• Acute Ethanol;
  • Fetal Alcohol Spectrum Disorders;
  • Hippocampal Formation;
  • Synaptic Plasticity;
  • Long-Term Potentiation


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References


Prenatal ethanol (EtOH) exposure results in a spectrum of structural, cognitive, and behavioral abnormalities, collectively termed “fetal alcohol spectrum disorders” (FASDs). The hippocampal formation, an area of the brain strongly linked with learning and memory, is particularly vulnerable to the teratogenic effects of EtOH. Prenatal EtOH exposure can lead to long-lasting impairments in the ability to process spatial information, as well as produce long-lasting deficits in the ability of animals to exhibit long-term potentiation (LTP), a biological model of learning and memory processing. These deficits also have the ability to facilitate EtOH and/or other drug abuse later in life. This study sought to determine prenatal EtOH exposure altered the effects of acute EtOH application on synaptic plasticity.


Prenatal EtOH exposure was modeled using a liquid diet where dams were given 1 of 3 diets: (i) a liquid diet containing EtOH (35.5% EtOH-derived calories), (ii) a liquid diet, isocaloric to the EtOH diet, but with maltose–dextrin substituting for the EtOH-derived calories, and (iii) an ad libitum diet of standard rat chow. Extracellular recordings from transverse brain slices (350 μm) prepared from 50- to 70-day-old rats, following prenatal EtOH exposure (gestational day 1 to 21). LTP was examined in the dentate gyrus following acute EtOH exposure (0, 20, or 50 mM) in these slices.


Prenatal EtOH exposure attenuated LTP in the adult dentate gyrus. In control offspring, acute application of EtOH in adulthood attenuated (20 mM) or blocked (50 mM) LTP. Conversely, the effect of acute EtOH application on LTP was not as pronounced in prenatal EtOH offspring.


Prenatal EtOH exposure alters the sensitivity of the adult dentate gyrus to acute EtOH application producing a long-lasting tolerance to the inhibitory effects of EtOH. This decreased sensitivity may provide a mechanism promoting the formation of drug-associated memories and help explain the increased likelihood of developing an alcohol dependency often observed in individuals with FASDs.

Alcohols are a family of organic chemical compounds that contain 1 or more hydroxyl groups attached to a carbon. Of the many types of alcohols, only 1, ethanol (EtOH), is consumed on a regular basis by humans. EtOH is classified as a psychoactive substance and depressant drug that also acts as a teratogen. The World Health Organization (2011) recently reported that 55% of all adults have consumed EtOH, making EtOH the most widely used psychoactive substance in the world (McKenzie et al., 2011). Furthermore, EtOH is the world's third largest risk factor for disease, contributing to at least 60 different types of diseases/disorders (World Health Organization, 2011). One of the more prevalent disorders, fetal alcohol spectrum disorders (FASDs), are a group of developmental disorders resulting from maternal consumption of EtOH during pregnancy.

Individuals who were exposed to EtOH during in utero development exhibit a myriad of central nervous system abnormalities (Hamilton et al., 2003; Streissguth et al., 1990). Associated behavioral and cognitive deficits increase in severity throughout these individuals’ lifetime, as more complex tasks are taken on (Streissguth et al., 1994; Willford et al., 2004). Furthermore, as an individual with FASD matures, many secondary disabilities begin to emerge, including drug abuse (Koren et al., 2003). Prenatal EtOH exposure is a known risk factor for the development of EtOH-drinking problems later in life (Baer et al., 2003). Upward of 47% of individuals with FASD report EtOH and other drug problems in adulthood (Streissguth et al., 2004; Yates et al., 1998). Interestingly, Baer and colleagues (2003) found that prenatal EtOH exposure association with EtOH problems at 21 years of age was independent from family history of alcohol problems, nicotine exposure, other prenatal exposures, and postnatal environmental factors. Therefore, this increased risk for EtOH abuse may be the result of alterations in the neurological response to EtOH exposure. During adulthood, rats prenatally exposed to EtOH exhibited a decreased hypothermic response to intoxicating doses of EtOH (Molina et al., 1987). Additionally, prenatal EtOH exposure leads to preference for and increased tolerance of EtOH in adulthood (Abel et al., 1981; Molina et al., 1987; Reyes et al., 1993). As suggested, reduced sensitivity to EtOH may lead to the higher abuse of EtOH (Grobin et al., 1998). Thus, a persistent alteration in the pharmacological effect of EtOH on the central nervous synaptic may underlie the high incidence of EtOH-drinking problems in individuals with FASD.

