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.

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

1. 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. 2. Focally evoked pilocarpine-induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10 mM for 40 min at a flow rate of 2 microl min(-1)) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, gamma-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 (20 mM), the sodium channel blocker tetrodotoxin (1 microM) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine maleate, 100 microM). The effectiveness of locally (600 microM) or systemically (10 mg kg(-1) day(-1)) applied lamotrigine against the pilocarpine-induced convulsions was evaluated. 3. 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. 4. 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.

The olivocerebellar system. I. Delayed and slow inhibitory effects: an overlooked salient feature of cerebellar climbing fibers.

(1) Chemical destruction of the inferior olive (ION), or midline section interrupting the climbing fibers (CFs) rapidly resulted in marked modifications of Purkinje cell (P. cell) simple spike (SS) firing rate and pattern. (2) After CF deafferentation, P. cells at first about doubled their SS frequency which further increased for the next 10 min. (3) Besides the increase in the firing rate, the spike train became much more regular, which in part seemed to be linked to mass oscillations of the neuronal circuitry, as revealed by strong oscillations of background noise. (4) After ION destruction CF activity could be supplied for by juxtafastigial (JF) stimulation which reduced SS frequency again while the firing became much less regular. These effects were shown to be due to the all-or-nothing activity of the CF and not to the simultaneous stimulation of mossy fibers (MFs) or P. cell axons. Neither were they ascribable to CF collaterals. The differences between this new powerful inhibitory action of the CF system on the P. cell and the well documented pause mechanism is discussed. (5) A quantitative relationship has been established between complex spikes (CSs) and SS firing rates. A steady 2/sec CS frequency was shown to effectively silence the P. cell. (6) When CF stimulation was discontinued, an "off" effect was described. It consisted of an initial rise in SS frequency developing in 9 sec, and a delayed further increase unfolding in about 10 min. (7) When CF stimulation began, an "on" effect was observed, which evolved with an exponential-like kinetic of very variable time-constant seemingly depending on past history.

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.

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.

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.

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.

Properties of cortical somatosensory evoked potentials in the awake rabbit.

By appropriate experimental procedures it was possible to distinguish the components of the contra- and ipsilateral cerebral somatosensory potentials evoked by electrical stimulation of an extremity in the unrestrained rabbit. The components of the contralateral response occur in the following order; an early positive primary wave (P1), a positive associative wave (P2), and ultimately a late negative N wave. The components of the ipsilateral response occur in the following order; a small positive wave P, the latency of which is intermediate between those of the contralateral P1 and P2 waves, and a large negative wave N, similar to the contralateral N wave. The different topographical distributions of these waves were elucidated by the use of insulated, chronically implanted electrodes glued onto the cortical surface. The properties of the waveform components were studied by various methods such as varying the stimulation parameters, simultaneous application of somesthetic and acoustic stimuli, and administration of narcotic drugs. The properties of P1 were similar to those of P28 in humans; the properties of P2 can be compared to those of P45; and, finally, the N wave resembles the late negative components observed in humans. Inconstant small positive waves of shorter latencies, which will be discussed in a following paper, may also be seen. Interestingly enough, no early negative wave such as that observed in humans (N20) was ever found. If, as is presently thought, this wave is, in fact, due to the folding of the cortical surface (Broughton, 1969), its absence is to be expected in the rabbit because the cortical surface of this species is lissencephalic and thus devoid of gyri.

The effect of acute anoxia on the cortical somatosensory evoked potential in the rabbit.

In the course of this study it was found that the functional state of the cortex during anoxic anoxia could be more accurately monitored by the cortical somatosensory evoked potentials (EPs) than by the electrocorticogram (ECoG). Compared with the ECoG, the EPs yield more detailed information. Five different steps may be distinguished in the evolution of EPs; the ECoG, however, exhibits only two (almost trivial) abnormal patterns (epileptic pattern and flat response). Of particular practical interest are the two steps preceding the disappearance of the EPs: (1) a rounding of the P wave accompanied by an increased voltage, followed later by (2) a decrease of the latter. If, at this stage, anoxia is maintained for 20 seconds, then permanent dysfunction is observed, at least during the time interval of the experimentation.

Discriminant parameters representing cerebral cortical function during anoxic anoxia investigations.

In order to quantify the effect of specific drugs on the cerebral cortex an "in vivo" model has been developed for the induction and the observation of anoxic anoxia. Rabbits are used as test animals. Sensors for the assessment of local parameters are chronically implanted: ECoG electrodes are applied; pO2 electrodes are inserted into the cortical tissue. The derived somatosensory evoked potentials are used for evaluating the cerebral cortical function. Animals are cannulated with a tracheal tube, curarized and artificially ventilated. Anoxic anoxia, controlled by a special purpose microprocessor system, may then be induced and repeated in a reproducible way. During the experiments local (pO2, ECoG, SEP) as well as general parameters (ECG, heart rate, systemic blood pressure, rectal temperature) are recorded and stored on analog magnetic tape as well as digitized with the microprocessor system. The cortical pO2 is measured with a polarographic method, the SEP's are obtained on-line by time coherent averaging and the ECoG states (e.g. epilepsy during anoxia) are derived by using band-pass filters and rms detectors. Off-line the signals (pO2, heart rate, mean systemic blood pressure, temperature) are standardized and represented together with parameters derived from ECoG and SEP. SEP-waveform parameters indicating intensity (norm) and similarity with a reference SEP signal (correlation value) are used. The measuring and processing method is still being optimized; special attention is being paid towards the quality of the calculated SEP's which are to be used for the quantification of the cortical function during reference, anoxia and recovery period. As such, in order to improve the signal-to-noise ratio of the SEP's and, consequently, of the derived parameters, ECoG signals are digitized off-line and subjected to a preprocessing, implying filtering and spectral analysis procedures.

