W.M. Burnham

Long-term changes in regional brain cytochrome oxidase activity induced by electroconvulsive treatment in rats.

Quantitative cytochrome oxidase (CO) histochemistry was used to examine brain regional metabolic effects of electroconvulsive shock-induced seizures (ECS). Rats receive a course of either eight ECS or control treatments and were sacrificed either 24 h or 28 days after the last session. Regional CO activity (mumol/gT/min) was quantitated throughout the brain using internally calibrated standards. Twenty-four hours after the last ECS session there was no significant difference between ECS- and sham-treated brains in any of the 99 brain regions examined. In contrast, 28 days after the last session, ECS brains showed significant increases in CO activity in the interpeduncular nucleus (+20%), bed nucleus of the stria terminalis (+25%), dorsomedial hypothalamus (+20%), ventromedial hypothalamus (+12%), mammillary nucleus (+14%), pontine nucleus (+16%), basolateral amygdala (+14%), medial amygdala (+12%), piriform cortex (+12%) and ventromedial thalamus (+9%). These results suggest that ECS induces localized increases in brain CO activity which are long-lasting and may develop independently of additional stimulation. The fact that CO changes were predominantly in limbic areas suggests that they may be relevant to the antidepressant effects of ECS.

A ketogenic diet rescues the murine succinic semialdehyde dehydrogenase deficient phenotype.

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a heritable disorder of GABA degradation characterized by ataxia, psychomotor retardation and seizures. To date, there is no effective treatment for SSADH deficiency. We tested the hypothesis that a ketogenic diet (KD) would improve outcome in an animal model of SSADH deficiency, the SSADH knockout mouse (Aldh5a1-/-). Using a 4:1 ratio of fat to combined carbohydrate and protein KD we set out to compare the general phenotype, in vivo and in vitro electrophysiology and [35S]TBPS binding in both Aldh5a1-/- mice and control (Aldh5a1+/+) mice. We found that the KD prolonged the lifespan of mutant mice by >300% with normalization of ataxia, weight gain and EEG compared to mutants fed a control diet. Aldh5a1-/- mice showed significantly reduced mIPSC frequency in CA1 hippocampal neurons as well as significantly decreased [35S]TBPS binding in all brain areas examined. In KD fed mutants, mIPSC activity normalized and [35S]TBPS binding was restored in the cortex and hippocampus. The KD appears to reverse toward normal the perturbations seen in Aldh5a1-/- mice. Our data suggest that the KD may work in this model by restoring GABAergic inhibition. These data demonstrate a successful experimental treatment for murine SSADH deficiency using a KD, giving promise to the idea that the KD may be successful in the clinical treatment of SSADH deficiency.

An evaluation of the anticonvulsant effects of vitamin E

The anticonvulsant effects of D-alpha-tocopherol (vitamin E) were studied in 4 animal seizure models: the Metrazol threshold model (MET), the maximal electroshock model (MES), the kindling model (well-established seizures), and the ferrous chloride model. Vitamin E failed to antagonize seizures in the MES, MET, or the kindling models. It was, however, able to significantly delay the onset of electrographic seizures in the intracerebral ferrous chloride model. Thus, vitamin E shows activity in the ferrous chloride model, but not in the animal models commonly used to screen for anticonvulsant drug actions.

The effects of a ketogenic diet on ATP concentrations and the number of hippocampal mitochondria in Aldh5a1−/− mice

BACKGROUND Succinic semialdehyde dehydrogenase (SSADH) deficiency is an inborn error of GABA metabolism characterized clinically by ataxia, psychomotor retardation and seizures. A mouse model of SSADH deficiency, the Aldh5a1(-/-) mouse, has been used to study the pathophysiology and treatment of this disorder. Recent work from our group has shown that the ketogenic diet (KD) is effective in normalizing the Aldh5a1(-/-) phenotype, although the mechanism of the effect remains unclear. METHODS Here, we examine the effects of a KD on the number of hippocampal mitochondria as well as on ATP levels in hippocampus. Electron microscopy was performed to determine the number of mitochondria in the hippocampus of Aldh5a1(-/-) mice. Adenosine triphosphate (ATP) levels were measured in hippocampal extracts. RESULTS Our results show that the KD increases the number of mitochondria in Aldh5a1(-/-) mice. We also show that Aldh5a1(-/-) mice have significant reductions in hippocampal ATP levels as compared to controls, and that the KD restores ATP in mutant mice to normal levels. GENERAL SIGNIFICANCE Taken together, our data suggest that the KDs actions on brain mitochondria may play a role in the diets ability to treat murine SSADH deficiency.

