A new method for semiautomatic analysis of surface EMG in patients with oral parafunctions.

Sleep-related phenomena or disorders, including snoring and tooth grinding, can be investigated using polysomnography. This method, however, generates large amounts of synchronically recorded data that are often analyzed visually with subjective interpretation. The purpose of this study was to minimize the need for subjective evaluation by developing a computer program for analysis of EMG data linked with polysomnographic recordings in a standardized and semi-automatic way. The selected algorithm differs from the Root Mean Square (RMS) method by being based on the theory of "differentiated EMG" (DIFEMG), which relies on two principles. The first says that the activation of a larger number of motor units results in a greater force production. The second principle says that the force production will continue for some time after the muscle is no longer stimulated. After a visual check for artifacts in the basic EMG recordings, the computer program is used to analyze the corrected basic EMG signal. The results were that both methods yield identical results as far as the detected number of events is concerned. There is, however, a significant difference when the duration of the events is considered, because the start and end of an event can be more accurately determined with the new method presented here. The computer program described will make comparison of data from different studies easier.

Effect of NaCN on currents evoked by uremic retention solutes in dissociated mouse neurons.

Uremic retention solutes possibly contribute to neuronal hypoxia/ischemia and its consequences in patients with renal failure. We examined the in vitro effects of several uremic retention solutes on murine central neurons under chemically induced metabolic hypoxia by application of sodium cyanide (NaCN). Whole cell currents were recorded using the tight-seal whole-cell voltage clamp technique. Application of NaCN caused an inward whole-cell current. From all tested toxins, which included several indoles, guanidino compounds, polyamines, purines, phenols, DL-homocysteine, orotate and myoinositol, only creatinine (CTN), guanidine (G) and guanidinosuccinic acid (GSA) produced a significant current in control and hypoxic neurons. Current evoked by GSA was significantly increased in the chemical hypoxic condition, and a synergistic effect of GSA and spermine was observed in hypoxic neurons.

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation.

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.

Influence of a bite-plane according to Jeanmonod, on bruxism activity during sleep.

One way to look at nocturnal oral parafunction such as bruxism, is using polysomnography. Although in recent years several studies have been carried out, it is almost impossible to compare the respective results because of the lack of standardization in the methodology used. The nocturnal muscular activity of m. Masseter and m. Temporalis Anterior has been registered in 21 patients suffering from bruxism, prior and after treatment with a bite-plane made according to Jeanmonod. A group of five persons without any dental or muscular parafunction has been included as a control. A computer program based on the biomechanical principles of muscle contraction has been developed and was used to analyse the raw electromyography (EMG) signal. The data shows a significant decrease (P = 0.008) in nocturnal parafunctional muscle activity with the bite-plane in situ. The present study shows that by analysing the raw signal obtained via EMG using a specially designed computer program, it becomes possible to isolate the occurrence of parafunctional events in the EMG signal and to study the influence of a given therapy on this EMG signal.

Ca2+ involvement in the action potential generation of myenteric neurones in the rat oesophagus.

Intracellular recordings were used to study the physiological behaviour of rat oesophageal myenteric neurones, which are embedded in striated muscle. Injection of depolarizing pulses evoked action potentials with a clear 'shoulder' in all neurones. This shoulder disappeared under low Ca2+/high Mg2+ conditions. Tetrodotoxin (TTX; 1 micromol L-1) did not impede spike firing, whereas under combined TTX and low Ca2+/high Mg2+ conditions the action potentials were completely abolished, indicating that TTX- resistant action potentials are mediated by a Ca2+ current. Further experiments with omega-conotoxin GVIA (100 nmol L-1) revealed that these Ca2+ currents enter the cell via N-type voltage-activated Ca2+ channels (see also accompanying paper). Tetraethylammonium (10 mmol L-1) caused broadening of the action potentials, which probably resulted from prolonged Ca2+ influx due to blockade of the delayed rectifier K+ channel. Although Ca2+ appears to be involved in the spike generation of all rat oesophageal myenteric neurones, only a minority (14%) shows a slow afterhyperpolarization. Thus, no strict correlation exists between the presence of a shoulder and a slow afterhyperpolarization. Furthermore, morphological identification of 25 of the impaled neurones revealed that there was no strict correlation between morphology and electrophysiological behaviour. Consequently, rat oesophageal myenteric neurones appear to differ in several aspects from myenteric neurones in smooth muscle regions of the gastrointestinal tract.

