Behavioural Pharmacology
Improvement of pentylenetetrazol-induced learning deficits by valproic acid in the adult zebrafish

https://doi.org/10.1016/j.ejphar.2010.06.041Get rights and content

Abstract

Pentylenetetrazol (PTZ) has been shown to induce seizure-like behavior, learning deficits in passive avoidance response test, and an increase in hsp70 (heat shock protein 70) mRNA expression in the adult zebrafish; PTZ has been increasingly appreciated as an excellent model system for the study of seizures. In this study, we demonstrate that valproic acid (VPA), an antiepileptic drug, suppresses seizure-like behavior and improves learning ability in adult zebrafish treated with PTZ. Pretreatment with VPA significantly reduces rapid involuntary movement and abrupt changes in moving direction in the PTZ-treated zebrafish. PTZ-induced learning impairments were also improved in the zebrafish pretreated with 200 or 500 μM VPA. However, the scopolamine-induced impairments of learning ability were not improved by VPA pretreatment. It is worth noting that while the zebrafish treated with 500 μM VPA for 1–3 weeks learned the passive avoidance response, those treated with 1 or 2 mM VPA for 3 h didn't. Furthermore, the increased level of hsp70 expression induced by PTZ, a stress marker protein, was significantly reduced in the VPA-pretreated zebrafish brains. Collectively, our data show the antiepileptic effects of VPA in the adult zebrafish, which coincides with reduced hsp70 mRNA expression, rescued learning impairment under PTZ-treated conditions.

Introduction

Cognitive impairments in epileptic patients have been reported in a broad spectrum including semantic-associated verbal tasks, object-recognition memory, and psychosocial function (Dodrill, 1986, Elger et al., 2004, Giovagnoli et al., 2005). Rats subjected to proconvulsants such as pentylenetetrazol (PTZ) and pilocarpine were also shown to evidence impairments in avoidance response, spatial learning, and emotional reactivity (Becker et al., 1995, Mortazavi et al., 2005, Müller et al., 2009). Therefore, whether or not an antiepileptic drug successfully controls the seizure-induced cognitive impairment is an important factor to be considered in selecting antiepileptic drugs. For example, both ethosuximide and phenobarbital reduced seizures in PTZ-kindled rats, but only phenobarbital improved impaired learning ability in the active avoidance response test (Becker et al., 1995).

Valproic acid (VPA) has been extensively used as a major antiepileptic drug and a mood stabilizing drug, and has also been shown to exert neuroprotective effects. It has a protective effect against PTZ-kindled seizures in rats, and also significantly reduces PTZ-induced epileptiform activity in the rat hippocampus in vitro (Gupta and Malhotra, 1997, Piredda et al., 1986).

However, the effects of VPA on behavioral and cognitive function have been proven inconsistent in many studies, although VPA is generally well-tolerated. Consistent with reports of the positive effects of VPA, visual memory and fine motor fluency were improved in children when absence seizure was abolished by VPA medication (Sirén et al., 2007). VPA also ameliorated deficits in visuospatial learning and object recognition, coupled to a suppressive effect against spontaneous seizures, and also blocked the hippocampal lesions reported in the kainic acid (KA)-induced status epilepticus rats (Bolanos et al., 1998, Jessberger et al., 2007). By way of contrast, reports of adverse cognitive side effects have become increasingly commonplace. It has been reported in previous case studies that VPA induces marked impairments in abstract thinking, imagination, memory, and mathematic reasoning in human patients (Straussberg et al., 1998, Reif et al., 2003). Complicating the issue, VPA was also shown to impair spatial working memory, which was tested using a noble object location test in the normal rats (Umka et al., 2010).

The results of some recent studies have shown that the zebrafish constitutes an excellent model system, in several regards, for the investigation of these apparently inconsistent cognitive effects of VPA, allowing for a simple standard experimental design for the evaluation of drug effects on learning in relation to seizures. First, GABA receptor antagonists such as PTZ and bicuculline that function as proconvulsants in the rats can also cause a recapitulation of seizures in developing and adult zebrafish, presumably via the blockage of the GABAergic inhibitory synaptic transmission (Baraban et al., 2005, Kim et al., 2004). VPA has also been identified as one of the most effective antiepileptic drugs for the inhibition of PTZ-induced epileptiform activity in the exposed brains of agar-embedded zebrafish larvae, reducing epileptiform discharge amplitude in a concentration-dependent manner (Baraban et al., 2005). Secondly, both behavioral seizure recording and drug treatment can be conducted with convenience and accuracy, as the fish are placed in a water-filled container and can thus readily absorb the drug elements dissolved in the water through their gills. For this reason, zebrafish have been previously employed for the screening of potential anticonvulsant drugs as well as the evaluation of new drug candidates for seizure liability (Berghmans et al., 2007, Winter et al., 2008). Thirdly, they are being increasingly recognized as a useful model organism for the analysis of specific drug-induced behavioral and molecular changes (Guo, 2004, Ninkovic and Bally-Cuif, 2006). For example, PTZ-treated adult zebrafish showed deficits in the acquisition and maintenance of passive avoidance response (Kim et al., 2009). Additionally, the rate of heat shock protein 70 (hsp70) mRNA expression was increased in the brains of PTZ-treated zebrafish. Heat shock proteins perform a molecular chaperone function for other proteins and prevent unwanted protein aggregation and protein conformation (Lanneau et al., 2008). Overall survival during convulsant PTZ-induced kindling was increased in mice overexpressing hsp70, which is believed to exert protective effects on the pathology of increased neuronal excitation (Ammon-Treiber et al., 2007).

Therefore, on the basis of the aforementioned studies, we attempted to determine whether VPA is capable of rescuing PTZ-induced learning deficits using passive avoidance response tests in the adult zebrafish, and then to determine whether acute or chronic treatment of VPA causes the impairments in learning ability. Additionally, the effects of VPA on hsp70 mRNA expression in the brains of adult zebrafish were analyzed.

Section snippets

Animals

Adult zebrafish (around 2.5 cm long), purchased from a local fish shop, were maintained at 28.0 ± 1.0 °C in aquarium containers with a 14 h light–10 h dark cycle. Tap water that passed through a multistage filtration system equipped with a sediment filter, a post-carbon filter, and a fluorescent UV light sterilizing filter, was supplied to the aquarium containers (Zebrafish AutoSystem, Genomic Design, Seoul, Korea). Water in the containers was aerated and maintained at pH 7.0–8.0. Zebrafish were fed

Suppression of pentylenetetrazol-induced seizure by valproic acid

The locomotion trace of the zebrafish indicated smooth exploratory circling swimming activity in the controls. However, seizure-like behaviors, such as rapid and involuntary whirlpool-like movements, were typically noted in the zebrafish treated with 10 mM PTZ, with a small twitch of the tail and abrupt changes in movement direction represented by a jagged locomotion trace. Notably, the PTZ-induced whirlpool-like movement disappeared in the zebrafish pretreated with 200 μM VPA for 1 h prior to PTZ

Discussion

In this study, the adult zebrafish showed seizure-like behaviors, such as excessive involuntary locomotion activity, under the PTZ-treated condition, as well as impairments in the learning of passive avoidance responses after PTZ treatment. Pretreatment with VPA, an antiepileptic drug, suppressed these adverse effects on locomotion activity and learning ability. Our results are consistent with previous reports demonstrating that epilepsy could induce behavioral and cognitive impairments in

Acknowledgements

This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2005-070-C00118).

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