All procedures were done in accordance with the University of Delaware Institutional Animal Care and Use Committee.
For experiment 1A, twelve Long Evans rat litters from timed pregnancies were obtained from Harlan on Gestational Day (GD5). After arrival at the animal facility these litters were housed and treated exactly as described below (Experiments 1B and 2).
For Experiment 1B, a total of 31 timed pregnancy litters from the animal housing colony at the University of Delaware's Office of Laboratory Animal Medicine facility were used. Females were housed overnight with breeder males and were checked for an ejaculatory plug the following morning. If a plug was found, that day was designated as gestational day (GD) 0. Pregnant females were housed in clear polypropylene cages (45×24×21 cm) with standard bedding and ad lib access to water and rat chow and maintained on a 12:12h light cycle. The date of birth was determined by checking for births during the light cycle. If births were detected, that day was designated postnatal day (PD) 0 (all births occurred on GD22). On PD3, litters were culled to eight pups (5 female, 3 males when possible) and pups received subcutaneous injections of a non-toxic black ink into one or more paws to aid in identification. A total of thirty-four male Long-Evan rat pups were used in this study. All pups were weaned on PD 23 and subsequently housed in social conditions of 2-3 rats per cage (45×24×17cm) with ad lib access to water and rat chow. On PD30, animals were housed in larger 45 × 24 × 21cm cages with ad lib access to food and water for the remainder of the study. All subjects were treated in accordance with a protocol approved by the Institutional Animal Care and Use Committee at the University of Delaware based on NIH standards. A total of thirty-seven male Long-Evans rat offspring were used in this study.
Experiment 2 followed the same breeding and housing procedures as Experiment 1B. A total of ninety-two Long Evans rats (46 females and 46 males) were derived from 15 time-bred litters in the UD animal colony.
4.2. Alcohol Exposure
Beginning on PD 4, pups were assigned to one of three conditions: suckle control (SC), sham intubated (SI) or alcohol-exposed (AE). SC rats were derived from separate litters than SI and AE rats, which were littermates randomly assigned (within each sex) to treatment group such that no more than one same-sex littermate was represented in a given postnatal sampling or behavioral condition. SC animals were weighed daily but otherwise left undisturbed. Intragastric intubations occurred from PD4-9 for both the SI and AE groups. SI animals were intubated, on the same schedule as the AE animals, and the tube was removed after approximately ten seconds without the infusion of any solution. AE animals were intubated and given a daily dose of 5.25g/kg of alcohol, (11.9% v/v in milk formula) which was divided into two feedings each day, two hours apart. Preparation of milk formula followed the protocol described previously (Kelly & Lawrence, 2008
). A third intubation of milk (no ethanol) was administered two hours after the second alcohol dose. Solely on PD 4, a fourth intubation of milk (no ethanol) was given four hours after the second alcohol dose in order to compensate for reduced milk intake.
4.3. Blood Alcohol Concentrations
Blood samples were collected from a tail clip of each AE and SI pup 90 minutes after the second alcohol intubation on PD 4 in order to determine the blood alcohol concentration (BAC). BACs for the AE animals were assayed from the plasma of each blood sample using an Analox GL5 Alcohol Analyzer (Analox Instruments, Boston, MA).
For experiments 1A and 1B, beginning on PD 59 (until PD 83) and on PD 94 (until PD 115) males were removed from the cage and the genital area was swabbed with a saline-moistened Puritan cotton-tipped applicator for approximately 5 seconds. This was done as a control for procedures for smear collection of females in another study not reported here.
For experiment 2, Beginning on PD59 (until PD 80) and then again on PD94 (~PD 117), vaginal swabs were collected from female rats daily at 0900AM. The tip of a Fisherbrand diagnostic swab (0.6mm) was moistened in 0.9% saline, gently inserted in the vagina and then rolled across a pre labeled microscopy slide. Slides were then immersed in 95% alcohol for 2 seconds and left overnight to dry before being stored. For the males, the animal was removed from the cage and the genital area was swabbed with a saline-moistened Puritan cotton-tipped applicator for approximately 5 seconds.
Vaginal smears were analyzed daily after collection in order to monitor the estrous cycle. Stages were determined based on the predominance of cell types found in the smear. A predominance of nucleated cells designated the rat was in proestrus. A predominance of cornified cells signified the stage of cycle to be estrus, while the presence of leukocytes signaled the rat was in diestrus. Swabs were collected to ensure that rats were cycling regularly and also to predict the stage of the estrous cycle for the next day in order to assure animals were preexposed during proestrus.
4.5. BrdU Injections
In Experiment 1A, rats were weighed and received a single injection of BrdU (200mg/kg in 0.9% sterile saline solution (20mg/ml), i.p.) at least two hours prior to perfusion. There were five days of perfusions (PD78-PD82). Injections typically occurred between 1100-1200 hrs on select days as indicated in .
