Neuroscience Letters 551 (2013) 7–11
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Omega-3 fatty acids can reverse the long-term deficits in hippocampal synaptic plasticity caused by prenatal ethanol exposure Anna R. Patten a,b , Helle M. Sickmann a,e , Roger A. Dyer f , Sheila M. Innis f , Brian R. Christie a,b,c,d,∗ a
Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, British Columbia, Canada Department of Biology, University of Victoria, Victoria, British Columbia, Canada c Brain Research Centre and Program in Neuroscience, University of British Columbia, Vancouver, British Columbia, Canada d Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada e Department of Drug Design and Pharmacology, Faculty of Health and Medicinal Sciences, University of Copenhagen, Copenhagen, Denmark f Department of Paediatrics, University of British Columbia, Vancouver, British Columbia, Canada b
h i g h l i g h t s • • • • •
PNEE negatively affects LTP in the dentate gyrus of male but not female animals. Enrichment with omega-3 fatty acids reverses the deficits in LTP in male animals. Enrichment with omega-3 fatty acids did not influence LTP in ethanol-exposed females. Enrichment with omega-3 fatty acids did not influence LTP in control animals. Omega-3 fatty acids may be a viable treatment option for alleviating some of the deficits associated with FASD.
a r t i c l e
i n f o
Article history: Received 22 January 2013 Received in revised form 15 May 2013 Accepted 18 May 2013 Keywords: Fetal alcohol spectrum disorders Sex-differences In vivo electrophysiology Long-term potentiation Omega-3 fatty acids
a b s t r a c t Fetal alcohol spectrum disorders result in long-lasting neurological deficits including decreases in synaptic plasticity and deficits in learning and memory. In this study we examined the effects of prenatal ethanol exposure on hippocampal synaptic plasticity in male and female Sprague-Dawley rats. Furthermore, we looked at the capacity for postnatal dietary intervention to rescue deficits in synaptic plasticity. Animals were fed an omega-3 enriched diet from birth until adulthood (PND55–70) and in vivo electrophysiology was performed by stimulating the medial perforant path input to the dentate gyrus and recording field excitatory post-synaptic potentials. LTP was induced by administering bursts of five 400 Hz pulses as a theta-patterned train of stimuli (200 ms inter-burst interval). Ethanol-exposed adult males, but not females, exhibited a significant reduction in LTP. This deficit in male animals was completely reversed with an omega-3 enriched diet. These results demonstrate that omega-3 fatty acids can have benefits following prenatal neuropathological insults and may be a viable option for alleviating some of the neurological deficits associated with FASD. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: ANOVA, analysis of variance; BAC, blood alcohol concentration; CNS, central nervous system; DHA, docosahexaenoic acid; FASD, fetal alcohol spectrum disorders; GD, gestational day; LTP, long-term potentiation; NMDA, Nmethyl-d-aspartate; PND, postnatal day; PNEE, prenatal ethanol exposure; PUFA, polyunsaturated fatty acid; SEM, standard error of the mean; TBS, theta-burst stimulation. ∗ Corresponding author at: Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada. Tel.: +1 250 472 4244; fax: +1 250 772 5505. E-mail address:
[email protected] (B.R. Christie). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.05.051
The consumption of alcohol during pregnancy can significantly damage the developing brain and result in a number of disorders that are grouped under the term fetal alcohol spectrum disorders (FASD). Recent reports have estimated the prevalence of FASD in young school children to be as high as 2–5% [11] and it is thought that FASD is the most common cause of intellectual disability and preventable birth defects [17]. In rodent models of FASD, it has been well documented that hippocampal synaptic plasticity is reduced in adult males following prenatal ethanol exposure (PNEE) [4,19–21]. Few studies have focused on the female brain but those that have, have shown
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Fig. 1. (A) Experimental Timeline. On GD 1 animals were assigned to one of three prenatal diets (Ethanol, Pair-fed or Ad libitum control). On GD 15 a blood sample was taken to assess blood alcohol concentration (BAC). When pups were born the dams were placed on either a regular chow diet or an omega-3 enriched diet. When pups were weaned at PND22 they were continued on the same postnatal diet as their mothers until they reached experimental age (PND55–70). At this point animals were used for in vivo electrophysiological experiments. (B) Pre-conditioning evoked responses were obtained by administering a pulse (0.12 ms in duration) at 0.067 Hz (Pre-stimulation). Once a stable baseline was observed for at least 15 min, LTP was induced by applying theta burst stimulation (TBS) consisting of 10 bursts of 5 pulses at 400 Hz with an inter-burst interval of 200 ms, which was repeated 4 times at 30 s intervals. The pulse duration was changed to 0.25 ms during TBS. Following TBS, baseline stimulation was resumed for 60 min at 0.067 Hz (Post Stimulation).
