| Learning plays pivotal roles in gaining new foraging skills and food preferences.However,the molecular mechanism underlying the effects of learning and mrmory on feeding habits is unclear.Chinese perch,as a demersal piscivore,has a unique feeding habit.Once the fry of Chinese perch start feeding,they only accept live prey fish in the wild and refuse dead prey fish or artificial diets.The results of our research group have shown that there are learning behaviors in Chinese perch,and the domestication of Chinese perch may be regulated by CaMKII and PKA learning and memory pathways.However,the regulation mechanism is still unclear of the learning and memory pathways of CaMKII and PKA on the domestication of Chinese perch.To investigate the effect of learning and memory on feeding habit transition in Chinese perch,we used the inhibitor of the CaMKII signaling pathway KN-93 in vivo and in vitro to explore how learning and mrmory can affect the acquirement of new feeding habit in Chinese perch.The overexpression vector of genes c-fos and zif268 was constructed,and pcDNA3.1-EGFP was chosen as the reporter plasmid and transferred into Chinese perch brain cells by electroporation.By analysis of gene expressions,protein phosphorylation,success rate,learning times,food intake,ChIP assay allowed us to gain insights into the molecular mechanism of learning and memory in acquisition new feeding habits in Chinese perch.In the experiment 1,after learning transcriptome sequencing,it is found that there are significant enrichment of representative signaling pathways related to learning and memory,including the signaling pathway Neurotrophin,cAMP,Long-term depression,Serotonergic synapse,Dopaminergic synapse,Glutamatergic synapse,Cholinergic synapse.ICV injection of the CaMKII inhibitor KN-93 in Chinese perch could further confirm the specific pathway of learning and mrmory involved in Chinese perch domestication.Compared with the W+PBS injection group,in the T+PBS injection group had a higher success rate,higher food intake of dead prey fish and lower learning times,suggesting that learning and mrmory can promote the development of new feeding habits in Chinese perch.Furthermore,the success rate and food intake of fish with learning and mrmory(T+PBS injection group)were decreased after the injection of the CaMKII inhibitor KN-93(T+KN-93 injection group),and learning times increased.In addition,there were no differences between the W+PBS and W+KN-93 injection groups or between the W+KN-93 and T+KN-93 injection groups,suggesting that KN-93 has neither a negative effect on food intake nor a positive effect without learning and mrmory.These results showed that CaMKII signaling might play an important role in the Chinese perch domestication through learning and mrmory.Consistent with the decreased learning and mrmory activity and domestication,inhibited CaMKⅡ signaling was also observed in Chinese perch after KN-93 injection.The protein level of p-CREB was significantly increased in the T+PBS group and then decreased after KN-93 injection.However,the p-CREB level showed no difference among the four groups.It has been confirmed that learning and mrmory can promote the acquisition of feeding habits in Chinese perch through the CaMKⅡ signaling pathway,but the phosphorylation of CREB is not involved.Regarding the IEG genes,we also found higher mRNA expression of the c-fos gene in the T+PBS injection group,and decreased expression in the T+KN-93 injection group.However,the mRNA expression levels of the zif268 gene were increased significantly in the T+PBS,W+KN-93 and T+KN-93 injection groups.For the target factors,such as appetite-related genes,we found that the mRNA expression level of the anorexigenic pomc gene was significantly decreased in the T+PBS injection group and increased in the T+KN-93 inj ection group.Meanwhile,the mRNA expression level of orexigenic mch gene was significantly increased in the T+PBS injection group and decreased in the T+KN-93 injection group.However,the expression level of the anorexigenic tac3a gene was decreased in the T+PBS injection group,but not increased in the T+KN-93 injection group.Compared with the T+PBS injection group,the expression in the W+PBS injection group was significantly increased.In addition,no significant changes in agrp,npy and cart gene expression levels were found between the W+PBS and T+PBS injection groups.We hypothesized that the CaMKⅡsignaling inhibited by KN-93 could contribute to the stimulation of the c-fos gene,which might be an important transcriptional factor to decrease the expression of the anorexigenic pomc gene and increase the expression of the orexigenic mch gene.It is suggested that learning and memory involved in the Chinese perch domestication could be attributed to the C aMKII-c-fos-pomc/mch signaling pathway.In the experiment 2,we used KN-93,the inhibitor of the CaMKⅡ signaling pathway,to explore how the CaMKⅡ pathway can affect the domestication of Chinese perch.The results showed that the p-CaMKⅡ and p-CREB protein level,the c-fos and the mch expression with KN-93 treatment were significantly lower than that in the control condition,the pomc expression were significantly increased,but no significant difference of the npy and the leptin a genes expression were found between groups.We showed that c-Fos,in addition to its well-known oncogenic function,has a novel regulatory function on food intake by the pomc gene.In the experiment 3,in order to study the influence of learning and memory pathways on the regulation of feeding in Chinese perch.The overexpression vector of genes c-fos and zif268 was constructed,and pcDNA3.1-EGFP was chosen as the reporter plasmid and transferred into Chinese perch brain cells by electroporation,and the optimal electroporation conditions were determined by testing the transfection efficiency with different voltages,plasmid amounts,transfection time,and numbers of-shocks.The results showed that learning and memory overexpression vector pcDNA3.1-c-fos-EGFP,pcDNA3.1-zif268-EGFP was successfully constructed.The maximum electroporation efficiency was achieved underthe following conditions in a 0.2 cm electroporation cuvette containing brain cells(1×106),Glectric voltage 240 V,pulse time 5 ms,plasmid 6 μg,one electric shock and transfection time 48 h.Showed that pcDNA3.1-EGFP was successfully transfected into Chinese perch brain cells in vitro.The mRNA expression levels of the appetite genes agrp,pomc,mch,npy were detected after overexpression of learning and memory genes.After overexpression of the c-fos gene,whereas the mRNA expression of the appetite genes pomc and wich was significantly higher.After overexpression of zif268 gene,there were no significant changes in the mRNA expression levels of the agrpy mch,npy and pomc genes in vitro.In summary,it has been confirmed that learning and mrmory can promote the acquisition of feeding habits in Chinese perch through the c-fos signaling pathway.As a transcription factor,c-Fos is likely to regulate the expression of appetite genes pomc and npy.Altogether,our results showed that learning and mrmory could promote the feeding habit domestication from live prey fish to dead prey fish through the CaMKⅡ-c-fos-pomc signaling pathway in Chinese perch. |
