Neuroprotective efficacy of caramiphen against soman and mechanisms of its action.

BACKGROUND AND PURPOSE: Caramiphen is a muscarinic antagonist with potent anticonvulsant properties. Here, we investigated the efficacy of caramiphen against behavioural seizures and neuropathology induced by the nerve agent soman, and revealed two mechanisms that may underlie the anticonvulsant efficacy of caramiphen. EXPERIMENTAL APPROACH: Rats were given caramiphen at 30 or 60 min after treatment with soman. Neuronal loss in the basolateral amygdala (BLA) and neuronal degeneration in the amygdala, hippocampus, piriform cortex, entorhinal cortex and neocortex, were investigated 24 h after soman, using design-based stereology and FluoroJade-C staining. The effects of caramiphen on NMDA-, AMPA- and GABA-evoked currents were studied in the BLA region of in vitro brain slices from un-treated rats, using whole-cell recordings. KEY RESULTS: Caramiphen given either 30 min or 60 min after soman, suppressed behavioural seizures within 10 min, but required 1∼4.5 h for complete cessation of seizures. Neuronal loss and degeneration were significantly reduced in the caramiphen-treated, soman-exposed rats. Postsynaptic currents evoked by puff-application of NMDA on BLA principal cells were reduced by caramiphen in a dose-dependent manner (100 µM, 300 µM and 1 mM), while GABA-evoked currents were facilitated by 100 µM and 300 µM, but depressed by 1 mM caramiphen. AMPA-evoked currents were not affected by caramiphen. CONCLUSIONS AND IMPLICATIONS: Caramiphen offered partial protection against soman-induced seizures and neuropathology, even when given 60 min after soman. NMDA receptor antagonism and facilitation of GABAergic inhibition in the BLA may play a key role in the anticonvulsive and neuroprotective properties of caramiphen.

Soman induces ictogenesis in the amygdala and interictal activity in the hippocampus that are blocked by a GluR5 kainate receptor antagonist in vitro.

Exposure to organophosphorus nerve agents induces brain seizures, which can cause profound brain damage resulting in death or long-term cognitive deficits. The amygdala and the hippocampus are two of the most seizure-prone brain structures, but their relative contribution to the generation of seizures after nerve agent exposure is unclear. Here, we report that application of 1 muM soman for 30 min, in rat coronal brain slices containing both the hippocampus and the amygdala, produces prolonged synchronous neuronal discharges (10-40 s duration, 1.5-5 min interval of occurrence) resembling ictal activity in the basolateral nucleus of the amygdala (BLA), but only interictal-like activity ("spikes" of 100-250 ms duration; 2-5 s interval) in the pyramidal cell layer of the CA1 hippocampal area. BLA ictal- and CA1 interictal-like activity were synaptically driven, as they were blocked by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. As the expression of the GluR5 subunit of kainate receptors is high in the amygdala, and kainate receptors containing this subunit (GluR5KRs) play an important role in the regulation of neuronal excitability in both the amygdala and the hippocampus, we tested the efficacy of a GluR5KR antagonist against the epileptiform activity induced by soman. The GluR5KR antagonist UBP302 reduced the amplitude of the hippocampal interictal-like spikes, and eliminated the seizure-like discharges in the BLA, or reduced their duration and frequency, with no significant effect on the evoked field potentials. This is the first study reporting in vitro ictal-like activity in response to a nerve agent. Our findings, along with previous literature, suggest that the amygdala may play a more important role than the hippocampus in the generation of seizures following soman exposure, and provide the first evidence that GluR5KR antagonists may be an effective treatment against nerve agent-induced seizures.

Peptides that mimic the carboxy-terminal domain of SNAP-25 block acetylcholine release at an Aplysia synapse.