To determine whether prenatal EtOH exposure (first and second trimester equivalent) results in an alteration in EtOH sensitivity, we investigated the combined effects of prenatal EtOH exposure and acute EtOH application in adulthood on long-term potentiation (LTP) in the hippocampal dentate gyrus, which is a widely used cellular model of learning and memory.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References


All animal experimentation was approved by the University of Victoria's Animal Care Committee and was performed in accordance with the guidelines established by the Canadian Council on Animal Care.

Breeding animals were obtained from Charles Rivers (Quebec, Canada) 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 (AL) access to food and water, except when being administered EtOH or pair-fed (PF) diets. Virgin female Sprague–Dawley rats were paired with breeding males in standard cages. A vaginal smear using 0.9% sodium chloride (NaCl) was performed at the beginning of each light cycle to detect the presence of sperm, indicating gestation day 1 (GD 1). During pregnancy, dams were weighed on GD 1, 7, 14, and 21. The day on which dams gave birth was designated as postnatal day 1 (PD 1). On PD 2/3, litters were culled to 10 pups, typically 5 males and 5 females. On PD 22/23, offspring were weaned and group housed according to gender. Due to unknown pregnancies, that is, failed detection of sperm on GD 1, some maternal data are missing for control/AL dams.

Prenatal Treatment

To model EtOH exposure during the first and second trimesters (prenatal ethanol exposure [PNEE1,2]) of human pregnancy, dams were given AL access to an EtOH liquid diet (35.5% EtOH-derived calories; 6.61% v/v) from GD 1 to 21 (Fig. 1). Dams were slowly introduced to the EtOH liquid diet during the first 3 days of administration by combining 1 of 3 EtOH diets with 2 of 3 PF diets on the first day, 2 of 3 EtOH diets with 1 of 3 PF diets on the second day, and 3 of 3 EtOH diets for the remainder of the diet administration period. Following the end of the specified diet administration period, dams were again given AL access to standard rat chow.


Figure 1. Timeline of experiments. Gestational day (GD) 1 was indicated by the presence of sperm in the vaginal smear. On GD 1, pregnant dams were individually housed and assigned to 1 of 3 diets (prenatal ethanol exposure (PNEE)1,2; pair-fed (PF)1,2; ad libitium (AL)). Liquid diets were replaced with standard rat chow on GD 22. Birth of pups indicated postnatal day (PD) 1. All offspring were reared normally and electrophysiological experiments were performed between PD 50 and 70.

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EtOH-fed rats generally consume less calories (10 to 40%) than those given AL access to normal rodent chow (Fisher et al., 1997). Therefore, 2 additional treatment groups were reared. (i) PF dams, offspring serve to distinguish the effects of EtOH and undernutrition (i.e., caloric restriction) on development were offered an equivalent quantity of food in g/kg that matched that consumed by an EtOH dam on the corresponding day of gestation. (ii) AL dams were given AL access to standard rat chow. All dams had AL access to water throughout gestation.

Blood EtOH Concentrations

To assess peak blood EtOH concentrations (BECs), blood samples were obtained by way of a tail clip from pregnant dams on GD 15. To determine peak BECs, blood samples were collected 5 hours after the presentation of the EtOH diet, approximately 2 hours after beginning of the dark phase when peak BECs are achieved. Blood was allowed to clot at 4°C for 24 hours after collection and then centrifuged. Plasma was collected and stored at −20°C until assay. BECs were analyzed using an Analox GL-5 Alcohol Analyzer (Analox Instruments, Lunenburg, MA).

Slice Preparation

Between PD 50 and 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, 255 to 305 mOsm/l). Transverse hippocampal slices (350 μm) were prepared using a Vibratome Sectioning System 1500 (Ted Pella, Redding, CA) and incubated in continuously oxygenated nACSF and maintained at 30°C. Sections were allowed to equilibrate for a minimum of 1 hour before recordings commenced.

