Amphetamine Induces Oxidative Stress, Glial Activation and Transient Angiogenesis in Prefrontal Cortex via AT1-R

Figures

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  FIGURE 1. Schematic representation of experimental protocols. VEH: vehicle, CAND: candesartan, SAL: saline, AMPH: amphetamine. Protocol 1: to study the effects of amphetamine and AT1-R blockade in oxidative stress (A). Protocol 2: to study the effects of amphetamine and candesartan on memory, glial reactivity and angioplasticity after 21 days of withdrawal (B). Protocol 3: to study the effects of amphetamine and candesartan on angioplasticity after 7 days of withdrawal (C). Schematic representation of prelimbic and infralimbic subdivisions of the rat prefrontal cortex (D). CxPL: prelimbic prefrontal cortex, CxIL: infralimbic prefrontal cortex.

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  FIGURE 2. Schematic representation of working memory and short-term memory assessment. Working memory assessment by Y-maze test (A). Sample session of novel object recognition test (B). Test session of novel object recognition test (C).

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  FIGURE 3. AT1-R blockade prevents amphetamine-induced oxidative stress. The graphs show the MDA and AOPP quantification as measurements of lipid and protein peroxidation, respectively, in the PFC for the four experimental groups of protocol 1. MDA quantification (A). Equivalents of chloramine T quantification (B). Values are the mean ± SEM; n = 4. *different from the rest of the groups (p < 0.05).

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  FIGURE 4. Amphetamine induces gliosis selectively in the prelimbic prefrontal cortex by AT1-R. The graphs show GFAP and CD11b expression and glial morphology in the PL- and IL-PFC as a measure of astrogliosis and microgliosis, respectively, for the four experimental groups of protocol 3. Schematic representation of GFAP and CD11b immunostaining assessment (A). GFAP immunoreactive area in the PL- and IL-PFC (B), left and right panel, respectively. CD11b positive area in the PL- and IL-PFC (C), left and right panel, respectively. Summed of astroglia branches and average branches length in PL-PFC (D), left and right panel, respectively. Summed of astroglia branches and average branches length in IL-PFC (E), left and right panel, respectively. Summed of microglia branches and average branches length in PL-PFC (F), left and right panel, respectively. Summed of microglia branches and average branches length in IL-.PFC (G), left and right panel, respectively. Values are the mean ± SEM; n = 6–9. *different from VEH-SAL and CAND-AMPH (p < 0.05; Figure 4A) or different from VEH-SAL (p < 0.05; Figure 4B) or CAND-SAL and CAND-AMPH (p < 0.05; Figure 4F panel left).

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  FIGURE 5. Amphetamine induces transient angiogenesis in the prefrontal cortex. The graphs show VWF expression in the PL-PFC and IL-PFC as a measure of angioplasticity for the four experimental groups of experimental protocols 2 and 3. Schematic representation of the VWF immunostaining assessment (A). The number of branching points and vascular tortuosity in IL-PFC after 7 days of withdrawal (experimental protocol 2) (B), left and right panel, respectively. The number of branching points and vascular tortuosity in IL-PFC after 21 days of withdrawal (experimental protocol 3) (C), left and right panel, respectively. Values are the mean ± SEM; n = 5–8. *different from VEH-SAL and CAND-AMPH (p < 0.05).

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  FIGURE 6. Candesartan prevented amphetamine-induced working memory deficit and amphetamine did not induce changes in short-memory recognition performance. The graph shows working memory performance assessed by the Y-maze paradigm (A). The main graph shows the percentage of spontaneous alternations in a Y-maze arena for the four experimental groups of protocol 3. Inset graph shows the number of entries to each arm in the Y-maze test. Values are the mean ± SEM, *different from the rest of the groups (p < 0.05); n = 10-11. The graph shows short-term memory performance evaluated as the time exploring each object in the one trial recognition task (B). The results were expressed as the preference index for the four experimental groups in the test session of protocol 3. The inset figure shows the preference index for the sample session. Values are the mean ± SEM; n = 7-8.

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  FIGURE 7. Hypothetical transient angiogenesis induced by amphetamine in the prefrontal cortex. The graph shows hypothetical angiogenic changes induced by amphetamine in the PL-PFC (A) and IL-PFC (B) subdivisions. The darker lines show the described vascular microarchitecture changes in both brain structures (in days 7 and 21 after the last amphetamine exposure), meanwhile the translucent lines show hypothetical changes in the gaps among these periods.