| Research BackgroundGiven the profound effects that early life experiences may have upon personalistic and psychosocial function later in life, it is funny that most adults fail to recall events from their infancy or early childhood. Infantile amnesia refers to the phenomenon that people can not recall the autobiographical events before a certain age, some researches describe infantile amnesia as the phenomenon of spontaneously accelerated forgetting during infancy or childhood (i.e. childhood infantile) as compared with the adult. The boundary age of infantile amnesia in human is undefined and the researches on human infantile amnesia still focus on the neuropsychology studies. Since Campbell found that infantile amnesia also existed in rats in 1962, it provided a neurobiological way to know more about the infantile amnesia. Using an active-avoidance task in Campbell’s study, rats from five different ages were tested after training; all groups exhibited the similar escape latencies when tested immediately.7 days after training, the P18 rats forgot rapidly, however, the P54 and the P100 rats still exhibited good memory retention after 42 days. This general finding that younger animals forgot more rapidly than older ones had since been replicated numerous times in different species with different learning procedures after Campbell’s experiment.The infantile amnesia is unlikely to be explained by a unitary theory. The brain regions included the hippocampus and the medical prefrontal cortex, the related neurotransmitter systems included the NMDA and GABA, the related hormones systems included the stress hormones, which might play an important role on learning and memory are likely to be involved.The hippocampus is a late maturing structure and belongs to the limbic system. Hippocampal neurogenesis, synaptogenesis, the development of neurotransmitters or hormones receptors and also the myelination which undergo major anatomical and physiological changes after birth occur in the hippocampus. Passive-avoidance task and contextual fear conditioning task are hippocampus-dependent memories, and both of them should need the complete hippocampal function. To test the spatial memory from different ages of the children and contrast them with the age-related structure changes in the hippocampus of the primates, Lavenex P found that the developing changes of the hippocampus were consistency of the infantile amnesia boundary age, inferred that there was a causal relationship between the structural development of the hippocampus and the infantile amnesia. Josselyn SA also considered that the hippocampal neurogenesis might play an important role on the infantile amnesia. The hippocampal neurogenesis can facilitate the acquisition of new hippocampus-dependent memories in some instances, however, when it comes to the memories that have already been formed, the continual integration of new neurons into the existing circuits may weaken the old memory and cause forgetting. The dentate gyrus (DG) is one of the brain regions that the neurogenesis persists throughout life, but rate of which declines dramatically with age. The high rate of neurogenesis observed during development may contribute to high rates of forgetting in infancy, this was also confirmed in Akers’s experiment. To use the contextual fear conditioning task and immunofluorescent staining, Akers found that the hippocampal neurogenesis was much higher in P17 than P60 mice, what’s more, the P17 mice exhibited time-dependent forgetting while the P60 mice still exhibited perfect memory retention even 28 days after training. This experiment also found that genetic and pharmacological decreased in neurogenesis might mitigate forgetting in P17 mice while the voluntary running increased neurogenesis in P60 mice could promote forgetting. There was an inverse relationship between the hippocampal neurogenesis and context-induced freezing observed, in other words, the hippocampal neurogenesis and memory persistence were causally related.The mammalian target of rapamycin (mTOR) is a conservative serine/threonine kinase contains of 2549 amino acids. The mTOR signaling pathway accepts the signal inputs from the hormones, growth factors, nutritional factors or energy, exerts an influence on controlling the genetic transcription, protein translation initiation, cell cycle regulation, cell apoptosis and metabolism through the PI3K/Akt/mTOR or AMPK pathways. Rapamycin is an inhibitor of the mTOR pathway, which can block the phosphorylation of the mTOR pathway downstream effectors p70S6K or 4EBPs, inhibit the transcription and translation of the related protein thus play an important role on the immunosuppressive, cell proliferation, cell apoptosis and autophagy. For example, rapamycin might affect to the cell proliferation of new neurons in the dentate gyrus of hippocampus in infant mice.Rapamycin can also affect to the synaptic plasticity thus play an important role on learning and memory. Local injection or intraperitoneal injection with rapamycin might impact the long-term memories of the hippocampal-dependent spatial memory, the mPFC-dependent or amygdala-dependent fear memory and the gustatory cortex-dependent taste aversion memory, but do not affect the short-term memories in this animal experiments. However, the low dose rapamycin by oral in rats might enhance the spatial memory in the adult mice and memory retention in the aged mice, improve the memory impairment on a mouse mode of Alzheimer and increase the average life expectancy in the elderly mice. Therefore, the rapamycin may exhibit different influences which depend on the physical condition, age or administration pathways of the experiment designs. Few researches were focused on the influences of rapamycin injection on learning and memory in infant rats. As the rapamycin can affect to the cell proliferation of new neurons in the dentate gyrus of hippocampus in infant rats, the hippocampal neurogenesis is associated with the memory retention, we suggest that rapamycin may affect the infantile amnesia through the effect to the hippocampal