Interactions between Aβ oligomers and presynaptic cholinergic signaling: age-dependent effects on attentional capacities

Abstract

Substantial evidence suggests that cerebral deposition of the neurotoxic fibrillar form of amyloid precursor protein, β-amyloid (Aβ), plays a critical role in the pathogenesis of Alzheimer's disease (AD). Yet, many aspects of AD pathology including the cognitive symptoms and selective vulnerability of cortically projecting basal forebrain (BF) cholinergic neurons are not well explained by this hypothesis. Specifically, it is not clear why cognitive decline appears early when the loss of BF cholinergic neurons and plaque deposition are manifested late in AD. Soluble oligomeric forms of Aβ are proposed to appear early in the pathology and to be better predictors of synaptic loss and cognitive deficits. The present study was designed to examine the impact of Aβ oligomers on attentional functions and presynaptic cholinergic transmission in young and aged rats. Chronic intracranial infusions of Aβ oligomers produced subtle decrements in the ability of rats to sustain attentional performance with time on task, irrespective of the age of the animals. However, Aβ oligomers produced robust detrimental effects on performance under conditions of enhanced attentional load in aged animals. In vivo electrochemical recordings show reduced depolarization-evoked cholinergic signals in Aβ-infused aged rats. Moreover, soluble Aβ disrupted the capacity of cholinergic synapses to clear exogenous choline from the extracellular space in both young and aged rats, reflecting impairments in the choline transport process that is critical for acetylcholine (ACh) synthesis and release. Although aging per se reduced the cross-sectional area of BF cholinergic neurons and presynaptic cholinergic proteins in the cortex, attentional performance and ACh release remained unaffected in aged rats infused with the control peptide. Taken together, these data suggest that soluble Aβ may marginally influence attentional functions at young ages primarily by interfering with the choline uptake processes. However, age-related weakening of the cholinergic system may synergistically interact with these disruptive presynaptic mechanisms to make this neurotransmitter system vulnerable to the toxic effects of oligomeric Aβ in robustly impeding attentional capacities.

Keywords: Aging; Alzheimer's disease; Attention; Cholinergic; Presynaptic; Soluble amyloid-beta.

Figure 1

Composition of synthetic Aβ oligomers and the experimental design. (A) The soluble Aβ1-42 preparation used for chronic infusions consisted of monomers, dimers, trimers, tetramers, and a series of large oligomeric peptides as detected by 6E10 antibody using western blotting. (B) Rats trained to criterion in the sustained attention task were prepared for chronic administration of Aβ oligomers. Oligomers or control peptide (Aβ_42-1) was chronically administered into the ventricles using an implanted cannula connected to a mini-osmotic Alzet pump that released oligomeric Aβ solution (50 ng/hr) for 28 days. Attentional performance was assessed throughout this period following which the animals were prepared either for amperometric recordings or immunoblotting studies.

Figure 2

Effects of chronic intracranial infusions of Aβ oligomers on SAT performance in young and aged rats. Average SAT scores (A) and hits (E) were signal duration-dependent across all groups. Segmenting average SAT scores by block demonstrated no difference between groups in the initial block (B). However, a significant decrease in SAT scores of the Aβ groups compared to the controls was noted specifically on the 500 ms signal in block 2 (C) and the 25 ms signal in block 3 (D). A similar pattern was observed when analyzing only the performance on signal trials (%hits; F-H). Overall SAT scores and hit rates did not differ between young and aged rats. Performance on non-signal trials, as demonstrated by correct rejections, did not vary across block or groups (I). Response latencies did not differ between groups for either signal (J) or non-signal trials (K). Omissions remained relatively low (< 10%) and were similar between the two age groups and manipulations (L). All data are mean ± SEM. (main effect of signal: ***, p < 0.001; post hoc comparisons: +, p < 0.05)

