Transient Morphological Alterations in the Hippocampus After Pentylenetetrazole-Induced Seizures in Rats.

The relationships between seizures, neuronal death, and epilepsy remain one of the most disputed questions in translational neuroscience. Although it is broadly accepted that prolonged and repeated seizures cause neuronal death and epileptogenesis, whether brief seizures can produce a mild but similar effect is controversial. In the present work, using a rat pentylenetetrazole (PTZ) model of seizures, we evaluated how a single episode of clonic-tonic seizures affected the viability of neurons in the hippocampus, the area of the brain most vulnerable to seizures, and morphological changes in the hippocampus up to 1 week after PTZ treatment (recovery period). The main findings of the study were: (1) PTZ-induced seizures caused the transient appearance of massively shrunken, hyperbasophilic, and hyperelectrondense (dark) cells but did not lead to detectable neuronal cell loss. These dark neurons were alive, suggesting that they could cope with seizure-related dysfunction. (2) Neuronal and biochemical alterations following seizures were observed for at least 1 week. The temporal dynamics of the appearance and disappearance of dark neurons differed in different zones of the hippocampus. (3) The numbers of cells with structural and functional abnormalities in the hippocampus after PTZ-induced seizures decreased in the following order: CA1 > CA3b,c > hilus > dentate gyrus. Neurons in the CA3a subarea were most resistant to PTZ-induced seizures. These results suggest that even a single seizure episode is a potent stressor of hippocampal neurons and that it can trigger complex neuroplastic changes in the hippocampus.

Complex action of tyramine, tryptamine and histamine on native and recombinant ASICs.

Proton-gated channels of the ASIC family are widely distributed in the mammalian brain, and, according to the recent data, participate in synaptic transmission. However, ASIC-mediated currents are small, and special efforts are required to detect them. This prompts the search for endogenous ASIC ligands, which can activate or potentiate these channels. A recent finding of the potentiating action of histamine on recombinant homomeric ASIC1a has directed attention to amine-containing compounds. In the present study, we have analyzed the action of histamine, tyramine, and tryptamine on native and recombinant ASICs. None of the compounds caused potentiation of native ASICs in hippocampal interneurons. Furthermore, when applied simultaneously with channel activation, they produced voltage-dependent inhibition. Experiments on recombinant ASIC1a and ASIC2a allowed for an interpretation of these findings. Histamine and tyramine were found to be inactive on the ASIC2a, while tryptamine demonstrated weak inhibition. However, they induce both voltage-dependent inhibition of open channels and voltage-independent potentiation of closed/desensitized channels on the ASIC1a. We suggest that the presence of an ASIC2a subunit in heteromeric native ASICs prevents potentiation but not inhibition. As a result, the inhibitory action of histamine, which is masked by a strong potentiating effect on the ASIC1a homomers, becomes pronounced in experiments with native ASICs.

Status epilepticus alters hippocampal long-term synaptic potentiation in a rat lithium-pilocarpine model.

Seizure-induced memory deficits are frequent in patients with temporal lobe epilepsy. However, the neural mechanisms responsible for this memory impairment are not entirely clear. Persistent changes in synaptic efficacy, long-term potentiation (LTP), and depression are considered a cellular substrate underlying the learning and memory processes. Using a lithium-pilocarpine model to induce status epilepticus (SE) in rats, the present study investigated whether the induction of LTP was altered in hippocampal slices obtained 3 h, 1, 3, and 7 days after SE. One week after SE, LTP induction was decreased in hippocampal slices. The reduced plasticity in post-SE tissue was attributable to N-methyl-D-aspartate receptor-dependent LTP. In contrast to control tissue, ifenprodil, a GluN2B-selective antagonist, did not reduce the LTP level in post-SE tissue, suggesting that SE disturbs the functional properties of GluN2B-containing N-methyl-D-aspartate receptors. These changes in synaptic transmission may contribute toward the genesis of epilepsy and seizure-associated memory deficits.

Histamine selectively potentiates acid-sensing ion channel 1a.

Although acid-sensitive ion channels (ASICs) play an important role in brain functions, the exact mechanism of their physiological activation remain unclear. A possible answer to the intriguing question is that some presently unknown endogenous ligand(s) positively modulate ASICs and enhance their responses to physiologically significant level. In the present work we found that histamine selectively potentiates ASIC1a homomers in CHO cells. Action of histamine was particularly pronounced at modest acidifications, which cause minor response. At these conditions micromolar concentrations of histamine have provided significant potentiation of ASIC1a response. We proposed that histamine and possibly some other endogenous amines can positively modulate ASICs functions.

Determinants of action of hydrophobic amines on ASIC1a and ASIC2a.