Benzodiazepine Receptor Binding Following Amygdala-Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes

The binding of [3H]flunitrazepam and [3H]RO5–4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala‐kindled and “yoked” control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of ‐γ‐aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.

Increases in seizure latencies induced by subcutaneous docosahexaenoic acid are lost at higher doses.

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) which has been found to have anticonvulsant properties. Our group has previously reported in a pilot study that the acute administration of subcutaneous (s.c.) DHA increases seizure latencies in the maximal pentylenetetrazole (PTZ) seizure test, however it loses its effect at higher doses. The purpose of the present experiments was (1) to confirm that DHA loses its effect at higher doses, (2) to correlate the anticonvulsant properties of DHA with DHA levels in the different lipid pools of serum and (3) to evaluate whether an anticonvulsant dose of DHA resulted in an increase in DHA release from the brain phospholipids following induction of seizure. In the first experiment, male Wistar rats were injected s.c. with 200, 300, 400 or 600 mg/kg of DHA, or 400mg/kg oleic acid (OA, isocaloric control), and seizure tested with the maximal PTZ test 1h post injection (Experiment 1). In a second experiment, subjects received either: (1) an effective dose of DHA (400mg/kg), (2) a higher, non-effective dose (600 mg/kg; based on the findings of Experiment 1), or (3) OA (400mg/kg). Subjects were sacrificed 1h post injection and blood was collected for fatty acid analysis (Experiment 2). In the third experiment, subjects were injected with either the effective dose of DHA (400mg/kg) or OA (400mg/kg). One hour post lipid injection, animals received either PTZ or saline, and animals were euthanized via microwave fixation. Brain were extracted and unesterified fatty acid concentrations were measured (Experiment 3). Experiment 1 confirmed that DHA loses its effects at higher doses in the maximal PTZ test. The 400mg/kg dose was maximally effective but effects were lost at 600 mg/kg. Experiment 2 showed that only the unesterified DHA pool in serum was statistically increased by an acute injection of s.c. DHA (P<0.05, as compared to OA), whereas esterified DHA pools were unchanged (P>0.05). Curiously, unesterified DHA levels were similar in both the 400mg/kg and 600 mg/kg dosage groups. Experiment 3 showed that an anticonvulsant dose of DHA (400mg/kg) did not increase DHA release from brain phospholipids following seizure induction (P>0.05). In conclusion, DHA has anticonvulsant properties when injected s.c., but these properties are lost at higher doses. The anticonvulsant effects of DHA are accompanied by increased levels of unesterified DHA in the serum, but not in increased DHA release from brain phospholipids.

The ketogenic diet causes a reversible decrease in activity level in Long–Evans rats

Individuals with epilepsy also often exhibit symptoms of attention deficit hyperactivity disorder (ADHD). The ketogenic diet, which is a high fat, low protein, and low carbohydrate diet used in the treatment of intractable epilepsy, also appears to improve symptoms of ADHD in individuals with both disorders. Previous research suggests that the diet decreases the activity level of rats. The purpose of the present research was to further investigate the effects of the ketogenic diet on activity level, using an animal model. Two experiments were conducted. The first experiment examined the time frame and reversibility of the effect of the diet on activity level. The second experiment examined the relationship between activity level and anxiety level. In both experiments, adult male Long-Evans rats were placed on either a ketogenic diet or a control diet. The results of the first experiment show that the ketogenic diet can cause a decrease in activity level within 24 h and that the results are reversible. The results of Experiment 2 show that the decrease in activity level is not linked to a change in anxiety level. The ketogenic diet may be of use in the treatment of ADHD.

