methylatropine and Epilepsy--Temporal-Lobe

To identify the modulation of Tandem of P-domains in a weak inwardly rectifying K(+) channel (TWIK)-related acid-sensitive K(+) (TASK)-2 channel expressions in epilepsy, we conducted a comparative analysis of TASK-2 channel immunoreactivity in the hippocampus of a pilocarpine-induced rat epilepsy model.. We performed and immunohistochemical study for TASK-2 and double immunofluorescent staining for TASK-2 and glial fibrillary acidic protein (GFAP) in the rat hippocampus of pilocarpine-induced epilepsy models.. In control animals, TASK-2 immunoreactivity was strongly detected in CA1-3 pyramidal layers and dentate granule cell layer. After status epilepticus (SE), TASK-2 immunoreactivity was increased in dentate granule cell layer and CA3 pyramidal cell layer, whereas its immunoreactivity was reduced in CA1 pyramidal cell layer. In addition, TASK-2 immunoreactivity is gradually increased in perivascular regions following SE. Double immunofluorescent study revealed that the enhancement of TASK-2 immunoreactivity in perivascular regions is caused by increase in the number of TASK-2 immunoreactive endfeet of perivascular astrocytes.. Our findings suggest that elevated TASK-2 immunoreactivity in neurons may contribute to rapid adaptive responses (presumably for extracellular alkalinization), which result in hyperpolarization and regulate seizure activity. In contrast, upregulated TASK-2 immunoreactivity in perivascular regions may be involved in abnormalities of blood flow regulation or brain-blood barrier impairment. These changes may contribute to acquisition of the properties of the epileptic hippocampus.

Topics: Animals; Astrocytes; Atropine Derivatives; Cerebral Ventricles; Disease Models, Animal; Epilepsy, Temporal Lobe; Glial Fibrillary Acidic Protein; Hippocampus; Image Processing, Computer-Assisted; Indoles; Male; Neurons; Pilocarpine; Potassium Channels, Tandem Pore Domain; Rats; Rats, Sprague-Dawley; Up-Regulation

Temporal lobe epilepsy is common and difficult to treat. Reduced inhibition of dentate granule cells may contribute. Basket cells are important inhibitors of granule cells. Excitatory synaptic input to basket cells and unitary IPSCs (uIPSCs) from basket cells to granule cells were evaluated in hippocampal slices from a rat model of temporal lobe epilepsy. Basket cells were identified by electrophysiological and morphological criteria. Excitatory synaptic drive to basket cells, measured by mean charge transfer and frequency of miniature EPSCs, was significantly reduced after pilocarpine-induced status epilepticus and remained low in epileptic rats, despite mossy fiber sprouting. Paired recordings revealed higher failure rates and a trend toward lower amplitude uIPSCs at basket cell-to-granule cell synapses in epileptic rats. Higher failure rates were not attributable to excessive presynaptic inhibition of GABA release by activation of muscarinic acetylcholine or GABA(B) receptors. High-frequency trains of action potentials in basket cells generated uIPSCs in granule cells to evaluate readily releasable pool (RRP) size and resupply rate of recycling vesicles. Recycling rate was similar in control and epileptic rats. However, quantal size at basket cell-to-granule cell synapses was larger and RRP size smaller in epileptic rats. Therefore, in epileptic animals, basket cells receive less excitatory synaptic drive, their pools of readily releasable vesicles are smaller, and transmission failure at basket cell-to-granule cell synapses is increased. These findings suggest dysfunction of the dentate basket cell circuit could contribute to hyperexcitability and seizures.

Topics: Analysis of Variance; Animals; Atropine Derivatives; Dentate Gyrus; Disease Models, Animal; Electric Stimulation; Epilepsy, Temporal Lobe; GABA Antagonists; In Vitro Techniques; Lysine; Male; Muscarinic Agonists; Muscarinic Antagonists; Nerve Net; Neurons; Patch-Clamp Techniques; Phosphinic Acids; Pilocarpine; Propanolamines; Rats; Rats, Sprague-Dawley; Synaptic Potentials; Synaptophysin; Time Factors