In Experiment 1B, all animals received a single injection of BrdU (200mg/kg in 0.9% sterile saline solution (20mg/ml), i.p.) on PD80. Injections occurred between 0900-1000 hrs (). Perfusions occurred 35 days after injection (PD115; ).
No BrdU injections occurred in Experiment 2.
4.6. Tissue Preparation
All animals assigned to neurogenesis experiments were anesthetized with a mixture of ketamine and xylazine. In Experiment 1A, this occurred two to three hours following injection with BrdU (see timeline in ). In Experiment 1B, this occurred 35 days post injection (0900hrs-1200hrs; timeline in ). All animals were transcardially perfused with heparinized 0.2M phosphate buffer followed by 4% paraformaldehyde in 0.2M phosphate buffer. Brains were stored in 30% sucrose in 4% paraformaldehyde solution. Serial horizontal sections (40μm) containing the dentate gyrus were obtained on a cryostat and collected in wells containing a cryoprotectant solution (glycerol and ethylene glycol in phosphate buffer solution). Brain tissue was stored at -20°C.
Every 16th section (640μm apart) of the entire dentate gyrus was chosen in a systematic random manner (first section chosen randomly from the first sixteenth consecutive sections containing the dentate gyrus) and processed for immunocytochemistry. Immunoreaction with Ki67, DCX and BrdU antibodies was visualized with diaminobenzidine.
For both Ki67 and BrdU visualization, sections were washed in Tris Buffer Solution (TBS) followed by incubation in 0.6% hydrogen peroxide and then washed in TBS. For the BrdU staining only, sections were then incubated in 50% Formamide in 2× concentrate of saline-sodium citrate buffer (SSC) at 65°C for 2hrs followed by a 2× SCC wash after which sections were quickly transferred into 2N hydrochloric acid for 30 mins at 37°C. This was followed by a 10 minute wash in 0.1M Boric acid in TBS wash and then a TBS wash. Next, for both stainings, sections were placed in blocking solution (3% normal goat serum-0.1% Triton X100 in 50mM TBS) for 1 hour. Sections were transferred into primary antibody (1:100 dilution, anti Ki67 (NCL-L-Ki67-MM1) Novacastra, Norwell, MA) (1:500 dilution, anti-BrdU made in rat (OBT0030) Accurate, Westbury, NY) in washing solution (3% normal goat serum in TBS) and left for 48hrs (Ki67) or blocking solution for 72hrs (BrdU) at 4°C. Next, sections were washed in TBS and then washing solution (Ki67 only), after which sections were incubated in secondary antibody (1:1000 dilution, goat anti-mouse biotinylated (BA-9200) Vector, Burlingame, CA) for 1hr or (1:250 dilution, goat anti-rat biotinylated (BA-9400) Vector, Burlingame, CA) for 2hrs at RT. This was followed by two TBS washes and one wash in washing solution (Ki67) or blocking solution (BrdU). Next, sections were incubated in ABC solution (Vector Laboratories, Burlingame, CA) for 1hr. Sections were then rinsed in TBS and then washed in DAB in TBS for at least five minutes (until reaction was visible under microscope). Sections were then rinsed quickly (1-2 sec) in TBS followed by two five minute TBS washes. Sections were then mounted onto slides and left to dry. The following day, slides were counterstained with 0.1% Pyronin Y and coverslipped using DPX mountant.
For DCX visualization, sections were washed with TBS followed by incubation in 0.3% hydrogen peroxide for 1r. Next, sections were washed with TBS and then transferred into primary antibody (1:500 dilution, goat anti-DCX(C-18) Santa Cruz Labs, CA). Twenty-four hours later, sections were rinsed in TBS and then incubated in secondary antibody (1:250 dilution, anti-goat biotinylated (Vector Labs, Burlingame, CA) for 1hr at RT. Next, sections were rinsed in washing solution, blocking solution and then incubated in ABC solution for 1hr. Sections were then rinsed in TBS and stained with DAB and counterstained according to methods described above.
Quantification of BrdU+, Ki67+ and DCX+ cells was performed in accordance with unbiased stereology approach. A series of sections (every 16th section) throughout the entire dentate gyrus was used for each staining batch. All cell counts were made on coded slides by an investigator blind to the treatment conditions. The whole granule cell layer including subgranular zone was outlined on the digitized image on each systematically random chosen section using StereoInvestigator software. Counts were made in an unbiased manner within a known volume of the dentate using the optical fractionator workflow (StereoInvestigator, MicroBrightField Inc., Williston, VT). The grid frame was set to 200 × 200 μm and the counting frame set to 200 × 200 μm. A guard zone of 2 μm and a dissector height of 16 μm were used. The frozen sections were originally cut at a nominal thickness of 40 μm. Immunostaining and mounting result in altered section thickness, which was measured at each counting site. An average section thickness was computed by the software and used to estimate the total volume of the DG sample region and total number of BrdU+ and Ki67+cells.
