Animals and Breeding
Breeding animals were obtained from Charles Rivers and housed in standard cages in colony rooms kept at a constant temperature of 21°C and maintained on a 12 hour light/dark cycle. All animals were given ad libitum access to food and water, except when being administered ethanol or pair-fed diets. This study was performed in strict accordance with the guidelines established by the Canadian Council on Animal Care. The protocol was approved by the University of Victoria's Animal Care Committee (Protocol Numbers: 2010–012 and 2010–011).
Virgin female Sprague-Dawley rats were paired with breeding males in standard cages with the addition of a metal enrichment tube. Pregnancy checks were performed in the early morning via a vaginal swab and visualized on a microscope slide with an Olympus microscope (Olympus CX21, Center Valley, PA, USA) for the presence of sperm. The presence of sperm was used to indicate GD 1. During pregnancy, females were weighed on GD 1, 7, 14 and 21. The day on which females gave birth was designated as PD 1. On PD 2/3, litters were culled to 10 pups and both dams and pups were weighed on PD 8, PD 15 and PD 22. The pups generated were then weaned and group housed according to gender on PD 22/23. Due to unknown pregnancies, some maternal data is missing for control/ad libitum (AL) dams.
Administration of the Liquid Ethanol Diets
To model ethanol exposure during an equivalent human trimester of pregnancy, rat dams or pups were exposed to a liquid diet containing ethanol either during the (): (i) 1st trimester-equivalent
prenatal ethanol exposure 1 (PNEE1
), pregnant dams given ad libitum access to the ethanol liquid diet from GD 1–11; (ii) 2nd
prenatal ethanol exposure 2 (PNEE2
), pregnant dams given ad libitum access to the ethanol liquid diet from GD 11–21; or (iii) 3rd
ethanol exposure 3 (EE3
), pups were feed an ethanol liquid diet through intubation from PD 4–9. The following administration periods and doses were chosen because of their reported effects on hippocampal development and common use in the FASD field.
PNEE dams were given ad libitum access to a liquid diet containing ethanol (35.5% ethanol derived calories; 6.61% v/v). PNEE dams were slowly introduced to the ethanol liquid diet during the first three days of administration by combining 1/3 ethanol diet with 2/3 pair-fed diet on the first day, 2/3 ethanol diet with 1/3 pair-fed diet on the second day, and 3/3 ethanol diet for the remainder of the diet administration period. Following the end of the specified diet administration period, dams were again given ad libitum access to standard rat chow.
Intubation treatments were administered three times a day from PD 4 to PD 9. For each round of intubations, pups were removed from the dam as a litter and kept on a 37°C heating pad. A premeasured length of polyethylene tubing-10 was lubricated with corn oil and gently inserted down the pup's esophagus into the stomach 
pups were intubated twice daily, two hours apart, and infused with a milk/ethanol solution containing 11.9% (v/v) ethanol, totaling 5.25 g/kg of ethanol a day. This was followed by a milk only intubation, to supplement EE pups with calories that were lost due to a reduction in suckling.
Appropriate PF, SI, and AL animals were also reared. PF dams were offered an equivalent quantity of food in g/kg matching that consumed by a PNEE dam on the corresponding day of gestation. SI3
pups received intubations, without infusion of any solution, since milk infusions are known to abnormally accelerate the growth of SI pups 
. AL dams and pups were given ad libitum access to standard rat chow. All groups had ad libitum access to water throughout gestation.
Blood Ethanol Concentrations
To assess peak BECs, blood samples were obtained by way of a tail clip from pregnant dams or postnatal pups: on GD 9 from PNEE1 dams, GD 19 from PNEE2 dams, and from EE3 pups on PD 4. These samples were collected five hours after the presentation of the ethanol diet from PNEE dams and ninety minutes after the last milk/ethanol intubation from EE3 pups. Blood was centrifuged 24 hours after collection and plasma was collected and stored at −20°C until assay. BECs were analyzed using an Analox GL-5 Alcohol Analyzer (Analox Instruments, Lunenburg, MA, USA).
