TrkB and PLCγ agonists improve neuronal morphology in primary neurons obtained from Fmr1 KO mice
Since TrkB and PLCγ activation is down regulated and may cause the aberrant dendritic spines in Fmr1 KO mice [19], we asked if TrkB and PLCγ agonists could improve hippocampal neuronal morphology in vitro. Then we examined the dendritic length and branches in neurons treated with or without 7,8-DHF (TrkB agonist), m-3M3FBS (PLCγ agonist) or K252a (TrkB inhibitor). Firstly, we treated WT and KO primary neurons at 4 days in vitro (DIV 4) with 0.5, 1 or 5µM 7,8-DHF for 3 days. Additionally, to ensure that the morphological effect was specific through TrkB, we used the inhibitor K252a (50 nM or 100 nM) at DIV 7 for 24h.We observed that branches and total dendritic length were significantly decreased in KO cultures compared with WT cultures (branches: df = 98, t = 2.145,p = 0.034; length: df = 98, t = 2.115, p = 0.037). When treated with 0.5 µM 7,8 DHF, cultures KO cultures exhibited a significant increase in numbers of branches and dendritic length compared to vehicle-KO (branches: df = 98, t = 9.777, p < 0.001; length: df = 98, t = 14.35, p < 0.001). Whereas treated with 100 nM K252a, both KO an WT cultures showed decreased dendritic length and reduced branches (branches: df = 98, t = 4.951, p < 0.001; length: df = 98, t = 4.488, p < 0.001) (Fig. 1A, 1B, and 1C), which suggest the pivotal role of TrkB acting on neuron mature. To further examine the effect of TrkB downstream activation in neuron maturation, we chose PLCγ, which is decreased in Fmr1 KO mice. PLCγ agonist 25µM m-3M3FBS, when used at DIV 5 for 3h, significantly increased the number of branches and dendritic length in vehicle-KO mice to levels that comparable to the vehicle-WT group (branches: df = 98, t = 5.648, p < 0.001; length: df = 98, t = 7.604, p < 0.001, Fig. 3D, 3E, and 3F). Two-way ANOVA of branches and dendritic length showed a main effect of genotype [F (1,196) = 10.20, p = 0.002; F (1,196) = 14.02, p < 0.001], treatment [F (1,196) = 9.692, p = 0.002; F(1,196) = 131.1, p < 0.001], and genotype×treatment interaction [F (1,196) = 7.522, p = 0.007; F(1,196) = 5.829, p = 0.017]. Therefore, activation of TrkB-PLCγ pathway contributes to enhanced neuron maturation in Fmr1 KO mice.
TrkB phosphorylation and downstream PLCγ-CaMKII signaling pathway are normalized by TrkB agonist 7,8-DHF in Fmr1 KO mice
Our previous study has suggested that BDNF/TrkB-PLCγ1-CaMKII signaling is downregulated at the early stages of postnatal development in Fmr1 KO mice [19]. To investigate whether aberrant TrkB and the downstream signaling pathways would be rescued by TrkB agonist 7,8-DHF, we first studied the activation status of p-TrkB, p-PLCγ, and p-CaMKII after treating P14 mice with 5 mg/kg 7,8-DHF daily for continuous 16 days (Fig. 2A). Quantitative analysis revealed higher activation of p-TrkBY816 in 7,8-DHF-treated Fmr1 KO mice than in vehicle-treated KO mice, whereas p-TrkBY515 kept unchanged (Fig. 2A and 2B). Two-way ANOVA of p-TrkBY816 levels showed a genotype × treatment interaction [F(1,28) = 5.727, p = 0.024], a main effect of genotype [F(1,28) = 4.410, p = 0.0449], and a main effect of treatment [F(1,28) = 9.308, p = 0.005]. In addition, levels of p-PLCγ and p-CaMKII were significantly increased in 7,8-DHF-treated KO mice compared with vehicle-treated KO mice (Fig. 2A and 2C). Two-way ANOVA of p-PLCγ and p-CaMKII levels showed a genotype × treatment interaction [F(1,28) = 9.578, p = 0.004; F(1,28) = 4.646, p = 0.040, respectively], a main effect of genotype [F(1,28) = 14.79, p < 0.001; F(1,28) = 5.300, p = 0.029, respectively], and a main effect of treatment [F(1,28) = 4.919, p = 0.035; F(1,28) = 4.313, p = 0.047, respectively]. Collectively, these results indicate that TrkB agonist 7,8-DHF rescued the deficient TrkB-PLCγ-CaMKII signaling in Fmr1 KO mice.
