Implications of Oligomeric Amyloid-Beta (oAβ42) Signaling through α7β2-Nicotinic Acetylcholine Receptors (nAChRs) on Basal Forebrain Cholinergic Neuronal Intrinsic Excitability and Cognitive Decline

Andrew A. George; Jaime M. Vieira; Cameron Xavier-Jackson; Michael T. Gee; John R. Cirrito; Heather A. Bimonte-Nelson; Marina R. Picciotto; Ronald J. Lukas; Paul Whiteaker

doi:10.1523/JNEUROSCI.0876-20.2020

Abstract

Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-β (Aβ) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-β (oAβ₄₂), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAβ₄₂ activates both homomeric α7- and heteromeric α7β2-nAChR subtypes while preferentially enhancing α7β2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAβ₄₂ showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the β2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7β2-nAChR in mediating the effects of oAβ₄₂ on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7β2-nAChR in oAβ₄₂-induced cognitive decline.

Significance Statement

Aberrant neural activity can occur years before amyloid-β (Aβ) plaque deposition. Recent evidence has shifted focus toward the epileptogenic potential of soluble, oligomeric forms of Aβ_1-42 (oAβ₄₂) and its role in Alzheimer's disease (AD)-related cognitive decline. This study provides insight into the underling mechanisms mediating oAβ₄₂-induced hyperexcitation in neurons particularly susceptible to degeneration in AD. Using single-channel and whole-cell patch-clamp recordings, we demonstrate the following: (1) oAβ₄₂ interacts with α7β2-containing nicotinic receptors, altering the intrinsic excitability of specific populations of basal forebrain cholinergic neurons; and (2) α7β2-nAChR signaling contributes to spatial reference memory deficits in the APP/PS1 mouse model of AD. Together, these findings reveal a unique role for α7β2-nAChR signaling during early, AD-related pathologic events.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease afflicting >50 million individuals worldwide (projected to be >130 million by 2050). AD is classically diagnosed using postmortem histopathological biomarkers of neuritic amyloid-β (Aβ) plaques and neuronal/glial fibrillary tangles of hyperphosphorylated tau protein (Scheltens et al., 2016; Jack et al., 2018). Attempted treatments targeting amyloid precursor protein (APP) processing and Aβ aggregates have failed (Nicoll et al., 2019; Panza et al., 2019), perhaps because of intervention too late in disease progression. This has revealed gaps in understanding of molecular and cellular-level changes underlying AD etiopathogenesis. Recent studies suggest that early changes in AD are triggered by a soluble, oligomeric form of Aβ_1-42 (oAβ₄₂) (Yang et al., 2017). This form is elevated early in AD and has been targeted in promising clinical trials (Jongbloed et al., 2015; Hey et al., 2018). Many factors contribute to cognitive decline in AD (Selkoe, 2002; Pereira et al., 2005; Nimmrich and Ebert, 2009). Prominently, basal forebrain cholinergic neurons (BFCNs) and their projections modulate circuitry involved in cognitive processing (Picciotto et al., 2012; Zaborszky et al., 2012; Mesulam, 2013) and degenerate during the mild-cognitive impairment phase of AD (Grothe et al., 2012). This neuronal loss also could account for degeneration of hippocampal and cortical regions receiving BFCN innervation, and associated memory deficits (Grothe et al., 2014; Schmitz et al., 2016; X. Q. Chen and Mobley, 2019; Hampel et al., 2019). However, triggers for BFCN neurodegeneration are unknown, as is the potential role of elevated oAβ₄₂ in BFCN loss in early AD.

Network hyperexcitability is a feature of AD and has been reported in numerous mouse models of AD pathology (Minkeviciene et al., 2009; Vossel et al., 2013). Importantly, oAβ₄₂'s ability to alter neuronal and network-level function and, ultimately, cognition has been linked to functional interactions between oAβ₄₂ and nicotinic acetylcholine receptors (nAChRs) containing the α7 subunit (α7*-nAChR) (Puzzo et al., 2008; Gulisano et al., 2019; van Goethem et al., 2019). In many brain regions, α7*-nAChRs mediate synaptic transmission and regulate intrinsic neuronal excitability (Kawai et al., 2002; Liu et al., 2013; Dao et al., 2014). In most regions, α7*-nAChRs are homomers containing only α7 subunits. However, a small fraction also contain β2 subunits (α7β2-nAChR). These heteromeric α7β2-nAChRs form functional receptors (Khiroug et al., 2002; Murray et al., 2012), are highly sensitive to functional modulation by Aβ (Liu et al., 2009, 2012), and are enriched in specific populations of cholinergic and noncholinergic neurons of the basal forebrain (Khiroug et al., 2002; Azam et al., 2003; Thinschmidt et al., 2005). Together, these findings suggest that selective expression of the α7β2-nAChR subtype on BFCN neurons might underlie the pathologic effects of oAβ₄₂ on modulation of BFCN function through heightened or maladaptive activation of α7*-nAChRs.

We find that, similar to the endogenous ligand acetylcholine (ACh), a pathophysiologically relevant concentration of oAβ₄₂ (100 nm) (Yang et al., 2017) directly activates both human α7- and α7β2-nAChR but preferentially enhances α7β2-nAChR α7β2-nAChR single-channel open-dwell times. Furthermore, we demonstrate that BFCNs chronically exposed to oAβ₄₂ exhibit enhanced action potential firing rates, and altered BFCN action potential waveforms (reduced time to spike, accelerated action potential repolarization, and reduced action potential medium afterhyperpolarization [mAHP]). These alterations in the intrinsic mechanisms mediating BFCN excitability are normalized through pharmacological antagonism of α7*-nAChR or genetic deletion of the β2-nAChR subunit gene. Last, we demonstrate that oAβ₄₂/α7β2-nAChR interactions likely reduce acquisition and retention of spatial reference memory, using the well-established APP/PS1 transgenic AD mouse model. This study is the first to demonstrate specific molecular and intrinsic-level mechanisms through which oAβ₄₂ enhances BFCN excitability, and provides a potential explanation for the selective vulnerability of these neurons in early AD. These findings also expand on findings that neuronal activity is increased by Aβ, including oAβ₄₂ (Walsh et al., 2002; Palop et al., 2007; Busche et al., 2008; Palop and Mucke, 2009) and suggest novel strategies to ameliorate cellular processes contributing to BFCN loss and cognitive impairment.

Materials and Methods

Construct encoding a human α7-nAChR subunit-mCherry fusion protein

Human α7-nAChR subunits were engineered to express the red fluorescent protein mCherry as a fusion protein. This allowed direct visualization of nAChR expression in vitro. Using a DNA synthesis approach (GeneArt, Thermo Fisher Scientific), the mCherry sequence was inserted between the native, human nAChR α7 subunit's second, large intracellular domain amino acid residues C412 and S413 (numbering from the translation start methionine), while avoiding interruption of post-translational modification and/or regulatory sequences (Nashmi et al., 2003). Correct modification was confirmed by DNA sequencing (Thermo Fisher Scientific). The nucleotide sequence of this mCherry-tagged α7-nAChR subunit was optimized for expression in vertebrate expression systems. Optimizations included minimization of high GC content sequence segments, improved codon usage, reduction of predicted RNA secondary structure formation, and removal of sequence repeats and possible alternative start and splice sites. This construct is referred to as “unlinked α7-nAChR-mCherry,” to differentiate it from the concatenated α7*-nAChR constructs described next, and was subcloned into the mammalian expression vector pcDNA 3.1-Zeocin.

Construct for the nAChR chaperone NACHO

The human sequence for the α7*-nAChR chaperone protein NACHO (Gu et al., 2016) was subcloned into the bicistronic mammalian expression vector pIRES (Addgene), facilitating simultaneous, constitutive expression of both NACHO and the GFP ZsGreen1. This construct was engineered to facilitate the cell-surface expression of α7- and α7β2-nAChR constructs and the visual identification of SH-EP1 cells expressing NACHO.

Constructs encoding concatenated homomeric α7 or heteromeric α7β2-nAChR

Fully pentameric α7*-nAChR concatemers were constructed, encoding three different arrangements of subunits: 5′-α7-α7-α7-α7-α7-3′ [α7 concatemer], 5′-α7-α7-β2-α7-α7-3′ [α7β2(P3) concatemer], or 5′-α7-β2-α7-β2-α7-3′ [α7β2(P2,P4) concatemer]. These were engineered largely as previously described (George et al., 2017), with the exception that the nucleotide sequences for α7 subunit genes expressed in the fifth subunit position were modified to include the mCherry sequence, as described in the preceding section for the unlinked α7-nAChR-mCherry construct. All other features of the constructs, including linker lengths and composition, placement of unique restriction sites within the nucleotide sequences encoding these linkers, and positioning of Kozac, signal peptide, and stop sequences, are as described in our previous publication (George et al., 2017). Schematics of these linear constructs and their assembled format (including locations of agonist binding sites) are provided in Figure 1A and Figure 1B, respectively. Sequences of all subunits, together with their mCherry fluorophore and associated linkers, were confirmed by DNA sequencing (Thermo Fisher Scientific), and correct assembly of each translated pentamer was verified at the cDNA level by restriction digest. These validated, fully pentameric, concatenated α7- and α7β2-nAChR-mCherry constructs were subcloned into the mammalian expression vector pcDNA 3.1-Zeocin. As shown by our published work (Moretti et al., 2014), concatenated α7- or α7β2-nAChR form functional receptors that recapitulate pharmacological and single-channel functional properties of native α7*- and α7β2-nAChR (Fu and Jhamandas, 2003; Andersen et al., 2016; Corradi and Bouzat, 2016; Bouzat and Sine, 2018; Nielsen et al., 2018).

Figure 1.

Design, engineering, and functional expression of human α7- and α7β2-nAChR. A, Schematic illustration of α7 and α7β2-nAChR DNA constructs (from top to bottom): Single α7 subunit gene (α7 unlinked) fused to mCherry, nAChR chaperone protein NACHO coexpressed with ZsGreen1, concatenated α7-nAChR (containing only α7 subunits (α7 homopentamer); α7-mCherry fusion in position 5) concatenated α7β2(P3)-nAChR (containing a single β2 subunit in position 3; α7-mCherry fusion in position 5), and concatenated α7β2(P2,P4)-nAChR (containing two β2 subunits in position 2 and 4; α7-mCherry fusion in position 5). B, Illustration of assembly and stoichiometry of unlinked α7-nAChR homopentamer, concatenated α7-nAChR homopentamer, concatenated α7β2(P3)-nAChR, and concatenated α7β2(P2,P4)-nAChR. C, Fluorescent imaging of α7- and α7β2-nAChR constructs transiently expressed in SH-EP1 cell lines. Top panels, NACHO expression (ZsGreen1). Middle panels, α7*-nAChR expression (mCherry). Bottom panels, Cells coexpressing NACHO and α7*-nAChR (merged; yellow). D, Cell-attached single-channel responses from α7- and α7β2-nAChR. Single-channel responses were elicited with ACh (300 μm; gray bar below each representative trace). Closed (Clsd) and open receptor states (O1-O3; dashed lines) are indicated for each nAChR subtype. Perfusion of the α7-selective PAM PNU 120596 during ACh application (3 μm; purple bar below each representative trace) enhances α7- and α7β2-nAChR single-channel activity, producing longer-lasting bursts of openings.

Cell culture

The unmodified SH-EP1 human epithelial cell line (nAChR null) was maintained as previously described (Fryer and Lukas, 1999; Eaton et al., 2014). Briefly, DMEM (high glucose, bicarbonate-buffered, with 1 mm sodium pyruvate and 8 mm L-glutamine) was supplemented with 10% horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Invitrogen) plus 5% FBS (Hyclone) on 100-mm-diameter plates in a humidified atmosphere containing 5% CO₂ in air at 37°C. SH-EP1 cells were passaged once per week as described previously (Lukas, 1993; Eaton et al., 2003) and maintained at 80% confluence.

Transient transfection SH-EPI cells

Forty-eight hours before transfection, SH-EP1 cells were split (1:50) and plated on 35 mm cell culture dishes coated with poly-d-lysine. NACHO-ZsGreen1 and nAChR-mCherry cDNA constructs (unlinked α7-nAChR subunits, α7-nAChR concatemer, α7β2(P3)-nAChR concatemer, or α7β2(P2,P4)-nAChR concatemer) were cotransfected at a 1:1 ratio (1 µg NACHO cDNA: 1 µg nAChR cDNA) using QIAGEN's Effectene transfection kit. Following transfection, cells were incubated at 37°C for 2 d in complete DMEM without antibiotic selection. On the day of single-channel recordings, cells were rinsed twice with prewarmed extracellular solution (described in Preparation of oAβ₄₂) to remove residual DMEM and allowed to equilibrate at 22°C for 5 min before recording.

Preparation of oAβ₄₂

Preparation of synthetic human or mouse oAβ₄₂ strictly adhered to methods previously described (Stine et al., 2011). Briefly, 1 mm Aβ stock solutions were made by solubilizing lyophilized human or mouse Aβ_1-42 powder (California Peptide Research) in 1,1,1,3,3,3-hexafluoro-2-propanol. Aβ peptide films were stored over desiccant in glass jars at −20°C. Before use, Aβ_1-42 peptide films were removed from −20°C freezer and warmed to 22°C. oAβ₄₂ was prepared under sterile conditions by resuspension of Aβ_1-42 peptide films in DMSO and sonicated for 10 min before dilution in cold ACSF (for single-channel recordings) or neurobasal media (for organotypic basal forebrain slice preparations) to a final stock concentration of 100 μm. Samples were vortexed (15 s), spun down, and transferred to 4°C for 24 h. To avoid protofibril formation, samples were not used for more than the day of a given experiment. This protocol yields oAβ₄₂ based on the evaluation of similarly prepared samples via negative-staining electron and atomic force microscopy (Liu et al., 2013).

