Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors

Neuropeptide Y (NPY) prominently inhibits epileptic seizures in different animal models. The NPY receptors mediating this effect remain controversial partially due to lack of highly selective agonists and antagonists. To circumvent this problem, we used various NPY receptor knockout mice with the same genetic background and explored anti-epileptic action of NPY in vitro and in vivo. In Y2 (Y2-/-) and Y5 (Y5-/-) receptor knockouts, NPY partially inhibited 0 Mg2+-induced epileptiform activity in hippocampal slices. In contrast, in double knockouts (Y2Y5-/-), NPY had no effect, suggesting that in the hippocampus in vitro both receptors mediate anti-epileptiform action of NPY in an additive manner. Systemic kainate induced more severe seizures in Y5-/- and Y2Y5-/-, but not in Y2-/- mice, as compared to wild-type mice. Moreover, kainate seizures were aggravated by administration of the Y5 antagonist L-152,804 in wild-type mice. In Y5-/- mice, hippocampal kindling progressed faster, and afterdischarge durations were longer in amygdala, but not in hippocampus, as compared to wild-type controls. Taken together, these data suggest that, in mice, both Y2 and Y5 receptors regulate hippocampal seizures in vitro, while activation of Y5 receptors in extra-hippocampal regions reduces generalized seizures in vivo.     

Y5 neuropeptide Y receptor overexpression in mice neither affects anxiety- and depression-like behaviours nor seizures but confers moderate hyperactivity

Neuropeptide Y (NPY) has been implicated in anxiolytic- and antidepressant-like behaviour as well as seizure-suppressant effects in rodents. Although these effects appear to be predominantly mediated via other NPY receptors (Y1 and/or Y2), several studies have also indicated a role for Y5 receptors. Gene therapy using recombinant viral vectors to induce overexpression of NPY, Y1 or Y2 receptors in the hippocampus or amygdala has previously been shown to modulate emotional behaviour and seizures in rodents. The present study explored the potential effects of gene therapy with the Y5 receptor, by testing effects of recombinant adeno-associated viral vector (rAAV) encoding Y5 (rAAV-Y5) in anxiety- and depression-like behaviour as well as in kainate-induced seizures in adult mice. The rAAV-Y5 vector injected into the hippocampus and amygdala induced a pronounced and sustained increase in Y5 receptor mRNA expression and functional Y5 receptor binding, but no significant effects were found with regard to anxiety- and depression-like behaviours or seizure susceptibility. Instead, rAAV-mediated Y5 receptor transgene overexpression resulted in moderate hyperactivity in the open field test. These results do not support a potential role for single transgene overexpression of Y5 receptors for modulating anxiety-/depression-like behaviours or seizures in adult mice. Whether the induction of hyperactivity by rAAV-Y5 could be relevant for other conditions remains to be studied.     

Anti-epileptic effects of neuropeptide Y gene transfection into the rat brain

Neuropeptide Y gene transfection into normal rat brain tissue can provide gene overexpression,which can attenuate the severity of kainic acid-induced seizures.In this study,a recombinant adeno-associated virus carrying the neuropeptide Y gene was transfected into brain tissue of rats with kainic acid-induced epilepsy through stereotactic methods.Following these transfections,we verified overexpression of the neuropeptide Y gene in the epileptic brain.Electroencephalograms showed that seizure severity was significantly inhibited and seizure latency was significantly prolonged up to 4 weeks after gene transfection.Moreover,quantitative fluorescent PCR and western blot assays revealed that the mRNA and protein expression of the N-methyl-D-aspartate receptor subunits NR1,NR2A,and NR2B was inhibited in the hippocampus of epileptic rats.These findings indicate that neuropeptide Y may inhibit seizures via down-regulation of the functional expression of N-methyl-D-aspartate receptors.     

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.     

