Oligomeric amyloid-β peptide disrupts olfactory information output by impairment of local inhibitory circuits in rat olfactory bulb

Neurobiology of Aging 51 (2017) 113e121

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Oligomeric amyloid-b peptide disrupts olfactory information output by impairment of local inhibitory circuits in rat olfactory bulb Bin Hu a, b, c, Chi Geng b, c, Xiao-Yu Hou a, b, c, * a

Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Jiangsu, China Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Jiangsu, China c Research Center for Biochemistry and Molecular Biology, Xuzhou Medical University, Jiangsu, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2016 Received in revised form 22 November 2016 Accepted 5 December 2016 Available online 14 December 2016

Although early olfactory dysfunction has been found in patients with Alzheimer’s disease, the underlying mechanisms remain unclear. In this study, we investigated whether and how oligomeric amyloid-b peptide (Ab) affects the responses of mitral cells (MCs). We found that oligomeric Ab1e42 increased spontaneous and evoked firing rates but decreased the ratio of evoked to spontaneous firings in MCs. Ab1e42 oligomers showed no impact on the hyperactivity exerted by pharmacological blockage of GABAA receptors, suggesting an involvement of GABAergic inhibitory transmission in Ab1 to 42einduced over-excitability. It was further determined that Ab1e42 oligomers inhibited the frequency of spontaneous inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents, as well as the amplitude of miniature inhibitory postsynaptic currents in MCs. Both recurrent and lateral inhibition of MCs, which are critical for odor discrimination, were also disrupted by Ab1e42 oligomers. The above data indicate that Ab impairs local inhibitory circuits and thereby leads to perturbations of olfactory information output in the olfactory bulb. This study reveals a cellular and synaptic basis of olfactory deficits associated with Alzheimer’s disease. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Alzheimer’s disease Amyloid-b peptide Gamma-aminobutyric acid Inhibitory neuronal circuit Mitral cell Olfactory bulb

1. Introduction Alzheimer’s disease (AD), the most common neurodegenerative disorder in the elderly, is characterized clinically by progressive declines in memory and cognitive functions and by behavioral and neuropsychiatric symptoms. It is well known that the 3 pathological hallmarks of AD are neuronal loss, neurofibrillary tangles, and extracellular senile plagues composed of aggregated amyloid-b peptides (Ab). In recent decades, impairments in the sense of smell have been found to be a common early symptom in patients with AD (Mesholam et al., 1998; Roberts et al., 2016; Serby et al., 1991; Velayudhan et al., 2013). Severe defects in odor identification and discrimination develop in the early stages of AD, prior to the appearance of cognitive and neuropathology dysfunction and correlating with cognitive decline (Devanand et al., 2015; Djordjevic et al., 2008; Lehrner et al., 2009; Velayudhan et al., 2013). Understanding the progress of olfactory damages,

* Corresponding author at: Jiangsu Key Laboratory of Brain Disease Bioinformation, Research Center for Biochemistry and Molecular Biology, Xuzhou Medical University, 209 Tongshan Road, Jiangsu 221004, China. Tel.: 86-516-83262620; fax: 86-516-8583-4231. E-mail address: [email protected] (X.-Y. Hou). 0197-4580/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2016.12.005

particularly in the early stages, may provide potential therapeutic interventions for AD. A compelling hypothesis of the AD mechanism is that imbalances in Ab production and/or clearance cause Ab accumulation and aggregation in the brain. Ab is a short peptide derived from the sequential proteolytic cleavage of amyloid-b precursor protein by b- and g-secretases. The most common isoforms of Ab are Ab1e42 and Ab1e40. Postmortem studies have shown that Ab-related pathology extensively exists in the olfactory system of AD cases (Attems et al., 2014; Kovacs et al., 1996). Correspondingly, senile Ab plaques extending from the olfactory neurepithelium, olfactory bulb (OB), anterior olfactory nucleus, prepiriform cortex, entorhinal cortex, amygdala, and then to the hippocampus and other brain regions are found in transgenic AD mice, and correspond with deficits in odor detection, discrimination, and memory (Cassano et al., 2011; Roddick et al., 2016; Wesson et al., 2010, 2011; Wu et al., 2013). It has been found that the soluble oligomeric form of Ab, but not fibrillar Ab aggregates, is associated with the olfactory impairments of AD (Wesson et al., 2010; Wu et al., 2013; Xu et al., 2015). In addition, inhibitors of Ab generation or Ab immunotherapy rescue olfactory deficits (Cramer et al., 2012; Wesson et al., 2013). Overall, previous studies implicate a close linkage between the spatial and temporal patterns of Ab and olfactory impairments in AD. However, the local mechanisms related to Ab-associated

