Alterations in Piriform and Bulbar Activity/Excitability/Coupling Upon Amyloid-β Administration in vivo Related to Olfactory Dysfunction

Abstract

Background:

Deficits in odor detection and discrimination are premature symptoms of Alzheimer’s disease (AD) that correlate with pathological signs in the olfactory bulb (OB) and piriform cortex (PCx). Similar olfactory dysfunction has been characterized in AD transgenic mice that overproduce amyloid-β peptide (Aβ), which can be prevented by reducing Aβ levels by immunological and pharmacological means, suggesting that olfactory dysfunction depends on Aβ accumulation and Aβ-driven alterations in the OB and/or PCx, as well as on their activation. However, this possibility needs further exploration.

Objective:

To characterize the effects of Aβ on OB and PCx excitability/coupling and on olfaction.

Methods:

Aβ oligomerized solution (containing oligomers, monomers, and protofibrils) or its vehicle were intracerebroventricularlly injected two weeks before OB and PCx excitability and synchrony were evaluated through field recordings in vivo and in brain slices. Synaptic transmission from the OB to the PCx was also evaluated in slices. Olfaction was assessed through the habituation/dishabituation test.

Results:

Aβ did not affect lateral olfactory tract transmission into the PCx but reduced odor habituation and cross-habituation. This olfactory dysfunction was related to a reduction of PCx and OB network activity power in vivo. Moreover, the coherence between PCx-OB activities was also reduced by Aβ. Finally, Aβ treatment exacerbated the 4-aminopyridine-induced excitation in the PCx in slices.

Conclusion:

Our results show that Aβ-induced olfactory dysfunction involves a complex set of pathological changes at different levels of the olfactory pathway including alterations in PCx excitability and its coupling with the OB. These pathological changes might contribute to hyposmia in AD.

Keywords

Alzheimer’s disease coherence hyperexcitability local field potential main olfactory bulb olfactory function piriform cortex

INTRODUCTION

Olfactory processing relies on a recurrent network pathway that relays first in the olfactory bulb (OB) and then in the piriform cortex (PCx) [1 –3]. Disruption of either of these circuits impairs odor perception [1 –3], which is an early sign of Alzheimer’s disease (AD) [4, 5]. Olfactory dysfunction in AD precedes cognitive impairments [5 –7] and includes deficits in odor detection and discrimination, possibly due to alterations in different olfactory circuits [7 –11]. Similar olfactory dysfunctions observed in AD transgenic mice that overproduce amyloid-β peptide (Aβ) [12 –15] correlate with neuroanatomical changes in the OB and PCx [16 –20].

Histopathological markers of AD are observed throughout the olfactory pathway [21 –23]. Neurofibrillary tangles [21, 24] and Aβ deposits observed in the OB and PCx [23, 24] correlate with clinical symptoms in AD patients [22]. A similar scenario, but with some variability, can be found in AD transgenic mice that overproduce Aβ [19 , 25–28]. For instance, Aβ deposition in the OB and PCx has been observed in Tg2576 transgenic mice [28, 29] and APP/PS1 double transgenic mice [16, 30]. Alternatively, triple transgenic mice (3xTg-AD) exhibit Aβ accumulation in the PCx but not in the OB [31], while PLB4 transgenic mice exhibit Aβ oligomer accumulation in the PCx, with no signs of plaque deposition [32]. Despite the differences in Aβ presence, these AD transgenic mice exhibit olfactory dysfunctions [26 , 28–32].

Olfactory dysfunction in AD transgenic mice that overproduce Aβ can be prevented by reducing Aβ levels or neutralizing Aβ through pharmacological means [28, 33] or immunization [34, 35], respectively, suggesting that olfactory dysfunction in AD transgenic mice depends on Aβ accumulation [28 , 34–36]. To further support the notion that Aβ is responsible for olfactory dysfunction in AD, we have shown that direct intrabulbar microinjection of Aβ oligomers [37 –39] induces a reduction in odor detection [37, 39], a function that mostly relies on the OB [40 –42]. Interestingly, Aβ oligomers also reduce odor habituation and discrimination [38, 39], which rely on both PCx activity and PCx–OB recurrent interactions [40 –42].

Odor habituation and discrimination are affected in AD patients [43] and AD transgenic mice that overproduce Aβ [29 , 33–36], which correlates with 1) Aβ accumulation in the PCx [29 , 2]) functional imaging alterations in the PCx [43, 44 and 3]) hyper- and hypoexcitability in the PCx of AD transgenic mice that overproduce Aβ [28]. Thus, it is likely that Aβ could directly affect PCx excitability and/or its interaction with the OB. To test this possibility, we evaluated the effects of Aβ on PCx activity (simultaneously recorded with the OB), PCx–OB interactions and on their excitability. We found that Aβ intracerebroventricular administration reduces odor habituation and discrimination while reducing PCx and OB network activity and their coherence. We also found that Aβ exacerbates the PCx response to the pro-convulsant 4-aminopyridine (4-AP) [45 –47].

MATERIALS AND METHODS

Ethics statement

The experimental procedures were approved by the Bioethics Committee of the Institute of Neurobiology at UNAM and were performed in accordance with the guidelines of the Official Mexican Norm for the Use and Care of Laboratory Animals (Norma Oficial Mexicana NOM-062-ZOO-1999) and the Institutional Animal Care and Use Committee Guidebook (NIH publication 80–23, Bethesda, MD, USA, 1996).