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release, presumably by cleaving SNAP-25, a protein involved in docking of synaptic vesicles with the presynaptic plasma membrane. Three excitation-secretion uncoupling peptides (ESUPs), which mimic the carboxy-terminal domain of SNAP-25 and span or adjoin the cleavage sites for BoNT/A and BoNT/E, also inhibit transmitter release from permeabilized bovine chromaffin cells. In this study, these peptides were tested for effects on acetylcholine (ACh) release at an identified cholinergic synapse in isolated buccal ganglia of Aplysia californica. The presynaptic neuron was stimulated electrically to elicit action potentials. The postsynaptic neuron was voltage-clamped, and evoked inhibitory postsynaptic currents (IPSCs) were recorded. The ESUPs were pressure-injected into the presynaptic neuron, and their effects on the amplitude of the IPSCs were studied. Acetylcholine release from presynaptic cells, as measured by IPSC amplitudes, was gradually inhibited by the ESUPs. All three peptides caused ca. 40% reduction in IPSC amplitude in 2 h. Random-sequence peptides of the same amino acid composition had no effect. Injection of BoNT/E, in contrast, caused ca. 50% reduction in IPSC amplitude in 30 min and almost complete inhibition in 2 h. These results are the first demonstration that ESUPs block neuronal cholinergic synaptic transmission. They are consistent with the concept that ESUPs compete with the intact SNAP-25 for binding with other fusion proteins, thus inhibiting stimulus-evoked exocytosis of neurotransmitter.

The 26-mer peptide released from SNAP-25 cleavage by botulinum neurotoxin E inhibits vesicle docking.

Botulinum neurotoxin E (BoNT E) cleaves SNAP-25 at the C-terminal domain releasing a 26-mer peptide. This peptide product may act as an excitation-secretion uncoupling peptide (ESUP) to inhibit vesicle fusion and thus contribute to the efficacy of BoNT E in disabling neurosecretion. We have addressed this question using a synthetic 26-mer peptide which mimics the amino acid sequence of the naturally released peptide, and is hereafter denoted as ESUP E. This synthetic peptide is a potent inhibitor of Ca2+-evoked exocytosis in permeabilized chromaffin cells and reduces neurotransmitter release from identified cholinergic synapses in in vitro buccal ganglia of Aplysia californica. In chromaffin cells, both ESUP E and BoNT E abrogate the slow component of secretion without affecting the fast, Ca2+-mediated fusion event. Analysis of immunoprecipitates of the synaptic ternary complex involving SNAP-25, VAMP and syntaxin demonstrates that ESUP E interferes with the assembly of the docking complex. Thus, the efficacy of BoNTs as inhibitors of neurosecretion may arise from the synergistic action of cleaving the substrate and releasing peptide products that disable the fusion process by blocking specific steps of the exocytotic cascade.

Suppression of drug-induced epileptiform discharges by cyclic AMP in rat hippocampus.

The effect of cyclic adenosine 3',5'-monophosphate (cAMP) on epileptiform activity in rat hippocampal slices was investigated. Bath-applied cAMP reversibly decreased the frequency of extracellularly recorded discharges in the CA3 subfield induced by bethanechol- or theophylline-containing solutions. Because cAMP was presumed to be relatively membrane impermeant, we developed and tested the hypothesis that this cAMP-mediated effect occurred extracellularly through the catabolic conversion of cAMP to 5'-AMP and, in turn, to adenosine, a known inhibitory neuromodulator. Three predictions derived from this catabolic hypothesis were tested. First, blockers of the enzymes involved were predicted to antagonize this effect of cAMP. In contrast, the coapplication of a cAMP-phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or a 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate (AMP-CP), enhanced the cAMP-induced suppressive effect. Second, the nonhydrolyzable cAMP analogs, dibutyryl- and 8-bromo-cAMP, were predicted to be ineffective. Low concentrations (5-40 microM) of these two derivatives, however, also suppressed bethanechol-induced discharges, while, at a higher concentration (100 microM), both analogs increased discharge frequencies. Third, enzymatic catabolism of adenosine was predicted to antagonize cAMP's effect, but coapplying adenosine deaminase (10 U/mL) did not diminish this action. Because these data did not support the catabolic hypothesis, other, as yet undefined, mechanisms must be responsible for the discharge-suppressant effect of cAMP.

Anticonvulsant effects of memantine and MK-801 in guinea pig hippocampal slices.