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Fig. 1. Oligomeric Ab1e42 increases MC spontaneous and evoked firings, but decreases signal-to-noise ratio in cell-attached configuration. (A) Oligomeric Ab1e42 (500 nM), but not Ab42e1, increases spontaneous firing rates of MCs. (B) Quantitative analysis of spontaneous firing rates in a period of 3 minutes. Paired t-test, n ¼ 8 cells, from 5 and 4 rats, respectively; **p < 0.01. (C) Oligomeric Ab1e42 (500 nM), but not Ab42e1, increases the olfactory nerve-evoked firing rates of MCs. (D) Quantitative analysis of olfactory nerve-evoked firings in a 1 second recording period. Paired t-test, n ¼ 7 cells, 5 and 4 rats, respectively; **p < 0.01. (E) Ab1e42 (500 nM), but not Ab42e1, decreases the ratio of olfactory nerve-evoked action potentials (eAPs) to spontaneous APs (sAPs). (F) Quantitative analysis of the ratio of eAPs in 1 second and sAPs in 5 seconds prior to olfactory nerve stimulation. Paired t-test, n ¼ 6 cells, 5 rats, respectively; *p < 0.05. Abbreviations: APs, action potentials; MC, mitral cell; n.s., not significant.

pathophysiology in involved olfactory regions remain largely unknown. In the OB, the first crucial relay station for olfactory perception, odorant signals are transformed into the informational output of the OB to the rest of the brain. This process is precisely tuned by local neuronal circuitry and then conveyed to the associated olfactory cortex and subcortical regions. As the principal output neurons of the OB microcircuits, mitral cells (MCs) receive odorant-evoked glutamatergic excitatory inputs from olfactory sensory neurons and a large number of GABAergic inhibitory inputs from surrounding interneurons. The spontaneous activity of MCs is mainly driven by local inhibitory granule cells (GCs) and periglomerular cells rather than excitatory olfactory sensory neurons (Duchamp-Viret and Duchamp, 1993; Stakic et al., 2011). Odor signal-activated MCs excite local interneurons that subsequently inhibit the activity of same MCs, this process is called recurrent inhibition of MCs. Recurrent inhibition is thought to increase the frequency of the odor-induced oscillations and odor discrimination (Abraham et al., 2010; Nunes and Kuner, 2015). Local interneurons also mediate lateral inhibition whereby MCs inhibit the activity of nearby MCs. Lateral inhibition between MCs functions as a means for contrast enhancement, by which the odorant-stimulated responses of MCs are sharpened or filtered and the sharpened output signals subsequently transmit to the deeper parts of the brain (Geramita et al., 2016; Yu et al., 2014). Indeed, local GABAergic inhibitory circuitry in the OB sculpts the network as a pattern separator thereby altering odor detection thresholds, discrimination, and learning (Gschwend et al., 2015; Mwilaria et al., 2008). Although Ab has been implicated in early olfactory dysfunction in AD, less is known about the impact of Ab on the OB microcircuits.

In the present study, we sought to discover how oligomeric Ab is detrimental to the processing of olfactory information. We used patch-clamp electrophysiological recordings in acute rat OB slices to examine the effects of Ab oligomers on excitatory outputs of MCs and inhibitory modulation of local interneurons onto MCs. We provided evidence that oligomeric Ab disrupts the spontaneous activity of MCs and is involved in the disturbance of MC excitatory outputs. Our findings also revealed that the disruption of local inhibitory processes is responsible for Ab-induced hyperactivity of MCs.

2. Methods 2.1. Reagents and animals Ab1e42 or reverse control peptide Ab42e1 (Sangon Biotech) was dissolved in sterile water at a concentration of 1 mM. The peptide solution was then incubated at 37
C for 7 days in order to form oligomers (Li et al., 2009). The Naþ channel blocker tetrodotoxin (TTX) was purchased from Tocris and GABAA receptor antagonist (þ)-bicuculline from Enzo. Other chemicals were from Sigma-Aldrich. All chemicals were diluted with artificial cerebrospinal fluid (ACSF; in mM: 124 NaCl, 3 KCl, 2 CaCl2 1.3 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, and 10 glucose) freshly before application. Sprague-Dawley rats were housed on a normal light cycle and had ad libitum access to food and water. All experimental procedures were conducted in accordance with the guidelines described in the revised Regulations for the Administration of Affairs Concerning Experimental Animals (2011) in China and approved by the Institutional Animal Care and Use Committee at Xuzhou Medical University.