Subjects

Adult CD1 mice (8 weeks old; 30–35 g) were obtained from the breeding colony of the animal facility at the Institute of Neurobiology, UNAM. The mice were housed in groups of 3-4 animals in transparent acrylic cages and maintained in a vivarium with controlled temperature (22±1°C), as well as with food and water ad libitum. Animals were kept under a normal 12 h light-12 h dark cycle.

Aβ preparation

Aβ_1–42 was obtained from American Peptide (Sunnyvale, CA). Oligomerization was performed as previously described [39, 48]. Briefly, solid Aβ_1–42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a final peptide concentration of 1 mM and incubated for 60 min at room temperature. HFIP was evaporated overnight, and DMSO was added to prepare a 5 mM solution. Such solution was then diluted with F12 medium to reach a final concentration of 100μM. This solution was incubated at 5°C for 24 h and then centrifuged at 14,000 rpm at 4°C for 10 min. The supernatant was collected and maintained at 4°C until used for experiments. The composition of the oligomerized Aβ_1–42 solution used for this study was corroborated with standard electrophoresis followed by silver staining, which exhibits the presence of Aβ oligomers, monomers, and protofibrils (Fig. 1C); inset [39, 48]. For this study, we administered Aβ into the cerebrospinal fluid (CSF) at a dose of 500 pmoles [49]. We chose this dose based on three previous experimental observations. First, we showed that 200 pmoles of Aβ induces olfactory dysfunction when applied directly into the OB [37, 39]. Secondly, we already know that, when applied into the CSF, increasing Aβ dose by 2.5-fold produces similar effects than those induced by its local application [50, 51]. Moreover, intraventricular injection of Aβ 500 pmoles is enough to induce olfactory dysfunction in mice, by reaching different circuits, while a lower dose did not induce alterations [49].

Fig. 1

Synaptic transmission from the OB to the PCx ex vivo is not affected by intracerebral Aβ injection. A) Representative fEPSPs recorded in the PCx layer II evoked by LOT stimulation (in OB-PCx slices) under basal conditions and in the presence of synaptic blockers as follows: bicuculline (Bic) 50μM, APV 100μM, CNQX 6μM, CdCl₂ 200μM, and lidocaine 1 mM (Lido). B) Quantification of the remnant of the normalized fEPSP amplitude (basal set as 100%) after the sequential application of synaptic blockers in slices obtained from control animals (injected with F12 medium; CTL; black) and from Aβ-injected animals (gray). *p < 0.05 denotes a significant difference versus basal. C) Stimulus-response curves of the EPSPs recorded in slices obtained from CTL and Aβ-injected animals (same color code). Inset: Silver-stained gel of the electrophoretic pattern of the Aβ oligomerized solution (right lane; monomers, dimers, trimers/tetramers, and heptamers/octamers are the main oligomeric forms) along with a molecular-weight (MW) size marker (left lane). D) Paired pulse ratio (P2/P1) as a function of the interstimulus interval (ISI) in slices obtained from CTL and Aβ-injected animals (same color code). No differences were observed between the groups in any of the stimulation intensities or interstimulus intervals used.

Aβ administration, electrode implantation, and recording in vivo

Animals were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (4 mg/kg) (i.p.). Mice were also administered (s.c) saline solution (0.9%) (15 ml/Kg). After verifying anesthesia depth (absence of tail pinch and pedal reflexes), animals were affixed to a stereotaxic frame (Stoelting Co. IL). Aβ (500 pmoles) or its vehicle (F12 medium) was intracerebroventricularly microinjected at the following coordinates: AP = –0.2, L =±1, V = 2.5 [52]. A stainless-steel needle connected to a Hamilton microsyringe (10μl) (Hamilton Company) by plastic tubing was used for the microinjection. An infusion volume of 5μl at 0.5μl/min was controlled by a microinfusion pump (WPI 220i). After the infusion, the needle remained inside the ventricle for at least 5 min to ensure proper diffusion of the injected content. A subgroup of injected animals was sutured, treated with an analgesic (meloxicam; 2 mg/kg; i.m.), and allowed to recover from the surgical procedure for 2 weeks before any behavioral or electrophysiological evaluation. After microinjection of either Aβ or its vehicle, a subgroup of injected animals was implanted with recording electrodes, as follows, to perform electrophysiological evaluations in vivo while awake. A bipolar electrode, made of two parallel strands of stainless steel (California Fine Wire), insulated except for their tip, was implanted into the PCx at the following coordinates: AP = 1.94, L =±2.1, V = 4.8 [52]. A second bipolar electrode, with identical characteristics, was implanted into the OB at the following coordinates: AP = 4.8, L =±0.7, V = 1.6 [52]. These electrodes were attached to a male connector pin for subsequent connection to the electrophysiological recording system. Two stainless steel screws were threaded into the cranium over the cerebellum (coordinates AP = –10.0, L =±3.0 and V = 1.0) to ground the signal and support the implanted electrodes. The arrangement of electrodes, screws and connector was fixed to the skull with dental acrylic (MDC Dental-NicTone R3V). Animals were treated with an analgesic (meloxicam; 2 mg/kg; i.m.) and allowed to recover from the surgical procedure for 2 weeks before any further experimental manipulation.

