Impaired synaptic tagging LTP observed in the ACC of middle-aged mice
We employed a 64-channel field potential recording system and analyzed the induction probability and properties of synaptic tagging in vitro ACC slices from middle-aged male mice (50–60 weeks). Two different channels were stimulated in different layers of the ACC (S1: superficial layer; S2: deep layer). Simultaneously, the evoked multi-channel fEPSP around the stimulation sites were recorded (Figs. 1a, 1b). Specifically, we first applied a strong TBS (4x5x5) to site S1, and 30 min later, a weak TBS (tagging TBS, 4x5x1) was delivered to site S2. We found that normal LTP can be induced by strong TBS in middle-aged mice (Fig. 1f). There are 2 channels showing E-LTP and 6 channels showing L-LTP in 8 activity channels around site S1. However, the synaptic tagging LTP induced by weak TBS was impaired in middle-aged mice, although there were 2 channels showing tagging-like response, while the other 4 activated channels only showed the E-LTP on site S2 (Fig. 1h).
A novel fEPSP signal modeling and visualization system was developed to monitor the spatiotemporal properties tagging LTP. We assumed that the 8x8 observations from the MED64 system, corresponding to the 63 recording channels plus the 64th channel as the stimulation input, are only a sparse observation of the complete fEPSP signal field residing in latent state space. Such latent state space could then project into another observation space with NxN resolution where N is usually greater than 8 and was set to 256 in our experiments. In this way, the originally recorded MED64 values of fEPSP slopes were reconstructed into high-density 3D signal sequences (Fig. 1d) As shown in Fig. 1e and Fig. 1g, the peak intensity and spatial distribution of the fEPSP slopes were significantly increased at 120 min and 240 min after strong TBS (Fig. 1e). However, the peak intensity and spatial distribution ware slight increased at 120 min after tagging TBS in middle-aged mice (Fig. 1g). These 3D maps clearly showed that the synaptic tagging was impaired in the ACC of middle-aged mice.
Next, we compared synaptic tagging response in adult mice (6–8 weeks) and middle-aged (50–60 weeks) mice. At site S1, strong TBS induced late-phase LTP (L-LTP) in all 5 adult mice, but only 3 mice showed L-LTP in 5 recorded middle-aged mice (Fig. 2a). The fEPSP slopes of the last 30 min were 148.01 ± 5.79% in adult mice and 128.04 ± 6.88% in middle-aged mice (Fig. 2c). At site S2, most channels showed tagging-like response after tagging TBS in all 5 adult mice. However, in middle-aged mice, only 3 mice out of 5 mice showed 1 or 2 tagging L-LTP, and most activated channels showed the E-LTP (Fig. 2b). The fEPSP slopes were 175.20 ± 8.92% in adult mice and 114.35 ± 7.75% in middle-aged mice (* * p < 0.01, n = 5 mice for each group). The conclusion could also be supported by analyzing the percentage of channels with tagging-like responses over all activated channels. The ration of the induction of tagging LTP was significantly higher in the adult group (73.33 ± 4.67%) than in the middle-aged group (33.33 ± 4.24%, Fig. 2d).
Less recruitment of inactive responses after synaptic tagging in middle-aged male mice
Previous studies have shown that the strong TBS-induced L-LTP could recruit newly-activated channels in the ACC [31, 36]. Consistently, we also found that there was clear enlargement of the response areas surrounding the strong TBS at site S1 both in adult mice and middle-aged mice (Fig. 3a). After tagging TBS on site S2, the enlargement of response areas can be observed in adult mice, but not in middle-aged mice (Fig. 3b). In adult mice, the number of recruited channels both on sites S1 and S2 gradually increased after TBS induction (Figs. 3c and 3d, n = 5 mice). However, in middle-aged mice, the tagging TBS on site S2 failed to increase many recruited channels. The recruited channels around site S2 are less than those in adult mice. (*p < 0.05, **p < 0.01, n = 5 mice)
The time courses of the changed fEPSP slope in the recruited channels were further shown in Figs. 3g and 3h. In these recruited channels, strong TBS-induced fEPSPs on site S1 were gradually potentiated, and the amplitude finally became as large as 24.79 ± 0.81 µV in adult mice and 23.85 ± 0.79 µV in middle-aged mice at 4.5 h after TBS induction (Fig. 3g). It is worth noting that the amplitudes of fEPSPs in tagging-TBS recruited channels were no significant difference in adult mice (23.90 ± 0.87 µV) and in middle-aged mice (24.09 ± 0.81 µV) when the channels occurred. (Fig. 3h).