Native/PAGE Western blotting to validate Aβ₄₂ oligomers

To confirm the preservation of oAβ₄₂ assemblies in basal forebrain organotypic slice cultures, basal forebrain organotypic cultures were prepared from male and female ChAT-EGFP mice (P7) as described above and exposed to neurobasal media containing 100 nm oAβ₄₂ at 37°C. Neurobasal media containing oAβ₄₂ was harvested immediately after exposure (time point 0; T0) and after a 24 h exposure period (time point 24; T24). Western blot analysis was performed to on samples from T0 and T24 to determine the predominant form(s) of Aβ (see Fig. 5G). Native PAGE for oAβ₄₂ assemblies was performed using a 12-well 4%-12% RunBlue Bis-Tris gels (Expedeon) under native conditions with 8 μl of media loaded per lane using a Tris-glycine running buffer. Independent samples of media were used at T0 and T24. Nitrocellulose blots were probed with mouse-anti-APP/Aβ (m6E10; BioLegend), followed by a sheep anti-mouse antibody conjugated to peroxidase (Cytiva). Bands were detected with Lumigen-TMA6 (GE Healthcare) and captured digitally using the Kodak ImageStation 440CF. Densitometry was performed using the Kodak 1D Image Analysis software.

Single-channel electrophysiology

Cells cotransfected with both the α7- or α7β2-nAChR constructs and the chaperone protein NACHO were selected for recording. These appeared yellow under fluorescence microscopy because of coexpression of the red mCherry and ZsGreen1 tags associated with the nAChR and NACHO constructs, respectively (illustrated in Fig. 1C). Single-channel α7- and α7β2-nAChR-mediated currents were recorded from SH-EPI cells under cell-attached configuration similar to that previously described for nAChR single-channel recordings from Xenopus laevis oocytes (George et al., 2017; Weltzin et al., 2019). All single-channel recordings were performed at room temperature (22°C). Patch pipettes were fabricated from thick-walled borosilicate glass (WPI), and tips were microforged to a final resistance of 15-20 mΩ. To elicit single-channel events, patch pipettes were filled with extracellular solution containing the following (in mm): 119 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 24 NaHCO₃, 12.5 glucose, 2 CaCl₂·4H₂O, 2 MgSO₄·7H₂O, and 1.5 atropine) that contained either ACh, corresponding to EC₅₀ values for each construct (α7- and α7β2-nAChR constructs = 300 μm) (Moretti et al., 2014) or oAβ₄₂ (100 nm; falling within the range of pathophysiologically relevant concentrations previously described in humans) (Yang et al., 2017) and in rodent models of AD (Fá et al., 2016; Koppensteiner et al., 2016). The concentrations of ACh and oAβ₄₂ used in our study were based, in part, on the practical consideration of choosing a concentration of oAβ₄₂ that resulted in the robust single-channel activation of both α7- or α7β2-containing nAChR without resulting in open-channel blockade of either receptor subtype. Patch pipettes also contained 100 nm charybdotoxin to block endogenous large conductance (BK) voltage and Ca²⁺-dependent potassium channels. These ACh and oAβ₄₂ concentrations produced sufficient open-channel events for analysis without producing an excessive overlap of unitary events or open-channel blockade of receptors (which can occur in the presence of very high agonist concentrations) (Papke and Oswald, 1989). Recordings were performed using an Axopatch 200B amplifier (Molecular Devices). For quality control, patches with seal resistance <10 GΩ were immediately discarded. Patches were clamped at a transmembrane potential of 100 mV. Current recordings were sampled at 50 kHz using pClamp10.7 (Molecular Devices) and low-pass filtered at 10 kHz. A Gaussian digital filter with a final cutoff frequency of 5 kHz was used during analysis. All single-channel recordings were analyzed using QuB software (version 1.4.0.132; https://qub.mandelics.com/). QuB software was used for preprocessing, which included baseline correction and idealization of single-channel events according to a half-amplitude, threshold-crossing criterion (George et al., 2017; Weltzin et al., 2019).

Experimental design

Promptly on seal formation, single-channel events (elicited by ACh or oAβ₄₂) were recorded for 15 min before bath perfusion (rate = 200 µl/s) of the nonselective nAChR antagonist mecamylamine (Meca; 1 mm) or the competitive antagonist methyllycaconitine (MLA; 10 nm, an α7*-nAChR-selective concentration). Single-channel recordings proceeded in the presence of Meca or MLA for an additional 15 min before the same patches were exposed to the Type II, α7*-nAChR-specific, positive allosteric modulator (PAM), PNU 120596 for an additional 5 min to validate α7- or α7β2-nAChR single-channel events. Single-channel events were recorded for an additional 5 min in the presence of PNU 120596 before termination of the recording. ACh or oAβ₄₂-elicited single-channel events from α7- or α7β2-nAChR could be sustained for ∼30 min before observing significant rundown in single-channel activity in the absence of PNU 120596.

Single-channel openings from α7- or α7β2-nAChR exhibit a broad distribution of current amplitudes that mainly result from limited time resolution of the inherently brief α7 and/or α7β2 openings (Andersen et al., 2013; Nielsen et al., 2018). However, these single-channel events may also represent a subpopulation of distinct subconductance states (Andersen et al., 2013). To define the conductance state for α7- and α7β2-nAChR constructs used in this study, we used the α7-specific PAM PNU-120596 to isolate the main conductance state for both α7- and α7β2-nAChR. Amplitude stability plots were generated from single-channel events elicited in the presence of ACh & PNU-120596 or oAβ₄₂ & PNU-120596 for α7-containing (linked and concatenated) nAChR (main amplitude of 4.8 ± 0.02 pA and 4.5 ± 0.03 pA; elicited with ACh or oAβ₄₂, respectively), α7β2(P3) nAChR (main amplitude of 5.2 ± 0.03 pA and 6.8 ± 0.06 pA; elicited with ACh or oAβ₄₂, respectively), and α7β2(P2,P4) nAChR (main amplitude of 4.7 ± 0.02 pA and 6.6 ± 0.08 pA; elicited with ACh or oAβ₄₂, respectively). Single-channel bursts corresponding to these precise amplitudes were segregated from isolated openings and only bursts were used for single-channel analysis (described below).

All recordings were analyzed using a Gaussian digital filter with a final cutoff frequency of 5 kHz. Single-channel amplitudes were derived from the idealized trace by fitting the raw data to a simple closed–open (C↔O) kinetic model. Closed- and open-dwell time distributions were generated for each recording and fitted by the sum of exponential functions by maximum likelihood. Closed-dwell time distributions were best fit with four components, and open-dwell time distributions were best fit with two components. Bursts of single-channel activity were defined as a series of openings separated by closures shorter than the minimum interburst closed duration (or T_crit) and separated from others by closed times longer than T_crit (Colquhoun and Sakmann, 1985). For all groups tested, the minimum interburst closed duration, or T_crit, was calculated using QuB software. Bursts containing overlapping currents, which indicate two simultaneously active channels, were rare and were discarded from analysis. The advantage of using bursts was to unequivocally determine that all the openings in a burst come from the same individual channel, and that the closed-dwell times within bursts can be interpreted in terms of channel mechanisms, even under conditions where there is an unknown number of channels in the patch. Under these conditions, no single-channel bursts were observed in untransfected SH-EP1 cells, which were exposed to ACh or oAβ₄₂, or in SH-EPI cells that were doubly transfected with any of the α7- or α7β2-nAChR constructs together with NACHO, but recorded from in the absence of ACh or oAβ₄₂.

Single-channel closed-dwell times were determined from individual patches, and time constants for each closed- and open-dwell time component were averaged across multiple patches. Averaged closed- and open-dwell times for each nAChR construct were compared in the presence of ACh or oAβ₄₂. Single-channel stability plots for amplitudes and closed- and open-dwell time distributions were determined for each individual patch, and means for single-channel amplitudes and open-dwell times were compared in the presence of ACh or oAβ₄₂. To determine whether oAβ₄₂ performed as an allosteric modulator at α7- and/or α7β2-nAChR, both ACh (300 μm) and oAβ₄₂ (100 nm) were added to the pipette simultaneously. For each group, single-channel burst durations were pooled from multiple patches. Single-channel open-dwell time distributions were generated from bursts of single-channel activity only, and therefore did not include isolated openings. All burst open-dwell time histograms were best fit with 2 exponentials as previously described (George et al., 2017). Each individual exponential and their respective time constants (τ) for single-channel open-dwell times were calculated using Qub software. Time constants of each exponential (i.e., short and long burst durations) were compared between α7- and α7β2-nAChR constructs in the presence of ACh or oAβ₄₂.

Single-channel electrophysiology statistical analysis

Group data for single-channel burst rates (bursts/s) and open-dwell times (τ values; ms) were analyzed using one-way ANOVA with Tukey's post hoc test for multiple comparisons. Two-way ANOVA was used (statistical significance set at p < 0.05) to compare differences in burst rates among all α7*-nAChR and between ACh and oAβ₄₂ and followed by a Tukey's post hoc test for multiple comparisons where applicable (GraphPad software). Data are mean ± SEM.

Basal forebrain organotypic slice preparations

Basal forebrain organotypic slice cultures were prepared according to methods previously described (Stoppini et al., 1991; Ting et al., 2014; Buendia et al., 2016). Initially, brains were removed from ChAT(BAC)-EGFP and nonlittermate β2 nAChR subunit KO mice (of either sex; postnatal day 7; see Animal husbandry, breeding, and safeguards) and placed immediately in ice-cold cutting solution composed of the following (in mm): 92 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO₄^.7H₂O, 0.5 CaCl₂^.2H₂O, 100 kynurenic acid, and 100 U/ml penicillin and 0.100 mg/ml streptomycin. Organotypic slices containing the medial septum diagonal band (MSDB), horizontal diagonal band (HDB), or nucleus basalis (NB) were sectioned at 400 µm and immediately placed in ice-cold neurobasal media [supplemented with 25% heat-inactivated horse serum, ascorbic acid (500 μm)], 2 mmol/l l-glutamine, B-27 supplement and NGF (Sigma Millipore, 10 ng/ml), and 100 U/ml penicillin and 0.100 mg/ml streptomycin. Slices were transferred to Millicell 0.4 μm culture inserts within each well of a six-well culture tray containing neurobasal media supplemented with B-27 and NGF in the presence or absence of oAβ₄₂ (100 nm). As a control, a scrambled version of the oAβ₄₂ peptide was prepared following the same methodology as the oligomeric isoform (methods strictly adhering to Stine et al., 2011). To avoid protofibril formation, neurobasal media containing oAβ₄₂ or scrambled oAβ₄₂ were exchanged every 24 h. Basal forebrain organotypic slices were incubated at 37°C with 5% CO₂ for 9 d before whole-cell patch-clamp recordings.

Whole-cell patch-clamp electrophysiology

Basal forebrain organotypic slice cultures were transferred from Millicell inserts to a recording chamber perfused with oxygenated ACSF composed of the following (in mm): 124 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 24 NaHCO₃, 5 HEPES, 13 glucose, 2 MgSO₄^.7H₂O, 2 CaCl₂^.2H₂O (osmolarity 300-310; pH 7.3) and maintained at room temperature throughout the experiment. BFCNs were identified under epifluorescence illumination using a BX50-WI Olympus microscope with a 40× water immersion objective. BFCNs from ChAT-EGFP transgenic mice were identified within the MSDB, HDB, and NB based on their neuroanatomical location and functional expression of EGFP. Additional features (e.g., low firing frequency, lack of membrane potential sag, and prominent spike AHP) were consistently associated with this neuronal population and were used to provide further validation of their neuroanatomical location and distinct electrophysiological characteristics (Liu et al., 2009; Unal et al., 2012; McKenna et al., 2013; Hedrick et al., 2016). BFCNs from β2 nAChR subunit KO mice were identified based on their neuroanatomical location, soma size (>25 µm), and distinct electrophysiological characteristics (above), all of which matched those of neurons positively identified in the ChAT-EGFP line.

For whole-cell recordings, patch pipettes were microforged to a final resistance of 8-10 mΩ and were filled with the following (in mm): 135 K-gluconate, 10 HEPES, 2 MgCl₂, 0.5 CaCl₂, 2 Mg-ATP, 0.1 Na-GTP, 10 phosphocreatine, and 5 EGTA, pH 7.3. Current-clamp recordings were corrected for 11.8 mV liquid junction potential between the intracellular and recording solutions (Neher, 1992). All voltage signals were amplified using an Axopatch 200B patch-clamp amplifier and digitized at a rate of 50 kHz (DigiData1440A, Molecular Devices). BFCNs were current-clamped at −65 mV, and access resistance was continuously monitored during recordings using pClamp10 software. To eliminate the contribution of spontaneous excitatory and inhibitory synaptic inputs onto BFCNs, recordings were performed in the presence of the synaptic blockers CNQX (25 μm in ACSF), AP-5 (50 μm in ACSF), and gabazine (SR-95 531 hydrobromide at 10 μm in ACSF), all purchased from Tocris Bioscience.

Experimental design

BFCN resting membrane potential (RMP, mV) was measured in current-clamp mode at I = 0, and RMP was measured immediately after whole-cell formation. To assess the effects of oAβ₄₂ exposure on BFCN intrinsic activity, we initially presented BFCNs with a ramp current injection (0.1 pA/ms; 1 s) from a constant holding command of −65 mV. We used this ramp protocol to determine action potential threshold, latency to first spike, and maximal rates of voltage change (dV/dt_max) during spike depolarization and repolarization. Next, we presented BFCNs with a step current injection protocol (−100 pA to 100 pA; δ level: 20 pA, duration: 1 s) to examine BFCN firing rates and input resistance. Last, we used a single-step current injection protocol (500 pA; duration: 1 s) to measure BFCN mAHP.

