Introduction

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The organophosphates sarin, soman, VX, and tabun are the most powerful agents of chemical mass destruction (for review, see Reutter, 1999). These compounds, commonly referred to as nerve agents, are extremely toxic and their toxicity has long been attributed to their ability to block cholinesterases irreversibly (Taylor, 1996). The therapeutic strategy used to prevent and/or counteract the toxic effects of nerve agents has been designed primarily to recover cholinesterase activity and thereby reduce manifestation of overstimulation of nicotinic and muscarinic receptors by excessive accumulation of acetylcholine (ACh) at cholinergic synapses. However, persistence of neurological sequella despite the use of this cholinesterase-based therapy emphasizes the significance of nerve agents' effects on a wider range of targets (for review, see Moore, 1998). Thus, the actions and effects of nerve agents and their antidotes in the central nervous system (CNS) continue to be investigated extensively.

Reportedly, doses of soman that are close to or higher than its LD₅₀ cause long-lasting epileptic seizure activity that is associated with severe brain damage in rats (McLeod et al., 1984; Lallement et al., 1997; McDonough and Shih, 1997). At much lower doses, soman induces behavioral alterations that are not accompanied by gross histopathological changes in the brain of rats (Sirkka et al., 1990; Moore, 1998; Baille et al., 2001). The epileptic activity induced by high doses of soman in laboratory animals can be prevented to a great extent by pretreatment with atropine (a muscarinic receptor antagonist), pralidoxime (an oxime that reactivates cholinesterases by removing the phosphate group bound to the esteratic site of cholinesterases), and benzodiazepines and/or reversible cholinesterase inhibitors that readily cross the blood-brain barrier such as huperzine (Lennox et al., 1992; Lallement et al., 1997; Carpentier et al., 2000).

Pretreatment of rats with pyridostigmine bromide (PB), a cholinesterase inhibitor normally included in the antidotal therapy for intoxication by nerve agents, does not prevent seizure activity induced by high doses of soman in rats (Lallement et al., 1997). However, it does improve the sensorimotor performance of rats exposed to low doses of sarin and the cognitive impairment induced by an acute, subtoxic treatment of rats with the organophosphate diisopropylfluorophosphate (Stone et al., 2000; Abou-Donia et al., 2002). There have been no studies directed at investigating whether PB can prevent and/or reverse neurological alterations associated with exposure to low levels of soman.

Although the quaternary ammonium structure of PB prevents its easy access to the brain, low levels of PB have been detected by means of radioimmunoassay in the brain of rats treated with a single dose of 1 mg/kg PB intramuscularly (Miller and Verma, 1989), and CNS-related effects have been observed in laboratory animals and humans treated with therapeutic doses of PB. These effects include enhanced CNS arousal in humans (Borland et al., 1985), increased startle response in Wistar Kyoto rats (Servatius et al., 1998), and sensorimotor alterations that are accompanied by increases in acetylcholinesterase activity and binding of the selective type 2 muscarinic receptor ligand AFDX 384 in specific areas of the brain of Sprague-Dawley rats (Abou-Donia et al., 2002).

The present study was designed to determine the effects of soman and low concentrations of PB on neurotransmission in the hippocampus and to investigate possible interactions between PB and soman at the cellular level. To this end, the whole-cell mode of the patch-clamp technique was used to record spontaneous and field stimulation-evoked postsynaptic currents from neurons in the CA1 pyramidal layer of rat hippocampal slices before, during, and after their exposure to soman and/or PB. The results presented herein demonstrate that GABAergic transmission in the CA1 field of the hippocampus is inhibited by soman (1-100 nM) and potentiated by PB (100 nM) and that preexposure of hippocampal slices to 100 nM PB can effectively counteract the effects of 1 nM soman on GABAergic transmission. Evidence is also provided that the effects of soman and PB on GABAergic transmission are not related to cholinesterase inhibition. Instead, they are the result of the direct interactions of soman and PB with m2 and m3 receptors, respectively, located on GABAergic neurons/fibers synapsing onto the neurons under study. It is, therefore, concluded that, acting primarily via m3 receptors, PB can prevent some of the toxic effects arising from the interactions of soman with m2 receptors in the CNS.

	Materials and Methods

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Rat Hippocampal Slices. Hippocampal slices of 250-µm thickness were obtained from 15- to 25-day-old Sprague-Dawley rats according to the procedure described previously (Alkondon et al., 1999). The slices were kept in a holding chamber containing artificial cerebrospinal fluid (ACSF) bubbled with 95% O₂ and 5% CO₂, at room temperature. Each slice, as needed, was transferred to a recording chamber (capacity of 2 ml) and held submerged by two nylon fibers. The recording chamber was continuously perfused with bubbled ACSF, which had the following composition: 125 mM NaCl, 25 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, and 25 mM glucose (osmolarity ~340 mOsM).