Electrophysiological recordings in vivo

Two weeks after Aβ or F12 medium was ad-ministered, the local field potential (LFP) was simultaneously measured in the OB and PCx while animals were freely moving. These animals were introduced into a new cage, with identical characteristics as their home cage but with clean sawdust, located inside a Faraday cage. LFPs were recorded for 30 min while animals remained at rest in this new cage. The signals were amplified, filtered (high-pass, 0.1 Hz; low-pass, 0.75 kHz; Grass Instruments) and digitized at 10 kHz (Digidata, Molecular Devices).

OB-PCx slice preparation

Two weeks after either Aβ or F12 medium was administered, the non-implanted animals were euthanized with an overdose of sodium pentobarbital (70 mg/kg, i.p.) and perfused transcardially with cold modified artificial cerebrospinal fluid (aCSF), containing 238 mM sucrose, 3 mM KCl, 2.5 mM MgCl₂, 25 mM NaHCO₃, and 30 mM D-glucose, pH 7.4 and bubbled with carbogen (95%O₂ and 5%CO₂). The brain was extracted and dissected in ice-cold normal aCSF containing 119 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 1 mM MgCl₂, 25 mM NaHCO₃, and 30 mM D-glucose, pH 7.4 and bubbled with carbogen. OB-PCx slices were obtained as previously described [53]. Briefly, one hemisphere was mounted on a plastic block, with the characteristics described by McGinley and Westbrook [53], to obtain a single parasagittal 400μm thick slice preserving the connectivity between the ventral anterior PCx and the OB through the lateral olfactory tract (LOT). The slices were allowed to recover in bubbled aCSF at room temperature for at least 60 min before any experimental manipulation.

Electrophysiological recordings in vitro

For LFP recordings, brain slices were transferred to a chamber that was continuously perfused with oxygenated aCSF maintained at 30–32°C. LFP was recorded with borosilicate electrodes (1 MΩ) filled with a CSF and positioned on anterior PCx layer II and the OB granular cell layer [37]. LFP was recorded for 20 min to obtain the basal network activity in both structures. Thereafter, 4-AP (100μM) [47, 54] was added to the bath and its effects were evaluated for 45 min. Finally, 1 mM lidocaine was applied to block neural activity and confirm the viability of each slice [55]. The signals were amplified, filtered (high-pass, 0.1 Hz; low-pass, 0.75 kHz; Grass Instruments) and digitized at 10 kHz (Digidata, Molecular Devices).

Synaptic transmission between the OB and PCx, and its short-term plasticity, was evaluated by recording the field excitatory postsynaptic potential (fEPSP) using the paired pulse protocol in vitro [55, 56], in slices obtained from Aβ and F12 medium microinjected animals. In the same OB-PCx slices, a concentric bipolar platinum electrode (75μm diameter at the tip) was placed on the LOT, near its origin in the OB, while evoked fEPSPs were recorded in PCx layer ll upon LOT voltage square pulse (100μs, 0.1 Hz) stimulation. Any given stimulation was repeated 5 times and the averaged fEPSP was included in each data set. The stimulus intensity was increased from 0.3 to 1.5 V (by 0.1 V steps) to evaluate the input/output (I/O) relationship in this synaptic connection [55, 56]. With the stimulus intensity adjusted to induce an fEPSP with 30–50%of the maximal amplitude, paired pulses were applied with an interstimulus interval (ISI) between 30 ms and 300 ms to evaluate the short-term plasticity of this synaptic connection [55, 56]. The paired pulse ratio (PPR) was calculated as follows: P2/P1, where P2 is the amplitude of the second fEPSP and P1 is the amplitude of the first one [55, 56]. After recording the fEPSP under basal conditions, at 30–50%of the maximal amplitude, several drugs were bath applied to evaluate the synaptic components of the fEPSP. To reveal a contribution of GABA_A-dependent transmission to the fEPSP, we bath applied its antagonist bicuculline (Bic 50μM; Sigma-Aldrich) [57, 58] and measured its effect for 10 min. Then, to evaluate the contribution of NMDA glutamate receptors to the fEPSP, we bath applied their antagonist APV (100μM; Sigma-Aldrich) [59, 60] and measured its effect for 10 min. To evaluate the contribution of non-NMDA glutamate receptors to the fEPSP, we bath applied their antagonist CNQX (6μM; Sigma-Aldrich) [61, 62] and measured its effect for 10 min. In an independent set of experiments, performed in OB-PCx slices obtained from naïve animals, the same set of fEPSPs and evaluations were recorded under control conditions as well as 60 min after the continuous presence of Aβ (30 nM) in the recording bath. At the end of all evaluations, CdCl₂ (200μM) and lidocaine (1 mM) were sequentially added to the bath to block synaptic transmission and neural activity, respectively [63]. The signals were amplified, filtered (high-pass, 0.1 Hz; low-pass, 0.75 kHz; Grass Instruments) and digitized at 10 kHz (Digidata, Molecular Devices).

