Interhemispheric EEG differences in olfactory bulbectomized rats with different cognitive abilities and brain beta-amyloid levels

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –1 94

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Interhemispheric EEG differences in olfactory bulbectomized rats with different cognitive abilities and brain beta-amyloid levels Natalia Bobkova a,1 , Vasily Vorobyov a,b,⁎,1 , Natalia Medvinskaya a , Irina Aleksandrova a , Inna Nesterova a a b

Institute of Cell Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia Cardiff School of Biosciences, Museum Avenue, PO Box 911, Cardiff CF10 3US, UK

A R T I C LE I N FO

AB S T R A C T

Article history:

Alterations in electroencephalogram (EEG) asymmetry and deficits in interhemispheric

Accepted 9 July 2008

integration of information have been shown in patients with Alzheimer's disease (AD).

Available online 19 July 2008

However, no direct evidence of an association between EEG asymmetry, morphological markers in the brain, and cognition was found either in AD patients or in AD models. In this

Keywords:

study we used rats with bilateral olfactory bulbectomy (OBX) as one of the AD models and

Learning

measured their learning/memory abilities, brain beta-amyloid levels and EEG spectra in

Alzheimer's disease

symmetrical frontal and occipital cortices. One year after OBX or sham-surgery, the rats

Morris test

were tested with the Morris water paradigm and assigned to three groups: sham-operated

Frequency spectra

rats, SO, and OBX rats with virtually normal, OBX(+), or abnormal, OBX(−), learning (memory) abilities. In OBX vs. SO, the theta EEG activity was enhanced to a higher extent in the right frontal cortex and in the left occipital cortex. This produced significant interhemispheric differences in the frontal cortex of the OBX(−) rats and in the occipital cortex of both OBX groups. The beta1 EEG asymmetry in SO was attenuated in OBX(+) and completely eliminated in OBX(−). OBX produced highly significant beta2 EEG decline in the right frontal cortex, with OBX(−) N OBX(+) rank order of strength. The beta-amyloid level, examined by post-mortem immunological DOT-analysis in the cortex–hippocampus samples, was about six-fold higher in OBX(−) than in SO, but significantly less (enhanced by 82% vs. SO) in OBX(+) than in OBX(−). The involvement of the brain mediatory systems in the observed EEG asymmetry differences is discussed. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Alzheimer's disease (AD) is characterized by the loss of cognitive abilities, neurodegeneration, pathological forma-

tion of extracellular amyloid plaques, and the intraneuronal aggregation of hyperphosphorylated tau into neurofibrillary tangles (for review, see Andreasen et al., 2003). To monitor the progression of AD and its treatment, different brainimaging techniques can be used. In particular, several lines

⁎ Corresponding author. Cardiff School of Biosciences, Museum Avenue, PO Box 911, Cardiff CF10 3US, UK. Fax: +44 29 2087 4094. E-mail address: [email protected] (V. Vorobyov). 1 N.B. and V.V. contributed equally to this work. 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.07.036

186

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –19 4

of evidence indicate that frequency spectra of the electroencephalogram (EEG) are closely associated with the evolution of this pathology and cognitive impairment (see e.g., Stevens et al., 2001). Furthermore, substantial alterations in cortical EEG asymmetry and deficits in interhemispheric integration of information have been shown in AD (Lakmache et al., 1998; Wada et al., 1998; Pogarell et al., 2005). MRI revealed asymmetrical atrophy in different brain structures in such patients, which was highly correlated with different stages in their cognitive decline (Apostolova et al., 2007). This is supposed to be a reliable in vivo tracking technique for AD, usable for evaluation during therapy. However, for understanding the mechanisms underlying ADlinked cognition impairment, a combined analysis of EEG asymmetry, AD morphological markers, and cognitive abilities seems to be a more suitable and universal approach. It is advantageous because it allows the discovery of changes in the functioning of neuronal circuits involved in AD and in its models. One such model uses bilateral olfactory bulbectomy (OBX) to produce AD-like symptoms both in mice and rats (Otmakhova et al., 1992; Yamamoto et al., 1997; Hozumi et al., 2003). This has been used, in particular, to search for therapeutic pharmacological solutions to AD-associated memory impairment (Ostrovskaya et al., 2007). However, only a few EEG studies have been performed on OBX animals, and in these cases EEG registration was performed either during sleep or shortly after OBX-surgery (Sakurada et al., 1976; Watanabe et al., 1980). Neither approach appears to be optimal for the study of AD mechanisms. Firstly, the structure of sleep has been shown to be fundamentally altered in AD patients (Montplaisir et al., 1998). Although a variety of clinical and basic research settings have demonstrated the relationships between sleep and cognition (for review, see Roth et al., 2001) this interaction is just one of the functional processes involved in the mechanisms of cognition. Secondly, EEG recorded shortly after OBX-surgery seems to interfere with acute changes in the emotional state of the animals (Wrynn et al., 2000) and causes possible side effects by re-evolution (for up to several months) of mediatory and hormonal systems which were disturbed by OBX (for review, see Song and Leonard, 2005). On the other hand, AD-like symptoms have been shown to be observed in extended periods (up to 1 year) after OBX in mice (Novoselova et al., 2003). Thus, we performed our experiments 1 year after OBX in rats and combined EEG recording with preliminary testing of cognitive abilities using the Morris water paradigm and postmortem examination of brain beta-amyloid level, as a typical marker for AD (for review, see Hardy and Selkoe, 2002). We compared EEG in the symmetrical frontal-parietal (somatosensory) cortical areas, which are well known to be associated closely with learning/memory mechanisms and are highly affected by OBX (Hozumi et al., 2003). Moreover, we registered EEG from symmetrical occipital (visual) cortices which have been shown to be affected by AD (Kavcic et al., 2006) and are accompanied by drastic changes in interhemispheric coherence (Kikuchi et al., 2002). The EEG results obtained have been reported in abstract form elsewhere (Bobkova et al., 2003) while the results of histological and