Seizures and neuronal damage in mice lacking vesicular zinc

Synaptically released zinc has neuromodulatory capabilities that could result in either inhibition or enhancement of neuronal excitability. To determine the net effects of vesicular zinc release in the brain in vivo, we examined seizure susceptibility and seizure-related neuronal damage in mice with targeted disruption of the gene encoding the zinc transporter, ZnT3 (ZnT3-/- mice). ZnT3-/- mice, which lack histochemically reactive zinc in synaptic vesicles, had slightly higher thresholds to seizures elicited by the GABA(A) antagonist, bicuculline, and no differences in seizure threshold were seen in response to pentylenetetrazol or flurothyl. However, ZnT3-/- mice were much more susceptible than wild-type mice to limbic seizures elicited by kainic acid, suggesting that the net effect of hippocampal zinc on acute seizures in vivo is inhibitory. The hippocampi of ZnT3-/- mice showed typical seizure-related neuronal damage in response to kainic acid, demonstrating that damage to the targets of zinc-containing neurons can occur independently of synaptically released zinc. Mice lacking the neuronal zinc-binding protein metallothionein III (MT-III) are also more susceptible to kainic acid-induced seizures. Double knockout (ZnT3 and MT3) mice show the same response to kainic acid as ZnT3-/- mice, suggesting that ZnT3 and MT-III function in the same pathway.     

Sigma 1 receptor-mediated increase in hippocampal extracellular dopamine contributes to the mechanism of the anticonvulsant action of neuropeptide Y

The potent anticonvulsant properties of neuropeptide Y (NPY) are generally attributed to a Y2 receptor-mediated inhibition of glutamatergic synaptic transmission. Independent studies have shown that NPY increases brain dopamine content, possibly via interaction with sigma 1 receptors. Recently, we showed that increased extracellular hippocampal dopamine attenuates pilocarpine-induced limbic seizures via activation of hippocampal D2 receptors. Our aim in this study was to elucidate the role of increased hippocampal dopamine in the mechanism of the anticonvulsant action of NPY and to investigate the involvement of Y2 and sigma 1 receptors in this process. Limbic seizures were evoked in freely moving rats by intrahippocampal administration of pilocarpine via a microdialysis probe. NPY was administered intracerebroventricularly, intrahippocampally via the microdialysis probe, or coadministered intrahippocampally with the D2 receptor antagonist remoxipride, the Y2 receptor antagonist BIIE0246 or the sigma 1 receptor antagonist BD1047. Changes in hippocampal extracellular dopamine were monitored, and behavioural changes indicative of seizure activity were scored. Intracerebroventricular (10 nmol/3 microL) and intrahippocampal (20-50 microm) NPY administration increased hippocampal dopamine and attenuated pilocarpine-induced seizures. Hippocampal D2 receptor blockade (4 microm remoxipride) reversed the anticonvulsant effect of NPY. Y2 receptor blockade (1 microm BIIE0246) reversed the anticonvulsant effect of NPY but did not prevent NPY-induced increases in hippocampal dopamine. Sigma 1 receptor blockade (10 microm BD1047) abolished NPY-induced increases in hippocampal dopamine and reversed the anticonvulsant effect of NPY. Our results indicate that NPY-induced increases in hippocampal dopamine are mediated via sigma 1 receptors and contribute to the anticonvulsant effect of NPY via increased activation of hippocampal D2 receptors. This novel mechanism of anticonvulsant action of NPY is separate from, and may be complementary to, the well established Y2 receptor-mediated inhibition of hippocampal excitability.     

Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice

Neuropeptide Y (NPY) exerts anxiolytic- and antidepressant-like effects in rodents that appear to be mediated via Y1 receptors. Gene therapy using recombinant viral vectors to induce overexpression of NPY in the hippocampus or amygdala has previously been shown to confer anxiolytic-like effect in rodents. The present study explored an alternative and more specific approach: overexpression of Y1 receptors. Using a recombinant adeno-associated viral vector (rAAV) encoding the Y1 gene (rAAV-Y1), we, for the first time, induced overexpression of functional transgene Y1 receptors in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data indicate that rAAV-induced overexpression of Y1 receptors in the hippocampus could confer anxiolytic-like effect accompanied by a moderate proconvulsant adverse effect. Further studies are clearly needed to determine whether Y1 gene therapy might have a future role in the treatment of anxiety disorders.     

Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures

We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y in the hippocampus strengthened the seizure-suppressant effect of transgene Y2 receptors. Here we show for the first time that another neuropeptide Y receptor, Y5, can also be overexpressed in the hippocampus. However, unlike Y2 receptor overexpression, transgene Y5 receptors in the hippocampus had no effect on kainate-induced motor seizures in rats. However, combined overexpression of Y5 receptors and neuropeptide Y exerted prominent suppression of seizures. This seizure-suppressant effect of combination gene therapy with Y5 receptors and neuropeptide Y was significantly stronger as compared to neuropeptide Y overexpression alone. These results suggest that overexpression of Y5 receptors in combination with neuropeptide Y could be an alternative approach for more effective suppression of hippocampal seizures.     