Field Recordings

Slices were transferred to recording chambers and superfused with oxygenated nACSF. Using an Olympus BX51 microscope and motorized micromanipulators (Siskiyou Design, Grants Pass, OR), electrodes were placed in the medial molecular layer of the dentate gyrus, approximately 200 μm apart. Field recordings were collected using an Axon MultiClamp 700B amplifier and Clampex 10.2 software (Molecular Devices, Sunneyvale, CA). Field excitatory postsynaptic potentials (fEPSPs) were elicited by delivering a 120 μs (10 to 40 μA) current pulse to the medial perforant path by way of a digital stimulus amplifier (Getting Instruments, San Diego, CA) and a single concentric bipolar stimulating electrode (FHC, Bowdoinham, ME). fEPSPs were recorded using a single glass recording electrode (0.5 to 1.5 MΩ) filled with nACSF. A modified input/output (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.

Baseline measurements were collected using fEPSPs evoked every 15 seconds. The GABAA receptor antagonist bicuculline methiodide (BMI; 10 μM; Sigma-Aldrich, Oakville, ON, Canada) was included in the nACSF during baseline and conditioning stimulus recordings for all experiments. In select experiments, EtOH (20 or 50 mM) was also included in the BMI + nACSF solution by diluting 100% EtOH in BMI + nACSF prior to each experiment. A stable baseline of 5 minutes was required before the addition of EtOH. Slices were perfused with the BMI + nACSF + EtOH solution for 15 minutes before the application of the conditioning stimulus, an exposure similar to that previously used in other experiments examining the acute effects of EtOH on LTP (Givens and McMahon, 1995; Izumi et al., 2007; Morrisett and Swartzwelder, 1993).

Following baseline acquisition, theta burst stimulation (TBS; i.e., conditioning stimulus) was used to induce LTP. TBS consisted of 4 pulses at 100 Hz followed 200 ms later by another burst of 4 pulses, occurring 5 times with a 30-second intertrain interval. Immediately following TBS, baseline stimulation parameters were returned to and fEPSPs were recorded for a minimum of 60 minutes in nACSF. At the end of the experiment, an I/O experiment was conducted by increasing stimulation magnitude (30 to 300 μs pulse width; 15-second intervals).

Statistical Analysis

All electrophysiological data analysis was conducted with Axon ClampFit 10.2 software (Molecular Devices). Data acquired from slices were used for statistical comparisons of electrophysiological experiments. The initial slope of the fEPSP was measured and used for all data analysis. I/O curves were calculated by normalizing recordings to the value of the fifth pulse and reported as a percent change, with the fifth 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 to 60 minutes) of the post-TBS recording. For all studies, data are presented as means ± standard error of the mean (SEM).

Statistical differences were examined with a 3-way factorial analysis of variance (ANOVA): prenatal treatment (AL, PF1,2, PNEE1,2) × acute EtOH application (0, 20, 50 mM) × 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. When no significant interactions between factors were obtained, data were pooled together for graphing purposes. Student's t-tests were also used to compare between groups, when appropriate. Additionally, Student's t-tests were used to determine whether LTP was induced by comparing the average of the last 20 traces (i.e., 15 to 20 min) of baseline to the last 20 traces (i.e., 55 to 60 min) of the post-TBS recording. Results were processed for statistical analysis using Statistica 7.0 (Statsoft, Inc., Tulsa, OK), and differences were considered significant when p < 0.05.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Maternal and Offspring Data

A total of 11 AL, 11 PF1,2, and 10 PNEE1,2 dams were used for generation of experimental offspring. All pregnant dams continued to gain body weight throughout gestation, regardless of diet, F(2, 23) = 1.22, p = 0.342 (Table 1). Furthermore, there was no significant difference in litter size, F(2, 28) = 0.15, p = 0.863, or gestation length, F(2, 22) = 0.73, p = 0.492. The average BEC was 145.32 ± 5.31 mg/dl for the EtOH treated dams. These BEC levels are similar to those found in previously published studies (Christie et al., 2005).

Table 1. Maternal and Offspring Data
Treatment groupDamsOffspring
% Weight gainLength of gestation (days)Litter size (pup #)BEC (mg/dl)PD 2/3 (g)aPD 50 to 70 (g)b,c
  1. AL, ad libitum; BEC, blood ethanol (EtOH) concentration; PD, postnatal day; PF, pair-fed; PNEE, prenatal EtOH exposed.

  2. a

    Prenatal treatment groups (PF1,2 and PNEE1,2) weighed less than AL group; < 0.01.