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Fig. 2. Oligomeric Ab1e42 increases current injection-evoked APs of MCs in whole-cell patch-clamping mode. (A) Oligomeric Ab1e42 (500 nM) but not Ab42e1 increases the AP frequency of MCs elicited by positive current injection (400 ms, 50 pA). (B) Quantitative analysis of evoked firings. Paired t-test, n ¼ 7 cells, 5 and 6 rats, respectively; *p < 0.05. (C) The resting membrane potentials of MCs remain stable during recording. n ¼ 7. (D) Oligomeric Ab1e42 increases the firing rates elicited by different positive currents injection. (E) Quantitative analysis of evoked firings under different currents. Two-way repeated measures ANOVA, n ¼ 6 cells, 4 rats, F3, 15 ¼ 14.5; *p < 0.05 compared with control. (F) The resting membrane potentials remain unchanged during the recording, n ¼ 6. Abbreviations: APs, action potentials; MC, mitral cell; n.s., not significant.

2.2. Slice preparation In brief, rats (P14e20) were deeply anesthetized with chloral hydrate (300e350 mg/kg, intraperitoneal injection). OBs were quickly removed and immersed in ice-cold ACSF oxygenated with 95% O2/5% CO2. Horizontal slices (320 mm) were cut with a Leica VT1000s vibratome, recovered at 37
C for 30 minutes and then maintained at 30
C for electrophysiological recordings. 2.3. Electrophysiological recordings OB slices were transferred to the recording chamber and superfused with oxygenated ACSF (2 mL/min). MCs were visualized with infrared optics using an upright microscope equipped with a

60 water-immersion lens (BX51WI; Olympus) and infraredsensitive charge-coupled device camera. For cell-attached mode, recording pipettes were pulled from borosilicate glass capillaries (Sutter Instrument Co) with resistances of 6e8 MU and filled with ACSF. For recording of the olfactory nerve-evoked response, monophasic square pulse (200 ms, 100e200 mA) was delivered through a concentric electrode that was placed on the most outside layer of the OB, near the recorded MC. For whole-cell action potential recording, pipettes were filled with the solution containing in mM: 140 K-methylsulfate, 4 NaCl, 10 N-(2-hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid), 0.2 ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid, 4 MgATP, 0.3 Na3GTP, and 10 phosphocreatine (pH was adjusted to 7.4 with KOH).

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Fig. 3. Ab1e42-induced hyperactivity of MCs requires GABAA receptor activation. (A) Oligomeric Ab1e42 is unable to increase the elevated spontaneous firing rates of MCs in the presence of bicuculline (10 mM). (B) Quantitative analysis of spontaneous firing rates in a period of 3 minutes. One-way repeated measures ANOVA, n ¼ 8 cells, 5 rats, F2, 12 ¼ 9.4, * p < 0.05. (C) Oligomeric Ab1e42 is unable to change eAPs or signal-to-noise ratio in the presence of bicuculline. (D) Quantitative analysis of eAPs in 1 second. One-way repeated measures ANOVA, n ¼ 7 cells, 3 rats, respectively, F2, 12 ¼ 4.7, *p < 0.05. (E) Quantitative analysis of the ratio of eAPs and sAPs. One-way repeated measures ANOVA, n ¼ 7 cells, 3 rats respectively, F2, 12 ¼ 9.4, *p < 0.05. (F) Oligomeric Ab1e42 is unable to increase the AP frequency of MCs elicited by positive current injection (50 pA, 400 ms) in the presence of bicuculline. (G) Quantitative analysis of current injection-evoked firings. One-way repeated measures ANOVA, n ¼ 6 cells, 3 rats respectively, F2, 10 ¼ 15.8, **p < 0.01. Abbreviations: AP, action potential; eAPs, evoked action potentials; MC, mitral cell; n.s., not significant; sAPs, spontaneous action potentials.

Inhibitory postsynaptic currents (IPSCs) were recorded with a CsCl-base intracellular solution containing (mM): 135 CsCl, 10 N-(2-hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid), 0.2 ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid, 2 Na2ATP, 0.3 Na3GTP, and 10 glucose. To isolate the GABAergic currents, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/ N-methyl-D-aspartate receptors were blocked with 1,2,3,4-tetrahydro6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (20 mM) and DL-2-amino-5-phosphonovaleric acid (50 mM), respectively. TTX (1 mM) was also included during recording of miniature IPSCs (mIPSCs). The holding potential was 65 mV. Lateral inhibition recording was obtained according to procedures described previously (Whitesell et al., 2013). Briefly, a concentric bipolar stimulating electrode was placed into the glomerulus next to the neighboring one correlated with the recorded MC and a stimuli with moderate intensity (100e200 mA) was applied to avoid directly stimulating the recorded MC and linked glomerulus. Single or paired pulses of 200 ms were delivered at 0.05 Hz then synchronized using a Master-8 stimulator (A.M.P.I). Alexa 488 dye (100 mM, Sigma-Aldrich) was added to the intracellular solution allowing visualization of cell morphology. All signals were acquired with a MultiClamp 700B amplifier (Molecular Devices), filtered at 2 kHz, and sampled at 10 kHz with a Digidata 1440A interface (Molecular Devices) using Clampex 10.2 (Molecular Devices). Data were accepted when series resistance fluctuated within 15% of initial values (15e25 MU).