beta-amyloid analyses have been published recently (Nesterova et al., 2008).

2.

Results

2.1.

Water maze acquisition

Mean latencies to reach the visible platform in sham-operated (SO, n = 9) and bulbectomized (OBX, n = 12) rats were approximately equal (10.1 ± 1.9 s and 14.4 ± 2.3 s, respectively;

Fig. 1 – Behaviour characteristics (mean ± SEM) of sham-operated (SO) and bulbectomized (OBX) rats in training session days (A) and during testing (B, C) in the Morris water paradigm. A—latency of correct attempts to find the hidden platform in seconds (filled diamond is the significant (p < 0.001) difference between OBX groups); B—time spent by rat in each water maze quadrant (I and T are “Indifferent” and “Target” ones, respectively) in seconds; C—the number of inward movements into a quadrant in % of crossings of all quadrants. ♦, ♦♦ and ♦♦♦ on B and C are significant differences (p < 0.05, 0.01 and 0.001, respectively) vs. the value for the target quadrant.

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –1 94

187

Fig. 2 – Representative fragments of baseline EEG and their frequency spectra in frontal and occipital cortices (black and gray lines, respectively) in rats from different groups: sham-operated (A) and bulbectomized with virtually normal (B) or abnormal (C) learning/ memory abilities. Radial presentation of the spectra (AU—arbitrary units) in different frequency subranges (marked in the outer part of the circle with their central values, in hertz). Time calibration (horizontal bars)—1 s, amplitude calibration (vertical bars)—100 μV.

188

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –19 4

F1,355 = 2.8, p N 0.05). In the training trials with the hidden platform, a day-dependent evolution of the latencies was evident in all groups (F5,210,138,138 = 202, 150, 47, for SO, OBX(+),

OBX(−), respectively, p < 0.001, for all), however, the differences between the latencies in different groups were revealed (Fig. 1A). Although the evolution curves for SO and

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –1 94

one group of OBX rats (OBX(+), n = 6) were very similar (with the exception of difference (p < 0.001) seen on day 2), the post hoc analyses showed a weak but significant (p < 0.05) difference between mean latencies in these groups. In contrast to this, another group of OBX rats (OBX(−), n = 6) demonstrated strong (p < 0.001) differences from both OBX(+) and SO. Memory testing showed that the trained SO(−) and OBX(+) rats spent more time (T) in the target quadrant (Fig. 1B) and visited it more frequently (F) (Fig. 1C) than the other quadrants (F3,32 = 29.7(T) and 12.4(F) for SO, and F3,20 = 33.2(T) and 12.2(F) for OBX(+), respectively; p < 0.001 for each pair). This was not the case for OBX(−) rats (F3,20 = 1.4(T) and 0.8(F), respectively; p N 0.05 for each pair).

2.3. Beta-amyloid content and histology evaluation (adapted from Nesterova et al., 2008) The beta-amyloid content in the brain samples of OBX rats was significantly higher compared to sham-operated animals (mean ± SEM, μg/g): 1.304 ± 0.221 (SO), 2.378 ± 0.202 (OBX(+), p < 0.05), and 7.379 ± 0.546 (OBX(−), p < 0.01). The beta-amyloid level was significantly less (p < 0.01) in OBX(+) than in OBX(−) rats. The results of morphometric analysis in the brains of rats from different groups are presented in Table 1.

3. 2.2.