Figure 3

Attentional performance under distracting conditions. dSAT sessions consisted of the presentation of visual distractors (flashing house light @ 0.5 Hz) during block 2 of the session, as indicated by green shading, while block 1 and block 3 were similar to normal SAT conditions. (A) dSAT scores declined significantly during the distractor block and rebounded slightly during block 3. (B) As observed in SAT, the experimental groups did not differ by signal duration in block 1. (C) During the distractor block, decreases in dSAT scores were observed across all groups and all signal durations. (D) However, in the post-distractor block, interactions between signal duration, Aβ treatment, and age were observed. dSAT scores were significantly lower in aged rats infused with the control peptide on 500 ms signal as compared to young animals. Chronic infusions of soluble Aβ produced robust impairments in aged rats on both 500 ms and 50 ms signals in block 3. Conversely, no significant effect of Aβ was observed in young rats. dSAT performance for the 25 ms signal approached chance levels for all groups. (E) The proportion of hits, on the other hand, displayed a steady decline from block 1 to block 3, with no differences seen between groups during the first block (F) but significant interactions emerging during the distractor and post-distractor blocks (G and H). Aged rats chronically treated with Aβ displayed reduced hits in the 500ms signal trials in block 2 and 3, as well as a significant decrease in hits in 50 ms signal trials in block 3 in comparison to aged controls and young Aβ-infused rats. Hit rates did not differ between the young and aged control groups indicating that minor decrements in dSAT performance in the latter might have occurred due to variations in non-signal trial performance. (I) Correct rejections decreased during the distractor block but recovered in the post-distractor block. (J-L) No differences in correct rejections were observed between the groups during the blocks. All data are mean ± SEM. (main effect of signal: *, **, *** p < 0.05, 0.01, 0.001; post hoc comparisons: +, ++ p < 0.05, 0.01)

Figure 4

Prefrontal ACh release and choline clearance capacity assessed using choline-sensitive microelectrodes and fixed-potential amperometry in vivo. Representative traces depicting choline spikes following brief depolarizing pulses of potassium in the medial PFC of young (A) and aged (B) rats that received chronic i.c.v. infusions of either oligomeric Aβ or control peptide. These signals reflect ACh release and occur as a consequence of rapid hydrolysis of ACh by acetylcholinesterase. The hydrolyzed choline is oxidized by the enzyme choline oxidase present on the platinum recording site at a fixed potential to generate increases in current. (C) Depolarization-evoked cholinergic signal amplitudes were lower in aged as well as Aβ-infused rats. Examples of current traces illustrate the effects of either Aβ oligomers or control peptide on clearance kinetics of exogenously applied choline in young (D) and aged (E) rats. Choline uptake rates were calculated from the hemicholinium-sensitive component of the clearance curve that accounts for the decrease in choline concentration from 40% to 80% of the peak amplitudes. (F) The capacity to clear choline from the extracellular space declined in soluble Aβ-infused rats and this effect was more prominent in aged animals (refer statistical analysis from 2 × 2 ANOVA in results). Data are Mean ± SEM. Aβ (−) depict chronic infusions with the control peptide while Aβ (+) indicate animals infused with soluble Aβ in (C) and (F), respectively.

Figure 5

Effects of age and chronic Aβ infusions on BF cholinergic neurons and cortical presynaptic cholinergic proteins. (A; upper panel) Representative coronal sections depicting ChAT-immunopositive neurons (marked by black arrowheads) from the nucleus basalis of young and aged rats infused with either Aβ or control peptide. (A; lower panel) The cross-sectional area of cholinergic neurons robustly declined with age but not by chronic Aβ infusions. (B; upper panel) Photographs illustrate sampled ChAT-immunostained fibers from the prelimbic region of the PFC. (B; lower panel) The density of cortical cholinergic processes was significantly lower in aged rats. However, prefrontal ChAT fiber counts in soluble Aβ-infused rats did not differ from the control animals. Immunoblot analyses of cortical CHT (C) and VAChT (D). These presynaptic protein markers for cholinergic terminals were estimated in homogenates prepared from prefrontal cortices. Representative immunoblots (top) show CHT- and VAChT-immunoreactive bands at 55 kDa and 68 kDa respectively. Bar charts (bottom) show CHT and VAChT densities normalized to β-tubulin. A downward trend in prefrontal CHT density was observed in aged animals (p = 0.06 vs. young). Likewise, VAChT expression also significantly reduced with age. However, neither CHT nor VAChT protein expression was affected by soluble Aβ. Data are Mean ± SEM. (main effects: *, **, *** p < 0.05, 0.01, 0.001).