BFCN firing rates were averaged for each current injection amplitude, and comparisons were drawn across experimental groups for each current injection between 20 and 100 pA. The mAHP (mV) amplitude was defined as the difference between baseline and the AHP potential measured 100 ms after cessation of the current stimulus, averaged across individual recordings, and measured from the baseline before the AP burst. Phase-plane portraits were generated for each BFCN by deriving the membrane potential with respect to time (dV/dt) and plotting it as a function of BFCN membrane potential. The maximal rate of voltage change (dV/dt_max) was measured during spike depolarization and repolarization for each of the first 10 action potentials generated using the ramp current-injection protocol and then averaged across BFCNs within each forebrain nucleus. Action potential threshold was experimentally determined and defined by the membrane potential at which dV/dt of the first action potential of the phase plane plot crossed 20 mV/ms (Yu et al., 2008). The maximal rate of voltage change (dV/dt_max) during spike depolarization and repolarization was calculated for the first 10 action potentials generated during the ramp current-injection protocol. Action potentials were individually compared across groups. Latency to first spike was measured for each BFCN and defined as the period between the start of the recording and arrival at the first action potential threshold. Action potential amplitude was defined as the peak voltage from threshold to the peak of the action potential. Passive membrane properties (Table 1) were measured from MSDB, HDB, and NB BFCNs during each experimental condition (i.e., scrambled oAβ₄₂ control, oAβ₄₂ alone, MLA + oAβ₄₂, and β2 nAChR KO slices + oAβ₄₂). Cell capacitance (pF) was determined using pClamp's automatic whole-cell compensation function. BFCNs were excluded from further analysis if the RMP > −55 mV or if the access resistance fluctuated >10%. Input resistance was measured by calculating the slope of the voltage change in response to current injections ranging from −100 pA to −20 pA (δ level = 20 pA).

View this table:

Table 1

Passive membrane properties of MSDB, HDB, and NB BFCNs

Whole-cell patch-clamp electrophysiology statistical analysis

Comparisons of BFCN firing rates and mAHP amplitudes were evaluated across all groups using one-way ANOVA followed by a Tukey's post hoc test for multiple comparisons. Differences in dV/dt_max across groups were analyzed using two-way ANOVA with statistical significance set at p < 0.05, followed by a Tukey's post hoc test for multiple comparisons. Action potential threshold, time to first spike, and action potential amplitude were analyzed using one-way ANOVA with Tukey's post hoc test (GraphPad software). Data are mean ± SEM.

Animal husbandry, breeding, and safeguards

All procedures were performed in accordance with the National Institutes of Health's Guide for the care and use of laboratory animals and institutional guidelines established by the Animal Care and Use Committee at the Barrow Neurologic Institute. Mice were maintained in standard housing on a 12 h light/dark cycle. C57BL/6J WT male and female mice expressing enhanced GFP under the control of the ChAT promoter (ChAT(BAC)-EGFP, The Jackson Laboratory; stock #007902), male and female β2 nAChR subunit KO mice [β2 nAChR KO; generously provided by Marina Picciotto, Yale University (Picciotto et al., 1995)], male and female APP/PS1-129/SvJ [generously provided by Drs. Antonella Caccamo and Salvatore Oddo; Arizona State University; (Caccamo et al., 2017)] were used. The latter two lines were used to generate mice used for behavioral testing (see below).

Generation of mice used for whole-cell patch-clamp recordings and Morris water maze (MWM) behavioral testing

For whole-cell patch-clamp recordings, ChAT(BAC)-EGFP and β2 nAChR KO colonies used were each maintained through homozygous × homozygous mating. For MWM, generation of β2 nAChR KO, APP/PS1, and APP/PS1-β2 nAChR KO mice was accomplished by backcrossing APP/PS1-129/SvJ mice (heterozygous for APP/PS1 transgene) to β2 nAChR KO/C57Bl6 mice for 10 generations to produce the following littermates: β2 nAChR KO mice, APP/PS1 mice, and APP/PS1-β2KO mice on a defined C57Bl6 background. The resulting littermates could be genotyped as follows: (A) hemizygous for the APP/PS1 transgene and genetically null for β2 nAChR subunit expression (i.e., APP/PS1-β2KO-C57Bl6), (B) hemizygous for the APP/PS1 transgene and homozygous for β2 nAChR subunit expression (i.e., APP/PS1-C57Bl6), or (C) absent of the APP/PS1 transgene and homozygous for β2 nAChR subunit expression (i.e., β2 nAChR KO-C57Bl6). As an additional control, WT 129/SvJ mice were backcrossed with WT C57Bl6 mice (i.e., β2 positive-C57Bl6) for the same number of generations to control for strain differences.

Genotyping

Genomic DNA was isolated from ear punches (postnatal day 21) using Phire Animal Tissue Direct PCR Kit (Thermo Fisher Scientific). For β2 nAChR subunit gene expression, male and female mice were genotyped using primers specific for β2 nAChR subunit gene expression and the β-galactosidase transgene (LacZ) (Picciotto et al., 1995) as follows: Lac-Z reaction primers (Lac-Z-5′: CAC TAC GTC TGA ACG TCG AAA ACC CG) and (Lac-Z-3′: CGG GCA AAT ATC GGT GGC CGT GG); for β2 reaction primers as follows: (B2-5′: CGG AGC ATT TGA ACT CTG AGC AGT GGG GTC GC) and (B2-3′: CTC GCT GAC ACA AGG GCT GCG GAC); for PS1 transgene reaction primers as follows (PS1-5′: AAT AGA GAA CGG CAG GAG CA) and (PS1-3′: GCC ATG AGG GCA CTA ACA T). Mice positive for the APP/PS1 transgene showed a product of 650 bp. Mice homozygous for β2 nAChR subunit expression showed a product at 650 bp (β-galactosidase transgene) and 350 bp (β2 nAChR expression). Animals lacking LacZ expression while positive for β2 nAChR expression (i.e., heterozygous) were excluded from behavioral testing.

MWM

Spatial reference memory was tested using a modified version of the MWM as previously described (M. T. Williams et al., 2003; Koebele et al., 2019). Briefly, mice (males and females; aged 10 months) were tested in a circular tub (188 cm in diameter) filled with water (22°C) tinted with white, nontoxic paint. Animals were randomized into groups to be placed in the maze from one of four cardinal locations (North, South, East, or West). Using visual cues, mice then had 60 s to locate a platform, which was submerged 1.5 cm beneath the surface of the water and remained in a fixed location in the southwest quadrant. Data acquisition was performed using a video camera mounted on the ceiling, and swim path was recorded and analyzed using EthoVision XT tracking software (Noldus Information Technology).

Experimental design

Spatial reference memory

Animals were tested from four cohorts, each representing a unique genotype (described above). If a mouse failed to find the platform that was located in the southwest quadrant within 60 s, it was guided to the platform and maintained on the platform for 15 s before being placed into a heated cage until the following trial. Mice received 4 trials/day for 6 consecutive days, with an intertrial interval of ∼10 min. A fifth probe trial was administered on day 6 in which the platform was removed to evaluate whether the mice used spatial cues to locate the platform. Latency and distance to the platform were measured for each genotype and compared across days. For the probe trial, target quadrant frequency (frequency of crossings into platformed quadrant) and percent of the total swim distance in the target quadrant (% of total) were compared with the quadrant diagonally opposite (northeast) and follow methods previously described (Bimonte-Nelson et al., 2015). To reassociate the original platform location with escape, a seventh trial was given, identical to Trials 1-4. Further, we assessed the effects of β2 nAChR subunit expression on overnight forgetting by comparing latency to the platform from the last test trial on the first day (Trial 4 on day 1) to the first test trial the next day (Trial 1 on day 2), and for the overnight interval from day 2 to 3, etc. (Braden et al., 2010).

Visible platform test

Following spatial reference memory testing, animals were given a visual platform test to rule out the possibility that the observed spatial learning deficits were a product of impaired vision or abnormal motor function necessary to solve a water-escape maze task. Mice were given six trials/platform location with the platform location varied across three different locations. Mice were dropped off from the same location across trials. Animals remained on the platform for 15 s after finding the platform before being placed back into a heated cage before the subsequent trial (intertrial interval of ∼10 min).

MWM statistical analysis

Behavioral data were evaluated using two-way ANOVAs with statistical significance set at p < 0.05, followed by a Fisher's LSD post hoc correction for multiple comparisons where applicable (StatView software). Omnibus repeated-measures ANOVAs, with one level repeated (day of testing) and one level between subjects (genotype), were used as a statistical measure for MWM training sessions. Differences in MWM probe test behavior were tested using two-way ANOVA followed by a Fisher's LSD multiple comparisons test and using Student's t test to compare within groups (GraphPad Software). Data are mean ± SEM.

Results

Single-channel electrophysiological recordings of ACh-induced openings of α7- and α7β2-nAChR can be obtained consistently from transiently-transfected SH-EP1 cells

We used a single-channel recording strategy, using transiently transfected, native nAChR-null, human SH-EP1 cells expressing concatenated human homomeric α7- or heteromeric α7β2-nAChR (Fig. 1A,B) to determine whether oAβ₄₂ modulation of α7- or α7β2-nAChR is dependent on the position and stoichiometric arrangement of α7 and β2 subunits within functional pentamers.

To validate these cells for single-channel electrophysiological experiments, we first stimulated with ACh at 300 μm, a typical EC₅₀ concentration for homomeric α7- and heteromeric α7β2-nAChR (Moretti et al., 2014). Cells visualized under fluorescence microscopy to coexpress nAChR-mCherry and NACHO-ZsGreen1 (Gu et al., 2016; Matta et al., 2017) were chosen and consistently expressed functional α7*-nAChR responding to ACh (Fig. 1C). Single-channel openings were seen as typical bursts of activation (examples shown in Fig. 1D) interspersed within longer periods of inactivity (corresponding to closed or desensitized states) (Nielsen et al., 2018). Channel openings also typically had a range of amplitudes and were short-lived. Because the latter could lead to underestimates of conductance state(s), and conductance state determinations could be confounded by simultaneous openings of more than one channel per patch, we chose to analyze openings only of the lowest-amplitude state (O1) consistently observed throughout this study because this could be guaranteed to correspond to a unitary conductance. Moreover, recordings done in the presence of bath-applied PNU 120596 (3 μm) aided determination of the amplitude of O1 by enhancing single-channel open probability and open-dwell times (Fig. 1D), as previously noted (Lasala et al., 2019).

We next used a pharmacological approach to determine whether openings elicited by ACh were genuinely produced by activation of α7- or α7β2-nAChR. Example data from individual patches are shown in Figure 2A–C. Single-channel events for each construct tested could be induced by ACh (300 μm) and could be suppressed by application of either the nonselective nAChR antagonist Meca (1 mm; Fig. 2A) or the α7*-nAChR selective antagonist MLA (10 nm; Fig. 2B). In either case, subsequent application of the α7*-selective PAM PNU 120596 enabled recovery of function, even in the presence of Meca or MLA. These results show that single-channel responses of human, heterologously expressed α7-nAChR formed from loose or linked subunits, or of human α7β2-nAChR heterologously expressed as two isoforms from concatenated subunits, are all similarly activated by exposure to ACh. Sensitivity to functional blockade by either Meca or MLA, or restoration of responses by PNU 120596 all demonstrate that the effects of ACh in this system are mediated via human α7*-nAChR.

Figure 2.

ACh and oAβ₄₂ evoke similar single-channel activated bursts from α7- and α7β2-nAChR. A, Representative recordings of the frequency of cell-attached single-channel bursts elicited with ACh (300 μm) from unlinked homomeric α7-nAChR, concatenated homomeric α7-nAChR, concatenated α7β2(P3)-nAChR, and concatenated α7β2(P2,P4)-nAChR. ACh is administered throughout the recording, via the patch pipette. Horizontal lines within each panel indicate the duration of Meca administration (orange line indicates 1 mm; bath administration at 15 min) and PNU 120596 (purple line indicates 3 μm; bath administration at 30 min). B, Same as in A, except with bath application at 15 min of MLA (red line indicates 10 nm). C, Representative traces of single-channel recordings from each of the four α7*-nAChR populations, responses elicited with ACh (gray traces) and verified with PNU 120596 administration (ACh + PNU; purple traces). C′, High-resolution images of single-channel recordings for each of the four α7*-nAChR elicited with ACh and verified with PNU 120596. Examples of single-channel bursts are boxed in each trace. Bursts were segregated from isolated events, and only bursts were used in all single-channel analyses. D, Representative recordings of the frequency of cell-attached single-channel bursts elicited with oAβ₄₂ (100 nm) from unlinked homomeric α7-nAChR, concatenated homomeric α7-nAChR, concatenated α7β2(P3)-nAChR, and concatenated α7β2(P2,P4)-nAChR. oAβ₄₂ is administered throughout the recording, via the patch pipette. Horizontal lines within each panel indicate the duration of Meca administration (orange line indicates 1 mm; bath administration at 15 min) and PNU 120596 (purple line indicates 3 μm; bath administration at 30 min). E, Same as in D, except with bath application at 15 min of MLA (red indicates 10 nm). F, Representative traces of single-channel recordings from each of the four α7*-nAChR populations, elicited with oAβ₄₂ (green traces) and verified with PNU 120596 administration (oAβ₄₂ + PNU; purple traces). F′, High-resolution images of single-channel recordings for each of the four α7*-nAChR elicited with oAβ₄₂ and verified with PNU 120596. Single-channel bursts are boxed in each trace. These bursts were segregated from isolated events and only bursts were used in all single-channel analyses.

oAβ₄₂ directly activates α7- and α7β2-nAChR

To address the lack of consensus about functional interactions between oAβ₄₂ and α7*-nAChR, we began by conducting single-channel recordings of SH-EP1 cells expressing human homomeric α7- or heteromeric α7β2-nAChR that were exposed to oAβ₄₂ (100 nm) in the patch pipette (Fig. 2D,E). We used exactly the same pharmacological approach as applied to the analysis of ACh-induced responses to demonstrate that these oAβ₄₂-evoked single-channel events also correspond to activation of α7*-nAChR. Similar to ACh exposure, oAβ₄₂-induced responses could be blocked by bath application of either Meca or MLA (Fig. 2D and Fig. 2E, respectively). Receptor function suppressed by Meca- or MLA-mediated blockade of oAβ₄₂-induced single-channel activity was restored by bath application of PNU 120596 (Fig. 2A,B and 2D,E, respectively).

