Early olfactory dysfunction has been consistently reported in both Alzheimer’s disease (AD) and in transgenic mice that reproduce some features of this disease. In AD transgenic mice, alteration in olfaction has been associated with increased levels of soluble amyloid beta protein (Aβ) as well as with alterations in the oscillatory network activity recorded in the olfactory bulb (OB) and in the piriform cortex. However, since AD is a multifactorial disease and transgenic mice suffer a variety of adaptive changes, it is still unknown if soluble Aβ, by itself, is responsible for OB dysfunction both at electrophysiological and behavioral levels. Thus, here we tested whether or not Aβ directly affects OB network activity in vitro in slices obtained from mice and rats and if it affects olfactory ability in these rodents. Our results show that Aβ decreases, in a concentration- and time-dependent manner, the network activity of OB slices at clinically relevant concentrations (low nM) and in a reversible manner. Moreover, we found that intrabulbar injection of Aβ decreases the olfactory ability of rodents two weeks after application, an effect that is not related to alterations in motor performance or motivation to seek food and that correlates with the presence of Aβ deposits. Our results indicate that Aβ disrupts, at clinically relevant concentrations, the network activity of the OB in vitro and can trigger a disruption in olfaction. These findings open the possibility of exploring the cellular mechanisms involved in early pathological AD as an approach to reduce or halt its progress.
Citation: Alvarado-Martínez R, Salgado-Puga K, Peña-Ortega F (2013) Amyloid Beta Inhibits Olfactory Bulb Activity and the Ability to Smell. PLoS ONE 8(9): e75745. https://doi.org/10.1371/journal.pone.0075745
Editor: Nadine Ravel, Université Lyon, France
Received: March 13, 2013; Accepted: August 20, 2013; Published: September 26, 2013
Copyright: © 2013 Alvarado-Martínez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by Consejo Nacional de Ciencia y Tecnología: Grants 151261 and 181323 (to FPO) and a scholarships to KSP and RAM, by Dirección General de Asuntos del Personal Académico–UNAM: Grants IACODI1201511 and IB200212 (to FPO), and by Alzheimer´s Association Grant NIRG-11-205443. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In recent decades, it has been demonstrated that at very early stages, Alzheimer’s patients find it difficult to detect, discriminate, and identify odors [1-6]. Such dysfunction has been correlated with early pathological findings such as sparse accumulation of amyloid beta protein (Aβ) in the olfactory bulb (OB) [7-11]. Similar observations of reduced olfaction [12-20] as well as early histopathological changes in the OB [15,20-24] have been reported in transgenic mice that overexpress proteins associated with familial AD. More recently, a strong correlation has been established between the accumulation of soluble forms of Aβ in the OB and very early olfactory dysfunction in both AD patients and transgenic mice . A similar correlation was found in young human subjects exposed to ambient pollution, such as that found in Mexico City, who exhibit reduced olfactory abilities along with Aβ accumulation in the OB [25,26]. Accordingly, experimental strategies that reduced the amount of Aβ also reduced the olfactory dysfunction observed in AD transgenic mice [17,19]. Taken together, this evidence suggests a causal relationship between Aβ and olfactory dysfunction. However, the effect of Aβ applied directly onto the different centers involved in the olfactory neural pathway has not been tested.
The OB is the first relay center and integrator in the olfactory information-processing pathway [27,28]. Starting with the pioneering studies performed by Adrian (1950) more than 7 decades ago, it has been shown that the OB generates a variety of network population activities that seem to be involved in the detection and signal processing of olfactory information [29-40]. For example, it was shown that the magnitude of the fast oscillatory activity generated by the OB is proportional to the olfactory task demand or performance [30,38-40]. Moreover, alterations in several of the cellular mechanisms involved in generating patterns of population activity by the OB, such as the dendrodendritic interaction between mitral and granular cells, affect the ability of the OB to represent and process olfactory information [30,32-35,41]. In addition, several experimental manipulations that either increase or decrease OB network activity improve or reduce, respectively, the rodents’ olfactory function [42-44].
Due to the facts that soluble Aβ is able to disrupt network activity in several circuits throughout the brain [45-51] and that such disruption is associated with deficits in behaviors regulated or commanded by such networks (i.e., learning and memory in the hippocampus [46,50]), it is likely that Aβ disrupts OB network activity and therefore affects olfactory function . We tested this hypothesis by applying clinically relevant concentrations of Aβ directly onto OB slices of mice and rats [52-55], while recording their population activity in the granule cell layer and by testing olfactory performance in rodents injected with Aβ. Our results show that Aβ decreases, in a concentration- and time-dependent manner, the network activity of OB slices at clinically relevant concentrations (low nM) and in a reversible manner. Moreover, we found that intrabulbar injection of Aβ decreases the olfactory ability of rodents two weeks after application, an effect that persists for at least two more weeks, correlating with the presence of Aβ deposits. These findings indicate that Aβ plays a major role in the alterations observed in OB function and olfactory performance in both AD transgenic mice and patients.
Materials and Methods
Experiments were approved by the Committee on Bioethics of the Instituto de Neurobiología, UNAM and were carried out according to the Norma Oficial Mexicana de la Secretaría de Agricultura (SAGARPA NOM-062-ZOO-1999), which complies with the guidelines in the Institutional Animal Care and Use Committee Guidebook (NIH publication 80-23, Bethesda, MD, USA, 1996).
Wistar rats (8 weeks old) as well as CD-1 mice of 3, 6, and 8 weeks of age were obtained from the animal facility of the Institute of Neurobiology, UNAM, where they were kept under a normal 12-h light: 12-h dark cycle (lights on at 7:00 a.m.) with free access to food and water. Three triple transgenic mice  were obtained from the same source and used as positive controls for thiazine red staining.
Aβ was obtained from Bachem (Heidelberg, Germany) and oligomerized using a standard protocol described earlier [52,57]. Briefly, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was added to solid amyloid beta1-42 at a final peptide concentration of 1 mM and incubated for 60 min at room temperature. HFIP was allowed to evaporate overnight, and a 5 mM solution was prepared by adding DMSO. This solution was diluted with F12 medium to reach a final concentration of 100 µM and then incubated at 5°C for 24 h. Then the solution was centrifuged at 14,000 × g for 10 min in the cold. The supernatant, containing the amyloid beta oligomers, was collected and used for the experiments. Characterization of this solution by electrophoresis showed the presence of both amyloid beta monomers and oligomers .
In Vitro Experiments
Olfactory Bulb Slice Preparation
To obtain OB slices, mice and rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally (i.p.)) and perfused transcardially with cold protective saline containing (in mM): 250 glycerol, 3 KCl, 36 NaHCO3, 5 KH2PO4, 10 glucose, 0.7 CaCl2, and 2 MgSO4, pH 7.4, bubbled with carbogen (95% O2 and 5% CO2). Then, animals were decapitated; the olfactory bulbs were removed and dissected in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 25 NaHCO3, and 30 D-glucose, pH 7.4, and bubbled with carbogen (95% O2 and 5% CO2). One olfactory bulb was glued to an agar block, and 400 µm thick parasagittal slices were cut with a vibratome (Vibratome, St. Louis, MO, U.S.A.). Slices were allowed to recover in aCSF at room temperature for at least 60 min before any further experimental manipulation.
For extracellular field recordings, the OB slices were transferred to a submerged recording chamber continuously perfused at 17-20 ml/min with oxygenated aCSF maintained at 30-32 °C. The field recordings were obtained with suction electrodes filled with aCSF and positioned on the granule cell layer using a stereoscopic microscope and a micromanipulator. The signal was amplified and filtered (highpass, 0.5 Hz; lowpass, 1.5 KHz) with a wide-band AC amplifier (Grass Instruments, Quincy, MA, U.S.A.). OB activity was recorded for 15-20 min to obtain the control recording in each slice before adding Aβ to the bath at three concentrations (3 nM, 10 nM, and 30 nM) to test its effect on network activity for 1 h. Finally, 1 mM lidocaine was added to the bath to block any neural activity, as a control for the viability of the slice. In some experiments, after testing the effect of 30 nM Aβ for 1 h, Aβ was washed out of the bath for at least 1 h to test for the reversibility of the Aβ effect. In another set of slices, we evaluated the effect of bath application of 30 nM inverse amyloid beta42-1 on OB activity network. All recordings were digitized at 3-9 KHz and stored on a personal computer with an acquisition system from National Instruments (Austin, TX, U.S.A.) using custom-made software designed in the LabView environment.
