform, is not affected by the non-stationary nature of the signals.Themainmodificationsofthealgorithmdeveloped by Stigsby et al. [41] were several types of frequency spectra normalization. The integrated power in twenty selected narrow EEG frequency bands and power ratios for each band over the integrated power in the 0.25–30.5 Hz range were calculated. The bands are marked in the text and figures by their center (mean) frequency values. The EEG spectra were averaged for every successive 10-min interval. The terms “lower” and “higher” in “classical”EEGbandsdelta(0.5–3.6Hz),theta(3.8–8.4 Hz), alpha (8.6–12.4 Hz), beta1 (12.9–16.8), and beta2 (18.4–26.2 Hz), are used below to differentiate corresponding frequency subranges of each band relative to its center frequency. For more details, see [42]. Morphological and histochemical evaluation of neuronal viability

EEG recording and APO treatment Between Day 4 and Day 11 after surgery, the rats were adapted, for 1 h/day, both to an experimental cage (Perspex, 25 × 25 × 30 cm) placed in an electrically shielded chamber and to handling (connecting/disconnecting the animal to/from the recording cable). EEG experiments were performed on Day 11 and on Day 14. Baseline EEGs were recorded for 10 min, starting about 20 min after placing the animal in the cage. During the baseline period, animals were in a relaxed posture, with closed eyes, and EEG recording

In an additional five groups of six rats in each group (na¨ive and infused into CA1 either with saline, C60 HyFn, A␤25-35 , or C60 HyFn+A␤25-35 ), we evaluated the distribution of surviving pyramidal neurons (which are most vulnerable to A␤ [43]), in the hippocampal slices on Day 14 versus Day 2 (three rats for the day from each group). A␤25-35 was chosen since this peptide, usually used in a synthetic form, on the one hand replicates A␤1-42 toxicity [44, 45] while on the other hand, it is not involved directly in recovery mechanisms associated with A␤1-42 [46].

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After decapitation under diethyl ether, one sample of brain was fixed in Carnow’s mixture (ethanol, chloroform, and acetic acid, at ratios of 6:3:1, respectively), whereas another one was put in 4% paraformaldehyde with PBS and then embedded in paraffin. Prepared blocks were 7-␮m sectioned on Leica’s microtome (Germany), and 25–30 sections from each animal were scanned and transferred through a digital camera to Axio Imager M1 optical microscope (Zeiss, Germany) for further analyses with use of Image J 1.44 (USA) software. Neuronal degeneration was evaluated after Nissl staining of the sections with cresyl violet (SigmaAldrich, St. Louis, USA). The immunohistochemical study was performed according to a standard technique [43]. To enhance sensitivity to A␤, the sections were treated with 70% formic acid for 4 min. To block the endogenous peroxidase activity, they were incubated in 0.5% H2 O2 /ethanol mixture, and 5% normal goat serum was used to block unspecific binding. The sections were further incubated with primary antibodies: rabbit polyclonal anti-A␤1-40 (1:400, Sigma) at a temperature of +4◦ C for 15 h. The antibodies were recognized by anti-rabbit IgG (1:800, Sigma) which were identified with StreptABComplex/HRP (1:800, Sigma). The reaction was visualized with 3,3’-diaminobenzidine. For control sections, the primary antibodies were replaced with PBS. For more details, see [28]. Statistics Differences in the averaged EEG spectra were evaluated by either a two-tailed non-parametric Wilcoxon test (p < 0.05 was considered statistically significant) or by one- or two-way ANOVA for repeated measures, when appropriate. Morphological data were analyzed by a Student’s t-test (p < 0.05 was deemed statistically significant).

