J. Manil

NMDA receptor-mediated pilocarpine-induced seizures: characterization in freely moving rats by microdialysis.

Pilocarpine administration has been used as an animal model for temporal lobe epilepsy since it produces several morphological and synaptic features in common with human complex partial seizures. Little is known about changes in extracellular neurotransmitter concentrations during the seizures provoked by pilocarpine, a non‐selective muscarinic agonist. Focally evoked pilocarpine‐induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10mm for 40min at a flow rate of 2μl min−1) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, γ‐aminobutyric acid (GABA) and dopamine levels were monitored and simultaneous electrocorticography was performed. The animal model was characterized by intrahippocampal perfusion with the muscarinic receptor antagonist atropine (20mm), the sodium channel blocker tetrodotoxin (1μm) and the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801 (dizocilpine maleate, 100μm). The effectiveness of locally (600μm) or systemically (10mg kg−1 day−1) applied lamotrigine against the pilocarpine‐induced convulsions was evaluated. Pilocarpine initially decreased extracellular hippocampal glutamate and GABA levels. During the subsequent pilocarpine‐induced limbic convulsions extracellular glutamate, GABA and dopamine concentrations in hippocampus were significantly increased. Atropine blocked all changes in extracellular transmitter levels during and after co‐administration of pilocarpine. All pilocarpine‐induced increases were completely prevented by simultaneous tetrodotoxin perfusion. Intrahippocampal administration of MK‐801 and lamotrigine resulted in an elevation of hippocampal dopamine levels and protected the rats from the pilocarpine‐induced seizures. Pilocarpine‐induced convulsions developed in the rats which received lamotrigine perorally. Pilocarpine‐induced seizures are initiated via muscarinic receptors and further mediated via NMDA receptors. Sustained increases in extracellular glutamate levels after pilocarpine perfusion are related to the limbic seizures. These are arguments in favour of earlier described NMDA receptor‐mediated excitotoxicity. Hippocampal dopamine release may be functionally important in epileptogenesis and may participate in the anticonvulsant effects of MK‐801 and lamotrigine. The pilocarpine‐stimulated hippocampal GABA, glutamate and dopamine levels reflect neuronal vesicular release.

Neuromotor alterations and cerebellar deficits in aged arylsulfatase A-deficient transgenic mice

Arylsulfatase A (ASA)-deficient (-/-) mice and ASA(+/+) controls were constructed as a transgenic model for the lysosomal storage disease, metachromatic leukodystrophy (MLD). One-year-old ASA(-/-) mice showed impaired rotarod performance and altered walking pattern characterized by a shorter pace, later evolving into more severe ataxia with tremor in 2-year-old mice. Examination of cerebellar histology showed that 2-year-old ASA(-/-) mice have lost most of the calbindin immunoreactivity from their Purkinje cell dendrites and show simplified dendritic architecture. Additionally, ASA-deficient mice lost a substantial proportion of their Purkinje cells. Recordings of unitary potentials and stimulation of climbing fibers on cerebellar slices from 2-year-old mice indicated that, although the main cerebellar synapses seem to be present and functioning physiologically, the climbing fibers of ASA-deficient mice may have enhanced effects on Purkinje cell activity. It is concluded that ambulatory dysfunctions in ASA(-/-) mice might be explained by an imbalance in the consequences of climbing fiber signals upon Purkinje cell activity due to selective neurodegeneration within the cerebellum.

Effects of diazepam on extracellular brain neurotransmitters in pilocarpine-induced seizures in rats

The present study was undertaken to gain insights into the mechanism of action of diazepam in focally-evoked pilocarpine-induced seizures by concomitantly assessing the changes produced in the extracellular levels of glutamate, GABA (gamma-aminobutyric acid) and dopamine. In vivo microdialysis, coupled to continuous monitoring of electrocorticographic (ECoG) recordings, was performed in freely moving rats. Intrahippocampal perfusion with 10 mM pilocarpine (40 min, 2 microl/min) produced limbic seizures. A single dose of intraperitoneal diazepam (5 mg/kg) was administered 2 h after pilocarpine perfusion was started. Dialysates were sampled both from hippocampus and cerebellum and analysed by microbore liquid chromatography. Diazepam produced instant inhibition of behavioural and ECoG seizure activity. Pilocarpine-induced increases in the extracellular levels of glutamate and dopamine in hippocampus were promptly reduced by diazepam. No concurrent alterations in pilocarpine-induced increases in the extracellular levels of GABA in either hippocampus or cerebellum were seen. Pilocarpine enhanced cerebellar glutamate levels only transiently and levels returned to baseline before diazepam administration. No further changes in cerebellar glutamate levels were observed with diazepam. Our findings suggest that the anti-convulsant action of diazepam against pilocarpine-induced seizures is associated with a prompt attenuation of extracellular hippocampal glutamate overflow without concurrent alteration of pilocarpine-induced increases in endogenous GABA levels. Diazepam also significantly decreased pilocarpine-induced increases in extracellular dopamine levels within the hippocampus. No immediate alterations of the basal levels of the neurotransmitters monitored were observed with diazepam.

