2. Materials and Methods
2.1. Animals
Thirty-six newborn SD rats were selected. All neonatal rats were randomly assigned to three groups (12 SD rats in each group), That is, 1 control group and 2 mother-infant separation groups. Onegroup of mother-infant separated group was given normal drinking water and the other group of mother-infant separated group was given normal drinking water and probiotics, Provided by Laboratory Animal Center, Southwest Medical University, China. The final concentration of probiotics in drinking water was109 CFU/mL. The animal room is well ventilated, the environment is at 22-24°C constant temperature, 55%-60% constant humidity, 12h/12h light and dark cycle, regular feeding, free diet. The date of completion of delivery of the female rat is 0 days after birth. In PND2-21, the offspring rats participating in the mother-infant separation were separated from their corresponding mothers for 3 hours per day (9:00-12:00), the offspring pups of the mother-infant separation group were transferred toa new cage each day and returned to the original cage after 3 hours for 21 days. to establish a mother-infant separation model. The experimental animals used in this study were conducted in strict accordance with the standards of the Ethical Review of Animal Experiments at Southwest Medical University.
2.2. Behavioral Assessments
The SD rats were transferred to the experimental room 30 minutes before the formal testing in order to get familiar with the environment, and at the end of the experiment, the SD rats were put back into the cage and transferred to the breeding room. Behavioral tests were performed using the sugar water preference test, the open field test, and the forced swim test.
2.2.1 Sugar water preference test
Pleasure deficit is one of the main symptoms of depression, and we generally assess the symptoms of pleasure deficit in SD rats using sugar-water preference experiments[29]. Prior to the assay, the animals were trained in PND50 in a quiet environment to adapt to water containing sucrose, while two bottles of water containing 1% sucrose were placed in each cage; After 24 h, one bottle with 1% sucrose water and one bottle with distilled water were replaced. In order to avoid site preference of SD rats, the location of the sucrose water and distilled water bottles needed to be changed at 12 h intervals during the experiment; PND52 was given to SD rats with food and water fasting for 24h; The test was performed in PND53 by giving 1% sucrose water and distilled water, and the water bottle was removed after 2h. The intake of sucrose water and distilled water in 2h was weighed and recorded, and the sugar water preference index was calculated. Sugar water preference rate = sugar water consumption / (sucrose water consumption + distilled water consumption) × 100%.
2.2.2 Open field experiments
The open-field test, also known as the open-box test, is a classic method for detecting spontaneous activity, exploration, and anxiety-like behavior in mice and rats [30]. The device is composed of a 100cm x 100 cm x 100 cm open observation box with black surrounding walls and bottom. The experiment was conducted in PND54, and the experimenter grasped the SD rats’ tails near the root third in a quiet environment and put them into the center of the open field. Using the video tracking system, the movement changes of each SD rat in 5 min were automatically recorded, including the totaldistance, the active distance in the central zone, and the active timein the central zone. Data analysis was performed by behavioral video software. Each SD rat was tested only once, and each SD rat was required to clean up the secretion in the field after the test was completed, and the alcohol wipe was cleaned to prevent the residual odor from affecting the action trajectory of the next SD rat.
2.2.3 Forced swimming test
Forced swimming experiment is an experiment to assess depression-like behavior in experimental animals[31]. In PND55 for acclimatization training, in a quiet environment the experimenter graspedthe SD rats by the tail near the root third, and put the SD rats into a transparent tall bottle of 50 cm height, 25 cm caliber, and 23-25°C water temperature, with the water depth of the SD rats just touching the bottom at the tip of the tail; after 15 min remove the rats, wipe them with a towel and then dry the hair of the SD rats with a hair dryer and put PND56 was used for formal testing, and the rest of the conditions were the same as before. Cell phone video recording of each SD rat swimming for 6 min, the main statistics in the test after 4 min SD rats stationary time, each SD rat test once, after the test need to change the water in the tall cylinder bottle.
2.3 Nissler staining
The fixed brain tissue was removed and placed in an embedding box, washed under running water and placed in 75% alcohol overnight. On day 2, they were dehydrated sequentially in different concentrations of alcohol, 85%→95%Ⅰ→95%Ⅱ→100%Ⅰ→100%Ⅱ, respectively. After transparency, paraffin embedding was performed. After embedding, continuous sections were made by microtome, andthe section thickness was 4 μm. After conventional dewaxing, Nissl staining was performed with methyl violet staining solution, and conventional dehydration and sealing were performed after differentiation. The morphology of Nissl vesicles in the CA1 region of hippocampus was observed and photographed by light microscopy, and thenumber was counted.