Immunohistochemical localization of voltage-activated calcium channels in the rat oesophagus.

Voltage-activated calcium channels play an important role in the physiology of the enteric nervous system. To determine which types of voltage-activated calcium channels are present in the rat oesophagus, an immunohistochemical study was performed using specific antibodies for the alpha1 subunits of Cav2.1 (P/Q-type), Cav2.2 (N-type), Cav1.2 and Cav1.3 (L-type) calcium channels. All myenteric cell bodies showed Cav2.2 immunoreactivity, whereas labelling for this N-type channel was absent in nerve fibres. Cav1.2 immunoreactivity was found on nerve fibres in the myenteric plexus and on fibres innervating the striated muscle of the rat oesophagus, whereas no labelling was detected on neuronal somata. Immunoreactivity against Cav1.3 was not detected in the myenteric plexus or at the level of the striated muscle. Labelling for Cav2.1 was absent at the level of the myenteric plexus, but present in the striated muscle layer at the level of the motor endplates. Comparison with recent literature data from rat small intestine reveals region-specific distribution patterns of the various subtypes of voltage-activated calcium channels within the enteric nervous system. In addition, the present immunohistochemical data corroborate our physiological data (see accompanying paper), which indicate that the Cav2.2 (N-type) channel is the predominant channel involved in the generation of the calcium-dependent action potential evoked by intrasomatic depolarizing current pulses in all rat oesophageal myenteric neurones.

Excitatory synaptic inputs on myenteric Dogiel type II neurones of the pig ileum.

The synaptic input on myenteric Dogiel type II neurones (n = 63) obtained from the ileum of 17 pigs was studied by intracellular recording. In 77% of the neurones, electrical stimulation of a fibre tract evoked fast excitatory postsynaptic potentials (fEPSPs) with an amplitude of 6 +/- 5 mV (mean +/- S.D.) and lasting 49 +/- 29 ms. The nicotinic nature of the fEPSPs was demonstrated by superfusing hexamethonium (20 microM). High-frequency stimulation (up to 20 Hz, 3 seconds) did not result in a rundown of the fEPSPs, and did not evoke slow excitatory or inhibitory postsynaptic potentials. The effects of neurotransmitters, possibly involved in these excitatory responses, were investigated. Pressure microejection of acetylcholine (10 mM in pipette) resulted in a fast nicotinic depolarisation in 67%(18/27) of the neurones (13 +/- 9 mV, duration 7.0 +/- 7.2 seconds) as did 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) application (10 mM; 14 +/- 10 mV, duration 4.1 +/- 2.8 seconds) in 76% of the cells. The fast nicotinic response to acetylcholine was sometimes (6/27) followed by a slow muscarinic depolarisation (8 +/- 4 mV; duration 38.7 +/- 10.8 seconds). Immunostaining revealed 5-hydroxytryptamine hydrochloride (5-HT)- and calcitonin gene-related peptide (CGRP)-positive neuronal baskets distributed around and in close vicinity to Dogiel type II neuronal cell bodies. Microejection of 5-HT (10 mM) resulted in a fast nicotinic-like depolarisation (12 +/- 6 mV, duration 3.0 +/- 1.3 seconds) in 4 of 8 neurones tested, whereas microejection of CGRP (20 mM) gave rise to a slow muscarinic-like depolarisation (6 +/- 2 mV, duration 56.0 +/- 27.5 seconds) in 8 of 12 neurones tested. In conclusion, myenteric Dogiel type II neurones in the porcine ileum receive diverse synaptic input. Mainly with regard to the prominent presence of nicotinic responses, these neurones behave contrary to their guinea pig counterparts.

Endogenous guanidino compounds as uremic neurotoxins.

Epileptic and cognitive symptomatologies are among the most typical manifestations of uremic encephalopathy. Several guanidino compounds (GCs) may play an important role in the etiology of uremic encephalopathy. Four GCs appeared to be highly increased as well in serum, cerebrospinal fluid, and brain of uremic patients, whereas the levels of other metabolically relevant GCs were not or only moderately increased and others were even decreased. These highly increased compounds or "uremic" GCs are creatinine (CTN), guanidine (G), guanidinosuccinic acid (GSA), and methylguanidine (MG). All four compounds were shown to be experimental convulsants in brain concentrations similar to those found in uremic brain. We have described a possible mechanism for the contribution of GCs to uremic hyperexcitability, referring to the in vitro effects of uremic GCs on inhibitory and excitatory amino acid receptors. The excitatory effects of uremic GCs on the central nervous system may be explained by the activation of N-methyl-D-aspartate (NMDA) receptors by GSA, concomitant inhibition of GABA(A) receptors by uremic GCs, and other depolarizing effects. These effects might also indicate the putative contribution of uremic GCs to the etiology of uremic encephalopathy.