Electrophysiological Recordings. By means of the whole-cell mode of the patch-clamp technique, spontaneous or field stimulation-evoked postsynaptic currents (PSCs) were recorded from neurons of the CA1 pyramidal layer of rat hippocampal slices. Test solutions were applied to the slices through a set of coplanar-parallel glass tubes (400-µm i.d.) glued together and assembled on a motor-driven system (Newport Corporation, Irvine, CA) controlled by microcomputer. The tubes were placed at a distance of approximately 100 to 150 µm from the slice, and the gravity-driven flow rate was adjusted to 1.0 ml/min. Each tube was connected to a different reservoir filled with test solution. Evoked PSCs were recorded after application of a supramaximal 20- to 60-µs electrical stimulus to afferent fibers via a bipolar electrode made of thin platinum wires (50-100 µm in diameter). The stimulus was delivered by an isolated stimulator unit (Digitimer Ltd., Garden City, England) connected to a digital-to-analog interface (TL-1 DMA; Axon Instruments, Union City, CA). The platinum electrode was positioned at the Schaffer collaterals. Possible changes in series resistance were detected by applying, online, a 5-mV hyperpolarizing pulse before the test pulse.

Data Analysis. Peak amplitude, 10 to 90% rise time, and decay-time constant of field stimulation-evoked PSCs were determined using the pClamp6 software. Spontaneously occurring currents were analyzed using the Continuous Data Recording software (Dempster, 1989). All the analyses were made on fixed 3-min recordings. Unless otherwise stated, data are presented as mean ± S.E.M. The Student's t test was used for pairwise comparison of results obtained in a test group and its respective control. In addition, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare results of repeated measures and multiple groups.

Cholinesterase Assay. Cholinesterase activity was measured by a two-phase radiotopic assay (Johnson and Russell, 1975) using 0.788 mM [³H]ACh iodide, which was sufficient to produce 100,000 cpm when totally hydrolyzed. The reaction (100 µl) was terminated by addition of 100 µl of a termination mixture (1 M monochloroacetic acid, 2 M NaCl, and 0.5 M NaOH) to which 4 ml of scintillation mixture (10% isoamyl alcohol, 0.5% diphenyloxazole, and 0.02% dimethylphenyloxazolylbenzene in toluene) was added. The hydrolyzed, acidified [³H]acetate partitioned into the organic phase and was subsequently counted. All samples were assayed in the presence of the butyrylcholinesterase inhibitor tetraisopropylpyrophosphoramide (10⁴ M). Protein assays were performed using a micro BCA kit (Pierce Chemical, Rockford, IL) according to the manufacturer instructions. Two sets of experiments were carried out. In one set, hippocampal slices were first perfused for 15 min with ACSF containing no drug, 1 nM soman, or 100 nM PB and subsequently washed three times with drug-free ACSF for 1.5 min. Each slice was transferred to a microfuge tube containing 50 µl of extraction buffer (pH 7.4; 1 M NaCl, 0.019 M NaH₂PO₄ · H₂O, 0.081 M NaHPO₄ · 7H₂O, and 1% Triton X-100) and snap frozen. Cells within the slices were lysed by five rounds of freeze-thaw, lasting approximately 5 min, and homogenates were centrifuged at 14,000 rpm at 4°C. Cholinesterase activity measured in the supernatant was normalized to the protein contents of the pellets. In the other set of experiments, hippocampal slices were first extracted as described above and cholinesterase activity was measured in the supernatants after 0-, 15-, 30-, and 60-min exposure to 100 nM PB.

Drugs and Biological Hazards. Soman (1,2,2-trimethylpropyl methylphosphonofloridate) was obtained from the U.S. Army Medical Research and Development Command. Atropine sulfate, PB, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonovaleric acid (APV), picrotoxin, and 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) methiodide were purchased from Sigma-Aldrich (St. Louis, MO). 11-[[[2-Diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AFDX-116) was purchased from Tocris Cookson (St. Louis, MO). Methyllycaconitine (MLA) citrate was a gift from Prof. M. H. Benn (Department of Chemistry, University of Calgary, Calgary, AB, Canada). Dihydro--erythroidine (DHE) hydrobromide was a gift from Merck (Rahway, NJ). A 250 mM stock solution of picrotoxin was made in dimethyl sulfoxide, and dilutions were made in the ACSF. NaOH was used to dissolve CNQX and APV (the 10 mM stock solution of CNQX had 12.5 mM NaOH and the 50 mM stock solution of APV had 0.5 M of NaOH). [³H]ACh iodide (specific activity 55 mCi/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA).