2.4 Enzyme-linked immunosorbent assays
Blood was collected from the abdominal aorta, centrifuged at 3400 rpm for 20 min, and the supernatant was collected and stored at -80°C for further analysis. The levels of 5-HT, CORT, interleukin-1β, IL-6, and TNF-α were measured using a commercial ELISA kit (ab133053, Abcam, Cambridge, UK). Briefly, different concentrations of standard solutions and sample solutions were added to the ELISA plate. All ELISA measurements were performed in two replicates.
2.5 Immunohistochemistry
(1) Dewaxing of paraffin sections to water: the fixed brain tissue was removed and soaked in a cup with formalin, and the liquid was changed every 10 min, keeping the temperature at 37°C for 20minduring the process. alcohol dehydration was performed accordingto the gradient type: the sections were placed in xylene Ⅰ, Ⅱ andⅢin turn for 15 min each, anhydrous alcohol Ⅰ and Ⅱ for 5 mineach, 85% alcohol, the time takes 5min, 75% alcohol, time takes 5min, followed by distilled water to wash.
(2) Antigen repair: Place the sections in a box with buffer, heat for10 min, leave for 8 min, heat for 10 min, then leave until cool, drench the sections with buffer, hold for 5 min, and wash 3 times repeatedly.
(3) Blocking: put the section into a cup with hydrogen peroxide at room temperature, soak for 10 min, remove and dry the excess water, then rinse with PBS buffer 3 times, each time lasting 3 min.
(4) Closure: add closure solution dropwise and wait for 30min.
(5) Dropwise addition of primary antibody: Drain excess solution from the sections, add primary antibody, and then place them in an incubation box overnight in a 4°C refrigerator.
(6) Dropwise addition of secondary antibody: Take out the box containing the sections from the refrigerator, put the sections on the slide stand, wait for rewarming, then wash with PBS for 3 min and repeat 3 times. Then put the sections back into the incubation box, add secondary antibody dropwise, cover the sections, and put them into a thermostat with 37℃ for 0.5h incubation time.
(7) DAB color development: take out the sections from the thermostat, wash them with PBS for 3 min, and repeat 3 times. Reconfigure the DAB solution, need to add the configured solution drop by drop on the tissue for color development, microscopic view and pay attention to the length of color development, if the appearance of the brownish yellow, considered positive, and finally wash with distilled water and stop color development.
(8) Re-staining cell nuclei: use hematoxylin staining for 3 min, rinse with water, and continue to rinse with running water after returning to blue.
(9) Dehydration sealing: The slices were placed in 75%, 85%, 95%,99.5% alcohol and xylene, respectively, and kept for 10 min, and sealed using neutral resin.
2.6 Protein Extraction and Western Blot
(1) Protein sample preparation 
①Protein extraction: take out the seahorse tissue sample, add the sample, 3mm steel beads and lysate in 2ml grinding tube in order (according to the size of the sample ratio: lysate = 1:10), debug the equipment, grind using the grinder, keep the temperature of the grinder at -20℃, grind 4 times for a total of 4min; take out from the grinder, put it into 4℃ refrigerator, wait for 30min and then take out and put it into the centrifuge instrument machine (4℃, 12000rpm centrifugation, 10min); after the operation, aspirate the upper layer, and then use the kit to measure the protein concentration of this sample.
②Protein quantification (BCA method): Configure the protein standard solution according to the instruction, the amount is 1.2ml, adsorb it into the protein standard (30mg BSA) tube, shake it several times to make it homogeneous so that the final standard solution is 25mg/mL. Take the appropriate amount of standard solution and dilute it into 0.5mg/mL of protein solution. Add the protein standard to the 96-well plate and fill it with ultrapure water if it is less than 20ul. Prepare the appropriate amount of solution according to the size of the sample and in the ratio of 50:1 and mix thoroughly;add 200 μL of BCA solution to each well; shake and mix well, then place at 37°C for 0.5 h. Set well 0 as the control and measure the OD of the sample protein wavelength at 562 nm; according to the OD value, find out the protein content corresponding to it(µg)。
③Sampling: Take 50μL amount of each group, add 5×Loding buffer according to certain ratio, shake well, set the temperature of thermal cycling machine to 95℃ for 15min, wait for cooling after the end, then centrifuge and sample, and finally store in -20℃ refrigerator for easy access.