Habituation/dishabituation test

Odor habituation and discrimination were evaluated through the habituation-dishabituation test, as described previously [38]. Briefly, mice were habituated to the experimental cage for 30 min. Next, a swab attached to a removable holder was presented at the side of the cage for 3 min. The tip of the swab was impregnated with either water (5μl) or an odor (acetic acid or vanilla; 5μl). This procedure was repeated several times with 1 min intervals. In the first two exposures the swab contained water. In the third trial the swab randomly contained either acetic acid (100%v/v; Herdez Company) or vanilla odor (100 %v/v; Herdez Company). The odor selected for the third trial was repeated in the fourth and fifth trials (always with 1 min intervals between trials), constituting an odor block. At the end of the first odor block, in the sixth trial, the other odor was presented. The odor selected for the sixth trial was repeated in the seventh and eighth trials (second odor block). To quantify the exploration time of each stimulus, exploration was considered when the mouse’s nose remained sniffing within 2 cm of swab. The habituation phase of the test evaluates the reduction in time that mice spend smelling on each repeated stimulus with the same odor, whereas the dishabituation phase of the test evaluates whether the mice were able to spontaneously recognize a novel odorant stimulus by spending more time smelling the swab that contains it [38].

Data analysis and statistics

Data distribution was assessed with the Shapiro-Wilk normality test. When the assumptions for parametric testing were not reached, no-parametric tests were used. Electrophysiological recordings were analyzed off-line. The amplitude of the fEPSP was measured from the end of the stimulus artifact to the fEPSP peak with Clampfit software (v.10.7, Molecular Devices). For the pharmacological experiments, the amplitude of the fEPSP recorded under basal conditions was set as 100%and the amplitude of the subsequent fEPSP in the presence of any drug was measured as %of basal. For statistical comparisons of these pharmacological experiments, an ANOVA test followed by Dunnett’s correction was performed. For the comparisons of the I/O curves or the PPR curves of both experimental groups (F12 medium versus Aβ) we used a two-way ANOVA test. To compare the effects of Aβ bath application on fEPSP amplitude or PPR, we used a t-test for independent groups.

For the spontaneous network activity, a power spectrum analysis was performed using the Rapid Fourier Transform Algorithm with a Hamming window using Clampfit. Ten segments (10 s long) were analyzed and averaged. The power of the broad-frequency band (1-55 Hz) of each individual experiment under basal conditions (i.e., activity before 4-AP application) was set as 100%and the activity in the presence of 4-AP 100μM was quantified as %of basal. Then, a Mann-Whitney U test was performed to compare the power before and after 4-AP application. Spontaneous population spikes induced by 4-AP were detected manually and their amplitude and frequency were measured with Clampfit. The power of population spikes was also normalized to basal condition (defined as the 100%) and analyzed using a Mann-Whitney U test. The in vivo LFP power spectra were segmented into the following frequency bands: delta (1–3 Hz), theta (3–12 Hz), beta (13–35 Hz), and gamma (36–55 Hz). Aside from raw power, relative power was calculated as the percentage contributed by each frequency to the total power in the broad-frequency band (1–55 Hz). These data were analyzed with the Mann-Whitney U test. The coherence between the activities of the OB and the PCx was measured using the function “mscohere” in MATLAB software (R2020a). Coherence was averaged in the following frequency bands: delta (1–3 Hz), theta (3–12 Hz), beta (13–35 Hz), and gamma (36–55 Hz). Coherence was analyzed with an ANOVA followed by Sidak’s multiple comparisons.

Behavioral parameters were analyzed using a two-way ANOVA followed by Fisher’s LSD post-hoc test to evaluate the habituation between the first odor stimulus and the last stimulus of each odor block and the cross-habituation comparing the third odor stimulus of a block with the first stimulus of the second block. The cross-habituation index was also calculated by subtracting the normalized exploratory time of the last stimulus of an odor block to the normalized investigatory time of the next odor block [33]. These cross-habituation values were compared using a Mann-Whitney U test. GraphPad Prism (version 8.0) software was used for graphs and statistical analysis. Data are presented as the mean±EEM. Differences were considered significant at p < 0.05.

RESULTS

Synaptic transmission between the OB and the PCx is not affected by injection of Aβ in vivo

Stimulation of the LOT in OB-PCx slices evoked a typical glutamatergic fEPSP in PCx layer II (Fig. 1A), which was mediated by non-NMDA receptors, but not by NMDA receptors, and with no contribution of GABAergic transmission (Fig. 1A, B). This fEPSP was blocked by bath application of the non-NMDA glutamate receptor CNQX 6μM (p < 0.05, n = 5; Fig. 1A, B), but was not altered by the previous sequential application of the GABA_A antagonist bicuculline 50μM (p = 0.9, n = 5; Fig. 1A, B) and the NMDA glutamate receptor antagonist APV 100μM (p = 0.9, n = 5; Fig. 1A, B). As expected, the fEPSP was blocked by the bath application of both CdCl₂ 200μM and lidocaine 1 mM (p < 0.05, n = 5; Fig. 1A, B). As mentioned, CNQX 6μM caused a significant decrease in the fEPSP amplitude of 79±7%in the control group (injected with F12 medium) and 84±7%in the group injected with Aβ (p < 0.05, n = 5; Fig. 1B). These reductions in fEPSP amplitude induced by CNQX were not different between groups (p > 0.05, n = 5; Fig. 1B). No significant differences were observed between groups in the presence of any of the other transmission blocker tested (p > 0.05, n = 5; Fig. 1B). The Aβ group was previously injected intracerebroventricularlly with an oligomerized solution containing oligomers, monomers and protofibrils (Fig. 1C); inset [39, 48].