2.4. Data analysis The frequency of firing was defined as numbers/time window. A 3-minute window was used for spontaneous firing rate analysis, and the first 1-second window after the stimulation of olfactory nerve was counted as evoked responses in cell-attached mode. Analysis of the spontaneous and miniature events was performed using Mini Analysis software (Synaptosoft, ver. 6.07). Data are presented as mean  standard error of the mean. A paired t-test and repeated measures analysis of variance (ANOVA) were used for statistical comparison of differences, with p < 0.05 considered significant. 3. Results 3.1. Ab1e42 oligomers induce the over-excitability of MCs First, we evaluated whether oligomeric Ab1e42 disturbs MC spontaneous activity by patch-clamp recordings in cell-attached configuration. The results showed that the firing rates of MC spontaneous action potentials (sAPs) were dramatically enhanced within 10 minutes following perfusion with Ab1e42 oligomers (500 nM) (Fig. 1A and B, left, from 5.2  1.0 to 10.4  2.1 Hz, paired t-test, n ¼ 8, p ¼ 0.008). In contrast, the same dose of control reverse sequence peptide Ab42e1 presented no influence on spontaneous firings (Fig. 1A and B, right, from 4.8  0.3 to 4.8  0.6 Hz, paired t-test, n ¼ 8, p ¼ 0.974). To assess the dose effect of oligomeric

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Fig. 4. Ab1e42 impairs GABAergic transmission in olfactory bulb. (A) The frequencies but not amplitudes of sIPSCs are decreased during bath application of oligomeric Ab1e42 instead of Ab42e1. Quantitative analyses of sIPSCs frequencies (B) and amplitudes (C) in a 5-minute recording period. Paired t-test, n ¼ 7 cells, 5 and 4 rats, respectively; **p < 0.01. (D) Oligomeric Ab1e42 but not Ab42e1 decreases the frequencies and amplitudes of mIPSCs. Quantitative analyses of mIPSCs frequencies (E) and amplitudes (F) in a 5-minute recording period. Paired t-test, n ¼ 7 cells, 6 and 5 rats, respectively; *p < 0.05. Abbreviations: mIPSCs, miniature inhibitory postsynaptic currents; n.s., not significant; sIPSCs, spontaneous inhibitory postsynaptic currents.

Ab1e42, a dose response curve was generated (Fig. S1), which showed a statistically significant effect on sAP firings after treatment beginning at 200 nM. Ab1e42 oligomers elicited the strongest response at 500 nM Ab1e42. For further experiments, 500 nM oligomeric Ab1e42 and the same dose of Ab42e1 were used, unless otherwise specified. Next, we examined whether oligomeric Ab1e42 also affects MC spike responses elicited by stimulation of the olfactory nerve in cell-attached mode. Again, Ab1e42 was found to significantly

increase the firing rates of evoked action potentials (eAPs) (Fig. 1C and D, left, from 12.1  1.1 to 15.0  1.4 Hz, paired t-test, n ¼ 7, p < 0.001), while Ab42e1 did not cause marked alteration of evoked firings (Fig. 1C and D, right, from 12.0  1.3 to 11.9  1.3 Hz, paired ttest, n ¼ 7, p ¼ 0.881). Interestingly, the increment of spontaneous activity was larger than that of olfactory nerve-evoked firings, indicating a possible role of Ab in altering signal-to-noise ratio. Therefore, eAPs and sAPs, which are presumed to reflect the signal and noise respectively,

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Fig. 5. Oligomeric Ab1e42 reduces recurrent inhibition of MCs. (A) Responses of MCs elicited by 11 APs before (control) and during Ab1e42 bath application. (B) Peak amplitudes of IPSPs are reduced by Ab1e42. Paired t-test, n ¼ 9 cells, 6 rats; *p < 0.05. (C) Decay time constants of IPSPs are also reduced by Ab1e42. Paired t-test, n ¼ 9 cells, 6 rats; *p < 0.05. (D) The resting membrane potentials remain unchanged during the recording. n ¼ 9. Abbreviations: APs, action potentials; IPSPs, inhibitory postsynaptic potentials; MC, mitral cell; n.s., not significant.

