Dietary Omega-3 Polyunsaturated Fatty Acid Deprivation Does Not Alter Seizure Thresholds but May Prevent the Anti-seizure Effects of Injected Docosahexaenoic Acid in Rats

Abstract

Background: Brain concentrations of omega-3 docosahexaenoic acid (DHA, 22:6n-3) have been reported to positively correlate with seizure thresholds in rodent seizure models. It is not known whether brain DHA depletion, achieved by chronic dietary omega-3 polyunsaturated fatty acid (PUFA) deficiency, lowers seizure thresholds in rats. Objective: The present study tested the hypothesis that lowering brain DHA concentration with chronic dietary n-3 PUFA deprivation in rats will reduce seizure thresholds, and that compared to injected oleic acid (OA), injected DHA will raise seizure thresholds in rats maintained on n-3 PUFA adequate and deficient diets. Methods: Rats (60 days old) were surgically implanted with electrodes in the amygdala, and subsequently randomized to the AIN-93G diet containing adequate levels of n-3 PUFA derived from soybean oil or an n-3 PUFA-deficient diet derived from coconut and safflower oil. The rats were maintained on the diets for 37 weeks. Afterdischarge seizure thresholds (ADTs) were measured every 4-6 weeks by electrically stimulating the amygdala. Between weeks 35 and 37, ADTs were assessed within 1 h of subcutaneous OA or DHA injection (600 mg/kg). Seizure thresholds were also measured in a parallel group of non-implanted rats subjected to the maximal pentylenetetrazol (PTZ, 110 mg/kg) seizure test. PUFA composition was measured in the pyriform-amygdala complex of another group of non-implanted rats sacrificed at 16 and 32 weeks. Results: Dietary n-3 PUFA deprivation did not significantly alter amygdaloid seizure thresholds or latency to PTZ-induced seizures. Acute injection of OA did not alter amygdaloid ADTs of rats on the n-3 PUFA adequate or deficient diets, whereas acute injection of DHA significantly increased amygdaloid ADTs in rats on the n-3 PUFA adequate control diet as compared to rats on the n-3 PUFA deficient diet (P < 0.05). Pyriform-amygdala DHA percent composition did not significantly differ between the groups, while n-6 docosapentaenoic acid, a marker of n-3 PUFA deficiency, was significantly increased by 2.9-fold at 32 weeks. Conclusion: Chronic dietary n-3 PUFA deficiency does not alter seizure thresholds in rats, but may prevent the anti-seizure effects of DHA.

Keywords: DHA; after-discharge seizure threshold; amygdala; omega-3 deficiency; pentylenetetrazol.

Figure 1

General experimental design for implanted (A) and non-implanted (B) rats. The amygdala-implanted rats (B) were randomized to an n-3 PUFA adequate or deficient diet for a total of 37 weeks. They were subjected to repeated ADT measurements, once every 4–6 weeks, for 34 weeks. ADTs were measured between weeks 35–37, following i.p., injection with 600 mg/kg of oleic acid (OA) or docosahexaenoic acid (DHA). The non-implanted rats (B) were randomized to the same diets for 33 weeks. Two-thirds of the animals were sacrificed following 16 and 32 weeks of dietary treatment, and one-third was subjected to pentylenetetrazol-induced seizures after 33 weeks of dietary treatment.

Figure 2

Effects of chronic n-3 PUFA deficiency on body weight over time. Rats were placed on a n-3 PUFA adequate (control) or n-3 PUFA deficient diet. Data are mean ± SD of n = 5 n-3 PUFA adequate controls and 4 n-3 PUFA deficient rats. Two-way repeated measures ANOVA showed a significant effect of time on body weight (P < 0.0001). There was no significant effect of diet on body weight (P > 0.05). Subjects' weights increased over time, regardless of diet.

Figure 3

Effect of chronic n-3 PUFA deficiency on (A) ADT (μA), (B) percent change in ADT over time and (C) seizure duration. ADTs and seizure duration were recorded for 34 weeks from rats maintained on the n-3 PUFA adequate diet (n = 5) or n-3 PUFA deficient diet (n = 4). Two-way repeated measures ANOVA showed a significant effect of time but no effect of treatment or interaction between time and treatment on ADT, % change in ADT and seizure duration. ADT (A) and the % change in ADT (B) decreased gradually over time in both n-3 adequate and deficient rats, whereas seizure duration increased over time (C).

Figure 4

Change in ADT 1 h post s.c. injection of OA or DHA (600 mg/kg) to rats fed an n-3 PUFA adequate or deficient for 37 weeks. Rats of each dietary group randomly received 600 mg/kg of oleic acid (OA) or docosahexaenoic acid (DHA) approximately 1 week after the last ADT was taken on week 34. ADTs and seizure duration were measured within 1 h of fatty acid treatment. One week later, the treatments were switched (i.e. rats that received OA now received DHA), and ADTs were measured within 1 h of fatty acid treatment. The difference in ADT following OA and DHA treatment of each subject, per diet, was determined (i.e., DHA ADT—OA ADT) using the prior ADT as a reference point. As shown in (A), two-way ANOVA revealed no main effect of diet or fatty acid treatment on the change in ADT. A significant diet and fatty acid treatment interaction was detected (P < 0.05). Post hoc analysis of the means with Bonferroni's post-hoc test indicated that the change in ADT following DHA treatment was significantly greater in rats on the n-3 adequate diet as compared to rats on the n-3 PUFA deficient diet (^*P < 0.05). No significant main effects or interaction were detected for seizure duration (B).

Figure 5

Latency to the onset of myoclonic jerks and tonic clonic seizures following PTZ administration (110 mg/kg, i.p.) to rats treated with an n-3 PUFA adequate or deficient diet for 33 weeks. One rat from the n-3 PUFA deficient group was excluded because it did not seize within the 10 min observation period. Data are mean ± SD of n = 7 n-3 PUFA adequate and n = 5 n-3 PUFA deficient subjects. (A) Latency to the onset of myoclonic jerks. A Mann-Whitney U was used to compare latencies. The latency to the onset of myoclonic jerks did not differ significantly between the groups (P = 0.15). (B) Latency to the onset of tonic-clonic seizures. A Mann-Whitney U was used to compare latencies. The latency to the onset of tonic-clonic seizures was lower in the n-3 deficient group as compared to the adequate group. This difference, however, was not statistically significant (p = 0.06).

Figure 6

DHA (A) and n-6 DPA (B) % of total fatty acids in piriform-amygdala of rats fed an n-3 PUFA adequate or deficient diet for 16 and 32 weeks. Data are mean ± SD of n = 7 per diet group per time-point for each fatty acid, except n-6 DPA, for which n = 6 in control rats at 32 weeks because it was not detected in one sample. 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 group was significant at 32 weeks (^**P < 0.01).