  3. b

    Male PF1,2 offspring weighed less than AL offspring; < 0.01.

  4. c

    Males weighed more than females; < 0.01.

  5. The values represent mean ± SEM.

AL29.95 ± 4.0722.20 ± 0.2015.18 ± 1.198.68 ± 0.288.26 ± 0.18395.12 ± 9.10260.58 ± 9.10
PF1,223.71 ± 4.0622.00 ± 0.2114.40 ± 1.127.03 ± 0.207.12 ± 0.18345.38 ± 10.41250.93 ± 9.70
PNEE1,231.84 ± 4.8522.30 ± 0.1515.20 ± 1.21145.31 ± 5.317.40 ± 0.186.92 ± 0.39388.54 ± 10.03228.27 ± 11.32

Offspring were weighed across the postnatal period until their experimental use in adulthood (Table 1). Following birth, weights taken on the litter cull date (PD 3/4) were significantly affected by prenatal treatment, F(2, 81) = 22.93, p < 0.001, but not gender, F(1, 81) = 1.83, p > 0.251. PNEE1,2 (p < 0.001; n = 25) and PF1,2 (p < 0.001; n = 28) offspring had significantly reduced body weights compared to AL (n = 34). At the time of electrophysiological experimentation, there was a main effect of prenatal treatment, F(2, 81) = 5.02, p = 0.008, and gender, F(1, 81) = 253.95, p < 0.001, on body weight. Additionally, there was a significant interaction between prenatal treatment and gender, F(2, 81) = 5.184, p = 0.008, on body weight. Upon assessment, AL males (n = 17) weighed more than PF1,2 males (p = 0.007; n = 13) at adulthood (i.e., PD 50 to 70). Additionally, males (n = 44) weighed significantly more than females (n = 43, p < 0.001), a common gender difference at this age (River, 2012).

Long-Term Potentiation

To determine whether prenatal EtOH exposure alters the response of the adult dentate gyrus to acute EtOH application, hippocampal slices from all 3 prenatal treatment groups were exposed to various concentrations of EtOH (0, 20, or 50 mM) prior to LTP induction. Table 2 provides a summary of all LTP data.

Table 2. Offspring Numbers and Long-Term Potentiation Data
GenderPrenatal treatment groupAcute ethanol (EtOH) concentration (mM)Slice (n), animal (a), litter (l) numberLong-term potentiation
  1. n, slice number; a, animal number; AL, ad libitum; PF, pair-fed; PNEE, prenatal EtOH exposed.

  2. The values represent mean field excitatory postsynaptic potential slope (% change relative to preconditioning responses) ± SEM.

MaleAL0n = 9, a = 7, l = 660.02 ± 9.18
20n = 8, a = 5, l = 316.72 ± 4.73
50n = 9, a = 7, l = 4−1.30 ± 4.11
PF1,20n = 8, a = 6, l = 323.78 ± 3.71
20n = 8, a = 7, l = 49.75 ± 2.80
50n = 7, a = 6, l = 33.13 ± 4.07
PNEE1,20n = 8, a = 7, l = 422.68 ± 5.10
20n = 7, a = 6, l = 421.72 ± 8.86
50n = 7, a = 7, l = 412.73 ± 6.77
FemaleAL0n = 9, a = 8, l = 546.12 ± 5.13
20n = 7, a = 6, l = 118.61 ± 3.45
50n = 9, a = 6, l = 40.06 ± 4.05
PF1,20n = 9, a = 6, l = 440.84 ± 5.60
20n = 6, a = 5, l = 221.33 ± 5.04
50n = 8, a = 7, l = 324.67 ± 6.25
PNEE1,20n = 10, a = 7, l = 537.38 ± 6.28
20n = 9, a = 4, l = 225.34 ± 5.90
50n = 8, a = 5, l = 216.91 ± 4.53

Statistical analyses revealed a main effect of acute EtOH application, F(2, 128) = 42.79, p < 0.001, as EtOH significantly reduced LTP in a concentration-dependent manner (0 mM vs. 20 mM, p < 0.001; 0 mM vs. 50 mM, p < 0.001; 20 mM vs. 50 mM, p = 0.009) and a main effect of gender, F(1, 128) = 6.58, p = 0.011, indicating higher levels of LTP in females compared to males (p = 0.009). There was no main effect of prenatal treatment found; however, there was a significant interaction between prenatal treatment and gender, F(2, 128) = 4.77, p = 0.010, and between prenatal treatment and acute EtOH application, F(4, 128) = 8.26, p < 0.001. Each interaction is presented below.