189

Discussion

Baseline EEG

In SO, the baseline cortical EEG was characterized by highamplitude mixed slow/fast waves with unstable periods (Fig. 2A, flanking columns) and, correspondingly, by small and relatively wide peaks in delta and beta frequency bands (Fig. 2A, central column). In OBX(+), these peaks were sharpened (Fig. 2B), while in OBX(−), the main activity was concentrated in higher theta–lower alpha frequency range with significant bias to the right hemisphere (Fig. 2C). In averaged spectra of baseline EEG registered for the first 10 min (Fig. 3), the bulbectomy vs. sham surgery attenuated the lowest delta in both cortices and enhanced the higher theta–lower alpha with domination of the latter in OBX(−) right frontal cortex (Fig. 3D). The beta EEG activity was also gradually diminished by OBX in this area (Figs. 3B and D) while in the left occipital cortex this effect was very similar in both OBX groups (Figs. 3E and G). The main OBX-dependent changes in the whole spectra mentioned above were confirmed for EEG amplitudes averaged in the “classical” frequency bands (Fig. 4): a) an enhancement of both the theta (B and G, two-way ANOVA, F23,144 = 3.3 and 3.2, respectively, P < 0.001 in both) and the alpha (C and H, F23,144 = 3.2, P < 0.001 in both); b) an attenuation of the beta2 in the right frontal cortex (E, one-way ANOVA, F23,72 = 2.5, P < 0.01); and, finally c) significant interhemispheric differences in the theta from the frontal cortex in OBX(−) group (B, two-way ANOVA, F1,48 = 7.3, P < 0.01) rather than in OBX(+) group (two-way ANOVA, F1,48 = 3.8, P = 0.06). In contrast to the frontal cortex, where the latter effect was due to an intensification of activity in the right hemisphere, the interhemispheric differences in the occipital cortex (Fig. 4G) were provoked by higher activity in the left hemisphere and observed in both OBX groups (two-way ANOVA, F1,48 N 4.1, P < 0.05 in both). Finally, significant interhemispheric asymmetry revealed in the frontal beta1 activity in SO (Fig. 4D, two-way ANOVA, F1,48 = 10.4, P < 0.01) was attenuated in OBX(+) and completely eliminated in OBX(−).

Our experiments on bulbectomized rats revealed an association between the extent of increased brain beta-amyloid level, impaired learning/memory ability, and re-distribution of EEG activities in symmetrical frontal cortices: enhanced right-side bias in the theta frequency band and attenuated initial interhemispheric EEG difference in the beta1 band. These effects on OBX rats are similar to EEG power redistribution observed in AD patients (Montplaisir et al., 1998) and they are in line with such symptoms of the Alzheimer's type dementia as typified by increased theta power and slowed alpha rhythms (Stevens et al., 2001). Thus, the EEG changes in OBX rats mimic those in AD, in contrast to EEG data obtained on transgenic “AD” mice (Wang et al., 2002). Moreover, OBX-induced changes in EEG asymmetry are in line with evidence of disturbances in interhemispheric integration of information in AD patients (Lakmache et al., 1998). One possible mechanism underlying such EEG asymmetry phenomena may be associated with well known abnormalities observed in the cholinergic system in AD patients (see e.g., Lehéricy et al., 1993; Knott et al., 2000) and with EEG data that this system can be involved in hemispheric interplay (Vorobyov and Ahmetova 1998). Furthermore, deficits in the cholinergic system have been revealed in AD models on animals (Wang et al., 2002; German et al., 2003), in particular, after OBX in mice (Bobkova et al., 2001; Hozumi et al., 2003) and in rats (Yamamoto et al., 1997; Hallam et al., 2004). Beta-amyloid has been shown to play a role in regulating the function and survival of cholinergic neurons (for review, see Kar et al., 2004) that corroborates such a suggestion. In OBX(−) rats, we found a drastic enhancement of the brain betaamyloid level and a significant right hemisphere bias in the theta band power of EEG from frontal cortex (see Fig. 3D). This area is well known to be associated with learning/memory mechanisms and is enriched with cholinergic receptors (McKinney and Coyle, 1982; Sokolovsky, 1984). However, systemic injection of physostigmine in naïve rats has been

Fig. 3 – Frequency spectra differences in baseline EEG from frontal (A–D) and occipital (E–H) cortices in bulbectomized (OBX) rats (black lines) vs. sham-operated (SO) rats (grey lines) in EEG amplitudes and their ratios (bars). OBX(+) and OBX(−) are groups of rats with virtually normal and abnormal learning/memory abilities, respectively. The abscissa is a frequency subrange marked with its mean value in hertz; the left ordinate is an amplitude of EEG in arbitrary units, the right ordinate is a ratio of the EEG amplitudes, calculated as (OBX − SO) / SO, in %. Vertical lines are ± 1 SEM; filled gray bars are significant (P < 0.05, Wilcoxon test) differences. Greek symbols label traditional EEG frequency bands indicated by interrupted horizontal black bars.

190

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –19 4

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –1 94

191

Table analysis in the in brains rats from groups: sham-operated, and bulbectomized, normal, OBX Table1 1– –Morphometric Morphometric analysis theofbrains ofdifferent rats from different groups: SO, sham-operated, SO, with andvirtually bulbectomized, (+), andvirtually abnormal, OBX(−), learning/memory abilities OBX(−), learning/memory abilities with normal, OBX(+), and abnormal,

Values in coloured cells are significantly different (light gray p < 0.05, dark gray p < 0.01, black p < 0.001, Student's criterion) from those either in SO 2 group (colour in the “Mean” column) or in OBX(+) group (colour in the “SE” column). The cell density values are number per mm .

