Development of two types of calcium channels in cultured mammalian hippocampal neurons.

Calcium influx through voltage-gated membrane channels plays a crucial role in a variety of neuronal processes, including long-term potentiation and epileptogenesis in the mammalian cortex. Recent studies indicate that calcium channels in some cell types are heterogeneous. This heterogeneity has now been shown for calcium channels in mammalian cortical neurons. When dissociated embryonic hippocampal neurons from rat were grown in culture they first had only low voltage-activated, fully inactivating somatic calcium channels. These channels were metabolically stable and conducted calcium better than barium. Appearing later in conjunction with neurite outgrowth and eventually predominating in the dendrites, were high voltage-activated, slowly inactivating calcium channels. These were metabolically labile and more selective to barium than to calcium. Both types of calcium currents were reduced by classical calcium channel antagonists, but the low voltage-activated channels were more strongly blocked by the anticonvulsant drug phenytoin. These findings demonstrate the development and coexistence of two distinct types of calcium channels in mammalian cortical neurons.

A large multiple endocrine neoplasia type 1 family with clinical expression suggestive of anticipation.

We describe a large multigenerational multiple endocrine neoplasia Type 1 (MEN1) family with clinical expression suggestive of anticipation. In the second and third generations, two deceased obligate gene carriers died at the ages of 85 and 76 without the history of MEN1, whereas two other living gene carriers above the age of 65 have had no clinical evidence of MEN1 to date. In the fourth generation, eight members were affected, with four having severe MEN1-related and atypical malignancies: a case of metastatic endocrine pancreatic tumor, two cases of metastatic thymic carcinoids, and a case of spinal ependymoma. In the fifth generation, all five patients were below the age of 22 when the disease was detected. MEN1 was confirmed in the family by linkage analysis using MEN1-linked microsatellite markers and by identification of a nonsense mutation in the MEN1/menin gene. Alleotyping showed loss of heterozygosity (LOH) involving the wild-type alleles in seven tumors in the family including the ependymoma, which is the first MEN1-related case that shows genetic abnormality in chromosome 11q13, suggesting that MEN1 gene might be involved in the tumorigenesis of a subset of ependymomas. In relation to clinical anticipation, repeated expansion studies were carried out but failed to detect any expansion. We conclude that this is a unique MEN1 family and that an unknown genetic mechanism might be contributing to the anticipation phenomenon. We demonstrate in this family that all gene carriers, including the very young members, will need close and careful follow-up.

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library.

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.

Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus.

The laminar distribution of decreases in extracellular free calcium and concomitant field potentials induced by repetitive orthodromic stimulation, ionophoretic application of N-methyl-D-aspartate and quisqualate, was studied in the CA1 field of rat hippocampal slices, at two different stages during postnatal development. While stimulation-elicited and quisqualate-induced signals remain maximal in stratum pyramidale during the first postnatal month, the laminar profiles of responses to N-methyl-D-aspartate (NMDA) depend on age: the responses to this agonist are maximal in stratum pyramidale in 5-9-day-old rats and in stratum radiatum in 12-30-day-old rats. Our findings suggest that, during the second postnatal week, the apical dendrites of pyramidal neurons in area CAl become more sensitive to NMDA, which is expressed by big influxes of calcium at this level.

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o in the hippocampal slice.

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K+]o) and calcium ([Ca2+]o) were investigated with ion selective-reference electrode pairs in area CA1 of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1-5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca2+]o induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-D-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca2+ entry. When synaptic transmission was blocked by lowering [Ca2+]o (0.2 mM) and elevating [Mg2+]o (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca2+]o was lowered to 0.2 mM, without elevating [Mg2+]o. The amplitudes of the rises in [K+]o induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K+]o signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.

Voltage-dependency of the responses of cerebellar Purkinje cells to excitatory amino acids.

The voltage-dependency of the responses of Purkinje cells to excitatory amino acids was examined in rat cerebellar slices, using intrasomatic recordings with the single electrode voltage-clamp. In standard perfusion medium, the depolarizations evoked in these neurones by ionophoretic pulse applications (less than 300 ms) of L-glutamate, L-aspartate and quisqualate in their dendritic fields had underlying inward currents which did not increase or even decreased, as the holding potential was shifted to values more negative than -65 mV. This 'abnormal' voltage-dependency was still present in Mg2+ -free solution but was abolished in the presence of CsCl2 (10 mM) in the perfusion medium. When TTX (5 microM) and CdCl2 (0.1 mM) were further added to the bath in order to block regenerative conductances, thus broadening the range of the clamp voltages to more positive values than -50 mV, the current-voltage relation between -80 and 0 mV for responses to L-glutamate and L-asparate was almost linear. Our results support the view that low doses of both amino acids act on Purkinje cells essentially via the activation of receptors which are not of the N-methyl-D-aspartate type.