The interaction between prenatal treatment and acute EtOH exposure indicates that prenatal treatment groups were affected differentially by acute EtOH in adulthood. In the absence of acute EtOH (0 mM), a robust and persistent enhancement of the fEPSP was observed in AL control offspring (53% change in the fEPSP 60 minutes following TBS). However, LTP levels were significantly smaller in PNEE1,2 (42% reduction, p = 0.001) and PF1,2 (38% reduction, p = 0.006) offspring than in AL offspring (Fig. 2). LTP levels did not differ between PF1,2 and PNEE1,2 offspring (p = 0.999).


Figure 2. Prenatal ethanol exposure (PNEE) results in long-term alterations in long-term potentiation (LTP). (A) Time course of LTP, prior to and after induction (at time 0 minute). (B) LTP was reduced in both PNEE1,2 and pair-fed (PF)1,2 offspring compared to ad libitum (AL) offspring. Insert illustrates samples of traces obtained from corresponding groups; field excitatory postsynaptic potentials (fEPSPs) recorded before (black) or 1 hour (gray) after conditioning stimulation are superimposed. *corresponds to significance level p < 0.05 from AL. Data presented as mean fEPSP slope (% change relative to preconditioning responses) ± SEM.

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When EtOH was administered acutely to the hippocampal slice prior to and during LTP induction, 20 mM EtOH significantly reduced LTP in AL offspring (67% reduction from 0 mM, p < 0.001), whereas 50 mM EtOH completely blocked LTP, t(34) = 0.222, p = 0.825; 101% reduction from 0 mM, p < 0.001. Furthermore, LTP was significantly affected by the acute application of EtOH in a concentration-dependent manner (20 mM vs. 50 mM, p = 0.032; Fig. 3A).


Figure 3. Effects of acute ethanol (EtOH) application on long-term potentiation (LTP). EtOH (black horizontal bar) was administered acutely to hippocampal slices prior to and during LTP induction. (A) Time course of LTP in slices from ad libitum offspring (white). LTP was reduced by 20 mM EtOH (triangle) and blocked by 50 mM EtOH (square). (B) Time course of LTP in slices from pair-fed (PF)1,2 offspring (gray). LTP was attenuated following application of 20 and 50 mM EtOH (triangle and square, respectively). (C) Time course of LTP in slices from prenatal EtOH exposure (PNEE)1,2 offspring (black). LTP in PNEE1,2 offspring was not affected by acute EtOH application. (D) Summary of LTP experiments examining the interactions between prenatal treatment and acute EtOH application. (E) Samples of traces obtained from corresponding groups; field excitatory postsynaptic potentials (fEPSPs) recorded before (black) or 1 hour (gray) after conditioning stimulation are superimposed. Symbols represent acute EtOH concentration, whereas colors represent prenatal treatment group. Black bar signifies the presence of EtOH for 15 minutes. *corresponds to significance level p < 0.05 from 0 mM EtOH slices within the respected prenatal treatment group. Data presented as mean fEPSP slope (% change relative to preconditioning responses) ± SEM.

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In contrast to the observations in AL offspring, 50 mM EtOH failed to block LTP in PF1,2, t(28) = −2.817, p < 0.001, Fig. 3B, or PNEE1,2, t(28) = −4.059, p < 0.001, Fig. 3C, offspring. However, LTP levels did not significantly differ across the prenatal treatment groups (PF1,2 vs. AL, p = 0.144; PNEE1,2 vs. AL, p = 0.124; PNEE1,2 vs. PF1,2, p = 1.00). Similarly, LTP levels did not significantly differ across prenatal treatment groups following acute exposure to 20 mM EtOH (PF1,2 vs. AL, p = 0.999; PNEE1,2 vs. AL, p = 0.999; PNEE1,2 vs. PF1,2, p = 1.00; Fig. 3D).