Neuropeptide Y and seizures: effects of exogenously applied ligands

The endogenous NPY system in the brain is centrally involved in seizure regulation. The present paper reviews the evidence that exogenously applied NPY receptor ligands can inhibit epileptic seizures in various rodent in vitro and in vivo models. Agonists at Y2 and/or Y5 receptors and antagonists at Y1 receptors appear to inhibit seizures, depending on the seizure model studied. Although progress has been made, further studies are needed using transgenic animals as well as novel selective agonists and antagonists to firmly identify the NPY receptors mediating antiepileptic effects. This may lead to the development of future antiepileptic drug treatments targeting the NPY system.     

Behavioral changes and expression of heat shock protein hsp-70 mRNA, brain-derived neurotrophic factor mRNA, and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid

Kainic acid-induced seizures in rats represent an established animal model for human temporal lobe epilepsy. However, it is well-known that behavioral responses to the systemic administration of kainic acid are inconsistent between animals. In this study, we examined the relationship between expression of genes, neuropathological damage, and behavioral changes (seizure intensity and body temperature) in rats after systemic administration of kainic acid. The considerable differences in the response to kainic acid-induced seizures were observed in rats after a single administration of kainic acid (12 mg/kg i.p.). There was no detection of the expression of heat shock protein hsp-70 mRNA and HSP-70 protein in brain of vehicle-treated controls and in animals exhibiting weak behavioral changes (stage 1–2). A moderate expression of hsp-70 mRNA was detected throughout all regions (the pyramidal cell layers of CA1–3 and dentate gyrus) of the hippocampus, the basolateral, lateral, central and medial amygdala, the piriform cortex, and the central medial thalamic nucleus of rats that developed moderate seizures (stage 3–4). Marked expression of hsp-70 mRNA was detected in the all regions (cingulate, parietal, somatosensory, insular, entorhinal, piriform cortices) of cerebral cortex and all regions of hippocampus, and the central medial thalamic nucleus of the rats that developed severe seizures (stage 4–5). In addition, marked HSP-70 immunoreactivity was detected in the pyramidal cell layers of CA1 and CA3 regions of hippocampus, all regions (cingulate, parietal, somatosensory, insular, piriform cortices) of cerebral cortex, and the striatum of rats that developed severe seizures (stage 4–5). Furthermore, a marked expression of cyclooxygenase-2 (COX-2) mRNA and brain-derived neurotrophic factor (BDNF) mRNA levels by kainic acid-induced behavioral seizures (stage 3–4 or stage 4–5) was detected in all hippocampal pyramidal cell layers, granule layers of dentate gyrus, piriform cortex, neocortex, and amygdala. The present study suggest that the behavioral changes (seizure intensity and body temperature) and neuropathological damage after systemic administration of kainic acid are inconsistent between animals, and that these behavioral changes (severity of kainic acid-induced limbic seizures) might be correlated with gene expression of hsp-70 mRNA, COX-2 mRNA, and BDNF mRNA in rat brain.     

Kv4.2 knockout mice demonstrate increased susceptibility to convulsant stimulation

Purpose:   Kv4.2 subunits contribute to the pore-forming region of channels that express a transient, A-type K⁺ current (A-current) in hippocampal CA1 pyramidal cell dendrites. Here, the A-current plays an important role in signal processing and synaptic integration. Kv4.2 knockout mice show a near elimination of the A-current in area CA1 dendrites, producing increased excitability in this region. In these studies, we evaluated young adult Kv4.2 knockout mice for spontaneous seizures and the response to convulsant stimulation in the whole animal in vivo and in hippocampal slices in vitro.
Methods:   Electroencephalogram electrode-implanted Kv4.2 knockout and wild-type mice were observed for spontaneous behavioral and electrographic seizures. The latency to seizure and status epilepticus onset in Kv4.2 knockout and wild-type mice was assessed following intraperitoneal injection of kainate. Extracellular field potential recordings were performed in hippocampal slices from Kv4.2 knockout and wild-type mice following the bath application of bicuculline.
Results:   No spontaneous behavioral or electrographic seizures were observed in Kv4.2 knockout mice. Following kainate, Kv4.2 knockout mice demonstrated a decreased seizure and status epilepticus latency as well as increased mortality compared to wild-type littermates. The background strain modified the seizure susceptibility phenotype in Kv4.2 knockout mice. In response to bicuculline, slices from Kv4.2 knockout mice exhibited an increase in epileptiform bursting in area CA1 as compared to wild-type littermates.
Discussion:   These studies show that loss of Kv4.2 channels is associated with enhanced susceptibility to convulsant stimulation, supporting the concept that Kv4.2 deficiency may contribute to aberrant network excitability and regulate seizure threshold.     