4.8. Contextual Fear Conditioning
4.8.1. Apparatus and stimuli
The apparatus and stimuli of the current study have been previously described (Burman, et al. 2009; Murawski & Stanton, 2010
). Fear conditioning occurred over three consecutive phases spaced 24h apart: preexposure; training; and testing. During preexposure subjects were exposed to one of two contexts (Context A or Context B). Context A was one of four clear Plexiglas chambers (16.5×21.1×21.6 cm) as described by Murawski & Stanton (2010)
All behavior in Context A was recorded by a video camera and fed into a Dell computer that ran FreezeFrame software (Actimetrics, Wilmette, IL) for analysis of freezing behavior (Murawski & Stanton, 2010
). Context B consisted of separate chambers (22×22×26 cm) as described previously (Murawski & Stanton, 2010
). All training and testing occurred in Context A.
4.8.2. Design and procedures
Subjects from each of the three dosing conditions were further assigned to one of two behavioral groups. Group Pre was preexposed to the training context (Context A). Group No Pre was preexposed to the alternate context (Context B). No more than one same-sex litter mate per dosing condition was assigned to any behavioral condition.
Starting on PD107, all subjects were handled for 3 minutes per day. For each subject, handling continued until preexposure (minimum of 5 days). Preexposure to Context A or B started on PD112. Female rats were only preexposed during the proestrus phase of their cycle (as close to PD122 as possible). Swabbing continued each day until a female rat was found to be in proestrus and then it was preexposed to either Context A or B. Male rats were yoked to females so that both sexes were preexposed on similar days. During preexposure, rats were transported two at a time in yellow ice buckets and placed into either Context A or B where they were allowed to freely explore the context for a 5 minute preexposure period, after which they were returned to their home cage. During training, all subjects were briefly placed into Context A where they received an immediate 2s 1.5mA footshock (<5s upon placement) and were then returned to their home cage. Finally, during testing, subjects were again placed into Context A and levels of contextual freezing were monitored over a 5 minute testing period. Context A was wiped down with a 5% ammonium hydroxide solution following removal of subjects during each phase of testing.
4.9. Data analysis
For Experiment 1, the data were imported into PASW Statistics 18 data analysis software. The level of significance was set at p < 0.05 for all tests. Body weight analysis utilized a repeated measure ANOVA with between-subjects factor of dosing condition and the within-subjects factor of age for PD4 and PD9 weights. BrdU analysis was performed through a Univariate ANOVA at both the proliferation and survival time points in order to examine the role of dosing condition on BrdU+ cell counts. Post-hoc tests (Tukey HSD) was used to characterize treatment effects at the survival timepoint. Ki67 analysis also required a Univariate ANOVA at both the proliferation and survival time points.
For Experiment 1A, a total of 2 pups were excluded from analysis as they died due to improper intubations. The total N's for the study were: SC: 12; SI: 12; AE: 10. For experiment 1B, a total of 4 pups were excluded from analysis as they died due to improper intubations. The total N's for the study were: SC: 8; SI: 9; AE: 9.
For Experiment 2, the data were analyzed using FreezeFrame software (Actimetrics, Wilmette IL). The bout length was set at 0.75 s and the freezing threshold (change in pixels/frame) was initially set as described in the instructions. A human observer blind to the animal groups verified the setting by watching the session and adjusting the threshold if necessary to ensure that small movements were not recorded as freezing. Freezing behavior was scored as a percentage of time spent freezing during the 5min testing session.
The data were imported into Statistica 8 data analysis software. Body weight analysis utilized a repeated measure ANOVA with between-subjects factors of sex and dosing condition and the within-subjects factor of age for PD4 and PD9 weights. Adult (preexposure) weights were analyzed with a factorial ANOVA with Sex and Dosing Condition as between subjects' factors. Freezing behavior was analyzed with a 2 (sex) ×3 (dosing condition) ×2 (preexposure group) factorial ANOVA. No significant main effects or interactions concerning the factor of sex appeared (p>0.07) and so the remaining statistics utilized a 3×2 (dosing condition×preexposure) factorial design. Post-hoc analyzes (Newman–Keuls) were used to characterize treatment and interaction effects, when statistically significant (alpha set at p≤0.05, two-tailed).
A total of 18 animals were excluded from analysis. Six rats died due to improper intubations. Four animals were excluded due to equipment failure/experimenter error. Outliers were defined a priori as individual freezing scores that differed by ± 2 standard deviations from the mean of the other rats in their respective group. Eight animals meet the criteria for outliers and were thus excluded from further analysis. Outliers included 2 animals each from Groups SC-Pre and SI-No-Pre, and one animal from each of the following Groups: SC-No-Pre, SI-Pre, AE-Pre, and AE-No-Pre. Statistics were run on the remaining 74 rats.