Effects Of Early Maternal Deprivation On Hippocampal Neurogenesis And The Expressions Of BDNF/CREB In Adult Rat

Objectives:Depression is a prevalent, highly debilitating mental disorder affecting up to15%of the population at least once in their lifetime, with huge costs for society. Although there is consensus in depression about interplay between genetic and envirormiental factors, neurobiological mechanisms of depression are still not well known. The monoamine hypothesis of depression, based on a deficiency or imbalance of mono-amine neurotransmitters, such as serotonin, noradrenaline and dopamine, has been the leading explanation for the disorder for the last50years. The hypothesis is supported by the enhancing effect of all antidepressants on the monoaminergic systems. However, in clinical, part of patients are poor responders to antidepressant, even to more recently discovered medications. Furthermore, clinical response only occurs following weeks to months of treatment. These phenomena suggested that other than acute effects on mono-aminergic systemsare needed for antidepressant efficacy. The reduction of monoamines may be only the initiation of the mechanism of depression.Clinical studies indicate that early adversity in the form of childhood lead to an increased vulnerability to develop depression later in life. Preclinical studies in rodents have shown that early stress results in depressive-like behaviour in adulthood. Mean-while, compared with controls, patients with depression are reported to present with abnormal structure of hippocampus (i.e.reduced hippocampal volume, reduced neurons volume, reduced density of neurons and glial cell). Recently, a number of researches prove that there are new neurons in hippocampus, added to the older cells in the dentate gyrus of hippocampus, maintaining the specific functions of this brain structure. The researchers gradually realized that an impairment of neural plasticity in specific areas of CNS may be part of a possible mechanism linking multiple susceptibility genes and environmental factors.One thing to be noted is that BDNF and CREB are involved in cellular proliferation, migration, differentiation and maintenance in the developing brain, preventing neuron death, Improving the neuron pathological state and promoting the regeneration of neurons and differentiationo It is necessary to the survival and normal phy-siological function of mature central neurons. The reduction of BDNF mRNA and CREB mRNA expression and protein level has negative effctor in neural plasticity and normal function of hippocampus. Given the relationship of early life stress, hippocampal neural plasticity and the mechanism of depression, we hypothesize that early life stress alter hippocampal neurogenesis, BDNF and CREB mRNA expression and protein level, leading to increased vulnerability to develop depression. The depression would occur when subject stress in adulthood.The aim of the present study was therefore to investigate the effect of early MD and the double effects of MD and following CUMS on neurogenesis, BDNF and CREB mRNA expression and protein level of in hippocampus in adult rats.Methods:1. GroupsMale-female pairs were housed under normal conditions, to allow mating. Immediately after the rat pups were born, the adult males were removed, while the mothers remained with the pups. Newborn male rats were randomly divided into four groups. For these animals, birth was designated as postnatal day (PND)1:①Control group:normal feeding form birth to the90th day;②MD group:deprived from their mother3hours per day during PND2to PND4, normal feeding form PND15to PND90;③CUMS group:normal feeding form birth to PND90, received21days CUMS after PND90;④Double stress group:deprived from their mother3hours per day during PND2to PND4, normal feeding form PND15to PND90, received21days CUMS after PND90.2. Stress models①MD model:Maternal deprivation occurred from PND2until PND14for180mins between09:30-12:30h each day.The newborn male rats were removing the home cage to clean box with sawdust from home cage and the same temperature as home cage. After MD, the dam was placed back with the pups and returned to the colony housing room.②CUMS model:Rats were subjected to various mild stressors for3weeks after PND90:water deprivation (24h), food deprivation (28h), tail clamping (1min), hot stress (45℃,5min), inversion of the light/dark cycle (24h), restraint (2hours,twice), ice water swimming (4℃,5min). Animals were exposed to stressors singly. Stressors were never presented simultaneously. 3. Behavior observation①Sucrose preference tests:Sucrose preference tests consisted of first depriving the food and water from each rat’s cage for a period of24h. Then, Water and1%sucrose were placed on the cages in standard drinking bottles with a5cm stainless-steel spout, and were presented for60min. The preference ratios were calculated as the amount of sucrose consumed divided by the total fluid intake. Anhedonia is defined as a reduction insucrose, which is a core symptom of depression. The sucrose preference tests were performed in control group and MD group at PND90, in CUMS group and double stress group after CUMS.