ASIV enhanced the memory of mice in both RAMT and SBT
To explore the effect of ASIV on the memory of mice, the mice were subjected to the RAMT and SBT after ASIV administration. For RAMT, the working memory and reference memory that refer to short-term memory and long-term memory respectively were analyzed. As shown in Fig. 1B and Fig. 1C, ASIV treated mice performed better in the RAMT as their working memory error times (Fig.1B, for 1-week, U = 62.00, P = 0.0782; for 2-week, U = 54.50, P =0.0358) and reference memory error times were reduced significantly (Fig. 1C, for 1-week, U = 46.00, P = 0.1276; for 2-week, U = 33.00, P =0.0174), compared with the control mice. Moreover, ASIV markedly increased the active avoidance times of mice in SBT (Fig. 1D, U = 30.50, P = 0.0012). These results showed that ASIV could enhance the memory of mice.
ASIV enervated hippocampal GABAergic system in mice
To determine the effect of ASIV on the GABAergic system, the concentration of GABA and the expression of GAD65 in hippocampi of mice were examined, respectively. As shown in Fig. 2A, hippocampal GABA level in ASIV treated mice was reduced when compared with that of the control mice (for 2-week, t (20) = 5.003, P = 0.0000; for 5-week, t (25) = 2.507, P = 0.0190). Accordingly, the expression of GAD65 in hippocampus of ASIV treated mice was significantly lower than that of the control mice (Fig. 2B and C, for 2-week, t (4) = 3.349, P = 0.0286; for 5-week, t (4) = 2.779, P = 0.0499). To further investigate if ASIV could affect the strength of inhibitory synaptic transmission in hippocampus, untrathin sections obtained from blocks (1 mm ×1 mm ×1 mm) of CA3 region were subjected to electron microscopy observation. According to the reports, there are two major morphologic types of synapses, i.e. asymmetric and symmetric synapses [35, 36]. Excitatory synapses are asymmetrical synapses with significant postsynaptic density, while inhibitory synapses are symmetrical synapses with thinner postsynaptic density. And there is also another type of synapse with oblique synaptic cleft and associated membrane density that is considered to be uncharacterized synapses [37]. As displayed in Fig. 2D, excitatory and inhibitory synapses could be clearly identified in the electron microscopy image. ASIV treatment for 2 weeks and 5 weeks decreased the ratio of inhibitory synapse remarkably in CA3 region (Fig. 2E, for 2-week, t (5) = 2.632, P = 0.0464; for 5-week, t (5) = 2.769, P = 0.0394). Since GABAergic system mainly participates in the synaptic inhibition, we next investigated the impact of ASIV on the GABAergic synaptic transmission on CA1 pyramidal neurons by whole-cell patch clamp recording in the presence of d-APV and NBQX, the antagonists for AMPA and NMDA receptors, respectively. As shown in Fig. 3A-B, ASIV treated mice showed reduced amplitude (t (15) = 2.762, P = 0.015) and frequency (t (15) = 3.304, P = 0.005) of the GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) compared with the control mice. Therefore, these results suggested that ASIV could enervate hippocampal GABAergic neurotransmission.
ASIV suppressed the expression of GAD65 through regulating the BDNF/TrkB signaling pathway
To explore the possible action mechanism of ASIV, the expressions of proteins, such as BDNF and TrkB that actively involved in the modulation of memory were detected. In our experiments, the expression of BDNF as well as its receptor, TrkB, was also decreased in mouse hippocampus after treated with ASIV for 2 weeks and 5 weeks (Fig.5A and B, for 2-week BDNF, t (4) = 4.294, P = 0.0127; for 5-week BDNF, t(4) = 2.794, P = 0.0491; Fig.5C and D, for 2-week TrkB t(4) = 3.512, P = 0.0249; for 5-week TrkB, t(4) = 3.203, P = 0.0328). Treatment with K-252a reduced the inhibitory effect of ASIV on GAD65 expression (Fig.4 E and F, F (3, 8) = 8.219, for Control vs ASIV, P = 0.0022; for K-252a vs K-252a + ASIV, P = 0.2465). The results suggested that ASIV exerted its function on GAD65 through regulating the BDNF/TrkB signaling pathway.