Within PF1,2 offspring, 20 mM (55% reduction from PF1,2 0 mM, p = 0.048) and 50 mM (55% reduction from PF1,2 0 mM, p = 0.038) EtOH had similar effects on LTP, significantly attenuating LTP as compared to levels observed in the absence of EtOH (0 mM; Fig. 3B). LTP levels did not differ following 20 and 50 mM application (p = 1.00). Within PNEE1,2 offspring, LTP levels were not significantly affected by acute EtOH exposure (20 mM: 22% reduction from PNEE1,2 0 mM, p = 0.937; 50 mM: 51% reduction from PNEE1,2 0 mM, p = 0.109; Fig. 3C).

The interaction between prenatal treatment and gender indicates that male and female offspring were differently affected by prenatal treatment. Post hoc analysis revealed that LTP levels were significantly larger in PF1,2 females as compared to PF1,2 males (p = 0.003; data not shown). It is important to note that the interpretation of this finding is limited as it does not take into consideration the effects of acute EtOH application. It is important to note that although no other significant gender differences were found, it does appear as though prenatal treatment and acute EtOH application had less of a negative effect on LTP levels in females than in males (see Table 2 for a breakdown of LTP levels).

Input/Output Function

To test whether acute EtOH exposure affected slice health, I/O function was recorded at the end of each LTP recording. I/O curves were generated from the application of increasing stimulus pulse width. In all slices, the slope of the fEPSP significantly increased with increasing stimulation, repeated measures ANOVA, F(8, 920) = 1,871.59, p < 0.001. There was no main effect of prenatal treatment, acute EtOH exposure, or gender. Furthermore, there were no significant interactions between any of the variables, demonstrating that the attenuation of LTP by acute application of EtOH is not due to a rundown in the health of the slice (Fig. 4).


Figure 4. Input/output curves. (A) Output population response as a function of pulse width for all prenatal treatment groups and acute ethanol (EtOH) concentrations. Neither prenatal treatment nor acute EtOH application had any effect on the population response. Data presented as mean field excitatory postsynaptic potential (fEPSP) slope (% change relative to the response induced from the fifth pulse). (B) Samples of traces obtained from corresponding groups; fEPSPs recorded at each test pulse are superimposed. Symbols represent acute EtOH concentration, whereas colors represent prenatal treatment group.

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

To our knowledge, this is the first study to examine the effects of acute EtOH application on synaptic plasticity in prenatal EtOH-exposed offspring in adulthood. There are 3 prominent observations from this study. First, prenatal EtOH exposure (combination of first and second trimester) attenuated LTP in the adult dentate gyrus. Our findings are in agreement with those previously reported (Brady et al., 2013; Christie et al., 2005; Sutherland et al., 1997; Varaschin et al., 2010). However, this is the first time these results have been shown in an adult hippocampal slice preparation using this particular animal model of FASD. Second, in AL (i.e., control) animals, LTP is attenuated by acute EtOH exposure in a concentration-dependent manner. Third, prenatal EtOH exposure results in reduced sensitivity to acute EtOH exposure in adulthood. These results demonstrate the negative effects that acute EtOH exposure has on memory-related mechanisms and the profound long-term neurological abnormalities associated with prenatal EtOH exposure.

Attenuation of LTP Following Prenatal EtOH Exposure

In the absence of acute EtOH, exposure to EtOH throughout the first and second trimester equivalent of human pregnancy resulted in a reduction in LTP as compared to AL offspring. These findings are in agreement with those previously observed in vivo (Christie et al., 2005; Sutherland et al., 1997; Varaschin et al., 2010) and in hippocampal slices following limited access, voluntary consumption model (Brady et al., 2013). Together, these results demonstrate long-lasting alterations in synaptic plasticity as the result of developmental EtOH exposure. Furthermore, these results allude to a malfunction of neural circuits within the hippocampal formation, perhaps relating to the learning and memory deficits observed in individuals with FASDs.

Attenuation of LTP in Control Animals Following Acute EtOH Application

Acute EtOH exposure negatively alters LTP in the adult dentate gyrus of control (i.e., AL) male and female rats. This is in agreement with previous studies examining the effects of acute EtOH on LTP within the dentate gyrus in adolescent and awake adult rats (Givens and McMahon, 1995; Morrisett and Swartzwelder, 1993). Additionally, we showed that LTP was attenuated following the application of both moderate (20 mM) and high (50 mM) levels of EtOH. Accordingly, the gradual decrease in the magnitude of LTP as the result of an increase in EtOH concentration has been reported by several groups in the CA1 (Izumi et al., 2007; Lovinger et al., 1990; Sugiura et al., 1995).