enhanced synaptic plasticity in adolescence [20]. There are very few studies that have tried to rectify the deficits in synaptic plasticity following PNEE [4,21]. Omega-3 fatty acids are polyunsaturated fatty acids (PUFAs) that are found in high concentrations in neuronal membranes [16]. The benefits of omega-3 fatty acid supplementation on synaptic plasticity have been shown in both the dentate gyrus and Cornu Ammonis region of the hippocampus, but only in the aged brain or following some sort of insult (i.e. trauma) [3,8–10,12]. In the healthy brain, omega-3 fatty acid supplementation does not appear to affect LTP, but may prevent age related declines in LTP from occurring [3,10,12]. It is thought that omega-3 supplementation can improve LTP through reducing oxidative stress, inhibiting apoptosis and enhancing membrane fluidity [3,9,10,12,15]. Since PNEE decreases brain concentrations of omega-3 fatty acids [2,22] and omega-3 fatty acid supplementation has been shown to improve synaptic plasticity in other disease or injury models, this study aimed to determine whether feeding an omega3 fatty acid-enriched diet from birth is able to overcome the deficits in synaptic plasticity that occur with PNEE. 2. Materials and methods Four male (300–350 g) and 24 virgin female (250–275 g) Sprague-Dawley rats were obtained from Charles River Laboratories (Quebec, Canada). Females were paired with males for breeding purposes and when sperm was detected through vaginal lavage the female was placed onto one of three prenatal diets (Ad libitum control, pair-fed or ethanol). The methods for administration and composition of the diets are exactly as found in [14] and are shown in Fig. 1A. A blood sample was taken on GD 15 to determine blood alcohol concentration (BAC). On the final day of pregnancy (GD 21), half of the animals were switched to regular rat chow while the
other half were placed on a semi-synthetic diet containing fish oil (kindly supplied by Dr. Shelia Innis, University of British Columbia, Canada) as described in [14]. Litters were culled to ten pups on postnatal day (PND) 2 and were weaned at 22 days of age and housed in pairs (based on sex) in standard caging. Each pup was maintained on either the regular chow diet or the omega-3 enriched diet depending on the diet their mother was assigned to (Fig. 1A). Animals were given ad libitum access to the diet from weaning and until they were used for electrophysiological experiments between the age of PND55–70 (n = 10 per group). In vivo electrophysiology procedures were carried out as outlined in [20]. Briefly, a stimulating electrode was placed in the medial perforant path, and a recording electrode was placed into the hilus region of the dentate gyrus. Basal recordings were obtained by administering a pulse (0.12 ms in duration) at 0.067 Hz. Once a stable baseline was observed for at least 15 min, LTP was induced by applying theta burst stimulation (TBS) consisting of 10 bursts of 5 pulses at 400 Hz with an inter-burst interval of 200 ms, which was repeated 4 times at 30 s intervals. The pulse duration was changed to 0.25 ms during TBS. Following TBS, baseline stimulation resumed for 60 min. Excitatory synaptic transmission was characterized through input/output (I/O) function. For analysis of LTP, the slope of the rising phase (10–80%) of the field EPSP at 55–60 min post stimulation was used to determine alterations in the level of synaptic efficacy. All EPSP slope data are presented as the mean percent change from the pre-conditioning baseline. The protocol for these experiments is summarized in Fig. 1B. Females were not used if they were in proestrous, where estrogen levels are highest, as high levels of this hormone can affect LTP and we wanted to reduce biological variation [13,18]. To assess the stage of the estrous cycle, a vaginal lavage with 0.9% sodium chloride was performed each morning and examined with an Olympus Microscope with a 10× objective (Olympus CX21, Center Valley, PA, USA).
A.R. Patten et al. / Neuroscience Letters 551 (2013) 7–11 Table 1 Gestational data for Ad libitum, pairfed and ethanol dams. All dams (8 per condition) had comparable weight gain over the course of their pregnancies [F(2,22) = 1.267, p = 0.301] and litter sizes were comparable between groups [F(2,22) = 0.213, p = 0.81].
