Neurotrophin-3 promotes the survival of oligodendrocyte precursors in embryonic hippocampal cultures under chemically defined conditions.

embryonic rat hippocampal cells were cultured in basal medium with or without addition of the neurotrophin NT-3. After culturing in these extreme conditions, the effects of NT-3 on the neuronal and on the glial components were assessed. Neurons survived even in the absence of NT-3 but failed to reach terminal differentiation. On the other hand, NT-3 promoted the survival but not the proliferation and/or the differentiation of oligodendrocytes precursors present in the same culture, an effect that was reversed by the addition of neutralizing antibodies against NT-3. Type I or II astrocytes were not affected by NT-3. These results reinforce the role for NT-3 in oligodendrocyte lineage development and allow to dissect the roles of this neurotrophin in survival and in proliferation/differentiation of oligodendrocytes.

Automatic speech recognition in vitreo-retinal surgery. A project for a prototypal computer-based voice-controlled vitrectomy machine.

In the past half decade automatic speech recognition techniques, software and hardware technology have matured enough to support sophisticated medical applications. The project described aimed at introducing a computer-based, voice-controlled prototype system in a simulated vitreo-retinal surgery scenario. The aim was to provide the surgeon with a tool that could significantly improve the quality and ease of work and shorten the duration of intervention. The speech recognition system allows voice entry of simple commands to simulate surgical instrument control, including the infusion pump, vitreous cutter and diathermy. The project relies on a Markov-based, speaker-dependent, commercial isolated-word recognizer, and consists of a specific recognition vocabulary and application software, created and developed by the authors. Results have been encouraging. The system performed well under the test conditions, proving robust, simple to use and accurate (over 97% average word recognition rate). On the basis of their experience, the authors believe that automatic speech recognition technology, though suffering from some limitations such as the need for training, speaker dependence and a relatively small vocabulary, and requiring extensive testing under operating conditions, merits further development and opens new perspectives for a possible new generation of surgical instruments.

Neurofilament-negative neurones exhibit HVA Ca2+ currents with faster inactivation kinetics.

The whole cell configuration of the patch clamp technique was used to study the biophysical and pharmacological properties of voltage activated Ca2+ channel currents on hippocampal neurones cultured in the presence or absence of foetal calf serum. In the presence of serum cells were intensively immunostained with neurofilament (NF) antibodies. In the absence of serum, cells were non-immunoreactive; they were identified as neurones by their ability to generate action potentials. NF negative cells still expressed both high (HVA) and low (LVA) voltage activated Ca2+ channels. However, in comparison to NF positive neurones, HVA Ca2+ currents present in NF negative neurones had a faster inactivation kinetics.

Selective increase in T-type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy.

The properties of voltage-dependent calcium currents were compared in thalamic neurons acutely dissociated from a rat model of absence epilepsy, designated as Genetic Absence Epilepsy Rat from Strasbourg (GAERS), and from a Nonepileptic Control strain (NEC). Two populations of neurons were isolated: thalamocortical relay neurons of the nucleus ventrobasalis (VB) and neurons of the nucleus reticularis (RT) of the thalamus. Whole-cell patch-clamp analysis demonstrated an increase in the amplitude of the calcium (Ca2+) current with a low threshold of activation (IT) in RT neurons of GAERS in comparison to that of the seizure-free rat strain (-198 +/- 19 pA and -128 +/- 14 pA, respectively), whereas the sustained component (IL) was not significantly different. The kinetic properties, voltage dependence, and basic pharmacological sensitivity of the Ca2+ conductances were similar in the two populations of neurons. The amplitude of both IT and IL in RT neurons increased after birth, and differences in IT between GAERS and NEC attained significance after postnatal day 11. At corresponding ages, the Ca2+ currents in VB thalamocortical relay neurons were not altered in GAERS in comparison to those in NEC. We conclude that the selective increase in IT of RT neurons enhances the probability of recurrent intrathalamic burst activity, thereby strengthening the synchronizing mechanisms in thalamocortical systems, and, as such, represents a possible primary neuronal dysfunction that relates to the pathological increase in synchronization underlying the generation of bilateral and synchronous spike and wave discharges (SWDs) in an established genetic model of generalized epilepsy.

Inactivation of single Ca2+ channels in rat sensory neurons by extracellular Ca2+.

1. Single Ca2+ channels conducting 20 mM Ba2+ from adult rat dorsal root ganglion cells were characterized using the two-electrode patch-clamp technique configuration. 2. Channels demonstrating specific characteristics of conductance, voltage dependence and dihydropyridine sensitivity were classified as high-threshold or L-type Ca2+ channels. 3. Mean single-channel current in 20 mM Ba2+ did not show inactivation, but inactivation occurred when using Ca2+ as a permeating ion. 4. Stimulus protocols were delivered alternately in the cell-attached and whole-cell electrode, while recording single-channel activity and total Ca2+ current simultaneously. 5. A mean single-channel Ba2+ current from a stimulated patch did not show inactivation. However, stimulation of a physiological whole-cell Ca2+ current induced a marked inactivation of mean single-channel Ba2+ current. 6. Complete Ca2+ current block by the addition of 200 microM Cd2+ in the external solution removed single-channel inactivation in patches stimulated through a whole-cell electrode.

Fluoride reversibly blocks HVA calcium current in mammalian thalamic neurones.

