Chun-Yao Lee

Lamotrigine inhibits postsynaptic AMPA receptor and glutamate release in the dentate gyrus

Purpose: The dentate gyrus (DG) is a gateway that regulates seizure activity in the hippocampus. We investigated the site of action of lamotrigine (LTG), an effective anticonvulsant, in the regulation of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) and N‐methyl‐D‐aspartic acid (NMDA) receptor‐mediated excitatory synaptic transmission on DG.

Levetiracetam inhibits glutamate transmission through presynaptic P/Q-type calcium channels on the granule cells of the dentate gyrus

Background and purpose:  Levetiracetam is an effective anti‐epileptic drug in the treatment of partial and generalized seizure. The purpose of the present study was to investigate whether levetiracetam regulates AMPA and NMDA receptor‐mediated excitatory synaptic transmission and to determine its site of action in the dentate gyrus (DG), the area of the hippocampus that regulates seizure activities.

Mossy fiber sprouting in pilocarpine-induced status epilepticus rat hippocampus: a correlative study of diffusion spectrum imaging and histology.

Mossy fiber sprouting (MFS) is the main characteristic of temporal lobe epilepsy (TLE), which is highly correlated with the frequencies of recurrent seizures as well as degrees of severity of TLE. A recent MRI technique, referred to as diffusion spectrum imaging (DSI), can resolve crossing fibers and investigate the intravoxel heterogeneity of water molecular diffusion. Being able to achieve higher accuracy in depicting the complex fiber architecture, DSI may help improve localization of the seizure-induced epileptic foci. In this study, two indices of DSI, which represented the mean diffusivity (MSL) and diffusion anisotropy (DA), were proposed. A correlative study between diffusion characteristics and the severity of MFS was investigated in the pilocarpine-induced status epilepticus (SE) rat model. Nine SE rats and five control rats were studied with MRI and histological Timms staining. For MSL, no significant correlation was found in the dentate gyrus (DG), r=-0.36; p=0.2017, and positive correlation was found in cornu ammonis (CA3), r=0.62; p=0.0174. The correlation between DA and Timms score showed positive correlation in DG, r=0.71; p=0.0047, and negative correlation in CA3, r=-0.63; p=0.0151. Our results were compatible with the previous reports on fiber architecture alterations in DG and CA3 subregions. In conclusion, the histological correspondence of DSI indices was demonstrated. With DSI indices, longitudinal follow-up of hippocampal fiber architecture can be achieved to elucidate the pathophysiology of TLE, which might be helpful in disease localization.

Studies on the [3H] choline uptake in rat phrenic nerve-diaphragm preparations

Abstract The uptake of [ 3 H] choline in the isolated rat phrenic nerve-diaphragm preparation was studied. The uptake was shown to be a saturable process temperature dependent against a concentration gradient and to be inhibited by sulfhydryl reagents and ouabain. It was concluded that at concentrations not higher than 2 × 10 −4 M choline is taken up by an active process. At 2 × 10 −6 M 3 H] choline appeared to be mostly localized at muscle fibers while at 1·2 × 10 −8 M approximately 12% [ 3 H] choline was probably incorporated into nerve endings. It was found that denervated diaphragms still retained the ability to incorporate choline as in the case of the innervated muscle. Stimulation of the phrenic nerve treatment with neostigmine or combination of both procedures did not increase [ 3 H] choline uptake at the nerve endings. No significant amount of labeled acetylcholine could be found. Hemicholinium-3 bretylium and high concentration of D -tubocurarine inhibited the uptake of choline. Selective localization of [ 3 H] choline at the motor nerve endings was not achieved by the present experiments.