Weight gain over pregnancy (%) Number of pups per litter
Ad Libitum
Pairfed
Ethanol
51.3 ± 5.2 15.7 ± 0.7
42.8 ± 2.9 15.6 ± 1.1
45.2 ± 3.7 14.6 ± 1.6
Statistical analyses were performed using Statistica 7.1 analytical software (StatSoft Inc., Tulsa, OK, USA). All data are presented as mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) was used to determine the effect of prenatal treatment on maternal weight gain across pregnancy and on litter size. A repeated measures ANOVA for prenatal diet (Ethanol, Pairfed, Ad Libitum) and postnatal diet (Omega-3, regular diet) was used for developmental data of pup weight taken on PNDs 2, 8, 15 and 22. The weights of the experimental animals between PND55–70 were initially analyzed with a three-way ANOVA with prenatal diet, postnatal diet, and sex as between-subjects factors. Since a significant main effect of sex was detected [F(1,123) = 778.36, p < 0.0001], male and female data were analyzed separately using a two-way ANOVA with prenatal and postnatal diet as between-subjects factors. A three-way ANOVA with prenatal diet, postnatal diet, and sex as between-subjects factors was initially performed for the electrophysiological data. Again and in agreement with previous studies [20], a significant main effect of sex was found [F(1,123) = 5.74, p = 0.018], and male and female data were subsequently analyzed separately using a two-way ANOVA. Post hoc analyses were conducted using Tukey’s test. A p value ≤ 0.05 was considered to be statistically significant. 3. Results 3.1. Developmental data The percentage weight gain over the course of pregnancy did not differ among prenatal treatment groups [F(2,22) = 1.27, p = 0.30; Table 1]. Litter size was also comparable among prenatal treatment groups [F(2,22) = 0.21, p = 0.81; Table 1]. Peak BAC levels were measured from a blood sample taken 2 h after the dark cycle commenced on GD 15 of pregnancy. The mean BAC level was 135.40 ± 7.54 mg/dl. Offspring weight was determined during the lactation period on PNDs2, 8, 15 and 22. No differences in sex were observed [F(4,38) = 0.92, p = 0.46]. Although all groups gained weight during this period, statistical analysis revealed a significant main effect of prenatal diet [F(8,123) = 5.03, p = 0.0001],
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with ethanol-exposed offspring weighing significantly less than ad libitum offspring at all time points measured (p < 0.01; Table 2). A significant main effect of postnatal diet was also obtained [F(4,62) = 4.97, p = 0.004], with animals on omega-3 diet weighing significantly less than animals on regular chow diet (p < 0.01; Table 2). When animals reached experimental age (PND55–70) a significant effect of sex was observed [F(1,123) = 778.36, p < 0.0001], so males and females were analyzed separately. A main effect of postnatal diet was detected for both males [F(1,62) = 230.13, p < 0.001] and females [F(1,61) = 69.40, p < 0.0001], with animals on the omega-3 diet weighing significantly less than animals on regular diet (p < 0.001 for both males and females; Table 2). 3.2. Electrophysiology 3.2.1. I/O Analysis Excitatory synaptic transmission was characterized through I/O function. In all animals, the slope of the fEPSP significantly increased with increasing stimulation [F(4,376) = 267.1, p < 0.0001]. Prenatal diet [F(2,94) = 0.51, p = 0.61], postnatal diet [F(1,94) = 0.60, p = 0.44] or sex [F(1,94) = 0.08, p = 0.78] had no significant effect on I/O function. 3.3. Long-term potentiation In males, a two-way ANOVA showed significant main effects of both prenatal diet [F(2, 62) = 3.75, p < 0.05] and postnatal diet [F(1,62) = 5.59, p < 0.05], as well as a significant interaction between both factors [F(2,62) = 3.08, p < 0.05]. Post hoc analyses revealed that PNEE males had significantly reduced LTP compared to their ad libitum (p < 0.01) and pair-fed (p < 0.05) controls (Fig. 2B) and that in PNEE males, omega-3 intervention from birth until adulthood led to a significant increase in LTP to control levels (p < 0.05, Fig. 2C and D). In females, a two-way ANOVA revealed no significant main effects of either prenatal or postnatal diets, indicating that dentate gyrus LTP is not significantly affected by PNEE or postnatal omega-3 intervention in adult female animals (Fig. 2E). 4. Discussion The results of this study demonstrate that adult male PNEE animals show long lasting deficits in synaptic plasticity in the dentate gyrus of the hippocampus. Intervention with an omega-3 rich diet from birth until adulthood was able to overcome these deficits in LTP to the point where PNEE males could no longer be distinguished from their control counterparts. On the other hand, female PNEE
Table 2 Offspring developmental data. Please see text for statistical details.