	Results

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Characteristics of EPSCs and IPSCs Evoked by Field Stimulation of Schaffer Collaterals and Recorded from Neurons in the CA1 Pyramidal Layer of Rat Hippocampal Slices. Application of a supramaximal stimulus to Schaffer collaterals in the stratum radiatum in the CA1 hippocampal field via a bipolar platinum electrode generated PSCs that could be recorded from the CA1 pyramidal neurons. The finding that these currents were blocked after perfusion of the slices with ACSF containing the GABA type A receptor antagonist picrotoxin (100 µM) and the glutamate receptor antagonists CNQX (20 µM) and APV (50 µM) indicated that they were mediated by GABA or glutamate released from field-stimulated fibers onto the neurons under study. Using these receptor antagonists, IPSCs and EPSCs were pharmacologically isolated. Evoked IPSCs were recorded from neurons in slices that were continuously perfused with ACSF containing 20 µM CNQX and 50 µM APV. On the other hand, evoked EPSCs were recorded from neurons in hippocampal slices continuously perfused with ACSF containing 100 µM picrotoxin. Under control conditions, evoked IPSCs or EPSCs recorded from a single neuron had variable amplitudes. However, up to 20 min, field stimulation-evoked IPSCs or EPSCs did not show any significant rundown (Fig. 1). In most experiments, recordings under control conditions lasted 3 to 5 min, after which time a given drug was applied to the hippocampal slice via the multibarrel perfusion system. In this system, solutions were exchanged within 20 to 25 s, as determined by the 4-aminopyridine-induced enhancement of the amplitudes of evoked PSCs.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Time-dependent changes in the amplitudes of EPSCs and IPSCs evoked by stimulation of the Schaffer collaterals and recorded from CA1 pyramidal neurons in rat hippocampal slices. Plot of the amplitudes of IPSCs and EPSCs evoked by field stimulation of the Schaffer collaterals at 0.2 Hz and recorded from CA1 pyramidal neurons versus recording time. Recordings of IPSCs were obtained from a neuron in a hippocampal slice continuously perfused with ACSF containing the glutamate receptor antagonists CNQX (20 µM) and APV (50 µM), whereas recordings of EPSCs were obtained from a neuron in another slice perfused with ACSF containing the GABAA receptor antagonist picrotoxin (100 µM). Holding potential, 60 mV.

Effects of Soman on IPSCs and EPSCs Evoked by Field Stimulation of Schaffer Collaterals and Recorded from CA1 Neurons in Hippocampal Slices. Perfusion of hippocampal slices with ACSF containing soman (0.1-100 nM) resulted in a significant reduction of the amplitudes of evoked IPSCs recorded from neurons in the CA1 pyramidal layer (Fig. 2A). The effect was concentration-dependent; it was statistically significant at 1 nM and reached its maximum at 10 nM (Fig. 2B). Inhibition of evoked IPSCs became apparent at 1 min after beginning of the perfusion of the slices with soman-containing ACSF. In addition, the effect of soman on evoked IPSCs was not fully reversed even after 10 min of washing the preparations with soman-free ACSF (Fig. 2A). The kinetics of evoked IPSCs was not significantly altered by 1 to 100 nM soman. The rise times of evoked IPSCs recorded under control conditions and in the presence of 100 nM soman were 4.54 ± 0.63 and 4.86 ± 0.23 ms, respectively (n = 3 neurons, each from a different slice). Likewise, the decay-time constants of the currents recorded in the absence and in the presence of 100 nM soman were 71.7 ± 1.43 and 66.86 ± 7.66 ms, respectively (n = 3 neurons, each from a different slice).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Soman reduces the amplitude of IPSCs evoked by field stimulation of the Schaffer collaterals and recorded from CA1 pyramidal neurons in rat hippocampal slices. A, representative recording samples of evoked IPSCs obtained 1) under control conditions; 2) in the presence of 0.1, 1, or 10 nM soman; and 3) after 10-min washing of the slices with soman-free ACSF. The neurons were exposed for 10 min to soman. All experiments were carried out in the presence of 20 µM CNQX and 50 µM APV. Membrane potential, 60 mV. B, concentration-dependent effect of soman on evoked IPSCs. Each concentration was tested on a slice that had not been previously exposed to soman. The amplitudes of events evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitude of 45 events recorded in the absence of soman was taken as 100% and used to normalize the averaged amplitude of events recorded at the same frequency for 3 min in the presence of the organophosphate. Graph and error bars represent mean and S.E.M., respectively, of results obtained from four neurons. According to the unpaired Student's t test, results are significantly different from control with *, p < 0.05 or **, p < 0.01.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   EPSCs evoked by field stimulation of the Schaffer collaterals and recorded from CA1 pyramidal neurons are not affected by soman. A, representative recording samples of evoked EPSCs obtained from neurons before (left traces) and during exposure to 1 and 50 nM soman (right traces). All experiments were carried out in the presence of 100 µM picrotoxin. Membrane potential, 60 mV. B, quantitative analysis of the effect of soman on evoked EPSCs. Each concentration was tested on a neuron that had not been previously exposed to soman. The amplitudes of events evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitude of 45 events recorded under control condition was taken as 100% and used to normalize the averaged amplitude of events recorded at the same frequency for 3 min in the presence of the drug. Each column and error bar represent mean and S.E.M., respectively, of results obtained from four neurons.