Between PD 50–70, offspring were anesthetized with isoflurane, rapidly decapitated, and their brains removed in oxygenated (95% O2/5% CO2), ice-cold normal artificial cerebral spinal fluid (nACSF). nACSF contained (in mM) 125.0 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25.0 NaHCO3, 2.0 CaCl2, 1.3 MgCl2, and 10.0 dextrose (pH 7.3). Transverse hippocampal slices (350 μm) were generated using a Vibratome Sectioning System 1500 (Ted Pella, Redding, CA, USA). Slices were kept in order using a modified 24-well plate and incubated in continuously oxygenated nACSF maintained at 30°C. Sections were allowed to rest for a minimum of 1 hour before recordings commenced.
Field recordings were collected in nACSF using an Axon MultiClamp 700B amplifier and Clampex 10.2 software (Molecular Devices, CA, USA). Using an Olympus BX51 microscope and motorized micromanipulators (Siskyou Design, OR, USA), electrodes were placed in the medial molecular layer of the DG, approximately 200 µm apart. Field excitatory postsynaptic potentials (fEPSPs) were elicited by delivering a 120 µs (10–40 µA) current pulse to the medial perforant path by way of a digital stimulus amplifier (Getting Instruments, CA, USA) and a single concentric bipolar stimulating electrode (FHC, Bowdoin, ME, USA). fEPSPs were recorded using a single glass recording electrode (0.5–1.5 MΩ) filled with nACSF. A modified I/O experiment was conducted in which the stimulation magnitude was increased until a maximal response prior to population spike appearance was acquired. Stimulation magnitude was then set to elicit approximately 50% of the maximal response. A PP experiment was conducted using an inter-pulse interval of 50 ms (5x; 15 s between pairings).
Baseline measurements were collected using fEPSPs evoked every 15 seconds. A stable baseline of 20 minutes was required before a conditioning stimulus could be applied to the slice. Baseline stimulation parameters were returned to immediately following the conclusion of the conditioning stimulus and fEPSPs were recorded for a minimum of 60 minutes. An I/O experiment was then conducted with increasing stimulation magnitude (30 to 300 µs pulse width; 15 s intervals).
Conditioning Stimulus Protocols
LTP of fEPSPs was induced using one of two conditioning stimuli: (i) HFS or (ii) TBS. HFS consisted of four trains of 50 pulses at 100 Hz, 30 seconds apart; whereas, TBS consisted of four pulses at 100 Hz followed 200 ms later by another burst of four pulses, occurring five times with a 30 second inter-train interval. The GABAA receptor antagonist bicuculline methiodide (Sigma-Aldrich, Oakville, ON, Canada) was included in the nACSF during baseline and CS recordings for all experiments. Bicuculline methiodide was prepared as a concentrated stock solution and diluted with nACSF to a concentration of 10 μM prior to each recording.
Data and Statistical Analysis
All electrophysiological data analysis was conducted with Axon ClampFit 10.2 software (Molecular Devices, CA, USA). The initial slope of the fEPSP was measured and used for all data analysis. PP ratios were calculated by dividing the slope of the second fEPSP by the first and converting this number into a percent change. I/O curves were calculated by normalizing recordings to the value of the 5th pulse and reported as a percent change, with the 5th pulse equaling 100%. For all other experiments, recordings were normalized to the average value of the 20 minute baseline and reported as percent change from baseline. LTP was calculated by averaging the last 20 traces (i.e., 55–60 min) of the post-CS recording.
Overall effects across trimester equivalents were examined with a two-way factorial analysis of variance (ANOVA): developmental treatment (AL, PNEE1, PF1, PNEE2, PF2, EE3, SI3) x gender (male, female). In the case of I/O analysis, a repeated measures ANOVA was conducted. Significant main effects and interactions were further analyzed with Tukey-HSD post hoc tests. T-tests were also used to compare between groups, when appropriate. For all studies, data was presented as means ± standard error of the mean (SEM). For graphing purposes, data from both male and female offspring were pooled together since no main effect of gender was obtained. Results were processed for statistical analysis using Statistica 7.0 (Statsoft, Inc., Tulsa, OK, USA) and differences were considered significant when p<0.05.