Dentate granule cell axon (mossy fiber) sprouting is a common abnormality in patients with temporal lobe epilepsy. Mossy fiber sprouting creates an aberrant positive-feedback network among granule cells that does not normally exist. Its role in epileptogenesis is unclear and controversial. If it were possible to block mossy fiber sprouting from developing after epileptogenic treatments, its potential role in the pathogenesis of epilepsy could be tested. Previous attempts to block mossy fiber sprouting have been unsuccessful. The present study targeted the mammalian target of rapamycin (mTOR) signaling pathway, which regulates cell growth and is blocked by rapamycin. Rapamycin was focally, continuously, and unilaterally infused into the dorsal hippocampus for prolonged periods beginning within hours after rats sustained pilocarpine-induced status epilepticus. Infusion for 1 month reduced aberrant Timm staining (a marker of mossy fibers) in the granule cell layer and molecular layer. Infusion for 2 months inhibited mossy fiber sprouting more. However, after rapamycin infusion ceased, aberrant Timm staining developed and approached untreated levels. When onset of infusion began after mossy fiber sprouting had developed for 2 months, rapamycin did not reverse aberrant Timm staining. These findings suggest that inhibition of the mTOR signaling pathway suppressed development of mossy fiber sprouting. However, suppression required continual treatment, and rapamycin treatment did not reverse already established axon reorganization.

Topics: Animals; Anticonvulsants; Atropine Derivatives; Axons; Dentate Gyrus; Disease Models, Animal; Epilepsy, Temporal Lobe; Immunohistochemistry; Infusions, Parenteral; Injections, Intraperitoneal; Male; Mossy Fibers, Hippocampal; Muscarinic Agonists; Neural Inhibition; Neurons; Parasympatholytics; Pilocarpine; Protein Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Staining and Labeling; Status Epilepticus; Time Factors; TOR Serine-Threonine Kinases

Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were <40% of controls, and the frequency of GABA(A) receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was <50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin- and somatostatin-immunoreactive interneuron numbers and the frequency of both fast- and slow-rising GABA(A) receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

Topics: Action Potentials; Animals; Atropine Derivatives; Cell Count; Dentate Gyrus; Disease Models, Animal; Electric Stimulation; Epilepsy, Temporal Lobe; Evoked Potentials; In Vitro Techniques; Interneurons; Male; Membrane Potentials; Neural Inhibition; Neurons; Patch-Clamp Techniques; Pilocarpine; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Sensory Thresholds; Status Epilepticus

The most common type of epilepsy in adults is temporal lobe epilepsy. After epileptogenic injuries, dentate granule cell axons (mossy fibers) sprout and form new synaptic connections. Whether this synaptic reorganization strengthens recurrent inhibitory circuits or forms a novel recurrent excitatory circuit is unresolved. We labeled individual granule cells in vivo, reconstructed sprouted mossy fibers at the EM level, and identified postsynaptic targets with GABA immunocytochemistry in the pilocarpine model of temporal lobe epilepsy. Granule cells projected an average of 1.0 and 1.1 mm of axon into the granule cell and molecular layers, respectively. Axons formed an average of one synapse every 7 microm in the granule cell layer and every 3 microm in the molecular layer. Most synapses were with spines (76 and 98% in the granule cell and molecular layers, respectively). Almost all of the synapses were with GABA-negative structures (93 and 96% in the granule cell and molecular layers, respectively). By integrating light microscopic and EM data, we estimate that sprouted mossy fibers form an average of over 500 new synapses per granule cell, but <25 of the new synapses are with GABAergic interneurons. These findings suggest that almost all of the synapses formed by mossy fibers in the granule cell and molecular layers are with other granule cells. Therefore, after epileptogenic treatments that kill hilar mossy cells, mossy fiber sprouting does not simply replace one recurrent excitatory circuit with another. Rather, it replaces a distally distributed and disynaptic excitatory feedback circuit with one that is local and monosynaptic.

Topics: Animals; Atropine Derivatives; Axons; Dendrites; Disease Models, Animal; Epilepsy, Temporal Lobe; Feedback; gamma-Aminobutyric Acid; Interneurons; Lysine; Male; Mossy Fibers, Hippocampal; Pilocarpine; Rats; Rats, Sprague-Dawley; Synapses