Experimental procedures followed the Canadian Council on Animal Care guidelines, and were approved by the Animal Care Committee of the Faculty of Medicine of the University of Toronto.

Male Wistar rats (Charles River, La Prairie, QC, Canada), aged 53–60 days, were housed individually in transparent plastic cages with corn-cob bedding in a 12 h light-dark cycle vivarium maintained at 21°C. Food (Teklad Global, 2018 18% Protein Rodent Diet) and water were available ad libitum. All subjects were handled for a period of 7 days, following arrival from the breeding farm.

Two parallel experiments were then initiated as outlined in Figures 1A,B and described in detail below. The first experiment involved electrode implantations into the basolateral amygdala of 20 rats, followed by repeated seizure threshold measurements over a 34 week period during which the subjects were fed an n-3 PUFA adequate or deficient diet (Figure 1A). These rats were also treated with OA or DHA to test their effects on seizure threshold under the two dietary conditions. The second experiment involved a parallel group of non-implanted rats (n = 42) that were randomized to the n-3 PUFA adequate and deficient diets. These subjects were sacrificed after 16 or 32 weeks of dietary treatment to measure the effects of diet on brain DHA levels, or subjected to PTZ seizure testing after 33 weeks of treatment (Figure 1B).

Twenty rats were surgically implanted with stainless steel bipolar electrodes (MS303/1, Plastics One, Roanoke, VA, USA) aimed at the right basolateral amygdala (n = 20), under isoflorane anesthesia. The amygdala coordinates were as follows: anterior-posterior, −2.8; medial-lateral (bregma), 4.6 (bregma); and dorsal-ventral, −8.6 (skull surface at bregma). The incisor bar was set and maintained in the horizontal position by aligning bregma and lambda to the horizontal plane. The electrodes were fixed to the skull with 3–4 stainless steel anchor screws and acrylic dental cement (Nuweld, LD, Caulk). All subjects were subcutaneously injected with buprenorphine analgesic (0.05 mg/kg) and physiological saline (1 ml/kg) for rehydration following surgery.

Baseline afterdischarge thresholds (ADTs) in the amygdala were measured 8 weeks following surgery, using the ascending series method (Figure 1A). Subjects were placed in a corn-bedded open field chamber and connected to a Grass model S-88 stimulator (Grass Instruments, Quincy, MA, USA), which delivered pulses through the recording electrode. The subjects received a one-second train of stimulation pulses at a frequency of 60 Hz, composed of a 1 ms positive and 1 ms negative phase separated by 0.5 ms. The initial stimulus intensity was 40 μA. The current was increased in steps of 20 μA up to 400 μA, and then in steps of 40 μA from 400 μA onwards, until an afterdischarge was evoked. The interval between stimulations was 5 min. The same electrodes were used for stimulating and recording focal electroencephalographic (EEG) activity.

Baseline ADT was measured again, 2 weeks later, after which, the animals were randomized to the n-3 PUFA adequate or deficient diets (see next section). ADTs were measured once every 4–6 weeks thereafter for 34 weeks. The second baseline ADT measurement was used as the reference point of comparison for assessing subsequent changes in seizure thresholds because the first baseline threshold measurements are known to drop drastically (but plateau to some extent) following the first stimulation (16).

Amygdaloid subjects were started on the control n-3 PUFA adequate diet or the experimental n-3 PUFA deficient diet, 1 week after the second baseline ADT measurement. The diets were mixed every 2–3 days in our laboratory, and stored at 4°C. The cornstarch and sucrose components of the diets were obtained from Disley Food Services (Scarborough, ON, Canada). The oils were obtained from Loblaws Supermarkets (Toronto, ON, Canada). Other ingredients (protein, fiber, vitamins, minerals and antioxidant) were obtained from Dyets Inc. (Bethlehem, PA, USA).

The control AIN-93G diet contained (g/kg): casein (200), cornstarch (530), sucrose (100), soybean oil (70), cellulose (50), vitamin mix (10), mineral mix (35), L-cysteine (3), choline bitartrate (2.5) and tertbutyl hydroquinone (0.014). The n-3 PUFA deficient diet contained identical macronutrient composition, but the source of fat was derived from 24 g/kg of safflower oil and 46 g/kg of coconut oil in lieu of the soybean oil. The fatty acid composition of the AIN-93G control and n-3 PUFA deficient diets is presented in Table 1.