As suggested by previous research, the attenuation of LTP following acute EtOH exposure is likely due to an inhibition of NMDA receptors (Hicklin et al., 2011; Schummers and Browning, 2001). Morrisett and Swartzwelder (1993) reported that the effects of acute EtOH on long-term changes in synaptic strength in the rat dentate gyrus are largely due to an action at the NMDA receptor–channel complex. Although the mechanism of EtOH-induced NMDA receptor inhibition is unclear, recently it has been suggested that EtOH binds at transmembrane (TM) domain 3 of the NR1 subunit and TM4 of the NR2A subunit (Ren et al., 2008). Evidence from cortical and hippocampal studies indicates that acute EtOH modulates the phosphorylation and expression of NR1 and NR2 subunits of the NMDA receptor as well as downstream signaling cascades, such as ERK1/2 (Chandler and Sutton, 2005; Ferrani-Kile et al., 2003; Hicklin et al., 2011; Roh et al., 2011). Additionally, pharmacological and behavioral studies have shown that EtOH inhibition of NMDA receptors is involved in acute intoxication (Hicklin et al., 2011; Wilson et al., 1990). Thus, there is considerable evidence to suggest that attenuation of LTP in control animals is a result of EtOH's inhibitory action on NMDA receptor function, possibly through changes in phosphorylation of specific receptor subunits.

Although the NMDA receptor is a likely candidate for the locus of acute EtOH's inhibition of LTP, there are other possible sites. EtOH not only acts as an NMDA receptor antagonist but also as a GABAA receptor agonist (Harris et al., 1997; Hoffman et al., 1992; Ikonomidou et al., 2000; Lovinger et al., 1989). However, BMI concentrations similar to those used in this study have been shown to block currents elicited by GABA and alphaxalone (a neuroactive steroid with a similar binding site as EtOH; Ueno et al., 1997). Thus, it is unlikely that our reported results are influenced by EtOH's actions on GABAA receptors.

EtOH is known to inhibit L-type Ca2+ channels and AMPA receptor-mediated transmission (Dildy-Mayfield and Harris, 1992). Although AMPA receptors are inhibited by EtOH to a lesser extent than NMDA receptors, they do account for the majority of the fEPSP (Blake et al., 1988; Dildy-Mayfield and Harris, 1992). Additionally, evidence suggests that LTP causes a larger increase in the portion of the fEPSP associated with AMPA receptors than that of NMDA receptors (Clark and Collingridge, 1995). Thus, it is possible that the EtOH-mediated decrease in LTP may be partially due to inhibition of AMPA receptors. Whole-cell analysis of the change in AMPA-mediated currents would provide important information on how acute EtOH exposure affects LTP. On the other hand, acute exposure to EtOH does not seem to affect basal synaptic transmission prior to the induction of LTP nor does it influence the maintenance of LTP (Givens and McMahon, 1995). It is also possible that this decrease is due EtOH inhibition of L-type Ca2+ channels, as they are known to play an important role in the induction (select forms) and maintenance of LTP (Grover et al., 2009; Kapur et al., 1998; Morgan and Teyler, 1999).

Prenatal EtOH-Exposed Offspring are Unaffected by Application of Acute EtOH

In the absence of acute EtOH, exposure to EtOH throughout the first and second trimester equivalent of human pregnancy resulted in a reduction in LTP as compared to AL offspring. We also showed that LTP evoked in the adult dentate gyrus of PNEE1,2 offspring was not affected by the acute application of EtOH, whereas control rats were negatively affected by acute EtOH exposure. These findings indicate that prenatal EtOH exposure is associated with the reduced pharmacologic effect of acute EtOH.

The cellular and molecular mechanisms of prenatal EtOH exposure's influence on EtOH neurotoxicity remains unclear. Previous research indicates that prenatal EtOH exposure is associated with long-lasting alterations of NMDA receptors, including altered receptor subunit composition, binding, and receptor function (Costa et al., 2000; Hughes et al., 1998; Morrisett et al., 1989, 1992; Toso et al., 2005). These results indicate that the reduction in synaptic plasticity seen after prenatal EtOH exposure and the altered pharmacological effect of acute EtOH may be due to alterations in the NMDA receptor–channel complex. Specifically, alterations in binding affinity and binding site availability suggest that prenatal EtOH exposure may also result in the decreased binding of EtOH to the NMDA receptor in adulthood. However, this is speculative and requires investigation. Further study is needed to determine the mechanisms that contribute to the altered sensitivity to acute exposure to EtOH.

