Article Figures & Data
Figures
- Figure 1.
Effects of MgT on delay and trace fear conditioning. A, Top, Experimental design showing, after 4 weeks of MgT treatment, the fear conditioning (day 1) and an LTM test (day 2). Bottom, Illustration of the delay and trace fear conditioning protocols. B, Left, Freezing behavior of control and MgT-treated rats during baseline (BL) and three trials of delay fear conditioning. Right, Freezing behavior during LTM test before tone (Pre-CS) and after tone (Post-CS) of control and MgT-treated rats (n = 8). C, Freezing behavior during LTM test before and after tone of control and MgT-treated rats (n = 9). Test was conducted 24 h after a single trial of delay fear conditioning. D, Left, Freezing behavior of control and MgT-treated rats during baseline and three trials of trace fear conditioning (trace interval = 15 s). Right, Freezing behavior during LTM test before and after tone of control and MgT-treated rats (n = 10). E, Freezing behavior during LTM test before and after tone of control and MgT-treated rats (n = 9). The LTM test was conducted 24 h after trace fear conditioning with trace interval = 30 s. *p < 0.05. Data presented as mean ± SEM.
- Figure 2.
Effects of MgT on extinction learning, retention, and retrieval. A, Top, Experimental design to test extinction (Ext.) learning and retention. Bottom left, Freezing behavior of MgT-treated and control rats during the first long-term memory test (LTM1) conducted 24 h after fear conditioning (Cond.; day 2). Middle, Freezing behavior of MgT-treated and control rats during extinction learning conducted 48 h after conditioning (day 3). Right, Freezing behavior of MgT-treated and control rats (n = 8) during a retention of extinction test (LTM2) conducted 3 d after extinction learning (day 6). B, Top, Experimental design to test the effects of MgT treatment on retention of extinction when treatment was given after fear conditioning for 4 weeks. Bottom, Freezing behavior of rats during conditioning, LTM1, extinction learning, and LTM2. In the LTM2 test, MgT-treated rats exhibited significantly lower freezing behavior than controls (n = 8). C, Top, Experimental design to test the effects of MgT on retrieval of the extinction memory. Bottom, Freezing behavior of untreated rats during three trials of fear conditioning (day 1) and 10 trials of extinction (day 2). Freezing behavior of rats assigned as control (white bar) and MgT-treated (lined bar) during retention of extinction test (LTM1) conducted 24 h after extinction learning (day 3). Right, Freezing behavior of MgT-treated (n = 13) and control (n = 14) rats during LTM2 conducted 4 weeks after the beginning of MgT treatment (day 32). *p < 0.05. Data presented as mean ± SEM.
- Figure 3.
Effects of MgT on spontaneous recovery, renewal and reinstatement. A, Spontaneous recovery test. Freezing behavior of MgT-treated (n = 9) and control (n = 10) rats during fear conditioning (Cond.; day 1), extinction learning (Ext.; day 2; average of the first and the last two of 14 trials is presented), LTM test (day 3), and spontaneous recovery test (day 30). All experiments were performed in the same context, namely context A. B, Renewal test. Freezing behavior of MgT-treated (n = 17) and control (n = 18) rats during memory tests performed in the context where extinction learning (14 trials) was performed (context B) and in the context where fear conditioning was performed (context A). C, Reinstatement test. Freezing behavior of MgT-treated (n = 13) and control (n = 10) rats, during the last two trials of extinction performed on day 2 and during a reinstatement test performed 24 h (day 4) after exposure to five unsignaled foot-shocks (5-US) in the same context. *p < 0.05; **p < 0.01. Data presented as mean ± SEM.
- Figure 4.
Effects of MgT on expression of NMDAR subunits, activation of NMDAR signaling, and BDNF expression in the prefrontal cortex and amygdala. A, Western blot analysis of expression of NR2B, NR2A, and NR1 subunits and activation of downstream molecules in the PFC. MgT treatment significantly increased NR2B expression only (n = 7) without increasing NR2A (n = 7) or NR1 (n = 7) subunits. The ratios of p-α-CaMKII/α-CaMKII (n = 6) and p-CREB/CREB (n = 9) were significantly higher in the prefrontal cortex of MgT-treated rats. Data are presented as the percentage of mean control and were statistically compared with the respective control (dashed line) for each experiment. β-actin was used as a loading control. B, Quantitative analysis of BDNF protein expression in the PFC using ELISA. BDNF level was significantly higher in MgT-treated rats in comparison with controls (n = 10). C, D, Same as A and B, but in the basolateral amygdala. MgT treatment did not alter NR2B (n = 7), NR2A (n = 6), or NR1 (n = 6) expression levels, p-CaMKII/CaMKII ratio (n = 7), p-CREB/CREB ratio (n = 7), or BDNF expression (n = 8) in the basolateral amygdala. *p < 0.05. Data presented as mean ± SEM.