after averaging in 10-min intervals (Fig. 2) and were relatively stable for 60 min of continuous recording (Table 1). Relative stability of the EEG spectral differences in other groups of rats was observed on Day 11, although their profiles were dependent on the treatment protocols (Table 2). On Day 14, individual baseline EEGs and their spectra in both the “saline + saline” (Fig. 3a and 3A, respectively) and “C60 HyFn + saline” (Fig. 3b and 3B, respectively) groups resembled those observed in the “na¨ıve” rats (Fig. 1). Intrahippocampal injection of A␤1-42 two weeks before EEG recordings (“saline + A␤1-42 ” group) dramatically slowed hippocampal and cortical baseline EEGs and made them similar both in individual 12-s fragments (Fig. 3c) and in their spectra (Fig. 3C). In contrast, pretreatment with the fullerene (“C60 HyFn + A␤1-42 ” group) practically eliminated this effect of A␤1-42 (Fig. 3D). The hippocampal-cortical differences observed in the individual baseline EEG spectra (Fig. 3) were more evident in those averaged for 10-min intervals. Briefly, in the groups of “saline + saline” (Figs. 4A, 5A) and “C60 HyFn + saline” (Figs. 4B, 5B), the main differences in cortical and hippocampal baseline EEG were associated with significant predominance of theta activity in the hippocampus and beta activity in the cortex (2-way ANOVA (brain areas X frequency subranges): F(1,36) = 18.2 and F(1,24) = 17.9, respectively, p < 0.001, in both). In the “saline + A␤1-42 ” group (Figs. 4C, 5C), the cortex-hippocampus EEG differences were negligible (2-way ANOVA: F(1,36) and F(1,24) 0.2, in both), moreover, they tended to be inverted in the beta range. In the “C60 Hy Fn + A␤1-42 ” group (Figs. 4D, 5D), the EEG differences between the cortex and the hippocampus were fully recovered in the beta band (2-way ANOVA: F(1,24) = 7.4, p = 0.012) and partly in the theta range. Apomorphine EEG effects

RESULTS Baseline EEG In “na¨ıve” rats, the baseline EEGs recorded on Day 14 from the cortex and the hippocampus were characterized by patterns of high amplitude slow waves, spindle activity and theta oscillations (Fig. 1A). The patterns were represented in EEG frequency spectra by peaks in the delta, beta, and theta bands, respectively, with theta activity dominating in the hippocampus and beta in the cortex (Fig. 1B). The cortical-hippocampal differences in EEG spectra reached significant values

To evaluate the APO specific effects on cortical versus hippocampal interrelations, relative differences between cortical and hippocampal EEG spectra after APO injection were normalized to those revealed in the baseline EEGs (Table 3). This made the enhanced sensitivity of EEG beta activity to APO in the “saline + A␤1-42 ” group more evident. Furthermore, the evolution of APO effects over time examined in the classical frequency bands showed the enhanced sensitivity to APO of the fullerene- versus saline-treated rats in both thethetaandthebetaband(Fig.6Aand6C,respectively; 2-way ANOVA: F(1,552) = 20 and F(1,360) = 60.4,

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Fig. 1. Typical patterns in baseline 12-s EEG fragments (A) and their frequency spectra (B) from the cortex and the hippocampus of the left hemisphere in a na¨ive rat. Ordinate on B is mean EEG amplitude in arbitrary units (AU), in 20 frequency subranges marked with their central (mean) value (in Hertz) on the abscissa. White and gray vertical bars on B mark the “classical” frequency bands in the EEG spectra (from left to right): delta, theta, alpha, and beta. Time calibration in A is 1 s; amplitude calibration (vertical bars left to the EEG recordings) is 100 ␮V. Table 1 Cortical versus hippocampal EEG interplay in na¨ive rats (n = 6) two weeks after electrode implantation

Values are calculated as a ratio of averaged baseline EEG amplitude differences per frequency subrange for (Cortex - Hippocampus)/Hippocampus (in %) in 10-min intervals (−10–0 – baseline interval, see text for details). Filled cells indicate significant prevalence (Wilcoxon test, p < 0.05) of the EEG rhythms in the cortex (dark colour) or in the hippocampus (grey colour). Twenty subranges in EEG frequency spectra are marked with their mean values, while Greek symbols (see the bottom line) denote so-called “classical” EEG frequency bands.