Effects of Soman on Spontaneously Occurring IPSCs and Miniature IPSCs and EPSCs Recorded from Neurons in the CA1 Pyramidal Layer of Hippocampal Slices. In the absence of the Na⁺-channel blocker tetrodotoxin and in the continuous presence of the glutamate receptor antagonists CNQX (20 µM) and APV (50 µM), spontaneously occurring IPSCs were recorded from neurons in the pyramidal layer of the CA1 field of hippocampal slices. When the Schaffer collaterals were not stimulated, 0.1 and 1 nM soman caused no significant changes in the frequency, amplitude, or kinetics of the spontaneously occurring IPSCs (Fig. 4A). Only at 100 nM did soman cause significant reduction of the peak amplitude and frequency of these events (Fig. 4A). In contrast, soman at 1 or 10 nM caused a significant reduction in the amplitudes of spontaneous IPSCs recorded from CA1 pyramidal neurons between two subsequent field stimuli applied to Schaffer collaterals (Fig. 4B). The frequency of spontaneous IPSCs recorded under the latter experimental condition was also significantly decreased by 10 nM soman (Fig. 4B).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Soman decreases the amplitude and frequency of spontaneously occurring IPSCs recorded from CA1 pyramidal neurons in rat hippocampal slices. A, quantitative analysis of the effects of soman on rise time, decay-time constant, amplitude, and frequency of spontaneous IPSCs recorded from CA1 pyramidal neurons in slices where the Schaffer collaterals had not been field stimulated. The average rise time and decay-time constant of spontaneous IPSCs recorded for 3 min from neurons before their exposure to soman were 2.5 ± 0.55 and 7.5 ± 1.44 ms, respectively. B, quantitative analysis of the effects of soman on rise time, decay-time constant, amplitude, and frequency of spontaneous IPSCs recorded from CA1 pyramidal neurons in slices where the Schaffer collaterals were stimulated at 0.2 Hz. The average rise time and decay-time constant of spontaneous IPSCs recorded for 3 min under control conditions were 2.03 ± 0.39 and 8.16 ± 1.73 ms, respectively. Values of average rise time, average decay-time constant, average amplitude, and frequency of events recorded from a neuron in the presence of a given concentration of soman are expressed as percentage of the values obtained before exposure of that neuron to soman. In the graphs, each column and error bar represent mean and S.E.M., respectively, of results obtained from five neurons. Holding potential, 60 mV. According to the unpaired Student's t test, results are significantly different from control with *, p < 0.05.

Effects of the Nonselective Muscarinic Receptor Antagonist Atropine and of Nicotinic Receptor Antagonists on Soman-Induced Inhibition of Evoked IPSCs in Hippocampal Slices. In an attempt to elucidate the mechanisms underlying the effect of soman on evoked IPSCs, the following facts were taken into account: 1) nicotinic and muscarinic receptors are known to modulate GABA release from hippocampal neurons (Pitler and Alger, 1994; Alkondon et al., 1999); 2) organophosphates, via cholinesterase inhibition, can indirectly alter the activity of muscarinic and nicotinic receptors; and 3) organophosphates can interact directly with different subtypes of nicotinic and muscarinic receptors (Albuquerque et al., 1988; Lockhart et al., 2001).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of the muscarinic receptor antagonist atropine and the nAChR antagonists MLA and DHE on soman-induced inhibition of evoked IPSCs in rat hippocampal slices. A, quantitative analysis of the effect of atropine on evoked IPSCs and on soman-induced inhibition of evoked IPSCs. Evoked IPSCs were recorded from CA1 pyramidal neurons in hippocampal slices that were exposed first to 1 nM atropine for 5 to 8 min and subsequently to the admixture of 1 nM atropine plus 1 nM soman for an additional 10 min. At the end of the experiments, the preparations were washed for 10 min with drug-free ACSF. The amplitudes of events evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitudes of events recorded in the presence of atropine or atropine plus soman and in the washing phase are expressed as percentage of the averaged amplitudes of events recorded at the same frequency for 3 min before exposure of the neurons to any drug. B, quantitative analysis of the effect of MLA and DHE on evoked IPSCs and on soman-induced inhibition of evoked IPSCs. Evoked IPSCs were recorded from CA1 pyramidal neurons in hippocampal slices that were exposed first to 50 nM MLA plus 10 µM DHE for 5 to 8 min and subsequently to the admixture of the antagonists and 1 nM soman for an additional 10 min. At the end of the experiments, the preparations were washed for 10 min with drug-free ACSF. The amplitudes of events evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitudes of events recorded in the presence of drugs and in the washing phase are expressed as percentage of the averaged amplitudes of events recorded at the same frequency for 3 min under control conditions. In the graphs, each column and error bar represent mean and S.E.M., respectively, of results obtained from four neurons. All experiments were carried out in the presence of CNQX and APV. Holding potential, 60 mV. According to the unpaired Student's t test, results are significantly different from control with *, p < 0.05.