In Vivo Experiments
Adult male Wistar rats (300-350 g at the time of surgery) were acclimated to the laboratory vivarium for at least one week before surgery. There, animals were housed individually in a temperature-controlled (24°C) room and maintained on 12-h/12-h light/dark cycle (lights on at 7:00 a.m.). Food and water were provided ad libitum throughout the experiment. On the day of Aβ or vehicle (F12 medium) microinjection into the olfactory bulbs, the animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.), given an extra application of atropine sulfate (1 mg/kg i.p.), and secured in a stereotaxic frame (Stoelting Co., IL) as previously described . The co-ordinates used were: AP 9.4 from bregma, L ± 1.3 from midline, and V -2.5 from dura . A 1-µl volume containing 100 pmoles of Aβ was bilaterally injected at a rate of 0.2 µl/min (200 pmoles of Aβ per animal); 5 min later, the needle was withdrawn and the skin sutured.
Seven days after surgery, animals were tested for olfactory function [20,59-61]. The fed rats were placed in a cage (24 × 20 × 45 cm) with clean sawdust covering the floor. In each test, a 50-mg piece of chocolate (TRIKI-TRAKES®) was randomly placed at one of the four corners, hidden under the sawdust. The time the animal took to reach the chocolate was recorded. The maximum test time allowed was 600 s. For statistical analysis only the rats in which the perfusion injectors were located in the OB region were included. Based on previous reports, the test does not require food or water deprivation [59-62], although it can be performed after food-deprivation [20,61,62] with no evident differences between the two conditions . In all cases, the inability to find (see pp 15-16 for explanation) the hidden food is interpreted as an alteration in the main olfactory bulb function [20,59-61]. Alternatively, to exclude both motor deficits and motivational failure, we repeated the same “olfactory test” with a food pellet (normal chow) in a visible position in front of the rat . Moreover, to rule out any motivational contribution that could be interfering with the olfactory assessment, the hidden food test was performed in other groups of animals injected either with vehicle or with the same amount of Aβ, while food-deprived during the third week post-injection or while fed during the fourth week post-injection. The same animals were also tested for food consumption  by measuring the amount of food eaten in 30 min and 120 min. All rats were placed individually in clean sawdust cages with 50 g of chow. We quantified the amount of food eaten by the animals at the end of the third post-injection week in 24-h food-deprived animals and repeated the quantification at the end of the fourth post-injection week in the same animals maintained with food ad libitum. We also measured food consumption during one 24-h period, once a week (which did not differ between groups, data not shown), and quantified weight gain throughout the four weeks post-injection to test for changes in feeding behavior that would alter our olfactory test observations .
Histological evaluation of the injection sites [64-66], as well as thiazine red staining [67-69] were performed as follows: Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.), transcardially perfused with 250 ml of 0.9% NaCl followed by 250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and brains were removed and prepared for the histological procedure, as described previously [64,65]. Sagittal sections (50 µm thick) were stained either with toluidine-blue  to verify the injection sites or with thiazine red to look for Aβ deposits [67-69].
The recordings obtained were analyzed off-line by performing classical power spectrum analysis [70-72]. Segments of 20 s every 5 min were analyzed using a Rapid Fourier Transform Algorithm with a Hamming window, in Clampfit (Molecular Devices). The power spectra, from 1 to 50 Hz, were averaged and plotted for the last two segments of each experimental condition. We also evaluated the power spectra by measuring different frequency bands such as theta (2-12 Hz), beta (13-35 Hz) and gamma (35-50 Hz). The spectra were integrated and normalized to the control, i.e., the control power spectrum was integrated, and this value was arbitrarily set as 100%. Finally, peak frequency was determined as the frequency with the maximal power in each spectrum.
We used the Prism and Sigma Plot programs to prepare the graphs and the descriptive and inferential statistics. A Kruskal-Wallis analysis was used for statistical comparisons between experimental conditions. Upon finding a significant difference, we performed the Dunn’s test, and values of p < 0.05 were considered significant. In all cases reported in the text, the data are reported as means ± S.E.M. To compare the effects of the three Aβ concentrations tested at the three different ages, we used the Friedman test followed by Dunn’s test. Additionally, to evaluate the effects of Aβ compared with its own control the Wilcoxon test was used. Since the olfactory capacity test was truncated at 600 s, for these experiments only, the data is represented in the figures as the median and the interquartile range.
Aβ reduces OB network activity in a reversible and specific manner
The OB network activity recorded in the granule cell layer of slices obtained from 8-week-old mice shows low-voltage neuronal activity (Figure 1A, top trace) that contains a broad range of frequency components (Figure 1B,C) with a peak frequency (frequency with maximal power) of 13.3 ± 2.5 Hz (n = 7). The activity described here has very similar characteristics to that reported in previous studies in vitro [35,73-76] and resembles that recorded in vivo [38,77,78]. Continuous bath application of 30 nM Aβ for 1 h induces a significant inhibition of OB network activity (Figure 1A, middle trace) with proportional reductions in the activity at the frequencies corresponding to theta (to 38.2 ± 15.8% of control, Figure 1C), beta (to 52.8 ± 23.4% of control, Figure 1C), and gamma bands (to 45.9 ± 0.36% of control, Figure 1C). Consequently, Aβ application does not affect peak frequency of the OB network activity (10.7 ± 3.8 Hz, Figure 1B). Quantification of the OB network activity over the whole range between 1 and 50 Hz shows that after 1 h of continuous Aβ application, such activity is reduced to 40.1 ± 11.3% of control (Figure 1D,E). The time-course of the Aβ-induced inhibition of OB network activity shows that the reduction can already be observed after 15 min of continuous Aβ application (to 75.7 ± 12.5% of control, Figure 1D) and that it reaches significance after 25 min of continuous Aβ application (to 58.8 ± 11.7% of control, Figure 1D). The Aβ-induced inhibition of OB network activity described for slices obtained from mice (Figure 1E) is identical to that observed in slices obtained from rats of the same age (to 51.3 ± 13.7% of control, Figure 1E). The use of this in vitro approach allows us to demonstrate that, after washout of Aβ from the bath the OB network activity recovers to control levels in slices obtained from both species (109.3 ± 24.7% of control in mice and 108.0 ± 16.2% of control in rats; Figure 1C-E).
A, Representative recordings of the OB spontaneous network activity in slices, filtered at highpass = 0.5 Hz and lowpass = 1.5 KHz, obtained from 8-week-old mice in control conditions (upper trace), after 60 min of Aβ exposure (middle trace), and after 30 min of washout (lower trace). B, Averaged power spectra (± standard error; shaded area) of the slices under the three experimental conditions shown in A. Note that OB spontaneous network activity includes a broad variety of frequencies and that Aβ application produces a generalized and reversible reduction of the power without preferentially affecting any frequency range. C, Quantification of the power of OB network activity (as % of control) for different frequency bands (theta, beta, and gamma) for the experimental conditions represented in A. Note that Aβ reversibly reduces the power of the OB network activity to the same degree for all frequencies. D, Time-course of Aβ-induced inhibition of OB network activity, as % of control, over a frequency range of 1 to 50 Hz, and its washout. E, Comparison of Aβ-induced inhibition of OB network activity and its washout recorded in slices obtained from mice and rats. Note that the phenomenon is identical in slices obtained from both species. * Denotes a significant difference (p < 0.05) relative to the control (n = 7).
Two control experiments indicate that these effects are due to specific actions of Aβ on the OB network. First, we demonstrated that long-lasting recording of OB slices does not affect network activity. The power of OB network activity remained unaltered after 60 min (99.5 ± 8.4% of control), 120 min (104.4 ± 2.3%), or 180 min (97.1 ± 2.9% control) of continuous recording (Figure 2A). Similarly, the peak frequency also remained unaltered (13.2 ± 0.8 Hz; 12.8 ± 1.4 Hz; 13.8 ± 1.6 Hz, after 60, 120, and 180 min, respectively). Moreover, we demonstrated that the application of the Aβ-inverse sequence (,42) does not affect OB network activity (Figure 2C,D). After 1 h of continuous bath application of the Aβ-inverse sequence, the power of OB network activity (99.6 ± 5.4% of control, n = 6) (Figure 2D) as well as its peak frequency (16.6 ± 2.8 Hz) remained unaltered (Figure 2C).
A, Averaged power spectra (± standard error) of the slices in control conditions (dark gray) and after 3 h of continuous recording under the same control conditions (light gray). The insets shows representative recordings, filtered at highpass = 0.5 Hz and lowpass = 1.5 KHz for both conditions using the same color code. B, Quantification of the power of OB network activity (as % of control) over a frequency range of 1 to 50 Hz at three different time points. Note that OB network activity remains unaltered for at least 3 h of recording. C, Averaged power spectra (± standard error) of the slices in control conditions (dark gray) and after 1 h of continuous application of the Aβ inverse sequence (42-1, light gray). The insets show representative recordings for both conditions (using the same color code). D, Quantification of the power of OB network activity (as % of control) over a frequency range of 1 to 50 Hz 1 h after application of the inverse Aβ sequence, showing no effect of the peptide.
