Changes in mouse brain metabolism following a convulsive dose of soman: a proton HRMAS NMR study.

Soman, an irreversible organophosphorus cholinesterase inhibitor, induces status epilepticus and, in sensitive brain areas, seizure-related brain damage (e.g. brain edema and neuronal loss). The brain metabolic disturbances associated with these events are ill known. In the present study, we thus evaluated these changes in a murine model of soman-induced status epilepticus up to 7 days after intoxication. Mice, protected by HI-6 and atropine methyl nitrate, were poisoned with soman (172 microg/kg) and then sacrificed at set time points, from 1 h to 7 days. Brain biopsies from the piriform cortex (Pir) and cerebellum (Cer) were analyzed by 1H HRMAS NMR spectroscopy. Spectra were then analyzed using both a supervised multivariate analysis and the QUEST procedure of jMRUI for the quantification of 17 metabolites. The multivariate analysis clearly showed the metabolic differences between a damaged structure (Pir) and a structure with less prominent changes (cerebellum) and helped to globally assess the time course of metabolic changes. Analysis of the individual metabolites showed that the major changes took place in the piriform cortex but that cerebellum was not change-free. The most prominent changes in the former were an early (1-4 h) increase in alanine and acetate, a delayed increase in lactate, glycerophosphocholine and glutamine as well as a delayed decrease in myo-inositol and N-acetylaspartate. A week after poisoning, some metabolic disturbances were still present. Further research will be necessary to clarify what could be the involvement of these metabolites in physiological processes and how they might become useful surrogate markers of brain damage and repair.

Intrahippocampal cholinesterase inhibition induces epileptogenesis in mice without evidence of neurodegenerative events.

The mechanisms of epileptogenesis remain largely unknown and are probably diverse. The aim of this study was to investigate the role of focal cholinergic imbalance in epileptogenesis. To address this question, we monitored electroencephalogram (EEG) activity up to 12 weeks after the injection of a potent cholinesterase (ChE) inhibitor (soman) at different doses (0.53, 0.75, 1, 2, 2.8, 4 and 11 nmol) into the right dorsal hippocampus of C57BL/6 mice. Different parameters were used to choose the dose for a focal model of epileptogenesis (mainly electrographic patterns and peripheral ChE inhibition). The pattern of neuronal activation was studied by Fos immunohistochemistry (IHC). Brain damage was evaluated by hemalun-phloxin, neuronal nuclei antigen IHC and silver staining. Glial fibrillary acidic protein IHC was used to evaluate astroglial reaction. Finally, long-term behavioral consequences were characterized. At the highest dose (11 nmol), soman quickly evoked severe signs, including initial seizures and promoted epileptogenesis in the absence of tissue damage. With lower doses, late-onset seizures were evidenced, after 1-4 weeks depending on the dose, despite the absence of initial overt seizures and of brain damage. Only a weak astroglial reaction was observed. Following injection of 1 nmol, Fos changes were first evidenced in the ipsilateral hippocampus and then spread to extrahippocampal areas. A selective deficit in contextual fear conditioning was also evidenced two months after injection. Our data show that focal hypercholinergy may be a sufficient initial event to promote epilepsy and that major brain tissue changes (cellular damage, edema, neuroinflammation) are not necessary conditions.

Quantitation with QUEST of brain HRMAS-NMR signals: application to metabolic disorders in experimental epileptic seizures.

Quantitation of High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) signals enables establishing reference metabolite profiles of ex vivo tissues. Signals are often contaminated by a background signal originating mainly from macromolecules and lipids and by residual water which hampers proper quantitation. We show that automatic quantitation of HRMAS signals, even in the presence of a background, can be achieved by the semi-parametric algorithm QUEST based on prior knowledge of a metabolite basis-set. The latter was quantum-mechanically simulated with NMR-SCOPE and requires accurate spin parameters. The region of interest of spectra is a small part of the full spectral bandwidth. Reducing the computation time inherent to the large number of data-points is possible by using ER-Filter in a preprocessing step. Through Monte-Carlo studies, we analyze the performances of quantitation without and with ER-Filtering. Applications of QUEST to quantitation of 1H ex vivo HRMAS-NMR data of mouse brains after intoxication with soman, are demonstrated. Metabolic profiles obtained during status epilepticus and later when neuronal lesions are installed, are established. Acetate, Alanine, Choline and gamma-amino-butyric acid concentrations increase in the piriform cortex during the initial status epilepticus, when seizures are maximum; Lactate and Glutamine concentrations increase while myo-Inositol and N-acetylaspartate concentrations decrease when neuronal lesions are clearly installed.