p  0.001, in both). A␤1-42 produced robust effect in the beta band (c.f., Fig. 6D and 6C, black and grey lines, respectively; 2-way ANOVA: F(1,360) = 137,

p  0.001, for rows, and F(11,360) = 4.6, p < 0.001, for interaction). The C60 HyFn pretreatment eliminated this effect of A␤1-42 (Fig. 6D, grey line; 2-way

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V. Vorobyov et al. / Fullerene and EEG in AD Model Table 2 Cortical versus hippocampal baseline EEG interplay in rats eleven days after intrahippocampal microinjections of saline, C60 HyFn, and A␤1-42 at different combinations (n = 4, in each group)

Values are calculated as a ratio of averaged baseline EEG amplitude differences per frequency subrange, (Cortex - Hippocampus) / Hippocampus (in %) in 10-min intervals (−10–0 – baseline interval, see text for details). Filled cells indicate significant prevalence (Wilcoxon test, p < 0.05) of the EEG rhythms in the cortex (dark color) or in the hippocampus (grey color). Twenty subranges in EEG frequency spectra are marked with their mean values, while Greek symbols (see the bottom line) denote so-called “classical” EEG frequency bands.

ANOVA: F(1,360) = 47.8, p < 0.001, for rows, and F(11,360) = 3.8, p < 0.001, for interaction). Interestingly, the fullerene effect in the theta band was diminished by A␤1-42 (c.f. Fig. 6A and 6B, black and grey lines, respectively; 2-way ANOVA: F(1,552) = 11, p < 0.001). Although the fullerene effect (versus saline) in the beta band (Fig. 6C, black line; 2-way ANOVA: F(1,360) = 46.7, p  0.001) tended to be attenuated by A␤1-42 (Fig. 6D, grey line), the difference did not reach significance (2-way ANOVA: F(1,360) = 3.4, p = 0.07). Finally, the APO effect in the beta band was more powerfully expressed (2-way ANOVA: F(1,360) = 25, p  0.001, for rows, and F(11,360) = 5.3, p  0.001, for interaction) in rats treated with A␤1-42 alone (Fig. 6D, black line) than in

those treated with the fullerene alone (Fig. 6C, black line). Behavior During the baseline period, animals were in a relaxed posture, with closed eyes, that was temporarily interrupted when they woke up. APO (0.1 mg/kg, s.c.) injection provoked, with a short delay (1-2 min), similar changes in behavior and in particular yawning, in rats from different groups. The time-course of these patterns of behavior in individual rats varied in phase and duration, indicating therefore that the change in behavior is unlikely to be the main cause of the EEG changes revealed in our study.

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Fig. 2. Differences in the frequency spectra of 12-s baseline EEG fragments averaged for 10-min intervals in the cortex (gray lines) and hippocampus (black lines) in six na¨ive rats. Ordinate on A is mean EEG amplitude in arbitrary units (AU) in 20 frequency subranges marked with their central (mean) value (in Hertz) on the abscissa. Ordinate on B is relative difference of the EEG amplitudes between cortex and hippocampus calculated as ratio (Cortex - Hippocampus) / Hippocampus, in %. White and gray vertical bars on A–D mark the “classical” frequency bands in the EEG spectra (from left to right): delta, theta, alpha, and beta. Diamonds on A denote significant difference between the cortex and hippocampus (p < 0.05, Wilcoxon test). Error bars show 1 SEM.

Morphology and histochemistry On Day 14, pyramidal neurons from CA1 in na¨ive rats had uniformly stained cytoplasm and nucleoplasm, distinct plasmatic and nuclear membranes, and well-

defined nuclei (Fig. 7A, B). Similar “healthy” images were typical for the hippocampi of rats infused with saline (Fig. 7C, D) or C60 HyFn (Fig. 7E, F), whereas A␤25-35 produced neuronal necrosis expressed in swelling of the cytoplasm and its vacuolization,

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Fig. 3. Typical patterns in baseline 12-s EEG fragments (a–d) and their frequency spectra (A–D) from the cortex and the hippocampus in individual rats from different groups: “saline + saline” (a & A), “C60 HyFn + saline” (b & B), “saline + A␤1-42 ” (c & C), and “C60 HyFn + A␤1-42 ” (d & D). Ordinate on A – B is mean EEG amplitude in arbitrary units (AU), in 20 frequency subranges marked with their central (mean) value (in Hertz) on the horizontal axis (abscissa). White and gray vertical bars on A–D mark the “classical” frequency bands in the EEG spectra (from left to right): delta, theta, alpha, and beta. Time calibration on a-d is 1 s; amplitude calibration (vertical bars left to the EEG recordings) is 100 ␮V.