In order to evaluate the consequences of Aβ exposure in vivo on synaptic transmission in the OB-PCx connection ex vivo (in slices), the I/O relationships were evaluated in OB-PCx slices obtained from control animals (injected with F12 medium) and Aβ-injected animals (Fig. 1C). We found no significant differences in the I/O relationships between groups (p = 0.9, n = 9; Fig. 1C). In order to evaluate the effects of Aβ exposure in vivo on short-term plasticity of the OB-PCx connection ex vivo, the PPR as a function of inter-stimulus interval (ISI) relationships were evaluated in slices obtained from control animals (injected with F12 medium) and Aβ-injected animals (Fig. 1D). We found no significant differences in the PPR/ISI relationships between groups (p = 0.8, n = 9; Fig. 1D).

Synaptic transmission between the OB and the PCx is not affected by acute application of Aβ in vitro

Considering that Aβ microinjection in vivo did not affect synaptic transmission between the OB and the PCx two weeks after its application, we evaluated the sensitivity of this connection to the bath application of Aβ in vitro. In OB-PCx slices obtained from naïve animals, the amplitude of the fEPSP recorded in the PCx layer-II exhibited an amplitude of 6.7±0.7μV (Fig. 2A, C) and a PPR of 1.5±0.07 (Fig. 2A, B) which remained unaltered 1 h after the continuous presence of Aβ 30 nM, since the fEPSP maintained an amplitude of 6.8±0.7μV (101±4 %of basal; p > 0.7, n = 7; Fig. 2A-D) and a PPR of 1.5±0.09 (p > 0.9, n = 7; Fig. 2A, B).

Fig. 2

Synaptic transmission from the OB to the PCx is not sensitive to Aβ application in vitro. A) Representative fEPSPs recorded in the PCx layer II evoked by a paired LOT stimulation (in OB-PCx slices) under basal conditions 30 min and 1 h after Aβ 30 nM bath application and after CdCl₂ 200μM application. B) Quantification of the paired pulse ratio of the paired fEPSPs recorded under basal conditions and 1 h after Aβ application. C) Quantification of the fEPSP amplitude of the first EPSP recorded under basal conditions and 1 h after Aβ application. D) Normalized fEPSP amplitude recorded under basal conditions (set as 100%) and 1 h after Aβ application. “ns” (p > 0.05) denotes the absence of a significant difference between conditions.

Aβ in vivo treatment exacerbates 4-AP-induced hyperexcitability only in the PCx ex vivo

To evaluate the effect of Aβ on PCx excitability we recorded its spontaneous LFP and induced hyperexcitation by bath application of 4-Aminopyridine (4-AP 100μM) [47, 54]. Bath application of 4-AP induces hyperexcitation in slices obtained from control animals (injected with F12 medium) and Aβ-injected animals (Fig. 3 A), leading to the generation of spontaneous population spikes in the PCx, more potently in slices obtained from Aβ-injected animals than in slices obtained from control animals (Fig. 3A). In the slices obtained from Aβ-injected animals, the 4-AP-induced population spikes exhibited a significantly higher amplitude (200.9±58.3 μV; n = 8; Fig. 3C), frequency (0.18±0.04 Hz; n = 8; Fig. 3D) and increase in power (7.8±4.0×10⁴ %of basal; n = 8; Fig. 3B) than the population spikes obtained from control animals (70.1±16.8μV, 0.07±0.02 Hz and 7.3±2.8×10³ %of basal, respectively, n = 9; p < 0.05; Fig. 3).

Fig. 3

Aβ treatment exacerbates 4-AP-induced hyperexcitability in the PCx. A) Representative traces of the PCx local field potential in the presence of 4-AP (100μM) in OB-PCx slices obtained from control animals (injected with F12 medium; CTL; upper trace) and Aβ-injected animals (Aβ; medium trace). The lower traces showed a representative population spike from each group. B) Normalized population spike power measured as %of basal activity (set as 100%) for the CTL (black) and Aβ (gray) groups. C) Mean amplitude of the population spikes for both groups (same color code). D) Quantification of the population spike frequencies for both groups (same color code). *p < 0.05 denotes a significant difference between experimental groups.

In contrast to what we observed in the PCx, 4-AP-induced hyperexcitation tended to be smaller in OB-PCx slices obtained from control animals (injected with F12 medium) than slices obtained from Aβ-injected animals (Fig. 4). In the OB, 4-AP-induced excitation lead to medium-voltage slow activity that increased the power to 3.2±2.4×10⁴ %of basal in OB-PCx slices obtained from control animals, which was higher but not significantly different (p = 0.1) than the increase in power (to 2.2±1.1×10³ %of basal) in OB-PCx slices obtained from Aβ-injected animals.

Fig. 4

Lack of effect of Aβ treatment on 4-AP-induced hyperexcitability in the OB. Representative traces of OB local field potential under basal conditions (upper traces) and in the presence of 4-AP 100μM (lower traces) in OB-PCx slices obtained from control animals (injected with F12 medium; left traces) and Aβ-injected animals (right traces). The quantification of normalized power, measured as %of basal activity (set as 100%), during 4-AP-induced hyperexcitability for both experimental groups is also shown. “ns” (p = 0.1) denotes the absence of a significant difference between experimental groups.