Synaptic loss and synaptic plasticity in Fmr1 KO mice were restored by TrkB agonist 7,8-DHF
To test whether TrkB agonist 7,8-DHF also restore synaptic structure and function in Fmr1 KO mice since it rescued deficient hippocampal TrkB-PLCγ-CaMKII signaling, we first examined spine density of pyramidal neurons in hippocampal CA1 using Golgi staining. Dendritic spines were classified into the following categories: mushroom, stubby, thin and branched [33, 41]. Vehicle-KO mice exhibited markedly increased thin, filopodia-like spines and decreased mushroom spines compared with vehicle-WT mice, which was noticeably rescued by 7,8-DHF treatment (Fig. 3A and 3B). Two-way ANOVA of thin and mushroom percentage showed a genotype × treatment interaction [F (1,16) = 28.48, p < 0.001; F (1,16) = 16.64, p = 0.002, respectively], a main effect of genotype [F (1,28) = 84.82, p < 0.001; F(1,28) = 174.6, p < 0.001, respectively], and a main effect of treatment [F(1,28) = 18.56, p < 0.001; F(1,28) = 30.68, p < 0.001, respectively]. The numbers of stubby spines in CA1 regions were similar between vehicle-WT and vehicle-KO mice. The length of dendritic spines was significantly increased in vehicle-KO mice compared to vehicle-WT mice but was remedied by 7,8-DHF treatment (Fig. 3A and 3C). Two-way ANOVA of spine length showed a genotype × treatment interaction [F (1,16) = 10.70, p = 0.005], a main effect of genotype [F(1,16) = 72.30, p < 0.001], and a main effect of treatment [F(1,16) = 24.06, p < 0.001].
We then utilized transmission electron microscopy (TEM) to compare the synaptic density and structure between groups in hippocampal CA1 of FXS mice. We found that Vehicle-KO hippocampus have less synapse, thinner PSD and shorter synaptic active zone (Fig. 3E, 3G and 3H) but wider synaptic cleft than Vehicle-WT group (Fig. 3F). Notably, 7,8-DHF treatment was sufficient to revert the decrease of synaptic density, PSD thickness, and length of the synaptic active zone, and also revert the increase of synaptic cleft width (Fig. 3E, 3F, 3G and 3H). Two-way ANOVA of synaptic density, PSD thickness, length of the synaptic active zone, and synaptic cleft width showed a genotype × treatment interaction [F (1,361) = 329.2, p < 0.001; F (1,361) = 3.38, p = 0.067; F (1,361) = 69.35; F (1,361) = 17.22, p < 0.001, respectively], a main effect of genotype [F (1,361) = 38.55, p < 0.001; F (1,361) = 37.05, p < 0.001; F (1,361) = 96.03, p < 0.001; F (1,361) = 65.53, p < 0.001,respectively], and a main effect of treatment [F (1,361) = 241.2, p < 0.001; F(1,361) = 17.66, p < 0.001; F (1,361) = 33.76, p < 0.001; F (1,361) = 30.36, p < 0.001, respectively].
We next examined levels of synaptophysin (presynaptic markers) and PSD95 (postsynaptic markers) using western blot. Results showed a significant reduction in synaptopsin and PSD95 in vehicle-KO mice, which was normalized in 7,8-DHF-KO mice (Fig. 3I, 3J and 3K). Two-way ANOVA of synaptophysin levels showed a genotype × treatment interaction [F (1,28) = 4.354, p = 0.046], a main effect of genotype [F(1,28) = 8.314, p = 0.008], and a main effect of treatment [F(1,28) = 6.858, p = 0.014]. Two-way ANOVA of PSD95 levels showed a genotype × treatment interaction [F(1,28) = 4.400, p = 0.045], a main effect of treatment [F(1,28) = 6.173, p = 0.019], and no main effect of genotype. In all, these results suggest that 7,8-DHF treatment recovered abnormal synaptic structure in the hippocampus of Fmr1 KO mice.