Effects on LTP in PF Offspring

The reduction in LTP following prenatal caloric restriction in is in agreement with previously reported findings utilizing models of undernutrition (Jordan and Clark, 1983). Experimental evidence indicates that prenatal undernutrition alters excitatory glutamatergic activity and leads to a decrease in glutamate binding in the adult brain (Rotta et al., 2003). Furthermore, a decrease in the glutamatergic activity shown through a reduction in Na+-independent 3H-glutamate binding in cellular membranes following undernutrition has been reported (Rotta et al., 2003). Therefore, alterations in the glutamatergic system may account for deficits in LTP observed in PF1,2 offspring.

Following prenatal caloric restriction (i.e., undernutrition), acute EtOH application had no effect on LTP in adulthood. Correspondingly, previous research has shown altered responses to EtOH in adulthood following undernutrition and malnutrition. Protein deprivation results in higher metabolism of EtOH, decreased behavioral impairment, and increased sensitivity to NMDA antagonists (Martin et al., 1989; Tonkiss et al., 1998, 2000). Additionally, there is evidence that prenatal malnutrition alters GABAergic response to EtOH (Borghese et al., 1998).

In the current study, we found that LTP was negatively affected in the PF1,2 offspring similar to those observed in PNEE1,2 offspring. Furthermore, the effects of acute EtOH application on PF1,2 offspring were also similar to those observed in PNEE1,2 offspring. Although the liquid diet given to PF1,2 dams provides adequate nutrition, the quantity of food is limited to correspond to that consumed by PNEE1,2 dams. Therefore, PF1,2 offspring serve as a model of undernutrition and provide a means of distinguishing the effects of EtOH and caloric restriction on development. Because of the similarities in results between PNEE1,2 and PF1,2 offspring, we cannot exclude the possibility that the our PNEE1,2 results are due to undernutrition. This then implicates that nutritional alterations that are caused by EtOH consumption (since PNEE1,2 dams are given AL access to their liquid diet) may be a causal factor in LTP alterations in PNEE1,2 offspring.

Interestingly, others who have investigated the effects of PNEE1,2 on LTP in the adult dentate gyrus that included a nutritional control in their studies found that there was no effect of PF1,2 on LTP (Brady et al., 2013; Christie et al., 2005; Sutherland et al., 1997). Thus, the results seem to indicate that the additional procedures required to generate in vitro recordings may uncover subtle nutritional deficits not apparent in vivo, perhaps masked by the modulatory influences available in the intact brain (i.e., in vivo). In support of this, experiments performed in vivo in littermates from the present study failed to demonstrate deficits in LTP in PF1,2 offspring were not observed (H. Sickmann, unpublished data). Therefore, our current findings highlight the need for further investigation between the differences in in vivo and in vitro methodologies and caution when comparing results obtained in vivo to those in vitro.

In the FASD field, there are numerous rodent models which aim to mimic the human condition. These models are classified by the method in which EtOH is administered and include a liquid diet, oral gavage/intubation, vapor inhalation, artificial rearing, and subcutaneous or intraperitoneal injection. The first 4 are the more common methods currently used in FASD research. Unfortunately, there is no perfect model, with each being associated with different advantages and disadvantages (reviewed in Gil-Mohapel et al., 2010). The liquid diet model that we use in this current study which includes a nutritional control group illustrates the importance of prenatal nutrition and demonstrates the need for a refinement in the liquid diet animal model of FASD; one in which undernutrition and FASD may be further teased apart.


In summary, in the absence of acute EtOH, LTP was attenuated in offspring prenatally exposure to EtOH as compared to controls. However, following acute exposure to EtOH in adulthood, a pronounced reduction in LTP was observed in controls but not in prenatally treated offspring. Further investigation is needed to uncover the implications of the reduced pharmacologic effect of acute EtOH in prenatal EtOH-exposed offspring. These findings may relate to the emergence of secondary disabilities common in FASD individuals and could provide insight into the potential treatment of or prevention of alcohol/drug abuse.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

This study was funded by CIHR Grant Number 36331.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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