ADTs were measured between weeks 35–37 in the amygdaloid implanted subjects following OA or DHA injection (Figure 1A). The rats received a subcutaneous injection of DHA (600 mg/kg) or an equivalent dose of OA control 1 week after the last ADT was measured on week 34. A week later, treatments were switched, meaning that rats that received OA, received DHA. ADTs were measured following injection as described above. ADT measurements were initiated within 15 min post-injection. This ensured that each subject reached its expected ADT by ~1 h post-injection, in view of a study showing that it takes 1 h for DHA to increase seizure threshold (35).

The rationale for the 600 mg/kg dose is based on body weight. We had previously reported that DHA raises seizure thresholds in the PTZ seizure model 1 h following injection at a dose of 300–400 mg/kg in rats weighing 200–300 g (35–37). The rats in the present study weighed ~834 g at the time of seizure testing. Because unesterified DHA has a short plasma half-life and high volume of distribution associated with increased adiposity in heavier rats, the higher DHA dose of 600 mg/kg was selected to account for the greater body weight that likely increases the volume of distribution.

To compare the effects of OA and DHA on seizure thresholds, we subtracted the ADT measured on weeks 35 and 36, following OA or DHA injection, from the previous ADT on week 34, and the ADT following OA or DHA injection on week 37 from the ADT measured on weeks 35 and 36. In other words, the change in ADT was measured by subtracting the ADT following OA or DHA injection, from the prior ADT level.

A parallel group of subjects were obtained from the breeding farm and placed on the n-3 PUFA adequate (n = 21) or deficient diets (n = 20) at the same time as the implanted animals (Figure 1B). They were handled in a similar manner upon arrival and throughout the course of the experiment, being placed in an open field for 30 min once a month. Two-thirds of these subjects were sacrificed at 16 and 32 weeks (n = 7 per group per time-point) post diet initiation with CO₂ asphyxiation. The remaining one-third (n = 6–7 per group) was sacrificed at 33 weeks as described in the following section. The rationale for sacrificing the rats after 16 or 32 weeks of dietary treatment is based on the 4–12 weeks half-life of brain DHA, which led us to predict that a duration of 3–4 half-lives would be required to observe measurable effects of diet on brain DHA levels (31–33). The brains were excised immediately after CO₂ asphyxiation, and dissected to separate piriform-amygdala from a 1 mm coronal section of the left hemisphere. The dissected pyriform-amygdala samples were stored in a minus 80°C freezer until they were subjected to fatty acid analysis as described below.

The PTZ seizure test was performed on the remaining group of subjects with no implanted electrodes following 33 weeks of dietary treatment (Figure 1B; n = 14). Subjects were injected intraperitoneally with 110 mg/kg of PTZ, and observed in an open field for 10 min. This dose was chosen because it reliably induced tonic-clonic seizures in a separate group of subjects that were of the same age. The latency to the onset of myoclonic jerks and tonic-clonic seizures was determined by two observers, of whom one was blinded and the other was handling the animals. Subjects were euthanized with sodium pentobarbital (100 mg/kg) upon visibly showing tonic-clonic convulsions.

Electrode-implanted subjects were deeply anesthetized with sodium pentobarbital (100 mg/kg), and subjected to a direct current of 100 μA for 30 s in order to lesion the site of the electrode implant for subsequent histological evaluation of the position of the electrode tip. The subjects were then decapitated, and the brains were dissected quickly and stored in formalin for a few weeks to ensure complete fixation of the tissue. The brains were then transferred to 20% sucrose solution containing 0.1% sodium azide and stored at 4°C for a few weeks until they were histologically examined.

The right hemisphere that contained the implanted electrode was used for histological confirmation as previously described. In brief, the hemispheres were chilled in isopentane on dry ice and sectioned using a cryostat (Leica Instruments, Willowdale, Ontario, Canada) maintained at −25°C. Coronal sections were obtained at a thickness of 40 μm and mounted onto gelatin coated glass slides. The electrode tract was visible to the naked eye, so sections were collected close to where the tract ended, and subsequently confirmed under light microscopy (Research Analysis System Model 421251; Amersham, MI). Subjects with misplaced electrodes were excluded from subsequent data analysis.