Y2 and Y4 Receptor Signalling Attenuates the Skeletal Response of Central NPY

Both the neuropeptide Y (NPY) and the leptin systems have been shown to be important central mediators of bone metabolism. However, the interaction between these two systems is complex and not fully understood. Here, we show that a unique interaction exists between Y2 and Y4 receptors in the regulation of bone homeostasis that is not evident when combined with lack of Y1 signalling. Despite the hypoleptinaemia shown in male Y2/Y4 double knockout (Y2^−/−Y4^−/−) mice, when on the leptin-deficient ob/ob background, these mice display reduced cancellous bone mass. However, combined Y2/Y4 deletion enhances the effect of leptin deficiency on the cortical bone compartment. By replicating the enhanced central NPY expression evident in ob/ob mice using virally mediated overexpression of NPY in the hypothalamus of Y receptor knockout mice, we demonstrate that Y2^−/−Y4^−/− mice have an exaggerated response to the anti-osteogenic effects of elevated hypothalamic NPY in both cancellous and cortical bone and that this effect appears to be dependent on Y1 receptor signalling. This study highlights the complex interaction between Y receptors in the control of bone mass. Moreover, it suggests that the reduction in cortical bone observed in the absence of leptin is due to the anti-osteogenic effect of elevated hypothalamic NPY levels.     

Neuropeptide Y suppresses absence seizures in a genetic rat model primarily through effects on Y receptors

Neuropeptide Y (NPY) potently suppresses absence seizures in a model of genetic generalized epilepsy, genetic absence epilepsy rats of Strasbourg (GAERS). Here we investigated the Y-receptor subtype(s) on which NPY exerts this anti-absence effect. A dual in vivo approach was used: the cumulative duration of seizures was quantified in adult male GAERS in 90-min electroencephalogram recordings following intracerebroventricular (i.c.v.) injection of: (i) subtype-selective agonists of Y1 ([Leu31Pro34]NPY, 2.5 nmol), Y2 (Ac[Leu(28,31)]NPY24-36, 3 nmol), Y5 receptors [hPP1(-17),Ala31,Aib32]NPY, 4 nmol), NPY (3 nmol) or vehicle; and following (ii) i.c.v. injection of antagonists of Y1 (BIBP3226, 20 nmol), Y2 (BIIE0246, 20 nmol) and Y5 (NPY5RA972, 20 nmol) receptors or vehicle, followed by NPY (3 nmol). Injection of the Y1- and Y5-selective agonists resulted in significantly less mean seizure suppression (37.4% and 53.9%, respectively) than NPY (83.2%; P < 0.05), while the Y2 agonist had similar effects to NPY (62.3% suppression, P = 0.57). Food intake was not increased following injection of the Y2 agonist, while significant increases in food intake were seen following NPY and the other Y-subtype agonists. Compared with vehicle, NPY injection suppressed seizures following the Y1 and Y5 antagonists (45.3% and 80.1%, respectively, P < 0.05), but not following the Y2 antagonist (5.1% suppression, P = 0.46). We conclude that NPY Y2 receptors are more important than Y1 and Y5 receptors in mediating the effect of NPY to suppress absence seizures in a genetic rat model. Y2 receptor agonists may represent targets for novel drugs against genetic generalized epilepsies without resulting in appetite stimulation.     

Selective increase of dark phase water intake in neuropeptide-Y Y2 and Y4 receptor knockout mice

