Mode of seizure inhibition by sodium channel blockers, an SV2A ligand, and an AMPA receptor antagonist in a rat amygdala kindling model.

PURPOSE: A number of antiepileptic drugs (AEDs) with a variety of modes of action, are effective in treating focal seizures. Several AEDs, such as perampanel (PER), levetiracetam (LEV), lacosamide (LCM), lamotrigine (LTG), and carbamazepine (CBZ), have been shown to elevate the seizure threshold in kindling models. These AEDs are clinically effective, but differences exist in the anti-seizure profiles of drugs with similar modes of action. Therefore, we hypothesized that there are differences in how these AEDs affect seizures. Here, we evaluated the effects of AEDs on various seizure parameters in a rat amygdala kindling model upon stimulation at the after-discharge threshold (ADT) and at three-times the ADT (3xADT) to characterize the differences in the effects of these AEDs. METHODS: PER, LEV, LCM, LTG, CBZ, or vehicle was administered intraperitoneally to fully kindled rats. Changes in Racine seizure score, after-discharge duration (ADD), and latency to Racine score 4 generalized seizure (S4L) were measured to assess differences in the modes of seizure inhibition among the AEDs. Stimulation at 3xADT was used to eliminate the influence of any AED-induced elevation of the seizure threshold on these parameters. RESULTS: PER, LEV, LCM, LTG, and CBZ significantly reduced the seizure score from Racine score 5 after stimulation at the ADT; this effect was lost with LEV and LTG after stimulation at 3xADT. PER and LEV significantly shortened the ADD when the seizure focus was stimulated at the ADT, whereas LCM, LTG, and CBZ did not. LEV, LCM, LTG, and CBZ failed to shorten the ADD upon stimulation at 3xADT. PER dose-dependently and significantly increased S4L, even at doses that were ineffective for seizure score reduction, after stimulation at both the ADT and 3xADT. LEV and LTG significantly increased S4L after stimulation at the ADT, whereas LCM and CBZ did not significantly increase S4L at any of the doses tested. CONCLUSIONS: The sodium channel blockers (LCM, LTG, and CBZ) appeared to act by elevation of the seizure threshold via reduction of neuronal excitability, whereas the AMPA receptor antagonist (PER) and the SV2A ligand (LEV), as well as LTG, exerted their effects through the weakening of synaptic transmission in neuronal networks at the seizure focus. Maintenance of the effect of PER even at 3xADT suggests direct and strong modulation of excitatory synaptic transmission by PER, both at the focus and along the seizure propagation route. These findings may provide further rationale for usage of AEDs beyond their respective modes of action.

LIM homeobox 8 (Lhx8) is a key regulator of the cholinergic neuronal function via a tropomyosin receptor kinase A (TrkA)-mediated positive feedback loop.

Basal forebrain cholinergic neurons play an important role in cognitive functions such as learning and memory, and they are affected in several neurodegenerative diseases, including Alzheimer disease and Down syndrome. Despite their functional importance, the molecular mechanisms of functional maturation and maintenance of these cholinergic neurons after the differentiation stage have not been fully elucidated. This study demonstrates that the LIM homeobox 8 (Lhx8) transcription factor regulates cholinergic function in rat septal cholinergic neurons in primary cultures from E18.5 embryos and in the adult brain. Lhx8 expression modulated tropomyosin receptor kinase A (TrkA) expression in septal cholinergic neurons in vitro and in vivo, resulting in regulated acetylcholine release as an index of cholinergic function. In addition, Lhx8 expression and function were regulated by nerve growth factor (NGF), and the effect of NGF was potentiated by Lhx8-induced TrkA expression. Together, our findings suggest that positive feedback regulation between Lhx8, TrkA, and NGF is an important regulatory mechanism for cholinergic functions of the septum.

Perampanel: a novel, orally active, noncompetitive AMPA-receptor antagonist that reduces seizure activity in rodent models of epilepsy.

PURPOSE: To assess the pharmacology of perampanel and its antiseizure activity in preclinical models. Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile] is a novel, orally active, prospective antiepileptic agent currently in development for refractory partial-onset seizures. METHODS: Perampanel pharmacology was assessed by examining changes in intracellular free Ca(2+) ion concentration ([Ca(2+) ](i) ) in primary rat cortical neurones, and [(3) H]perampanel binding to rat forebrain membranes. Antiseizure activity of orally administered perampanel was examined in amygdala-kindled rats and in mice exhibiting audiogenic, maximal electroshock (MES)-induced, pentylenetetrazole (PTZ) -induced, or 6 Hz-induced seizures. KEY FINDINGS: In cultured rat cortical neurones, perampanel inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced increases in [Ca(2+) ](i) (IC(50) 93 nm vs. 2 µm AMPA). Perampanel had a minimal effect on N-methyl-d-aspartate (NMDA)-induced increases in [Ca(2+) ](i) , and only at a high concentration (30 µm). [(3) H]Perampanel binding to rat forebrain membranes was not significantly displaced by glutamate or AMPA but was displaced by the noncompetitive AMPA receptor antagonists CP465022 (K(i) 11.2 ± 0.8 nm) and GYKI52466 (K(i) 12.4 ± 1 µm). In mice, perampanel showed protective effects against audiogenic, MES-induced, and PTZ-induced seizures (ED(50) s 0.47, 1.6, and 0.94 mg/kg, respectively). Perampanel also inhibited 6 Hz electroshock-induced seizures when administered alone or in combination with other antiepileptic drugs (AEDs). In amygdala-kindled rats, perampanel significantly increased afterdischarge threshold (p<0.05 vs. vehicle), and significantly reduced motor seizure duration, afterdischarge duration, and seizure severity recorded at 50% higher intensity than afterdischarge threshold current (p<0.05 for all measures vs. vehicle). Perampanel caused dose-dependent motor impairment in both mice (TD(50) 1.8 mg/kg) and rats (TD(50) 9.14 mg/kg), as determined by rotarod tests. In mice, the protective index (TD(50) in rotarod test/ED(50) in seizure test) was 1.1, 3.8, and 1.9 for MES-induced, audiogenic, and PTZ-induced seizures, respectively. In rat, dog, and monkey, perampanel had a half-life of 1.67, 5.34, and 7.55 h and bioavailability of 46.1%, 53.5%, and 74.5%, respectively. SIGNIFICANCE: These data suggest that perampanel is an orally active, noncompetitive, selective AMPA receptor antagonist with potential as a broad spectrum antiepileptic agent.

