| Objectives:To investigate the inhibitory effect of the isoflurane-exposure on the formation of contexual fear conditioning(CFC) memory in mice and whether this kind effect is related to altered histone acetylation in the hippocampus and altered memory-related gene expression which were mediated by histone acetylation. To reveal the relationship between isoflurane-induced changes of histone acetylation during memory formation and amnesia caused by isoflurane.Metheds:1.72adult male C57BL mice(8weeks old) were randomLy divided into6groups(n=12):group Air+CFC:After exposure to oxygen-enriched air for30min, the mice were immediately underwent the CFC training procedure; group0.2%ISO+CFC:After exposure to0.2%isoflurane which was carried by30%O2, the mice were immediately underwent the CFC training procedure; group0.4%ISO+CFC:the same as above but the concentration of isoflurane was0.4%; group0.6%ISO+CFC:the same as above but the concentration of isoflurane was0.6%; group Air+MT(mock training):After exposure to oxygen-enriched air for30min, the mice were immediately underwent the mock CFC training procedure,which was same as CFC training but did not receive a footshock; group0.4%ISO+MT:After exposure to0.4%isoflurane which was carried by30%O2, the mice were immediately underwent the mock CFC training session.2.36adult male C57BL mice(8weeks old) were randomLy divided into6groups(n=6):group Air+Naive:the mice were transported to the laboratory and housed in gas-delivery chamber for4days before air exposure,the duration of air exposure was30min; group ISO+Naive:the mice were transported to the laboratory and housed in gas-delivery chamber for4days before isoflurane exposure,the duration of0.4%isoflurane exposure was30min; group Air+MT:the mice were exposed to oxygen-enriched air for30min and then received the experimental manipulations of mock training as described above; group ISO+MT:the mice were exposed to30%O2+0.4%ISO for30min and then received the experimental manipulations of mock training; group Air+CFC:the mice were exposed to oxygen-enriched air for30min and then received the experimental manipulations of CFC training; group ISO+CFC:the mice were exposed to30%O2+0.4%ISO for30min and then received the CFC training.The mice in each group were sacrificed1h after completing the procedure described above, the tissues of hippocampal CA1,CA2/3,DG areas were micro-dissected for Western-blot analysis of H3K9,H3K14,H4K5,H4K12acetylation and c-Fos,BDNF protein.3.104adult male C57BL mice(8weeks old) were randomLy divided into4groups(n=26):group NS+Air:the mice received an once-daily intraperitoneal injection of saline for27days, then were exposed to oxygen-enriched air for30min; group SB (sodium butyrate)+Air:the mice received an once-daily intraperitoneal injection of SB for27days, then were exposed to oxygen-enriched air for30min; group NS+ISO:the mice received an once-daily intraperitoneal injection of saline for27days, then were exposed to30%O2+0.4%isoflurane for30min; group SB+ISO: the mice received an once-daily intraperitoneal injection of SB for27days, then were exposed to30%O2+0.4%isoflurane for30min. After completing the procedure above,the mice were immediately underwent the CFC training. At1h after CFC training,12mice from each group were sacrificed by dislocation of the neck, six of them for immunohistochemistry analysis of H3K9, H3K14, H4K5, H4K12acetylation and c-Fos protein and the rest were micro-dissected the hippocampal CA1areas for western-blot analysis of H3K9, H3K14, H4K5, H4K12acetylation and c-Fos, BDNF protein and RT-PCR analysis of c-Fos, BDNF gene expression.The other14mice in each group were housed in cages and received the last injection, the CFC test were arranged at24h after CFC training. Results:1.During CFC training, when a single1.0-mA footshock was given, all mice in group Air+CFC showed the visible nociceptive reflexive behaviours, including screaming, jumping, and withdrawal. There was no difference in visible nociceptive reflexive behaviours among group Air+CFC,0.2%ISO+CFC and0.4%ISO+CFC when footshock was given. Compared with group Air+CFC, the mice in group0.6%ISO+CFC showed less jumping and withdrawal behaviour. Compared with CFC test, the mice from group Air+CFC,0.2%ISO+CFC and0.4%ISO+CFC showed less freezing behaviour during CFC training, but in group0.6%ISO+CFC, the situation was opposite to that. During CFC training, there was no difference in the proportion of time spent freezing among group Air+CFC,0.2%ISO+CFC and0.4%ISO+CFC and the freezing behaviour of group0.6%ISO+CFC was more than group Air+CFC. During CFC test, the proportion of time for freezing in each group was as following:group Air+CFC>group0.2%ISO+CFC>group0.4%ISO+CFC> group0.6%ISO+CFC.2. Compared with group air+naive, the levels of H3K9, H3K14, H4K12acetylation and c-Fos protein in the hippocampal CA1area of group air+MT were significantly increased, and the levels of H3K9, H3K14, H4K5, H4K12acetylation and c-Fos, BDNF protein in the hippocampal CA1area of group air+CFC were significantly increased. There was no difference in above molecular markers between air+naive/ISO+naive group. There was also no difference in those molecular markers between air+MT/ISO+MT group. Compared with group air+CFC, the levels of H3K14, H4K5, H4K12acetylation and c-Fos, BDNF protein in hippocampal CA1area of group ISO+CFC were significantly decreased.3. At1h after CFC training, The levels of H3K14, H4K5, H4K12acetylation in hippocampal CA1area of group NS+ISO were significantly lower than in group NS+Air, using the western-blot analysis and immunohistochemistry analysis, but these molecular markers in group SB+ISO were higher than in group NS+ISO. In western-blot, immunohistochemistry and RT-PCRanalysis, we found that the c-Fos gene expression of hippocampal CA1area in group NS+ISO was the lowest among the four groups at1h after CFC training and the chronic injection of SB was shown to be able to increase the c-Fos level in response to CFC training. Using western-blot and RT-PCR analysis, we also found that the BDNF expression of hippocampal CA1area in group NS+ISO was the lowest among the four groups at1h after CFC training and the chronic injection of SB could improve BDNF expression1h after CFC training. Additionally, the proportion of freezing time during CFC test in group NS+ISO was lower than in group NS+Air, and the group SB+ISO showed larger proportion of freezing time than group NS+ISO.Conclusions: 1.The low concentration inhalation of isoflurane can induce the amnesia.0.4%isoflurane inhalation can effectively inhibit the formation of CFC memory with negligible effect on animals’mobility and nociceptive reflexive behaviour and was applicable for amnesia model of mice.2.The formation of CFC memory is related to hippocampal H3K9, H3K14, H4K5, H4K12acetylation and memory-related gene expression, c-Fos and BDNF, which receives the regulation of hitone acetylation.0.4%isoflurane inhalation can inhibit the H3K14, H4K5, H4K12acetylation and downstream c-Fos, BDNF gene expression in response to CFC traming.3. The chronic intraperitoneal injection of SB can antagonize the inhibitory effect on CFC memory induced by0.4%isoflurane inhalation, and the underlying mechanism may be because the injection of SB rescues the repression of hippocampal H3K14, H4K5, H4K12acetylation and c-Fos, BDNF gene expression following CFC training caused by isoflurane inhalation. |
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