Lovastatin Modulates Glycogen Synthase Kinase-3β Pathway and Inhibits Mossy Fiber Sprouting after Pilocarpine-Induced Status Epilepticus

This study was undertaken to assay the effect of lovastatin on the glycogen synthase kinase-3 beta (GSK-3β) and collapsin responsive mediator protein-2 (CRMP-2) signaling pathway and mossy fiber sprouting (MFS) in epileptic rats. MFS in the dentate gyrus (DG) is an important feature of temporal lobe epilepsy (TLE) and is highly related to the severity and the frequency of spontaneous recurrent seizures. However, the molecular mechanism of MFS is mostly unknown. GSK-3β and CRMP-2 are the genes responsible for axonal growth and neuronal polarity in the hippocampus, therefore this pathway is a potential target to investigate MFS. Pilocarpine-induced status epilepticus animal model was taken as our researching material. Western blot, histological and electrophysiological techniques were used as the studying tools. The results showed that the expression level of GSK-3β and CRMP-2 were elevated after seizure induction, and the administration of lovastatin reversed this effect and significantly reduced the extent of MFS in both DG and CA3 region in the hippocampus. The alteration of expression level of GSK-3β and CRMP-2 after seizure induction proposes that GSK-3β and CRMP-2 are crucial for MFS and epiletogenesis. The fact that lovastatin reversed the expression level of GSK-3β and CRMP-2 indicated that GSK-3β and CRMP-2 are possible to be a novel mechanism of lovatstain to suppress MFS and revealed a new therapeutic target and researching direction for studying the mechanism of MFS and epileptogenesis.

Paraquat inhibits postsynaptic AMPA receptors on dopaminergic neurons in the substantia nigra pars compacta

Parkinsons disease (PD) is a neurodegenerative disease that mainly affects dopaminergic (DA-ergic) neurons in the substantia nigra pars compacta (SNc). Glutamate modulates neuronal excitability, and a high concentration of glutamatergic receptors is found on DA-ergic neurons in the SNc. Paraquat (PQ) is a putative causative agent for PD. Its effects on synaptic glutamate transmission in SNc DA-ergic neurons were evaluated using whole-cell voltage-clamp recording in brain slices from 7- to 14-day-old Wistar rats. In the presence of bicuculline (BIC), strychnine, and dl-aminophosphonovaleric acid, PQ reversibly suppressed AMPA receptor-mediated evoked excitatory postsynaptic currents (eEPSCs) in a concentration-dependent manner (P<0.05). In the presence of tetrodotoxin (1 microM), PQ (50 microM) significantly reduced the amplitudes, but not the frequencies, of miniature EPSCs in the SNc, suggesting PQ inhibited eEPSCs through a postsynaptic mechanism. Exogenous application of AMPA to induce AMPA-mediated inward currents excluded involvement of a presynaptic response. The AMPA-induced currents in the SNc were significantly reduced by PQ (50 microM) to 74% of control levels (P<0.05), supporting that PQ acts on postsynaptic AMPA receptors. No effect of PQ on eEPSCs was seen in the LD thalamic nucleus and hippocampus, showing PQ specifically inhibited DA-ergic neurons in the SNc. Our results demonstrate a novel mechanism of action of PQ on glutamate-gated postsynaptic AMPA receptors in SNc DA-ergic neurons. This effect may attenuate the excitability and function of DA-ergic neurons in the SNc, which may contribute to the pathogenesis of PD.

PKA-mediated phosphorylation is a novel mechanism for levetiracetam, an antiepileptic drug, activating ROMK1 channels.