Neuropeptide-Y (NPY) is involved in the regulation of ingestive behaviour and energy homeostasis. Since deletion of the NPY Y2 and Y4 receptor gene increases and decreases food intake, respectively, we examined whether water intake during the light and dark phases is altered in Y2 and Y4 receptor knockout mice. The water consumption of mice staying in their home cages was measured by weighing the water bottles at the beginning and end of the light phase during 4 consecutive days. Control, Y2 and Y4 receptor knockout mice did not differ in their water intake during the light phase. However, during the dark phase Y2 and Y4 receptor knockout mice drank significantly more (46-63%, P<0.05) water than the control mice. The total daily water intake over 24 h was also enhanced. The enhanced water intake during the dark phase was not altered by the beta-adrenoceptor antagonist propranolol or the angiotensin AT1 receptor antagonist telmisartan (each injected intraperitoneally at 10 mg/kg). These data indicate that NPY acting via Y2 and Y4 receptors plays a distinctive role in the regulation of nocturnal water consumption. While beta-adrenoceptors and angiotensin AT1 receptors do not seem to be involved, water intake in Y2 and Y4 receptor knockout mice may be enhanced because presynaptic autoinhibition of NPY release and inhibition of orexin neurons in the central nervous system are prevented.     

Anticonvulsant properties of BIBP3226, a non‐peptide selective antagonist at neuropeptide Y Y1 receptors

Several lines of evidence indicate that neuropeptide Y (NPY)-mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. We have investigated the role of NPY Y₁ receptor subtypes, which are enriched in the dentate area of the hippocampus, on EEG seizures induced by a local injection of 0.04 μg kainic acid in rats. Intrahippocampal administration of 10 μg BIBP3226 (N²- (diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]d -arginamide), a non-peptide selective antagonist at the NPY Y₁ receptors, increased threefold on average (P < 0.01) the time to onset of seizures and reduced the number of seizures and the total time in seizures three- and fourfold, respectively (P < 0.01). Its inactive S-enantiomer BIBP3435 was ineffective on seizure activity. One microgram [Leu³¹,Pro³⁴]NPY, an agonist at Y₁ receptors, did not modify per se the EEG sequelae induced by kainic acid but it antagonized the anticonvulsant effect of BIBP3226. These results indicate that NPY Y₁ receptors in the hippocampus are involved in epileptic phenomena and suggest that selective Y₁ receptor antagonists may be of value for attenuating limbic seizures.     

Structural consequences of Kcna1 gene deletion and transfer in the mouse hippocampus

PURPOSE: Mice lacking the Kv1.1 potassium channel alpha subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1(-/-) (knock-out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. METHODS: Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1(-/-) animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1(-/-) brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E. coli lacZ reporter gene. Vector-injected mice were examined to determine the extent of neuronal infection. RESULTS: Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1(-/-) mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed "ectopic" Kv1.1 alpha channel subunit expression. CONCLUSIONS: Kcna1 deletion in mice results in a seizure disorder that resembles--electrographically and neuropathologically--the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection.     

Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus.

Cyclooxygenase-2 (COX-2) in the brain is expressed constitutively and also increased in pathological conditions such as seizure, cerebral ischemia, and inflammation. This study examined the role of COX-2 in kainic acid-induced seizure and in the following neuronal death by using selective inhibitors. Systemic kainate injection (50 mg/kg; i.p.) in mice evoked seizure within 15 min and led to 29% mortality within 2 h. TUNEL-positive neuronal death peaked at 3 days after injection and was prominent in CA(3a) regions of the hippocampus. NS-398 or celecoxib (10 mg/kg, COX-2 selective inhibitor) and indomethacin (5 mg/kg, nonselective inhibitor) exaggerated kainic acid-induced seizure activity and mortality. COX-2 selective inhibitors induced the seizure at earlier onset and more severe mortality within the first hour than indomethacin and aspirin. NS-398 also aggravated kainic acid-induced TUNEL positive neuronal death and decreased Cresyl violet stained viable neurons, and extended lesions to CA(1) and CA(3b). Kainic acid increased the levels of PGD(2), PGF(2a) and PG E(2) in the hippocampus immediately after injection. Indomethacin attenuated the production of basal and kainic acid-induced prostaglandins. In contrast, NS-398 failed to reduce until the first 30 min after kainic acid injection, during which the animals were severely seizured. It has been challenged the endogenous PGs might have anticonvulsant properties. Thus, COX-2 selective inhibitor, including nonselective inhibitor such as indomethacin, aggravated kainic acid-induced seizure activity and the following hippocampal neuronal death even with variable prostaglandin levels.     