To quantify the outcomes shown in typical traces for ACh-induced (Fig. 2A–C′) and oAβ₄₂-induced (Fig. 2D–F′) α7*-nAChR single-channel openings, a summary of data pooled across multiple patches containing homomeric α7- or heteromeric α7β2-nAChR isoforms is shown in Figure 3. Exposure to Meca (1 mm) significantly reduced the single-channel burst rate in the presence of ACh for all α7*-nAChRs tested (Fig. 3A). Further, for all α7*-nAChR isoforms studied, application of PNU 120596, subsequent to block by Meca alone, resulted in the recovery of ACh-induced single-channel bursting to a level indistinguishable from that before Meca administration. Similar results were observed for ACh-induced single-channel responses in the presence of MLA (Fig. 3B). Perfusion of MLA significantly reduced (>90%) single-channel burst rates in the presence of ACh for all α7*-nAChR isoforms, whether expressed as unlinked or concatenated subunits. Further, for all α7*-nAChR isoforms studied, application of PNU 120596 subsequent to block by Meca alone, resulted in the recovery of single-channel activity and bursting rates to be statistically indistinguishable to those before MLA application. Since MLA and PNU 120596 had to be prepared in a DMSO solution, we also tested whether DMSO at the final concentration used in these experiments (0.002% v/v; vehicle control) had any effect on bursting rate (Fig. 3C). No differences in single-channel burst rates were observed for unlinked α7, concatenated α7, concatenated α7β2(P3), or concatenated α7β2(P2,P4) nAChR. Following DMSO application, the addition of PNU 120596 increased bursting rate significantly over that induced by ACh alone.

Figure 3.

Analysis of ACh- and oAβ₄₂-induced single-channel burst rates. A, Single-channel burst rates (bursts/s) in the presence of ACh (300 μm) were calculated for patches containing unlinked homomeric α7-nAChR (red), concatenated homomeric α7-nAChR (orange), concatenated α7β2(P3)-nAChR (green), and concatenated α7β2(P2,P4)-nAChR (blue). Burst rates were recorded under the following experimental conditions: before Meca application, during Meca (1 mm) application simultaneously with ACh stimulation, and during coapplication of both Meca and PNU 120596 (3 μm) simultaneously with ACh stimulation. Application of Meca significantly reduced the mean single-channel burst rates in the presence of ACh for all α7*-nAChR populations investigated: unlinked α7-nAChR homopentamers (69.9 ± 2.9 to 2.0 ± 0.1; p = 0.00006; n = 5), concatenated α7-nAChR homopentamers (26.0 ± 1.0 to 3.0 ± 0.4; p = 0.00004; n = 5), concatenated α7β2(P3)-nAChR (24.8 ± 1.3 to 4.0 ± 0.5; p = 0.00003; n = 5), and concatenated α7β2(P2,P4)-nAChR (39.2 ± 2.5 to 4.9 ± 0.4; p = 0.00008; n = 5). Subsequent coapplication of PNU 120596 with Meca to the same patches reversed the effects of Meca blockade. ACh-induced single-channel burst rates were returned to values indistinguishable from those before Meca was applied for all α7*-nAChR populations studied: α7-nAChR (unlinked α7-nAChR = 72.6 ± 3.1, p = 0.73; concatenated α7-nAChR = 30.2 ± 1.6, p = 0.54) and α7β2-nAChR (α7β2(P3)-nAChR = 15.6 ± 1.6, p = 0.51; α7β2(P2,P4) = 40.7 ± 4.1 p = 0.92). B, Same as in A, except with the selective α7*-nAChR antagonist MLA (10 nm). Similar to Meca, MLA significantly reduced the ACh-induced single-channel burst rate for unlinked α7-nAChR (70.1 ± 4.6 to 3.4 ± 0.7, p = 0.00002, n = 6), concatenated α7-nAChR (21.8 ± 1.8 to 2.3 ± 0.4, p = 0.005, n = 4), concatenated α7β2(P3)-nAChR (28.9 ± 3.8 to 8.3 ± 1.1, p = 0.003, n = 4), and concatenated α7β2(P2,P4) nAChR (32.5 ± 3.5 to 4.3 ± 0.8, p = 0.00006, n = 5). Subsequent coapplication of MLA and PNU 120596 to the same patches reversed the MLA-induced reductions in burst rate, returning rates to levels indistinguishable from those before MLA was applied: unlinked α7-nAChR = 56.8 ± 3.2, p = 0.76; concatenated α7-nAChR = 18.1 ± 5.0, p = 0.68; concatenated α7β2(P3)-nAChR = 28.1 ± 2.6, p = 0.98; concatenated α7β2(P2,P4)-nAChR = 34.8 ± 3.3 p = 0.83). C, Same as in A, B, except with DMSO (vehicle) control (0.002%) instead of antagonist (Meca or MLA) application. No differences in mean single-channel burst rate were observed when moving from buffer only condition to the presence of the vehicle control for any of the α7*-nAChR constructs: unlinked α7-nAChR (54.4 ± 5.4 to 51.4 ± 3.2, p = 0.9, n = 5), concatenated α7-nAChR (26.5 ± 4.3 to 30.5 ± 5.0, p = 0.75, n = 5), concatenated α7β2(P3)-nAChR (21.0 ± 4.5 to 26.5 ± 4.1, p = 0.88, n = 4) or concatenated α7β2(P2,P4)-nAChR (40.9 ± 4.3 to 46.0 ± 4.8, p = 0.56, n = 5). Subsequent addition of PNU (3 μm) in the presence of the vehicle control increased single-channel burst rate significantly over those induced by ACh alone (α7 unlinked = 73.0 ± 2.6, p = 0.011, n = 5; α7 linked = 55.7 ± 2.8, p = 0.0007, n = 5; α7β2(P3) = 52.5 ± 5.2 p = 0.005, n = 4; α7β2(P2,P4) = 64.8 ± 5.4, p = 0.012, n = 5). D, Single-channel burst rates (bursts/s) in the presence of oAβ₄₂ (100 nm) were calculated for patches containing unlinked homomeric α7-nAChR (red), concatenated homomeric α7-nAChR (orange), concatenated α7β2(P3)-nAChR (green), and concatenated α7β2(P2,P4)-nAChR (blue). Burst rates were recorded under the following experimental conditions: before Meca application, during Meca (1 mm) application simultaneously with oAβ₄₂ stimulation, and during coapplication of both Meca and PNU 120596 (3 μm) simultaneously with oAβ₄₂ stimulation. Application of Meca significantly reduced the mean single-channel burst rates in the presence of oAβ₄₂ for all α7*-nAChR populations investigated: unlinked α7-nAChR homopentamers (63.5 ± 4.4 to 3.4 ± 1.1; p = 0.00003; n = 7), concatenated α7-nAChR homopentamers (23.0 ± 2.2 to 3.3 ± 1.4; p = 0.00001; n = 7), concatenated α7β2(P3)-nAChR (21.6 ± 1.3 to 3.0 ± 1.3; p = 0.00003; n = 7), and concatenated α7β2(P2,P4)-nAChR (38.0 ± 3.8 to 4.1 ± 1.5; p = 0.0003; n = 7). Subsequent coapplication of PNU 120596 with Meca to the same patches reversed the effects of Meca blockade. oAβ₄₂-induced single-channel burst rates were returned to values indistinguishable from those before Meca was applied for all α7*-nAChR populations studied: α7-nAChR (unlinked α7-nAChR = 65.7 ± 3.8, p = 0.63; concatenated α7-nAChR = 28.4 ± 2.2, p = 0.26) and α7β2-nAChR (α7β2(P3)-nAChR = 19.3 ± 1.7, p = 0.46; α7β2(P2,P4) = 42.3 ± 2.4 p = 0.22). E, Mean oAβ₄₂-induced single-channel burst rates calculated for patches containing unlinked α7-nAChR, concatenated α7-nAChR, concatenated α7β2(P3)-nAChR, and concatenated α7β2(P2,P4)-nAChR before and after application of the selective α7*-nAChR antagonist MLA (10 nm). Similar to Meca, MLA significantly reduced the single-channel burst rate for unlinked α7-nAChR (59.9 ± 2.9 to 4.5 ± 2.3, p = 0.00004, n = 7), concatenated α7-nAChR (22.8 ± 3.2 to 3.7 ± 1.8, p = 0.00003, n = 7), concatenated α7β2(P3)-nAChR (23.1 ± 2.0 to 1.2 ± 0.8, p = 0.00004, n = 7), and concatenated α7β2(P2,P4)-nAChR (36.8 ± 2.6 to 2.2 ± 0.9, p = 0.00002, n = 7). Subsequent coapplication of MLA and PNU 120596 to the same patches reversed the MLA-induced reductions in burst rate, returning rates to levels indistinguishable from those before MLA was applied: unlinked α7-nAChR = 58.5 ± 2.9, p = 0.13; concatenated α7-nAChR = 23.2 ± 2.4, p = 0.61; concatenated α7β2(P3)-nAChR = 30.3 ± 1.7, p = 0.54; concatenated α7β2(P2,P4)-nAChR = 42.5 ± 2.3 p = 0.58). F, Same as in A, B, except with DMSO (vehicle) control (0.002%) instead of antagonist (Meca or MLA) application. No differences in mean single-channel burst rate were observed when moving from buffer only condition to the presence of the vehicle control for any of the α7*-nAChR constructs: unlinked α7-nAChR (56.6 ± 4.9 to 49.0 ± 3.2, p = 0.25, n = 7), concatenated α7-nAChR (10.5 ± 1.2 to 10.3 ± 1.1, p =0.62, n = 7), concatenated α7β2(P3)-nAChR (25.7 ± 1.9 to 25.1 ± 1.5, p = 0.5, n = 7), or concatenated α7β2(P2,P4)-nAChR (24.3 ± 2.2 to 21.5 ± 2.0, n = 7, p = 0.56). Subsequent addition of PNU (3 μm) in the presence of the vehicle control increased single-channel burst rate significantly over those induced by oAβ₄₂ alone (α7 unlinked = 56.6 ± 25.0 to 75.2 ± 3.3, p = 0.003, n = 7; α7 linked = 10.5 ± 1.2 to 38.3 ± 4.3, p = 0.00005, n = 7; α7β2(P3) = 25.7 ± 1.9 to 56.5 ± 2.6 p = 0.00004, n = 7; α7β2(P2,P4) = 24.3 ± 2.2 to 58.5 ± 3.7, p = 0.00006, n = 7). For each group, n indicates individual patches. Bursts were segregated from isolated events, and only bursts were used in all single-channel analyses. All within-group comparisons were analyzed using one-way ANOVA with Tukey's post hoc test. Across-group comparisons between ACh and oAβ₄₂ were analyzed using two-way ANOVA with Tukey's post hoc test. A–F, Data are mean single-channel burst rate ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Comparable to the results observed for ACh, perfusion of Meca (1 mm) significantly reduced the single-channel burst rate in the presence of oAβ₄₂ for all α7*-nAChR isoforms tested (Fig. 3D; >85% suppression). Furthermore, application of PNU 120596, subsequent to block by Meca alone, resulted in the recovery of single-channel bursting to a level indistinguishable from that before Meca administration. Nearly equivalent results were observed for oAβ₄₂-induced responses in the presence of MLA (Fig. 3E). MLA significantly reduced (>85%) single-channel burst rates in the presence of oAβ₄₂ for all α7*-nAChR constructs. Further, for all α7*-nAChR isoforms studied, application of PNU 120596 subsequent to block by Meca alone resulted in the recovery of single-channel activity and bursting rates to be statistically indistinguishable to those before MLA application. Similar to the effects observed for ACh, no differences in single-channel burst rates were observed for unlinked α7, concatenated α7, concatenated α7β2(P3), or concatenated α7β2(P2,P4) nAChR in the presence of DMSO (0.002% v/v). Again, following DMSO application, the addition of PNU increased bursting rate significantly over that induced by oAβ₄₂ alone. Furthermore, an analysis of burst rate of all α7*-nAChR subtypes before drug application revealed no main effect of ligand (i.e., ACh vs oAβ₄₂), and no significant interaction was observed between construct and ligand. These results indicate that both ACh versus oAβ₄₂ are equally efficacious in activating the α7*-nAChR subtypes.