[Which neurophysiologic effects at low level 2.45 GHz RF exposure?]. / Quels effets neurophysiologiques pour un champ électromagnétique de faible puissance à 2,45 GHz?

The LS electromagnetic band (1-4 GHz) is widely used both in domestic and industrial domains. Several studies suggested that the biological systems would exhibit a specific sensitivity to the 2.45 GHz microwaves (water resonance frequency). Potential human health hazards and especially a disruption of the cholinergic system have been reported, due to exposure to microwaves even at low power density. This work presents a multiparametric study of freely moving rat where neurophysiology was investigated during 70 hours using neurochemical (microdialysis technique), electrophysiological, behavioral (vigilance stages quantification) and thermophysiological approaches. The rats were exposed 24 hours to a 2.45 GHz pulsed electromagnetic field at low power density. In this exposure conditions, no significant effect have been reported.

Neurophysiologic effects at low level 1.8 GHz radiofrequency field exposure: a multiparametric approach on freely moving rats.

Deleterious effects on healthcare and particularly disruption of the cholinergic system have been reported after exposure to radiofrequency field at low power density. This work presents a 72 hours multiparametric study, where cholinergic system was investigated using a neurochemical, electrophysiological and physiological approaches. Free moving rats were exposed 24 hours to RF GSM signal at 1.8 GHz at low power density (1.2 and 9 W/m(2)). Acetylcholine (ACh) release in the hippocampus was simultaneously monitored using the microdialysis technique, electroencephalogram (EEG), electromyogram (EMG) and subcutaneous temperature. A spectral analysis of EEG was also performed and sleep stages were determined. After experimental time, the animals were sacrificed and a NMR study was performed on lipid brain extract. No significant parameters modification was observed under RF exposure. The only significant difference was the lack of increase in time spent in REM sleep, the third day, for the 1.2 W/m(2) group. This observation appeared difficult to explain and could not be reasonably related with RF exposure. Similarly, the NMR study also failed to show any effect of RF.

Hydrolytic properties of per (3,6-anhydro, 2-O-carboxymethyl) alpha cyclodextrin complexes of Ce (III) and Eu (III): application to soman (GD) degradation.

Per (3,6-anhydro-2-O-carboxymethyle) alpha-cyclodextrin ([ACX]) is a polydentate analog of EDTA, a well-known cation chelating reagent. ACX exhibits strong affinities in vitro for uranyl, cobalt and also for lanthanids such as Europium and Cerium. The hydrolytic activities of ACX-Eu and ACX-Ce complex were directly tested on an organophosphorous compound: the neurotoxic Soman (GD), an inhibitor of acetylcholinesterase (ACHE from rat brain). It was found a three fold reduction of soman activity when measured in the presence of Ce-ACX complex. Conversely, Eu-ACX effect did not result in soman inhibition variation under physiological conditions. It is suggested that, considering usual organometallic complex of cyclodextrin, such direct complexes would be of interest in the design of pseudo-enzyme systems for phosphoester hydrolysis.

Effects of exposure to low level radiofrequency fields on acetylcholine release in hippocampus of freely moving rats.

Some central cholinergic effects have been reported in animals after acute exposure to radiofrequency electromagnetic field at low intensity. We studied acetylcholine (ACh) release in the brain of freely moving rats exposed for 1 h during the day to a 2.45 GHz continuous wave radiofrequency field (RF) (2 or 4 mW/cm(2)) or exposed for 1 or 14 h during the night to a 800 MHz field modulated at 32 Hz (AM 200 mW/cm(2)). Measurements were performed by microdialysis using a membrane implanted through the upper CA1 region of the hippocampus. After irradiation with the 2.45 GHz RF, rats exposed at 2 mW/cm(2) did not show a significant modification of Ach release, whereas those exposed at 4 mW/cm(2) showed a significant 40% decrease in mean ACh release from hippocampus. This decrease was maximal at 5 h post exposure. Exposure to the 800 MHz RF for 1 h did not cause any significant effect, but exposure for 14 hrs induced a significant 43% decrease in ACh release during the period 11 p.m.-4 a.m. compared to control rats. In the control group we observed an increase of ACh release at the beginning of the night, which was linked to the waking period of rats. This normal increase was disturbed in rats exposed overnight to the 800 MHz RF. This work indicates that neurochemical modification of the hippocampal cholinergic system can be observed during and after an exposure to low intensity RF.