Effects of d-amphetamine and apomorphine in a new animal model of petit mal epilepsy.

Abstractd-Amphetamine is effective in controlling seizures in petit mal epilepsy. The flash-evoked afterdischarge (FEAD) in rats has been proposed as a model of the petit mal seizure. The experiments reported here investigated the dose response relationship for the suppression of FEAD by d-amphetamine, and compared its effects with those of the dopamine-mimetic, apomorphine. Significant suppression of FEAD was observed at doses of d-amphetamine greater than 0.2 mg/kg. A maximum decrease of 60% occurred at 1.2 mg/kg. Higher doses did not result in any further suppression. In contrast, apomorphine had no effect on the FEAD event at doses that induced intense stereotypic behavior. In other experiments, administration of either the dopamine antagonist pimozide or the α-adrenergic antagonist phenoxybenzamine exacerbated FEAD and also prevented the suppression of FEAD by d-amphetamine. The results of these experiments support the hypothesis that the FEAD is a valid model of the petit mal seizure. Furthermore, they provide evidence that norepinephrine is necessary for the seizuresuppressant action of d-amphetamine.

A minimum of 3 months of dietary fish oil supplementation is required to raise amygdaloid afterdischarge seizure thresholds in rats - implications for treating complex partial seizures

Complex partial seizures, which typically originate in limbic structures such as the amygdala, are often resistant to antiseizure medications. Our goal was to investigate the effects of chronic dietary supplementation with n-3 polyunsaturated fatty acids (PUFAs) derived from fish oil on seizure thresholds in the amygdala, as well as on blood and brain PUFA levels. The acute effects of injected n-3 PUFAs--eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)--were also tested in the maximal pentylenetetrazol (PTZ) seizure model. In amygdala-implanted subjects, fish oil supplementation significantly increased amygdaloid afterdischarge thresholds, as compared with controls at 3, 5, and 7 months after the start of supplementation. Fish oil supplementation also increased serum EPA and DHA concentrations. DHA concentration in the pyriform-amygdala area increased in the fish-oil treated group by 17-34%, but this effect did not reach statistical significance (P=0.065). DHA significantly increased the latency to seizure onset in the PTZ seizure model, whereas EPA had no significant effect. These observations suggest that chronic dietary fish oil supplementation can raise focal amygdaloid seizure thresholds and that this effect is likely mediated by DHA rather than by EPA.

Intraperitoneal administration of docosahexaenoic acid for 14 days increases serum unesterified DHA and seizure latency in the maximal pentylenetetrazol model

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (n-3 PUFA) which has been shown to raise seizure thresholds following acute administration in rats. The aims of the present experiment were the following: 1) to test whether subchronic DHA administration raises seizure threshold in the maximal pentylenetetrazol (PTZ) model 24h following the last injection and 2) to determine whether the increase in seizure threshold is correlated with an increase in serum and/or brain DHA. Animals received daily intraperitoneal (i.p.) injections of 50mg/kg of DHA, DHA ethyl ester (DHA EE), or volume-matched vehicle (albumin/saline) for 14days. On day 15, one subset of animals was seizure tested in the maximal PTZ model (Experiment 1). In a separate (non-seizure tested) subset of animals, blood was collected, and brains were excised following high-energy, head-focused microwave fixation. Lipid analysis was performed on serum and brain (Experiment 2). For data analysis, the DHA and DHA EE groups were combined since they did not differ significantly from each other. In the maximal PTZ model, DHA significantly increased seizure latency by approximately 3-fold as compared to vehicle-injected animals. This increase in seizure latency was associated with an increase in serum unesterified DHA. Total brain DHA and brain unesterified DHA concentrations, however, did not differ significantly in the treatment and control groups. An increase in serum unesterified DHA concentration reflecting increased flux of DHA to the brain appears to explain changes in seizure threshold, independent of changes in brain DHA concentrations.