Decreased amount of slow wave sleep in nocturnal bruxism is not improved by dental splint therapy.

OBJECTIVE: To test the efficacy of dental treatment of bruxism on sleep quality, using slow wave sleep as the primary outcome parameter. METHODS: The study design consisted of an open label, unpaired comparison between normals and patients and a paired comparison between pre- and post-treatment patient recordings. Twenty patients suffering from bruxism (13 male, 7 female, mean age 35 years) and 6 normal volunteers (3 male, 3 female, mean age 30 years) participated in the study. Polysomnographic recordings were performed in a sleep laboratory in a general hospital both before and after treatment. The treatment was derived from a model that ascribes bruxism to a dental malocclusion, and consisted solely of dental therapy (Jeanmonood 1988). RESULTS: The untreated bruxism group had worse sleep than normals when comparing slow wave sleep (21% versus 32% slow wave sleep percentage in sleep period time) during the second polysomnographic recording, after one night adaptation. Therapy did not improve sleep quality; bruxism patients showed only minor, non-significant differences in sleep quality when comparing pre- and post-treatment recordings.

Electrophysiological features of morphological Dogiel type II neurons in the myenteric plexus of pig small intestine.

By intracellular recording, 99 myenteric neurons with Dogiel type II morphology were electrophysiologically characterized in the porcine ileum and further subdivided into three groups based on their different types of afterhyperpolarization (AHP). In response to a depolarizing current injection, a fast AHP (fAHP; duration 34 +/- 11 ms; amplitude -11 +/- 6 mV; mean +/- SD) immediately followed every action potential in all neurons. In 32% of the neurons, this fAHP was the sole type of hyperpolarization recorded. Statistical analysis revealed the presence of two neuronal subpopulations that displayed either a long-lasting medium AHP (mAHP; duration after a single spike 773 +/- 753 ms; 51% of neurons) or a slow AHP (sAHP; 4, 205 +/- 1,483 ms; 17%). Slow AHP neurons also differed from mAHP neurons in the delayed onset of the AHP (mAHP 0 ms; sAHP 100-200 ms), as well as in maximum amplitude values and in the time to reach this amplitude (t(max); 148 +/- 11 ms vs. 628 +/- 108 ms). Medium AHP neurons further differed from the sAHP neurons in the occurrence of the AHP following subthreshold current injection and in their resting membrane potential (mAHP, -53 +/- 8 mV; sAHP, -62 +/- 10 mV). Medium AHP and sAHP behaved similarly in that a higher number of spikes increased their amplitude and duration, but not t(max). The majority of neurons fired multiple spikes (up to 25) in response to a 500-ms current injection (81/99) and showed a clear TTX-resistant shoulder on the repolarizing phase of the action potential (77/99), irrespective of the presence of sAHP or mAHP. These results demonstrate that the porcine Dogiel type II neurons differ in various essential electrophysiological properties from their morphological counterparts in the guinea pig ileal myenteric plexus. The most striking interspecies differences were the low occurrence of sAHP (17% vs. 80-90% in guinea pig) with relatively small amplitude (-5 vs. -20 mV), the high occurrence of mAHPs (unusual in guinea pig) and the ability to fire long spike trains (up to 25 spikes vs. 1-3 in guinea pig). In fact, Dogiel type II neurons in porcine ileum combine distinct electrophysiological features considered typical of either S-type or sAHP-type neurons in guinea pig. It can therefore be concluded that in spite of a similar morphology, Dogiel type II neurons do not behave electrophysiologically in a universal way in large and small mammals.

Species-dependent features of Dogiel type II neurones in the mammalian enteric nervous system.

