thapsigargin and Epilepsy

Dentate granule cell (DGC) swelling was studied by imaging changes in light transmittance from hippocampal slices in the rat pilocarpine model of epilepsy and human epileptic specimens. Brief bath-application of N-methyl-D-aspartic acid (NMDA) induced swelling in the control rat DGC (physiological swelling). Physiological swelling was short-lasting, and rapidly recovered upon removal of NMDA. In contrast, the swelling induced in the pilocarpine-treated rat hippocampus and human epileptic hippocampus (epileptic swelling) was long-lasting, and often recovered slowly over an hour. Both types of swelling were blocked by the NMDA receptor (NMDAR) antagonist, D-APV, suggesting that they shared the same induction mechanism. However, the swellings differed in their sensitivity to a calcium chelator, 1.2-bis(2-aminophenoxy)ethane-N,N,N,N-tetra-acetate (BAPTA), and an endoplasmic reticulum (ER) Ca2+-ATPase inhibitor, thapsigargin (TG). BAPTA and TG affected only epileptic swelling, and physiological swelling was spared. This suggested that the NMDAR-induced epileptic swelling might involve an additional mechanism for its maintenance, likely recruiting ER Ca2+ stores. Brain-derived neurotrophic factor (BDNF) slightly attenuated physiological swelling, and blocked epileptic swelling. The present study suggests a functional link between the activation of NMDAR and a release of Ca2+ from internal stores during the induction of epileptic swelling, and a neuroprotective role of BDNF on the NMDAR-induced swelling in the epileptic hippocampus.

Topics: Adenosine Triphosphatases; Animals; Brain-Derived Neurotrophic Factor; Calcium; Cell Size; Chelating Agents; Cytosol; Dentate Gyrus; Egtazic Acid; Epilepsy; Excitatory Amino Acid Agonists; Hippocampus; Light; Male; N-Methylaspartate; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Thapsigargin

Pharmacological modulation of the epileptiform electric activity induced by caffeine, 10 mM (CAF) on rat hippocampal slices was studied upon field potential recordings in CA3 area of the slices. This concentration of CAF, reportedly releasing Ca2+ ions from the endoplasmic reticulum, led single fimbrial stimuli to evoke repetitive population spikes (PSs) and induced periodic spontaneous field bursts. Carbamazepine, 50 microM reduced (by <40%) the number of repetitive PSs and the rate of spontaneous bursting, with no significant effect on the amplitude of evoked and spontaneous bursts. Valproate, 1 mM reduced only the number (by approximately 25%), but not the amplitudes, of repetitive PSs. Clonazepam, 1 microM consistently reduced the number of repetitive PSs (by approximately 45%), their amplitudes (by 30-60%), and the amplitude of spontaneous bursts (by approximately 70%). The adenosine receptor agonists 2-chloroadenosine, 5 microM and R(-) N6-(2-phenylisopropyl)adenosine, 1 microM had only scanty anti-CAF activity. The depletor of intracellular Ca2+ stores, thapsigargin, 2 microM transiently inhibited the number of evoked PSs and spontaneous bursting. The blocker of ryanodine receptor opening, ruthenium red had an anti-CAF effect, modest at 30 microM, but very strong at 40 microM. Nifedipine, 20 microM opposed CAF-induced spontaneous bursting, but not the evoked PSs. Flunarizine, 50 microM presented only a transient tendency to delay spontaneous bursting. In conclusion, this in vitro slice model appears readily able to reveal antiepileptic properties, though it does not support unequivocal mechanistic interpretation. Nevertheless, anti-CAF activity in this model would suggest the likely involvement of the neuronal ryanodine receptor-related traffic of calcium.

Topics: 2-Chloroadenosine; Animals; Anticonvulsants; Caffeine; Calcium; Calcium Channel Blockers; Calcium Signaling; Central Nervous System Stimulants; Electrophysiology; Enzyme Inhibitors; Epilepsy; Evoked Potentials; Flunarizine; Hippocampus; In Vitro Techniques; Male; Perfusion; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Ruthenium Red; Ryanodine Receptor Calcium Release Channel; Thapsigargin

The transition from an interictal to an ictal pattern of epileptiform activity is a strategic target for antiepileptic drug (AED) action. Both the muscarinic agonist pilocarpine and the selective group I metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) produce prolonged synchronous activity in the hippocampal slice that resembles ictal discharges. We evaluated the role of synaptic mechanisms and release of calcium from intracellular stores in the generation of prolonged ictal oscillations.. Pilocarpine (10 microM) in 7.5 mM[K+]o or DHPG (100 microM) in 5 mM[K+]o artificial cerebrospinal fluid (ACSF) were bath applied to hippocampal slices, and extracellular recordings were made from the CA3 region. The pattern of activity was characterized as ictal if prolonged oscillations of discharges occurred at >2 Hz lasting for >3 s. The pattern of epileptiform activity was characterized and compared with the pattern observed after bath application of pharmacologic agents.. The AMPA/kainic acid (KA) glutamate receptor blocker DNQX (20 microM) dampened and stopped ictal oscillations; however, antagonism of N-methyl-d-aspartate (NMDA) or gamma-aminobutyric acid (GABAA) receptors had minimal effects on ictal patterns. Ictal discharges were suppressed by dantrolene (30-100 microM), which blocks release of calcium from intracellular stores, or thapsigargin (1-5 microM), which inhibits the adenosine triphosphatase (ATPase) that maintains intracellular calcium stores. The L-type calcium channel antagonist nifedipine (1 microM) blocked ictal activity produced by pilocarpine or DHPG.. Ictal discharges produced by pilocarpine or DHPG depended on intact synaptic transmission mediated by AMPA/KA receptors, release of calcium from intracellular stores, and L-type calcium channel activation. The results suggest that muscarinic and group I mGluRs activate a positive-feedback system that creates calcium oscillations and prolonged neuronal synchronization mediated by recurrent excitatory synaptic connections in the CA3 region of the hippocampus.