ASIV decreased the expressions of EGR-1 in mouse hippocampus
To explore the possible action mechanism of ASIV, the expression of EGR-1, which actively involves in the modulation of memory, was detected. In Fig. 4A-C, ASIV treatment for 2 weeks and 5 weeks significantly suppressed the expressions of hippocampal EGR-1 at both mRNA (for 2-week, t (10) = 2.730, P = 0.0212; for 5-week, t (5) = 3.260, P = 0.0225) and protein levels (for 2-week, t (4) = 2.817, P = 0.0480; for 5-week, t (4) = 3.587, P = 0.0247). The results suggested that ASIV might enhance the memory of mice through EGR-1.
EGR-1 KO abrogated the memory beneficial effect of ASIV on mice in both RAMT and SBT
To reveal the role of EGR-1 in the memory beneficial effect of ASIV on mice, EGR-1 KO mice were treated with ASIV and subjected to the RAMT and SBT using the same timeline as illustrated in Fig. 1A. As shown in Fig. 6B and C, ASIV treatment did not reduce the working memory (for 1-week, U = 19.00, P = 0.2228; for 2-week, U = 43.00, P =0.6158) and reference memory errors (for 1-week, U = 23.00, P = 0.4815; for 2-week, U = 35.00, P =0.2775) of EGR-1 KO mice in RAMT. And ASIV treatment also did not change the active avoidance times of EGR-1 KO mice in SBT (Fig. 6D, U = 44.00, P =0.6684). These results confirmed that EGR-1 played an indispensable role in the memory beneficial effect of ASIV.
EGR-1 KO abolished the inhibition of ASIV on GABAergic synaptic transmission in mice
To further corroborate the role of EGR-1 in hippocampal GABAergic system of ASIV treated mice, the sIPSCs of EGR-1 KO mice treated with ASIV for 2 weeks were recorded. As shown in Fig. 7A-B, ASIV treatment failed to alter the amplitude (t (17) = 0.337, P = 0.740) and frequency (t (17) = 1.362, P = 0.191) of sIPSCs. These results demonstrated that EGR-1 KO abolished the inhibition of ASIV on hippocampal GABAergic synaptic transmission.
EGR-1 KO blocked the inhibition of ASIV on GAD65, BDNF and TrkB in mouse hippocampus
To further validation of EGR-1 in the inhibitory effect of ASIV on GABAergic synaptic transmission, the hippocampal protein expression of GAD65, BDNF and TrkB were examined in EGR-1 KO mice treated with ASIV for 5 weeks. As shown in Fig. 8, EGR-1 KO decreased GAD65 expression (Fig.8A and B, t (4) = 5.517, P = 0.0067) but did not affect expression of BDNF (Fig.8E and F, t (4) = 0.3035, P = 0.7766) and TrkB (Fig.8 E and F, t (4) = 0.6084, P = 0.5758) in hippocampus. ASIV treatment did not change the expression levels of GAD65 (Fig. 8C and D, t (6) = 0.6829, P = 0.5202), BDNF (Fig. 8G and H, t (6) = 1.331, P = 0.2317) and TrkB (Fig. 8G and H, t (6) = 0.2750, P = 0.7958) in hippocampus of EGR-1 KO mice, suggesting that ASIV modulated GABAergic synaptic transmission through EGR-1.
ASIV increased basic synaptic transmission and enhanced mRNA expression of EGR-1 in response to external stimuli in mice
To test the effects of ASIV on basic synaptic transmission in the CA1 region, the I/O function of ASIV treated mice was evaluated. As illustrated in Fig. 9A, the PS amplitude of ASIV-treated mice showed the increasing tendency, especially when the stimulus current was at 1.2 mA, 1.3 mA, 1.5 mA and 1.6 mA (P < 0.1), suggesting that ASIV might enhance hippocampal synaptic transmission in mice.
To further understand the physiological significance of the inhibitory effect of ASIV on EGR-1, the mice-treated with ASIV for 2 weeks were subjected to a single-trial SBT together with the control mice. Consequently, the hippocampal mRNA expression of EGR-1 was analyzed. In Fig. 9B, mRNA expression level of EGR-1 in both control and ASIV-treated mice was elevated. However, the increase amplitude of the mRNA expression level of EGR-1 in ASIV treated mice was greater than that in the vehicle-treated control mice (t (11) = 2.327, P = 0.0400). These results suggested ASIV could increase the response of EGR-1 to external stimuli.