- Figure 5.
Effects of MgT on presynaptic boutons in the infralimbic and prelimbic prefrontal cortex and in basolateral amygdala. A, Left, Illustration of the medial prefrontal cortex showing the prelimbic and infralimbic regions (green). Middle, Syn+ puncta in the IL-PFC of control and MgT-treated rats. Right, Quantitative analysis of the density of Syn+ puncta in control and MgT-treated rats (n = 5). B, Quantitative analysis of the density of Syn+ puncta in the PrL-PFC of control and MgT-treated rats (n = 5). C, Left, Illustration of the lateral and basal amygdala (LA and BA, respectively; green). Middle, Syn+ puncta in the lateral amygdala of control and MgT-treated rats. Right, Quantitative analysis of the density of Syn+ puncta in the lateral amygdala of control and MgT-treated rats (n = 6). D, Quantitative analysis of the density of Syn+ puncta in the basal amygdala of control and MgT-treated rats (n = 6). The density was estimated as the number of immunostained puncta per 1000 μm2. ***p < 0.001. Data presented as mean ± SEM.
- Figure 6.
Effects of MgT treatment on synaptic plasticity in the infralimbic prefrontal cortex and lateral amygdala. A, Left, Long-term potentiation (as percentage of baseline) induced by the spike timing protocol (arrow) in pyramidal neurons in the infralimbic prefrontal cortex slices of control (n = 9) and MgT-treated (n = 5) rats. Insets, Representative traces of EPSCs are presented before (solid line) and after (dotted line) induction of long-term potentiation. Right, The magnitude of long-term potentiation (average over last 5 min). MgT treatment significantly increased the long-term potentiation in the infralimbic prefrontal cortex. B, Same as A, but in the lateral amygdala of control (n = 8) and MgT-treated (n = 6) rats. MgT treatment did not significantly change the long-term potentiation in the lateral amygdala. Dashed lines indicate the normalized basal synaptic responses. **p < 0.01. Data presented as mean ± SEM.
- Figure 7.
Effects of elevation of [Mg2+]o in vitro on synaptic NMDAR current and synaptic plasticity in the infralimbic prefrontal cortex and lateral amygdala. A, Left, Representative traces of AMPA receptor EPSC (gray trace) and NMDAR EPSC (black traces) recorded at membrane potentials of −60 and +50 mV, respectively, in the infralimbic prefrontal cortex. Right, The ratio of amplitude of NMDAR EPSCs to amplitude of AMPA receptors EPSCs (INMDA/AMPA) calculated for each cell in the infralimbic prefrontal cortex slices incubated (5 h) under physiological extracellular magnesium concentration (0.8-[Mg2+]o, n = 7) and elevated [Mg2+]o (1.2-[Mg2+]o, n = 7). Elevation of [Mg2+]o in vitro significantly increased the INMDA/AMPA in the infralimbic prefrontal cortex. B, Left, Long-term potentiation (as percentage of baseline) induced by the spike timing protocol (arrow) in the infralimbic prefrontal cortex slices (0.8-[Mg2+]o slices, n = 6; 1.2-[Mg2+]o slices, n = 9). Insets, Representative traces of EPSC are presented before (solid line) and after (dotted line) induction of long-term potentiation. Right, The magnitude of long-term potentiation (average over last 5 min) of 0.8-[Mg2+]o and 1.2-[Mg2+]o slices. Elevation of [Mg2+]o in vitro significantly increased the long-term potentiation in the infralimbic prefrontal cortex. C, Same as A, but in the lateral amygdala (0.8-[Mg2+]o, n = 7; 1.2-[Mg2+]o, n = 8). D, Same as B, but in the lateral amygdala (0.8-[Mg2+]o, n = 6; 1.2-[Mg2+]o, n = 5). Elevation of [Mg2+]o in vitro did not significantly change the INMDA/AMPA ratio or long-term potentiation in the lateral amygdala. Dashed lines indicate the normalized basal synaptic responses. *p < 0.05. Data presented as mean ± SEM.