OB and PCx network activity and coherence are depressed by injection of Aβ in vivo

When we evaluated the effect of Aβ injection in vivo on the spontaneous LFP in freely moving animals, we observed that two weeks after Aβ administration the activity of both the OB and the PCx exhibited a reduction in amplitude (Fig. 5A). The quantification of power in the theta (3–12 Hz), beta (13–35 Hz), and gamma (36–55 Hz) frequency bands exhibited a significant reduction in Aβ-injected animals compared to control animals (injected with F12 medium) (p < 0.05, n = 15; Fig. 5B, D). However, no differences in the delta frequency band (<3 Hz) were observed between groups (p > 0.1, n = 15; Fig. 5B, D). Moreover, the relative power in all frequency bands did not show differences between groups (p > 0.2, n = 15; Fig. 5C, E).

Fig. 5

PCx and OB network activity is depressed by Aβ injection in vivo. A) Representative traces of spontaneous network activity in the OB (left traces) and PCx (right traces) obtained from control (F12-injected; CTL; upper traces) and Aβ-injected (lower traces) freely moving animals. B) Quantification of the power of OB activity in CTL (black) and Aβ (gray) animals in the following frequency bands: delta (1–3 Hz), theta (3–12 Hz), beta (13–35 Hz), and gamma (36–55 Hz). C) OB Relative power for the same frequency bands and experimental groups (same color code). D) Same as B but for the PCx. E) Same as C but for the PCx. ^*p < 0.05; ^**p < 0.01 denotes a significant difference between experimental groups. “ns” (p > 0.05) denotes the absence of a significant difference between experimental groups.

As a proxy for functional communication between the OB and the PCx, we evaluated the effect of Aβ administration in vivo on their synchrony in freely moving animals. For this purpose, we measured the coherence of both signals (Fig. 6) and found that the coherence of OB-PCx activities was reduced in Aβ-injected animals in the fast frequency components of the spectrum (Fig. 6A). The control group showed a coherence value of 0.30±0.01 for the beta frequency band (13–35 Hz), which was significantly higher than the coherence recorded in Aβ-injected animals (0.19±0.01; p = 0.02, n = 15; Fig. 6B). This was similar for the gamma frequency band (36–55 Hz), as the control group showed a coherence value of 0.29±0.03, which was significantly higher than the coherence recorded in Aβ-injected animals (0.18±0.01; p = 0.04, n = 15; Fig. 6B). In addition, the theta band showed a tendency toward a lower coherence value for the Aβ-injected group (0.25±0.01) compared to the control group (0.35±0.03; p = 0.052, n = 15; Fig. 6B).

Fig. 6

Aβ reduces OB-PCx functional coupling. A) Coherence between OB-PCx local field potentials (LFPs) as a function of frequency obtained from control (F12-injected; CTL; black) and Aβ-injected (gray) freely moving animals. B) Quantification of the coherence between OB-PCx LFPs in the following frequency bands: delta (1–3 Hz), theta (3–12 Hz), beta (13–35 Hz), and gamma (36–55 Hz) for both experimental groups. ^*p < 0.05 denotes a significant difference between experimental groups. “ns” (p > 0.05) denotes the absence of a significant difference between experimental groups.

Aβ disrupts odor habituation and discrimination

Since proper communication between the OB and the PCx is necessary for discrimination and olfactory learning [28 , 64], we evaluated whether Aβ alters the habituation/dishabituation performance. We also evaluated if the implantation of electrodes in both structures affected these functions (Fig. 7). Control animals (injected with F12 medium) exhibited reduced exploration time after repeated presentations of the same odor (p < 0.05, n = 10; Fig. 7A), which indicates odor habituation [38]. In contrast, animals injected with Aβ did not exhibit a significant reduction in the exploration time between the first and last odor presentation in each block (p > 0.1, n = 10; Fig. 7A), which indicates that these animals are not capable of olfactory habituation [38]. We assessed odor discrimination by evaluating odor cross-habituation (i.e., difference between the explorations of a habituated odor and a newly presented odor) through the cross-habituation index [33]. Thus, the increased investigation time (higher cross-habituation index) reflects discrimination between odors, whereas lower investigation times (small cross-habituation index) reflect generalization or a failure to discriminate [33]. We found that control animals (injected with F12 medium) significantly increased the exploration time of a newly presented odor (p < 0.05; n = 10; Fig. 7A) and exhibited a high cross-habituation index (0.93±0.02, Fig. 7B). In contrast, Aβ-injected animals did not show a change in the investigation time of the new odor (Fig. 7 A) and exhibited a small cross-habituation index (0.11±0.3, p = 0.001, n = 10; Fig. 7B), indicating that they are not capable of odor discrimination. These results suggest that the animals treated with Aβ present deficiencies in odor habituation and olfactory discrimination [33]. An identical scenario was observed in implanted animals (n = 11; Fig. 7C, D), demonstrating that the damage produced by electrodes introduced in the OB and PCx do not interfere with the described phenomena.