The sensitivity of OB network activity to Aβ increases with age
The effects of Aβ seem to be time dependent ; therefore, we tested the effects of three clinically relevant concentrations of Aβ (3, 10, and 30 nM) on slices obtained from mice at three different ages (3, 6, and 8 weeks old). The OB network activity recorded in the granule cell layer of slices obtained from 3-week-old mice shows low-voltage neuronal activity (Figure 3A, insets) that contains a broad range of frequency components (Figure 3A) and a peak frequency of 12.4 ± 0.6 Hz (n = 7). Continuous bath application of 3 nM Aβ for 1 h does not affect either the power of OB network activity (96.7 ± 9.5% of control; Figure 3B) or its peak frequency (13.4 ± 0.7 Hz). Subsequent increase of the Aβ bath concentration to 10 nM for 1 h does not affect OB network activity either (Figure 3B), since the power of the activity remains at 80.4 ± 13.7% of control, and the peak frequency did not change (12.8 ± 1.5 Hz). In contrast, subsequent increase of the Aβ bath concentration to 30 nM for 1 h induces a significant reduction in OB network activity (Figure 3A,B) to 53.2 ± 10.7% of control. The inhibition of OB network activity induced by Aβ affects all frequency components proportionally (Figure 3A) and does not affect peak frequency (12.0 ± 0.4 Hz).
A, Averaged power spectra (± standard error) of the slices obtained from 3-week-old animals in control conditions (dark gray) and after 1 h of continuous application of 30 nM Aβ (light gray). The inset shows representative recordings, filtered at highpass = 0.5 Hz and lowpass = 1.5 KHz, for both conditions using the same color code. B, Quantification of the power of OB network activity (as % of control) over a frequency range of 1 to 50 Hz after the application of different concentrations of Aβ to slices obtained from 3-week-old animals. Note that Aβ reduces OB network activity only at the highest concentration tested. C and D, Same as A and B except the slices were obtained from 6-week-old animals. Note that Aβ reduces OB network activity at the two highest concentrations tested. D and F, Same as A and B except the slices were obtained from 8-week-old animals. Note that Aβ reduces OB network activity at the two highest concentrations tested and that 3 nM Aβ also tends to reduce the activity. * Denotes a significant difference relative to control, and # denotes a significant difference between conditions (p < 0.05).
The OB network activity recorded in the granule cell layer of slices obtained from 6-week-old mice showed low-voltage neuronal activity (Figure 3C, insets) that contained a broad range of frequency components (Figure 3C) and a peak frequency of 13.3 ± 2.6 Hz (n = 7). This activity was slightly more sensitive to the presence of Aβ than the activity recorded in slices obtained from younger animals. Continuous bath application of 3 nM Aβ for 1 h affected neither the power of OB network activity (93.2 ± 5.6% of control; Figure 3D) nor its peak frequency (10.0 ± 4.7 Hz). In contrast to slices obtained from younger animals, subsequent increase of Aβ bath concentration to 10 nM for 1 h induced a significant reduction in OB network activity (Figure 3D) to 54.2 ± 19.4% of control, inhibiting all frequency components proportionally but without altering peak frequency (7.8 ± 1.7 Hz). A subsequent increase of Aβ bath concentration to 30 nM for 1 h induced no further reduction in OB network activity (44.1 ± 11.8% of control; Figure 3C, D); however, the power of OB network activity in the presence of 30 nM Aβ was significantly lower than the power in the presence of 3 nM Aβ, but this was not the case for 10 nM Aβ (Figure 3D). The inhibition of OB network activity induced by Aβ 30 nM affected all frequency components proportionally but did not affect peak frequency (8.7 ± 1.73 Hz, Figure 3C).
The OB network activity recorded in the granule cell layer of slices obtained from 8-week-old mice shows low-voltage neuronal activity (Figure 3E, insets) that contains a broad range of frequency components (Figure 3E) with a peak frequency of 16.4 ± 1.7 Hz (n = 7). Such activity tends to be more sensitive to the presence of Aβ than the activity recorded in slices obtained from younger animals. Bath application of 3 nM Aβ induces reduction in OB network activity (Figure 3F) to 65.2 ± 4.9% of control; this reduction did not reach statistical significance when tested by Dunn’s test (after a Friedman test), but it was significantly different from the control when evaluated by the Wilcoxon test (p < 0.05). This inhibition affected all frequency components proportionally and did not affect peak frequency (19.0 ± 2.0 Hz). A subsequent increase of Aβ bath concentration to 10 nM for 1 h induced a significant reduction in OB network activity (Figure 3F) to 54.6 ± 5.2% of control, again inhibiting all frequency components proportionally without affecting peak frequency (15.7 ± 1.9 Hz). Increasing the Aβ bath concentration to 30 nM for 1 h induced a further reduction in OB network activity to 46.8 ± 4.1% of control (Figure 3E, F), again with proportional effects on all frequency components (Figure 3E) but no effect on peak frequency (12.6 ± 0.9 Hz). In the presence of 30 nM Aβ, but not of 10 nM Aβ, the power of the OB network activity was significantly lower than the power in the presence of 3 nM Aβ (Figure 3E). The effects of the different Aβ concentrations on slices obtained at different times (3, 6, and 8 weeks old) showed no significant differences when compared using the Friedman test followed by Dunn’s post test, considering both time and concentration as variables.
A single intrabulbar injection of Aβ induces a time-dependent loss of smell
To assess the behavioral consequence of Aβ-induced neural network alterations, we injected Aβ1-42 into the olfactory bulb (100 picomoles/side, n = 18; Figure 4) and tested olfactory function by the ability of the animals to find a hidden piece of chocolate [59-62] while either normally fed or food deprived. We verified that the tip of the microinjector reached the granule cell layer as a criterion to include the data obtained from a given animal in the behavioral analysis (Figure 4A,B). Moreover, we found that intrabulbar injection of Aβ induced thiazine red positive deposits in the olfactory bulb that can be observed four weeks after injection (Figure 4D) but that are absent in control animals (Figure 4D). As a positive control, we observed abundant thiazine red staining in the brain of the triple transgenic mice (Figure 4C), which strongly suggests that thiazine red is staining Aβ deposits [56,67-69]. In these animals, we found that intrabulbar injection of Aβ induced a time-dependent loss of smell. One week after Aβ application (n = 9), the ability of the animals to the hidden chocolate remained unaltered compared with animals injected with vehicle (medium F12; n = 10; 155.7 ± 43.5 vs. 120.1 ± 24.2 s, respectively; Figure 5). However, only two weeks after Aβ application, the ability of the animals to find the hidden chocolate was seriously compromised (431.9 ± 69.0 s). At this time, 5 out of 9 animals failed to locate the piece of chocolate, and the average latency to reach the hidden food was significantly longer compared both to the same animals one week after Aβ application and to the control animals two weeks after vehicle injection (Figure 5). A similar result was found three and four weeks after Aβ application when the ability of the animals to find the hidden chocolate remained seriously impaired (450.9 ± 63.0 and 520.0 ± 45.9 s, respectively); at these times, 4 and 5 out of 9 animals, respectively, failed to reach the piece of chocolate. In both cases, the average latency to reach the hidden food was significantly longer than that of the same animals one week after Aβ application, and it was also longer than that of the control animals three and four weeks after vehicle injection (Figure 5). In another set of animals (n = 9 for each group), we performed control measurements to test whether or not the differences in the time to reach the hidden chocolate was due to changes in motivation to seek food [61-63]. First, we observed that the differences in the ability to find the hidden chocolate between control animals and those injected with Aβ was the same with or without a motivation imposed by hunger (Deprived for 24 h vs. normally fed; Figure 6A). We also excluded the possibility that intrabulbar injection of Aβ induces motor deficits or motivational failure by finding no difference between control and experimental groups in the time required to reach a food pellet that was visible to the animals (Figure 6A ). Moreover, there was no difference between control animals injected with vehicle and animals with intrabulbar injection of Aβ when measuring parameters related to feeding, such as weight gain (Figure 6B) or food intake (Figure 6C ).
A, Photomicrographs of histological sections stained with toluidine-blue taken from both vehicle (left) and Aβ-injected (right) animals showing the lesion induced by the microinjector. Note that the microinjector reaches the granule cell layer. B, Schematic representation (adapted from Paxinos and Watson ) of the microinjector tip locations, represented as a gray circles, in both bulbi of all animals. B denotes the position of Bregma. C, Photomicrograph illustrating thiazine red positive plaques stained in the subiculum of a 19-month-old triple transgenic (3xTgAD) mouse. D, Photomicrographs of histological sections, stained with thiazine red, from both vehicle (left) and Aβ-injected (right) animals. Note that Aβ-treated animals exhibit thiazine red deposits, whereas vehicle-treated animals do not. The t denotes the track left by the microinjector. Scale-bars denote 1 mm for A, and 200 µm for C and for the micrographs in D.