Effects of anticonvulsants on spontaneous epileptiform activity which develops in the absence of chemical synaptic transmission in hippocampal slices.

Spontaneous epileptiform activity (SEA) develops in area CA1 of hippocampal slices, when the Ca2+ concentration in the perfusate is lowered to 0.2 mM, at which level evoked chemical synaptic transmission is blocked. We investigated the effects of different anticonvulsants on this autonomous activity, in order to determine whether the antiepileptic effect can be ascribed to an influence on neuronal excitability. Carbamazepine was the most effective to block SEA at concentrations of 1-15 microM. Phenobarbital and phenytoin depressed SEA at concentrations of 25 microM. Valproate was effective at concentrations of 2-5 mM. Midazolam, a water-soluble benzo-diazepine agonist and the N-methyl-D-aspartate antagonists, DL-alpha-aminoadipic acid and 2-amino-7-phosphonoheptanoic acid were ineffective in blocking SEA suggesting that they exert their antiepileptic action by interference with synaptic mechanisms.

Pharmacological blockade of the globus palidus-induced inhibitory response of subthalamic cells in the rat.

The aim of the present study was to investigate, with extracellular recording and microiontophoretic techniques, the possibility that gamma-aminobutyric acid (GABA) is the transmitter substance in the pallido-subthalamic (GP-STN) pathway. Experiments were carried out in 94 rats, anesthetized with ketamine. Among the 236 recorded STN neurons, 85 were inhibited by GP stimulation. This inhibition lasted 10--20 msec (mean duration +/- S.E.M. 13.45 +/- 0.5 msec). Control stimulations located either in the internal GP or in the striatum never elicited the same type of response as they did in the STN. STN neurons were inhibited by microiontophoretically applied GABA or muscimol. Iontophoretically applied bicuculline or picrotoxin reversibly blocked the GP-evoked inhibition at doses which blocked the GABA inhibitory responses but not those produced by glycine. The results are consistent with the hypothesis that GABA is the transmitter releasted by the inhibitory pallido-subthalamic pathway, and are in agreement with recent biochemical data. Nevertheless, on a few cells, strychnine blocked the GP-induced inhibition. This results is discussed in relation to the specificty of GABA and glycine antagonists.

GABA and baclofen reduce changes in extracellular free calcium in area CA1 of rat hippocampal slices.

Reductions in extracellular free Ca2+-concentration [( Ca2+]o) result from neuronal activation and can be induced by repetitive electrical stimulation or by application of excitatory amino acids. They reflect Ca2+-movements along its electrochemical gradient into pre- and postsynaptic elements. Amino acid-induced reductions in [Ca2+]o are predominantly caused by postsynaptic Ca2+ entry. Under conditions of blocked chemical synaptic transmission, a presynaptic component of extracellular Ca2+ loss becomes apparent during stimulation of the Schaffer collateral/commissural fibers system in stratum radiatum/moleculare. GABA, both iontophoretically and bath applied, always reduces pre- and postsynaptic components of [Ca2+]o changes. Baclofen regularly affects postsynaptic Ca2+ entry and has frequently also a suppressant action on presynaptic Ca2+ entry in area CA1.

An N-methyl-D-aspartate receptor-independent excitatory action of partial reduction of extracellular [Mg2+] in CA1-region of rat hippocampal slices.

Partial reduction of [Mg2+]o from 2 to 1 mM markedly enhanced neuronal responses evoked by Schaffer collateral-commissural fiber stimulation in the CA1-region of rat hippocampal slices. The amplitude of extracellular population potentials recorded in the CA1-pyramidal cell layer and maximum dV/dt of extracellular population EPSP's recorded in the CA1-pyramidal apical dendritic layer were both increased. However, unlike findings from slices where Mg2+ was completely removed from the bathing medium, there was no spontaneous or evoked epileptiform activity, and the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (2-APV) did not antagonize the enhancement of evoked responses. These results indicate that, in addition to the participation of NMDA receptors in the epileptiform activity observed when Mg2+ is completely removed from the bathing medium, there is also an NMDA receptor-independent excitatory action of partial reduction of [Mg2+]o in hippocampal slices.

Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium.

Reduction of [Mg2+]o induced spontaneous epileptiform activity consisting of 40-100-ms bursts of population spikes in hippocampal slices. This activity disappeared from area CA1 when the connections to area CA3 were cut, but persisted in isolated minislices of area CA3. Spontaneous activity was also observed in the dentate gyrus, provided that the connections to the subiculum and entorhinal cortex (EC) were intact. In the parasubiculum and EC longer lasting epileptiform events were observed which resembled seizure-like behaviour. The epileptiform activity was completely suppressed by 2-aminophosphonovalerate (30 microM) suggesting that N-methyl-D-aspartate receptors for excitatory amino acid transmitters participate in the generation of this activity. These findings show that the EC possesses properties which permit the generation of seizure-like activity in contrast to the hippocampus where the activity resembled recurrent interictal events.

[Does the placebo effect exist in newborn infants?]. / L'effet placebo existe-t-il chez le nouveau-né?

Although comparison with a placebo is necessary to demonstrate the "true" effect of a drug, neonatologists are usually reluctant to use a placebo. The reason given is the lack of placebo effect in neonates. We studied heart and respiration rates and behaviour in normal neonates during heelstick for diagnosis of phenylketonuria. In this open randomized study we compared no treatment with an "analgesic" treatment consisting of water and sucrose. There was no difference in heart and respiration rates and behaviour between the two groups. These results do not demonstrate a "suggested" placebo effect and can in part be explained by the model and tools used to measure pain. The results do not support the non-use of placebo in drug evaluation trials in children.

[Apolipoprotein E and bleomycin hydrolase. Polymorphisms: association with neurodegenerative diseases]. / Maladies neurodégénératives et polymorphisme des gènes de l'apolipoprotéine E et de la bléomycine hydrolase.

Several studies indicate a possible association between different genes and chronic neurodegenerative diseases including Alzheimer's disease (DTA). To further investigate, we have analyzed association between the apolipoprotein E (apo E) and bleomycin hydrolase (BH) polymorphisms and three groups of elderly patients: control subjects (T) (n = 68), late-onset sporadic DTA patients (DTAst) (n = 65) and other non vascular neurodegerative diseases (MNDA) (n = 52). Apo E-epsilon4 and BH-G alleles frequencies (%) are: 8.2 (T), 31.5 (DTAst), 16.4 (MNDA) and 41.4 (T), 35.6 (DTAst). No association has been observed between carrying the G allele and DTA in epsilon4 negative subjects but, our data have confirmed the earlier reports: carrying the epsilon4 allele is a dose-dependent risk factor for the DTAst (OR: 6.0, IC 95 %: 2.6-13.7) and decrease the age of symptom onset (p < 0.005). They have also suggested that apo E genotyping may be of interest to perform differential diagnosis of neurodegenerative diseases in elderly subjects.

[Pneumopathy due to sulfasalazopyrine. A case with a challenge test]. / Pneumopathie à la sulfasalazopyrine. Une observation avec test de réintroduction.

The complications of treatment with sulfasalazopyrine(SSP) are rare. We report a case of a 68 year old patient who was hospitalised for a hectic fever with weight loss, chest pain and dyspnoea of effort. This patient had been treated for 2 1/2 months for a haemorrhagic proctocolitis with 3 gms. per day of SSP. There was a leucocytosis of 23 700/mm3 of which there were 3,318 eosinophils/mm3. The pulmonary radiograph showed a predominantly interstitial pattern at the apices in the axillary region and in the costo-phrenic angle. The broncho alveolar lavage (LBA) was normal. A thoracic scan confirmed the interstitial syndrome and the existence of pleural thickening in the upper part of the lungs. As drug induced pulmonary disease was suspected SSP was stopped. The patient became apyrexial in 48 hours without any further treatment. Thirty days later the pulmonary radiograph and the laboratory investigations had returned to 'normal and a challenge test using the drug was given whilst the patient was still in hospital. After 24 hours there was a febrile peak of 38.7 degrees C and 36 hours later there was a leucocytosis once more of 19 700/mm3 of which there were 1,100 eosinophils/mm3. The pulmonary radiograph showed diffuse bilateral interstitial opacities. The treatment was stopped and the fever disappeared within 24 hours. After a review of the literature, two points of this case were discussed in particular: the frequency of pulmonary disease occurring as a complication of SSP and the indication for challenge test.