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Fig. 4. Differences in the frequency spectra of baseline 12-s EEG fragments averaged for 10-min intervals from the cortex (gray lines) and the hippocampus (black lines) in rats from different groups (n = 4, in each group): “saline + saline” (A), “C60 HyFn + saline” (B), “saline + A␤1-42 ” (C), and “C60 HyFn + A␤1-42 ” (D). Ordinate is mean EEG amplitude in arbitrary units (AU) in 20 frequency subranges marked with their central (mean) value (in Hertz) on the abscissa. White and gray vertical bars on A–D mark the “classical” frequency bands in the EEG spectra (from left to right): delta, theta, alpha, and beta. Diamonds denote significant difference between the cortex and hippocampus (p < 0.05, Wilcoxon test). Error bars show 1 SEM.

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Fig. 5. Cortical versus hippocampal EEG relative frequency spectra profiles averaged for 10-min baseline intervals in rats from different groups (n = 4, in each group): “saline + saline” versus “C60 HyFn + saline” (A, C), “saline + A␤1-42 ” versus “C60 HyFn + A␤1-42 ” (B, D). Ordinate is ratio of averaged EEG frequency band amplitudes from the cortex and the hippocampus calculated as (Cortex-Hippocampus)/Hippocampus, in %. Abscissa is frequency subrange marked with its central (mean) value, in Hertz. White and gray vertical bars mark the “classical” frequency bands in the EEG spectra (from left to right): delta, theta, alpha, and beta. Error bars show 1 SEM.

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forming huge clusters of indistinct and dark pyramidal neurons (Fig. 7G, H). In rats pretreated with C60 HyFn, A␤25-35 was obviously ineffective (Fig. 7I, J). These differences were confirmed by quantitative analyses of surviving cells distribution in the hippocampi of rats from different groups (Fig. 8A, Day 14). Interestingly, the distributions on Day 2 were practically identical in all groups (Fig. 8A, Day 2) that emphasized specificity of the A␤25-35 effect on Day 14. The immunohistochemical analyses of the brain sections on Day 14 after A␤25-35 infusion revealed significantly enlarged deposits of diffuse A␤1-42 in the cytoplasm of CA1 pyramidal neurons (Fig. 8B), whereas in salinepretreated rats, no immunostained cells were observed. In rats pretreated with C60 HyFn, the A␤25-35 effect was practically eliminated (Fig. 8B).

DISCUSSION In our study, A␤1-42 , infused in the hippocampus two weeks before EEG testing, eliminated both hippocampal theta and cortical beta wave predominance in baseline EEG spectra observed in both na¨ive and salinetreated rats. However, intrahippocampal pretreatment with hydrated fullerene C60 (C60 HyFn), which when infused alone tended to increase the baseline differences, diminished the effect of A␤1-42 . Following apomorphine-injection, the fullerene practically eliminated A␤1-42 -evoked changes in the beta range. Baseline EEG in our study was recorded during a relatively stable sleep-like state (see details in the Materials and Methods). The differences in frequency spectra of cortical and hippocampal baseline EEG in control rats (Fig. 2) are in line with the idea that various cortical and subcortical brain structures have specific sleep characteristics [47]. The effects of C60 HyFn (Figs. 4B, 5B) and A␤1-42 (Figs. 4C, 5C) on the baseline EEG indicate a rearrangement of neuronal networks in different brain areas involved in this physiological state. Sleep has been shown to play a role in memory consolidation that has been supposed to be based on an information transfer between the hippocampus and the neocortex through neurophysiological mechanisms that generate sleep-specific slow waves, spindles, and ripples (for review, see [48, 49]). Thus, the elimination/inversion of the beta predominance in the cortex after A␤1-42 treatment (Figs. 4C, 5C) is in line both with the memory/learning impairment shown in the A␤25-35 model of AD in rats [25, 26] and with the reduction of EEG sleep spindles observed in AD patients suffering disrupted performance [50].