The graph shows the time needed for control animals (injected with vehicle; white bars) and animals with intrabulbar injection of Aβ (gray bars) to reach a hidden piece of chocolate (50 mg). The horizontal axis indicates when the test was made, expressed as the number of weeks after surgery. The maximal time allowed for the search was 600 s. Note that the animals lose the ability to smell, and the loss increases with time after the Aβ injection. For these experiments the data are represented as the median and the interquartile range. * Denotes a significant difference relative to control (vehicle; p < 0.05).
A, Quantification of time needed to reach food for control animals (injected with vehicle; white bars) and animals with intrabulbar injection of Aβ (gray bars) in two conditions: Food-deprived and Fed. Additionally, animals were tested while food was either hidden (a chocolate piece) or in plain sight (normal chow). Note that the animals injected with Aβ exhibit a deficit in their ability to find the hidden food, regardless of their feeding status. In contrast, no difference was found for any experimental group when the food was visible. Note that just for these experiments the data are represented as the median and the interquartile range. B, Time-course of body weight increase, quantified as % of the original weight (on the day of surgery), for animals injected with vehicle and animals with intrabulbar injection of Aβ. C, Food intake of control animals injected with vehicle (white bars) and animals with intrabulbar injection of Aβ (gray bars) tested at two time points (30 min upper graph and 120 min, lower graph). * Denotes a significant difference relative to control (vehicle; p < 0.05).
Here, we found that soluble Aβ oligomers reversibly alter the activity of the OB in slices obtained from mice and rats and that their injection triggers a delayed loss of the ability to smell that might be related to the presence of Aβ deposits but not to a change in the motivation to seek food. We also found that Aβ-induced inhibition of OB network activity is time and concentration dependent, and occurs at concentrations similar to those found in AD patients [52-55]. Since the amyloid hypothesis indicates that the soluble Aβ oligomers are responsible for neural dysfunction [55,79] and because different varieties of neural functions correlate with the generation of certain types of spontaneous oscillatory activity [29-33,37,51], our results might provide the cellular basis of the early olfactory dysfunction observed both in AD patients and AD transgenic mice. Moreover, our findings confirm previous evidence that experimental manipulations alter the generation of synchronized activity by the OB and also produce olfactory deficits [42-44]. Importantly, as mentioned, Aβ induces OB network dysfunction at concentrations (low nanomolar) very close to those found in the brain of AD patients [53,54], and which closely correlate with the concentrations that produce oscillatory alterations in these patients [80,81].
The spontaneous OB network activity of slices recorded in our experimental conditions is very similar to that reported by other groups using a similar experimental approach [35,73-76]. Since this activity maintains the features of that generated by the OB in vivo [38,77,78], the OB slices can be used to test the effects of Aβ in controlled physiological conditions and at clinically relevant concentrations as well as the reversibility of these effects .
Another major finding of our study is that Aβ-induced OB network dysfunction is time dependent. The olfactory bulb is a network highly sensitive to AD pathology [1-10,15,20,23], and it is one of the first brain structures to accumulate Aβ; it shows functional deterioration even before other neural networks involved in cognitive performance, such as the hippocampus and the cholinergic nuclei [15,82]. As the brain becomes older, it becomes more sensitive to several insults. We found that the OB, like the hippocampus, becomes more sensitive to the effects of Aβ with age . In fact, this increase in sensitivity to the effects of Aβ is more pronounced in the OB. For instance, we found that the network activity generated by the OB can be reduced by the application of 30 nM Aβ in slices taken from 3-week-old animals (Figure 3), whereas it was necessary to apply 100 nM Aβ to induce a similar reduction in network activity of hippocampal slices taken from 2-week-old animals . Aging has been associated with loss of synaptic contacts [83,84], silencing of synapses [83-85], or a decrease of postsynaptic responsiveness [83,84,86,87] and synaptic dynamics [68, for review see 87], which is reflected in deficits in the generation of certain spontaneous oscillatory activities [52,88]. It remains to be determined which of these phenomena is responsible for the age dependency of Aβ-induced OB network dysfunction.
As observed for the hippocampus , we demonstrated that Aβ-induced inhibition of the OB network is concentration dependent and that this inhibition occurs at clinically relevant concentrations [53,54], which we believe do not necessarily cause neuronal loss. Previously, it was reported that Aβ oligomers can induce neuronal death [55,57], even at concentrations very close to those used in our experiments . Here, we have exploited one of the advantages of the in vitro approach to show that acute application of Aβ induces a reduction of spontaneous network activity in OB slices that is “reversible”, which strongly suggests that it does not involve cell damage. Thus, it is very likely that Aβ-induced neuronal network dysfunction is produced by an alteration of specific cellular mechanisms involved in generating spontaneous network activity . One such mechanism might be the well-known Aβ-induced reduction in synaptic transmission [45,51,52]. In the hippocampus, Aβ reduces both glutamatergic [45,51,52] and GABAergic synaptic transmission [51,90,91], which are known to be key elements for the generation of population activity in the OB [32,35,37,42,73,76,91-96]. Complex interactions among GABAergic inhibition provided by granular cells and interneurons [32,35,42,73,76,93-96] along with glutamatergic synapses both in the glomerulus and that provided by principal cells [35,42,73,76,96] are known to be essential for the generation of different patterns of activity in the OB [35,42,73,76,96]. Since Aβ disrupts both types of synaptic interactions in other neuronal networks [42,52] it is highly likely that Aβ-induced synaptic inhibition plays a major role in the alterations in OB network activity observed in this study. Interestingly, several studies in AD mouse models have suggested that a reduction in synaptic transmission occurs before any neuronal death and plaque formation and correlates with the neural dysfunction observed in these animals [15,55,97]. It remains to be determined to what extent the inhibition of OB network activity induced by acute application of Aβ to OB slices involves changes in the different synaptic interactions that occur in this network and whether these changes play a role in the long-term effects triggered by Aβ in the OB. It would be particularly interesting to test whether or not Aβ affects the dendrodendritic interaction between mitral and granular cells, which is a keystone of OB oscillatory capabilities [32,98-100].
Finally, we determined that a single application of Aβ in the OB affects olfaction two weeks after its injection (Figure 5), a phenomenon that is not accompanied by a change in motor performance or motivation to seek food (Figure 6). The observation that the advent of Aβ-induced smell loss occurs between the first and second week after its intrabulbar application strongly suggests that additional mechanisms, beyond the presence of the injected oligomers, are involved in this dysfunction (Figure 4). It is possible that injected Aβ oligomers trigger pathological events that need time to build up and that these secondary changes are responsible for sensory dysfunction. This finding is similar to previous observations that Aβ-induced impairment in learning and memory requires several days or weeks to develop [101-109]. Several processes can account for such delayed effects of Aβ microinjection. For instance, it is well known that Aβ inoculation can trigger central amyloidosis [102,105-117]. Accumulation and eventual precipitation of endogenous Aβ, such as that shown in Figure 4, can be achieved by the intracerebral inoculation of brain material containing Aβ seeds extracted from AD brains or from AD-transgenic mice [111-117] or by the microinjection of synthetic Aβ [102,105–110,116], but not of a scrambled Aβ sequence . In both cases, these amylodogenic processes can take several weeks, or even months , to be established and are associated with a variety of pathological changes such as astroglial [105,106,108,109,112,113,115,118,119] and microglial reactions [105,109,112,113,118,119], appearance of dystrophic dendrites [112,113] neuroinflammation [111,118,119], endocrine stress [118,119], reduction in BDNF [118,119], tau phosphorylation , and reduction in synaptic transmission [102,105]. Whether our intrabulbar microinjection of Aβ is inducing one or several of these mechanisms and how they contribute to the induction of smell loss need to be determined. In any case, we found evidence of thiazine red positive deposits, probably Aβ accumulations [67-69], four weeks after the oligomers injection (Figure 4). It would be important to consider that long-term overproduction of Aβ has already been related to the neurodegeneration of olfactory sensory neurons , which raises the possibility that intrabulbar Aβ application would eventually induce some neuronal damage in the OB or its related networks. An alternative, or complementary, explanation for the delayed effects of intrabulbar application of Aβ on smell is that the pathological alterations produced by the injected Aβ may require the delayed transsynaptic modification of other olfactory networks beyond the olfactory bulb. This possibility is supported by the observations that injection of Aβ in a specific neural network, or its over-production, induces progressive transsynaptic alterations that gradually spread from the area of injection or over-production to brain regions with which it has prominent efferent connections [120,121]. Regardless of the mechanisms involved in the delayed Aβ-induced smell loss, our results support previous observations that transgenic animals expressing an AD-like phenotype have deficits in a variety of olfactory-related behaviors [12-20] that might involve alterations not only in the OB but also in other neural networks [13-19]. In fact, previous findings have linked early accumulation of soluble Aβ in the OB with olfactory dysfunction [15,20]. Since transgenic mice suffer a variety of adaptive changes that preclude establishing a direct, unequivocal relationship between the genetic alteration and the phenotypic outcome, the relationship between Aβ and olfactory dysfunction can be considered only a correlation [15,20]. Here, we show direct evidence that Aβ oligomers trigger a delayed loss of the ability to smell (Figure 4), which may be due to the actions of soluble and/or insoluble Aβ. Thus, our data support previous work showing that Aβ alters the activity of the OB network, resulting somewhat later in an inability to process olfactory information, as occurs in patients with AD and in transgenic animals [15,20,24]. Our demonstration of Aβ-induced OB dysfunction indicates that studies of the cellular mechanisms involved will help to understand AD pathology and reveal potential therapeutic targets against this disease.