Total lipids were extracted from pyriform-amygdala by the method of Folch et al. (38) after being weighed to the nearest tenth of a milligram. The weighed samples were grinded in 6.5 ml of 0.9% KCl using a glass-grinder, and washed twice with 5 ml methanol, and twice with 10 ml of chloroform. Diheptadecanoyl L-α-phosphatidylcholine (Sigma, St. Louis, MO) in chloroform was added as an internal standard to the total lipid extracts, which were then dried under nitrogen and reconstituted in 2 ml of chloroform.

Total lipids directly methylated in 14% methanolic BF₃ (2 mL) and hexane (2 ml) at 100°C for 1 h. The samples were allowed to cool at room temperature for 10 min and centrifuged at 1,200 g following the addition of deionized water (2 ml). The upper hexane layer containing fatty acid metyl esters (FAMEs) was reconstituted in 50 μl hexane and analyzed by gas-chromatography as described in the following section.

FAMEs were analyzed on a Varian-430 gas chromatograph (Varian, Lake Forest, CA, USA) equipped with a Varian FactorFour capillary column (VF-23 ms; 30 m × 0.25 mm i.d. × 0.25 μm film thickness) and a flame-ionization detector. FAMEs were injected in splitless mode. The carrier gas was helium, set to a constant flow rate of 0.7 ml/min. The injector and detector ports were set at 250°C. FAMEs were eluted using a temperature program set initially at 50°C for 2 min, increased at 20°C/min to 170°C held at 170°C for 1 min, increased at 3°C/min to 212°C and held at 212°C for 5 min. Peaks were identified by retention times of authentic FAME standards of known composition (Nu-Chek-Prep, Elysian, MN).

The fatty acid composition of the n-3 PUFA adequate and deficient diets was also determined by gas-chromatography. Total lipids were first extracted from ~0.5 g of powdered diet in chloroform/methanol (2:1 v/v) after adding 2 mg of unesterified heptadecanoic acid as an internal standard (Sigma, St. Louis, MO). Saline (0.9% w/v, 2 ml) was added to separate polar compounds. The bottom layer containing total lipids was transferred to test-tubes. A portion of the extract was dried under nitrogen, reconstituted in 2 ml of hexane, and directly methylated with 2 ml of 14% boron trifluoride in methanol at 100°C for 1 h. The hexane layer was extracted and FAMEs were analyzed by gas-chromatography as described above.

The data are presented as means ± SD. Data analysis was performed using Sigma Stat v.3.2 (Jandel Corporation) or Graphpad Prism v 5.0 (La Jolla, CA). A two-way repeated measures analysis of variance (ANOVA) was used to determine the effects of diet and time on body weight, ADT, seizure duration and seizure score. A two-way ANOVA was used to test the effect of diet and time on pyriform-amygdala DHA and n-6 DPA composition. Due to the small sample size, the Mann-Whitney U-test was used to assess differences in the latency to PTZ-induced seizures between the n-3 PUFA adequate and deficient groups. Statistical significance was accepted at P < 0.05.

Although, the surgeries were aimed at placing the electrodes within the basolateral amygdala, subjects with electrodes falling within the amygdaloid complex were included in the analysis. Electrodes were successfully implanted within the amygdala in 5 out of 8 n-3 PUFA adequate control rats and 4 out of 8 n-3 PUFA deficient rats. Subjects with electrodes misimplanted outside the amygdaloid complex were, therefore, excluded from subsequent analyses.

In the successfully implanted subjects, electrodes were within the basolateral amygdala for 3 (out of 5) and 2 (out of 4) subjects within the n-3 PUFA adequate and deficient groups, respectively. The electrodes for the remaining subjects were within the amygdala, but slightly medial or anterior to the basolateral amygdala. We were not able to compare differences in ADTs within the different amygdala foci due to the small sample size, which is why we accepted subjects with successful implants anywhere within the amygdala. This is also consistent with our primary hypothesis, which aimed to test the effects of diet on amygdaloid seizure thresholds.

The data related to body weight of implanted rats from the start of the baseline ADT measurements and throughout the 34 weeks period of threshold measurements is shown in Figure 2. A two-way repeated measured ANOVA showed a significant effect of time (P < 0.0001) but not of dietary treatment (P > 0.05) on body weight. As shown in Figure 2, both n-3 PUFA adequate and deficient rats gained weight over time. There were no significant differences in body weight between the groups at any time point.

