A longitudinal study of cognition, proton MR spectroscopy and synaptic and neuronal pathology in aging wild-type and AβPPswe-PS1dE9 mice

Abstract

Proton magnetic resonance spectroscopy ((1)H MRS) is a valuable tool in Alzheimer's disease research, investigating the functional integrity of the brain. The present longitudinal study set out to characterize the neurochemical profile of the hippocampus, measured by single voxel (1)H MRS at 7 Tesla, in the brains of AβPPSswe-PS1dE9 and wild-type mice at 8 and 12 months of age. Furthermore, we wanted to determine whether alterations in hippocampal metabolite levels coincided with behavioral changes, cognitive decline and neuropathological features, to gain a better understanding of the underlying neurodegenerative processes. Moreover, correlation analyses were performed in the 12-month-old AβPP-PS1 animals with the hippocampal amyloid-β deposition, TBS-T soluble Aβ levels and high-molecular weight Aβ aggregate levels to gain a better understanding of the possible involvement of Aβ in neurochemical and behavioral changes, cognitive decline and neuropathological features in AβPP-PS1 transgenic mice. Our results show that at 8 months of age AβPPswe-PS1dE9 mice display behavioral and cognitive changes compared to age-matched wild-type mice, as determined in the open field and the (reverse) Morris water maze. However, there were no variations in hippocampal metabolite levels at this age. AβPP-PS1 mice at 12 months of age display more severe behavioral and cognitive impairment, which coincided with alterations in hippocampal metabolite levels that suggest reduced neuronal integrity. Furthermore, correlation analyses suggest a possible role of Aβ in inflammatory processes, synaptic dysfunction and impaired neurogenesis.

Figure 1. Localization of the spectroscopic volume of 1.0×1.0×1.6 mm placed in the hippocampus.

Figure 2. Representative image of synaptophysin-immunoreactive presynaptic boutons (SIPBs) in the hippocampus of a 12-month-old wild-type mouse.

A: In the hippocampus SIPBs were analyzed in the inner (yellow) and outer (red) molecular layer of the dentate gyrus, stratum radiatum (SR) of the CA1 area (blue), and stratum lucidum (SL) of the CA3 area (green). Scale bar = 200 µm. B: SIPBs were quantified with an 100× objective using image analysis from digitized photomicrographs of the synaptophysin-immunoreactivity. Scale bar = 10 µm.

Figure 3. Representative image of doublecortin-positive (Dcx+) cells in the hippocampus of a 12-month-old wild-type mouse.

A: Image taken using an 10× objective. Scale bar = 100 µm. B: Image taken with an 40× objective. Scale bar = 50 µm.

Figure 4. Representative images of the amyloid-β plaque load in the brain of a 12-month-old AβPP-PS1 mouse.

A: The Aβ plaque load was quantified in the prelimbic area at level +1.98 up to +1.78 anterior to bregma, B: in the anterior cingulate gyrus at level +1.10 up to +0.86 anterior to bregma, and C: in the dentate gyrus (DG), CA1 and CA3 areas of the hippocampus at level −2.18 up to −2.46 posterior to bregma, using one appropriate section per animal. Images were taken using a 2.5× objective. Scale bar = 500 µm.

Figure 5. Open field behavior in AβPP-PS1 and wild-type mice at 8 and 12 months of age.

Different open field parameters were measured within a 30 min period, and analyzed in three 10 min trial blocks. A and B: AβPP-PS1 mice (n = 5) did not differ from wild-type mice (n = 8) at 8 months of age (A), but spent slightly less time walking than wild-type mice at 12 months of age, # trend p = 0.058 (B). C and D: The duration of sitting was similar among wild-type and AβPP-PS1 mice at 8 (C) and at 12 months of age (D). E and F: AβPP-PS1 mice spent less time rearing than wild-type mice at 8 months of age, *p<0.05 (E), but did not differ from wild-type mice at 12 months of age (F). G and H: AβPP-PS1 mice traveled a longer distance than wild-type mice at 8 months of age, *p<0.05 (G), but did not differ from wild-type mice at 12 months of age (H). I and J: The duration of wall leaning was similar among wild-type and AβPP-PS1 mice at 8 months of age (I), but was increased in AβPP-PS1 mice at 12 months of age, *p<0.05 (J). K and L: Both at 8 (K) and 12 months of age (L), the time spent grooming was similar among wild-type and AβPP-PS1 mice. M and N: AβPP-PS1 mice did not differ from wild-type mice at 8 months of age (M), but spent less time in the center of the open field than wild-type mice at 12 months of age, *p<0.05 (N). O and P: AβPP-PS1 mice (n = 7) spent more time in the corners of the open field than wild-type animals (n = 9) at 12 months of age, *p<0.05 (P), but did not differ from wild-type at 8 months of age (O).