We would like to thank Mario Andrade Lozano, José Antonio Caldera Pérez and Karen Prieto Reyes for their help with some of the behavioral experiments. We also thank Drs. Wendy Portillo and Raúl Paredes for their advice regarding the behavioral experiments, José Luna for providing us with thiazine red, Jorge Larriva-Sahd for providing us with toluidine-blue and Dorothy Pless for reviewing the English version of the manuscript. Currently, KSP and RAM are enrolled in the Ph.D. program in Biomedical Sciences (INB, Universidad Nacional Autónoma de México).
Conceived and designed the experiments: FPO. Performed the experiments: RAM KSP. Analyzed the data: RAM KSP. Contributed reagents/materials/analysis tools: FPO. Wrote the manuscript: RAM FPO.
- 1. Thompson MD, Knee K, Golden CJ (1998) Olfaction in persons with Alzheimer’s disease. Neuropsychol Rev 8: 11-23. doi:https://doi.org/10.1023/A:1025627106073. PubMed: 9585920.
- 2. Warner MD, Peabody CA, Flattery JJ, Tinklenberg JR (1986) Olfactory deficits and Alzheimer’s disease. Biol Psychiatry 21: 116-118. doi:https://doi.org/10.1016/0006-3223(86)90013-2. PubMed: 3942798.
- 3. Mesholam RI, Moberg PJ, Mahr RN, Doty RL (1998) Olfaction in neurodegenerative disease: a meta-analysis of olfactory functioning in Alzheimer’s and Parkinson’s diseases. Arch Neurol 55: 84-90. doi:https://doi.org/10.1001/archneur.55.1.84. PubMed: 9443714.
- 4. Murphy C, Gilmore MM, Seery CS, Salmon DP, Lasker BR (1990) Olfactory thresholds are associated with degree of dementia in Alzheimer’s disease. Neurobiol Aging 11: 465-469. doi:https://doi.org/10.1016/0197-4580(90)90014-Q. PubMed: 2381506.
- 5. Serby M, Larson P, Kalkstein D (1991) The nature and course of olfactory deficits in Alzheimer’s disease. Am J Psychiatry 148: 357-360. PubMed: 1992839.
- 6. Schofield PW, Ebrahimi H, Jones AL, Bateman GA, Murray SR (2012) An olfactory 'stress test' may detect preclinical Alzheimer’s disease. BMC Neurol 12: 24. doi:https://doi.org/10.1186/1471-2377-12-24. PubMed: 22551361.
- 7. Price JL, Davis PB, Morris JC, White DL (1991) The distribution of tangles, plaques and related immunohistochemical markers in healthy aging and Alzheimer’s disease. Neurobiol Aging 12: 295-312. doi:https://doi.org/10.1016/0197-4580(91)90006-6. PubMed: 1961359.
- 8. Kovács T (2004) Mechanisms of olfactory dysfunction in aging and neurodegenerative disorders. Ageing Res Rev 3: 215-232. doi:https://doi.org/10.1016/j.arr.2003.10.003. PubMed: 15177056.
- 9. Kovács T, Cairns NJ, Lantos PL (1999) Beta-amyloid deposition and neurofibrillary tangle formation in the olfactory bulb in aging and Alzheimer’s disease. Neuropathol Appl Neurobiol 25: 481-491. doi:https://doi.org/10.1046/j.1365-2990.1999.00208.x. PubMed: 10632898.
- 10. Attems J, Lintner F, Jellinger KA (2005) Olfactory involvement in aging and Alzheimer’s disease: an autopsy study. J Alzheimers Dis 7: 149-157. PubMed: 15851853.
- 11. Arnold SE, Lee EB, Moberg PJ, Stutzbach L, Kazi H et al. (2010) Olfactory epithelium amyloid-beta and paired helical filament-tau pathology in Alzheimer disease. Ann Neurol 67: 462-469. doi:https://doi.org/10.1002/ana.21910. PubMed: 20437581.
- 12. Coronas-Sámano G, Medina-Aguirre I, Aguilar-Vázquez A, Portillo-Martínez W, González-Luna I et al. (2011) Conducta olfativa en un modelo murino de Alzheimer. Rev Dig Univ 12: 1067-1079.
- 13. Guérin D, Sacquet J, Mandairon N, Jourdan F, Didier A (2009) Early locus coeruleus degeneration and olfactory dysfunctions in Tg2576 mice. Neurobiol Aging 30: 272-283. doi:https://doi.org/10.1016/j.neurobiolaging.2007.05.020. PubMed: 17618708.
- 14. Young JW, Sharkey J, Finlayson K (2009) Progressive impairment in olfactory working memory in a mouse model of Mild Cognitive Impairment. Neurobiol Aging 30: 1430-1443. doi:https://doi.org/10.1016/j.neurobiolaging.2007.11.018. PubMed: 18242780.
- 15. Wesson DW, Levy E, Nixon RA, Wilson DA (2010) Olfactory dysfunction correlates with amyloid beta burden in an Alzheimer’s disease mouse model. J Neurosci 30: 505-514. doi:https://doi.org/10.1523/JNEUROSCI.4622-09.2010. PubMed: 20071513.
- 16. Wesson DW, Morales-Corraliza J, Mazzella MJ, Wilson DA, Mathews PM (2013) Chronic anti-murine Abeta immunization preserves odor guided behaviors in an Alzheimer’s beta-amyloidosis model. Behav Brain Res 237: 96-102. doi:https://doi.org/10.1016/j.bbr.2012.09.019. PubMed: 23000537.
- 17. Yang DS, Stavrides P, Mohan PS, Kaushik S, Kumar A et al. (2011) Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer’s disease ameliorates amyloid pathologies and memory deficits. Brain 134: 258-277. doi:https://doi.org/10.1093/brain/awq341. PubMed: 21186265.
- 18. Montgomery KS, Simmons RK, Edwards G 3rd, Nicolle MM, Gluck MA et al. (2011) Novel age-dependent learning deficits in a mouse model of Alzheimer’s disease: implications for translational research. Neurobiol Aging 32: 1273-1285. doi:https://doi.org/10.1016/j.neurobiolaging.2009.08.003. PubMed: 19720431.
- 19. Cramer PE, Cirrito JR, Wesson DW, Lee CY, Karlo JC et al. (2012) ApoE-directed therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models. Science 335: 1503-1506. doi:https://doi.org/10.1126/science.1217697. PubMed: 22323736.
- 20. Cao L, Schrank BR, Rodriguez S, Benz EG, Moulia TW et al. (2012) Abeta alters the connectivity of olfactory neurons in the absence of amyloid plaques in vivo. Nat Commun 3: 1009. doi:https://doi.org/10.1038/ncomms2013. PubMed: 22910355.
- 21. Lehman EJ, Kulnane LS, Lamb BT (2003) Alterations in beta-amyloid production and deposition in brain regions of two transgenic models. Neurobiol Aging 24: 645-653. doi:https://doi.org/10.1016/S0197-4580(02)00153-7. PubMed: 12885572.
- 22. Kameshima N, Nanjou T, Fukuhara T, Yanagisawa D, Tooyama I (2012) Correlation of Abeta deposition in the nasal cavity with the formation of senile plaques in the brain of a transgenic mouse model of Alzheimer’s disease. Neurosci Lett 513: 166-169. doi:https://doi.org/10.1016/j.neulet.2012.02.026. PubMed: 22343315.
- 23. Rey NL, Jardanhazi-Kurutz D, Terwel D, Kummer MP, Jourdan F et al. (2012) Locus coeruleus degeneration exacerbates olfactory deficits in APP/PS1 transgenic mice. Neurobiol Aging 33: 426.e1-426.e11. PubMed: 21109328.
- 24. Cheng N, Cai H, Belluscio L (2011) In vivo olfactory model of APP-induced neurodegeneration reveals a reversible cell-autonomous function. J Neurosci 31(39): 13699-13704. doi:https://doi.org/10.1523/JNEUROSCI.1714-11.2011. PubMed: 21957232.
- 25. Calderón-Garcidueñas L, Franco-Lira M, Torres-Jardón R, Henriquez-Roldán C, Barragán-Megía G et al. (2007) Pediatric respiratory and systemic effects of chronic air pollution exposure: nose, lung, heart, and brain pathology. Toxicol Pathol 35: 154-162. doi:https://doi.org/10.1080/01926230601059985. PubMed: 17325984.
