Blockade of neuropeptide Y(2) receptors and suppression of NPY's anti-epileptic actions in the rat hippocampal slice by BIIE0246.

Neuropeptide Y (NPY) has been shown to suppress synaptic excitation in rat hippocampus by a presynaptic action. The Y(2) (Y(2)R) and the Y(5) (Y(5)R) receptors have both been implicated in this action. We used the non-peptide, Y(2)R-selective antagonist, BIIE0246, to test the hypothesis that the Y(2)R mediates both the presynaptic inhibitory and anti-epileptic actions of NPY in rat hippocampus in vitro. NPY and the Y(2)R-selective agonist, [ahx(5-24)]NPY, both inhibited the population excitatory postsynaptic potential (pEPSP) evoked in area CA1 by stratum radiatum stimulation in a concentration-dependent manner. BIIE0246 suppressed the inhibitory effects of both agonists, suppressing the maximal inhibition without causing a change in the agonist EC(50), in a manner inconsistent with competitive antagonism. BIIE0246 washed out from hippocampal slices extremely slowly. Application of agonist at high concentrations (1 - 3 microM) for prolonged periods did not alter the rate of washout, but did partially overcome the antagonism, inconsistent with an insurmountable antagonism by BIIE0246. In the stimulus train-induced bursting (STIB) model of ictal activity in hippocampal slices, both NPY and [ahx(5-24)]NPY inhibited primary afterdischarge (1 degrees AD) activity. BIIE0246 (100 nM) completely suppressed the actions of NPY and [ahx(5-24)]NPY in this model. In contrast, the potent Y(5)R-selective agonist, Ala(31)Aib(32)NPY, affected neither 1 degrees AD activity in the presence of BIIE0246, nor, by itself, even the pEPSP in CA1. BIIE0246 potently suppresses NPY actions in rat hippocampus, suggesting a dominant role for Y(2)R there. The apparently insurmountable antagonism observed may result from the lipophilic nature of the antagonist.

Delayed kindling epileptogenesis and increased neurogenesis in adult rats housed in an enriched environment.

Environmental risk factors such as stressful experiences have long been recognized to affect seizure susceptibility, but little attention has been paid to the potential effects of improving housing conditions. In this study, we investigated the influence of an enriched environment on epileptogenesis. Epileptic susceptibility was assessed in animals housed in an enriched environment either before and during (group I) or only during (group II) a kindling procedure and in animals placed in isolated conditions (group III). The kindling paradigm provides a reliable assessment of the capacity to develop seizures following repeated daily low-frequency electrical stimulations. As both enriched environment and seizures are known to interfere with hippocampal neurogenesis, the number of newly generated dentate cells was assessed before and after the kindling procedure to investigate in more detail the relationship between epileptogenesis and neurogenesis. We found that susceptibility to developing epilepsy differed in animals housed in complex enriched environments and in those housed in isolated conditions. Kindling epileptogenesis occurred significantly later in animals housed in enriched conditions throughout the procedure (group I) than in animals from groups II and III. We also demonstrated that cells generated during kindling survived for at least 42 days and that these cells were more numerous on both sides of the brain following environmental enrichment than in rats housed in isolated conditions. As similar values were obtained regardless of the duration of the period of enrichment, these cellular changes may not play a major role in delaying kindling development. We suggest that the increase response in neurogenesis following seizures may be an adaptative rather an epileptogenic response.

A model of 'epileptic tolerance' for investigating neuroprotection, epileptic susceptibility and gene expression-related plastic changes.

Previous insult, for example, sustained epileptic seizures, confers a substantial temporary protection against the cellular damage induced by subsequent epileptic challenge. Here we describe a useful model of this so-called 'epileptic tolerance'. Expression of a status epilepticus was triggered by infusing the excitotoxic agent, kainate, into the right hippocampus of adult rats. An appropriate dose of kainate was used to cause brain damage in the homolateral, but not contralateral, hippocampus. At various times following this preconditioning insult, kainate was then re-administered into the lateral ventricle and neuroprotection was observed in the contralateral side between 1 and 15 days later. This model can be used to investigate the mechanisms of this endogenous neuroprotection. It is also particularly suitable for studying the epileptic susceptibility, as reflected by the modifications of the after-discharge threshold, as well as any changes in gene expression induced associated with the preconditioning episode.

A novel method for inducing focal ischemia in vitro.