Effects of PB on IPSCs and EPSCs Evoked by Field Stimulation of the Schaffer Collaterals and Recorded from CA1 Neurons in the Pyramidal Layer of Hippocampal Slices. In the continuous presence of CNQX and APV, perfusion of hippocampal slices with ACSF containing PB increased the amplitude of IPSCs evoked by field stimulation of Schaffer collaterals and recorded from CA1 pyramidal neurons (Fig. 6, A and C). This effect, which was apparent at 100 nM and became negligible at 1 µM PB (Fig. 6C), was completely reversed after 10 min of washing the preparations with PB-free ACSF (Fig. 6A). In contrast, perfusion of hippocampal slices with ACSF containing PB (30 nM-1 µM) resulted in no change in the amplitude of evoked EPSCs recorded from CA1 pyramidal neurons in the continuous presence of 100 µM picrotoxin (Fig. 6, B and C).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   PB increases the amplitude of IPSCs and does alter the amplitude of EPSCs evoked by field stimulation of the Schaffer collaterals and recorded from CA1 pyramidal neurons in rat hippocampal slices. A and B, sample recordings of evoked IPSCs (A) and EPSCs (B) obtained from two neurons under control conditions, in the presence of 100 nM PB and after 10 min of washing of the preparations with PB-free ACSF. The neurons were exposed to PB for 10 min. Evoked IPSCs were recorded in the presence of 20 µM CNQX and 50 µM APV, and evoked EPSCs were recorded in the presence of 100 µM picrotoxin. Holding potential, 60 mV. C, graph of PB concentrations versus the amplitudes of evoked EPSCs or IPSCs normalized to control conditions. The amplitudes of EPSCs or IPSCs evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitudes of events recorded in the presence of PB are expressed as percentage of the averaged amplitudes of events recorded at the same frequency for 3 min under control conditions. Each concentration was tested on a neuron that had not been previously exposed to PB. Each point and error bar represent mean and S.E.M., respectively, of results obtained from four neurons. According to the unpaired Student's t test, results are different from control with *, p < 0.05.

Effects of Atropine on PB-Induced Potentiation of Field Stimulation-Evoked IPSCs in the CA1 Layer of Hippocampal Slices. As aforementioned, exposure of hippocampal slices to 100 nM PB resulted in the enhancement of the amplitudes of IPSCs evoked by field stimulation of the Schaffer collaterals and recorded from neurons in the CA1 pyramidal layer of hippocampal slices. The effect reached its maximum at 30 to 60 s after beginning of the perfusion of the slices with PB-containing ACSF, remained constant for as long as the drug was present, i.e., 5 to 8 min (Fig. 7, A and B), and was blocked by subsequent exposure of the slices to ACSF containing 1 nM atropine (Fig. 7, A and C).