Figure 6. Morris water maze learning and memory in 8- and 12-month-old wild-type and AβPP-PS1 mice.

Spatial learning was measured in a 4-day acquisition phase, by determining the latency to find a hidden platform in the NE quadrant. Spatial memory was tested in the probe phase in which the percentage of time spent in the target NE quadrant was measured and the total number of platform crossings (where formerly the platform had been located). A: Both 8-month-old wild-type (n = 14) and AβPP-PS1 mice (n = 10) showed a decrease in latency during training. Overall latencies tended to be higher in AβPP-PS1 mice, although it did not reach statistical significance, # trend p = 0.060. B: During the probe trial, the 8-month-old AβPP-PS1 mice traveled a slightly longer distance than wild-type animals, although it did not reach statistical significance, # trend p = 0.056. C: No differences were observed between the 8-month-old mice in the percentage of time spent in the target NE quadrant, although only wild-type mice deviated from 25% chance performance level. D: 8-month-old AβPP-PS1 mice crossed the exact platform location less often than wild-type mice, *p<0.05. E: Both 12-month-old wild-type (n = 9) and AβPP-PS1 mice (n = 7) showed a decrease in latency during training. Overall latencies did not differ between the genotypes. F: During the probe trial, no differences were observed in the distance moved between 12-month-old wild-type and AβPP-PS1 mice. G: 12-month-old AβPP-PS1 mice spent less time in the target NE quadrant, although both groups performed above 25% chance level, *p<0.05. H: No differences were observed between the 12-month-old mice in the frequency of platform crossings.

Figure 7. Reverse Morris water maze learning and memory in 8- and 12-month-old wild-type and AβPP-PS1 mice.

Spatial learning with an extra episodic memory component was measured in a 2-day acquisition phase, by determining the latency to find a hidden platform in the SW quadrant. Spatial memory was tested in the probe phase in which the percentage of time spent in the target SW quadrant was measured and the total number of platform crossings (where formerly the platform had been located). A: Both 8-month-old wild-type (n = 14) and AβPP-PS1 mice (n = 10) showed a decrease in latency during training. Overall latencies did not differ between the genotypes. B: During the probe trial, the 8-month-old wild-type and AβPP-PS1 mice traveled a similar distance. C: No differences were observed between the 8-month-old mice in the percentage of time spent in the target SW quadrant, although only wild-type mice deviated from 25% chance performance level. D: No differences were observed between the 8-month-old mice in the frequency of platform crossings E: Only 12-month-old wild-type mice (n = 9) showed a decrease in latency during training. 12-month-old AβPP-PS1 mice (n = 7) did not improve their performance during acquisition. However, overall latencies did not differ between the genotypes. F: During the probe trial, the 12-month-old AβPP-PS1 mice traveled a slightly longer distance than wild-type animals, although it did not reach statistical significance, # trend p = 0.066. G: 12-month-old AβPP-PS1 mice tended to spent less time in the target SW quadrant, although it did not reach statistical significance, # trend p = 0.066. Only wild-type animals deviated from 25% chance performance level. H: 12-month-old AβPP-PS1 mice crossed the exact former platform location less often than wild-type animals, *p<0.05.

Figure 8. Neurochemical profile of the hippocampus measured by single voxel ¹H MRS at 7 Tesla.

A: Representative ¹H MR spectra acquired from the hippocampus of a 12-month-old wild-type mouse. B: Representative ¹H MR spectra acquired from the hippocampus of a 12-month-old AβPPswe-PS1dE9 transgenic mouse. Notice the decreased NAA peak in AβPP-PS1compared to wild-type. C: At 8 months of age, no differences between wild-type (n = 13) and AβPP-PS1 mice (n = 8) were observed in the hippocampal neurochemical profile. D: At 12 months of age, AβPP-PS1 mice (n = 4) had significantly lower concentrations of NAA than wild-type mice (n = 7), *p<0.05. tCho = choline-containing compounds; tCre = creatine and phosphocreatine; Glu = glutamate; Glx = glutamine and glutamate; mI+Gly = myo-Inositol and glycine; NAA = N-acetylaspartate; Tau = taurine.

Figure 9. Amount of synaptophysin-immunoreactive presynaptic boutons (SIPBs) per µm² in 12-month-old wild-type and AβPP-PS1 mice.

The amount of SIPBs were quantified in the hippocampal inner (IML) and outer molecular layer (OML) of the dentate gyrus, the stratum radiatum (SR) of the CA1 area, and the stratum lucidum (SL) of the CA3 area, and in the cortical prelimbic area (PLA) and anterior cingulate gyrus (ACg). No differences in the amount of SIPs between 12-month-old wild-type (n = 9) and AβPP-PS1 mice (n = 6) were observed in any region analyzed (p>0.10).