Effects of GABA and bicuculline on N-methyl-D-aspartate- and quisqualate-induced reductions in extracellular free calcium in area CA1 of the hippocampal slice.

Decreases in extracellular free calcium ([Ca2+]o) and concomitant field potentials were recorded from the dendritic and cell body layers of the CA1 field in transverse hippocampal slices. They were elicited by tetanic stimulation of Schaffer collaterals and commissural fibers or by iontophoretic application of the excitatory amino acids N-methyl-D-aspartate (NMDA) and quisqualate (Quis). Under control conditions, decreases in [Ca2+]o were found to be maximal in stratum pyramidale (SP). In stratum radiatum (SR), 100 micron away from SP, decreases in [Ca2+]o were half the size of those observed in SP. Bicuculline methiodide, bath-applied at concentrations of 10-100 microM, enhanced the reductions in [Ca2+]o, increased the field potentials in all layers and also induced "spontaneous" epileptiform activity. In the presence of bicuculline, the decreases in [Ca2+]o were particularly enhanced in SR and were often greater than those recorded in SP. This was the case for changes in [Ca2+]o induced either by repetitive electrical stimulation or by application of NMDA and Quis. When synaptic transmission was blocked by perfusing the slices with a low Ca2+ medium, all NMDA and Quis-induced changes in [Ca2+]o were predictably reduced but there was a relative enhancement of changes in [Ca2+]o in SR with respect to those in SP. We propose that, under normal conditions, an inhibitory control mediated by GABA limits the reductions of [Ca2+]o particularly in SR. In support of this proposal, we found that bath-applied GABA had a depressant action on changes in [Ca2+]o.

Nicotine inhibits slowly inactivating K+ currents in rat cultured striatal neurons.

As shown on cultured striatal neurons recorded in whole-cell configuration, both acetylcholine (in the presence of atropine) and nicotine reduced voltage-dependent outward currents. Although, at early postnatal ages, outward currents in these cells are mainly carried by rapidly and slowly inactivating K+ channels, these inhibitions resulted from a selective and reversible effect on the slowly inactivating K+ conductance (IK+). This action was blocked by the nicotinic antagonist dihydro-beta-erythroïdine and reproduced by nicotinic agonists. When neurons were recorded under current-clamp conditions, nicotine increased reversibly their firing rate generated by step depolarizations. Therefore, in addition to its well documented muscarinic effects, acetylcholine also controls K+ currents in striatal neurons through mechanisms mediated by nicotinic receptors.

Nicotine-induced inhibition of neuronal phospholipase A2.

A protective effect of nicotine against glutamate-induced neurotoxicity has previously been reported in cultured striatal and cortical neurons. The aim of this study was to investigate whether nicotine also inhibits glutamate-evoked arachidonic acid release from cultured striatal neurons. (-)-Nicotine selectively inhibited the release of [3H]-arachidonic acid induced by the joint stimulation of alpha-amino-3-isoxazol-5-propionic acid and metabotropic receptors, whereas the response evoked by the sole activation of N-methyl-D-aspartate receptors remained unchanged. The inhibitory effect of (-)-nicotine was not mediated by nicotinic receptors because it was neither reproduced by acetylcholine (in the presence of atropine) or 1,1-dimethyl-4-phenyl piperazinium, nor reversed by dihydro-beta-erythroidine or hexamethonium, two central nicotinic receptor antagonists. (-)-Nicotine, which induced rapidly desensitizing inward currents in 17% of striatal neurons, did not alter the alpha-amino-3-isoxazol-5-propionic acid-evoked currents. Moreover, (-)-nicotine did not inhibit the accumulation of inositol phosphate derivatives induced by agonists of glutamate metabotropic receptors. In fact, using the fluorogenic phospholipase A2 substrate 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine, (-)-nicotine was found to inhibit both particulate and soluble phospholipase A2 activities from striatal neurons. Therefore, (-)-nicotine can modulate a neuronal response (arachidonic acid release) evoked by glutamate but this process is not involved in the neuroprotective effect of the drug on glutamate-induced neurotoxicity.