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Atropine blocks PB-induced potentiation of IPSCs evoked by field stimulation of the Schaffer collaterals and recorded from CA1 pyramidal neurons. A and B, amplitudes of IPSCs evoked at 0.2 Hz and recorded from CA1 pyramidal neurons under different experimental conditions. In A, recordings were obtained from a neuron in the following sequence: 1) under control condition, 2) in the presence of 100 nM PB, 3) in the presence of 100 nM PB and 1 nM atropine, and 4) during the washout with drug-free ACSF. The experimental results shown in B were obtained from another neuron subjected to the same treatment as that described in A, except that the concentration of atropine was 100 nM. C and D, quantitative analyses of the effects of 1 nM (C) and 100 nM (D) atropine on PB-induced potentiation of evoked IPSCs. The amplitudes of IPSCs evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitudes of events recorded from a given neuron in the presence of PB or atropine plus PB and during the washing phase are expressed as percentage of the averaged amplitudes of events recorded at the same frequency for 3 min under control conditions. Each column and error bar represent mean and S.E.M., respectively, of results obtained from five neurons. All experiments were carried out in the presence of the glutamate receptor antagonists 20 µM CNQX and 50 µM APV. Holding potential, 60 mV. According to the ANOVA test, peak amplitudes of IPSCs recorded in the presence of PB before exposure of the neurons to 1 nM (C) or 100 nM atropine (D) are significantly different from control with **, p < 0.01. In addition, peak amplitudes of IPSCs recorded in the presence of 100 nM PB plus 100 nM atropine and after washout of both drugs are significantly different from control with *, p < 0.05. Finally, amplitudes of events recorded during exposure of neurons to PB plus atropine and after removal of both drugs were significantly different from those recorded in the presence of PB alone with **, p < 0.01.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Atropine blocks PB-induced increases in the amplitude and frequency of spontaneous IPSCs recorded from CA1 pyramidal neurons in hippocampal slices where the Schaffer collaterals were stimulated at 0.2 Hz. A, quantitative analyses of the effects of various concentrations of PB on the amplitude and frequency of spontaneous IPSCs. The frequency and average amplitude of IPSCs recorded for 3 min in the presence of PB are expressed as percentage of the frequency and average amplitude of events recorded under control condition. Each concentration was tested on a neuron that had not been previously exposed to PB. Each point and error bar represent mean and S.E.M., respectively, of results obtained from four neurons. Results are significantly different from those obtained under control conditions; *, p < 0.05 according to the unpaired Student's t test. B, analyses of the effects of atropine on PB-induced increase in the amplitude and frequency of spontaneous IPSCs. The neurons were first exposed to 100 nM PB for 5 to 8 min and subsequently to 100 nM PB plus 1 nM atropine. After this treatment, the neurons were perfused for 10 min with ACSF containing only PB and for an additional 10 min with ACSF containing no atropine or PB. The frequency and average amplitude of IPSCs recorded for 3 min under each experimental condition are expressed as percentage of the frequency and average amplitude of events recorded under control condition. Each graph and error bar represent mean and S.E.M., respectively, of results obtained from four neurons. Results were significantly different from those obtained under control conditions; *, p < 0.05 according to the unpaired Student's t test.

Effects of PB on Spontaneously Occurring IPSCs Recorded from CA1 Pyramidal Neurons in Rat Hippocampal Slices: Blockade by Atropine. In the continuous presence of the glutamate receptor antagonists CNQX and APV, spontaneously occurring IPSCs were recorded from CA1 pyramidal neurons in slices where the Schaffer collaterals had not been subjected to field stimulation. At 1 and 30 nM, PB had no apparent effect on the amplitude or frequency of spontaneously occurring IPSCs (Fig. 8A). At 100 nM, PB caused a small, albeit significant, enhancement in the frequency and amplitude of these events. These effects became negligible as concentration of PB was increased to 1 µM (Fig. 8A). PB (100 nM)-induced increase in the frequency and amplitudes of spontaneously occurring IPSCs started at 30 s after beginning of perfusion of the hippocampal slices with PB-containing ACSF and was fully reversed after 10 min of washing the slices with PB-free ACSF. In addition, PB at all concentrations tested caused no significant change on the decay-time constants of spontaneously occurring IPSCs (data not shown).

Effects of the Muscarinic Receptor Antagonists 4-DAMP and AFDX-116 on Soman-Induced Inhibition and PB-Induced Potentiation of GABAergic Transmission in Rat Hippocampal Slices. Numerous reports have indicated that PB and soman can interact directly with m2 and m3 receptors (Silveira et al., 1990; McBride et al., 1991; Ramnarine et al., 1996; Shibata et al., 1998; Lockhart et al., 2001). Thus, the m2- and m3-preferring antagonists AFDX-116 and 4-DAMP, respectively, were used in an attempt to identify the muscarinic receptor subtype underlying the effects of PB and soman on GABAergic transmission.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Effects of AFDX-116 and 4-DAMP on soman-induced inhibition and PB-induced potentiation of evoked GABAergic transmission in hippocampal slices. A, quantitative analysis of the effects of the m3 receptor-preferring antagonist 4-DAMP and of the m2 receptor-preferring antagonist AFDX-116 on evoked IPSCs and on soman-induced inhibition of evoked IPSCs. Evoked IPSCs were recorded from CA1 pyramidal neurons in hippocampal slices that were exposed first to 100 nM 4-DAMP for 5 to 8 min and subsequently to the admixture of 100 nM 4-DAMP plus 1 nM soman for an additional 10 min. The same protocol was carried out using AFDX-116 instead of 4-DAMP. At the end of the experiments, the preparations were washed for 10 min with drug-free ACSF. The amplitudes of events evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitudes of events recorded in the presence of a muscarinic receptor antagonist or that antagonist plus soman are expressed as percentage of the averaged amplitudes of events recorded at the same frequency for 3 min before exposure of the neurons to any drug. Each graph and error bar represent mean and S.E.M., respectively, of results obtained from three to six experiments. The amplitudes of currents recorded in the presence of 4-DAMP or 4-DAMP plus soman were significantly smaller than those recorded under control conditions; *, p < 0.05 and **, p < 0.01 according to the ANOVA test. In addition, the amplitudes of events recorded in the presence of 4-DAMP plus soman were significantly different from those recorded in the presence of 4-DAMP; *, p < 0.05 according to the ANOVA test. B, quantitative analysis of the effects of the m3 receptor-preferring antagonist 4-DAMP and of the m2 receptor-preferring antagonist AFDX-116 on evoked IPSCs and on PB-induced potentiation of evoked IPSCs. The analysis is similar to that described in A. Each graph and error bar represent mean and S.E.M., respectively, of results obtained from three to six experiments. Amplitudes of events recorded in the presence of 4-DAMP, 4-DAMP plus PB, and AFDX-116 plus PB were significantly different from those recorded under control conditions; *, p < 0.05; ** p < 0.01 according to the ANOVA test.