Although autonomic gastrointestinal reflex movements, which occur in all mammalian species, have been described almost a century ago, little was known on the mechanisms underlying this behaviour. Recently, however, intrinsic primary afferent neurones, functioning as the first relay in the reflex arches embedded in the intestinal wall, have been identified in the guinea pig ileum. In guinea pig, such neurones display a Dogiel type II morphology and behave electrophysiologically as slow AHP neurones. In other gastrointestinal regions, in both guinea pig and rat, Dogiel type II cells are also encountered, but the strong correlation with slow AHP neuronal features seems less strict. In large mammals, a correlation of the cellular morphology with intracellular el ectrophysiological recordings has only been obtained in the pig small intestine. Surprisingly, in these experiments aberrant electrophysiological behaviour of Dogiel type II neurones is even more striking since the majority of these cells display electrophysiological features considered typical of S neurones. Furthermore, in those rare cases in which a slow afterhyperpolarization (AHP) could be recorded in porcine Dogiel type II cells, its amplitudes were negligible. This has led us to the conclusion that the differences in electrophysiological behaviour of neurones with comparable morphology in different species are most probably due to the modulating influence of the neurotransmitter substances present. This seems to be the most likely hypothesis in view of the considerable differences in neurotransmitter content of neurones with comparable functions throughout the species.

Uraemic guanidino compounds inhibit gamma-aminobutyric acid-evoked whole cell currents in mouse spinal cord neurones.

Guanidine, creatinine (CTN), methylguanidine (MG) and guanidinosuccinic acid (GSA) are four endogenous guanidino compounds with proven neuroexcitatory actions, and putative pathophysiological significance as uraemic toxins. The effects of these uraemic guanidino compounds, were studied on whole-cell current evoked by gamma-amino butyric acid (GABA) on mouse spinal cord neurones in vitro. CTN, MG and GSA concentration dependently blocked GABA-evoked current with calculated IC50 values (+/-SE) of 9.6 +/- 0.9, 9.7 +/- 1.5 and 5.1 +/- 0.4 mM, respectively. CTN, MG and GSA were shown to block inward and outward currents to the same extent, demonstrating voltage independent block of GABA-evoked current by these compounds. Guanidine, however, evoked inward whole-cell currents, which were almost completely blocked by strychnine, indicating that the guanidine-evoked current might have been due to glycine receptor activation.

Effects of competitive NMDA receptor antagonists on excitatory amino acid-evoked currents in mouse spinal cord neurones.

The effects of CGP 37849 [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoate] and its ethylester CGP 39551 on whole-cell currents evoked by the endogenous excitatory amino acids, L-glutamate and L-aspartate, were studied in cultured mouse spinal cord neurones. Although CGP 37849 was the more potent compound, both antagonists inhibited 20 microM L-aspartate or 2 microM L-glutamate currents concentration-dependently and reversibly. We calculated IC50 values of 370 +/- 180 nM for CGP 37849 and 2200 +/- 140 nM for CGP 39551 (inhibition of L-aspartate current), and 210 +/- 25 nM for CGP 37849 and 6000 +/- 4700 nM for CGP 39551 (inhibition of L-glutamate current). Both CGP 37849 and CGP 39551 selectively blocked N-methyl-D-aspartate (NMDA)-evoked inward current. Current evoked by 5 microM kainate or 5 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unaffected by 10 microM CGP 39551. Current evoked by NMDA was concentration-dependently blocked by CGP 39551 with an IC50 of 2100 +/- 220 nM. After application of 10 microM CGP 37849, 17 +/- 6% of the current evoked by 5 microM L-glutamate remained. This residual current was due to non-NMDA receptor activation since application of 25 microM 2-amino-5-phosphonovalerate (APV) together with CGP 37849 did not significantly alter the residual current, whereas application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with CGP 37849 did significantly inhibit this current. Clamping cells at potentials ranging from -80 to +60 mV showed a linear potential--current relationship for the 20 microM L-aspartate-evoked current with reversal potential around 0 mV. The proportion of the L-aspartate current antagonized by CGP 37849 or CGP 39551 appeared to be independent of clamping potential. The concentration--current relationship of L-aspartate in the absence of the antagonists showed an EC50 of 49 +/- 14 microM. Upon application of 1 microM CGP 37849 and 10 microM CGP 39551, the L-aspartate concentration--current curve shifted to higher concentrations, and resulted in a 5- and 13-fold increase in the EC50 of L-aspartate, respectively, whereas Imax was not changed by application of the antagonists. Thus, the potent NMDA antagonists CGP 37849 and CGP 39551 were shown to inhibit excitatory amino acid responses specifically by competitive binding to the neurotransmitter recognition site of the NMDA receptor. Selective, competitive antagonism of L-glutamate- and L-aspartate-evoked NMDA receptor responses probably underlies the effects of CGP 37849 and CGP 39551 such as their anticonvulsant, neuroprotectant and antidepressant actions.