Next, we determined whether differences in single-channel burst rates were observed among α7*-nAChR subtypes tested or whether oAβ₄₂ differentially altered single-channel burst rates compared with ACh. A main effect of construct on single-channel burst rate was observed (F_(3,32) = 113.3; p = 0.00004). Further, we show that there is no main effect of ligand (F_(1,32) = 3.3; p = 0.08). Nor was a significant interaction observed between construct and ligand (F_(3,32) = 0.32; p = 0.81). These results indicate that ACh versus oAβ₄₂ are equally efficacious in activating the α7*-nAChR subtypes tested in this study. However, these experiments do not directly address whether oAβ₄₂ acts as a traditional agonist or as allosteric modulator. To address this question, we coapplied ACh (300 μM) and oAβ₄₂ (100 nm) to patches expressing α7- and α7β2-nAChR subtypes. Interestingly, the coapplication of ACh and oAβ₄₂ enhanced the single-channel burst rate of both α7- and α7β2-containing nAChR (F_(3,37) = 31.3; p = 0.00006). Post hoc analyses indicate that the coapplication of ACh and oAβ₄₂ increased the single-channel burst rate of both the α7 and α7β2(P2,P4) concatenated nAChR compared with ACh alone (24.9 ± 1.6 to 63.0 ± 7.3, p = 0.00008 and 37.5 ± 2.1 to 75.8 ± 9.1, p = 0.00005; respectively) or oAβ₄₂ alone (18.8 ± 1.8 to 63.0 ± 7.3, p = 0.00003 and 33.1 ± 2.1 to 75.8 ± 9.1, p = 0.00009; respectively). Collectively, these results demonstrate functional activation of human α7- and α7β2-nAChR by oAβ₄₂ as well as by ACh. Both Meca and MLA-mediated blockade, and restoration of function in the continued presence of antagonists by PNU 120596 support direct actions of oAβ₄₂ at α7*-nAChR. The lack of an effect of DMSO at the assay concentration used (0.002%) verifies that all of the observed outcomes are induced by administration of ACh, oAβ₄₂, or drug, but not vehicle.

oAβ₄₂ preferentially enhances the single-channel open-dwell times of α7β2-nAChR

Having demonstrated that oAβ₄₂ can directly activate α7- and α7β2-nAChR subtypes, we next examined whether the open-dwell times of oAβ₄₂-evoked single-channel events were similar to or different from those induced by the canonical agonist ACh. Single-channel open-dwell time distributions for all homomeric α7-nAChR and heteromeric α7β2-nAChR constructs were best fit with two open-dwell time components. This indicates the existence of two distinct populations of single-channel open durations in all cases, regardless of whether unlinked or concatenated nAChR subunit constructs were used (Fig. 4). This was true whether the openings were induced by ACh (Fig. 4A) or oAβ₄₂ (Fig. 4B).

Figure 4.

oAβ₄₂ preferentially enhances single-channel open-dwell times of α7β2-but not α7-nAChR. Single-channel open-dwell time distributions generated for α7 and α7β2- nAChR activated by (A) ACh (300 μm) or (B) oAβ_1-42 (100 nm). Single-channel open-dwell time distributions were best fit with 2 components for ACh-elicited single-channel bursts (solid green lines; dotted green line indicates overall fit) and oAβ₄₂-elicited single-channel bursts (solid red lines; dotted red line indicates overall fit). A, B, Vertical dashed lines indicate 1 ms open-dwell time duration, shown for reference. C, Mean single-channel open-dwell times were calculated for each open time constant (τ) observed for homomeric α7-nAChR in the presence of ACh or oAβ₄₂. No significant differences were seen in τ1 values (corresponding to shorter-duration openings) that were induced by ACh versus oAβ₄₂ (unlinked α7-nAChR, τ1 ACh = 0.3 ± 0.04 ms vs τ1 oAβ₄₂ = 0.26 ± 0.03 ms, p = 0.93, n = 7; concatenated α7-nAChR, τ1 ACh = 0.29 ± 0.04 ms vs τ1 oAβ₄₂ = 0.26 ± 0.04 ms, p = 0.90, n = 7). Nor were significant differences seen in τ2 values (corresponding to longer-duration openings) that were induced by ACh versus oAβ₄₂ (unlinked α7-nAChR, τ2 ACh = 1.4 ± 0.08 ms vs τ2 oAβ₄₂ = 1.8 ± 1.4 ms, p = 0.74, n = 7; concatenated α7-nAChR, τ2 ACh = 1.7 ± 0.12 ms vs τ2 oAβ₄₂ = 1.6 ± 0.14 ms, p = 0.91). One-way ANOVA summary: F_(3,24) range 85.1-68.3, p = 0.00008. D, Mean single-channel open-dwell times were also calculated for each open time constant (τ) observed for openings of heteromeric α7β2-nAChR in the presence of ACh or oAβ₄₂. Shorter-duration single-channel open-dwell times (indicated by τ1) evoked by the two ligands were indistinguishable for concatenated α7β2(P3)- or α7β2(P2,P4)-nAChR (α7β2(P3), τ1 ACh = 0.4 ± 0.06 ms vs τ1 oAβ₄₂ = 0.5 ± 0.06 ms, p = 0.87, n = 7; α7β2(P2,P4), τ1 ACh = 0.27 ± 0.06 ms vs τ1 oAβ₄₂ = 0.33 ± 0.05 ms, p = 0.94). However, oAβ₄₂ significantly enhanced the longer-duration openings (indicated by τ2) of concatenated α7β2-nAChR isoforms (α7β2(P3), τ2 ACh = 0.7 ± 0.07 ms vs τ2 oAβ₄₂ = 3.8 ± 0.18 ms, p = 0.0002, n = 7; α7β2(P2,P4), τ2 ACh = 0.92 ± 0.05 ms vs τ2 oAβ₄₂ = 4.8 ± 0.18 ms, p = 0.0007). One-way ANOVA summary: F_(3,24) range 245.1-430.6, p = 0.00009. Bursts were segregated from isolated events, and only bursts were used in all single-channel analyses. Individual data points represent mean open-dwell times for individual single-channel recordings. For each group, n indicates individual patches. All data analyzed using one-way ANOVA with Tukey's post hoc test. A-D, Data are mean single-channel burst rate ± SEM. ****p < 0.0001.

An analysis of the open-dwell time distributions of homomeric α7-nAChR (whether expressed from unlinked or concatenated subunits) revealed that single-channel open-dwell times for the shorter-duration population (τ1) evoked by either ACh or oAβ₄₂ were indistinguishable (Fig. 4C). Similarly, no differences in duration were observed for the longer-duration population of single-channel (τ2) openings in the presence of ACh versus oAβ₄₂. However, the heteromeric α7β2-nAChR presented a more complex picture. For α7β2(P3)- or α7β2(P2,P4)-nAChR, τ1 values (corresponding to the populations of shorter openings) were indistinguishable whether evoked by ACh or oAβ₄₂. However, τ2 values (corresponding to the populations of longer openings) were significantly extended in the presence of oAβ₄₂ compared with ACh (Fig. 4D; 3.1- and 3.9-fold, respectively). Together with the findings shown in Figure 3, these data unequivocally demonstrate that oAβ₄₂ activates both homomeric α7- and heteromeric α7β2-nAChR, and that it alters α7β2-nAChR kinetics by prolonging α7β2-nAChR open-dwell times compared with those evoked by the conventional agonist, ACh. Last, we determined whether coapplication of ACh and oAβ₄₂ altered the open-dwell times of α7*-nAChR, and we coapplied ACh (300 μM) and oAβ₄₂ (100 nm) to patches expressing concatenated α7- and α7β2-nAChR subtypes. Coapplication of ACh and oAβ₄₂ failed to modulate the single-channel open-dwell times of either α7 (τ1 = 0.192 ± 0.04 ms, p = 0.99, τ2 = 1.7 ± 0.33 ms, p = 0.99) or α7β2(P2,P4)-concatenated nAChR (τ1 = 0.224 ± 0.03 ms, p = 0.99, τ2 = 4.1 ± 0.36 ms, p = 0.99) compared with the single-channel open-dwell time elicited by oAβ₄₂ alone. In the case of the α7β2-nAChR, we would expect competitive antagonism to produce three distinct time constants (corresponding to short bursts produced by both ligands, medium duration bursts produced by ACh, and longer bursts produced by oAβ₄₂). Given these results, we conclude that oAβ₄₂ acts as ago-PAM (i.e., an agonist working through an allosteric site that is also capable of acting as a PAM in the presence of an agonist) at α7β2-containing nAChR. Since burst durations produced by both ligands at α7-nAChRs are indistinguishable, it remains an open question whether oAβ₄₂ also acts as an ago-PAM at this subtype.

Subnucleus-specific enhancement of BFCN excitability is mediated by oAβ₄₂/α7β2-nAChR interactions

The ability of Aβ to destabilize neuronal function has been well documented (Palop et al., 2007; Busche et al., 2012; Vossel et al., 2017). However, there is an inadequate understanding of (1) the molecular processes triggering BFCN dysfunction in response to elevations in oAβ₄₂ and (2) how BFCNs transition from stable neuronal activity to hyperactive dysfunction. First, we determined whether the interaction between oAβ₄₂ and α7*-nAChR leads to alterations in BFCN intrinsic excitability. We prepared basal forebrain organotypic slice cultures from ChAT-EGFP transgenic mice (postnatal day 7). These slices contained cholinergic neuronal populations from the MSDB (Fig. 5A), HDB (Fig. 5B), or the NB (Fig. 5C). Basal forebrain organotypic slices containing these nuclei were incubated for 9 d in oAβ₄₂ (100 nm), scrambled oAβ₄₂ (negative control; prepared under identical conditions to those used to prepare oAβ₄₂), oAβ₄₂ (100 nm) + MLA (50 nm; used to block all α7*-nAChR function). Since no highly selective α7β2-nAChR antagonist is available (Wu et al., 2016), organotypic slice cultures were also prepared from β2 nAChR KO mice to remove the possible contribution of α7β2-nAChR from recordings. Organotypic slices from β2 nAChR KO mice were also incubated for 9 d in oAβ₄₂ (100 nm). Following the 9 d incubation period, we used whole-cell current-clamp recordings and implemented a step current injection protocol (20 nA increments) using hyperpolarizing and depolarizing current pulses to measure voltage changes in BFCNs identified within these three regions. Representative current-clamp recordings are shown for the four conditions specified (Fig. 5A′–C′), and group data for numbers of action potentials elicited are shown (Fig. 5D–F).

Figure 5.

oAβ₄₂ induces BFCN hyperexcitation through the activation of α7β2-nAChR. A–C, Organotypic basal forebrain slice cultures prepared from ChAT-EGFP transgenic mice. BFCNs were identified for patch-clamp recordings from the MSDB (dashed circle; A), HDB (dashed circle; B), and the NB (dashed circle, C). A′-C′, Representative current-clamp recordings from MSDB (A′), HDB (B′), and NB (C′) cholinergic neurons. A current step protocol (−100 to 100 pA; 20 pA steps; below representative traces) was applied to MSDB, HDB, and NB BFCNs that were chronically incubated with a scrambled version of oAβ₄₂ (Scr; 100 nm; red), oAβ₄₂ (100 nm; green), or oAβ₄₂ + MLA (100 and 10 nm, respectively; orange). Organotypic slice cultures were also prepared from β2 nAChR subunit KO mice and incubated with oAβ₄₂ (100 nm; purple). D, MSDB BFCNs chronically exposed to oAβ₄₂ exhibited a significant increase in the number of action potentials generated across the entire range of depolarizing current injections compared with scrambled oAβ₄₂ controls (20 pA, oAβ₄₂ = 7.2 ± 1.2 vs scrambled oAβ₄₂ = 3.0 ± 0.6, F_(3,15) = 7.6, p = 0.003, Tukey's p = 0.02; 40 pA, oAβ₄₂ = 13.3 ± 1.9 vs scrambled oAβ₄₂ = 4.4 ± 1.2, F_(3,15) = 10.2, p = 0.0006, Tukey's p = 0.003; 60 pA, oAβ₄₂ = 16.8 ± 1.9 vs scrambled oAβ₄₂ = 8.2 ± 1.5, F_(3,15) = 7.6, p = 0.003, Tukey's p = 0.008; 80 pA, oAβ₄₂ = 19.0 ± 2.3 vs scrambled oAβ₄₂ = 10.6 ± 1.7, F_(3,15) = 5.3, p = 0.011, Tukey's p = 0.026; 100 pA, oAβ₄₂ = 21.3 ± 1.9 vs scrambled oAβ₄₂ = 12.8 ± 1.5, F_(3,15) = 7.4, p = 0.003, Tukey's p = 0.006; oAβ₄₂, n = 12; scrambled oAβ₄₂ controls, n = 12). The observed oAβ₄₂-induced increase in action potential firing rate was reversed with MLA or through genetic deletion of the β2 nAChR subunit (p > 0.05 for all current injections; compared with scrambled oAβ₄₂ controls; oAβ₄₂ + MLA, n = 10; β2 nAChR KO slices incubated with oAβ₄₂, n = 8). E, HDB BFCNs exposed to oAβ₄₂ also exhibited enhanced action potential firing rates across nearly all current injections (20 pA, oAβ₄₂ = 5.3 ± 1.6 vs scrambled oAβ₄₂ = 2.1 ± 0.5, F_(3,16) = 1.5, p = 0.26, Tukey's p = 0.29; 40 pA, oAβ₄₂ = 8.8 ± 1.4 vs scrambled oAβ₄₂ = 4.3 ± 0.8, F_(3,16) = 4.9, p = 0.014, Tukey's p = 0.033; 60 pA, oAβ₄₂ = 12.2 ± 1.8 vs scrambled oAβ₄₂ = 7.0 ± 0.8, F_(3,16) = 4.2, p = 0.025, Tukey's post hoc, p = 0.038; 80 pA, oAβ₄₂ = 15.6 ± 1.9 vs scrambled oAβ₄₂ = 9.3 ± 0.8, F_(3,16) = 6.6, p = 0.004, Tukey's p = 0.01; 100 pA, oAβ₄₂ = 19.3 ± 1.7 vs scrambled oAβ₄₂ = 11.0 ± 0.9, F_(3,16) = 11.1, p = 0.0003, Tukey's p = 0.0008; oAβ₄₂, n = 12; scrambled oAβ₄₂ controls, n = 12). Pharmacological antagonism with MLA or genetic deletion of the β2 nAChR subunit normalized this effect (Tukey's p > 0.05 for all current injections; compared with scrambled oAβ₄₂ controls; oAβ₄₂ + MLA, n = 12; β2 nAChR KO slices incubated with oAβ₄₂, n = 10). F, NB BFCNs exposed to oAβ₄₂ exhibited no differences in the number of action potentials generated under the same conditions compared with scrambled oAβ₄₂ controls (20 pA, no spikes elicited for oAβ₄₂ or scrambled oAβ₄₂ groups; 40 pA, oAβ₄₂ = 2.2 ± 0.3 vs scrambled oAβ₄₂ = 3.3 ± 0.3, F_(3,16) = 2.5, p = 0.10, Tukey's p = 0.11; 60 pA, oAβ₄₂ = 4.5 ± 0.5 vs Scrambled oAβ₄₂ = 4.5 ± 0.5, F_(3,16) = 0.03, p = 0.99, Tukey's p = 0.96; 80 pA, oAβ₄₂ = 5.5 ± 0.7 vs scrambled oAβ₄₂ = 6.3 ± 0.7, F_(3,16) = 1.3, p = 0.29, Tukey's p = 0.86; 100 pA, oAβ₄₂ = 7.2 ± 1.3 vs scrambled oAβ₄₂ = 7.8 ± 0.9, F_(3,16) = 0.47, p = 0.71, Tukey's p = 0.97; oAβ₄₂, n = 10; scrambled oAβ₄₂ controls, n = 12; oAβ₄₂ + MLA, n = 10; β2 nAChR KO slices incubated with oAβ₄₂, n = 10). G, Native/PAGE Western blotting to confirm soluble oAβ₄₂ assemblies. Neurobasal media was collected from organotypic slice cultures immediately (time point 0; T0) and 24 h after incubation (time point 24; T24). Large molecular weight oAβ₄₂ species (∼680 kDa) and their intermediates (∼250, 150, and 60 kDa) were present at both T0 and T24. Optical densitometry revealed no change in band intensity or shift in the relative size of soluble oAβ₄₂ assemblies at T0 and T24 (right), validating that basal forebrain organotypic cultures were consistently exposed to soluble, oAβ₄₂ and not insoluble protofibrillar forms of Aβ₄₂. All data analyzed using one-way ANOVA with Tukey's post hoc test. Data are the mean number of action potentials generated ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001.

