Rapid Aging in the Perforant Path Projections to the Rodent Dentate Gyrus

Abstract

Why layers II/III of entorhinal cortex (EC) deteriorate in advance of other regions during the earliest stages of Alzheimer's disease is poorly understood. Failure of retrograde trophic support from synapses to cell bodies is a common cause of neuronal atrophy, and we accordingly tested for early-life deterioration in projections of rodent layer II EC neurons. Using electrophysiology and quantitative imaging, changes in EC terminals during young adulthood were evaluated in male rats and mice. Field excitatory postsynaptic potentials, input/output curves, and frequency following capacity by lateral perforant path (LPP) projections from lateral EC to dentate gyrus were unchanged from 3 to 8-10 months of age. In contrast, the unusual presynaptic form of long-term potentiation (LTP) expressed by the LPP was profoundly impaired by 8 months in rats and mice. This impairment was accompanied by a reduction in the spine to terminal endocannabinoid signaling needed for LPP-LTP induction and was offset by an agent that enhances signaling. There was a pronounced age-related increase in synaptophysin within LPP terminals, an effect suggestive of incipient pathology. Relatedly, presynaptic levels of TrkB-receptors mediating retrograde trophic signaling-were reduced in the LPP terminal field. LTP and TrkB content were also reduced in the medial perforant path of 8- to 10-month-old rats. As predicted, performance on an LPP-dependent episodic memory task declined by late adulthood. We propose that memory-related synaptic plasticity in EC projections is unusually sensitive to aging, which predisposes EC neurons to pathogenesis later in life.SIGNIFICANCE STATEMENT Neurons within human superficial entorhinal cortex are particularly vulnerable to effects of aging and Alzheimer's disease, although why this is the case is not understood. Here we report that perforant path projections from layer II entorhinal cortex to the dentate gyrus exhibit rapid aging in rodents, including reduced synaptic plasticity and abnormal protein content by 8-10 months of age. Moreover, there was a substantial decline in the performance of an episodic memory task that depends on entorhinal cortical projections at the same ages. Overall, the results suggest that the loss of plasticity and related trophic signaling predispose the entorhinal neurons to functional decline in relatively young adulthood.

Keywords: TrkB; aging; entorhinal cortex; lateral perforant path; long-term potentiation; memory.

Figure 1.

Waveforms of LPP fEPSPs are comparable in early and later adulthood. A, B, Responses from individual slices were normalized to their peak amplitude and then averaged across all slices within a group. At left, mean ± SEM traces for the different age groups were superimposed for mouse (3-month-old, N = 8; 6-month-old, N = 9; 10-month-old, N = 7; A) and rat (3-month-old, N = 7; 8-month-old, N = 7; B); there were no obvious differences between 3-month-old and older rodents. Calibration: (in B) A, B, 50% peak fEPSP amplitude, 10 ms. At right, graphs show the initial slopes, and decay time constants (tau) did not differ between young and older mice (A; slope: p = 0.431; decay tau: p = 0.999, one-way ANOVA; 3-month-old, N = 7; 6-month-old, N = 8; 10-month-old, N = 7) and rats (B; slope: p = 0.159, t test; decay tau: p = 0.5406; 3-month-old, N = 7; 8-month-old, N = 7). C, D, Input/output (fiber volley vs fEPSP amplitude) curves were comparable across ages in mice (C; 3-month-old, N = 5; 6-month-old, N = 6, 10-month-old, N = 5) and rats (D; 3-month-old, N = 7; 8-month-old, N = 8); traces for the different age groups are shown at right. Calibration: (in D) C, D, 1 mV, 10 ms.

Figure 2.