Use-dependent block of Ih in mouse dorsal root ganglion neurons by sinus node inhibitors.

1. The sinus node inhibitors UL FS 49 and DK-AH 269 reduce heart rate by slowing diastolic depolarization rate in the sino-atrial (SA) node, which might originate from the use-dependent blockade of a hyperpolarization-activated current If. A hyperpolarization-activated current Ih, which is present in many types of neurons, is similar to If. We studied the effects of these drugs on Ih in cultured mouse dorsal root ganglion (DRG) neurons. 2. With the whole-cell patch-clamp technique use-dependent block of Ih was observed. The steady-state block following a voltage-clamp pulse train (1-s steps from -38 to -108 mV applied at 0.5 Hz) was dependent on drug concentration and showed an apparent Kd of 0.1 and 0.79 microM with DK AH 269 and UL-FS 49 respectively. 3. The rate of block increased linearly with drug concentration. The rate of recovery from block was, however, much slower compared to cardiac tissue. 4. There was no significant effect of UL-FS 49 on the activation curve. 5. At high concentrations of UL-FS 49 a clear association of the drug with the open channel was observed. 6. When the cell was stimulated at a frequency of 3 Hz, a distinct hyperpolarization was observed in the presence of extracellular Cs+ or when Ih was blocked with UL-FS 49, but not in the absence of Cs+ and UL-FS 49. 7. These results indicate that Ih protects the cell against hyperpolarizations and subsequent inexcitability. The action of the drugs on the hyperpolarization-activated current in cardiac and neuronal tissue show some similarities; however, some pronounced differences indicate that different subtypes of the channel might exist.

Effects of the competitive N-methyl-D-aspartate antagonist CGP 37849 and its ethylester CGP 39551 on N-methyl-D-aspartate-evoked whole-cell currents in cultured spinal neurones and on vestibular stimulation-induced seizures in EL mice.

The competitive N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CAS 127910-31-0, 4-methyl-APPA, CGP 37849) and its ethyl ester (CAS 127910-32-1, CGP 39551) potently block NMDA-evoked whole-cell current on mouse spinal neurones in primary dissociated cell cultures with IC50 (+/- SE) values of 189 +/- 9 nmol/l (CGP 37849) and 2100 +/- 220 nmol/l (CGP 39551), respectively. The compounds dose-dependently blocked vestibular stimulation-induced convulsions in EL mice, 2 h after oral administration, with ED50 (95% CI) values of 135 (78-236) mumol/kg (CGP 37849) and 65 (45-94) mumol/kg (CGP 39551). In male Swiss albino mice, performance in the step-through passive avoidance procedure was dose-dependently impaired with ED50 (95% CI) values of 85 (56-157) mumol/kg (CGP 37849) and 27 (18-42) mumol/kg (CGP 39551). In addition performance of these animals in the rotarod test of motor coordination was impaired, 2 h after oral administration of CGP 39551, with an ED50 (95% CI) of 142 (100-201) mumol/kg. These findings demonstrate anticonvulsant activity in these potent NMDA antagonists after oral administration with CGP 39551 possessing greater relative potency. However, the unfavourable ratio of therapeutic dose versus dose inducing memory or motor impairment supports the prevailing notion that such adverse effects of the presently available compounds preclude the use of NMDA antagonists as long-term therapies.

Effect of cAMP and ATP on the hyperpolarization-activated current in mouse dorsal root ganglion neurons.

In mouse dorsal root ganglion (DRG) neurons the activation curve of the hyperpolarization-activated current (Ih) shifted towards depolarized potentials when cAMP was present in the pipette. The relation between the midpoint potential and cAMP concentration could be described by a Hill function with a half-maximal concentration of 0.55 microM cAMP, reflecting a direct action of cAMP on the channel. With 5 mM ATP and a saturating concentration of cAMP an additional shift of the midpoint potential is observed which can be explained by phosphorylation. Application of Rp-cAMPS and Sp-cAMPS support the hypothesis of both a phosphorylation pathway and a direct effect exhibited by these molecules. The bell-shaped curves, relating the time constants for the slow and fast current components to the voltage, shifted towards positive membrane potentials when cAMP and ATP were in the pipette. The fully activated Ih/voltage relation and the reversal potential were not dependent on the presence of cAMP or ATP in the pipette. The mean resting membrane potential of -59 mV, using the perforated-patch configuration, hyperpolarized in the presence of extracellular CsCl. In the whole-cell configuration the resting membrane potential was significantly more negative at 0 microM cAMP (-61 mV) than at 100 microM cAMP (-57 mV). Thus, the activation of Ih, regulated by both the intracellular cAMP and the ATP concentration, may influence the excitability of DRG neurons.