Increased sensitivity to seizures in mice lacking cellular prion protein

PURPOSE: The physiologic role of the cellular prion protein (PrPc) is unknown. Mice devoid of PrPc develop normally and show only minor deficits. However, electrophysiologic and histologic alterations found in these mice suggest a possible role for PrPc in seizure threshold and/or epilepsy. METHODS: We tested the sensitivity of PrPc knockout mice to seizures induced by single convulsant or repeated subconvulsant (kindling) doses of pentylenetetrazol (PTZ), and to status epilepticus (SE) induced by kainic acid or pilocarpine. RESULTS: In PTZ kindling, seizure severity progressed faster in the PrPc knockout group, in which 92.8% reached stage 5 or death after 4 days of stimulation, as opposed to 38.4% in wild-type animals. After 10 injections, mortality was 85.7% among knockouts and 15.3% among controls. After a single PTZ injection (60 mg/kg), overall mortality due to seizures was 91% in knockout mice, but only 33% among wild-type animals. Pilocarpine-induced SE (320 mg/kg) caused an 86.7% mortality in knockouts, as opposed to 40% in wild-type animals. Finally, after kainic acid injections (10 mg/kg), 70% of the knockouts developed at least one severe seizure, and 50% showed repetitive seizures, whereas no wild-type animal exhibited observable seizures. CONCLUSIONS: Animals lacking cellular prion protein expression are more susceptible to seizures induced by various convulsant agents. This is perhaps the most striking alteration yet found in PrPc-null mice, who at first analysis appeared to be completely normal. A possible role for PrPc in chronic and idiopathic (familial), secondary, or cryptogenic epilepsies in humans remains to be investigated.     

Effective G-protein coupling of Y2 receptors along axonal fiber tracts and its relevance for epilepsy

Neuropeptide Y (NPY)-Y2 receptors are G-protein coupled receptors and, upon activation, induce opening of potassium channels or closing of calcium channels. They are generally presynaptically located. Depending on the neuron in which they are expressed they mediate inhibition of release of NPY and of the neuron's classical transmitter GABA, glutamate or noradrenaline, respectively. Here we provide evidence that Y2 receptor binding is inhibited dose-dependently by GTPγS along Schaffer collaterals, the stria terminalis and the fimbria indicating that Y2 receptors are functionally coupled to G-proteins along these fiber tracts. Double immune fluorescence revealed coexistence of Y2-immunoreactivity with β-tubulin, a marker for axons in the stria terminalis, but not with synaptophysin labeling presynaptic terminals, supporting the localization of Y2 receptors along axonal tracts. After kainic acid-induced seizures in rats, GTPγS-induced inhibition of Y2 receptor binding is facilitated in the Schaffer collaterals but not in the stria terminalis. Our data indicate that Y2 receptors are not only located at nerve terminals but also along fiber tracts and are there functionally coupled to G-proteins.     

Altered hippocampal expression of neuropeptides in seizure-prone GALR1 knockout mice

PURPOSE: Mice carrying a deletion of the GALR1 galanin receptor have recently showed spontaneous seizure phenotype with 25% penetrance. To better understand the role of neuropeptides, which are known to undergo complex plasticity changes with development of epileptic seizures, we characterized their expression in the hippocampal formation in GALR1- knockout (-KO) mice with or without seizures and in wild-type (WT) mice. MEHODS: Immunohistochemistry and in situ hybridization were used to study expression of galanin, neuropeptide Y (NPY), substance P, enkephalin, dynorphin, and cholecystokinin (CCK). RESULTS: In GALR1-KO mice that had been displaying seizures, a strong upregulation of galanin immunoreactivity (ir) and messenger RNA (mRNA) was found in the polymorph layer of the dentate gyrus; galanin-ir also appeared in a dense fiber network in the supragranular layer. A strong upregulation of enkephalin was found in the granule cells/mossy fibers, whereas dynorphin mRNA levels were modestly decreased. NPY was strongly expressed in the granule cells/mossy fibers, and an increase of NPY mRNA levels in the polymorph cells was paralleled by an increase of NPY-ir in the molecular layer. An upregulation of substance P-ir was confined to the fibers in the granule and molecular layers, whereas substance P mRNA was increased in the cells of the polymorph layer. Both CCK-ir and mRNA were strongly downregulated in the granule cell/mossy fiber system, but CCK-ir appeared increased in the supragranular and molecular layers. No changes in neuropeptide-ir were found in GALR1-KO mice not displaying seizures. CONCLUSIONS: Complex changes in neuropeptide expression in some principal hippocampal neurons and interneurons appear as a characteristic feature of the spontaneous-seizure phenotype in GALR1-KO mice. However, to what extent causal relations exist between this "epilepsia peptidergic profile" and development of seizures requires further clarification.