- 26. Calderón-Garcidueñas L, Franco-Lira M, Henríquez-Roldán C, Osnaya N, González-Maciel A et al. (2010) Urban air pollution: influences on olfactory function and pathology in exposed children and young adults. Exp Toxicol Pathol 62: 91-102. doi:https://doi.org/10.1016/j.etp.2009.02.117. PubMed: 19297138.
- 27. Nieuwenhuys R (1967) Comparative anatomy of olfactory centres and tracts. Prog Brain Res 23: 1-64. PubMed: 6020786.
- 28. Shepherd GM (1972) Synaptic organization of the mammalian olfactory bulb. Physiol Rev 52: 864-917. PubMed: 4343762.
- 29. Adrian ED (1950) The electrical activity of the mammalian olfactory bulb. Electroencephalogr Clin Neurophysiol 2: 377-388. doi:https://doi.org/10.1016/0013-4694(50)90075-7. PubMed: 14793507.
- 30. Beshel J, Kopell N, Kay LM (2007) Olfactory bulb gamma oscillations are enhanced with task demands. J Neurosci 27: 8358-8365. doi:https://doi.org/10.1523/JNEUROSCI.1199-07.2007. PubMed: 17670982.
- 31. Kay LM, Beshel J (2010) A beta oscillation network in the rat olfactory system during a 2 alternative choice odor discrimination task. J Neurophysiol 104: 829-839. doi:https://doi.org/10.1152/jn.00166.2010. PubMed: 20538778.
- 32. Kay LM, Beshel J, Brea J, Martin C, Rojas-Líbano D (2009) Olfactory oscillations: the what, how and what for. Trends Neurosci 32: 207-214. doi:https://doi.org/10.1016/j.tins.2008.11.008. PubMed: 19243843.
- 33. Rojas-Líbano D, Kay LM (2008) Olfactory system gamma oscillations: the physiological dissection of a cognitive neural system. Cogn Neurodyn 2: 179-194. doi:https://doi.org/10.1007/s11571-008-9053-1. PubMed: 19003484.
- 34. Friedman D, Strowbridge BW (2000) Functional role of NMDA autoreceptors in olfactory mitral cells. J Neurophysiol 84: 39-50. PubMed: 10899181.
- 35. Friedman D, Strowbridge BW (2003) Both electrical and chemical synapses mediate fast network oscillations in the olfactory bulb. J Neurophysiol 89: 2601-2610. doi:https://doi.org/10.1152/jn.00887.2002. PubMed: 12740407.
- 36. Murakami M, Kashiwadani H, Kirino Y, Mori K (2005) State-dependent sensory gating in olfactory cortex. Neuron 46: 285-296. doi:https://doi.org/10.1016/j.neuron.2005.02.025. PubMed: 15848806.
- 37. Wachowiak M, Shipley MT (2006) Coding and synaptic processing of sensory information in the glomerular layer of the olfactory bulb. Semin Cell Dev Biol 17: 411-423. doi:https://doi.org/10.1016/j.semcdb.2006.04.007. PubMed: 16765614.
- 38. Martin C, Gervais R, Chabaud P, Messaoudi B, Ravel N (2004) Learning-induced modulation of oscillatory activities in the mammalian olfactory system: the role of the centrifugal fibres. J Physiol Paris 98: 467-478. doi:https://doi.org/10.1016/j.jphysparis.2005.09.003. PubMed: 16274975.
- 39. Martin C, Gervais R, Messaoudi B, Ravel N (2006) Learning-induced oscillatory activities correlated to odour recognition: a network activity. Eur J Neurosci Volumes 7: 1801-1810. PubMed: 16623837.
- 40. Gervais R, Buonviso N, Martin C, Ravel N (2007) What do electrophysiological studies tell us about processing at the olfactory bulb level? J Physiol Paris 101: 40-45. doi:https://doi.org/10.1016/j.jphysparis.2007.10.006. PubMed: 18054211.
- 41. Stakic J, Suchanek JM, Ziegler GP, Griff ER (2011) The source of spontaneous activity in the main olfactory bulb of the rat. PLOS ONE 6: e23990. doi:https://doi.org/10.1371/journal.pone.0023990. PubMed: 21912614.
- 42. Nusser Z, Kay LM, Laurent G, Homanics GE, Mody I (2001) Disruption of GABA(A) receptors on GABAergic interneurons leads to increased oscillatory power in the olfactory bulb network. J Neurophysiol 86: 2823-2833. PubMed: 11731539.
- 43. Le Pichon CE, Valley MT, Polymenidou M, Chesler AT, Sagdullaev BT et al. (2009) Olfactory behavior and physiology are disrupted in prion protein knockout mice. Nat Neurosci 12: 60-69. doi:https://doi.org/10.1038/nn.2238. PubMed: 19098904.
- 44. Lepousez G, Mouret A, Loudes C, Epelbaum J, Viollet C (2010) Somatostatin contributes to in vivo gamma oscillation modulation and odor discrimination in the olfactory bulb. J Neurosci 30: 870-875. doi:https://doi.org/10.1523/JNEUROSCI.4958-09.2010. PubMed: 20089895.
- 45. Peña F, Ordaz B, Balleza-Tapia H, Bernal-Pedraza R, Márquez-Ramos A et al. (2010) Beta-amyloid protein (25-35) disrupts hippocampal network activity: role of Fyn-kinase. Hippocampus 20: 78-96. PubMed: 19294646.
- 46. Villette V, Poindessous-Jazat F, Simon A, Léna C, Roullot E et al. (2010) Decreased rhythmic GABAergic septal activity and memory-associated theta oscillations after hippocampal amyloid-beta pathology in the rat. J Neurosci 30: 10991-11003. doi:https://doi.org/10.1523/JNEUROSCI.6284-09.2010. PubMed: 20720106.
- 47. Colom LV, Castañeda MT, Bañuelos C, Puras G, García-Hernández A et al. (2010) Medial septal beta-amyloid 1-40 injections alter septo-hippocampal anatomy and function. Neurobiol Aging 31: 46-57. doi:https://doi.org/10.1016/j.neurobiolaging.2008.05.006. PubMed: 18547680.
- 48. Pena-Ortega F, Solis-Cisneros A, Ordaz B, Balleza-Tapia H, López-Guerrero J (2012) Amyloid beta 1-42 inhibits entorhinal cortex activity in the beta-gamma range: role of GSK-3. Curr Alzheimers 9: 857-863. doi:https://doi.org/10.2174/156720512802455403. PubMed: 22631612.
- 49. Peña-Ortega F, Bernal-Pedraza R (2012) Amyloid Beta Peptide slows down sensory-induced hippocampal oscillations. Int J Pept, 2012: 2012: 236289. PubMed: 22611415.
- 50. Verret L, Mann EO, Hang GB, Barth AM, Cobos I et al. (2012) Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell 149: 708-721. doi:https://doi.org/10.1016/j.cell.2012.02.046. PubMed: 22541439.
- 51. Peña-Ortega F (2013) Amyloid Beta-Protein and Neural Network Dysfunction. Neurodegener Dis: 2013: 657470.
- 52. Balleza-Tapia H, Huanosta-Gutiérrez A, Márquez-Ramos A, Arias N, Peña F (2010) Amyloid β oligomers decrease hippocampal spontaneous network activity in an age-dependent manner. Curr Alzheimer Res 7: 453-462. doi:10.2174/156720510791383859. PubMed: 20043810.
- 53. Näslund J, Haroutunian V, Mohs R, Davis KL, Davies P et al. (2000) Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA 283: 1571-1577. doi:https://doi.org/10.1001/jama.283.12.1571. PubMed: 10735393.
- 54. Lue LF, Kuo YM, Roher AE, Brachova L, Shen Y et al. (1999) Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer’s disease. Am J Pathol 155: 853-862. doi:https://doi.org/10.1016/S0002-9440(10)65184-X. PubMed: 10487842.
- 55. Peña F, Gutierrez-Lerma A, Quirzo-Baez R, Arias C (2006) The role of beta amyloid protein in synaptic function: implications for Alzheimer’s disease therapy. Curr Neurpharmacol 4: 149-163. doi:https://doi.org/10.2174/157015906776359531.
- 56. Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE et al. (2003) Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39: 409-421. doi:https://doi.org/10.1016/S0896-6273(03)00434-3. PubMed: 12895417.
- 57. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R et al. (1998) Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci USA 95: 6448-6453. doi:https://doi.org/10.1073/pnas.95.11.6448. PubMed: 9600986.
- 58. Paxinos G, Watson C (2007) The rat brain: in stereotaxic coordinates. (P. G and W. C, Eds.) (sixth edit., pp. 456) Elsevier/Academic Press, New York..
- 59. Edwards DA, Walter B, Liang P (1996) Hypothalamic and olfactory control of sexual behavior and partner preference in male rats. Physiol Behav 60: 1347-1354. PubMed: 8916193.