Selective block of N-methyl-D-aspartic acid (NMDA)-evoked whole-cell currents in mouse cultured spinal neurones by CGP 40116.

1. CGP 40116 is the active (R)-enantiomer of the most potent N-methyl-D-aspartic acid (NMDA) receptor antagonist presently available: 2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849). In this study, we describe the effect of CGP 40116 on whole-cell currents induced by excitatory amino acids in cultured mouse spinal cord cells by use of the whole-cell patch-clamp technique. 2. We found that application of CGP 40116 in the nM range, concentration-dependently inhibited whole-cell current evoked by 20 microM NMDA in mouse cultured spinal neurones (IC50 +/- s.e. mean 48 +/- 8 nm CGP 40116). 3. The compound appeared to be highly selective for the NMDA current. At concentrations as high 1 microM, currents evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid were not affected by CGP 40116. The threshold concentration for antagonism of NMDA-induced responses was 10 nM suggesting a selectivity ratio of > or = 100 fold for NMDA receptors versus AMPA or kainate receptors. 4. CGP 40116 produced a parallel rightward displacement of the NMDA log concentration-current curve indicating competitive antagonism at the transmitter recognition site of the NMDA receptor complex. An apparent dissociation constant for the antagonist was calculated from the displacement of the agonist concentration-current curve: 117 +/- 53 nM CGP 40116 (estimated Kd +/- s.e.). Like other competitive NMDA antagonists, CGP 40116 blocked NMDA-evoked current in a voltage-independent manner.

Electrophysiology of porcine myenteric neurons revealed after vital staining of their cell bodies. A preliminary report.

Due to practical limitations in visualizing and getting access to the ganglionic components of large mammals, electrophysiology of the enteric nervous system has been restricted mainly to small laboratory animals, more particularly the guinea-pig. The use of the vital dye 4-(4-diethylaminostyry1)-N-methylphyridinium iodide (4-Di-2-ASP), however, overcomes some of these difficulties. A 20-min incubation period with this dye, followed by a minimum period of 4 h in Krebs solution, suffices to stain the neuronal cell bodies, permitting selection of a neuron and positioning of the microelectrode for impalement and recording. The method has been applied to pig ileum and guinea-pig large and small bowel myenteric neurons. Impalements of untreated guinea-pig myenteric neurons were compared with those of 4-Di-2-ASP-pretreated ones. According to our preliminary data, the staining did not suppress the expression of apparently normal electrophysiological activity. Moreover, the procedure permitted impalement and recording of myenteric plexus neurons in pig ileal tissue with a rate of success equalling blind impalement on guinea-pig tissue. In contrast with formerly published results whereby staining of the neuronal cell bodies only occurred when the cells had been chemically damaged, our experiments suggest a possible correlation between fluorescence and cell viability.

Anticonvulsant activity of piperine on seizures induced by excitatory amino acid receptor agonists.

In traditional Chinese medicine, a mixture of radish and pepper is used to treat epilepsy. The presumptive effectiveness of this prescription might be due to the anticonvulsant actions of the principal component of pepper, the alkaloid piperine (CAS 94-62-2). The effects of piperine on convulsions induced in mice by agonists at different excitatory amino acid receptor subtypes were studied. Piperine was shown to significantly block convulsions induced by intracerebroventricular injection of threshold doses of kainate, but to have no or only slight effects on convulsions induced by L-glutamate, N-methyl-D-aspartate or guanidinosuccinate. Piperine suspensions, injected intraperitoneally, 1 h before injection of the threshold intracerebroventricular dose of kainate for the induction of clonic convulsions (1 nmol), blocked these convulsions with an ED50 (and 95% confidence interval) of 46 (25-86) mg/kg. Although piperine did block convulsions, induced by kainate, the compound does not appear to act as a kainate receptor antagonist. Whole-cell currents induced by the application of kainate to spinal cord cells in primary dissociated cultures were not affected by co-application of piperine.

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.