neurogenesis.Therefore, this study attempted to investigate how intraperitoneal injection with rapamycin could affect the passive-avoidance task, as well as the cell proliferation, neurogenesis and cell apoptosis of the hippocampus in infant rats. To discuss how rapamycin might affect to the infantile amnesia.Studies included the following two parts:Part I:Effects of rapamycin injection on passive-avoidance task in infant ratsObjectiveUsing a passive-avoidance task to train 17-day-old and 60-day-old rats to show the phenomenon of infantile amnesia. To study how intraperitoneal injection with rapamycin can affect to the memory retention on a passive-avoidance task in infant rats.Methods(1) 17-day-old(P17) and 60-day-old(P60) male Sprague-Dawley rats were trained in a passive-avoidance task, each of the two ages was divided into the shocked group and non-shocked group, all rats were tested immediately,2 days and 7 days after training for memory retention; (2) P17 rats were randomly divided into three groups:vehicle group,20mg/kg rapamycin group (Rap20 group) and 40mg/kg rapamycin group (Rap40 group), each of the three groups was divided into the shocked group and non-shocked group. Rats were intraperitoneal injected with either vehicle or rapamycin immediately after passive-avoidance training at P17, memory retention were evaluated after 2 days (P19) and 7 days (P24).Results(1) The crossing latency of adaptive training were no significant differences between the P17 rats and P60 rats (P>0.05). A 2×3 factorial analysis of variance (ANOVA) revealed a significant main effect of Age (P<0.05) and Retention Interval (P<0.05) in the shocked groups. When the shocked rats were tested at the immediate retention interval, comparable levels of crossing latencies were observed in the P17 and P60 rats (P>0.05). When being tested after 2 days and 7 days, the P17 rats forgot more rapidly than P60 rats (P<0.05). There were no age-related differences in crossing latency between the non-shocked groups (P>0.05).(2) The crossing latency of adaptive training were no significant differences among the vehicle group, Rap20 group and Rap40 group (P>0.05). A 3×2 factorial analysis of variance (ANOVA) revealed a significant main effect of Dose (P<0.05) and Retention Interval (P<0.05) in the shocked groups. Three shocked groups exhibited comparable levels of crossing latencies when being tested 2 days after training(P>0.05), however, administration of rapamycin could enhance the memory retention 7 days after training(P<0.05). There were no crossing latency differences between the Rap20 and Rap40 shocked group (P>0.05). There were no dose-related differences in crossing latency among the non-shocked groups (P>0.05).ConclusionsThe 17-day-old rats exhibited an accelerated forgetting phenomenon in a passive-avoidance task to show the infantile amnesia. The intraperitoneal injection with rapamycin may enhance the memory retention in the 17-day-old rats.Part II:Effects of rapamycin injection on cell proliferation, neurogenesis and cell apoptosis in hippocampus in infant rats.ObjectiveTo study how intraperitoneal injection with rapamycin can affect to the cell proliferation, neurogenesis and cell apoptosis of hippocampus in P17 rats.Methods(1) P17 rats were randomly divided into four groups:normal group, vehicle group,20mg/kg rapamycin group (Rap20 group) and 40mg/kg rapamycin group (Rap40 group). Rats were intraperitoneal injected with either vehicle or rapamycin at P17.44 hours after vehicle or rapamycin injection, BrdU was intraperitoneal injected in all groups (100mg/kg), immunofluorescence staining of BrdU was detected at P19 to observe the cell proliferation of hippocampus 4 hours after BrdU injection. (2) P17 rats were randomly divided into four groups:normal group, vehicle group, Rap20 group and Rap40 group. Rats were intraperitoneal injected with either vehicle or rapamycin at P17,12 hours after vehicle or rapamycin injection, BrdU was intraperitoneal injected in all groups(50mg/kg×5 times, every 6 hours),12 hours and 132 hours after the final injection of BrdU, immunofluorescence staining of BrdU/DCX was detected to observe the hippocampal neurogenesis at P19 and P24. (3) P17 rats were randomly divided into four groups:normal group, vehicle group, Rap20 group and Rap40 group. Rats were intraperitoneal injected with either vehicle or rapamycin at P17, immunohistochemical staining of caspase3 was detected to observe the cell apoptosis of hippocampus 48 hours(P19) after vehicle or rapamycin injection.Results(1)The BrdU-positive cells were significant differences between the vehicle group and the Rap20 group, Rap40 group (P<0.05). Less BrdU-positive cells were found in the DG of hippocampus at P19 after the administration of rapamycin. The BrdU-positive cells were no significant differences between the normal group and vehicle group (P>0.05) and showed significant differences between the Rap20 group and Rap40 group (P<0.05). (2) The BrdU-positive and BrdU/DCX-positive cells were significant differences between the vehicle group and the Rap20 group, Rap40 group at each time (P<0.05). Less BrdU-positive and BrdU/DCX-positive cells were found in the DG of hippocampus at P19 and P24 after administration of rapamycin. The BrdU-positive and BrdU/DCX-positive cells were no significant differences between the normal group and vehicle group (P>0.05) and showed significant differences between the Rap20 group and Rap40 group (P<0.05) at each time. (3) The caspase3-positive cells were significant differences between the vehicle group and the Rap20 group, Rap40 group (P<0.05). More caspase3-positive cells were found in the DG of hippocampus at P19 after administration of rapamycin. The caspase3-positive cells were no significant differences between the normal group and vehicle group (P>0.05) and showed significant differences between the Rap20 group and Rap40 group (P<0.05).ConclusionsRapamycin might reduce the cell proliferation and neurogenesis of hippocampus and enhance the cell apoptosis of hippocampus in P17 rats. |
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