Interactive Effects of PB and Soman on Evoked IPSCs Recorded from CA1 Neurons in the Pyramidal Layer of Rat Hippocampal Slices. The results presented herein demonstrate that whereas PB facilitates, soman inhibits GABAergic transmission between the Schaffer collaterals and CA1 neurons in the pyramidal layer of rat hippocampal slices. Reduction of the GABAergic tone in the hippocampus can underlie the proconvulsant effects of soman in animals and humans. Numerous studies have indicated that the effectiveness of PB as part of the antidotal regimen against poisoning by organophosphates is only evident when PB is administered before exposure to the toxicant. Thus, GABAergic transmission was analyzed in hippocampal slices that were exposed first to PB and subsequently to soman and in slices that were exposed first to soman and subsequently to PB. All the results described below were obtained from hippocampal slices in which the Schaffer collaterals had been subjected to field stimulation.

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Preexposure of the hippocampal slices to 100 nM PB masks the inhibitory effect of soman on evoked IPSCs. A, quantitative analyses of the effects of soman on evoked IPSCs recorded in the continuous presence of 100 or 300 nM PB. The IPSCs were recorded from hippocampal slices that were exposed first to 100 nM PB, subsequently to 1, 3, or 10 nM soman plus 100 nM PB, and finally to PB alone. In another set of experiments, IPSCs were recorded from neurons that were exposed first to 300 nM PB and subsequently to 300 nM PB plus 3 or 10 nM soman. Each perfusion lasted 5 to 8 min. The amplitudes of IPSCs evoked at a frequency of 0.2 Hz for 3 min were averaged. The averaged amplitudes of events recorded from a given neuron in the presence of the test compounds are expressed as percentage of the averaged amplitudes of events recorded at the same frequency for 3 min under control conditions. Each column and error bar represent mean and S.E.M., respectively, of results obtained from three neurons. B, comparison of the effects of 1 and 10 nM soman on evoked IPSCs recorded from slices that were not preexposed to 100 nM PB and from slices that had been preexposed for 5 to 8 min to 100 nM PB. Data were extracted from Figs. 2B and 10A to allow mutual comparison. C, inhibitory effect of soman on evoked IPSCs was unaltered when hippocampal slices were first exposed to 1 nM soman and subsequently to 1 nM soman plus 100 nM PB. Soman-induced inhibition of IPSCs remained after 10-min washing of the preparations with drug-free ACSF solution. Analyses of the results were done according to the protocol described in A. Each graph and error bar represent mean and S.E.M., respectively, of results obtained from four neurons. All experiments were carried out in the presence of 20 µM CNQX and 50 µM APV. Holding potential, 60 mV. Wherever indicated in the graphs of A, B, and C, amplitudes of events recorded in the presence of a given drug were significantly different from those of events recorded under control conditions; *, p < 0.05 and **, p < 0.01, according to the unpaired Student's t test.