Fig. 7

Aβ disrupts odor habituation and discrimination. A) Mean investigation time for the sequential and repetitive presentation of water and two odors (acetic acid and vanilla) to control (F12-medium injected; CTL; black) and Aβ-injected (gray) animals. Odor habituation is reflected as a reduction in the investigation time after repeated presentation of the same odor. Cross habituation is reflected as an increase in the exploration time when a new odorant was presented. B) Cross-habituation index obtained by subtracting the normalized investigation time of the last trial of an odor block to the normalized investigation time of the first trial of the next odor block, for both experimental groups (same color code). C) Same as A, but for animals implanted with electrodes. D) Same as B, but for animals implanted with electrodes. ^*p < 0.05; ^*^*^*p < 0.001; ^*^*^*^*p < 0.0001 denotes a significant difference between the first and the last trial of an odor block. ^###p < 0.001; ^####p < 0.0001 denotes a significant difference between the last trial of one odor block and the first trial of the next odor block (indicated by horizontal lines). “ns” (p > 0.05) denotes the absence of significant difference between the indicated exploration times.

DISCUSSION

In the present study, we show that intracerebral administration of an Aβ oligomerized solution, containing oligomers, monomers and protofibrils (Fig. 1 C; inset) [39, 48] disrupts odor habituation and discrimination while reducing both OB and PCx network activity, as well as their coherence (Fig. 8). We also found that after Aβ treatment the PCx exhibited an exacerbated 4-AP-induced excitation, while the OB tended to be less excitable. Our results indicate that Aβ-induced olfactory dysfunction involves complex changes at different levels of the olfactory pathway, including modifications in PCx excitability and its coupling with the OB (Fig. 8). As we have done before [50, 63], F12 medium injection was used for control experiments. Despite that the injection of the reverse Aβ sequence would constitute a more accurate test for Aβ specificity [39, 55], vehicle application still allows to control for a variety of experimental manipulations used in studies like ours [49 , 65].

Fig. 8

Aβ disrupts OB and PCx activity and coupling, inducing olfactory dysfunction. Scheme of the complex changes in the olfactory network, including the olfactory bulb (OB) and piriform cortex (PCx), leading to olfactory dysfunction. By recording OB and PCx excitability and coherence, we found that olfactory dysfunction was related to a reduction of PCx and OB network activity and coupling, which could contribute to hyposmia in Alzheimer’s disease.

Our finding that intracerebral Aβ administration reduces odor habituation and discrimination coincides with similar observations in AD patients [43] and AD transgenic animals that overproduce Aβ [29 , 33–36], but goes a bit further to show that a two-week-long increase in Aβ is enough to induce this disruption in OB and PCX network activity (Fig. 8). A similar timeline was observed for the olfactory disruption induced by intrabulbar Aβ application [37, 38] or by intraventricular Aβ application [49], which correlates with signs of oxidative stress and neurodegeneration in the OB and the hippocampus [49]. Studies in AD transgenic animals, which are a closer proxy to the chronic and insidious nature of AD neuropathology and of long-term Aβ-induced effects [66 –68], have shown that olfactory dysfunction coincides with Aβ accumulation in the PCx [29 , 36] and that this disruption can be reversed by reducing Aβ levels [28 , 34–36]. Our data, and those just described, strongly suggest that Aβ accumulation is sufficient and necessary for the induction of olfactory dysfunction in AD animal models and perhaps in AD (Fig. 8). It is likely that these Aβ-induced olfactory deficits are the consequence of a variety of functional alterations, although cell-damage cannot be discarded [49], that are going to be discussed next. Before, is worth mentioning that Aβ injection in vivo or its bath application in vitro did not affect synaptic transmission from the OB to the PCx, which is similar to the lack of effect of Aβ in basal synaptic transmission in different circuits [63 , 69–71].

The observation that Aβ induces a reduction in the power of neural network activity in both the OB and the PCx (Fig. 8), coincides with previous findings that acute [37 , 73] and chronic [38 , 74] Aβ exposure reduces neural network activity, in vitro and in vivo, in a variety of circuits including the hippocampus [48 , 73], the entorhinal cortex [75] and the prefrontal cortex [76]. Moreover, we have previously shown that acute [37, 39] and chronic [38] Aβ presence reduces network activity in the OB. However, here we demonstrated for the first time that this phenomenon is also present in the PCx and might contribute to olfactory dysfunction (Fig. 8). It is likely that this reduction in PCx network activity involves changes in synaptic transmission [48 , 77–79] and in intrinsic neuronal properties [55 , 80–82] that need to be specifically characterized in the PCx.

As observed for Aβ in the present study, similar alterations of PCx activity induced by APOE4 expression also correlate with olfactory dysfunction [83]. Thus, the PCx is likely a neural circuit that is highly sensitive to pathological conditions [83], including Aβ accumulation [84]. In fact, Aβ application reduces antioxidant defenses in the PCx [84], which could make it prone to dysfunction and/or to oxidative stress and neurodegeneration [49]. Another mechanism that might contribute to Aβ-induced PCx dysfunction is neuroinflammation [32, 85], since the hypometabolic activity in the PCx observed in AD transgenic mice [44], and the associated olfactory dysfunction [32], have been closely related to gliosis. Neuroinflammatory conditions can be induced directly by Aβ and can contribute to the induction of neural network dysfunction [85]. Alternatively, Aβ-induced PCx depression could also involve the activation of pathological intracellular pathways leading to the recruitment of GSK3β [19 , 86]. In fact, GSK3β is accumulated in the PCx of AD transgenic mice that overproduce Aβ [19] and is activated upon Aβ presence [38 , 86]. Moreover, pharmacological and non-pharmacological GSK3β inhibition can prevent the Aβ-induced depression of neural network activity in a variety of neural circuits [38 , 86], including the OB [38]. It is likely that GSK3β contributes to AD pathology, including olfactory loss. All these mechanisms, and others, need to be evaluated to reveal the cellular and molecular mechanism underlying Aβ-induced olfactory dysfunction.