- 60. Portillo W, Paredes RG (2004) Sexual incentive motivation, olfactory preference, and activation of the vomeronasal projection pathway by sexually relevant cues in non-copulating and naive male rats. Horm Behav 46: 330-340. doi:https://doi.org/10.1016/j.yhbeh.2004.03.001. PubMed: 15325233.
- 61. Mucignat-Caretta C, Bondí M, Rubini A, Calabrese F, Barbato A (2009) The olfactory system is affected by steroid aerosol treatment in mice. Am J Physiol Lung Cell Mol Physiol 297: L1073-L1081. doi:https://doi.org/10.1152/ajplung.00014.2009. PubMed: 19801453.
- 62. Badonnel K, Lacroix MC, Monnerie R, Durieux D, Caillol M et al. (2012) Chronic restricted access to food leading to undernutrition affects rat neuroendocrine status and olfactory-driven behaviors. Horm Behav 62: 120-127. doi:https://doi.org/10.1016/j.yhbeh.2012.05.010. PubMed: 22633909.
- 63. Baunez C, Amalric M, Robbins TW (2002) Enhanced Food-Related Motivation after Bilateral Lesions of the Subthalamic Nucleus. J Neurosci 22: 562-568. PubMed: 11784803.
- 64. Peña F, Tapia R (2000) Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate- and GABA-mediated neurotransmission and of ion channels. Neuroscience 101: 547-561. doi:https://doi.org/10.1016/S0306-4522(00)00400-0. PubMed: 11113304.
- 65. Peña F, Tapia R (1999) Relationships among seizures, extracellular amino acid changes, and neurodegeneration induced by 4-aminopyridine in rat hippocampus: a microdialysis and electroencephalographic study. J Neurochem 72: 2006-2014. PubMed: 10217278.
- 66. Larriva-Sahd J (2012) Cytological organization of the alpha component of the anterior olfactory nucleus and olfactory limbus. Front Neuroanat 6: 23. PubMed: 22754506.
- 67. Bacskai BJ, Hickey GA, Skoch J, Kajdasz ST, Wang Y et al. (2003) Hyman Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-β ligand in transgenic mice. Proc Natl Acad Sci USA 14; 100: 12462–12467.
- 68. Luna-Muñoz J, Peralta-Ramirez J, Chávez-Macías L, Harrington CR, Wischik CM et al. (2008) Thiazin red as a neuropathological tool for the rapid diagnosis of Alzheimer’s disease in tissue imprints. Acta Neuropathol 116: 507-515. doi:https://doi.org/10.1007/s00401-008-0431-x. PubMed: 18810470.
- 69. Condello C, Schain A, Grutzendler J (2011) Multicolor time-stamp reveals the dynamics and toxicity of amyloid deposition. Sci Rep 1: 19. PubMed: 22355538.
- 70. Gutiérrez-Lerma AI, Ordaz B, Peña-Ortega F (2013) Amyloid Beta Peptides Differentially Affect Hippocampal Theta Rhythms In Vitro. Int J Pept, 2013: 2013; 328140. PubMed: 23878547.
- 71. Ramírez-Jarquín JO, Lara-Hernández S, López-Guerrero JJ, Aguileta MA, Rivera-Angulo AJ et al. (2012) Somatostatin modulates generation of inspiratory rhythms and determines asphyxia survival. Peptides 34: 360-372. doi:https://doi.org/10.1016/j.peptides.2012.02.011. PubMed: 22386651.
- 72. Adaya-Villanueva A, Ordaz B, Balleza-Tapia H, Márquez-Ramos A, Peña-Ortega F (2010) Beta-like hippocampal network activity is differentially affected by amyloid beta peptides. Peptides 31: 1761-1766. doi:https://doi.org/10.1016/j.peptides.2010.06.003. PubMed: 20558221.
- 73. Lagier S, Carleton A, Lledo PM (2004) Interplay between local GABAergic interneurons and relay neurons generates gamma oscillations in the rat olfactory bulb. J Neurosci 24: 4382-4392. doi:https://doi.org/10.1523/JNEUROSCI.5570-03.2004. PubMed: 15128852.
- 74. Lagier S, Panzanelli P, Russo RE, Nissant A, Bathellier B et al. (2007) GABAergic inhibition at dendrodendritic synapses tunes gamma oscillations in the olfactory bulb. Proc Natl Acad Sci USA 104: 7259-7264. doi:https://doi.org/10.1073/pnas.0701846104. PubMed: 17428916.
- 75. Sassoè-Pognetto M, Panzanelli P, Lagier S, Fritschy JM, Lledo PM (2009) GABA receptor heterogeneity modulates dendrodendritic inhibition. Ann N Y Acad Sci 1170: 259-263. doi:https://doi.org/10.1111/j.1749-6632.2009.03882.x. PubMed: 19686144.
- 76. Igelström KM, Shirley CH, Heyward PM (2011) Low-magnesium medium induces epileptiform activity in mouse olfactory bulb slices. J Neurophysiol 106: 2593-2605. doi:https://doi.org/10.1152/jn.00601.2011. PubMed: 21832029.
- 77. Chapman CA, Xu Y, Haykin S, Racine RJ (1998) Beta-frequency (15–35 Hz) electroencephalogram activities elicited by toluene and electrical stimulation in the behaving rat. Neuroscience 86: 1307–1319. doi:https://doi.org/10.1016/S0306-4522(98)00092-X. PubMed: 9697135.
- 78. Li A, Gong L, Xu F (2011) Brain-state-independent neural representation of peripheral stimulation in rat olfactory bulb. Proc Natl Acad Sci USA 108: 5087-5092. doi:https://doi.org/10.1073/pnas.1013814108. PubMed: 21321196.
- 79. Selkoe DJ (1993) Physiological production of the beta-amyloid protein and the mechanism of Alzheimer’s disease. Trends Neurosci 16: 403-409. doi:https://doi.org/10.1016/0166-2236(93)90008-A. PubMed: 7504355.
- 80. Hughes JR, Shanmugham S, Wetzel LC, Bellur S, Hughes CA (1989) The relationship between EEG changes and cognitive functions in dementia: a study in a VA population. Clin Electroencephalogr 20: 77-85. PubMed: 2706793.
- 81. Schreiter-Gasser U, Gasser T, Ziegler P (1994) Quantitative EEG analysis in early onset Alzheimer’s disease: correlations with severity, clinical characteristics, visual EEG and CCT. Electroencephalogr Clin Neurophysiol 90: 267-272. doi:https://doi.org/10.1016/0013-4694(94)90144-9. PubMed: 7512907.
- 82. Busciglio J, Andersen JK, Schipper HM, Gilad GM, McCarty R et al. (1998) Stress, aging, and neurodegenerative disorders. Molecular mechanisms. Ann N Y Acad Sci 851: 429-443. doi:https://doi.org/10.1111/j.1749-6632.1998.tb09021.x. PubMed: 9668637.
- 83. Rosenzweig ES, Barnes CA (2003) Impact of aging on hippocampal function: plasticity, network dynamics, and cognition. Prog Neurobiol 69: 143-179. doi:https://doi.org/10.1016/S0301-0082(02)00126-0. PubMed: 12758108.
- 84. Araki T, Kato H, Shuto K, Itoyama Y (1997) Age-related changes in [3H]nimodipine and [3H]rolipram binding in the rat brain. J Pharm Pharmacol 49: 310-314. doi:https://doi.org/10.1111/j.2042-7158.1997.tb06802.x. PubMed: 9231352.
- 85. Barnes CA, Rao G, Orr G (2000) Age-related decrease in the Schaffer collateral-evoked EPSP in awake, freely behaving rats. Neural Plast 7: 167-178. doi:https://doi.org/10.1155/NP.2000.167. PubMed: 11147459.
- 86. Thibault O, Landfield PW (1996) Increase in single L-type calcium channels in hippocampal neurons during aging. Science 272: 1017-1020. doi:https://doi.org/10.1126/science.272.5264.1017. PubMed: 8638124.
- 87. Mostany R, Anstey JE, Crump KL, Maco B, Knott G et al. (2013) Altered Synaptic Dynamics during Normal Brain Aging. J Neurosci 33: 4094-4104. doi:https://doi.org/10.1523/JNEUROSCI.4825-12.2013. PubMed: 23447617.
- 88. Vreugdenhil M, Toescu EC (2005) Age-dependent reduction of gamma oscillations in the mouse hippocampus in vitro. Neuroscience 132: 1151-1157. doi:https://doi.org/10.1016/j.neuroscience.2005.01.025. PubMed: 15857717.
- 89. Ramirez JM, Tryba AK, Peña F (2004) Pacemaker neurons and neuronal networks: an integrative view. Curr Opin Neurobiol 14: 665-674. doi:https://doi.org/10.1016/j.conb.2004.10.011. PubMed: 15582367.