Topics: Animals; Calcium; Calcium Channels, L-Type; Dantrolene; Electrophysiology; Enzyme Inhibitors; Epilepsy; Excitatory Amino Acid Agonists; Glycine; Hippocampus; In Vitro Techniques; Male; Muscarinic Agonists; Neural Inhibition; Pilocarpine; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, Glutamate; Resorcinols; Thapsigargin

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.

Topics: Animals; Boron Compounds; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum; Epilepsy; Glutamic Acid; Hippocampus; Homeostasis; Microscopy, Fluorescence; Neuronal Plasticity; Neurons; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

The present study was performed to investigate the effects of depleting intracellular Ca(2+) stores on bicuculline- or gabazine-induced epileptiform excitability. Studies were performed on monolayer rat hippocampal neuronal networks utilising a system that allowed simultaneous multiple extracellular single-unit recordings of neuronal activity. Hippocampal neuronal networks were prepared from enzymatically dissociated hippocampi from 18-day-old fetal Wistar rats. The cells were cultured in Neurobasal medium with B27 serum-free supplements directly onto the surface of planar multiple microelectrode arrays with a central recording array of 64 (4 x 16) indium-tin thin-film recording electrodes. All cells recorded at 21 days-in-vitro exhibited spontaneous discharge activity with firing rates between 0.3-30.7 Hz. gamma-aminobutyric acid (GABA) produced a concentration-dependent decrease in firing (EC(50)=9.1 microM) which could be blocked by pre-application of bicuculline methobromide (10 microM). Addition of the GABA(A)-receptor antagonists gabazine (10 microM) or bicuculline (10 microM) resulted in the rapid generation of synchronised bursting within all the cells recorded. Bicuculline exhibited heterogeneity of action on firing rate, whereas gabazine always increased firing. Pre-incubation with thapsigargin, which depletes intracellular calcium stores, resulted in a decrease in the amount of neuronal excitation produced by bicuculline, but not by gabazine, suggesting that bicuculline-induced neuronal excitation requires release of Ca(2+) from intracellular stores.

Topics: Animals; Bicuculline; Calcium; Cells, Cultured; Convulsants; Electrophysiology; Enzyme Inhibitors; Epilepsy; GABA Antagonists; Hippocampus; Immunohistochemistry; Microelectrodes; Nerve Net; Neurons; Pyridazines; Rats; Rats, Wistar; Thapsigargin

1. Previous work has suggested that presynaptic effects of adenosine may be dependent on divalent cations. The present study was undertaken to determine whether a similar requirement existed at postsynaptic sites. 2. Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum or antidromic stimulation of the alveus. In antidromic stimulation experiments, CaCl2 was omitted (calcium-free medium) or reduced to 0.24 mM (low calcium medium) and in some experiments MgSO4 was increased to 2 mM. Kynurenic acid at concentrations of 1 and 5 mM in calcium-free medium and 1 mM in low calcium medium had no effect on secondary spike size. 3. Adenosine and baclofen induced a concentration-dependent reduction in the amplitude of orthodromic potentials with maximum effects at 20 and 5 microM respectively. 4. In nominally calcium-free medium, bursts of multiple population spikes were obtained in response to antidromic stimulation. Adenosine had little effect in reducing the secondary spike amplitude. At high concentration (2 mM) an initial depression was seen which declined within 3-5 min. 5. Sensitivity to adenosine was restored in low calcium medium or by raising magnesium. Although raising the divalent cation concentration increased the inhibitory effect of adenosine, desensitization was still seen. 6. 2-Chloroadenosine (100-500 microM) and R-PIA (50 microM), which are not substrates for either the nucleoside transporters or adenosine deaminase, were inactive in the absence of calcium. S-(2-hydroxy-5 nitrobenzyl)-6-thioinosine, an adenosine uptake blocker, at a concentration 100 MicroM had no effect on secondary potential size and did not restore adenosine sensitivity in calcium-free medium.7. Thapsigargin, which discharges intracellular calcium stores, had no significant effect at 1 MicroM on the bursts of action potentials and did not change the effect of 0.5 mM adenosine in calcium-free medium.8. Unlike adenosine, baclofen concentration-dependently reduced the secondary spike size in calcium free medium and no sign of recovery was observed during maintained superfusion for up to 45 min. No cross-desensitization was seen between baclofen and adenosine.9. Applications of adenosine locally by pressure to neuronal somata or dendrites still resulted in desensitized responses in calcium-free medium.10. It is concluded that the postsynaptic sensitivity to ade

Topics: Adenosine; Animals; Baclofen; Calcium; Calcium-Transporting ATPases; Electric Stimulation; Epilepsy; Evoked Potentials; Hippocampus; Male; Purines; Pyramidal Cells; Rats; Rats, Wistar; Synapses; Terpenes; Thapsigargin