were recorded in cell-attached mode simultaneously. As shown in Fig. 1E and F, treatment with Ab1e42, but not Ab42e1 (right, from 3.4  0.3 to 3.3  0.2, paired t-test, n ¼ 6, p ¼ 0.837), significantly reduced the ratio between eAP and sAP firings (left, from 3.0  0.2 to 2.1  0.1, paired t-test, n ¼ 6, p ¼ 0.011), suggesting that Ab1e42 causes a deterioration of OB processing. To further explore the roles of Ab1e42 oligomers in the modulation of MC responses, action potentials (APs) were elicited by a positive current injection (50 pA, 400 ms) in whole-cell patchclamping mode. As shown in Fig. 2A and B, the AP frequency of MCs was elevated after incubating with oligomeric Ab1e42 (left, from 24.6  3.3 to 30.0  3.5 Hz, paired t-test, n ¼ 7, p ¼ 0.011) instead of Ab42e1 (right, from 24.6  2.5 to 24.3  4.1 Hz, paired t-test, n ¼ 7, p ¼ 0.881). More importantly, Ab1e42 increased the activities of MCs elicited by different current injections ranging from 50 to 200 pA in whole-cell patch-clamping mode (Fig. 2D and E, 2-way repeated measures ANOVA, n ¼ 6, F3, 15 ¼ 14.5, p ¼ 0.013). The increased activities of current injection-elicited APs were not due to alterations of resting membrane potentials (Fig. 2C and F).

from 1.8  0.1 to 2.6  0.3, 1-way ANOVA, n ¼ 6, F2, 15 ¼ 11.5, p ¼ 0.006). The absence of a synergistic effect of Ab1e42 and bicuculline may be attributable to their being a similar molecular mechanism between them. To confirm this hypothesis, lower concentrations of bicuculline (2 mM) and Ab1e42 (200 nM) were used. It was found that Ab1e42 (200 nM) augmented the elevated firings exerted by bicuculline (2 mM) (Fig. S2B, normalized sAP frequency from 1.4  0.1 to 2.1  0.1, 1-way ANOVA, n ¼ 6, F2, 15 ¼ 11.9, p < 0.001). Furthermore, bicuculline increased the firings of eAPs and lessened the ratio of eAPs to sAPs in cell-attached mode (Fig. 3CeE, from 11.3  1.7 to 13.6  1.6 Hz, 1-way repeated measures ANOVA, n ¼ 7, F2, 12 ¼ 4.7, p ¼ 0.030; from 2.8  0.5 to 2.1  0.4, n ¼ 7, F2, 12 ¼ 9.4, p ¼ 0.010, respectively), and this did not seem to be affected by Ab1e42 (Fig. 3CeE, from 13.6  1.6 to 13.1  2.1 Hz, p ¼ 0.695; from 2.1  0.4 to 2.1  0.4, p ¼ 0.963, respectively). In addition, the frequency of APs elicited by positive current injection (50 pA, 400 ms) in whole-cell patch-clamping mode increased strikingly in the presence of bicuculline (Fig. 3F and G, 1-way repeated measures ANOVA, from 21.3  4.0 to 33.3  3.4 Hz, n ¼ 6, F2, 10 ¼ 15.8, p ¼ 0.004), while Ab1e42 exhibited no further effect (Fig. 3F and G, from 33.3  3.4 to 33.8  3.6 Hz, p ¼ 0.895). These data indicate that the dysfunction of GABAergic transmission accounts for the Ab1e42-caused hyperactivity of MCs and the reduction of signal-to-noise ratio. Next, AP-dependent spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in whole-cell patch-clamping mode. The application of oligomeric Ab1e42 instead of Ab42e1 decreased the frequency of sIPSCs (Fig. 4A and B, from 1.1  0.2 to 0.8  0.2 Hz, paired t-test, n ¼ 7, p ¼ 0.002), but showed no effect on the amplitude of sIPSC (Fig. 4A and C, from 37.3  4.3 to 36.2  3.0 pA, paired t-test, n ¼ 7, p ¼ 0.649). This result further proved that Ab1e42 impairs GABAergic transmission. To further investigate whether presynaptic and/or postsynaptic mechanisms are involved in Ab1e42 impairment of spontaneous GABAergic transmission, AP-independent mIPSCs were measured in the presence of TTX in whole-cell patch-clamping mode. As shown in Fig. 4DeF, the decline of mIPSC frequency was found to be associated with a small but significant reduction in amplitude after oligomeric Ab1e42 incubation (paired t-test, n ¼ 7, from 0.5  0.1 to 0.3  0.1 Hz, p ¼ 0.012; 32.6  2.9 to 30.0  2.6 pA, p ¼ 0.030, respectively), while control peptide Ab42e1 had no effect on either frequency or amplitude (paired t-test, n ¼ 7, from 0.5  0.1 to 0.4  0.1 Hz, p ¼ 0.487; 24.7  2.7 to 25.8  3.5 pA, p ¼ 0.265, respectively). These findings illustrate that Ab1e42 attenuates both presynaptic and postsynaptic inhibitory transmission, which contributes to the hyperexcitability of MCs.