Effects of Huperzine used as pre-treatment against soman-induced seizures.

Huperzine A (HUP), an alkaloid isolated from the Chinese club moss, Huperzia serrata is a reversible inhibitor of cholinesterases which crosses the blood-brain barrier and shows high specificity for acetylcholinesterase (AChE) and a prolonged biological half-life. We tested, in vivo, its efficiency in protecting cortical AChE from soman inhibition and preventing subsequent seizures. The release of acetylcholine (ACh) was also followed in the cortex of freely moving rats using microdialysis techniques. We previously found that soman-induced seizures occurred in rodents only when the cortical AChE inhibition was over 65% and when the increase of ACh level was over 200 times the baseline level. This was verified in the present study in control animals intoxicated by 1 LD50 of soman (90 microg/kg). Using the same dose of soman in rats pre-treated with 500 microg/kg of HUP, we observed that 93% of the animals survived and none of them had seizures. This dose of HUP reduced AChE inhibition to 54% and increase of ACh level to 230 times baseline value. HUP thus appears as a promising compound to protect subjects against organophosphorus intoxication.

Triggering of soman-induced seizures in rats: multiparametric analysis with special correlation between enzymatic, neurochemical and electrophysiological data.

Soman, an anticholinesterasic neurotoxic drug, induces epileptic seizures during severe intoxication. The neuropathological lesions then observed are linked to the appearance of these seizures, but their trigger conditions still remain unknown and a great variability between animals is observed. We have developed a technique allowing, in freely moving rats, the in vivo determination of three sets of neurophysiological data, before and during a soman intoxication. For the same rat, we associated cortical acetylcholinesterase (AChE) activity by microdialysis with both the assay of extracellular acetylcholine (ACh) concentrations and electroencephalographic (EEG) recording and power spectrum analysis (gamma band). Data have been analyzed to define the critical parameters which lead to the epileptic fit. Although we found thresholds for seizure occurrence, AChE inhibition having to be over 65% and ACh over 200-fold the baseline, these two criteria are not sufficient to predict the appearance of seizures. Only animals with no increase of energy in the gamma band early after soman poisoning will then exhibit an epileptic fit. Gamma band energy is modulated by noradrenergic activity and might be related to the sympathetic response to stress. So we can hypothesize, that the variation of energy in gamma band after intoxication, which might be related to stress adaptation strategy, may determine whether or not the animal will exhibit an epileptic fit.

Evidence for homeostatic adjustments of rat somatosensory cortical neurons to changes in extracellular acetylcholine concentrations produced by iontophoretic administration of acetylcholine and by systemic diisopropylfluorophosphate treatment.

We describe the responses of single units in the awake (24 cells) or urethane-anesthetized (37 cells) rat somatosensory cortex during repeated iontophoretic pulses (1.0 s, 85 nA) of acetylcholine, both before and after systemic treatment with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (i.p., 0.3-0.5 LD50). The time-course of the response to acetylcholine pulses differed among cortical neurons but was characteristic for a given cell. Different time-courses included monophasic excitatory or inhibitory responses, biphasic (excitatory-inhibitory, inhibitory-excitatory, excitatory-excitatory, and inhibitory-inhibitory), and triphasic (excitatory-excitatory-inhibitory, inhibitory-inhibitory-excitatory, and inhibitory-excitatory-inhibitory) responses. Although the sign and time-course of the individual responses remained consistent, their magnitude fluctuated across time; most cells exhibited either an initial increase or decrease in response magnitude followed by oscillations in magnitude that diminished with time, gradually approaching the original size. The time-course of the characteristic response to an acetylcholine pulse appeared to determine direction and rate of change in response magnitude with successive pulses of acetylcholine. Diisopropylfluorophosphate treatment, given 1 h after beginning repeated acetylcholine pulses, often resulted in a gradual increase in spontaneous activity to a slightly higher but stable level. Superimposed on this change in background activity, the oscillations in the response amplitude reappeared and then subsided in a pattern similar to the decay seen prior to diisopropylfluorophosphate treatment. Our results suggest that dynamic, homeostatic mechanisms control neuronal excitability by adjusting the balance between excitatory and inhibitory influences within the cortical circuitry and that these mechanisms are engaged by prolonged increases in extracellular acetylcholine levels caused by repeated pulses of acetylcholine and by acetylcholinesterase inhibition. However, this ability of neurons in the cortical neuronal network to rapidly adjust to changes in extracellular levels of acetylcholine questions the potential efficacy of therapeutic treatments designed to increase ambient levels of acetylcholine as a treatment for Alzheimer's disease or to enhance mechanisms of learning and memory.