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The preservation of the sleep-dependent cortical beta predominance from the A␤1-42 -produced toxicity by C60 HyFn (Figs. 4D, 5D) may additionally highlight its protective and therapeutic potential for neurodegenerative diseases [28, 51, 52] and open the gate for the understanding of its neurophysiological mechanisms. In particular, our data indicate that C60 HyFn is able to enhance some specific activities in the cortex and in the hippocampus (c.f. Figs. 4B, 5B and Figs. 4A, 5A, in the beta and the theta range, respectively). This is in line with in vitro experiments on hippocampal slices where C60 HyFn-enhanced excitability of pyramidal neurons has been demonstrated [53]. The hippocampal theta predominance in baseline EEG spectra from control (na¨ive) rats (Fig. 2, Table 1) seems to reflect an optimal level of interrelation between the cortex and the hippocampus tuned to play a role, after awaking, in the sensory-motor integration [54] and in the neural coding of place, in particular (for review, see [55, 56]). The elimination of the hippocampal theta predominance in the A␤1-42 -treated rats (Figs. 4C, 5C) and its partial preservation from the A␤1-42 -produced toxicity by C60 HyFn (Figs. 4D, 5D) are in line with the results on spatial learning obtained in the A␤25-35 model of AD in rats [25, 26, 57]. Apomorphine, an agonist of DA receptors, has been shown to stimulate postsynaptic receptors at moderate to high doses (0.5–1.0 mg/kg) whereas it activates presynaptic receptors at low doses, tending to cause sleep [32] (for review, see [58]). In our study, a low dose of APO (0.1 mg/kg), produced significant suppression of the cortical beta dominance in rats treated with A␤1-42 alone, with two phases in the time-course of the effect (Table 3, Fig. 6D, black line). This might provide additional support for the involvement of presynaptic DA receptors in A␤1-42 -treated rats since immediate and delayed effects of APO at low doses have been shown in previous studies [59, 60]. Suppression of the cortical beta dominance in baseline EEG from A␤1-42 -treated rats (Fig. 5C) and its potentiation by APO (Fig. 6D, black line, Table 3) are thought to have common mediatory mechanism(s) and might be associated, in particular, with an inflammatory ability of A␤1-42 [12]. Indeed, bilateral intrahippocampal injection of aggregated A␤1-42 has been shown to produce protracted (above 30 days) inflammatory-associated impairments in rat’s operant behavior [13]. Inflammatory reactions are well known to be important factors in AD pathogenesis, however, microglia activated through receptors for GABA, acetylcholine, and adrenaline has been revealed to suppress inflammatory responses whereas its activation

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V. Vorobyov et al. / Fullerene and EEG in AD Model Table 3 Cortical versus hippocampal EEG interplay after subcutaneous injections of apomorphine (0.1 mg/kg) in rats from different groups (n = 4, in each group)

Values are calculated as a ratio of averaged EEG amplitude differences per frequency subrange, (Cortex - Hippocampus) / Hippocampus (in %) in 10-min intervals after injection of apomorphine (normalized versus baseline values). Filled cells indicate significant prevalence (Wilcoxon test, p < 0.05) of the EEG rhythms in the cortex (dark color) or in the hippocampus (grey color). Twenty subranges in EEG frequency spectra are marked with their mean values, while Greek symbols (bottom line) denote so-called “classical” EEG frequency bands.