Within the MSDB, BFCNs chronically exposed to oAβ₄₂ exhibited an increase in the number of action potentials generated across the entire range of depolarizing current injections compared with scrambled oAβ₄₂ controls (Fig. 5D). The observed oAβ₄₂-induced increase in spike rate was normalized either through pharmacological antagonism of α7*-nAChR with MLA or through genetic deletion of the β2 nAChR subunit. Similarly, organotypic slices incubated in oAβ₄₂ showed enhanced HDB BFCN action potential firing rates for nearly all current injections compared with scrambled oAβ₄₂ (Fig. 5E). Similar to MSDB neurons, inhibition of α7- and α7β2-nAChR with MLA or genetic deletion of the β2 nAChR subunit normalized the induced increase in HDB BFCN action potential firing rate compared with scrambled oAβ₄₂ controls.

By contrast, BFCNs within NB that were exposed to oAβ₄₂ exhibited no difference in spike number compared across NB BFCNs in organotypic slices incubated in scrambled oAβ₄₂, slices coincubated with oAβ₄₂ + MLA, or β2 nAChR KO slices incubated with oAβ₄₂ (Fig. 5F). As demonstrated by our single-channel studies (Fig. 2), activation by oAβ₄₂ can persist for >30 min, compared with acute macroscopic activation induced by a bolus of conventional agonist (which typically persists for milliseconds) (D. K. Williams et al., 2011). Indeed, the typically accepted explanation for extended closed-dwell times between bursts of openings is that these represent periods of desensitization (and that bursts of activity arise when the receptor recovers from desensitization). So, it seems safe to conclude that α7*-nAChR can exhibit a persistent pattern of desensitization and then recovery in the extended presence of oAβ₄₂. For these reasons, we hypothesized that, in the continued presence of oAβ₄₂, the persistent activation of α7β2-nAChR (exacerbated by prolonged open-dwell time induced by oAβ₄₂ at this subtype) contributes to BFCN decline through alterations in BFCN intrinsic excitability. Further, these results indicate an important distinction between BFCNs from these cholinergic nuclei, demonstrating that oAβ₄₂, interacting with α7*-nAChR, leads to enhanced BFCN intrinsic excitability within the MSDB and HDB but not within the NB. The most parsimonious explanation for the indistinguishable outcomes between MLA administration (which blocks function of all nAChR containing α7 subunits) and nAChR β2 subunit deletion is that the oAβ₄₂ effects seen in MSDB and HDB are dependent on oAβ₄₂ interactions with α7β2-nAChR and not homomeric α7-nAChR. It is possible that the absence of both α4β2- and α7β2-nAChR oAβ₄₂ can still increase the functional activity of the remaining homomeric α7-nAChR, in turn enhancing BFCN output and thereby rescuing cognitive deficits observed in either β2-nAChR KO or APP/PS1 mice. Complementary to our findings that APP/PS1-β2 KO mice show less impairment compared with APP/PS1 mice alone, genetic deletion of the α7-nAChR subunit in another mouse model of AD recovers learning and memory deficits associated with increased amyloid load (Dziewczapolski et al., 2009). This further supports the hypothesis that the critical α7*-nAChR involved in mediating the deleterious effects of oAβ₄₂ is the α7β2-nAChR subtype.

Alterations in BFCN spike AHP contribute to enhanced BFCN firing rate and are dependent on oAβ₄₂/α7β2-nAChR interactions

Next, we determined the effects of oAβ₄₂/α7β2-nAChR interactions on altering the medium phase of BFCN spike mAHP, a process that contributes to regulation of neuronal firing rate and is a key determinant in regulating neuronal and network-level excitability (Santos et al., 2009; S. Chen et al., 2014; Mateos-Aparicio et al., 2014; Deng et al., 2019). Organotypic slice preparations were prepared under the same experimental conditions as noted in the preceding section. As before, these slices contained BFCNs within the MSDB (Fig. 6A), HDB (Fig. 6B), and NB (Fig. 6C) cholinergic nuclei. Following the 9 d incubation period, we presented a single depolarizing current pulse (500 nA; 1 s) to BFCNs in each region and measured the magnitude of the mAHP (Fig. 6A′–C′). In two of the regions examined, MSDB and HDB, BFCNs exposed to oAβ₄₂ exhibited a significant reduction in the magnitude of the mAHP compared with scrambled oAβ₄₂ controls (Fig. 6A′-B′). However, the effect of oAβ₄₂ exposure was absent in MSDB and HDB BFCNs that were either coincubated in the presence of MLA or recorded from organotypic slices prepared from β2 nAChR KO mice. In these further controls, MSDB and HDB BFCNs showed no significant alteration in mAHP magnitude and outcomes were similar to those in the scrambled oAβ₄₂ controls (Fig. 6A′–B′).

Figure 6.

oAβ₄₂ enhancement of BFCN intrinsic excitability results from abrogated mAHP. A single step current injection (1 s; 500 pA; shown below representative traces) was used to measure mAHP magnitude from (A) MSDB, (B) HDB, or (C) NB BFCNs from ChAT-EGFP organotypic slice cultures incubated with oAβ₄₂ (green traces), oAβ₄₂ + MLA (orange traces), or scrambled oAβ₄₂ (gray traces). Organotypic slice cultures harvested from β2 nAChR subunit KO mice were incubated with oAβ₄₂ (purple trace) or scrambled oAβ₄₂ (gray trace). Insets, Area within dashed circle. A′, MSDB BFCNs exposed to oAβ₄₂ exhibited a significant reduction in the magnitude of the mAHP compared with scrambled oAβ₄₂ controls (oAβ₄₂, 8.2 ± 0.5 mV, n = 10; scrambled oAβ₄₂, 10.8 ± 0.4 mV, n = 9, F_(3,34) = 4.9; p = 0.006, Tukey's p = 0.022). This effect was reversed in MSDB BFCNs incubated with oAβ₄₂ in the presence of MLA or MSDB BFCNs recorded from β2 nAChR KO organotypic slice cultures incubated with oAβ₄₂ (oAβ₄₂ + MLA, n = 11, 10.6 ± 0.6 mV, Tukey's p = 0.99; β2 nAChR KO + oAβ₄₂, n = 9, 10.7 ± 0.7 mV, Tukey's p = 0.99). B′, HDB BFCNs exhibit similar, significant, reductions in mAHP amplitude after oAβ₄₂ exposure compared with scrambled oAβ₄₂ controls (oAβ_42, 8.8 ± 0.4 mV, n = 10; scrambled oAβ₄₂, 11.0 ± 0.4 mV, n = 10, F_(3,36) = 4.7, p = 0.007, Tukey's p = 0.012). HDB BFCNs incubated with oAβ₄₂ in the presence of MLA or recorded from β2 nAChR KO slices incubated with oAβ₄₂ showed similar mAHP magnitude compared with scrambled oAβ₄₂ controls (oAβ₄₂ + MLA, 10.7 ± 0.5 mV, n = 11, Tukey's p = 0.96; β2 nAChR KO + oAβ₄₂, 10.9 ± 0.6 mV, n = 10, Tukey's p = 0.99). C′, NB BFCNs showed no attenuation in mAHP amplitude after oAβ₄₂ treatment and were similar to control groups (oAβ₄₂, 8.9 ± 0.4 mV, n = 10; scrambled oAβ₄₂, 9.6 ± 0.5 mV, p = 0.56, n = 10; MLA, 8.7 ± 0.6 mV, p = 0.96, n = 10; β2 nAChR KO + oAβ₄₂, 9.2 ± 0.6 mV, p = 0.9, n = 8; F_(3,34) = 0.72; p = 0.54). Symbols represent individual recordings. All data analyzed using one-way ANOVA with Tukey's post hoc test. Symbols represent individual recordings. Data are mean mAHP amplitude ± SEM. *p < 0.05.

Conversely, BFCNs recorded from the NB exhibited no attenuation in mAHP amplitude after oAβ₄₂ treatment; mAHP amplitudes were statistically indistinguishable from those of each of the control groups (Fig. 6C′). Together, these results demonstrate that oAβ₄₂-induced enhanced firing rate of distinct cholinergic populations (as seen in Fig. 5) may result from a reduction in BFCN mAHP magnitude. As in the preceding section, the indistinguishable outcomes between MLA and β2 nAChR KO controls suggest strongly that oAβ₄₂-induced attenuation in MSDB and HDB BFCN mAHP magnitude is dependent on the interaction between oAβ₄₂ and α7β2-nAChR. Furthermore, these data potentially link oAβ₄₂-α7β2-nAChR interactions to functional modulation of intrinsic ionic mechanisms [e.g., small (SK) and/or large (BK) potassium channels] that are known to mediate neuronal firing rates (Bean, 2007).

oAβ₄₂ exposure leads to alterations in BFCN action potential dynamics and is dependent on α7β2-nAChR activation

Next, we investigated the effects of oAβ₄₂ on BFCN action potential waveform by measuring the maximal rates of membrane voltage change during BFCN spike depolarization and repolarization. As in the preceding two sections, basal forebrain organotypic slices were prepared from ChAT-EGFP and β2 nAChR KO nonlittermates containing MSDB, HDB, and NB nuclei, and the same 9 d incubation protocols were applied. A ramp current injection protocol (0.1 pA/ms; 100 pA max) was used to elicit a train of action potentials from BFCNs, and phase-plane portraits were generated for MSDB, HDB, and NB BFCNs from ChAT-EGFP and β2 nAChR KO mice by plotting the maximal first-order derivative of the BFCN somatic membrane potential (dV/dt_max) as a function of BFNC membrane potential (Fig. 7A–C).

Figure 7.