The effect of intracellular fluoride ions on voltage-dependent calcium currents was tested during whole-cell voltage-clamp recordings in thalamic neurones acutely dissociated from young adult rats. It is demonstrated that 5-30 mM intracellular fluoride selectively and reversibly suppresses the high voltage-activated, dihydropyridine-sensitive calcium current, without affecting the transient, low voltage-activated calcium current. Intracellular diffusion of a fluoride-free solution restores the blocking effect on the slow inactivating current induced by a transitory fluoride perfusion obtained by filling the patch microelectrode tip with caesium fluoride.

P2Y purinoceptors in normal NIH 3T3 and in NIH 3T3 overexpressing c-ras.

The ability of purinergic agonists to induce Ca2+ responses has been tested in two lines of murine fibroblasts: normal NIH 3T3 fibroblasts and NIH 115.14, a clone expressing high levels [1] of the c-ras protooncogene. Both kinds of cells are responsive to ATP in the range 1 microM-1 mM; ADP and ATP gamma S are almost as potent as ATP, while AMP is unable to elicit a response. Ca2+ measurements performed in single cells by image analysis show great variability among cells but in each individual responding cell the Ca2+ rise occurs in an all-or-none fashion. The transient Ca2+ response does not depend on influx from the extracellular medium. Electrophysiological experiments reveal the activation of an outward current (at -50 mV) by ATP, probably due to Ca(2+)-activated K+ channels, confirming the absence of a substantial Ca2+ influx. Finally, stimulation by ATP produces a small but significant increase in the production of inositol phosphates. These results indicate that these cell lines possess purinergic receptors which are not integral membrane channels and which are coupled to InsP3 formation and may be therefore classified as P2Y.

[Ca2+]i recordings and the inactivation of the high-voltage activated Ca2+ currents in the adult rat sensory neuron.

Fast, single cell, measurement of the average cytosolic [Ca2+]i with the Fura-2 technique suggests that the depolarization induced [Ca2+]i rise is entirely due to entry through the voltage-activated Ca2+ channels. Involvement of a Ca(2+)-induced Ca(2+)-release process is not evident. Under physiological cytosolic buffering the current-induced [Ca2+]i rise persists for seconds and decays exponentially (tau = 7 s). Analysis of the [Ca2+]i changes during two-pulse protocols indicates that the purely voltage-dependent inactivation of the high voltage-activated (HVA) channels, in the range -80/+70 mV, is a slow process (0.2-1 s) which removes at most 40% of the current. On the contrary, Ca(2+)-dependent inactivation acts in a fast way and it is therefore responsible for the fast inactivating phase of the current; this phase disappears under sustained [Ca2+]i loads, and reappears when redistribution of free Ca2+ takes place. A suitable correction may be devised to compensate for the Ca(2+)-dependent inactivation.

Characterization of Ca2+ transients induced by intracellular photorelease of InsP3 in mouse ovarian oocytes.

Ca2+ transients (measured with Fluo-3) were induced in single mouse ovarian oocytes by photolytic liberation of InsP3. The time course of cytosolic Ca2+ changes induced in this way is composed of distinct phases: upstroke, fast decline, slow declining plateau and fast decline to rest level. All the phases reflect mainly intracellular redistributions of the ion and not influx, since they are not strongly dependent on external Ca2+ or on changes in transmembrane potential. Often sustained Ca2+ oscillations followed the first InsP3-induced Ca2+ transient. These persisted for several minutes in the absence of external Ca2+. The initial rate of Ca2+ rise and the delay between the InsP3 stimulus and Ca2+ upstroke are correlated with the amount of liberated InsP3. A second InsP3 stimulation, applied during the plateau, causes only small Ca2+ elevations, lacking the upstroke phase. A second, full sized, transient could be elicited only after a complete return to the basal level. Vanadate, applied intracellularly, appeared to inhibit the re-uptake phase into the stores, stabilizing the plateau level. The present observations suggest that in mouse oocytes the InsP3-sensitive stores provide only a small and graded Ca2+ release which may then act as a trigger for a more substantial Ca(2+)-induced Ca2+ release (CICR) process.

Cytosolic calcium responses induced by photolytic release of 1,4,5-inositol trisphosphate in single human fibroblasts.

We have used the whole cell technique to microinject human fibroblasts with either 1,4,5-inositol trisphosphate (InsP3) or 'caged' InsP3, in order to study the mechanisms of transmembrane signalling related to mitogenic stimulations. Cytosolic Ca2+ elevations in response to 1,4,5 InsP3 diffusing from the patch pipette were difficult to detect, while 1,4,5 InsP3, photoreleased after loading the cell with its inactive precursor, was capable of generating not only a single cytosolic Ca2+ rise but sometimes triggered an oscillatory calcium response, similar to that often observed under mitogenic stimulation. We estimated that less than 100 nM InsP3 was sufficient to generate Ca2+ responses. The Ca2+ rise produced by the photoreleased InsP3 could fully activate the K+ channels present in the plasma membrane of human fibroblasts.

Cross-talk between receptors coupled to calcium currents in adult but not neonatal rat sensory neurons.

In adult rat sensory neurons Ca2+ currents were studied with the whole-cell patch-clamp technique. Two categories of neuromodulators, known to activate different 2nd messenger systems: 1) angiotensin II (AII), bovine serum albumin (BSA), Acetylcholine (ACh) and 2) GABA, stimulated the low-voltage activated (LVA) and inhibited the high voltage activated (HVA) currents, respectively. The simultaneous application of the two types of drugs failed to inhibit the HVA current via a putative cross-talk between the two 2nd messengers.