View source for Mussulini 2015 Abstract MiPschool London

{{Abstract
|title=Changes of brain substrates preference and metabolism during seizure induced by pentylenetetrazole in adult zebrafish.
|authors=Mussulini BH
|year=2015
|event=MiPschool London 2015
|abstract=Epilepsy is a neurological disorder characterized by recurrent spontaneous epileptic seizures associated with energy depletion. Although certain metabolic characteristics such as ictal and interictal glucose hypometabolism are well established, other important substrates (glutamate, lactate, glutamine, acid hydroxibutiric), and their role during seizure episodes remains unclear. One reliable way to define it is to measure the impact of these molecules under oxygen consumption, which can be achieved by pharmacological approach using the Oroboros Oxygraph-2k. Furthermore, answer this goal in a model highly used to develop new treatments is of great relevance because 30% of epileptic patients still suffer with refractory epilepsy. In this context, the model of seizure induced by pentylenetetrazole in adult zebrafish (''Danio rerio'') appears to be an excellent choice. However the literature lacks precise characterization of adult zebrafish brain metabolism. 

Thus, our aim is to increase the knowledge about the adult zebrafish brain metabolism in front of glucose, glutamine, glutamate, lactate and acid hydroxibutiric using basal respirometry protocol in the Oroboros Oxygraph-2k under control and seizure conditions. Therefore, adult zebrafish brain was dissected. A gentle pressure was applied to the tissue and the brains were set into the Oroboros chamber. A curve of oxygen consumption per weight was performed, and we found a linear curve from 2 mg (1/2 brain) to 18 mg of brain tissue. In order to evaluate membrane stability after dissociation we add succinate to the chamber [10 mM], the basal respiration increased ±20%. To all next steps we used 8 mg of brain tissue. Oligomicine was tested from 0.5 µg/ml to 8 µg/ml, and the best inhibition of ATP syntheses coupled to oxygen consumption without overall respiration impairment was 4 µg/ml resulting in an inhibition of ±35%. The FCCP was tested from 0.025 to 0.5 µM in classical step by step addition of 1 µL. The best concentration without toxic effect was 0.25 µM with an increase of basal respiration of ±280%. The KCN was tested from 0.5 to 2 mM with best concentration of 1 mM showing ±93% inhibition of maximal respiration.
}}
{{Labeling
|area=Respiration, Instruments;methods, Patients
|diseases=Other
|organism=Zebrafish
|tissues=Nervous system
|preparations=Intact organ
|topics=Substrate
|couplingstates=ROUTINE, ET
|pathways=N, S
|instruments=Oxygraph-2k
}}
== Abstract continued ==
The buffer used in all experiments was Hank’s balanced salt solution (HBSS-HEPES buffer) containing: 137 mM NaCl; 0.63 mM Na2HPO4; 3.0 mM NaHCO3; 5.36 mM KCl; 0.44 mM KH2PO4; 1.26 mM CaCl2; 0.90 mM MgSO4; 5.55 mM glucose; and 20 mM HEPES, pH 7.4. Then, the substrates glutamine, glutamate, lactate and acid hydroxibutiric, were added in the presence of glucose in a step by step addition of 10 µL (1, 4, 45, 50, 150, 250, 500 µM). To mimic seizure concentration lactate curve presented an additional concentration of 9 mM. The control and glutamine presented a similar basal respiration. Glutamate addition increased routine respiration ±65% and the consumption of O<sub>2</sub> to ATP production to ±50%. Lactate addition increased routine respiration ±33%, but reduced the O<sub>2</sub> to ATP production to ±10%. The acid hydroxibutiric modulated only extramitocondrial O<sub>2</sub> consumption to ±99% of inhibition. Seizure was induced by pentylenetetrazole in adult zebrafish and basal respiration was performed in 2.5, 5, 20 min of exposure, 1 and 3h after removel of the animal from the solution. Glucose hipometabolism was detected after 20 min of seizure considering consumption of O<sub>2</sub> to ATP production reduced to ±8% (control ±35%). At this moment each of the other substrates was tested. Glutamate (1 mM) presented a reduction impact under routine respiration when compared to glutamate control but was able to increase O<sub>2</sub> consumption to ATP production similar to control conditions. Same results were found with glutamine (500 µM) and  acid hydroxibutiric (700 µM). Despite the fact that lactate (10 mM) inhibited O<sub>2</sub> consumption to ATP production under control conditions, it was able to reverse seizure effects presenting no different from control in any aspect. We speculate it can be due to signalization instead of just metabolic effects. Our perspective is to pharmacologically modulate respirometry with specific agonist of lactate receptor. At this moment, we can conclude that this particular model resembles to human condition in what concerns glucose hypometabolism, as well as glutamate hypometabolism when compared to control, which can justify the high levels of this neurotransmitter in liquor of patients. Furthermore, lactate metabolism in the concentration of 10 mM is able to return the respirometry to normal condition, fact not seen in control. All these aspects indicate a change in the brain substrates preference and metabolism during seizure induced by pentylenetetrazole in adult zebrafish.

== Affiliations ==
Dept Bioquímica, Univ Federal Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. - ben_hurmussulini@yahoo.com.br