Given its effect on synaptic structure, we next exaimined whether 7,8-DHF restored synaptic plasticity in Fmr1 KO mice. We tested low-frequency stimulation (LFS)- induced long-term depression (LTD) in each mice group, and observed significantly larger LTD in vehicle-KO than in vehicle-WT mice (87.24 ± 3.66% of baseline vs 62.07 ± 1.59% of baseline, df = 8, t = 6.308, p < 0.001, Fig. 4A and 4B). Whereas 7,8-DHF reduced LTD magnitude in KO mice (52.04 ± 6.63% of baseline, df = 8, t = 4.649, P = 0.002, compared to vehicle group, Fig. 4A and 4B) but showed no effect in LTD in WT mice (55.12 ± 6.56% of baseline, df = 8, t = 1.030, P = 0.333). Thus, 7,8-DHF appeared to rescue LTD deficit in Fmr1 KO mice with no significant impact on WT mice. No significant difference in hippocampal LTP was observed in WT and Fmr1 KO mice (data not shown). Taken together, synaptic structure and synaptic plasticity in Fmr1 KO mice were restored by BDNF mimic and TrkB agonist, 7,8-DHF, suggest BDNF-TrkB dysregulation in Fmr1 KO mice is the underlying mechanism of synaptic development.
Learning and memory deficits in Fmr1 KO mice were rescued by TrkB agonist 7,8-DHF
We first investigated the rescue effect in hippocampus-dependent learning and memory of 7,8-DHF, Morris water maze (MWM) was tested on 2-month-old Fmr1 KO mice. The learning phase lasted 5 days. Two-way ANOVA of escape latency showed a main effect of genotype [F (1,44) = 9.71, p = 0.003], treatment [F (1,44) = 6.521, p = 0.014], and genotype×treatment interaction [F (1,44) = 5.512, p = 0.024]. During the learning phase (Fig. 5A), Fmr1 KO mice showed significantly longer escape latencies, which suggested impaired spatial memory acquisition. 7,8-DHF-KO mice located the hidden platform more quickly than vehicle-KO mice. In fact, 7, 8-DHF restored the performance of KO mice to the level of vehicle-WT mice (Fig. 5A). After 5 days of training sessions, the probe test was used to evaluate spatial memory by measuring the time spent in the correct quadrant after the removal of the hidden platform. Two-way ANOVA of time spending and platform crossings in the target quadrant showed a main effect of genotype [F (1,44) = 9.857, p = 0.003; F (1,44) = 20.61, p < 0.001], treatment [F (1,44) = 7.118, p = 0.011; F(1,44) = 10.05, p = 0.003], and genotype×treatment interaction [F (1,44) = 5.932, p = 0.019; F(1,44) = 17.45, p < 0.001]. Vehicle-KO mice showed no preference for the correct quadrant and less platform crossings, whereas 7, 8-DHF-KO mice spent more time in the correct quadrant and crossed the previous location of the hidden platform as frequently as vehicle-WT mice did (Fig. 5B and 5C). In the visible-platform test, comparable motor and visual functions were observed among the four groups (Fig. 5D and 5E). Two-way ANOVA of escape latency and swimming speed showed no effect of genotype [F (1,44) = 0.0003, not significant (ns); F (1,44) = 0.80, ns], treatment [F (1,44) = 0.528, ns; F(1,44) = 0.0003, ns], or genotype×treatment interaction [F (1,44) = 1.236, ns; F(1,44) = 0.515, ns]. Therefore, variations in vision ability and swimming speeds did not cause behavioral differences among the groups.
We next tested whether 7,8-DHF normalizes the fear memory deficiencies in Fmr1 KO mice. In fear conditioning test, 7,8-DHF showed a significant main effect of genotype [Hb: F (1, 36) = 0.9259, ns; CS-US1: F(1,36) = 0.182, ns; CS-US2: F(1,36) = 22.37, p < 0.001; CS-US3: F(1,36) = 11.94, p = 0.002] and treatment [Hb: F (1, 36) = 0.3707, ns;CS-US1: F(1,36) = 0.021, ns; CS-US2: F(1,36) = 13.23, p < 0.001; CS-US3: F(1,36) = 5.581, p = 0.024] as well as significant interactions [F(1, 36) = 0.160, ns; CS-US1: F(1,36) = 0.087, ns; CS-US2: F(1,36) = 10.82, p = 0.002; CS-US3: F(1,36) = 7.200, p = 0.011] with fear acquisition in the training session. Freezing time in vehicle-KO mice was significantly lower compared to vehicle-WT mice during the second and third tone-shock pairs (Fig. 6A). During inter-trial intervals, Vehicle-KO mice showed significantly less freezing time (Fig. 6B). Vehicle-KO mice also exhibited less freezing time when tested 24h later for contextual fear and 48h later for cued fear (Fig. 6C and Fig. 6D). In 7,8-DHF-KO mice, freezing time was normalized in the training session (Fig. 6B), the contextual fear test (Fig. 6C), and the cued fear test (Fig. 6D).