Seizures were successfully recorded at baseline (“week 0”) and at 4, 10, 16, 20, 22, 26 and 34 weeks thereafter. Recordings were obtained for all subjects with amygdala implants, except for one n-3 PUFA adequate control rat on weeks 22, 26, and 34 and one n-3 deficient rat on weeks 10, 16, and 20. These two subjects did not show an afterdischarge when measured during these time periods, probably due to a transient (but unconfirmed) infection. We were not able to retrieve raw ADT files for one control rat on week 16 and one n-3 deficient rat on week 20. ADTs that were not successfully obtained for these 4 rats during the 1–3 time-points were not included in the statistical analysis. Thus, ADTs successfully recorded for these 4 rats during other weeks, as well as the rest of the amygdala-implanted subjects, were included in the statistical analysis.

The data related to ADT (μA), percent change in ADT from baseline and seizure duration over the 34 week measuring period are presented in Figures 3A–C. Two-way repeated measures ANOVA showed a significant effect of time but no effect of treatment or interaction between time and treatment for ADT, % change in ADT and seizure duration. As shown in Figure 3, ADT (Figure 3A) and the % change in ADT (Figure 3B) decreased gradually over time in both n-3 adequate and deficient rats, whereas seizure duration increased over time (Figure 3C). A peculiar observation is that absolute ADT values appeared to decrease more for the n-3 PUFA adequate group at 26 and 34 weeks (Figure 3A), but after correcting for the small but insignificant differences in baseline ADT between the two groups, this effect was no longer seen (Figure 3B).

OA or DHA (600 mg/kg) were injected subcutaneously to n-3 PUFA adequate and deficient rats between weeks 35 and 37, to test whether DHA raises ADT. In particular, we wanted to address whether ADTs increased in n-3 deficient rats following DHA injection.

Figure 4 shows the change in ADT and seizure duration following s.c., injection of n-3 PUFA adequate and deficient rats with OA or DHA. Two-way ANOVA revealed a significant interaction between diet and fatty acid injection (P = 0.048), but no significant main effect of diet or injection on ADT (Figure 4A). Post-hoc analysis with Bonferroni's post-hoc test indicated that the change in ADT following DHA treatment was significantly higher in rats on the n-3 adequate group as compared to rats on the n-3 deficient group (P < 0.05). The change in ADT following OA injection was not significant between n-3 adequate or deficient rats. Also, no significant differences between OA and DHA injection were observed.

Two-way ANOVA showed no significant effect of diet or fatty acid (OA or DHA) injection on seizure duration (Figure 4B). Also, no significant interaction was detected.

Body weights measured at the time of seizure testing were not significantly different between the two groups (n-3 PUFA adequate, 873 ± 125 g, n = 7; n-3 PUFA deficient 789 ± 116 g, n = 6, P = 0.29 Mann-Whitney U-test).

The data related to the onset of myoclonic jerks and tonic-clonic seizures following PTZ administration to non-implanted rats maintained on the n-3 PUFA adequate or deficient diet are presented in Figure 5. One rat from the PUFA n-3 deficient group did not seize within the 10 min observation period so it was not included in the statistical analysis. As shown in Figure 5A, the latency to mycolonic jerks in the rats that seized did not differ significantly between the groups (P = 0.15 by Mann-Whitney U-test). The latency to tonic-clonic seizures (Figure 5B) was lower by 57% in the n-3 PUFA deficient group as compared to controls, but the difference was not statistically significant (P = 0.06).

Piriform-amygdala DHA and n-6 DPA were measured in a parallel group of non-electrode implanted subjects administered the n-3 PUFA adequate or deficient diets for 16 and 32 weeks. Figure 6 shows piriform-amygdala DHA (6-A) and n-6 DPA (6-B) composition, expressed as percentage of total fatty, of rats fed an n-3 PUFA adequate or deficient diet for 16 and 32 weeks. A two-way ANOVA followed by Benferroni's post-hoc was used to assess the effect of diet and time on pyriform-amygdala DHA and n-6 DPA% composition.

There was no significant effect of diet, time or interaction between diet and time on DHA percent composition. There was a significant effect of time (P = 0.0261) and diet (P = 0.0006) on n-6 DPA percent composition, but no significant interaction was detected (P = 0.0997). Post-hoc comparison of the means indicated that the difference between the n-3 adequate and deficient groups at 32 months was statistically significant. N-6 DPA was 2.9-fold higher in the n-3 PUFA deficient group as compared to the n-3 PUFA adequate group.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors AT.