LTP but not frequency facilitation is impaired in the LPP of older adults. A, A train of 10 stimulation pulses was delivered to the LPP at 10, 20, or 40 Hz, and LPP fEPSPs were recorded in the rat DG outer molecular layer. Representative traces above each graph (calibration: 1 mV, 50 ms) show the first five responses for 10 Hz and all responses for 20 and 40 Hz. For 10 and 40 Hz, within-train fEPSP slope facilitation was comparable in 8- to 10-month-old and 3-month-old rats (10 Hz: p = 0.123, F_(9,189) = 1.39; 40 Hz: p = 0.055, F_(9,189) = 1.897; RM ANOVA). For 20 Hz, facilitation was greater in 8- to 10-month-old versus 3-month-old old rats (p = 0.024, F_(9,189) = 2.204; 3-month-old, N = 11; 8- to 10-month-old, N = 12 for all frequencies). B, Following HFS (100 Hz, 1 s) in mice, LPP potentiation (assessed by initial fEPSP slope) was greater and more stable in 3-month-old versus 6- and 8- to 10-month-old mice; representative traces at right show superimposed baseline (darker line) with post-HFS waveforms (calibration: 1 mV, 10 ms; 3-month-old, N = 5; 6-month-old, N = 6; 8- to 10-month-old, N = 7). C, Mean percentage of potentiation over baseline, during the last 5 min of recording after HFS for LTP or during the first 2 min post-HFS for STP, shows that LTP was lower in the two older mouse groups, whereas STP was comparable across groups (for LTP: F_(2,39) = 8.283, p = 0.001; **p < 0.01 and ***p < 0.001 vs 3-month-old, post hoc Bonferroni; STP: n.s.). D, lppLTP was greater in 3- versus 8- to 10-month-old rats (calibration: 1 mV, 10 ms; 3-month-old, N = 7; 8- to 10-month-old, N = 7); superimposed traces also show the difference in LTP size with age. E, The mean percentage of LTP at 55–60 min post-HFS was approximately twofold greater in 3- versus 8- to 10-month-old rats (***p = 0.0009, t test), whereas STP was equivalent between groups.

Figure 3.

Age-dependent effects of treatments that influence endocannabinoid signaling on synaptic responses and lppLTP in rat. A, Physostigmine (30 μm, gray bar) reduced the slopes of fEPSPs elicited by single-pulse LPP stimulation to a greater extent in slices from 3- versus 8-month-old rats. Traces show representative fEPSPs pre-infusion (dark line) and postinfusion (3-month-old, N = 14; 8-month-old, N = 18; calibration: 1 mV, 10 ms). B, The percentage of reduction in fEPSP slopes (from data in A) at the 60 min time point (****p < 0.0001 vs 3-month-old, t test). C, D, PPF of fEPSP slopes (50 ms interpulse interval) was comparable between age groups before physostigmine administration (C; p = 0.736) but was greater after physostigmine administration in slices from 3-month-old versus 8-month-old rats (D; **p = 0.0071; 3-month-old, N = 6; 8-month-old, N = 9); representative traces shown at left. Calibration: 1 mV, 10 ms. E, JZL184 (JZL; 1 μm in slice bath) enhanced lppLTP in 8- to 10-month-old rats. Above, representative traces from before (dark line) and after HFS. Calibration: 1 mV, 10 ms [vehicle (veh), N = 5; JZL184, N = 10]. F, Graphs summarize, from E, the percentage of LTP and the percentage of STP at 55–60 and 0–2 min after HFS, respectively (**p = 0.01 vs veh, t test).

Figure 4.

Early aging alters presynaptic markers in the LPP terminal zone. A, Section through hippocampus shows the two zones in the DG outer molecular layer (OML) used for analysis of synaptic proteins. B, Deconvolved immunofluorescence images show the localization of p-FAK (red) and vesicular protein synaptophysin (SYN; green) in rat. Arrows point to double-labeled puncta. Scale bar, 10 µm. C, FDT analysis of the density of p-FAK immunoreactivity (ir) colocalized with synaptophysin. Measurements were made for >100,000 individually reconstructed, double-labeled (2×) synapses per slice. Values for each slice were plotted as the percentage of 2× synapses (y-axis) fitting into an ascending series of labeling-density bins (x-axis). The resultant curves for individual slices were then averaged for a group and plotted as mean ± SEM values. There was a small but significant left shift in the curve for the 8-month-old group (N = 12) relative to that for the 3-month-old group (N = 15). Scatter plot summarizes the percentage of 2× synapses with high-density p-FAK-ir (right of vertical dotted line; *p < 0.05). D, Same analysis as in C but for the density of synaptophysin-ir: there was a pronounced rightward skew in the plot for 8-month-old rats (toward greater synaptic densities) versus the 3-month-old rats, and a large increase in the proportion of terminals with dense synaptophysin-ir (scatter plot; ***p < 0.001). E, Deconvolved images show dual immunofluorescence for TrkB (red) and synaptophysin (green) in mouse. Arrows point to 2× puncta. Scale bars: 4 µm; inset, 1 µm. F, Analysis of the density of TrkB-ir in LPP terminals in mice: there was a pronounced left shift in the density frequency distribution for the 8-month-old group relative to the 3-month-old group (N = 6/group), resulting in a large reduction in the proportion of terminals with dense TrkB-ir (scatter plot; ***p < 0.001).