View larger version (25K): [in this window] [in a new window]   Fig. 11.   Preexposure of the hippocampal slices to 100 nM PB masks the inhibitory effect of soman on spontaneous IPSCs recorded from CA1 neurons in hippocampal slices where the Schaffer collaterals were stimulated at 0.2 Hz. A, quantitative analyses of spontaneous IPSCs recorded from hippocampal slices exposed first to 100 nM PB and subsequently to 3 and 10 nM soman plus 100 nM PB and to 100 nM PB alone. B, analysis of results obtained using 300 nM in the same experimental protocol as that described in A. Each perfusion lasted 5 to 8 min. In A and B, the frequency and average amplitude of IPSCs recorded for 3 min in the presence of the test compounds are expressed as percentage of the frequency and average amplitude of events recorded under control condition. Each point and error bar represent mean and S.E.M., respectively, of results obtained from four neurons. Wherever indicated in A and B, results obtained during a given treatment were significantly different from those obtained under control conditions; *, p < 0.05 according to the unpaired Student's t test.

Cholinesterase Activity in Hippocampal Slices Exposed to Soman or PB. A radiometric assay was used to determine the extent to which cholinesterase activity was affected after exposure of hippocampal slices to 1 nM soman or 100 nM PB. Cholinesterase activity in intact slices that had been exposed for 15 min to 1 nM soman was approximately 99.5% lower than that measured in untreated slices (Table 2). On the other hand, cholinesterase activity in intact slices that had been exposed to 100 nM PB was similar to that measured in untreated slices (Table 2). When added to homogenates of hippocampal slices, 100 nM PB caused a time-dependent inhibition of cholinesterase. The cholinesterase activity in homogenates that were incubated with 100 nM PB for 15, 30, and 60 min was 68.5 ± 3.2, 56.3 ± 3.1, and 40.9 ± 1.8%, respectively, of that measured in control homogenates (mean ± S.D., n = 3 slices). The lack of inhibition of cholinesterase in intact hippocampal slices exposed for 15 min to PB is likely due to the difficult penetration of PB into the slices.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrates that the nerve agent soman and the carbamate PB at clinically relevant concentrations selectively affect GABAergic transmission between the Schaffer collaterals and CA1 pyramidal neurons in rat hippocampal slices. Although sparing glutamatergic transmission, 1 to 100 nM soman inhibits and 100 nM PB facilitates GABAergic transmission. As discussed hereafter, the effects of both agents are the result of their direct interactions with different muscarinic receptors present on GABAergic axons/terminals synapsing onto the neurons from which recordings were obtained and correlate well with their reported behavioral effects.

Mechanism of Action Underlying the Effects of Soman and PB on GABAergic Transmission in Rat Hippocampal Slices. At 1, 10, and 100 nM, soman reduced by approximately 20, 35, and 36%, respectively, the amplitude of IPSCs evoked by field stimulation of Schaffer collaterals and recorded from CA1 pyramidal neurons. Soman also decreased the amplitude and frequency of spontaneous IPSCs recorded from CA1 pyramidal neurons. In contrast, PB potentiated evoked and spontaneous GABAergic transmission in the CA1 field of hippocampal slices; these effects were evident at 100 nM and became negligible at 300 nM PB. Whereas the effects of soman on GABAergic transmission could not be reversed during washout, those of PB were promptly reversed. The finding that soman had no effect on the amplitude or kinetics of miniature IPSCs and the fact that only at 3 µM can soman or PB interact directly with GABA_A receptors (Gant et al., 1987; Swanson et al., 1997) indicate that soman-induced inhibition and PB-induced potentiation of GABAergic transmission result from presynaptic actions of the drugs.

Pretreatment, but not Post-Treatment with PB Can Mask Soman-Induced Inhibition of GABAergic Transmission. In hippocampal slices where GABAergic transmission had already been inhibited by 1 nM soman, 100 nM PB was devoid of any effect. An irreversible interaction of soman with m3 receptors could explain the ability of the organophosphate to inhibit the effect of PB. Alternatively, because there seems to be cross talk between m2 and m3 receptors at the level of second messenger (Dell'Acqua et al., 1993), it is possible that alterations induced by soman in the coupling of m2 receptors with G proteins impair signaling of the m3 receptors that subserve the effects of PB on GABAergic transmission.

Toxicological Relevance. Anxiety and hypolocomotion are common symptoms observed in animals treated with low doses of soman, and seizures are commonly observed in animals and humans exposed to high doses of soman (Moore, 1998). These symptoms correlate well with decreased GABA_A receptor activity in the CNS (Ungard et al., 2000; Trevitt et al., 2002) and can, therefore, be the result of the concentration-dependent inhibition of GABAergic transmission by soman. In addition, although the cationic structure of PB impairs its access to the brain, CNS-related symptoms have been reported in both laboratory animals and humans treated with clinically relevant doses of PB (Borland et al., 1985; Servatius et al., 1998; Abou-Donia et al., 2002). These symptoms, which include increased arousal in humans and increased startle response in rats, have also been observed under conditions of increased GABAergic activity (Fendt, 1998; Xi et al., 1999) and can thereby be the result of PB-induced facilitation of GABAergic transmission.