Offspring weight
Ad libitum Regular diet
Male PND2 PND8 PND15 PND22 PND55–70
7.8 18.8 35.7 57.2 382.0
± ± ± ± ±
0.2 2.1 3.3 5.4 7·4
7.1 15.4 24.5 44.1 294.9
± ± ± ± ±
0.2 0.8 2.5 3.3 9.4
6.4 13.9 28.9 46.5 371.5
± ± ± ± ±
0.3 0.4 1.3 3.8 8.9
6.6 14.7 26.3 43.8 260.0
± ± ± ± ±
0.2 0.8 2.0 5.1 11
6.3 12.9 27.5 43.5 388.2
± ± ± ± ±
0.2** 0.7** 0.5** 1.5** 8.2
6.7 13.7 26.0 41.0 282.8
± ± ± ± ±
0.2** 0.8** 1.8** 2.7** 6.1
Female PND2 PND8 PND15 PND22 PND55–70
7.4 18.0 34.1 53.7 246.2
± ± ± ± ±
0.4 3.1 4.7 7.3 5.0#
6.9 13.7 23.8 43.4 206.6
± ± ± ± ±
0.3 1.3 2.3 3.3 4.1#
6.5 14.8 30.2 47.8 243.6
± ± ± ± ±
0.1 0.5 1.9 3.5 4.1#
6.4 13.9 24.9 43.8 181.7
± ± ± ± ±
0.2 0.7 1.6 4.3 6.0#
6.0 13.1 28.2 46.9 236.8
± ± ± ± ±
0.1** 0.7** 1.5** 3.4** 8.9#
6.2 12.7 24.1 39.0 208.0
± ± ± ± ±
0.2** 1.0** 1.9** 3.5** 5.9#
** $ #
Omega-3 diet$
Significantly different to ad libitum offspring of same age/sex (p < 0.01). Significantly different to animals on the regular diet (p < 0.001). Significantly different to males of same condition/diet (p < 0.001).
Pairfed Regular diet
Omega-3 diet$
Ethanol Regular diet
Omega-3 diet$
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Fig. 2. The effects of PNEE on LTP in the dentate gyrus of adult rats. (A) Representative traces from ad libitum males on regular diet (A1) and omega-3 enriched diet (A2), ethanol-exposed males on regular diet (A3) and omega-3 diet (A4) and ethanol-exposed females on regular diet (A5) and on omega-3 enriched diet (A6). Darker traces were taken immediately prior to HFS and lighter traces were taken 55–60 min post-HFS. Scale bar: vertical: 5 mV, horizontal 10 ms. (B) PNEE causes a significant decrease in LTP in the dentate gyrus of adult male rats (n = 8 per condition). Although there is significant short-term plasticity in both groups, the PNEE group fails to sustain LTP as robustly as the control group. (C) Omega-3 supplementation from birth until adulthood can completely reverse the deficits in LTP caused by PNEE in the male brain (n = 8 per condition). Omega-3 supplementation does not increase LTP in control or pair-fed animals. (D) PNEE causes a significant decrease in LTP in males, which can be reversed with omega-3 supplementation. Results are presented as mean ± SEM and were considered statistically significant when p ≤ 0.05. *p < 0.05, **p < 0.01. (E) PNEE does not reduce LTP in females and omega-3 supplementation does not increase LTP in control pair-fed or PNEE animals (n = 8 per group).