Levetiracetam (LEV) is an effective antiepileptic drug (AED) with distinct mechanism from the conventional AEDs. The major physiological function of ROMK1 channels is to maintain the resting membrane potential (RMP). In this study, we investigated the mechanisms underling LEV on ROMK1 channels. Xenopus oocytes were injected with mRNA to express the wild-type or mutant ROMK1 channels. Giant inside-out patch clamp recordings were performed to study the effect of LEV on these channels. LEV increased the activity of ROMK1 channels in a concentration-dependent manner and enhanced both wild-type and pH(i) gating residue mutant (K80M) channels over a range of pH(i) values. LEV activated the mutated channels at PIP(2)-binding sites (R188Q, R217A and K218A) and PKC-phosphorylation sites channels (S4A, S183A, T191A, T193A, S201A and T234A). However, this drug failed to enhance the channel activity in the presence of PKA inhibitors and did not activate the mutants of PKA-phosphorylation sites on C-terminal (S219A, S313A) and the constructed mutants (S219D and S313D) that mimic the negative charge carried by a phosphate group bound to a serine. Our results demonstrated PKA-mediated phosphorylation is a novel mechanism for LEV activating ROMK1 channels. These findings show that LEV activates ROMK1 channels independently from pH(i) and not via a PIP(2)- or PKC-dependent pathway. The effects of LEV may come from the PKA-induced conformational change but not charge-charge interaction in ROMK1 channels. Enhancing the activity of ROMK1 channels may be an important molecular mechanism for the antiepileptic effects of LEV in restoring neuronal RMP to prevent seizure spreading.

Carisbamate (RWJ-333369) inhibits glutamate transmission in the granule cell of the dentate gyrus.

Carisbamate (CRS, RWJ-333369) is a novel antiepileptic drug awaiting approval for use in the treatment of partial and generalized seizures. Our aim was to determine whether CRS modulates synaptic transmission in the dentate gyrus (DG) and the underlying mechanism. The whole-cell patch-clamp method was used to record AMPA receptor- and NMDA receptor-mediated excitatory postsynaptic currents (EPSC(AMPA) and EPSC(NMDA)) and GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in granule cells of the DG in brain slices prepared from 3- to 5-week-old male Wistar rats. CRS (30-300 μM) inhibited the evoked EPSC(AMPA) and EPSC(NMDA) by the same extent (20%) with significantly altered CV(-2), suggesting presynaptic modulation. It did not significantly change the inward currents induced by AMPA application. The inhibitory effect of CRS on the evoked EPSC(AMPA) was not occluded by selective voltage-gated Ca(2+) channel blockers, ruling out the involvement of presynaptic Ca(2+) channels. The frequency, but not the amplitude, of spontaneous EPSC(AMPA) was significantly reduced by CRS. However, CRS did not alter either the frequency or the amplitude of TTX-insensitive miniature EPSC(AMPA), indicating an action potential-dependent mechanism was involved. In addition, CRS (100 or 300 μM) did not significantly change the amplitude of the evoked IPSCs. To summarize, our results suggest that CRS reduces glutamatergic transmission by an action potential-dependent presynaptic mechanism and consequently inhibits excitatory synaptic strength in the DG without affecting GABAergic transmission. This effect may contribute to the antiepileptic action observed clinically at therapeutic concentrations of CRS.

Gabapentin activates ROMK1 channels by a protein kinase A (PKA)‐dependent mechanism

Background and purpose: Gabapentin is an effective anticonvulsant. The major physiological function of renal outer medullary potassium (ROMK1) channels is to maintain the resting membrane potential (RMP). We investigated the effect of gabapentin on ROMK1 channels and the mechanism involved.

GABAergic tonic inhibition is regulated by developmental age and epilepsy in the dentate gyrus.

&ggr;-Aminobutyric acid (GABA) spillover from synaptic cleft activates extrasynaptic GABAA receptor and results in a tonic inhibition, which induces a background inhibitory effect to stabilize the membrane potential of the neuronal cells. However, the role of tonic inhibition and how it can be regulated during brain development and epileptic state remain elusive. By whole-cell patch-clamp recording on the granule cell in the dentate gyrus, we recorded tonic conductance to investigate the level of tonic inhibition in these two critical periods. According to our observation, an age-dependent increase in tonic conductance was observed. Furthermore, a change in tonic inhibition was also found in a chronic epileptic animal model, indicating that the alteration in tonic inhibition after epilepsy induction persists for a long duration to modulate neuronal activities. The present results show that tonic inhibition is altered during brain development and a chronic epileptic condition, indicating a role of the tonic inhibitory effect in both the critical periods.