N-type calcium channel in the pathogenesis of experimental autoimmune encephalomyelitis.

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

Caspase-3-like protease activity-independent apoptosis at the onset of neuronal cell death in the gerbil hippocampus after global ischemia.

To investigate the relationship between caspase-3-like protease activity, which has been suggested to be related to apoptosis, and DNA fragmentation, we measured changes in caspase-3-like activity and DNA fragmentation in the hippocampus of gerbils exposed to global ischemia induced by bilateral occlusion of the carotid arteries for 5 min. Caspase-3-like protease activity began to increase at day 4 post-ischemia, reached a peak at day 5, and declined thereafter. The levels of DNA fragmentation, evaluated in terms of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) staining and cytosolic nucleosomes, in the ischemic hippocampus began to increase significantly at day 3 after ischemia, reached a peak at day 4, and decreased thereafter. Our data suggest that DNA fragmentation in ischemic hippocampus of gerbils precedes caspase-3-like protease activation. Our results indicate that a caspase-3-like protease-independent apoptotic pathway operates, at least at the onset of neuronal cell death, in the hippocampus of gerbils after global ischemia.

Dopaminergic agonists and muscarinic antagonists improve lateralization in hemiparkinsonian rats in a novel exploratory Y-maze.

Parkinson's disease (PD) is characterized by the degeneration of nigrostriatal dopaminergic neurons. Its primary clinical symptoms are akinesia, tremor, and rigidity, which usually start from one side, resembling the lateralization in hemiparkinsonian rats having 6-hydroxydopamine-induced unilateral lesion of the medial forebrain bundle. A novel exploratory Y-maze was designed to detect the lateralization of hemiparkinsonian rats in terms of biased turns in the maze. Dopamine agonists levodopa (L-3,4-dihydroxyphenylalanine, 10-30 mg/kg) and apomorphine (0.1-0.3 mg/kg), but not methamphetamine (0.5-2 mg/kg), improved the lateralization in the rat model. However, high doses of the dopamine agonists, 30 and 0.3 mg/kg, respectively, caused small movements in the arms that seemed to parallel the increase in counts per turn in the Y-maze. Interestingly, the muscarinic antagonists trihexyphenidyl and scopolamine improved lateralization moderately, while increasing total turns, an index of locomotive activity. (-)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (0.3 mg/kg), an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, increased total counts, but did not alleviate the lateralization. The alpha2-adrenoceptor antagonist idazoxan (1 and 10 mg/kg) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (1 and 3 mg/kg), a non-NMDA glutamate receptor antagonist, did not affect any of the indices. These findings suggest that the clinical action of drugs on unbalanced movement in PD could be predicted by measuring their effects on lateralization of the 6-hydroxydopamine-lesioned rat model in this exploratory Y-maze.

Ataxia Jackson (ax(J)): a genetic model for apoptotic neuronal cell death.

Programmed cell death or apoptosis is an important process to form normal adult cytoarchitecture. But in vivo analysis of neuronal apoptosis has not been well advanced. Therefore, apoptotic cell death of a particular neuronal system or anatomical part in a mutant is an invaluable target to learn about a link between a gene and neuronal apoptosis. Ataxia (ax) is an autosomal recessive neurological mutant mouse. We recently investigated brains of homozygotes for ataxia Jackson (ax(J)), an allele of ax, using TUNEL method. A few TUNEL-positive cells were observed in the granular cell layer of the cerebellum, the dentate gyrus, and the olfactory bulb of phenotypically normal littermates (ax(J)/+ or +/+) aged at 23-38 days. In affected ax(J)/ax(J) mice, however, the number of TUNEL-positive cells was significantly increased in the cerebellum, particularly in the granular cell layer (p < 0.05). The ax(J) mouse will be an in vivo unique model for studies on the genetic basis of apoptotic neuronal cell death, and identification of the ax gene is desired to elucidate molecular basis of the apoptosis.

Altered expression of glutamate transporters in experimental autoimmune encephalomyelitis.

Amelioration of experimental autoimmune encephalomyelitis (EAE) by blockade of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), has been recently demonstrated [Nat. Med. 6 (2000) 67; Nat. Med. 6 (2000) 62]. However, the mechanisms underlying regulation of the extracellular glutamate concentration in EAE are unclear. To address this, we examined the expression of three distinct Na(+)-dependent glutamate transporters (GLT-1, GLAST and EAAC1) in the spinal cord of the Lewis rat EAE. EAE induced a dramatic increase in EAAC1 protein and mRNA levels, which corresponded closely with the course of neurological symptoms. In contrast, the levels of GLT-1 and GLAST protein were down-regulated in the spinal cord at the peak of disease symptoms, and no recovery was observed after remission. Furthermore, these changes in GLT-1, GLAST and EAAC1 expression were suppressed by treatment with NBQX. These results suggest that AMPA receptor activation precedes the altered expression of glutamate transporters, and that the dysregulation of extracellular glutamate concentration might play a critical role in pathological changes and neuronal dysfunction in EAE.