- 90. Xing C, Yin Y, Chang R, He X, Xie Z (2005) A role of insulin-like growth factor 1 in beta amyloid-induced disinhibition of hippocampal neurons. Neurosci Lett: 12-19;384(1-2): 93-7.
- 91. Nimmrich V, Grimm C, Draguhn A, Barghorn S, Lehmann A et al. (2008) Amyloid beta oligomers (A beta(1-42) globulomer) suppress spontaneous synaptic activity by inhibition of P/Q-type calcium currents. J Neurosci 28: 788-797. doi:https://doi.org/10.1523/JNEUROSCI.4771-07.2008. PubMed: 18216187.
- 92. Didier A, Carleton A, Bjaalie JG, Vincent JD, Ottersen OP et al. (2001) A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb. Proc Natl Acad Sci USA 98: 6441-6446. doi:https://doi.org/10.1073/pnas.101126398. PubMed: 11353824.
- 93. Mori K, Takagi SF (1978) An intracellular study of dendrodendritic inhibitory synapses on mitral cells in the rabbit olfactory bulb. J Physiol 279: 569–588. PubMed: 671363.
- 94. Gray CM, Skinner JE (1988) Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade. Exp Brain Res 69: 378–386. PubMed: 3345814.
- 95. Eeckman FH, Freeman WJ (1990) Correlations between unit firing and EEG in the rat olfactory system. Brain Res 528: 238-244. doi:https://doi.org/10.1016/0006-8993(90)91663-2. PubMed: 2271924.
- 96. Fourcaud-Trocmé N, Courtiol E, Buonviso N, Voegtlin T (2011) Stability of fast oscillations in the mammalian olfactory bulb: experiments and modeling. J Physiol 105: 59-70. PubMed: 21843638.
- 97. Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y et al. (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274: 99-102. doi:https://doi.org/10.1126/science.274.5284.99. PubMed: 8810256.
- 98. Freeman WJ (1978) Spatial properties of an EEG event in the olfactory bulb and cortex. Electroencephalogr Clin Neurophysiol 44: 586–605. doi:https://doi.org/10.1016/0013-4694(78)90126-8. PubMed: 77765.
- 99. Isaacson JS, Strowbridge BW (1998) Olfactory reciprocal synapses: dendritic signaling in the CNS. Neuron 20: 749-761. doi:https://doi.org/10.1016/S0896-6273(00)81013-2. PubMed: 9581766.
- 100. Rall W, Shepherd GM, Reese TS, Brightman MW (1966) Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. Exp Neurol 14: 44-56. doi:https://doi.org/10.1016/0014-4886(66)90023-9. PubMed: 5900523.
- 101. Cleary J, Hittner JM, Semotuk M, Mantyh P, O’Hare E (1995) Beta-amyloid(1-40) effects on behavior and memory. Brain Res 682: 69-74. doi:https://doi.org/10.1016/0006-8993(95)00323-I. PubMed: 7552329.
- 102. Giovannelli L, Casamenti F, Scali C, Bartolini L, Pepeu G (1995) Differential effects of amyloid peptides beta-(1-40) and beta-(25-35) injections into the rat nucleus basalis. Neuroscience 66: 781-792. doi:https://doi.org/10.1016/0306-4522(94)00610-H. PubMed: 7651609.
- 103. O’Hare E, Weldon DT, Mantyh PW, Ghilardi JR, Finke MP et al. (1999) Delayed behavioral effects following intrahippocampal injection of aggregated A beta (1-42). Brain Res 815: 1-10. doi:https://doi.org/10.1016/S0006-8993(98)01002-6. PubMed: 9974116.
- 104. Nakamura S, Murayama N, Noshita T, Annoura H, Ohno T (2001) Progressive brain dysfunction following intracerebroventricular infusion of beta(1-42)-amyloid peptide. Brain Res 912: 128-136. doi:https://doi.org/10.1016/S0006-8993(01)02704-4. PubMed: 11532428.
- 105. Stéphan A, Laroche S, Davis S (2001) Generation of aggregated beta-amyloid in the rat hippocampus impairs synaptic transmission and plasticity and causes memory deficits. J Neurosci 21: 5703-5714. PubMed: 11466442.
- 106. Richardson RL, Kim EM, Shephard RA, Gardiner T, Cleary J et al. (2002) Behavioural and histopathological analyses of ibuprofen treatment on the effect of aggregated AB(1-42) injections in the rat. Brain Res 954: 1-10. doi:https://doi.org/10.1016/S0006-8993(02)03006-8. PubMed: 12393227.
- 107. Yamada K, Takayanagi M, Kamei H, Nagai T, Dohniwa M et al. (2005) Effects of memantine and donepezil on amyloid beta-induced memory impairment in a delayed-matching to position task in rats. Behav Brain Res 162: 191-199. doi:https://doi.org/10.1016/j.bbr.2005.02.036. PubMed: 15904984.
- 108. Malm T, Ort M, Tähtivaara L, Jukarainen N, Goldsteins G et al. (2006) Beta-Amyloid infusion results in delayed and age-dependent learning deficits without role of inflammation or beta-amyloid deposits. Proc Natl Acad Sci USA 103: 8852-8857. doi:https://doi.org/10.1073/pnas.0602896103. PubMed: 16723396.
- 109. Klementiev B, Novikova T, Novitskaya V, Walmod PS, Dmytriyeva O et al. (2007) A neural cell adhesion molecule-derived peptide reduces neuropathological signs and cognitive impairment induced by Abeta25-35. Neuroscience 145: 209-224. doi:https://doi.org/10.1016/j.neuroscience.2006.11.060. PubMed: 17223274.
- 110. Frautschy SA, Yang F, Calderón L, Cole GM (1996) Rodent models of Alzheimer’s disease: Rat A beta infusion approaches to amyloid deposits. Neurobiol Aging 17: 311-321. doi:https://doi.org/10.1016/0197-4580(95)02073-X. PubMed: 8744413.
- 111. Kane MD, Lipinski WJ, Callahan MJ, Bian F, Durham RA et al. (2000) Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice. J Neurosci 20: 3606-3611. PubMed: 10804202.
- 112. Langer F, Eisele YS, Fritschi SK, Staufenbiel M, Walker LC et al. (2011) Soluble Aβ seeds are potent inducers of cerebral β-amyloid deposition. J Neurosci 31: 14488-14495. doi:https://doi.org/10.1523/JNEUROSCI.3088-11.2011. PubMed: 21994365.
- 113. Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer C et al. (2006) Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313: 1781-1784. doi:https://doi.org/10.1126/science.1131864. PubMed: 16990547.
- 114. Eisele YS, Bolmont T, Heikenwalder M, Langer F, Jacobson LH et al. (2009) Induction of cerebral beta-amyloidosis: intracerebral versus systemic Abeta inoculation. Proc Natl Acad Sci USA 106: 12926-12931. doi:https://doi.org/10.1073/pnas.0903200106. PubMed: 19622727.
- 115. Watts JC, Giles K, Grillo SK, Lemus A, DeArmond SJ et al. (2011) Bioluminescence imaging of Abeta deposition in bigenic mouse models of Alzheimer’s disease. Proc Natl Acad Sci USA 108: 2528-2533. doi:https://doi.org/10.1073/pnas.1019034108. PubMed: 21262831.
- 116. Nobakht M, Hoseini SM, Mortazavi P, Sohrabi I, Esmailzade B et al. (2011) Neuropathological changes in brain cortex and hippocampus in a rat model of Alzheimer’s disease. Iran Biomed J 15: 51-58. PubMed: 21725500.
- 117. Morales R, Duran-Aniotz C, Castilla J, Estrada LD, Soto C (2012) De novo induction of amyloid-β deposition in vivo. Mol Psychiatry 17: 1347-1353. doi:https://doi.org/10.1038/mp.2011.120. PubMed: 21968933.
- 118. Zussy C, Brureau A, Delair B, Marchal S, Keller E et al. (2011) Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid β fragment in rats. Am J Pathol 179: 315-334. doi:https://doi.org/10.1016/j.ajpath.2011.03.021. PubMed: 21703413.
- 119. Zussy C, Brureau A, Keller E, Marchal S, Blayo C et al. (2013) Alzheimer’s disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats. PLOS ONE 8: e53117. doi:https://doi.org/10.1371/journal.pone.0053117. PubMed: 23301030.
- 120. Sigurdsson EM, Lorens SA, Hejna MJ, Dong XW, Lee JM (1996) Local and distant histopathological effects of unilateral amyloid-beta 25-35 injections into the amygdala of young F344 rats. Neurobiol Aging 17: 893-901. doi:https://doi.org/10.1016/S0197-4580(96)00169-8. PubMed: 9363801.
- 121. Harris JA, Devidze N, Verret L, Ho K, Halabisky B et al. (2010) Transsynaptic progression of amyloid-β-induced neuronal dysfunction within the entorhinal-hippocampal network. Neuron 68: 428-441. doi:https://doi.org/10.1016/j.neuron.2010.10.020. PubMed: 21040845.