shown to produce the left side bias in the theta of frontal EEG (Vorobyov and Ahmetova, 1998). This correlates with natural asymmetry found in the cholinergic system (Glick, 1983). Thus, OBX, which has been shown to modify the density of cholinergic receptors (Hozumi et al., 2003), appears to reverse the initial asymmetry, affecting a cholinergic balancing in the hemispheres. The evidence of asymmetrical atrophy in different brain structures in AD (Apostolova et al., 2007) is in line with such a suggestion. Indeed, in OBX rats, both our morphological analyses (Nesterova et al., 2008) and MRI study (Wrynn et al., 2000) revealed some structural alterations in different brain areas, particularly in the hippocampus. Moreover, a greater reduction in cholinergic activity in the hippocampus vs. the frontal cortex in AD patients was observed (Geula, 1998). On the other hand, the blockade of the cholinergic system with high doses of scopolamine has been reported to depress the beta and to enhance the theta in EEG from both the hippocampus and frontal cortex (Podol'skii et al., 2001). Given a similarity between the effects of scopolamine and those in our OBX rats (maximally expressed in OBX(−) group), the AD-associated asymmetrical atrophy (see Apostolova et al., 2007) appears to be accompanied by more substantial disruptions of the cholinergic interplay in the right hemisphere. Alzheimer disease is a complex pathology, and the cholinergic system is one of those involved in its mechanisms (Dringenberg, 2000; Tadano et al., 2004; Slotkin et al., 2005). In particular, the well known clinical AD feature of depression has been shown to be associated with disturbances in different mediatory systems (for review, see Morilak and Frazer, 2004). Furthermore, a depressive-like state was observed after OBX in

mice and rats (see, e.g., Nesterova et al., 1997; Robichaud et al., 2001) that allowed the involvement of the model in studies of depression (for review, see Song and Leonard, 2005). These observations and our own data, reveal a close correlation between anterior cerebral EEG asymmetry and affective style (Davidson, 2004), and point towards a potentially effective supplementary assessment tool. Thus, monitoring EEG asymmetry in the frontal–parietal brain areas of individuals would provide an indication of both cognitive and emotional state, for a more adequate and balanced examination of AD in clinics. This approach appears to add a new dimension in the analyses of the relations between EEG and AD features which are invisible in the overall EEG (Wang et al., 2002). It should be mentioned however, that the association of the frontal EEG asymmetry and AD-like symptoms revealed in our OBX rats (memory impairments and enhanced beta-amyloid levels) is more sophisticated than a mere lack of afferentation from the chemosensory organs to the frontal cortex (for review, see Song and Leonard, 2005). OBX has been shown to result in a retrograde degeneration of the neuronal pathways to many brain areas, in particular, the hippocampus, amygdala, and locus coeruleus. In addition to these regions, our previous study of OBX rats has also demonstrated a substantial loss of raphe neurons (Nesterova et al., 1997). Thus, OBX-produced changes in behaviour and EEG appear to arise from an imbalance between different mediatory systems, each of which require a specific approach in order to clarify their involvement in these effects. The disturbances in spatial discrimination learning revealed in OBX rats (see Fig. 1) may be associated with changes in their navigational abilities that, in turn, depend on

Fig. 4 – Interhemispheric differences in EEG amplitudes in different frequency bands from frontal (A–E) and occipital (F–J) cortices in three groups of rats: sham-operated (SO) and bulbectomized with virtually normal, OBX(+), and abnormal, OBX(−), learning/memory abilities. Grey and black lines are the left and the right hemispheres, respectively. The abscissa represents successive 10-min intervals in different groups of rats. The ordinate is the value (arbitrary units, AU, ± 1 SEM)) of the EEG amplitudes summarized in a frequency band and normalized to the sum of the amplitudes from all bands. Horizontal filled bars indicate significant (two-way ANOVA) interhemispheric differences: light gray, P < 0.05; dark gray, P < 0.01; black, P < 0.001.

192

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –19 4

adequate functioning of the visual cortex. In this respect, the effects observed in occipital theta in OBX rats (see Fig. 4G) are in line with visual processing impairment revealed in AD patients (Kavcic et al., 2006). The similarity of changes in our OBX(+) and OBX(−) rats allows the possibility of a common source for these effects. This appears to be associated with impairments in visual attention (Rizzo et al., 2000) and/or in interhemispheric functional connectivity in the occipital cortex (Wada et al., 1998) which have been shown in AD. The latter conclusion, based upon the lowering of interhemispheric EEG coherence, is confirmed by increasing EEG asymmetry (i.e., uncorrelated/ incoherent changes in EEG) in our rats from both OBX groups (see Fig. 4G). Moreover, significant imbalance between occipital cortices in OBX(+) rats is in line with the fact that deterioration of visual attention abilities occurs in the early stages of AD (Rizzo et al., 2000), which provides useful clues during diagnosis. Thus, the hemispheric interplay in different cortices seems to be predominantly associated with different processes that are affected by OBX: in the occipital cortex — with visual attention, whereas in the frontal cortex — with cognition. The mechanism underlying the differences revealed in rats from OBX(+) and OBX(−) groups is uncertain. On first appearance, it could be linked with different extents of residual intracerebral connections after OBX (see Experimental procedures) and/or with possible individual peculiarities in the synaptic plasticity and neuronal regenerative capacity associated with prolonged recovery (1 year, in our study) and, respectively, with ageing (Slotkin et al., 2005). Morphological analysis in the cortex and the hippocampus has showed an increase of some pathological features (pyknosis, karyolysis, and vacuolation) with OBX(+) < OBX(−) rank order of strength (see Table 1). The deteriorative effect of OBX was evidently greater in the hippocampus than in the cortex and this is in line with the results obtained in AD patients (Wrynn et al., 2000). However, a significant reduction in cellular density, indicating a degradation of cells, was only revealed in the cortex. Together, these might be linked to post-OBX recovery of cell density in the hippocampus due to neurogenesis/ proliferation, which has also been shown in AD (Jin et al., 2004). In order to locate the actual source of OBX group separation morphometric analyses of each brain hemisphere performed at different time intervals after OBX is required. Regardless of possible mechanisms underlying group separation, OBX(+) rats with specific changes in behaviour, brain morphology, and EEG asymmetry in frontal and occipital cortices, appear to be a promising tool for use in studies of the early stages of AD and/or other related disorders.

