Deficits in synaptic function occur at medial perforant path-dentate granule cell synapses prior to Schaffer collateral-CA1 pyramidal cell synapses in the novel TgF344-Alzheimer's Disease Rat Model

Abstract

Alzheimer's disease (AD) pathology begins decades prior to onset of clinical symptoms, and the entorhinal cortex and hippocampus are among the first and most extensively impacted brain regions. The TgF344-AD rat model, which more fully recapitulates human AD pathology in an age-dependent manner, is a next generation preclinical rodent model for understanding pathophysiological processes underlying the earliest stages of AD (Cohen et al., 2013). Whether synaptic alterations occur in hippocampus prior to reported learning and memory deficit is not known. Furthermore, it is not known if specific hippocampal synapses are differentially affected by progressing AD pathology, or if synaptic deficits begin to appear at the same age in males and females in this preclinical model. Here, we investigated the time-course of synaptic changes in basal transmission, paired-pulse ratio, as an indirect measure of presynaptic release probability, long-term potentiation (LTP), and dendritic spine density at two hippocampal synapses in male and ovariectomized female TgF344-AD rats and wildtype littermates, prior to reported behavioral deficits. Decreased basal synaptic transmission begins at medial perforant path-dentate granule cell (MPP-DGC) synapses prior to Schaffer-collateral-CA1 (CA3-CA1) synapses, in the absence of a change in paired-pulse ratio (PPR) or dendritic spine density. N-methyl-d-aspartate receptor (NMDAR)-dependent LTP magnitude is unaffected at CA3-CA1 synapses at 6, 9, and 12months of age, but is significantly increased at MPP-DGC synapses in TgF344-AD rats at 6months only. Sex differences were only observed at CA3-CA1 synapses where the decrease in basal transmission occurs at a younger age in males versus females. These are the first studies to define presymptomatic alterations in hippocampal synaptic transmission in the TgF344-AD rat model. The time course of altered synaptic transmission mimics the spread of pathology through hippocampus in human AD and provides support for this model as a valuable preclinical tool in elucidating pathological mechanisms of early synapse dysfunction in AD.

Keywords: Alzheimer's disease; Hippocampus; Long-term plasticity; Spine density; Synaptic transmission; TgF344-AD rat model.

Fig. 1.

TgF344-AD synaptic strength is decreased at MPP-DGC synapses prior to CA3-CA1 synapses in both sexes. (A) At MPP-DGC synapses, basal synaptic strength was significantly decreased in TgF344-AD males (blue triangles) compared to WT males (black squares) at (A.1) 6 months (n = 9 slices/8 animals WT, n = 7 slices/6 animals Tg; *p<0.05), at (A.2) 9 months (n = 11 slices/11 animals WT, n = 11 slices/11 animals Tg; *p < 0.05), and at (A.3) 12 months (n = 11 slices/10 animals WT, n = 11 slices/11animals Tg; ** p < 0.01). (B) At CA3-CA1 synapses, basal synaptic strength was not different between TgF344-AD males compared to WT males at (B.1) 6 months (n = 10 slices/10 animals WT, n = 8 slices/7animals Tg, p>0.05), but was significantly decreased at (B.2) 9 months, (n = 11 slices/11 animals WT, n = 11 slices/11 animals Tg; *p<0.05), and at (B.3) 12 months (n = 9 slices/9 animals WT, n = 13 slices/13 animals Tg; *p<0.05). (C) At MPP-DGC synapses in OVX females, basal synaptic strength was decreased in TgF344-AD females (red triangles) compared to WT littermates (black circles) at (C.1) 6 months (n = 6 slices/6 animals WT, n = 8 slices/7 animals Tg; *p < 0.05), at (C.2) 9 months (n = 10 slices/10 animals WT, n = 6 slices/6 animals Tg; *p < 0.05), and at (C.3) 12 months (n = 13 slices/11 animals WT, n = 5 slices/5 animals Tg; *p < 0.05). (D) At CA3-CA1 synapses, basal synaptic strength in TgF344-AD females compared to WT littermates is not altered at (D.1) 6 months (n = 9 slices/8 animals WT, n = 10 slices/10 animals Tg; p>0.05), or at (D.2) 9 months (n = 12 slices/11 animals WT, n = 8 slices/8 animals Tg; p>0.05), but is significantly decreased at (D.3) 12 months (n = 11 slices/11 animals WT, n = 7 slices/7 animals Tg; *p < 0.05). Data represent mean ± SEM. Significance determined by unpaired Student’s t-test at 200 μA. (Insets) Example traces from TgF344-AD and WT age/sex matched littermates at 200 μA stimulus intensity. Scale bar 0.5 mv, 5ms.