animals show normal levels of LTP, and omega-3 fatty acid intervention from birth until adulthood did not further enhance synaptic plasticity in the dentate gyrus of these animals. As previously shown by our laboratory, LTP is reduced in PNEE males [4,20], but not in adult PNEE females (unpublished observations). These sex differences are not fully understood, but it is likely that females show a compensatory or protective mechanism that prevents the deficits in LTP from occurring. Further studies are warranted in order to elucidate the differences between the male and female PNEE brain that can account for these results. In this study we found that dietary enrichment with omega-3 did not increase LTP in animals showing normal levels of this form of synaptic plasticity (i.e., ad libitum and pair-fed animals as well as PNEE females, Fig 2D and E). Previous research has shown that the benefits of omega-3 fatty acids depend on the timing and length of supplementation as well as the brain region, age of the animal, and whether disease or injury has occurred [3,9,10,12]. In the adult normal (i.e., non-diseased) brain, omega-3 fatty acid supplementation does not enhance LTP in the dentate gyrus, even when supplementation is provided for as long as eight weeks [3,10,12], which is in line with our results. Nevertheless, it is possible that omega-3 supplementation can still be beneficial in animals showing normal levels of LTP by preventing any potential age-induced decreases in this form of synaptic plasticity. In this study we have shown that omega-3 supplementation from birth until adulthood is robust enough to reverse deficits in
LTP in the dentate gyrus of adult male PNEE rats (Fig. 2B and C). Since a reduction in omega-3 PUFAs is known to occur as a result of PNEE [2,22], it is possible that the observed reduction in LTP could in fact be due to a PNEE-induced decrease in the levels of these fatty acids in the male hippocampus. Thus, omega-3 fatty acid supplementation may enhance LTP in PNEE males by increasing the availability of PUFAs that were lost as a result of developmental exposure to ethanol and/or by reducing the levels of oxidative stress in these animals [14]. The form of LTP studied in these experiments is dependent on the NMDA receptor [20] and the function of the NMDA receptor relies on the thiol redox state of the cell, which is influenced by glutathione (GSH) concentration [7]. Previous results from our laboratory [1,14] indicate that GSH levels are diminished in the adult brain after PNEE and can be restored with omega-3 fatty acid supplementation [14]. Thus, it is possible that omega-3 fatty acids restore LTP in the dentate gyrus by increasing the intracellular content of GSH. It is important to note that animals supplemented with omega-3 fatty acids weighed significantly less than animals receiving regular chow (Table 2). Obesity can be detrimental for LTP, and restricted diets, which reduce weight, can restore LTP [5]. Furthermore, calorie restriction can prevent age-related declines in hippocampal LTP [6]. While we cannot be certain that it is the reduced weight observed in the animals supplemented with omega-3 that is causing the reversal in LTP deficits in ethanol-exposed males, the fact that control and pair-fed animals on the omega-3 enriched diets
A.R. Patten et al. / Neuroscience Letters 551 (2013) 7–11
did not show increases in LTP despite significant weight loss indicates that the beneficial effects are due to the mechanism of action of omega-3. While in most studies omega-3 fatty acids are administered concomitantly with the insult [3,9], in our study administration of an omega-3 enriched diet occurred postnatally, after ethanol exposure had occurred. This showcases the remarkable ability of omega-3 fatty acids to rescue rather than just prevent deficits in synaptic plasticity, as administration following PNEE was robust enough to restore LTP in PNEE males. While administering omega-3 fatty acids prenatally concomitantly with ethanol exposure would still be relevant, preventive strategies are less likely to be adhered to by alcohol-drinking mothers. Thus, the development of postnatal treatments is likely to have an increased clinical relevance for the treatment of children afflicted with FASD. In conclusion, we have shown that omega-3 fatty acids can rescue the deficits in synaptic plasticity observed in the dentate gyrus of adult male rats that were exposed to ethanol during gestation. These results extend previous findings from our laboratory showing that dietary enrichment with omega-3 fatty acids can also reduce oxidative stress and enhance antioxidant protection in PNEE animals [14], and indicate that these PUFAs may be a viable treatment option for the neurological deficits associated with FASD. Acknowledgements This work was funded by grants from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council (NSERC), the Michael Smith Foundation for Health Research (MSFHR), and the Canada Foundation for Innovation (CFI). AP acknowledges funding from NeuroDevNet and the University of Victoria. HMS was supported by The Alfred Benzon Foundation. B.R.C. is a Michael Smith Senior Scholar. The authors would like to thank Jennifer Graham for technical support and Ellie Parton, Kristin Stevens, Jennifer Jackson, Jennifer Helfer, Scott Sawchuk and James Dunbar for help with animal care. We are also thankful to Dr. Joana Gil-Mohapel and Dr. Patricia de Souza Brocardo for critical reading of the manuscript. References [1] P.S. Brocardo, F. Boehme, A. Patten, A. Cox, J. Gil-Mohapel, B.R. Christie, Anxietyand depression-like behaviors are accompanied by an increase in oxidative stress in a rat model of fetal alcohol spectrum disorders: Protective effects of voluntary physical exercise, Neuropharmacology (2011). [2] G.C. Burdge, A.D. Postle, Effect of maternal ethanol consumption during pregnancy on the phospholipid molecular species composition of fetal guinea-pig brain, liver and plasma, Biochim. Biophys. Acta 1256 (1995) 346–352.
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