Implication of gamma band in soman-induced seizures.

Soman, an anticholinesterasic neurotoxic drug, induces epileptic seizures during severe intoxication. Their trigger conditions still remain unknown and a great variability between animals is observed. The butterfly model in the catastrophe theory has been used to explain these triggering conditions. We have developed a technique allowing, in freely moving rats, the "in vivo" determination of three sets of neurophysiological data, followed before and during a soman intoxication. For the same rat, we associated cortical acetylcholinesterase (AChE) activity by microdialysis with both the assay of extracellular acetylcholine (ACh) concentrations and electroencephalographic (EEG) recording and power spectrum analysis (gamma band). Data have been analysed to define the critical parameters which lead to the epileptic fit. Although we found thresholds for seizure occurrence, AChE inhibition having to be over 65% and ACh over 200 fold the baseline, these two criteria are not sufficient to predict the appearance of seizures. Only animals with no increase of energy in the gamma band early after soman poisoning will then exhibit an epileptic fit. The butterfly model provides an original interpretation of these results and gives a particular role of the energy in gamma band as a survival attractor.

Simultaneous in vivo determination of acetylcholinesterase activity and acetylcholine release in the cortex of waking rat by microdialysis. Effects of VX.

We have designed a microdialysis technique to measure acetylcholinesterase (AChE) activity in the cortex of freely moving rats while simultaneously measuring the release of acetylcholine (ACh). Our approach was validated using ethyl S-2-di-isopropylaminoethyl-phosphonothiolate (VX), an irreversible inhibitor of AChE and comparing inhibition measured by this 'in vivo' method with traditional post-mortem assays of AChE activity 120 min after an intraventricular injection of VX. Maximum inhibition of AChE occurred 30 min after injection and was followed by a slow recovery. ACh release reached its maximum 60 min after treatment and then decreased towards normal levels. This method offers a new way to develop medications against poisoning with anticholinesterasic neurotoxic and allows the evaluation of the effects of cholinergic drugs for the treatment of Alzheimer's disease.

Acetylcholine release from frontal cortex in the waking rat measured by microdialysis without acetylcholinesterase inhibitors: effects of diisopropylfluorophosphate.

Acetylcholine (ACh) release was measured in frontal cortex of awake quietly resting rats by microdialysis without using cholinesterase blockers in the perfusate. Resting release was 16.61 +/- 2.05 fmol/h (+/- S.E.M., n = 18). Injection of sublethal doses of the acetylcholinesterase blocker, diisopropylfluorophosphate produced dose-dependent increases in ACh release, reaching 79.9 fmol/h with a dose of 0.7-times the LD50. Although this irreversible inactivation of acetylcholinesterase increased ACh recovery to more than 700% of control values, levels of ACh in the perfusate never reached those seen in physostigmine-treated animals. The relationship between the amount of acetylcholinesterase inactivation and the quantity of ACh in the perfusate suggests that the extracellular ACh concentrations are controlled by simple enzyme kinetics. Within 2 h after enzyme inactivation, extracellular choline levels fell significantly, suggesting that ACh degradation by acetylcholinesterase plays an important role in regulating the amount of choline in the extracellular space.

Stimulated release of acetylcholinesterase in rat striatum revealed by in vivo microspectrophotometry.

The microspectrophotometric technique allows a direct in vivo measurement of brain extracellular acetylcholinesterase. An optical probe associated with electrodes for stimulation was implanted in striatum of anaesthetized rats to determine the effects of neuronal excitation on the acetylcholinesterase activity. Electrical stimulations induced a reversible increase in acetylcholinesterase activity of about 30 to 50%, with a recovery to baseline occurring after 1 or 2 h. Furthermore, iterative electrical stimulation induced a progressive fading of this phenomenon. An enhancement of acetylcholinesterase activity was also observed by stimulations with potassium injections through a canal of the probe. These results suggest mainly an intracellular origin of the released enzyme and estimate its contribution at about 40% of the whole extracellular enzyme activity.