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Fig. 6. Evolution of cortical-hippocampal EEG interrelations in the theta and the beta frequency bands (A, B and C, D, respectively) after APO injection (0.1 mg/kg, s.c.) in rats from different groups (n = 4, in each group): “saline + saline” versus “C60 HyFn + saline” (A, C), “saline + A␤1-42 ” versus “C60 HyFn + A␤1-42 ” (B, D). Ordinate is the ratio of averaged EEG frequency band amplitudes in the cortex and the hippocampus calculated as (Cortex-Hippocampus)/ Hippocampus, in %. Normalized baseline values for each of the groups are denoted by “0” at ‘BL’ point on abscissa and extended as a horizontal dashed line. Abscissa shows time after apomorphine injection, marked in 10-min intervals. Diamonds denote significant difference between the groups (p < 0.05, Wilcoxon test). Error bars show 1 SEM.

through receptors for ATP and adenosine stimulates release of pro-inflammatory factors (for review, see [61]). Interestingly, glutamate and dopamine can be pro- or anti-inflammatory depending on the receptor subtype expressed in microglia. Pharmacological blockade of dopamine D2 receptors has been shown to suppress both tau aggregation and neurotoxicity [62] while their activation by rotigotine, an agonist with high affinity for the D2 /D3 receptors, results in both increased cortical excitability and restored central cholinergic transmission in AD patients [63]. The brain inflammation has been shown to be accompanied by increased concentration of serum phenylalanine, i.e., by its impaired conversion into the neurotransmitters, in particular, dopamine [64]. This dopamine

pool shrinkage reduces, in turn, the probability of spontaneous binding of dopamine to its receptors, thus opening the gate for different effects of exogenetic APO molecules depending on their agonist affinities for various types of dopamine receptors [65]. Significantly diminished beta predominance in the cortical EEG after injection of APO in A␤1-42 -treated rats (Fig. 6D, gray line) is in line with this suggestion and with supposedly potentiating effect of APO on A␤associated toxicity [62]. Finally, the enlarged deposits of diffuse A␤1-42 in the cytoplasm of CA1 pyramidal neurons revealed after pretreatment with A␤25-35 (Fig. 8B) seems to be a result of its inflammatory ability that is typical for A␤ (for review, see [66]), and for A␤1-42 in particular [12, 13].

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Fig. 7. Microphotographs of the hippocampal (CA1) sections stained with cresyl violet in rats from different groups on Day 14 (n = 3, in each group): na¨ive (A, B) and after intrahippocampal infusion of either saline (C, D), C60 HyFn (E, F), A␤25-35 , (G, H), or C60 HyFn + A␤25-35 (I, J).

Thus, the neuroprotective effect of C60 HyFn (Fig. 6D, grey line) against the A␤ toxicity, seemingly associated with the induction of diffuse A␤1-42 deposits in the neuronal cytoplasm (Fig. 8B), is thought to be produced by the well-known scavenging activity of fullerenes against free radicals (for review, see [67]) and, presumably, against the pro-inflammatory cytokines released by activated astrocytes. The latter

might allow both the disruption of a vicious inflammatory cycle of A␤ generation between astrocytes and the redirection of cytokines from their neurodegenerative to neuroprotective roles (for review, see [66]). Regardless of possible mechanisms underlying the effects of C60 HyFn, this approach could be one of potentially protective interventions forwarding future investigation in AD (for review, see [68]).

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Author’s disclosures available online (http://j-alz. com/manuscript-disclosures/14-2469r1). REFERENCES [1]

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Fig. 8. Intact cell density (A) in the hippocampal (CA1) sections from rats on Day 2 and Day 14 after intrahippocampal infusion of saline (I), C60 HyFn (II), A␤25-35 (III), or C60 HyFn + A␤25-35 (IV) (n = 3, in each group/day) and distribution of diffuse A␤1-42 deposits in the cytoplasm of CA1 pyramidal neurons on Day 14 in rats from different groups (n = 3, in each group). * marks significant difference (p < 0.001, Student’s t-test).

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Two weeks after intrahippocampal amyloid-␤1-42 infusion, cortical versus hippocampal EEG interplay was dramatically disrupted. Pretreatment with hydrated fullerene C60 significantly attenuated the amyloid-␤1-42 effect. Dopaminergic D1 /D2 receptors are involved in the A␤1-42 effects. The water-soluble fullerenes C60 have a neuroprotective potential.

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