N-methyl-D-aspartate receptor activation by guanidinosuccinate but not by methylguanidine: behavioural and electrophysiological evidence.

Levels of methylguanidine (MG) and guanidinosuccinate (GSA) are known to be highly increased in uraemic patients. In the present work, the effects of these uraemic guanidino compounds on the excitatory amino acid system were investigated in vivo and in vitro. It was found that convulsions induced by intracerebroventricular GSA injection in mice were antagonized by N-methyl-D-aspartate (NMDA) receptor blockade, whereas those induced by MG were not significantly altered. Application of GSA (between 25 and 10,000 microM) to mouse spinal cord neurones in primary dissociated cell cultures, evoked depolarizing, inward whole-cell currents in a dose-dependent fashion and with reversal potential at 0 mV; MG did not produce such effects. GSA-induced whole-cell currents were caused by NMDA receptor activation since NMDA receptor antagonists (2-amino-5-phosphonovalerate, Mg2+ and ketamine) blocked GSA-evoked whole-cell currents completely and reversibly, whereas co-application of a non-NMDA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) did not affect GSA-induced current. Evoked field potentials in CA1 region of rat hippocampal slices were completely abolished by GSA, and this effect was antagonized by NMDA receptor blockade. All data were consistent with selective agonist action of GSA upon the NMDA-type glutamate receptor. In view of the results presented here, it should be examined whether NMDA receptors contribute to the neurological complications of renal failure through GSA-induced inappropriate or excessive activation of NMDA receptors.

The uremic guanidino compound guanidinosuccinic acid induces behavioral convulsions and concomitant epileptiform electrocorticographic discharges in mice

As yet, the in vivo epileptogenic properties of guanidinosuccinic acid (GSA) remained highly conjectural, still requiring the demonstration of GSA-induced behavioral convulsions accompanied by epileptiform electrographic discharges. Therefore, Swiss mice were injected intraperitoneally (i.p.) with increasing doses of GSA. Full-blown clonic or clonic-tonic convulsions appeared in a dose-dependent manner, with a median latency of about 25 min. CD50 (convulsive dose of the drug in 50% of the animals), the LD50 (lethal dose in 50%), and their 95% confidence limits for GSA suspensions in i.p. administration were 363 (287-458) mg/kg and 579 (445-756) mg/kg, respectively. In addition, four-channel electrocorticographic (ECoG) recordings were made in freely moving mice following the injection of 700 mg/kg (CD97). Epileptiform ECoG discharges coincided with the behavioral manifestation of the GSA-induced convulsions starting with initial decrease in amplitude, occasional spike-waves (10-20 min after injection), eventually leading to sustained spiking and spike-wave activity (30-50 min after injection). Clonic convulsions induced by a CD97 dose of GSA were only moderately attenuated by high doses of i.p. phenobarbital (20, 40 and 80 mg/kg), while tonic extension and lethal effects were dose-dependently blocked. A dose of 1000 mg/kg (CD97 for tonic extension) induced tonic extension in 100% of the animals, following treatment with 20 mg/kg of phenytoin none of the animals displayed tonic extension, and following 10 mg/kg only 30% of the animals displayed tonic extension, while the occurrence of clonic convulsions was not significantly attenuated.

Formalin-induced central sensitization in the rat: somatosensory evoked potential data

Cortical somatosensory evoked potentials were used to measure secondary hyperaesthesia resulting from subcutaneous 1 and 5% formalin in unanesthetized rats with permanently implanted electrodes. Near field responses were evoked by contralateral non-noxious electrical stimulation of the middle third of the tail. 0.05 ml 5% formalin injected subcutaneously at the base of the tail increased the amplitude of P1-N1 a maximum of 158.5+/-10.91% and N2 a maximum of 150.4+/-21.40% compared to controls (P<0.05 and P<0.01). Amplitudes were increased from 5 min after injection to the end of the 70 min test period. The effect of 1% formalin was equivalent to 5% formalin. This increase was prevented by pretreatment with 5 mg/kg ketamine or 5 mg/kg morphine, in agreement with behavioral and electrophysiological data. Cortical somatosensory evoked potentials are objective measures of central sensitization which may usefully complement current behavioral models for the evaluation of analgesic drugs.