Early diagnosis of multiple sclerosis by combined multimodal evoked potentials: results and practical considerations.

It is shown that using four combined types of evoked potentials one can disclose new localisations in 62.5% of as yet undiagnosed M.S. patients. Brainstem auditory EP recordings must be considered in detail to avoid missing slight anomalies. Blink reflexes yielding bilateral R1 seem to bear the same significance as an augmented latency with respect to the problem of disclosing new localisations.

Effectiveness of vigabatrin against focally evoked pilocarpine-induced seizures and concomitant changes in extracellular hippocampal and cerebellar glutamate, gamma-aminobutyric acid and dopamine levels, a microdialysis-electrocorticography study in freely moving rats.

Limbic seizures were evoked in freely moving rats by intrahippocampal administration of the muscarinic agonist pilocarpine via the microdialysis probe (10 mM for 40 min at 2 microl/min). This study monitored changes in extracellular hippocampal gamma-aminobutyric acid (GABA), glutamate and dopamine levels after systemic (30 mg/kg/day) or local (intrahippocampal or intranigral, 5 mM or 600 microM for 180 min at 2 microl/min) vigabatrin administration, and evaluated the effectiveness of this antiepileptic drug against pilocarpine-induced seizure activity. Extracellular GABA and glutamate overflow in the ipsilateral cerebellum was studied simultaneously. Microdialysis was used as an in vivo sampling technique and as a drug-delivery tool. Electrophysiological evidence for the presence or absence of seizures was recorded with electrocorticography. The observed alterations in extracellular hippocampal amino acid levels support the hypothesis that muscarinic receptor stimulation by the intrahippocampal administration of 10 mM pilocarpine is responsible for the seizure onset, and that the amino acids maintain the sustained seizure activity. The focally evoked pilocarpine-induced seizures were completely prevented by intraperitoneal vigabatrin premedication for 7 days or by a single intraperitoneal injection. Effective protection was reflected in a lack of sustained elevations of hippocampal glutamate levels. Rats receiving vigabatrin intrahippocampally or intranigrally still developed seizures, although there appeared to be a partial protective effect. During the intrahippocampal perfusion with 5 mM vigabatrin, extracellular hippocampal GABA levels increased, whereas the extracellular glutamate and dopamine overflow decreased. The lack of a complete neuroprotection after local vigabatrin treatment is discussed.

The ouabain-sensitive fluxes of sodium and potassium in squid giant axons.

1. Fifty to ninety per cent of the Na efflux from axons of Loligo forbesi is inhibited by ouabain. The properties of the ouabain-sensitive component of the Na efflux are different from those of the ouabain-insensitive component.2. In unpoisoned axons with an average Na content of 75 m-mole/kg axoplasm the bulk of the ouabain-sensitive Na efflux is dependent on external K.3. In the presence of 460 mM Na in the external medium, raising the external K concentration from 0 to 100 mM increases the ouabain-sensitive Na efflux along a sigmoid curve which shows signs of saturating at high K concentrations.4. The curve relating ouabain-sensitive K influx to external K concentration is similar in shape to that for the ouabain-sensitive Na efflux. At all K concentrations examined the ouabain-sensitive K influx was less than the ouabain-sensitive Na efflux.5. Potassium-free sea water acts rapidly in reducing the Na efflux. There is no appreciable difference between the rates of action of K-free sea water on the Na pump and Na-free sea water on the action potential.6. Caesium and Rb can replace external K in activating the ouabain-sensitive Na efflux. Both the affinity and maximum rate of the Na efflux mechanism are lower when Cs replaces K as the activating cation.7. Isosmotic replacement of external Na by either choline or dextrose, but not Li, increases the affinity of the ouabain-sensitive Na efflux mechanism for external K without appreciably affecting the maximum rate of pumping. External Li behaves like external Na and exerts an inhibitory action on the Na efflux.8. There is a large ouabain-sensitive Na efflux into K-free choline or dextrose sea waters. Addition of either Na or Li to the external medium reduces this efflux along a section of a rectangular hyperbola. The properties of this efflux suggest that there is a residual K concentration of up to 2 mM immediately external to the pumping sites in the axolemma.9. Over the range of internal Na concentrations studied (16-140 m-mole/kg axoplasm) the ouabain-sensitive Na efflux increased linearly with Na concentration.10. Tetrodotoxin (10(-6) g/ml.) reduces the Na influx by about half, but does not affect the ouabain-sensitive Na efflux.11. Isobutanol (1% v/v) reversibly decreases both the ouabain-sensitive and ouabain-insensitive components of the Na efflux.12. Application of 2 mM cyanide to axons immersed in K-free sea water produces a transient rise in the Na efflux. This rise is not seen if ouabain is included in the sea water. The rise in efflux occurs at a time when the axons are partially poisoned and contain adenosine triphosphate (ATP) but no arginine phosphate (ArgP). A similar, but maintained rise can be obtained after application of dinitrophenol (DNP) at pH 8.0. The increased Na efflux in these partially poisoned axons is also inhibited by ouabain.13. Under conditions of partial-poisoning by alkaline DNP, there is a ouabain-sensitive Na influx from K-free sea water. The ouabain-sensitive Na influx is of similar size to the ouabain-sensitive Na efflux. These results show that in partially-poisoned axons immersed in K-free sea water intracellular Na exchanges with extracellular Na in a one-for-one manner by a ouabain-sensitive route. External Li cannot replace external Na in maintaining this process.14. Axons partially poisoned with alkaline DNP are not insensitive to external K. In the absence of external Na their response to external K is essentially the same as that seen in unpoisoned axons.15. Possible mechanisms are discussed for the appearance of Na-Na exchange in partially poisoned axons.