3.2. The Ab1 to 42einduced hyperactivity is ascribed to the impairment of GABAergic transmission 3.3. Ab1e42 oligomers impair recurrent inhibition onto MCs GABAA receptors are widely expressed in MCs. The GABAergic inputs to MCs regulate the output pattern of MCs. To define the role of GABAergic inhibitory synaptic transmission in the Ab1 to 42einduced hyperactivity of MCs, a selective GABAA receptor antagonist bicuculline (10 mM) was preadministrated 10 minutes before Ab1e42 oligomer incubation. It was observed that the firings of sAPs in MCs were elevated by bicuculline in cell-attached mode (Fig. 3A and B, from 4.7  0.9 to 9.1  1.5 Hz, 1-way repeated measures ANOVA, n ¼ 8, F2, 12 ¼ 9.4, p ¼ 0.010). Ab1e42 was unable to boost such elevated firings (Fig. 3A and B, from 9.1  1.5 to 9.0  1.3 Hz, p ¼ 0.957). To examine whether there is a limit to the firing rate increase, we used the Kþ channel blocker tetraethylammonium (25 mM) coapplied with bicuculline. Compared to a single treatment of bicuculline, tetraethylammonium coincubation induced an additive increase of sAP firings (Fig. S2A, normalized sAP frequency

Since oligomeric Ab1e42 impairs GABAergic inhibitory transmission in the OB, we examined the influence of Ab1e42 on recurrent inhibition of MCs. The prolonged hyperpolarization that occurs following AP firings results from recurrent inhibition, which was recorded in whole-cell patch-clamping configuration. This hyperpolarization was blocked by bicuculline with some spontaneous APs occurring instead (Fig. S3A), thus confirming that recurrent inhibition onto MCs is mediated by GABAA receptors. It is reported that the strength of recurrent inhibition depends on MC firing rates (Margrie et al., 2001); however, neither amplitudes nor decay times of recurrent inhibitory postsynaptic potentials (IPSPs) were up-regulated along with the Ab1e42-elicited increase of AP frequency (Fig. S3BeD, paired t-test, n ¼ 7, from 3.7  0.8 to 4.4  0.6 pA, p ¼ 0.235 and from 270.7  54.3 to 220.8  52.1 ms,

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tr Aβ ol 142

A

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100 pA 50 ms 1.5

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Fig. 6. Oligomeric Ab1e42 diminishes lateral inhibition without altering the paired-pulse ratio (PPR). (A) Schematic of lateral inhibition experimental configuration. Whole-cell recording of an MC (right) upon stimulus of an adjacent glomerulus (left). (B) The amplitudes of eIPSCs are decreased during bath application of Ab1e42 but not Ab42e1. (C) Quantitative analysis of eIPSCs amplitudes in a 1-minute recording period. Paired t-test, n ¼ 7 cells, 5 and 4 rats, respectively; **p < 0.01. (D) PPR of eIPSCs elicited at a 50-ms interval is unchanged during Ab1e42 application. Paired t-test, n ¼ 6 cells, 3 rats. Abbreviations: eIPSCs, evoked inhibitory postsynaptic currents; EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; MC, mitral cell; MCL, mitral cell layer; n.s., not significant; ONL, olfactory nerve layer.

p ¼ 0.388, respectively). One possible explanation is that Ab1e42 severely impedes recurrent IPSPs. To test above hypothesis, we next recorded recurrent IPSPs following a given frequency of APs during Ab1e42 bath application. As shown in Fig. 5A, during Ab1e42 perfusion, approximately 80 pA current injections had already elicited the same firing rate as that in control group. Remarkably, Ab1e42 oligomers diminished both recurrent IPSP amplitudes and decay time constants (Fig. 5AeC, paired t-test, n ¼ 9, from 5.7  0.7 to 4.5  0.7 pA, p ¼ 0.011 and from 531.9  99.7 to 252.2  43.2 ms, p ¼ 0.021, respectively). Moreover, the reductions in APs were not due to alterations of resting membrane potential (Fig. 5D). These results demonstrate that Ab1e42 weakens recurrent inhibition evoked by the same stimulus intensity onto MCs. 3.4. Ab1e42 oligomers disrupt lateral inhibition between MCs The local inhibitory network provides the basis of lateral inhibition between MCs, a process that is suggested to enhance the contrast of odor representations and contribute to odor discrimination. Here, we tried to detect the pathological consequence of Ab on lateral inhibition between MCs. Using Alexa 488 dye in the