Fig. 2.

Paired-pulse ratio is unaltered at MPP-DGC and CA3-CA1 synapses in TgF344-AD rats compared to WT littermates in both sexes. (A, B) PPR was not altered at MPP-DGC synapses (A.1, 6 months: n = 8 slices/7 animals WT, n = 7 slices/7 animals Tg; A.2, 9 months: n = 10 slices/9 animals WT, n = 9 slices/9 animals WT; A.3, 12 months: n = 6 slices/6 animals WT, n = 9 slices/9 animals Tg) or at CA3-CA1 synapses (B.1, 6 months: n = 11 slices/8 animals WT, n = 10 slices/6 animals Tg; B.2, 9 months: n = 13 slices/13 animals WT, n = 12 slices/12 animals Tg; B.3, 12 months: n = 10 slices/9 animals WT, n = 12 slices/12 animals Tg) in TgF344-AD males versus WT littermates. (C, D) The same is true at CA3-CA1 synapses (C.1, 6 months: n = 6 slices/6 animals WT, n = 5 slices/5 animals Tg; C.2, 9 months: n = 11 slices/11 animals WT, n = 6 slices/6 animals Tg; C.3, 12 months: n = 13 slices/11animals WT, n = 7 slices/7animals Tg) and at CA3-CA1 synapses (D.1, 6 months: n = 8 slices/7 animals WT, n = 13 slices/10 animals Tg; D.2, 9 month: n = 12 slices/11 animals WT, n= 8 slices/8 animals Tg; D.3, 12 months: n = 6 slices/6 animals WT, n = 9 slices/9animals Tg) in TgF344-AD females versus WT littermates. Data represent mean ± SEM. Statistical analysis was determined by repeated measures GLM; p>0.05 for all data sets.

Fig. 3.

NMDAR-dependent long-term potentiation (LTP) is selectively enhanced at MPP-DGC synapses in 6 month TgF344-AD rats compared to WT littermates. (A, B) The magnitude of High Frequency Stimulation (HFS) induced LTP at MPP-DGC synapses was significantly increased in (A) TgF344-AD males (blue triangles) compared to WT littermates (black squares) (n = 13 slices/13 animals WT, n = 7 slices/7 animals Tg; *p<0.05), and in (B) OVX TgF344-AD females (red triangles) compared to WT littermates (black circles) (n = 7 slices/7 animals WT, n = 12 slices/10 animals Tg; *p < 0.05) at 6 months. (C, D) The magnitude of Theta-Burst Stimulation (TBS) induced LTP is not different at CA3-CA1 synapses at 6 months in (C) TgF344-AD males compared to WT littermates (n = 9 slices/7animals WT, n = 6 slices/6 animals Tg; p>0.05) or in (D) OVX TgF344-AD females compared to WT littermates (n = 8 slices/7 animals WT, n = 12 slices/10 animals Tg; p>0.05). Data represent mean ± SEM. Significance determined by unpaired Student’s t-test.

Fig. 4.

Depolarization during tetanus is increased during the LTP inducing stimulation at MPP-DGC but not CA3-CA1 synapses in TgF344-AD rats compared to WT littermates. (A.1, B.1) Averaged traces during HFS from experiments in Fig. 3A-B show increased steady-state depolarization (defined as the difference between baseline and when the deflection reached steady state) during 4^th round of HFS tetanus at MPP-DGC synapses in TgF344-AD male (A.1, blue trace) and female (A.2, red trace) slices compared to WT (black traces). Scale bar 0.1mv, 25ms. (A.2) Pooled data from LTP experiments in Fig. 3A show the HFS steady-state depolarization is significantly increased in TgF344-AD males (n = 11 WT, 0.100 ± 0.002, n = 7 Tg, 0.152 ± 0.003; *p < 0.05). (A.3) Area Under the Curve (AUC) was additionally measured (n = 11 WT, 32.81 ± 3.0, n = 7 Tg, 42.65 ± 5.8; p = 0.08). (B.2) Pooled data from LTP experiments in Fig. 3B show the HFS steady-state depolarization is significantly increased in TgF344-AD females (n = 7 WT, 0.088 + 0.001, n = 10 Tg, 0.159 + 0.003; ** p < 0.01). (B.3) AUC was also measured (n = 7 WT, 28.24 ± 2.1, n = 10 Tg, 40.98 ± 3.4; ** p < 0.01). (C.1,D.1) Averaged traces during the 4^th bout of TBS from experiments in Fig. 3C-D show depolarization during tetanus is not altered at CA3-CA1 synapses in either TgF344-AD males (C.1, blue trace) or females (D.1, red trace) compared to WT littermates (black traces). Scale bar 0.25 mv, 25 ms. (C.2) Pooled data from LTP experiments in Fig. 3C do not show a difference in CA3-CA1 steady-state depolarization during tetanus in 6 month TgF344-AD males (n = 7 WT, 0.61± 0.043, n = 5 Tg, 0.63 ± 0.010; p>0.05). (C.3) AUC was not different (n = 7 WT, 29.79 ± 2.0, n = 5 Tg, 29.49 ± 2.1; p>0.05). (D.2) Pooled data from LTP experiments in Fig. 3D do not show a difference in CA3-CA1 steady-state depolarization during tetanus in 6 month TgF344-AD females (n = 7 WT, 0.56 ± 0.013, n = 10 Tg, 0.54 ± 0.032; p>0.05). (D.3) AUC was not significant (n = 7 WT, 32.05 ± 2.1, n = 10 Tg, 31.43 ± 2.2; p>0.05). Data represent mean ± SEM. Significance determine by unpaired Student’s t-test.