Influence of the radioprotective agent WR 2721 on the striatal acetylcholinesterase activity in the rat.

The radioprotective thiophosphate S-2(3 amino-propyl-amino) phosphorothioic acid (WR 2721) induced an early reduction of striatal acetylcholinesterase activity followed by an increase, when intraperitoneally injected to rats, although it does not cross the blood-brain barrier. These results were obtained using an original technique which allows the measurements in the same animal for several days. Transient general oxidative metabolism inhibition might affect the extra-cellular enzyme amount or its activity.

Rapid postmortem decrease in the ectocellular acetylcholinesterase activity in rat striatum as assessed by in vivo microspectrophotometry.

The acetylcholinesterase (AChE) activity in striatum rat was determined before and shortly after death using the in vivo microspectrophotometric method. This technique allowed us to monitor the Ellman colorimetric reaction directly inside the brain using an optical probe implanted in a live animal and to determine locally the AChE activity. Whatever the cause of the animals death, we observed a drastic postmortem decrease of the AChE activity of about 35-50%, 10 min after death. We have verified that the postmortem decrease of brain temperature or pH and postmortem optical properties changes could only explain a fraction of the AChE activity fall (16%). This phenomenon seems to be related to events strictly localized at the cellular level, since local injection of cyanide at the measuring site promotes a decrease of the enzymatic activity (40%) close to the levels observed after death. The origin of this rapid postmortem fall of the AChE activity is discussed. The technical properties of the microspectrophotometric method exclude a decrease of the ectocellular pool of enzyme after death. Our results allow us to envisage the existence of an in vivo endogenous regulation of the AChE activity which disappears shortly after death.

In vivo spectrophotometric determination of striatal acetylcholinesterase activity: the modulation induced by the antidepressant amitriptyline.

A new technology called in vivo spectrophotometry was applied to the quantitative determination of the variations in local acetylcholinesterase (AChE) activities. Repeated measurements of the enzyme activities in the same live animal allowed the study of the in vivo inhibition of AChE by amitriptyline. Interactions between AChE and this tricyclic antidepressant were investigated at the striatal level in anesthetized rats. In this anesthetized model, AChE assays were shown to be stable for approximately 8 h. The dose-effect relationship was explored in the 2.5- to 50-mg/kg amitriptyline range. A reversible inhibition was observed after acute amitriptyline administration. The maximum of inhibition appeared between 90 and 210 min after the intoxication and reached up to 22% for the 50-mg/kg dose. The threshold dose was established as 8 mg/kg. Evidence for an indirect interaction between tricyclic antidepressant and AChE was demonstrated when the total integrity of the biological system was preserved.

Demonstration of functional acetylcholinesterase on the soma of individual neurones of Aplysia by in vivo microspectrophotometry.

The presence of functional acetylcholinesterase is demonstrated in vivo on somatic membranes of single ganglionic neurones of Aplysia using concurrently microspectrophotometry and electrophysiology. The similarity of the effects of an irreversible blocker of acetylcholinesterase and of phospholipase C from Bacillus cereus suggests that acetylcholinesterase is anchored in the membrane via phosphatidylinositol.

Spectrophotometry in vivo, a technique for local and direct enzymatic assays: application to brain acetylcholinesterase.

In vivo enzymology is not widely studied due to the lack of a well-adapted technology. We have developed a system that allows local and long-term spectrophotometric assays in brain tissue of live animals. It utilizes a miniaturized optical probe consisting of a multibarrel micropipette for reagent injections and optical fibers for light absorption measurements. We have applied this system to the colorimetric determination of brain acetylcholinesterase activity in rats. The reproducibility of the assay was demonstrated by repetitive assays over 24 hr, its specificity was established through the use of a highly specific organophosphorus inhibitor, and the activities measured in different brain areas agreed with the known distribution of acetylcholinesterase. No electroencephalographic abnormalities and no change in vigilance level were observed in the experimental animals. This methodology should prove to be useful for the colorimetric measurement of different enzymes or metabolites in various organs.