Zebrafish as a Model for Epilepsy-Induced Cognitive Dysfunction: A Pharmacological, Biochemical and Behavioral Approach

Abstract

Epilepsy is a neuronal disorder allied with distinct neurological and behavioral alterations characterized by recurrent spontaneous epileptic seizures. Impairment of the cognitive performances such as learning and memory is frequently observed in epileptic patients. Anti-epileptic drugs (AEDs) are efficient to the majority of patients. However, 30% of this population seems to be refractory to the drug treatment. These patients are not seizure-free and frequently they show impaired cognitive functions. Unfortunately, as a side effect, some AEDs could contribute to such impairment. The major problem associated with conducting studies on epilepsy-related cognitive function is the lack of easy, rapid, specific and sensitive in vivo testing models. However, by using a number of different techniques and parameters in the zebrafish, we can incorporate the unique feature of specific disorder to study the molecular and behavior basis of this disease. In the view of current literature, the goal of the study was to develop a zebrafish model of epilepsy induced cognitive dysfunction. In this study, the effect of AEDs on locomotor activity and seizure-like behavior was tested against the pentylenetetrazole (PTZ) induced seizures in zebrafish and epilepsy associated cognitive dysfunction was determined using T-maze test followed by neurotransmitter estimation and gene expression analysis. It was observed that all the AEDs significantly reversed PTZ induced seizure in zebrafish, but had a negative impact on cognitive functions of zebrafish. AEDs were found to modulate neurotransmitter levels, especially GABA, glutamate, and acetylcholine and gene expression in the drug treated zebrafish brains. Therefore, combination of behavioral, neurochemical and genenetic information, makes this model a useful tool for future research and discovery of newer and safer AEDs.

Keywords: T-maze; anti-epileptic drugs; cognitive dysfunction; epilepsy; zebrafish model development.

FIGURE 1

The significance of using zebrafish as a screening model.

FIGURE 2

Experimental procedure and experiment tanks used. (A) Represents scheme for the experiment procedure used to carry out the study. (B) Represent the tank used for the locomotor behavior study. (C) Illustrates the typical size and specification of T-maze apparatus.

FIGURE 3

Seizure onset latency and behavior seizure score. (A) Represent seizure onset latency (score 4) of AEDs treated fish when compared with PTZ treated group. (B) Represent the effect of AEDs on PTZ induced seizure in zebrafish. Data are expressed as Mean ± SEM, n = 10 and statistical analysis by one-way ANOVA followed by Dunnett test. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

FIGURE 4

Pentylenetetrazole (PTZ) induced locomotor pattern and behavior. (A) Representative locomotion tracking patterns of vehicle control, PTZ treated and all the AEDs treated groups. (B) Represent the total distance traveled by the fish in experiment tank in different groups. (C,D) Represent the total time spent by each fish in the lower and upper half of the experimental tank. Data are expressed as Mean ± SEM, n = 10 and statistical analysis by one-way ANOVA followed by Dunnett test ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

FIGURE 5

T-maze analysis and tracking pattern. In (A) each plot represents locomotor pattern of vehicle control, PTZ treated and all the AEDs treated groups. (B,C) represent total time spent in the wrong arm of T-maze by each fish and total distance traveled to reach the deeper chamber. (D,E) represents graph plot of inflecion ratio at 24 and 3 h T-maze trial. Data are expressed as Mean ± SEM, n = 10 and statistical analysis by one-way ANOVA followed by Dunnett test ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

FIGURE 6

Concentration of neurotransmitters in zebrafish brain after T-maze test. Figure represents zebrafish neurotransmitter levels. (A) GABA, (B) Glutamate (C) Acetylcholine. Data are expressed as Mean ± SEM, n = 6 and statistical analysis by one-way ANOVA followed by Dunnett test ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

FIGURE 7

Gene expression in zebrafish brain determined by real time-PCR. (A) represents graph plot for BDNF mRNA expression in zebrafish brain, the level of BDNF fold change was found to be high in AEDs treated and control group as compared to PTZ treated group. (B) represents graph plot of CREB_1 mRNA expression levels in zebrafish brain, the level of CREB_1 fold change were found to be high in AEDs treated as compared to PTZ treated group. (C) represents graph plot of NPY mRNA expression in zebrafish brain, the level of NPY fold change were found to be high in DZP treated and control group as compared to PTZ treated group. This result shows that AEDs alters the gene expression fold change in the brain and affect the cognitive functioning of the brain. Data are expressed as Mean ± SEM, n = 6 and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.