Fig. 5.

NMDAR-dependent LTP is intact at MPP-DGC and CA3-CA1 synapses in 9 and 12 month TgF344-AD males and females. (A.1, A.2) LTP magnitude is not different between genotypes in 9 month (A.1, n = 3 slices/3 animals WT, n = 8 slices/8 animals Tg; p > 0.05) and 12 month males (A.2, n = 7 slices/6 animals WT, n = 5 slices/4 animals Tg; p > 0.05) at MPP-DGC synapses. (B.1-B.2) LTP magnitude is not different after TBS at CA3-CA1 synapses in (B.1) 9 month TgF344-AD males compared to WT littermates (n = 12 slices/9 animals WT, n = 16 slices/12 animals Tg; p > 0.05) or (B.2) 12 month males (n = 11 slices/10animal WT, n = 13 slices/13 animals Tg; p > 0.05). (C.1-C.2) LTP magnitude is not different at MPP-DGC synapses in OVX females at 9 (C.2, n = 5 slices/4 animals WT, n = 5 slices/5 animals Tg; p > 0.05) and 12 months (C.2, n = 11 slices/7 animals WT, n = 9 slices/6 animals Tg; p > 0.05). (D.1-D.2) The same is true at CA3-CA1 synapses, where there is no difference in LTP magnitude between genotypes in OVX females at 9 (D.1, n = 14 slices/12 animals WT, n = 14 slices/10 animals Tg; p > 0.05) and 12 months (D.2; n = 9 slices/8 animals WT, n = 12 slices/12 animals Tg, p > 0.05). Data represent mean ± SEM. Significance determine by unpaired Student’s t-test.

Fig. 6.

Dendritic spine density is unaltered on DGCs and CA1 pyramidal cells in TgF344-AD rats compared WT littermate controls. (A.1, A.2) Representative confocal images of Golgi-stained DGC middle molecular layer (MML) apical dendrites from 9-month male and OVX female TgF344-AD rats and WT littermates. (B.1) The number of MML apical dendritic spines per 10 μm is not different between genotypes in 9 month males (WT 17.8 ± 1.1 spines per 10 μm, 34 sections from 7 animals vs TgF344-AD 20.0 ± 0.8 spines per 10 μm, 46 sections from 10 animals; p>0.05) or (B.2) females (WT 14.0 ± 0.7/10 μm, 57 sections/11 animals vs TgF344-AD 13.8 ± 0.8/10 μm, 61 sections/13 animals; p>0.05) compared to WT littermates. (C.1, C.2) Representative confocal images of Golgi-stained CA1 pyramidal cell tertiary dendrites located in stratum radiatum from 9-month male and OVX female TgF344-AD rats and WT littermates. (D.1) The number of CA1 tertiary dendritic spines per 10 μm is not different between genotypes in 9 month males (WT 19.7 ± 1.2 /10 μm, 36 sections/7 animals vs TgF344-AD 19.0 ± 0.710 μm, 47 sections/10 animals; p>0.05) or (D.2) TgF344-AD females (WT 15.4 ± 0.6/10 μm, 59 sections/11 animals vs TgF344-AD 15.7 ± 0.8/10 μm, 64 sections/13 animals; p>0.05). Data represent mean ± SEM. Significance determine by unpaired Student’s t-test. Scale bar, 10 μm.