4.

Experimental procedures

Twenty one male Wistar rats bred in a colony (donated by Charles Rivers Laboratories, Wilmington, MA, USA) under control barrier conditions were used in this study. The animals were given food and water ad libitum and reared in a standard 12-h/12-h day/night cycle. All manipulations were carried out in accordance with the principles enunciated in the Guide for Care and Use of Laboratory Animals, NIH publication No. 85-23 with efforts made to minimize animal suffering and to reduce the number of subjects used.

4.1.

Olfactory bulbectomy

Two-month old rats (150–170 g) were anaesthetised with Nembutal (60 mg/kg, i.p.) and two holes (2 mm in diameter) were drilled symmetrically over the olfactory bulbs (8 mm anterior to the bregma and 2 mm laterally from the midline). The bulbs were aspirated carefully under visual control through a blunt needle attached to a water pump. Sham-operated rats were treated similarly with exception of the bulbectomy.

4.2.

Spatial discrimination testing

Twelve months after surgery, the learning/memory abilities of the rats were tested with the Morris water paradigm (Morris, 1984) in a circular water pool (diameter, 150 cm; height, 80 cm), filled to a depth of 50 cm with water (25 °C) made opaque with powdered milk. The pool was divided into four quadrants mentally. Each of the rats was tested on a visible platform (10 cm above the water surface) task to evaluate its sensorimotor ability by the time taken (latency) to reach the platform. A day later, all rats were trained (four trials/day for 6 days) to find a hidden platform (9 cm in diameter) centered in one of the quadrants, 2 cm below the water surface. The rats were allowed a maximum of 1 min to find the platform followed by a 15-s rest period once on the platform before being dried and relocated into the home cage. On the next day, after ending of the training trials, a 60-s test trial was given, with the platform removed, to assess memory for platform location. The latency to find the platform during training and both the total time spent in each quadrant and the number of the inward crossings of its borders during testing were computed (a video camera was fixed 2.4 m above the center of the water pool).

4.3.

Electrode implantation and EEG registration

After behavioural testing, the recording electrodes were implanted under Nembutal anesthesia epidurally over symmetrical somatosensory and visual cortices (AP −2.2, L 2.2 and AP −7.5, L 2.2, respectively) (Krieg, 1946). A reference electrode was placed in the nasal bone close to the midline and at the distance of 4–4.5 mm in front of the holes drilled for OBX. Moreover, the tip of the reference electrode was positioned at an optimal depth to exclude/minimize respiration artifacts. To achieve this a signal between the reference electrode and an electrode temporarily clamped on the ear was monitored during implantation. All electrodes (stainless steel wire 0.4 mm in diameter) were fixed to the skull with dental cement and linked to a microconnector. Between days 4–7 after surgery, the rats were adapted for 1 h/day to an experimental box (transparent perspex, 15 × 17 × 20 cm) in an electrically shielded chamber. On day 8, a baseline EEG was registered for 80 min, starting 30 min after placing the animal in the box.

4.4.

Computation of EEG spectra

The frequency spectra of successive 12-s EEG epochs were analyzed on-line in the range of 0.25–30.5 Hz (band-pass 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