Synaptic tagging LTP in middle-aged female mice
We further tested the synaptic tagging LTP in middle-aged female mice. Similar results were obtained in middle-aged female mice and male mice (Fig. 4). A summarized plot of the fEPSP slope showed that both strong TBS at the site S1 and tagging TBS at the site S2 can induce L-LTP in adult female mice (n = 5 mice). However, in the middle-aged female mice, there was no tagging LTP, and the L-LTP occurred only in site S1 in 4 slices from 5 mice (Figs. 4a and 4b). These results suggest that there is no gender difference for synaptic tagging LTP in middle-aged mice.
Next, we showed the spatial distribution of the active responses in the ACC before and after strong TBS and tagging TBS application across both male and female mice. The distribution of all observed activated channels was displayed by a polygonal diagram on a grid representing the channels (Figs. 4c- 4j).
In adult male mice, there were 55 channels exhibited clear synaptic responses from 5 slices (5 mice) at the baseline, and 26 new channels were recruited at 4.5 h after strong TBS in site S1 (Fig. 4c). Meanwhile, in middle-aged male mice, only 28 channels exhibited clear synaptic responses from 4 slices (4 mice) at the baseline, and 8 new channels were recruited at 4.5 h after strong TBS in site S1 (Fig. 4e). For tagging LTP, in adult male mice, 53 channels exhibited clear synaptic responses from 5 slices at the baseline, and 26 new channels were recruited at 4 h after tagging TBS in site S2 (Fig. 4d). In middle-aged male mice, 26 channels exhibited clear synaptic responses from 4 slices at the baseline, and only 3 new channels were recruited at 4 h after tagging TBS site S2 (Fig. 4f).
In adult female mice, 60 channels exhibited clear synaptic responses from 5 slices in 5 mice at the baseline, and 22 new channels were recruited at 4.5 h after strong TBS in site S1 (Fig. 4g). In middle-aged female mice, only 47 channels exhibited clear synaptic responses in 4 slices from 4 mice at the baseline, and 4 new channels were recruited at 4.5 h after strong TBS in site S1 (Fig. 4i). For the site S2, in adult female mice, 93 channels exhibited clear synaptic responses from 5 slices in 5 mice at the baseline, and 20 new channels were recruited at 4 h after tagging TBS (Fig. 4h). In the middle-aged female mice, 72 channels exhibited clear synaptic responses from 4 slices in 4 mice at the baseline, and only 1 new channel was recruited at 4 h after tagging TBS (Fig. 4j). Taken together, these results suggest tagging TBS can significantly enhance the spatial distribution of active responses in the adult mice, but failed to change spatial distribution of active responses in middle-aged mice.
Age difference of weak TBS induced L-LTP within ACC
In the above results, it was observed that synaptic tagging induced by tagging TBS (weak TBS) was more easily found in younger mice. Therefore, we wonder whether L-LTP could be induced by weak TBS alone, and whether is age dependent or not.
We attempted only delivering a single weak TBS, the same as tagging TBS (five trains of a burst with four 100 Hz pulses at 200 ms intervals), to the deep layer of ACC after obtaining a stable baseline for an hour. Interestingly, it was found that L-LTP could be induced by weak TBS in some channels in adult mice. However, there was no L-LTP induced by weak TBS in middle-aged mice (Fig. 5). In adult male mice, there were 99 channels activated in total after weak TBS, among which there were 21 channels showing E-LTP, 54 channels showing L-LTP and 24 channels showing non-LTP (n = 5 mice, Figs. 5a-c). The ratio of L-LTP channels was 54.55% in all activated channels (Fig. 5k). In middle-aged male mice, there were 34 channels activated, among which there were only 6 channels showing E-LTP and 28 channels showing non-LTP (n = 5 mice, Figs. 5d and 5e). No channel showed L-LTP in the middle-aged mice.