Olfactory dysfunction observed after Aβ microinjection also coincides with a reduction in PCx-OB coherence (Fig. 8), that is similar to the alterations in OB-PCx coherence [28, 33] and in OB-OB coherence in AD transgenic mice that overproduce Aβ [87]. Previous reports have shown that PCx-OB coherence is involved in olfactory processing [88] and that a disruption of either of these structures impairs odor perception [1 –3]. Since we found that Aβ microinjection did not affect the transmission from the OB through the PCx (via the LOT; Fig. 1), it is likely that the reduction in PCx-OB coherence is due to alterations in the centrifugal connection from the PCx to the OB. Since disruptions in centrifugal innervation to the OB affect olfactory discrimination [89] or odor-reward association [90], it remains to be determined whether Aβ affects centrifugal transmission into the OB. This possibility is highly likely since Aβ affects the long-range coupling between different brain circuits [76, 91], such as the hippocampal-prefrontal cortex coupling [76] or the cortico-cortical connections [91].

We also found that after Aβ treatment the PCx exhibited an exacerbated response to 4-AP, while the OB tended to be less hyperexcitable. This finding indicates that the PCx and the OB could be differentially sensitive to the presence of Aβ and even produce opposite effects in identical conditions, as described for synaptic transmission in the hippocampus and the prefrontal cortex [92, 93]. This differential sensitivity between circuits of the olfactory pathway has been previously described in AD transgenic animals that overproduce Aβ [27 , 94], where the PCx exhibits earlier and more abundant Aβ accumulation than the OB [27, 31]. Moreover, in 6–7-month-old Tg2576 mice, the increase in LFP power induced by odor exposure is exacerbated in the PCx and depressed in the OB [28], which is similar to our findings. Moreover, gamma activity is increased in the PCx of 6–7-month-old Tg2576 mice, whereas these oscillations are normal in the OB [28]. This differential sensitivity between neural circuits to the presence of Aβ is observed in the olfactory pathway and in other forebrain circuits (i.e., hippocampus and different cortical areas) [28 , 93]. The differential composition of neuronal types, their connectivity, and their sensitivity to Aβ could likely account for these differences in network excitability after Aβ presence [28, 93]. Moreover, differences in the interconnection with other circuits could also influence the differential response of some neural networks to the differences in excitability induced by Aβ [28, 76].

The fact that the PCx becomes more prone to hyperexcitability in Aβ-injected animals coincides with previous observations in transgenic AD animals that overproduce Aβ exhibiting signs of hyperexcitability [28, 95] and odor hyperresponsiveness [25], which have been associated with alterations in olfactory function [28, 95]. Moreover, the worsening of 4AP-induced excitability produced by Aβ injection is similar to the findings that some AD transgenic animals that overproduce Aβ exhibit an increased susceptibility to seizures [96, 97]. It is likely that the PCx hyperexcitability and hyperresponsiveness in the presence of Aβ might be due to the high susceptibility of the PCx to seizures [20] and alterations in intra-network inhibition caused by damage to different inhibitory interneurons (i.e., somatostatin- and calretinin-positive interneurons) within the PCx, already observed in AD transgenic animals that overproduce Aβ and in AD patients [16, 24].

As we have previously described in the hippocampus [51], the pro-epileptogenic effects of Aβ in the PCx are associated with a reduction in the power of spontaneous network activity and with a reduction in its coupling with the OB. It is possible that both phenomena have a common origin and are correlated. In fact, the reduction in oscillatory network activity has been associated with increased pyramidal cell activity [92 , 98–100] and with the inhibition of GABAergic interneurons [65 , 101–106], which are hallmarks of epileptic conditions [50, 107]. On the other hand, although it could be counterintuitive to associate a reduction in coupling with hyperexcitation, modeling and electrophysiological experiments have shown that weak coupling could lead neocortical networks to a state that favors the induction of epileptiform activity [108]. Thus, the Aβ-induced reduction in network activity and coupling could determine the pro-epileptic effects of Aβ [51 , 109] and, altogether, contribute to olfactory dysfunction when this complex phenomenon affects the OB-PCx network.

Footnotes

ACKNOWLEDGMENTS

Ignacio Martínez is a student from Programa de Maestría en Ciencias (Neurobiología) at Universidad Nacional Autónoma de México (UNAM). Rebeca Hernández and Benjamín Villasana are students from Programa de Doctorado en Ciencias Biomédicas at Universidad Nacional Autónoma de México (UNAM). All received fellowships from Consejo Nacional de Ciencia y Tecnología (CONACyT), México. The authors would like to thank Jessica González-Norris and Anaid Antaramian for the editorial comments.

This work was supported by Dirección General de Asuntos del Personal Académico (Grants IN202018 and IG20052) at UNAM and Conacyt (Grant A1-S-7540).

Author’s disclosures available online ().

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