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –1 94

330 Hz. Taking into account the non-stationary nature of EEG signals (e.g. Gray et al., 1992), we have used a modified version of period-amplitude analysis (Gal'chenko and Vorobyov, 1999) which, contrary to the Fourier transform, is not affected by this phenomenon. The main modifications were linked with two types of frequency power normalization: 1) identical ratios of frequency bands to their central values for all relatively narrow bands (twenty in this study) and 2) using weighted coefficients for each band which were equal to the ratio of the number of periods in that band to the duration of the analysed epoch. The program allowed both automatic and manual rejection of the EEG fragments containing artifacts during on-line registration. The main criteria were prolonged (N10% of the epoch duration) high-amplitude slow waves (more than two-fold of the mean baseline activity in the previous epoch and <0.2 Hz) or signal truncation (N10% of the epoch duration). However, it should be made clear that such artifacts were very rare, because tight connections in the recording cable sockets and insertion of the cable into a thin flexible grounded silvered shield provided protection of the EEG against so-called “capacity” artifacts. The integrated power in twenty selected narrow EEG frequency bands (in Hz ): 0.25–0.75 (0.5), 0.75–1.25 (1), 1.25–1.75 (1.5), 1.75–2.25 (2.0), 2.25–2.75 (2.5), 2.75–3.25 (3), 3.25–3.9 (3.6), 3.9–4.6 (4.3), 4.6–5.3 (5.0), 5.3–6 (5.7), 6–6.8 (6.4), 6.8–7.6 (7.2), 7.6–8.7 (8.2), 8.7–9.8 (9.3), 9.8–11 (10.4), 11–12.8 (11.9), 12.8–14.8 (13.8), 14.8–17.8 (16.3), 17.8–22.5 (20.2), 22.5–30.5 (26.5), and power ratios for each band over the integrated power in the 0.25–30.5 Hz range were calculated. The bands were marked in Figs. 2, 3 by their center (mean) frequency values (see values in brackets, above). The EEG spectra were averaged for every successive 5-min. After such detailed analyses of the EEG spectra the averaging of the individual spectra was performed in the ranges of “classical” EEG bands: delta (0.5–3.5 Hz), theta (3.6–7.5 Hz), alpha (7.6– 12.5 Hz), and beta (12.6–27.5 Hz). The terms of “lower” and “higher” are used below to differentiate corresponding frequency subranges of the band relative to its centre frequency.

4.5.

Histology evaluation and measure of beta-amyloid

At the termination of the experiment (next day after the last EEG session), the rats were euthanized by an overdose of ketamine (50 mg/kg, i/m, Sigma, USA), their brains perfused through the left cardiac ventricle with a phosphate-buffered solution and verified the extent of OBX lesion was verified. Only the brains with sufficiently lesioned OBX (N90% of their estimated volumes) and undisturbed frontal cortices were accepted for histological and beta-amyloid analyses. A “cortex–hippocampus” sample from one hemisphere was examined for beta-amyloid, while the other hemisphere was preliminarily fixed in 4% paraformaldehyde (dissolved in 0.1 M PBS, pH 7.4) for 24 h at RT, to be used for histological evaluation. Beta-amyloid was detected by modified immunological DOTanalysis. For all other details of the histological and beta-amyloid analyses see Nesterova et al. (2008).

5.

Statistics

Differences in behavioural parameters were evaluated by oneor two-way ANOVA for repeated measures (when appropri-

193

ate); in EEG—a two-tailed non-parametric Wilcoxon test (p < 0.05 was considered statistically significant) was used for the frequency spectra and one- or two-way ANOVA (when appropriate), for each of the frequency bands; in beta-amyloid and morphometric studies — by Student's criterion.

Acknowledgment We are sincerely grateful to Dr. Chris Howarth for helpful comments and corrections of English in our manuscript.

REFERENCES

Andreasen, N., Sjogren, M., Blennow, K., 2003. CSF markers for Alzheimer's disease: total tau, phospho-tau and Abeta42. World J. Biol. Psychiatry 4 (4), 147–155. Apostolova, L.G., Steiner, C.A., Akopyan, G.G., Dutton, R.A., Hayashi, K.M., Toga, A.W., Cummings, J.L., Thompson, P.M., 2007. Three-dimensional gray matter atrophy mapping in mild cognitive impairment and mild Alzheimer disease. Arch. Neurol. 64 (10), 1489–1495. Bobkova, N.V., Nesterova, I.V., Nesterov, V.V., 2001. The state of cholinergic structures in forebrain of bulbectomized mice. Bull. Exp. Biol. Med. 131 (5), 427–431. Bobkova, N.V., Vorobjov, V.V., Medvinskaya, N.I., Nesterova, I.V., 2003. Electrophysiological markers of neurodegenerative process on model of sporadic form of Alzheimer's disease. Materials of the 2nd International Conference: “High Medical Technologies in XXI century”, November 1–8, Spain, Benidorm, p. 100. Davidson, R.J., 2004. What does the prefrontal cortex “do” in affect: perspectives on frontal EEG asymmetry research. Biol. Psychol. 67 (1–2), 219–233. Dringenberg, H.C., 2000. Alzheimer's disease: more than a ‘cholinergic disorder’ — evidence that cholinergic–monoaminergic interactions contribute to EEG slowing and dementia. Behav. Brain. Res. 115 (2), 235–249. Gal'chenko, A.A., Vorobyov, V.V., 1999. Analysis of electroencephalograms using a modified amplitude-interval algorithm. Neurosci. Behav. Physiol. 29, 157–160. German, D.C., Yazdani, U., Speciale, S.G., Pasbakhsh, P., Games, D., Liang, C.L., 2003. Cholinergic neuropathology in a mouse model of Alzheimer's disease. J. Comp. Neurol. 462 (4), 371–381. Geula, C., 1998. Abnormalities of neural circuitry in Alzheimer's disease: hippocampus and cortical cholinergic innervation. Neurology 51 (1 Suppl 1), S18–S29. Glick, S.D., 1983. Cerebral lateralisation in the rat and tentative extrapolation to man. In: Myslobotsky, M. (Ed.), Heimsyndromes: Physiology, Neurology, Psychiatry. Academic Press, New York, pp. 7–26. Gray, C.M., Engel, A.K., König, P., Singer, W., 1992. Synchronization of oscillatory neuronal responses in cat striate cortex: temporal properties. Visual neuroscience 8 (4), 337–347. Hallam, K.T., Horgan, J.E., McGrath, C., Norman, T.R., 2004. An investigation of the effect of tacrine and physostigmine on spatial working memory deficits in the olfactory bulbectomised rat. Behav. Brain Res. 153 (2), 481–486. Hardy, J., Selkoe, D.J., 2002. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297 (5580), 353–356. Hozumi, S., Nakagawasai, O., Tan-No, K., Niijima, F., Yamadera, F., Murata, A., Arai, Y., Yasuhara, H., Tadano, T., 2003. Characteristics of changes in cholinergic function and impairment of learning and memory-related behavior