Current in vitro models of stroke involve applying oxygen-glucose deprived (OGD) media over an entire brain slice or plate of cultured neurons. Thus, these models fail to mimic the focal nature of stroke as observed clinically and with in vivo rodent models of stroke. Our aim was to develop a novel in vitro brain slice model of stroke that would mimic focal ischemia and thus allow for the investigation of events occurring in the penumbra. This was accomplished by focally applying OGD medium to a small portion of a brain slice while bathing the remainder of the slice with normal oxygenated media. This technique produced a focal infarct on the brain slice that increased as a function of time. Electrophysiological recordings made within the flow of the OGD solution ("core") revealed that neurons rapidly depolarized (anoxic depolarization; AD) in a manner similar to that observed in other stroke models. Edaravone, a known neuroprotectant, significantly delayed this onset of AD. Electrophysiological recordings made outside the flow of the OGD solution ("penumbra") revealed that neurons within this region progressively depolarized throughout the 75 min of OGD application. Edaravone attenuated this depolarization and doubled neuronal survival. Finally, synaptic transmission in the penumbra was abolished within 50 min of focal OGD application. These results suggest that this in vitro model mimics events that occur during focal ischemia in vivo and can be used to determine the efficacy of therapeutics that target neuronal survival in the core and/or penumbra.

Ethyl-eicosapentaenoate modulates changes in neurochemistry and brain lipids induced by parkinsonian neurotoxin 1-methyl-4-phenylpyridinium in mouse brain slices.

Evidence suggests a link between Parkinson's disease and the dietary intake of omega (n)-3 and n-6 polyunsaturated fatty acids (PUFAs). Presently, we investigated whether an acute dose of parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) affects brain n-3 and n-6 PUFA content and expression of fatty acid metabolic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) in brain slices from C57Bl/6 mice. Furthermore, we investigated whether feeding a diet of n-3 PUFA ethyl-eicosapentaenoate (E-EPA) to these mice can attenuate the MPP(+) induced changes in brain PUFA content and expression of cPLA2 and COX-2, and attenuate MPP(+) induced changes in neurotransmitters and metabolites and apoptotic markers, bax, bcl-2 and caspase-3. MPP(+) increased brain content of n-6 PUFAs linoleic acid and arachidonic acid, and increased the mRNA expression of cPLA2. MPP(+) also depleted striatal dopamine levels and increased dopamine turnover, and depleted noradrenaline levels in the frontal cortex. The neurotoxin induced increases in bax, bcl-2 and caspase-3 mRNA expression that approached significance. E-EPA by itself increased brain n-3 content, including EPA and docosapentaenoic acid (C22:5, n-3), and increased cortical dopamine. More importantly, E-EPA attenuated the MPP(+) induced increase in n-6 fatty acids content, partially attenuated the striatal dopaminergic turnover, and prevented the increases of pro-apoptotic bax and caspase-3 mRNAs. In conclusion, increases in n-6 PUFAs in the acute stage of exposure to parkinsonian neurotoxins may promote pro-inflammatory conditions. EPA may provide modest beneficial effects in Parkinson's disease, but further investigation is warranted.

The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors.

Neuropeptide Y (NPY) potently inhibits glutamate release and seizure activity in rodent hippocampus in vitro and in vivo, but the nature of the receptor(s) mediating this action is controversial. In hippocampal slices from rats and several wild-type mice, a Y2-preferring agonist mimicked, and the Y2-specific antagonist BIIE0246 blocked, the NPY-mediated inhibition both of glutamatergic transmission and of epileptiform discharges in two different slice models of temporal lobe epilepsy, stimulus train-induced bursting (STIB) and 0-Mg2+ bursting. Whereas Y5 receptor-preferring agonists had small but significant effects in vitro, they were blocked by BIIE0246, and a Y5 receptor-specific antagonist did not affect responses to any agonist tested in any preparation. In slices from mice, NPY was without effect on evoked potentials or in either of the two slice seizure models. In vivo, intrahippocampal injections of Y2- or Y5-preferring agonists inhibited seizures caused by intrahippocampal kainate, but again the Y5 agonist effects were insensitive to a Y5 antagonist. Neither Y2- nor Y5-preferring agonists affected kainate seizures in mice. A Y5-specific antagonist did not displace the binding of two different NPY ligands in WT or mice, whereas all NPY binding was eliminated in the mouse. Thus, we show that Y2 receptors alone mediate all the anti-excitatory actions of NPY seen in the hippocampus, whereas our findings do not support a role for Y5 receptors either in vitro or in vivo. The results suggest that agonists targeting the Y2 receptor may be useful anticonvulsants.

Neuropeptide Y and Epilepsy.

It is a central tenet of the epilepsy field that seizures result from the imbalance of excitation over inhibition (1). The bulk of excitation is mediated by the neurotransmitter glutamate, whereas inhibition results mainly from the actions of gamma-aminobutyric acid (GABA). In the neocortex and hippocampus, the intrinsic sources of GABA are the interneurons, which lately have come under intense scrutiny. It has become clear that a large number of distinct types of interneurons can be differentiated in part by the array of neuropeptides they coexpress (cf. (2)). Evidence is emerging that the neuropeptide complement of interneurons plays important roles in the way that interneurons regulate excitability. Here we discuss what is known about the relation of one well-characterized neuropeptide, neuropeptide Y (NPY), and epilepsy in experimental animals and humans, and suggest possible roles for the receptors as targets for the control of excessive excitation in epilepsy.