oAβ₄₂-induced alterations BFCN action potential dynamics. A–C, Representative phase plane portraits [i.e., maximal rate of voltage change (dV/dt_max) plotted as a function of membrane potential] of BFCN action potential waveforms from MSDB, HDB, or NB BFCNs chronically incubated with scrambled oAβ₄₂ (100 nm; red traces), oAβ₄₂ (100 nm; green traces), or oAβ₄₂ (100 nm) + MLA (50 nm; orange traces). Additional controls included MSDB, HDB, and NB BFCNs from β2 nAChR KO animals chronically incubated with oAβ₄₂ (100 nm; purple traces). Insets, Action potentials generated with a ramp current injection protocol (0.1 pA/s; 100 pA max). Vertical dashed lines indicate action potential threshold. Horizontal dashed lines indicate dV/dt_max = 0. A′–A′′, Comparisons of MSDB BFCN dV/dt_max for the first 10 spikes generated. Incubation in oAβ₄₂ progressively and significantly reduced dV/dt_max during the depolarizing phase in MSDB BFCNs compared with scrambled oAβ₄₂ controls (ANOVA summary: F_(3,38) = 100.2, p = 0.00008; Tukey's p values: spikes 1-10 = 0.000036; oAβ₄₂, n = 12; scrambled oAβ₄₂, n = 12). Conversely, MSDB BFCNs exposed to oAβ₄₂ exhibited a significant increase in dV/dt_max during spike repolarization (ANOVA summary: F_(3,36) = 39.9, p = 0.00003; Tukey's p values: spike 1 = 0.98, spike 2 = 0.14, and spikes 3-10 = 0.000073; oAβ₄₂, n = 12; scrambled oAβ₄₂, n = 12). B′, B′′, Comparisons of HDB BFCN dV/dt_max during spike depolarization and repolarization. HDB BFCNs exposed to oAβ₄₂ exhibited a progressive and significant decrease dV/dt_max during spike depolarization compared with scrambled oAβ₄₂ controls (ANOVA summary: F_(3,41) = 70.2, p = 0.00009; Tukey's p values: spikes 1-10 = 0.00005; oAβ₄₂, n = 12; scrambled oAβ₄₂, n = 11) and a significant increase in dV/dt_max during spike repolarization compared with scrambled oAβ₄₂ controls (ANOVA summary: F_(3,41) = 24.7, p = 0.00002; Tukey's p values: spike 1 = 0.86, spike 2 = 0.03, spike 3 = 0.0002, spikes 4-10 = 0.00008; oAβ₄₂, n = 12; scrambled oAβ₄₂, n = 11). C′, C′′, Comparisons of NB BFCN dV/dt_max during spike depolarization and repolarization. An effect of treatment on NB BFCN dV/dt_max was observed [ANOVA summary (depolarization): F_(3,40) = 13.5; p = 0.00006; ANOVA summary (repolarization): F_(3,40) = 5.4; p = 0.003]. However, post hoc comparisons revealed no differences in dV/dt_max between NB BFCNs exposed to oAβ₄₂ versus scrambled oAβ₄₂ controls during spike depolarization or repolarization [Tukey's p values (depolarization): spikes 1-10, p = 0.85; Tukey's p values (repolarization): spikes 1-10, p = 0.95; oAβ₄₂, n = 12; scrambled oAβ₄₂, n = 10]. D, MSDB, HDB, and NB BFCN threshold analysis revealed no differences in spike threshold after oAβ₄₂ treatment (MSDB, F_(3,38) = 0.26, p = 0.85, oAβ₄₂, −41.0 ± 1.0 mV vs scrambled controls −40.2 ± 0.5 mV, Tukey's p = 0.84; HDB, F_(3,42) = 4.9, p =0.005, oAβ₄₂, −43.0 ± 0.6 mV vs scrambled controls −43.1 ± 0.5 mV, Tukey's p = 0.99; NB, F_(3,38) = 0.78, p = 0.51, oAβ₄₂, −40.1 ± 0.5 mV vs scrambled controls −39.8 ± 0.7 mV, Tukey's p = 0.98). E, Incubation with oAβ₄₂ reduced time to first spike in MSDB (F_(3,38) = 10.2, p = 0.00002, oAβ₄₂, 151.0 ± 14.6 ms vs scrambled oAβ₄₂, 257.7 ± 16.5 ms; Tukey's p = 0.0001) and HDB BFCNs (F_(3,42) = 7.1, p = 0.0006, oAβ₄₂, 179.3 ± 8.6 ms, scrambled oAβ₄₂, 262.8 ± 20.4 ms; p = 0.001) but not NB BFCNs (F_(3,40) = 0.4, p = 0.76, oAβ₄₂, 282.0 ± 14.4 ms, scrambled oAβ₄₂, 256.3 ± 17.9 ms; p = 0.99). F, oAβ₄₂-induced reduction in spike amplitude was observed in MSDB neurons (F_(7,72) = 31.9, p = 0.00003, oAβ₄₂ last spike [closed red circles], 57.9 ± 1.6 mV, scrambled oAβ₄₂ last spike [closed green circles], 84.4 ± 2.2 mV; p = 0.0006) and HDB neurons (F_(7,72) = 35.5, p = 0.0008, oAβ₄₂ last spike, 64.1 ± 1.9 mV, scrambled oAβ₄₂ last spike, 92.2 ± 1.8 mV; p = 0.0003), but not NB neurons (F_(7,72) = 4.4, p = 0.0004, oAβ₄₂ last spike, 83.7 ± 1.8 mV, scrambled oAβ₄₂ last spike, 90.4 ± 1.7 mV; p = 0.0009). Differences in dV/dt_max across groups were analyzed using two-way ANOVA with Tukey's post hoc test. Action potential threshold, time to first spike, and action potential amplitude were analyzed using one-way ANOVA with Tukey's post hoc test. Data are mean mAHP amplitude ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Chronic incubation in oAβ₄₂ progressively and significantly reduced the dV/dt_max during the depolarizing phase of the spike train in MSDB (Fig. 7A′) and HDB BFCNs (Fig. 7B′). This effect of oAβ₄₂ was reversed in MSDB and HDB BFCNs incubated in the presence of scrambled oAβ₄₂, those coincubated with oAβ₄₂ + MLA, and was absent in those slices prepared from β2 nAChR KO mice. Notably, chronic incubation in oAβ₄₂ was without effect on dV/dt_max during the depolarizing phase of the spike train in BFNCs located in NB (Fig. 7C′). The effects of oAβ₄₂ were not limited to the action potential depolarization phase. Further analysis of outcomes in MSDB and HDB BFCNs revealed a progressive and significant oAβ₄₂-induced increase in the dV/dt_max during action potential repolarization in the spike train of MSDB (Fig. 7A′′) and HDB BFCNs (Fig. 7B′′) compared with basal forebrain organotypic slices incubated in the scrambled oAβ₄₂ control. The observed oAβ₄₂-induced increase in action potential repolarization rates was also reversed by coincubation with oAβ₄₂ + MLA, and absent in slices prepared from β2 nAChR KO mice, in MSDB (Fig. 7A′′) and HDB (Fig. 7B′′) BFCNs. Further, chronic incubation of NB BFCNs in oAβ₄₂ did not change dV/dt_max compared with that measured in the same neurons after chronic incubation with scrambled oAβ₄₂ (Fig. 7C′′).

While no significant changes were observed in BFCN action potential threshold after oAβ₄₂ administration (Fig. 7D), MSDB and HDB BFCNs exposed to oAβ₄₂ exhibited a significant reduction in the latency to spike compared with scrambled oAβ₄₂ controls (Fig. 7E). This effect was normalized in MSDB and HDB BFCNs coincubated in oAβ₄₂ + MLA, and absent in MSDB and HDB BFCNs recordings from slices prepared from β2 nAChR KO mice and incubated with oAβ₄₂ alone. Furthermore, MSDB and HDB BFCNs exposed to oAβ₄₂ showed a significant reduction in action potential amplitude comparing the amplitude of the first spike generated to the last spike generated in the train of action potentials (Fig. 7F). Again, these changes in action potential amplitude in MSDB and HDB BFCNs were lost following coincubation of slices with oAβ₄₂ + MLA, or in recordings from slices prepared from β2 nAChR KO mice incubated in oAβ₄₂. No alterations were seen in NB BFCNs incubated with oAβ₄₂, across any of these measures (action potential threshold, time to first spike, or action potential amplitude). Overall, these results demonstrate the ability of oAβ₄₂ to modulate several aspects of BFCN action potential waveform, including reduced rates of spike depolarization and repolarization, latency to generate action potentials, and reduced action potential amplitude. They also demonstrate a regional specificity to oAβ₄₂-induced changes, which were consistently seen in BFCNs of the MSDB and HDB, but not those from the NB.

Genetic deletion of the β2 nAChR subunit ameliorates deficits in spatial reference memory in the APP/PS1 mouse model of AD

Expression of human APP and Aβ in transgenic mice elicits several AD-like neuropathological phenotypes that correlate strongly with aberrant neuronal activity and impairments in learning and memory (Palop et al., 2007). To determine whether nAChR that contain β2 subunits (i.e., including α7β2-nAChR) mediate the cognitive deficits observed in the APP/PS1 mouse model of AD, we genetically deleted the β2 nAChR subunit gene (thus eliminating all α7β2-nAChR expression within the CNS) in the APP/PS1 transgenic mouse model. We then assessed the acquisition and overnight retention of spatial reference memory using the MWM test (Fig. 8).

Figure 8.

Genetic deletion of the β2 nAChR subunit gene alleviates spatial reference memory deficits in APP/PS1 mice. WT (green), β2 nAChR KO (purple), APP/PS1 (red), and APP/PS1-β2 KO (blue) groups were tested for spatial reference memory on days 1-6 (4 trials/day) with an additional fifth trial to test for spatial localization of the platform location. A, A′, Effect of genotype on latency to the platform across the first 6 d of testing and mean latency to platform collapsed across days, respectively. B, B′, Effect of genotype on swim distance across the first 6 d of testing and mean latency to the platform collapsed across days, respectively. C–E, Probe trial data (day 6, Trial 5) measuring number of target quadrant entries, swim duration (% of total), swim distance (% of total) in target (T) or opposite (O) quadrants of the MWM. F, Heat maps illustrating localization patterns in the maze on day 1 (first row), day 6 (second row), and the probe trial whereby the platform was removed (third row). G, Overnight forgetting calculated between days. H, H′, Visual platform tests (average latency for each group at three different maze locations) conducted following spatial reference memory testing to confirm that all animals possessed the visual and motor competence to solve the swimming maze task. C-E, H′, Data points represent individual animals. APP/PS1-β2 nAChR KO, N = 7; APP/PS1, N = 8; β2KO, N = 9; WT mice, N = 7. Data analyzed using omnibus repeated-measures ANOVA with Fisher's LSD post hoc test. Student's t test was used for within-group comparisons. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

For both latency to platform and distance traveled, repeated-measures ANOVA revealed a main effect of Genotype (Latency: F_(3,28) = 5.70, p = 0.006; Distance: F_(3,28) = 10.25, p = 0.00003) and Day (Latency: F_(5,140) = 27.27, p = 0.00004; Distance: F_(5,140) = 49.62, p = 0.00006). A significant Genotype × Day interaction was observed for Latency (Fig. 8A; F_(15,140) = 2.59, p = 0.0004), but not Distance (Fig. 8B; F_(15,140) = 1.53, p = 0.10). Post hoc tests for Latency revealed that the WT group differed from the β2 nAChR KO group and the APP/PS1 group, but not the APP/PS1-β2KO group (Fig. 8A′; p = 0.001, p = 0.0003, and p = 0.10, respectively). Furthermore, the APP/PS1 group differed from APP/PS1-β2KO group (Fig. 8A′; p = 0.018). Post hoc tests for Distance revealed that the WT group differed from β2 nAChR KO, APP/PS1, and APP/PS1-β2KO groups (Fig. 8B′; p = 0.00007, p = 0.00008, and p = 0.023, respectively). The APP/PS1 group also differed from the APP/PS1-β2KO group (Fig. 8B′; p = 0.033). Together, these data suggest that, in aged mice, genetic deletion of the β2 nAChR subunit in APP/PS1 mice resulted in improved learning during acquisition of the spatial reference memory task.

To test for spatial localization of the platform, a probe trial (whereby the platform was removed) was conducted on day 6 Trial 5. One-way ANOVA revealed a main effect of Genotype for target quadrant preference (Fig. 8C; F_(3,28) = 5.01; p = 0.004). Post hoc tests for number of target quadrant entries revealed that WT and APP/PS1-β2KO groups exhibited a greater number of target quadrant entries compared with opposite quadrant entries (Fig. 8C; p = 0.00002 and p = 0.0004, respectively). Target quadrant preference was absent in the APP/PS1 and the β2 nAChR KO groups (p = 0.35 and 0.54, respectively), indicating that these groups did not localize the platform. A main effect of Genotype was observed for swim duration in the target quadrant (Fig. 8D; F_(3,28) = 8.88; p = 0.00007). Post hoc test revealed that WT and APP/PS1-β2KO groups spent significantly more time in the target quadrant (T) compared with time spent in the opposite (O) quadrant (Fig. 8D; p = 0.00008 and p = 0.00005, respectively). APP/PS1-β2KO groups were similar to WT animals (p = 0.99). However, APP/PS1-β2KO mice spent significantly more time in the target quadrant than APP/PS1 mice (p = 0.0036). A main effect of Genotype was also observed for swim distance in the target quadrant (Fig. 8E; F_(3,28) = 7.62; p = 0.0006). Post hoc tests revealed that WT and APP/PS1-β2KO groups traveled further in the target quadrant (T) compared with the swim distance in the opposite (O) quadrant (Fig. 8E; p = 0.00006 and p = 0.019, respectively) and were indistinguishable from each other on this measure. Conversely, the β2 nAChR KO and APP/PS1 KO groups failed to localize to the target quadrant (p = 0.94 and 0.97, respectively). Interestingly, the APP/PS1-β2KO group swam a greater distance within the target quadrant compared with the APP/PS1 group (p = 0.48). These data demonstrate that the deficits in the acquisition of spatial reference memory and spatial localization of the platform location are ameliorated in APP/PS1 mice genetically null for the β2 nAChR subunit.

Next, we examined overnight forgetting by comparing Trial 4 of each day to Trial 1 of the next day, with these scores as repeated measures. A main effect of Trial (Fig. 8G; F_(3,84) = 5.60, p = 0.002) and Day (F_(5,140) = 27.27, p = 0.00007) was observed for Latency. A significant Trial × Day interaction was also observed for Latency (F_(15,420) = 1.75, p =0.013). Post hoc analysis revealed that no overnight forgetting was observed in WT groups or APP/PS1-β2KO groups (Fig. 8G; p = 0.76 and p = 0.25, respectively). However, overnight forgetting was observed for APP/PS1 groups (p = 0.022) and β2 nAChR KO groups (p = 0.021) between days 3 and 4 of testing. These results demonstrate an important role of β2-containing nAChR in mediating day-to-day spatial reference memory retention in the APP/PS1 rodent model of AD.

Discussion

These findings demonstrate oAβ₄₂, interacting with α7β2-nAChR, plays a crucial role in BFCN functional instability and may contribute to cognitive deficits commonly observed during the etiopathogenesis of AD. We show that oAβ₄₂ directly activates α7*-nAChR and preferentially enhances the open-dwell time kinetics of heteromeric α7β2-nAChR. Further, oAβ₄₂ interacting with both α7- and α7β2-nAChR induces neuronal hyperexcitation in specific BFCN subpopulations by increasing action potential firing rates. This effect is mediated, in MSDB and HDB BFCNs, by (1) altered action potential waveforms and (2) diminished action potential AHP. Both effects can be normalized through pharmacological antagonism or genetic deletion of α7β2-nAChR. Furthermore, we demonstrate that genetic deletion of β2-nAChR subunits (and, therefore, α7β2-nAChR subtypes) in vivo ameliorates spatial reference memory deficits in the APP/PS1 mouse AD model.