The anticonvulsant properties of memantine (Mem) were compared to those of MK-801. Extracellular field recordings were obtained from area CA1 of guinea pig hippocampal slices in a total submersion chamber at 32 degrees C in normal oxygenated artificial cerebrospinal fluid (ACSF). Evoked responses were elicited by 0.07 Hz stimulation of the Schaffer collateral and commissural fibers. Bath perfusion of slices with Mg(2+)-free ACSF and N-methyl-D-aspartate (NMDA)-containing ACSF induced epileptiform afterdischarges following evoked responses. Pretreatment of slices by bath application of 100 microM Mem for 18-20 min prevented epileptiform afterdischarges under both convulsant conditions. Perfusion with 100 microM Mem alone for up to 50 min had no discernible effect on evoked responses. MK-801 was as effective at < or = 10 microM and required application for over 15 min to suppress afterdischarges completely. Both Mem and MK-801 suppressed epileptiform activity when applied after such activity was induced by NMDA or MG(2+)-free ACSF. The EC50 of Mem was 16.6 microM and that of MK-801 was 0.19 microM for blocking NMDA-induced evoked response suppression. Thus, in the guinea pig hippocampal slice preparation, Mem appeared to have anticonvulsant properties qualitatively similar to those of MK-801, but was 10-100 fold less potent.

Brevetoxin depresses synaptic transmission in guinea pig hippocampal slices.

Extracellular recordings were obtained from area CA1 of guinea pig hippocampal slices. PbTx-3, a brevetoxin fraction isolated from the red tide dinoflagellate Ptychodiscus brevis, was applied by bath perfusion. The toxin produced a concentration-dependent depression of the orthodromically evoked population spike with an EC50 of 37.5 nM. Brevetoxin concentrations below 10 nM were without effect, and concentrations above 100 nM led to total inhibition of evoked responses. PbTx-3 did not produce spontaneous synchronous discharges but did induce afterdischarges following evoked responses in about 50% of the slices tested, particularly at concentrations between 10 nM and 100 nM. Orthodromically evoked responses were more sensitive to PbTx-3 than were those elicited by antidromic stimulation. High-Ca2+ solution, 4-aminopyridine, and tetraethylammonium failed to antagonize either orthodromic or antidromic effects of the toxin. Although the precise mechanism by which PbTx-3 depresses evoked responses is not certain, depolarization of the presynaptic nerve terminals leading to failure of transmitter release could explain the toxin's actions. This is the first report of the effects of brevetoxin applied directly to central nervous system tissue.

Paraoxon block of chloride conductance in cell R2 of Aplysia californica.

In the presence of paraoxon, the amplitudes of chloride currents activated by acetylcholine (ACh) or gamma-aminobutyric acid (GABA) were reduced in cell R2 of Aplysia californica. IC50 values were 12 and 9.7 microM for ACh and GABA responses, respectively. Paraoxon did not affect resting membrane potential, input resistance, or chloride reversal potential. Both the slopes and maxima of ACh and GABA concentration-response curves were reduced by paraoxon, suggesting that paraoxon antagonism of these responses is not competitive. The antagonism of ACh and GABA responses by paraoxon was not related to inhibition of acetylcholinesterase.

Effects of non-opioid antitussives on epileptiform activity and NMDA responses in hippocampal and olfactory cortex slices.

Three commonly used antitussive compounds were tested for their ability to block epileptiform activity recorded extracellularly from hippocampal and olfactory cortex slices maintained in vitro. Antitussives were bath-applied to brain slices either before or after epileptiform activity was induced. Dextromethorphan (DM) prevented electrically evoked epileptiform afterdischarges and arrested spontaneous bursting induced by exposure to added NMDA or to Mg2(+)-free medium. In contrast, caramiphen (CM) and carbetapentane (CB) were effective against epileptiform activity induced by Mg2(+)-free medium, but not by NMDA. Atropine was not effective in blocking epileptiform activity at concentrations 10 times the effective concentration of CM, which has known cholinolytic activity. Our results suggest that all these antitussives exert their anticonvulsant action at the DM binding site. Neither cholinolytic activity nor antagonism of the NMDA receptor-channel complex appears to be necessary for antitussives to prevent or arrest epileptiform activity. DM appears to have a separate NMDA-antagonist property in addition to its actions at the DM site. Our neurophysiological evidence supports the hypothesis that these antitussives have anticonvulsant properties independent of any action at the NMDA receptor-channel complex.