194

BR A I N R ES E A RC H 1 2 3 2 ( 2 00 8 ) 1 8 5 –19 4

induced by olfactory bulbectomy. Behav. Brain Res. 138 (1), 9–15. Jin, K., Peel, A.L., Mao, X.O., Xie, L., Cottrell, B.A., Henshall, D.C., Greenberg, D.A., 2004. Increased hippocampal neurogenesis in Alzheimer's disease. Proc. Natl. Acad. Sci. U S A. 101 (1), 343–347. Kar, S., Slowikowski, S.P., Westaway, D., Mount, H.T., 2004. Interactions between beta-amyloid and central cholinergic neurons: implications for Alzheimer's disease. J. Psychiatry Neurosci. 29 (6), 427–441. Kavcic, V., Fernandez, R., Logan, D., Duffy, C.J., 2006. Neurophysiological and perceptual correlates of navigational impairment in Alzheimer's disease. Brain 129 (Pt 3), 736–746. Kikuchi, M., Wada, Y., Takeda, T., Oe, H., Hashimoto, T., Koshino, Y., 2002. EEG harmonic responses to photic stimulation in normal aging and Alzheimer's disease: differences in interhemispheric coherence. Clin. Neurophysiol. 113 (7), 1045–1051. Knott, V., Engeland, C., Mohr, E., Mahoney, C., Ilivitsky, V., 2000. Acute nicotine administration in Alzheimer's disease: an exploratory EEG study. Neuropsychobiology 41 (4), 210–220. Krieg, W.J.S., 1946. Connections of the cerebral cortex. 1. The albino rat. A. Topography of the cortical areas. J. Comp. Neurol. 84 (2), 221–275. Lakmache, Y., Lassonde, M., Gauthier, S., Frigon, J.Y., Lepore, F., 1998. Interhemispheric disconnection syndrome in Alzheimer's disease. Proc. Natl. Acad. Sci. USA 95 (15), 9042–9046. Lehéricy, S., Hirsch, E.C., Cervera-Piérot, P., Hersh, L.B., Bakchine, S., Piette, F., Duyckaerts, C., Hauw, J.J., Javoy-Agid, F., Agid, Y., 1993. Heterogeneity and selectivity of the degeneration of cholinergic neurons in the basal forebrain of patients with Alzheimer's disease. J. Comp. Neurol. 330 (1), 15–31. McKinney, M., Coyle, J.T., 1982. Regulation of neocortical muscarinic receptors: effects of drug treatment and lesions. J. Neurosci. 2 (1), 97–105. Montplaisir, J., Petit, D., Gauthier, S., Gaudreau, H., Decary, A., 1998. Sleep disturbances and EEG slowing in Alzheimer's disease. Sleep Res. Online 1 (4), 147–151. Morilak, D.A., Frazer, A., 2004. Antidepressants and brain monoaminergic systems: a dimensional approach to understanding their behavioural effects in depression and anxiety disorders. Int. J. Neuropsychopharmacol. 7 (2), 193–218. Morris, R.G.M., 1984. Development of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Nesterova, I.V., Gurevich, E.V., Nesterov, V.I., Otmakhova, N.A., Bobkova, N.V., 1997. Bulbectomy-induced loss of raphe neurons is counteracted by antidepressant treatment. Prog. Neuropsychopharmacol. Biol. Psychiatry 21 (1), 127–140. Nesterova, I.V., Bobkova, N.V., Medvinskaya, N.I., Samokhin, A.N., Aleksandrova, I.Y., 2008. Morphofunctional state of neurons in the temporal cortex and hippocampus in relation to the level of spatial memory in rats after ablation of the olfactory bulbs. Neurosci. Behav. Physiol. 38 (4), 349–353. Novoselova, E.G., Bobkova, N.V., Sinotova, O.A., Ogai, V.B., Glushkova, O.V., Medvinskaya, N.I., Samokhin, A.N., 2003. The immune state of bulbectomized mice. Dokl. Biol. Sci. 393, 505–507. Ostrovskaya, R.U., Gruden, M.A., Bobkova, N.A., Sewell, R.D., Gudasheva, T.A., Samokhin, A.N., Seredinin, S.B., Noppe, W., Sherstnev, V.V., Morozova-Roche, L.A., 2007. The nootropic and neuroprotective proline-containing dipeptide noopept restores spatial memory and increases immunoreactivity to amyloid in an Alzheimer's disease model. J. Psychopharmacol. 21 (6), 611–619.