Properties of the Spontaneous Fluctuations in Cortical Oxygen Pressure

In previous publications (Manil et al., 1978; 1981; Bourgain et al., 1980) the spontaneous fluctuations of the cortical tissue pressure in oxygen (Po2) in the awake rabbit were described in detail. The mean amplitude of these irregular oscillations was 5% of the normal local Po2 where 100% is defined as the value recorded in normal control conditions and 0% as the value recorded when death occurred after sustained pure nitrogen breathing. Analysis of the frequency domain demonstrates that in control conditions the power of the spectrum decreased from 0.06 to 0.5 Hz.

Naloxone-reversible opiate-sensitivity of a nociceptive cortical component of the somesthetic evoked potential (SEP) in the rat

NALOXONE-REVERSIBLE OPIATE-SENSITIVITY OF A NOCICEPTIV CORTICAL COMPONENT OF THE SOMESTHETIC EVOKED POTENTIAL (SEP {m IN THE RAT. C. Van Laere*, J. Manil*. M. Vervaeck’, Ph. Lebrun*. M Sosnovski. A. Soebert* and F. Cohn*. Physiological Laboratories of the Free Universities of Brussels, B-1090 Brussels, Belgium Aim of the Investigation: We firstly defined the cortical SEP of the rat after electrical stimulation of its tail-base, with an intensity of 1.5 x motor threshold (MT). Secondly, we evidenced a nociceptive component using noxious stimuli, and analyzed its naloxonereversible sensitivity to fentanyl, in order to define an electrophysiological index for pain and opiate-analgesia evaluation. Methods: The SEPs were recorded extradurally at the parietal region, referred to the nostril (frequency bandpass: 0.1 to 700 Hz; sampling frequency for digitization: 2000 Hz). After 256 stimulations, the averaged raw data were sent to a VAX 11/75 (DEC) computer for further analysis and display. Fentanyl(15 microg/kg) and naloxone (0.12 mg/kg) were injected intramuscularly (IM). Results: Stimulation at 1.5 x MT elicited an early positive component (P), followed by a larger negative one (N). with peak-latencies of 10 and 15.5 msec, respectively, (PlON15.5). A number of rats showed a W-shaped early component (P6PllN15.6). The peak to peak amplitude was in the range of 100 microV. Noxious stimuli (10 x MT or more) increased the amplitude of these components until saturation occurred, and evoked a supplementary late negative component of longer duration, peaking at 26 msec. This nociceptive wave disappeared within 10 min after IM injection of fentanyl. The effect of fentanyl lasted for 15 mm. The plateau then gradually reappeared and reached its initial value 45 min after fentanyl injection. The IM injection of naloxone reversed the opiate-analgesia within 3 min: the animal displayed again an exacerbated nociceptive behaviour, and the late negative plateau in the SEP reappeared. Conclusions: We have evidenced a negative, nociceptive, opiate-sensitive and naloxone-reversible plateau in the SEP of the rat. It constitutes a new objective assessment for pain measurement and opiate analgesia evaluation.

Relations between local microflow and tissue pressure in oxygen of the cerebral cortex in the awake rabbit (1 figure)

AbstractPrevious experiments (Manil et al., 1983) have revealed spontaneous oscillations of the cortical tissue pressure in oxygen (CTpO2) in a frequency band going from 0.03 to 0.33 Hz. No correlation could be found with the fluctuations of the systemic blood pressure except in stress situation. These oscillations were still present even after the spontaneous electrocortical activity had completely disappeared as a result of deep barbiturate anaesthesia. It was shown that these oscillations had to be attributed to circumscribed areas of the cerebral cortex of about 2 mm. Maximal vasodilation induced by breathing 5% CO2 increased and stabilized the CTpO2. Pharmacological analysis showed that alpha-blocking agents tremendously enhanced this otherwise spontaneous phenomenon. It was thus tempting to attribute this phenomenon to some vasomotor mechanisms. However, it remained to be shown that the oscillations of the CTpO2 were linked to fluctuations of the local microflow.