(2) Glue making
① Rinse the glass plate with water to keep it clean, then prepare the sample comb, glass plate, and glue filling equipment and other utensils, add ddH2O to observe whether there is water leakage, and use filter paper to save the water on the glass plate dry.
② Mix TEMED and APS immediately, inject them into the glue-making glass plate, remove the air bubbles, and wait for some time to make sure the lower layer of glue is solidified.
③Take equal volume of upper layer glue solution and upper layer glue buffer, stir well with pipette, add a certain proportion of coagulant solution and mix thoroughly again; slowly and smoothly beat into the glass plate of glue making and insert the comb teeth; after waiting for a period of time, after the upper layer glue solidifies, you can pull out the comb carefully.
(3) Sample loading, electrophoresis
①When the upper layer of glue solidifies, hold both sides of the comb with both hands slowly and gently pull it upward, and then wait for 10min in the middle, while fixing the glass plate into the electrophoresis tank, and pour fresh buffer in the middle of the glass plate, and pour the old electrophoresis buffer in the surroundings of the tank, the solution should be on the marked line, and start to put on the sample.
② Calculate the amount of sample to be added. The glass plate is fixed in the electrophoresis tank, add the appropriate amount of electrophoresis solution in the tank, and add the required protein samples of each group and Marker in order according to the grouping (during the process of adding samples, try to go faster so as not to spread the samples) of the loading solution, the samples need to wait for rewarming when they are taken out from the refrigerator,then stir well with the gun, and add samples in order with the small gun, when adding samples, it is necessary to replace the new gun to avoid cross-contamination.
③ Connect the power supply, keep the voltage at 100V for 15min. After the Marker dye runs to the separation gel, set the voltage to 180V and continue electrophoresis until the target strip is completely run, then you can turn off the power supply and stop the electrophoresis operation.
(4) Transfer film
①According to the size of the separation gel, assemble the sponge, filter paper, protein gel, PVDF membrane, filter paper, and sponge in this order, in which the air bubbles on the surface need to be excluded. Then put it into the membrane transfer solution.
②Subsequently, the membrane transfer apparatus is placed in an ice bath, connected to positive and negative terminals, and the power is turned on at 200mA for 1-2h.
(5) Closure
Equipped with 5% skim milk solution first, when the transfer is finished, the PVDF membrane is removed and placed in the incubation box with milk solution, and then transferred to a shaker for 2h.
(6) Antibody hybridization
①Incubation of primary antibody: PVDF membrane was cleaned and put into the incubation box and added primary antibody (primary antibody concentration: BDNF 1:2000; CAMP 1:2000; CREB11:2000; β-actin 1:50,000), put the box on a shaker at 4℃ for overnight; after that, it was washed 3 times with TBST, each time lasting 5min. ② secondary antibody incubation: put the PVDF membrane into the incubation box with secondary antibody solution, put the box on a shaker and shake it for 2-3h; after that, wash it 3 times with TBST, each time lasts for 5min.
(7) development, fixation
The ECL luminescent solution is added dropwise in the box according to 1:1 equal volume, and the film paper is put into the box to make it mixed evenly for 60s, and then its film paper is laid flat on the exposure plate to ensure that the imager is carried out in the dark room, and the signal is waited for to be prompted for photo preservation.
(8) Image analysis
Using Image J software, graphical analysis was performed and the grayscale values of the target proteins were read and then compared with the internal reference to provide a basis for later data analysis.
2.7 Statistical Analysis
All data from this experiment were entered using excel sheets, and data analysis was performed using SPSS 23.0 statistical software, and Graphpad Prism 8.0 software was used for graphing. The data involved were measures and tested for normality using Shapiro-Wilk; all data were statistically described using the mean ± standard deviation (X±S) and analyzed using one-way ANOVA. If the differences between multiple groups were statistically significant, two-by-two comparisons would be made, and the LSD test would be used when the data met the chi-square, and the Dunnett-t3 test would be used if the chi-square did not, with α=0.05 as the test level in this study.