Acid-sensing ion channels (ASICs) are involved in numerous physiological and pathological processes in the central nervous system. Development of pharmacological tools capable to inhibit or potentiate these channels is important for our knowledge about roles of ASICs in the neuronal network and can be promising for treatment of some disorders. Recently we described four hydrophobic monoamines that potentiate and inhibit ASICs depending on subunit composition of the channel and peculiarities of the drug structure. In the present work we performed structure-activity relationship analysis using derivatives of adamantane, phenylcyclohexyl and 9-aminoacridine to reveal the main determinants of action of amine-containing compounds on recombinant ASIC1a and ASIC2a homomers expressed in CHO cells. We found that the most active compounds are monocations with protonatable aminogroup. In general, potentiators and inhibitors of ASIC1a we found, but only potentiators for ASIC2a. Flat aromatic structure of the headgroup determines inhibition of ASIC1a while "V-shape" structure of the hydrophobic moiety favors potentiation of ASIC2a. Moreover, for some series of monoamines there was a correlation between action on ASIC1a and ASIC2a, the weaker ASIC1a inhibition, the stronger ASIC2a potentiation. Decay of response was accelerated by ASIC1a inhibitors as well as by potentiators. All compounds potentiating ASIC2a slowed down desensitization. Our results suggest that hydrophobic amines cause complex action on ASICs.

Changes of AMPA receptor properties in the neocortex and hippocampus following pilocarpine-induced status epilepticus in rats.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in humans. The lithium-pilocarpine model in rodents reproduces some of the main features of human TLE. Three-week-old Wistar rats were used in this study. The changes in AMPA receptor subunit composition were investigated in several brain areas, including the medial prefrontal cortex (mPFC), the temporal cortex (TC), and the dorsal (DH) and ventral hippocampus (VH) during the first week following pilocarpine-induced status epilepticus (PILO-induced SE). In the hippocampus, GluA1 and GluA2 mRNA expression slightly decreased after PILO-induced SE and returned to the initial level on the seventh day. We did not detect any significant changes in mRNA expression of the GluA1 and GluA2 subunits in the TC, whereas in the mPFC we observed a significant increase of GluA1 mRNA expression on the third day and a decrease in GluA2 mRNA expression during the entire first week. Accordingly, the GluA1/GluA2 expression ratio increased in the mPFC, and the functional properties of the pyramidal cell excitatory synapses were disturbed. Using whole-cell voltage-clamp recordings, we found that on the third day following PILO-induced SE, isolated mPFC pyramidal neurons showed an inwardly rectifying current-voltage relation of kainate-evoked currents, suggesting the presence of GluA2-lacking calcium-permeable AMPARs (CP-AMPARs). IEM-1460, a selective antagonist of CP-AMPARs, significantly reduced the amplitude of evoked EPSC in pyramidal neurons from mPFC slices on the first and third days, but not on the seventh day. The antagonist had no effects on EPSC amplitude in slices from control animals. Thus, our data demonstrate that PILO-induced SE affects subunit composition of AMPARs in different brain areas, including the mPFC. SE induces transient (up to few days) incorporation of CP-AMPARs in the excitatory synapses of mPFC pyramidal neurons, which may disrupt normal circuitry functions.

N-methyl-D-aspartate receptor channel blockers prevent pentylenetetrazole-induced convulsions and morphological changes in rat brain neurons.

Alterations in inhibitory and excitatory neurotransmission play a central role in the etiology of epilepsy, with overstimulation of glutamate receptors influencing epileptic activity and corresponding neuronal damage. N-methyl-D-aspartate (NMDA) receptors, which belong to a class of ionotropic glutamate receptors, play a primary role in this process. This study compared the anticonvulsant properties of two NMDA receptor channel blockers, memantine and 1-phenylcyclohexylamine (IEM-1921), in a pentylenetetrazole (PTZ) model of seizures in rats and investigated their potencies in preventing PTZ-induced morphological changes in the brain. The anticonvulsant properties of IEM-1921 (5 mg/kg) were more pronounced than those of memantine at the same dose. IEM-1921 and memantine decreased the duration of convulsions by 82% and 37%, respectively. Both compounds were relatively effective at preventing the tonic component of seizures but not myoclonic seizures. Memantine significantly reduced the lethality caused by PTZ-induced seizures from 42% to 11%, and all animals pretreated with IEM-1921 survived. Morphological examination of the rat brain 24 hr after administration of PTZ revealed alterations in the morphology of 20-25% of neurons in the neocortex and the hippocampus, potentially induced by excessive glutamate. The expression of the excitatory amino acid transporter 1 protein was increased in the hippocampus of the PTZ-treated rats. However, dark neurons did not express caspase-3 and were immunopositive for the neuronal nuclear antigen protein, indicating that these neurons were alive. Both NMDA antagonists prevented neuronal abnormalities in the brain. These results suggest that NMDA receptor channel blockers might be considered possible neuroprotective agents for prolonged seizures or status epilepticus leading to neuronal damage.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Influence of external magnesium ions on the NMDA receptor channel block by different types of organic cations.