Figure 5.

Age-related changes in MPP potentiation in rats. A, With single-pulse stimulation the averaged decay tau and initial fEPSP slope were comparable for 3-month-old (N = 10) and 8-month-old (N = 12) rats (slope, p = 0.3542; decay tau, p = 0.4678; t test). B, The MPP input–output curve was not different between 3-month-old (N = 6) and 8-month-old (N = 12) rats. C, Following HFS, both initial and enduring MPP potentiation were smaller in slices from 8-month-old versus 3-month-old rats. Representative traces show baseline (dark line) and post-HFS responses. Calibration: 1 mV, 10 ms. D, Scatter plots show that for the MPP, the mean percentage of potentiation during the last 5 min of recording (LTP) and the 2 min post-HFS interval (STP) were both significantly lower for slices from 8-month-old versus 3-month-old rats (***p < 0.001; data from C). C, D: 3-month-old, N = 10; 8-month-old, N = 12.

Figure 6.

MPP presynaptic protein levels are altered with age. A, FDT analysis (plotted as in Fig. 4) of the density of postsynaptic GluN2A immunoreactivity (ir) for synapses double labeled (2×) for postsynaptic marker PSD-95 in the DG middle molecular layer (MML) of 3-month-old (N = 5) and 8-month-old (N = 5) rats; scatter plot shows that the percentages of 2× synapses with dense GluN2A-ir (≥95 U, x-axis) did not differ between age groups (p = 0.328, unpaired t test). B, FDT analysis in rats shows a marked right shift in the per-terminal density of synaptophysin-ir in the MPP, the predominant excitatory afferent to the MML; scatter plot shows the increase in the proportion of synapses with dense immunolabeling at 8 months of age versus 3 months of age (**p < 0.01). C, In CA1 stratum radiatum, the per-terminal density of synaptophysin-ir did not differ between age groups. B, C, 3-month-old, N = 15; 8-month-old, N = 12 rat hippocampal slices as assessed in Fig. 4B,D. D, Analysis of the density of presynaptic TrkB-ir in the MPP terminal field; there was a pronounced left shift in density frequency distribution for the 8-month-old group relative to the 3-month-old group (N = 6 mice/group), resulting in a large reduction in terminals with dense TrkB-ir (scatter plot; **p < 0.01).

Figure 7.

LPP-dependent episodic memory task performance declines from early to late phases of young adulthood in mouse. A, Mice were tested on two paradigms. The serial odors task entailed sampling a sequence of identical odor pairs (A:A, B:B, C:C) followed by a retention trial with a novel odor paired with a previously sampled one (A:D). In the two-odor control task, an identical odor pair (A:A) was followed by a retention trial pairing odor A with a novel odor (A:D). The delay between the first pair and the A:D retention test was the same in the two paradigms. B, The 3-month-old mice (N = 12) preferentially explored the novel versus the familiar odor in the serial odors task (****p< 0.0001, paired Student's t test), whereas 8- to 10-month-old mice (N = 11) did not (p = 0.471, paired Student's t test; n.s., not significant). C, Group measures for the discrimination index calculated from data presented in B (**p = 0.0013; unpaired t test). D, Total time sampling cues in the serial odor retention test did not differ between groups (p = 0.897). E, In the two-odor control task, mice in both age groups spent a greater proportion of sampling time exploring the novel cue (***p ≤ 0.0002, both groups). F, Control saliency tests demonstrated that both odors A and D were equally sampled by mice of both age groups (p = 0.4205 and p = 0.5377 for 3- and 8- to 10-month-old groups, respectively).