②Open field test:The tests were performed in a quiet room at9:00-12:00per day.Each experimental animal was placed in the center of a dimly illuminated rectangular cage (120cm×90cm×35cm).Behavioural responses were recorded using an automated video tracking system (Ethovision3.0, Noldus, the Netherlands) during a10min observational period in the open field. The bottom of field is divided into25same squares, outer16squares as outer zone and inner9squares as central zone. The frequencies of rearing (standing upright on the hind legs, while forepaws are free) was registered manually. Locomotor activity (the total and the central distance traveled) was quantified for10min using the video tracking system. Total travel, central distance traveled and the frequencies of rearing reflected the degree of excitement, the degree of anxiety and the exploration ability in rats, respectively. The Open field test were performed in control group and MD group at PND90,in CUMS group and double stress group after CUMS.4. BrdU mark and immunohistochemical method analysis hippocampal neurogenesisThe hippocampal neurogenesis in Control group (n=12) and MD (n=12)group were analysed by marked and immunohistochemical method at the second day after the Sucrose preference tests and the open field test.①BrdU mark were injected into rats abdominal (50mg/kg), Once every three hours, three times. Rats were anesthesia after24h, perfusion with physiological saline and4%Paraformaldehyde phosphate bufferand fixed. Then, the rats were beheaded, removed brain, fixed again, dehydrated and embed. The tissues were stored-70℃.②Immunohistochemical methodFrozen rats tissue were taked out. The tissues were cut into coronal slices,3interval pieces take1piece, each set of slice including5pieces,30μm per pieces. The first antibody is BrdU mice monoclonal antibody. The second antibody is biotin marked mice antibody., The number of BrdU marked cells in hippocampal dentate gyrus was counted using inmmunohistochemical method and microscope.5.Real time PCR examination of the expression level of BDNFmRNA、CREBmRNA in hippocampusControl group (n=12) and MD group (n=12) were beheaded at the second day after the Sucrose preference tests and the open field test. CUMS group (n=12) and double stress group (n=12) were beheaded at the second day after CUMS. Immediately after decapitation, the hippocampus were dissected from the brain on an ice-cold aluminium plate and stored-70℃. The samples were thawed and processed with RT-PCR measuring the mRNA expression levels of BDNF and CREB. GAPDH was used as internal standard for semiquantification. The values of BDNF and CREB product were normalized against the amount of PCR product for GAPDH obtained for the same RT sample.6. Western blot examination of the protein level of BDNF and CREBThe method of making specimens was same as RT-PCR. Pulverized frozen hippo-campus samples were prepared and analyzed by quantitative immunoblotting. The rabbit monoclonal anti-CREB antibody (sc-186, Santa Cruz Biotechnology) and rabbit monoclonal anti-BDNF antibody (sc-20981, Santa Cruz Biotechnology) were used. β-Actin was used as internal standard. The expression of BDNF protein, CREB protein was normalized to β-Actin expression.Results1. Effects of early maternal deprivation on behavior and hippocampal neurogenesis in adult male rats(1) The percentage of sucrose preference in MD group were significant less than that in the control group (P<0.01);(2) Behaviors in open field for10minutes:The total distance traveled, the central distance traveled, the frequencies of rearing in MD group were all significant less than that in the control group (P<0.05);(3)BrdU-labeled cells in the dentate gyrus in maternal deprivation group were significant less than that in contral group (P<0.01).2. MD on behavior and BDNFmRNA/protein, CREBmRNA/protein level of hippocampus in adult male rats(1) The percentage of sucrose preference in MD group were significant less than that in the control group (P<0.01);(2) Behaviors in open field for10minutes:The total distance traveled, the central distance traveled, the frequencies of rearing in MD group were all significant less than that in the control group (P<0.05);(3) MD group showed significant decrease in expression of BDNF/CREB mRNA and level of BDNF/CREB protein in hippocampus compared to control group in their adulthood (P<0.05).3. CUMS and double stress on on behavior and BDNFmRNA/protein and CREB mRNA/protein level of hippocampus in adult male rats(1) The percentage of sucrose preference in MD group, CUMS group and double stress group were significant less than that in control group (P<0.05); CUMS group were less than that double stress group (P<0.05);(2) Behaviors in open field for10minutes:The total distance traveled, the central distance traveled, the frequencies of rearing in MD group, CUMS group and double stress group were significant less than that in control group (P<0.05); CUMS group were less than that double stress group (P<0.05);(3) The expression of BDNF/CREB mRNA of hippocampus in MD group, CUMS group and double stress group were significant less than that in control group (P<0.05); CUMS group were less than that double stress group (P<0.05);(4) The level of BDNF/CREB protein of hippocampus in MD group, CUMS group and double stress group were significant less than that in control group (P <0.05); CUMS group were less than that double stress group (P<0.05)。Conclusions:(1) Early life stress, chronic mild stress in adulthood and double stress all could alter behavior and the level of hippocampal neurotrophic factor in adult rat;(2) Early life stress could alter hippocampal neurogenesis and the level of hippocampal neurotrophins in adult rat. It may have influence in susceptibility to depression disorders.(3) Early stress could alter the response to chronic mild stress in adulthood, including behavior and the regulation of hippocampal neurotrophic factor:resulting a slow response to chronic mild stress in adulthood. (4)Long-term effects of early stress on the hippocampus increased the susceptibility to depression. The incidence of depression also need further environmental factors.