Long-Term Valproate Treatment Increases Brain Neuropeptide Y Expression and Decreases Seizure Expression in a Genetic Rat Model of Absence Epilepsy

et al. (2013) Long-Term Valproate Treatment Increases Brain Neuropeptide Y Expression and Decreases Seizure Expression in a Genetic Rat Model of Absence Epilepsy. PLoS ONE 8(9): e73505. doi:10.1371/journal.pone.0073505 Long-Term Valproate Treatment Increases Brain Neuropeptide Y Expression and Decreases Seizure Expression in a Genetic Rat Model of Absence Epilepsy Johanna Elms 0 Kim L. Powell 0 Leena van Raay 0 Stefanie Dedeurwaerdere 0 Terence J. O'Brien 0 Margaret J. Morris 0 Alice Y. W. Chang, Kaohsiung Chang Gung Memorial Hospital, Taiwan 0 1 Pharmacology, School of Medical Sciences, University of New South Wales , Sydney , Australia , 2 The Department of Medicine, The University of Melbourne, Royal Melbourne Hospital , Melbourne , Australia , 3 Department of Translational Neurosciences, University of Antwerp , Belgium The mechanisms by which valproate, one of the most widely prescribed anti-epileptic drugs, suppresses seizures have not been fully elucidated but may involve up-regulation of neuropeptide Y (NPY). We investigated the effects of valproate treatment in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) on brain NPY mRNA expression and seizure control. GAERS were administered either valproate (42 mg.kg21 hr21) or saline continuously for 5 days. Electroencephalograms were recorded for 24 hrs on treatment days 1, 3 and 5 and the percentage of time spent in seizure activity was analysed. NPY mRNA expression was measured in different brain regions using qPCR. Valproate treatment suppressed seizures by 80% in GAERS (p,0.05) and increased NPY mRNA expression in the thalamus (p,0.05) compared to saline treatment. These results demonstrate that long-term valproate treatment results in an upregulation of thalamic expression of NPY implicating this as a potential contributor to the mechanism by which valproate suppresses absence seizures. - . These authors contributed equally to this work. Valproate is one of most commonly prescribed anti-epileptic drugs and is effective in a broad range of seizure types. Valproate is generally regarded as the first-line anti-epileptic drug for patients with generalised epilepsy syndromes [1]. However, its precise mechanism(s) of action is still not fully understood. Recently it has been proposed that the up-regulation of neuropeptide Y (NPY) in the brain may play a role in the anti-epileptic action of valproate based on the observation that healthy rats chronically treated with valproate for four days showed increased expression of NPY in the nucleus reticularis thalami (nRT) and the hippocampus [2]. A variety of mechanisms have been implicated as being involved in mediating valproates pharmacological effects. These include increased GABA synthesis and release resulting in increased GABAergic transmission [3,4,5], decreases excitatory synaptic activity through the modulation of postsynaptic non-NMDA receptors [6] and blockade of voltage-dependent sodium channels [7,8]. In vitro experiments show that valproate has an early effect to inhibit cellular excitability, which is exerted from the extracellular side of the neuronal membrane, and a more delayed effect resulting from intracellular actions [9,10]. These data indicate that there are both immediate and long-term actions of valproate on cellular excitability. NPY is a 36 amino acid peptide that is abundantly expressed in GABAergic interneurons of the mammalian central nervous system with highest expression seen in the cerebral cortex, dentate hilus, striatum, the reticular nucleus of the thalamus and the arcuate nucleus of the hypothalamus [11,12] and is known to be an endogenous suppressor of seizure activity [13]. NPY signals through identified Y1, Y2, Y4, and Y5 receptors that couple to Gproteins, inhibiting adenylate cyclase and thus decreasing intracellular calcium levels [14,15]. Multiple receptors have been implicated in mediating NPYs seizure suppression action, including Y1, Y2 and Y5 subtypes [16,17,18]. There is substantial evidence for an important role of NPY in the regulation of epileptic seizures. NPY immunoreactivity and Y2 receptor binding is increased in the hippocampus in patients with temporal lobe epilepsy [19] and NPY expression is increased in the rodent brain after recurrent seizures induced chemically or electrically [20,21,22,23,24,25]. Additionally, exogenously administered NPY has been shown to suppress seizure activity in cortical and hippocampal slices in vitro [26,27,28], in experimental models of epilepsy, including the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model of genetic generalised epilepsy [13,18,29], after kainic acid-induced seizures [30] and chronic infusions of NPY delays amygdala kindling epileptogenesis [31]. Further evidence for the importance of NPY in epilepsy has come from NPY knockout and transgenic mice. Mutant mice lacking NPY have an increased susceptibility to spontaneous and pharmacologically induced seizures [32] and are unable to terminate limbic motor seizures [33]. On the other hand, transgenic rats overexpressing NPY have reduced susceptibility to induced seizures [34]. This study tested the hypothesis that increases in NPY expression in the thalamus or cortex, the primary brain regions involved in the oscillatory neuronal network activity that underlies absence seizures, occur with long-term valproate administration in an animal model of genetic generalized epilepsy. We also examined whether NPY expression was increased in the arcuate nucleus of the hypothalamus, and the relationship of this to food intake. Ethics Statement All procedures on rats were approved by The University of Melbourne Animal Ethics Committee (Ethics Number 0705408) and conformed to National Health and Medical Research Council Guidelines for the ethical use of animals in scientific research. All efforts were made to minimize stress and the number of animals necessary to produce reliable data. Animals GAERS are an inbred Wistar rat strain that begin to manifest absence-like seizures after 46 weeks of age accompanied by 5 8 Hz spike-and-wave discharges (SWDs) recorded on the EEG [35]. The seizures in GAERS exhibit a similar pharmacological response to anti-epileptic drugs as for human absence epilepsy, being suppressed by valproate and ethosuximide and aggravated by carbamazepine [36,37,38]. For this study GAERS aged between 46 months were obtained from the breeding colonies at the Royal Melbourne Hospital and Ludwig Institute for Cancer Research Melbourne, Australia. After surgery, animals were placed in individual cages and handled daily to acclimatize them to handling. Animals were fed a normal diet of tap water and rat chow. All procedures were approved by the Melbourne Health Animal Ethics committee and followed the Australian Code of Practice for the care and use of animals for scientific purposes. Surgery Rats were anaesthetised with isoflurane (0.3 L.min21 oxygen and 0.2 L.min21 air) and placed in a stereo (...truncated)