whole-cell patch pipette and an infrared-sensitive charge-coupled device camera, the recorded MC corresponding to the target glomerulus was identified visually (Fig. 6A and Fig. S4). Monosynaptic evoked IPSCs (eIPSCs) of lateral inhibition were recorded in whole-cell patch-clamping mode after blocking ionotropic glutamatergic synaptic transmission. Strikingly, Ab1e42 application led to a decline of eIPSCs amplitudes (Fig. 6B and C, left, paired t-test, n ¼ 7, from 110.5  25.7 to 68.2  17.6 pA, p ¼ 0.005). This decline was not due to “run down” or other nonspecific effects, as control peptide Ab42e1 did not change their values at all (Fig. 6B and C, right, paired t-test, n ¼ 7, from 111.9  44.7 to 103.6  13.3 pA, p ¼ 0.862). In some experiments, eIPSCs were fully blocked by bicuculline (10 mM) at the end of the recordings, indicating that eIPSCs are mediated by GABAA receptors (Fig. 6B). To further determine whether Ab1e42 decreases eIPSCs by acting on presynaptic terminals, we characterized the paired-pulse ratios of eIPSCs in response to 2 stimulations at 50 ms interpulse intervals. No statistically difference in paired-pulse ratios was observed before or after Ab1e42 application (Fig. 6D, paired t-test, from 0.7  0.1 to 1.0  0.2, n ¼ 6, p ¼ 0.252). These data suggest postsynaptic receptors as the main target of Ab1e42 disruption in lateral inhibition.

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4. Discussion The incidence of AD has increased progressively with the growth of the aging population worldwide. Olfactory dysfunction has been found to be an early symptom of AD (Mesholam et al., 1998; Roberts et al., 2016; Serby et al., 1991; Velayudhan et al., 2013). Elucidating the mechanisms underlying early olfactory deficits is important for understanding the preclinical features of AD, and has implications for prevention and treatment. In the present study, Ab oligomers were found to increase the firing activity of MCs but decrease signal-to-noise ratio in the OB, one of the earliest brain regions involved in AD. These data also demonstrate that Ab oligomers compromise a wide spectrum of inhibitory synaptic circuits in the OB. The findings predict that the up-regulation of inhibitory neurotransmission may ameliorate olfactory dysfunction, providing a potential treatment strategy in the early stages of AD. MCs function as the primary output neurons in the OB microcircuitry. We report here that acute incubation of oligomeric Ab rapidly augments spontaneous firing rates of MCs in OB slices, which indicates enhancement of background noise. Although the electrical stimulation-elicited activity of MCs also rises following Ab incubation, the induced efficiency is much lower than that of spontaneous activity. In fact, Ab oligomers most likely downregulate the responsiveness of MCs to odor signals by interfering with signal-to-noise ratio in the OB. Whether and how the Ab-toxicityeinduced alteration of MC responsiveness observed here leads to olfactory detection deficits in AD requires further investigation. Neuronal hyperexcitability has been found in higher-order cortical regions of patients with AD and in transgenic AD model mice (Amatniek et al., 2006; Wesson et al., 2011; Xu et al., 2015). Moreover, the alteration of Naþ or Kþ currents of excitatory neurons appears to be involved in the Ab-induced hyperexcitability in higher-order cortical regions (Brown et al., 2011; Mayordomo-Cava et al., 2015; Scala et al., 2015; Tamagnini et al., 2015). Our data provide additional evidence that oligomeric Ab rapidly and selectively impairs GABAergic synaptic transmission in the OB, and subsequently causes early hyperactivity and decreases in signal-tonoise ratio in MCs. A recent study found that soluble Ab oligomers inhibit the population activity in the OB slices and the sensitivity of olfactory bulb network activity to oligomeric Ab increases with age (Alvarado-Martínez et al., 2013). In the OB, inhibitory GABAergic neurons greatly outnumber principal MCs. We propose that Ab exerts opposing influence on different types of OB neurons. Our article clarifies that reduced GABAergic transmission contributes to the Ab-induced hyperactivity of MCs. The decreases in amplitudes and frequencies of mIPSCs suggest that Ab acts on both presynaptic and postsynaptic sites of inhibitory synapses. MCs express GABAA receptor alpha 1 and gamma 2 mRNAs strongly, while alpha 2 mRNA is weakly expressed (Laurie et al., 1992; Zhang et al., 1991). Further studies are required to analyze the influences of Ab on postsynaptic GABA receptor subunit expression in MCs. More importantly, we found Ab-induced impairment of presynaptic GABA release in the OB. Previous studies indicated that interneuron populations in the olfactory system are selectively susceptible in AD (De la Rosa-Prieto et al., 2016; Saiz-Sanchez et al., 2016). It has also been demonstrated that GABAergic neurons in higher-order cortex are vulnerable to AD-related neuropathology (Albuquerque et al., 2015; Ramos et al., 2006; Verret et al., 2012). Thus, upregulating GABAergic transmission may provide potential treatment strategy for progressive neurodegeneration in patients with AD. The molecular mechanisms underlying the preferential vulnerability of interneurons remain to be elucidated. In this article, we provided the first evidence (to our knowledge) that oligomeric Ab disrupts both recurrent and lateral inhibition.