Similarly, in adult female mice, there were 91 channels activated, among which there were 22 channels showing E-LTP, 59 channels showing L-LTP, and 10 channels showing non-LTP (n = 5 mice, Figs. 5f-h), and the ratio of L-LTP channels was 65.56% (Fig. 5k). In the middle-aged female mice, there were 27 channels activated, among which there were 15 channels showing E-LTP and 12 channels showing non-LTP (Figs. 5i and 5j), and again no channel showed L-LTP. In summary, L-LTP can be induced by weak TBS in the ACC and shows an age-dependent manner in both male and female mice.
BDNF rescued synaptic tagging LTP in middle-aged mice
Our previous study reported that BDNF contributes to synaptic potentiation in the ACC of adult mice (Miao et al. 2021). In this study, we tested whether BDNF can rescue the impaired tagging LTP in middle-aged mice. As shown in Fig. 6a, all the BDNF incubated brain slices had synaptic tagging responses in middle-aged mice, and 35 channels (34.3%) exhibited clear synaptic tagging LTP. In control mice, only 2 brain slices from 5 middle-aged mice showed tagging-like response, and only 11channels (9.73%) exhibited synaptic tagging LTP. In site S1 of strong TBS, it was also found that BDNF improved the L-LTP in the middle-aged mice (Fig. 6b). All the BDNF incubated brain slices had L-LTP after strong TBS, while only 3 brain slices from 5 mice showed L-LTP in no BDNF application slices.
In addition, the change of tyrosine kinase receptor B (Trk B) and cAMP-response element binding protein (CREB) were also tested in the middle-aged mice slices after the application of BDNF. The result from the western blot showed that both the Trk B and the CREB in the ACC increased after BDNF incubation in the middle-aged mice (Figs. 6c and 6d). These results are consistent with the previous reports [37, 38].
Next, we compared the Spatio-temporal distribution of the synaptic tagging LTP after BDNF in middle-aged mice by using the developed modeling and visualization system (Figs. 6e and 6f). After incubating BDNF, it is then observed that the adopted tagging-TBS exhibits multiple L-LTP peaks (vertices), representing the spatial strength and spatial frequency of the synaptic tagging changes in brain slices after the application of BDNF. This is consistent with our previous work [31] that the network LTP is often formed as a ring distribution surrounding the stimulation site.
TrkB agonist R13 rescued synaptic tagging LTP in middle-aged mice
R13 is a prodrug for 7,8-dihydroxyflavone(7,8-DHF), which is a flavone found in plants and has a similar function to BDNF [39, 40]. We also tested the roles of R13 in the impaired tagging LTP in middle-aged mice. R13 was orally administered for 15 days in middle-aged mice, and then the brain slices were used for fEPSPs recording as above mentioned. As shown in Fig. 7, in R13-treated middle-aged mice, all the brain slices from 5 mice with R13 treatment had synaptic tagging response, while no brain slices from control mice showed tagging-like response (Fig. 7a). On site S1, we found that R13 enhanced the L-LTP in middle-aged mice (Fig. 7b). All the R13 treated mice shown L-LTP responses after strong TBS (n = 5 slices/ 5 mice), while only 4 mice showed E-LTP in control mice.
We also analyzed the recruited channels in R13-treated middle-aged mice. As shown in Figs. 7c and 7d, 4.5 hours after strong TBS on site S1, 2.4 ± 1.4 channels were recruited in each slice with R13 treated mice, and 1.6 ± 0.9 channels in untreated mice. After tagging TBS, 6.2 ± 2.6 channels were recruited in brain slices from R13 treated mice, and it was 0.4 ± 0.4 channels for the untreated mice (*p < 0.01, n = 5 mice for each group, Student’s t-test.). These results suggest that R13 enhances the network propagation of synaptic tagging responses in the ACC of middle-aged mice.
In addition, as revealed in Figs. 7e and 7f where the Spatio-temporal distribution of synaptic plasticity response after R13 oral administration was visualized as a 3D surface. Taken together, these results show that the TrkB agonist rescued synaptic tagging and LTP in middle-aged mice.