At the molecular level, studies examining α7*-nAChR macroscopic currents provide disparate evidence regarding α7*-nAChR activation by oAβ₄₂ (Dineley et al., 2002; Fu and Jhamandas, 2003; Liu et al., 2009). Macroscopic activation of α7*-nAChR by conventional agonists is sensitive to the timing and concentration of agonist application (Uteshev et al., 2002); this may be even more critical for oAβ₄₂. Accordingly, single-channel recordings provide a key technical advantage, as recently shown for homomeric α7-nAChR (Lasala et al., 2019). In our present study, we show unequivocally that oAβ₄₂ activates α7- and α7β2-nAChR. Here, oAβ₄₂-induced single-channel open-dwell times match those induced by ACh, with one exception. Openings of α7β2-nAChRs are prolonged by oAβ₄₂ compared with ACh. Our findings suggests that, while homomeric α7-nAChR certainly may be targets for pathogenic oAβ₄₂ effects (as also demonstrated by Lasala et al., 2019), the function of the narrowly distributed α7β2-nAChR population may be disproportionately enhanced by oAβ₄₂. Further, oAβ₄₂'s ability to lengthen α7β2-nAChR openings resembles that of a PAM (D. K. Williams et al., 2011; Andersen et al., 2016), indicating that oAβ₄₂ has PAM activity at α7β2-nAChR, but not α7-nAChR. An allosteric mechanism fits with spectroscopic studies suggesting that oAβ₄₂ does not occupy orthosteric binding sites (Lasala et al., 2019). It also suggests the novel possibility that introducing β2 subunits adds an allosteric oAβ₄₂ binding site, perhaps at α7/β2 subunit interfaces.

A wide range of oAβ₄₂ concentrations is observed in AD patients (low pM to low µM range), and the effects of oAβ₄₂ at 100 nm observed here fall within the neuropathologically confirmed range in AD (Yang et al., 2013, 2017). Synthetic oAβ₄₂ prepared under carefully defined conditions likely replicates the forms adopted by oAβ₄₂ in research animals or AD patients, and translational relevance is supported by studies using Aβ-containing extracts from human AD subjects (Shankar et al., 2008; Puzzo et al., 2017).

In most brain regions, α7*-nAChR contain only α7 subunits. However, α7β2-nAChRs are expressed in restricted neuronal populations, including BFCNs (Azam et al., 2003). Inspired by our finding that α7β2-nAChRs are sensitive to functional modulation by oAβ₄₂, we examined the effects of oAβ₄₂ on BFCN function. Our central novel observation was that long-term exposure to oAβ₄₂ enhanced spike firing rates of MSDB and HDB BFCNs. Critically, these oAβ₄₂ effects are blocked by the α7-nAChR-selective antagonist, MLA. This hyperexcitatory effect of oAβ₄₂ could be viewed as a maladaptive response to sustained stimulation by oAβ₄₂ of MSDB and HDB BFCNs expressing α7*-nAChR. Moreover, oAβ₄₂ effects are abrogated in organotypic cultures prepared from β2-nAChR KO mice. Since the outcomes of α7*- or α7β2-nAChR functional loss are indistinguishable, the most parsimonious explanation is that α7β2-nAChRs are necessary and sufficient to mediate oAβ₄₂-induced hyperexcitation of MSDB and HDB BFCNs. It is possible that interactions of oAβ₄₂ with both α7*- and α7β2-nAChR could be important. Unfortunately, no pharmacological agents exist to isolate α7- from α7β2-nAChR (Wu et al., 2016). As another example, interactions of oAβ₄₂ with other β2*-nAChR may contribute to the phenomena observed. Certainly, α4β2-nAChR expression seems likely on at least a subset of BFCNs, and such a nAChR population would be well placed to affect functional and behavioral outcomes.

Localized expression of α7β2-nAChR on BFCNs (Liu et al., 2009; Moretti et al., 2014) may significantly contribute to these neurons' loss in early AD. Elevated oAβ₄₂ may be particularly damaging to BFCNs by provoking longer-lasting openings of α7β2-nAChR than does ACh. This effect may be exacerbated by oAβ₄₂'s persistence versus transient exposure to ACh, potentially prolonging otherwise self-limiting responses to ACh. Thus, BFCNs will be especially vulnerable compared with the majority of neurons expressing only homomeric α7-nAChR.

Our observations also lend insights into the active mechanisms mediating oAβ₄₂-induced enhancement of BFCN firing rates. In BFCNs from MSDB and HDB, but not from NB, we observed that oAβ₄₂ (1) reduces mAHP amplitude, (2) alters the maximal rate of voltage change during BFCN spike depolarization and repolarization, and (3) reduces latency to spike generation. These processes, which contribute to the intrinsic electrical characteristics of many neurons, are shaped by the activity of specific Na⁺, Ca²⁺, and K⁺ channel subtypes (Bean, 2007). Prior studies point to the role of oAβ₄₂ in altering voltage- and calcium-dependent ion channels that govern neuronal excitability (Nimmrich et al., 2008; Alier et al., 2011; Gavello et al., 2018). Our findings, therefore, could reflect changes in the activity of Ca²⁺ and/or K⁺ channels following sustained oAβ₄₂ exposure.

In contrast, NB BFCNs are insensitive to oAβ₄₂-induced hyperexcitation. This might reflect reduced prevalence of α7β2-nAChR in NB BFCNs. While PCR-based studies confirm α4-nAChR mRNA expression in MSDB BFCNs (Liu et al., 2009), ISH indicates that this is much more prominent in NB BFCNs, and in noncholinergic cells of the medial septum and mesopontine tegmentum (Azam et al., 2003). Accordingly, a greater proportion of NB β2-containing-nAChRs are likely α4β2-nAChR, possibly reducing α7β2-nAChR expression in this region. Furthermore, clinical studies have documented α7-nAChR subunit mRNA upregulation in NB BFCNs of human AD patients (Counts et al., 2007). This could bias the expression toward homomeric α7-nAChR. The results shown here demonstrate subregion heterogeneity of BFCN functional output with potential clinical importance.

Previous studies have shown that α7*-nAChRs are involved in hippocampal-dependent synaptic plasticity, and the hippocampus receives extensive MSDB and HDB BFCN innervation (Fabian-Fine et al., 2001; Gu et al., 2012; Changeux et al., 2015). In contrat, NB cholinergic projections primarily innervate the neocortex (mPFC), mediating top-down saliency of attention and working memory formation (Gusnard et al., 2001). Our in vitro studies demonstrate susceptibility of MSDB and HDB, but not NB, BFCNs to oAβ₄₂-induced neuronal instability through α7β2-nAChR activation. Accordingly, we postulate that deleterious effects of elevated oAβ₄₂ levels in APP/PS1 transgenic mice, working through α7β2-nAChR expressed on MSDB and HDB cholinergic neurons, correlate with spatial reference memory deficits.

Importantly, other studies have shown deleterious effects of constitutive β2 KO alone on elements of memory in aged animals (Picciotto et al., 1995, 1998; Changeux et al., 1998; Zoli et al., 1999; Caldarone et al., 2000). This is compatible with our own observations of cognitive deficits in β2 KO mice relative to WT controls. While we believe that α7β2-nAChRs are key mediators of the cognitive deficits observed in APP/PS1 mice, the poor performance of β2-nAChR KO mice during MWM testing may be because of the absence of α4β2-nAChR. Indeed, α7*-nAChR activity enhances LTP and memory in rodent models (Puzzo et al., 2008) and physiological roles for Aβ-induced memory enhancement have been proposed (Morley et al., 2010). Our novel finding is that deficits in spatial memory, which are observed in β2-nAChR KO as well as APP/PS1 mice, are neutralized in APP/PS1-β2 KO mice. One possible explanation is, in the absence of both α4β2- and α7β2-nAChR, oAβ₄₂ can still increase the functional activity of the remaining homomeric α7-nAChR, in turn enhancing BFCN output and thereby rescuing cognitive deficits observed in either β2-nAChR KO or APP/PS1 mice. Complementary to our findings that APP/PS1-β2 KO mice show less impairment compared with APP/PS1 mice alone, genetic deletion of the α7 nAChR subunit in another mouse model of AD recovers learning and memory deficits associated with increased amyloid load (Dziewczapolski et al., 2009). This further supports the hypothesis that the critical α7*-nAChR involved in mediating the deleterious effects of oAβ₄₂ is the α7β2-nAChR subtype.

Our results clearly link α7β2-nAChR to BFCN hyperexcitation, expanding on prior findings that Aβ exposure increases neuronal activity (Palop et al., 2007; Busche et al., 2008) and that persistent neuronal excitability elevates Aβ levels (Bero et al., 2011). Thus, we provide a working model (Fig. 9) describing early elevations in oAβ₄₂ leading to activation of α7*-nAChR and persistent α7β2-nAChR functional enhancement. Enhanced α7β2-nAChR signaling alters the function of BFCN intrinsic ionic mechanisms (e.g., Ca²⁺ and/or K⁺ channels) mediating BFCN excitability, producing BFCN hyperexcitation. These mechanisms, coupled with increased levels of oAβ₄₂ possibly because of activity-dependent synaptic release (Cirrito et al., 2008), may induce a destabilizing feedback loop further elevating oAβ₄₂ levels and, in turn, lead to BFCN degeneration and memory decline. Bringing further potential translational relevance, such hyperexcitation may also contribute to observations that seizures and subclinical epileptiform activity are increased in early AD (Vossel et al., 2013), together with hyperexcitability in neuronal circuits (O'Brien et al., 2010; Petrache et al., 2019).

Figure 9.

Working model for the role of α7β2-nAChR in oAβ₄₂-induced BFCN hyperexcitation and cognitive decline. Early and sustained exposure to oAβ₄₂ causes the activation and functional enhancement of α7β2-nAChR (gray arrow). This leads to alterations in BFCN intrinsic mechanisms (e.g., BFCN AHP and spike waveform) possibly through the functional dysregulation of voltage and/or calcium-dependent ion channels (green arrow). As a result, these hyperexcited BFCNs may degenerate, leading to a decline in spatial reference memory processing (dotted red line). Simultaneously, BFCN hyperexcitability may lead to increased presynaptic release of oAβ₄₂ (Cirrito et al., 2008), perpetuating this vicious cycle, leading to further BFCN death.

Footnotes

This work was primarily supported by Arizona Biomedical Research Commission ADHS14-083003 to A.A.G., Arizona Alzheimer's Consortium to A.A.G., and Barrow Neurological Foundation endowment and capitalization funds to A.A.G. This work was also supported by the National Institutes of Health R21 AG067029 to A.A.G. and R01 DA 043567 to A.A.G. and P.W. and R01 DA042749 to P.W. We thank Jason A. Miranda and Dale J. Buskirk for critical feedback on the manuscript; and Linda M. Lucero for technical assistance with concatemer design and construction.
The authors declare no competing financial interests.
Correspondence should be addressed to Andrew A. George at Andrew.George{at}dignityhealth.org

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Implications of Oligomeric Amyloid-Beta (oAβ42) Signaling through α7β2-Nicotinic Acetylcholine Receptors (nAChRs) on Basal Forebrain Cholinergic Neuronal Intrinsic Excitability and Cognitive Decline

Abstract

Significance Statement

Introduction

Materials and Methods

Construct encoding a human α7-nAChR subunit-mCherry fusion protein

Construct for the nAChR chaperone NACHO

Constructs encoding concatenated homomeric α7 or heteromeric α7β2-nAChR

Cell culture

Transient transfection SH-EPI cells

Preparation of oAβ42

Native/PAGE Western blotting to validate Aβ42 oligomers

Single-channel electrophysiology

Experimental design

Single-channel electrophysiology statistical analysis

Basal forebrain organotypic slice preparations

Whole-cell patch-clamp electrophysiology

Experimental design

Whole-cell patch-clamp electrophysiology statistical analysis

Animal husbandry, breeding, and safeguards

Generation of mice used for whole-cell patch-clamp recordings and Morris water maze (MWM) behavioral testing

Genotyping

MWM

Experimental design

Spatial reference memory

Visible platform test

MWM statistical analysis

Results

Single-channel electrophysiological recordings of ACh-induced openings of α7- and α7β2-nAChR can be obtained consistently from transiently-transfected SH-EP1 cells

oAβ42 directly activates α7- and α7β2-nAChR

oAβ42 preferentially enhances the single-channel open-dwell times of α7β2-nAChR

Subnucleus-specific enhancement of BFCN excitability is mediated by oAβ42/α7β2-nAChR interactions

Alterations in BFCN spike AHP contribute to enhanced BFCN firing rate and are dependent on oAβ42/α7β2-nAChR interactions

oAβ42 exposure leads to alterations in BFCN action potential dynamics and is dependent on α7β2-nAChR activation

Genetic deletion of the β2 nAChR subunit ameliorates deficits in spatial reference memory in the APP/PS1 mouse model of AD

Discussion

Footnotes

References

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Neurobiology of Disease

Implications of Oligomeric Amyloid-Beta (oAβ₄₂) Signaling through α7β2-Nicotinic Acetylcholine Receptors (nAChRs) on Basal Forebrain Cholinergic Neuronal Intrinsic Excitability and Cognitive Decline

Preparation of oAβ₄₂

Native/PAGE Western blotting to validate Aβ₄₂ oligomers

oAβ₄₂ directly activates α7- and α7β2-nAChR

oAβ₄₂ preferentially enhances the single-channel open-dwell times of α7β2-nAChR

Subnucleus-specific enhancement of BFCN excitability is mediated by oAβ₄₂/α7β2-nAChR interactions

Alterations in BFCN spike AHP contribute to enhanced BFCN firing rate and are dependent on oAβ₄₂/α7β2-nAChR interactions

oAβ₄₂ exposure leads to alterations in BFCN action potential dynamics and is dependent on α7β2-nAChR activation