Otmakhova, N.A., Gurevich, E.V., Katkov, Y.A., Nesterova, I.V., Bobkova, N.V., 1992. Dissociation of multiple behavioral effects between olfactory bulbectomized C57Bl/6J and DBA/2J mice. Physiol. Behav. 52 (3), 441–448. Podol'skii, I.Y., Vorob'ev, V.V., Belova, N.A., 2001. Long-term changes in hippocampus and neocortex EEG spectra in response to pharmacological treatments affecting the cholinergic system. Neurosci. Behav. Physiol. 31 (6), 589–595. Pogarell, O., Teipel, S.J., Juckel, G., Gootjes, L., Moller, T., Burger, K., Leinsinger, G., Moller, H.J., Hegerl, U., Hampel, H., 2005. EEG coherence reflects regional corpus callosum area in Alzheimer's disease. J. Neurol. Neurosurg. Psychiatry 76 (1), 109–111. Rizzo, M., Anderson, S.W., Dawson, J., Myers, R., Ball, K., 2000. Visual attention impairments in Alzheimer's disease. Neurology 54 (10), 1954–1959. Robichaud, M., Beauchemin, V., Lavoie, N., Dennis, T., Debonnel, G., 2001. Effects of bilateral olfactory bulbectomy on N-methyl-D-aspartate receptor function: autoradiographic and behavioral studies in the rat. Synapse 42 (2), 95–103. Roth, T., Costa e Silva, J.A., Chase, M.H., 2001. Sleep and cognitive (memory) function: research and clinical perspectives. Sleep Med. 2 (5), 379–387. Sakurada, T., Shima, K., Tadano, T., Sakurada, S., Kisara, K., 1976. Sleep wakefulness rhythms in the olfactory bulb lesioned rat. Jpn. J. Pharmacol. 26, 605–610. Slotkin, T.A., Cousins, M.M., Tate, C.A., Seidler, F.J., 2005. Serotonergic cell signaling in an animal model of aging and depression: olfactory bulbectomy elicits different adaptations in brain regions of young adult vs aging rats. Neuropsychopharmacology 30 (1), 52–57. Sokolovsky, M., 1984. Muscarinic receptors in the central nervous system. Int. Rev. Neurobiol. 25, 139–183. Song, C., Leonard, B.E., 2005. The olfactory bulbectomised rat as a model of depression. Neurosci. Biobehav. Rev. 29 (4–5), 627–647. Stevens, A., Kircher, T., Nickola, M., Bartels, M., Rosellen, N., Wormstall, H., 2001. Dynamic regulation of EEG power and coherence is lost early and globally in probable DAT. Eur. Arch. Psychiatry Clin. Neurosci. 251 (5), 199–204. Tadano, T., Hozumi, S., Yamadera, F., Murata, A., Niijima, F., Tan-No, K., Nakagawasai, O., Kisara, K., 2004. Effects of NMDA receptor-related agonists on learning and memory impairment in olfactory bulbectomized mice. Methods Find. Exp. Clin. Pharmacol. 26, 93–97. Vorobyov, V.V., Ahmetova, E.R., 1998. Muscarinic elucidation of EEG asymmetry in freely moving rats. Brain Res. 794, 299–303. Wada, Y., Nanbu, Y., Koshino, Y., Yamaguchi, N., Hashimoto, T., 1998. Reduced interhemispheric EEG coherence in Alzheimer disease: analysis during rest and photic stimulation. Alzheimer. Dis. Assoc. Disord. 12 (3), 175–181. Wang, J., Ikonen, S., Gurevicius, K., van Groen, T., Tanila, H., 2002. Alteration of cortical EEG in mice carrying mutated human APP transgene. Brain Res. 943 (2), 181–190. Watanabe, S., Fukuda, T., Ueko, S., 1980. Changes in electroencephalogram of the rat following olfactory bulbectomy. Tohoku J. Exp. Med. 130 (1), 41–48. Wrynn, A.S., MacSweeney, C.P., Franconi, F., Lemaire, L., Pouliquen, D., Herlidou, S., Leonard, B.E., Gandon, J., de Certaines, J.D., 2000. An in-vivo magnetic resonance imaging study of the olfactory bulbectomized rat model of depression. Brain Res. 879 (1–2), 193–199. Yamamoto, T., Jin, J., Watanabe, S., 1997. Characteristics of memory dysfunction in olfactory bulbectomized rats and the effects of cholinergic drugs. Behav. Brain Res. 83 (1–2), 57–62.