Our findings suggest that Ab weakens lateral and recurrent inhibition of MCs, and therefore provides a local circuit basis for deficits in olfactory discrimination at early stages of AD. Recurrent and lateral inhibition onto MCs are mainly mediated by GCs that form dendrodendritic synapses with lateral dendrites of MCs in the external plexiform layer of the OB. In the OB of aged AD model mice, amyloid plaques almost exclusively and strongly localize within the GC layer (De la Rosa-Prieto et al., 2016; Wesson et al., 2010; Xu et al., 2015). We reason that soluble Ab mainly targets dendrodendritic synapses between MCs and GCs. Taken together, this study demonstrates that Ab disturbs local GABAergic neuronal circuits through both presynaptic and postsynaptic mechanisms, which affects the processing of odorant information and results in abnormal output signals from MCs. Our findings of local microcircuit impairments in the OB will be helpful for better understanding the cellular and synaptic mechanisms behind early olfactory dysfunction in AD and provide a potential strategy for treatment. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (81473185), a project funded by Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Qinglan Project for Innovative Teams. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2016.12.005. References Abraham, N.M., Egger, V., Shimshek, D.R., Renden, R., Fukunaga, I., Sprengel, R., Seeburg, P.H., Klugmann, M., Margrie, T.W., Schaefer, A.T., Kuner, T., 2010. Synaptic inhibition in the olfactory bulb accelerates odor discrimination in mice. Neuron 65, 399e411. Albuquerque, M.S., Mahar, I., Davoli, M.A., Chabot, J.G., Mechawar, N., Quirion, R., Krantic, S., 2015. Regional and sub-regional differences in hippocampal GABAergic neuronal vulnerability in the TgCRND8 mouse model of Alzheimer’s disease. Front Aging Neurosci. 7, 30. Alvarado-Martínez, R., Salgado-Puga, K., Peña-Ortega, F., 2013. Amyloid beta inhibits olfactory bulb activity and the ability to smell. PLoS One 8, e75745. Amatniek, J.C., Hauser, W.A., DelCastillo-Castaneda, C., Jacobs, D.M., Marder, K., Bell, K., Albert, M., Brandt, J., Stern, Y., 2006. Incidence and predictors of seizures in patients with Alzheimer’s disease. Epilepsia 47, 867e872. Attems, J., Walker, L., Jellinger, K.A., 2014. Olfactory bulb involvement in neurodegenerative diseases. Acta Neuropathol. 127, 459e475. Brown, J.T., Chin, J., Leiser, S.C., Pangalos, M.N., Randall, A.D., 2011. Altered intrinsic neuronal excitability and reduced Naþ currents in a mouse model of Alzheimer’s disease. Neurobiol. Aging 32, 2109.e1e2109.e14. Cassano, T., Romano, A., Macheda, T., Colangeli, R., Cimmino, C.S., Petrella, A., LaFerla, F.M., Cuomo, V., Gaetani, S., 2011. Olfactory memory is impaired in a triple transgenic model of Alzheimer disease. Behav. Brain Res. 224, 408e412. Cramer, P.E., Cirrito, J.R., Wesson, D.W., Lee, C.Y., Karlo, J.C., Zinn, A.E., Casali, B.T., Restivo, J.L., Goebel, W.D., James, M.J., Brunden, K.R., Wilson, D.A., Landreth, G.E., 2012. ApoE-directed therapeutics rapidly clear b-amyloid and reverse deficits in AD mouse models. Science 335, 1503e1506. De la Rosa-Prieto, C., Saiz-Sanchez, D., Ubeda-Banon, I., Flores-Cuadrado, A., Martinez-Marcos, A., 2016. Neurogenesis, neurodegeneration, interneuron vulnerability, and amyloid-b in the olfactory bulb of APP/PS1 mouse model of Alzheimer’s disease. Front Neurosci. 10, 227. Devanand, D.P., Lee, S., Manly, J., Andrews, H., Schupf, N., Doty, R.L., Stern, Y., Zahodne, L.B., Louis, E.D., Mayeux, R., 2015. Olfactory deficits predict cognitive decline and Alzheimer dementia in an urban community. Neurology 84, 182e189.

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