The NMDA type of ionotropic glutamate receptors plays a unique role in synaptic functions because of high permeability for calcium and because of a voltage-dependent block by endogenous Mg(2+). Activity and voltage dependence of the NMDA receptor channel block by organic cations are strongly affected by competition with magnesium ions for the binding site in the channel pore. It complicates prediction of action of NMDA receptor channel blockers in vivo. In the present work we studied the NMDA receptor channel block in the presence of Mg(2+) by several organic blockers with different characteristics of voltage dependence and mechanism of action. The action of NMDA receptor channel antagonists was studied in native NMDA receptors of hippocampus CA1 pyramidal neurons isolated from rat brain slices. It was demonstrated that the IC(50) values of NMDA receptor channel blockers at -30 mV are increased 1.5-5 times compared with magnesium-free conditions. The voltage dependence of the channel block is decreased, abolished or even inversed in the presence of magnesium. Although simple competition between magnesium ion and organic channel blockers provides a general explanation of the observed effects, certain disagreements were revealed. Diversity in Mg(2+) effects on the NMDAR channel block by different organic cations reported herein likely reflects interaction of NMDAR channel blockers with additional binding site(s) and suggests that individual analysis in the presence of Mg(2+) is required for newly developed NMDAR channel blocking drugs.

Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide.

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

Common binding site for externally and internally applied AMPA receptor channel blockers.

Adamantane derivative IEM-1676 (Ad-N(+)H(2)-(CH(2))(5)-N(+)Me(3)) causes open-channel block of Ca(2+)-permeable AMPA receptors when applied externally, but internal application results in both closed- and open-channel block. The relationships between blocking action of externally and internally applied IEM-1676 were studied using patch clamp technique. Extracellular action of IEM-1676 was decreased by its intracellular application, thus suggesting that the binding sites of the externally and internally applied drug coincide or at least overlap significantly. We demonstrated that internal closed-channel block is voltage-dependent and occurs at the region where the externally applied drug is trapped. We conclude that the selectivity filter of the closed AMPA receptor channel is not occluded and remains permeable for organic cations.

Blockade of NMDA receptor channels by 9-aminoacridine and its derivatives.

9-Aminoacridine is known as "foot-in-the-door" NMDA receptor channel blocker because its binding prevents channel closure. Structural determinants of this mechanism of block were studied using a series of 9-aminoacridine derivatives. Experiments were performed on native NMDA receptors of hippocampal pyramidal neurons, isolated from rat brain slices. The use-dependence of block and kinetics of recovery from block were used to characterize mechanism of block produced by the compounds. Modifications, which preserve the flat structure of the tricyclic 9-aminoacridine moiety, affect blocking activity and kinetics but not the foot-in-the-door mechanism. On the contrary, disruption of the flat structure changes the mechanism of block to trapping. It is concluded that flat aromatic structure is one of the critical determinants of the action mechanism of 9-aminoacridine.

Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneurons.

The inhibitory action of non-steroid anti-inflammatory drugs was investigated on acid-sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton-induced currents in hippocampal interneurons (IC(50) were 622 +/- 34 muM and 3.42 +/- 0.50 mM, respectively). This non-competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 muM were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton-evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage-independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady-state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification.

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium.

The effects of calcium and strontium on the quantal content of nerve-evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2-0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca(2+)](o) was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca(2+)](o). Hence, extracellular calcium affected mainly the late, 'asynchronous', portion of phasic release. The average quantal content grew exponentially from 0.09 +/- 0.01 to 1.04 +/- 0.07 with the increase in [Ca(2+)](o) without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2-1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) (25 micromol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 micromol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms.

Cholinergic regulation of the evoked quantal release at frog neuromuscular junction.

The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 x 10(-4) M), carbachol (CCh, 1 x 10(-5) M) or nicotine (5 x 10(-6) M) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90-120 microm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 x 10(-7) M (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content m(o) (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m(o) and dispersed the release (ACh, CCh and nicotine). The effects on m(o) were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.

Modeling of the pore domain of the GLUR1 channel: homology with K+ channel and binding of channel blockers.

Molecular models of the M2 segments of the GluR1 channel have been elaborated using a molecular mechanics approach. The models are based on the homology between pore-lining segments of AMPA receptor channels and the KcsA K+ channel and on cyclic H bonds at the Q/R site of the AMPA receptor channel. The N-terminal region of an M2 segment of the channel is assumed, like that of the K+ channel, to adopt a helical conformation. Due to a deletion, the C-terminal end of the M2 segment of the AMPA receptor is more stretched than that of the K+ channel. As a result, only a single oxygen ring may be exposed to the AMPA receptor channel pore. Data on the block of AMPA receptor channels by dicationic adamantane derivatives have been used to select the most relevant model. The model with the oxygen of a Gly residue (position +2 from the Q/R site) exposed to the pore best fits the experimental data. This model also fits experimental data for another class of AMPA receptor antagonists, the polyamine amides. According to the model, the side-chains of the C-terminal residues are involved in intra-receptor interactions that stabilize the structure of the channel rather than in interactions with ions in the pore.